DNA Structure and Genomics (pp 7-22) Flow of Genetic Information [pp 7 — 10) |. Distinguish between gene as conceptual unit of information and DNA as the physical material in which that information is encoded. @ Gene: The instructions for making a component of the cell i. Usually a protein or subunit of a protein b. The geneis physically embodied in DNA Analogy: Genes are to words as DNA is to a book Il, Describe the central dogma of molecular biology, including the informat nal roles of DNA, RNA, and protein. T™ —> RNA ——— > Proteins Transcription Translation Replication a. DNA= storage b. RNA = copies Protein = functional form I Define the processes of replication, transcription, and translation. a. Replication: DNA > new DNA copies ‘When cells undergo mitosis, the daughter cell needs a complete genome b. Transcription (T/sc): DNA > RNA i. Only the pieces of DNA needed are transcribed at a time & Translation (1/s)): RNA > Protein IV. Define the terms genome and chromosome. a. Genome: Entire collection of genes for a given organism b. Chromosome: Many genes spread along a single DNA molecule ‘The human genome has 23 pairs of chromosomes {ple attributes of the genetic code. Distinguish between degeneracy and un-ambiguity in V. Describe the pr the genetic code. a Genetic code is made of non-overlapping sets of 3 nucleotides b. Degeneracy: A3 nucleotide system with 4 bases yields 64 possible combinations (4 = 64) Therefore, there can be multiple codes for the amino acids and stop codons Un-ambiguity: Each code specifies for a single amino acid, not muttiple. VI. Given a DNA or RNA sequence, predict the corresponding protein structure. Using the table provided, read the code from 5 to 3° b. Find the first letter in the left column © Find the second letter in the top row d._ Find the third letter in the right column TL Page 1VII. Given a protein structure, identify one of more corresponding DNA or RNA sequences. a. Same as objective VI, but work backwards. b. Be careful to note that most amino acids have multiple potential sequences, due to degeneracy DNA Structure (pp 11 — 18} 4s 1. Describe the chemical bond that joins adjacent nucleotides in a nucleic acid polymer. . a. Phosphodiester bond \ / i. Bond of the 3’ OH of one nucleotide and oe theS’ phosphate of another nucleotide I. Given a diagram of the chemical structure of a nucleic acid, identify the 5’ and 3 ends. a. Primary structure > Il, Identify and describe the position of the bases, sugars, and phosphates in the double helix model of DNA structure. a. Secondary structure of DNA b. The sugar-phosphate backbone makes up the os outer portions of the double helix. ne The nucleic bases point inward, perpendicular to the axis of the double helix IV. Describe the forces that contribute to the stability of the double helix. Explain the cooperativity of DNA denaturation and renaturation by reference to these forces. Predict the relative melting temperatures of DNA molecules with various (guanine + cytosine) contents. a. The two stabilizing forces are: i. Hydrogen bonds: Found between the base pairs (between strands) 1. 2hydrogen bonds form between Adenine and Thymine 2. 3hydrogen bonds form between Cytosine and Guanine Hydrophobic interactions: Also between base pairs (on the same strand) 1. Bases are internal because of hydrophobic interactions 2. Thisis the major force in holding DNA structure together b. Denaturation: Separation of the two strands Renaturation: Rejoining (hybridization or re-annealing) of the two complementary strands ‘This is an important feature of DNA, that is driven by the hydrophobic forces This also leads to multiple conformations 1. DNA-DNA 2. DNA-RNA =transcription 3. RNA-RNA =tRNA d. Melting temperature: Because cytosine and guanine have more hydrogen bonds, they melt at a higher temperature i. Melting temperature can be used to calculate general nucleotide composition, TL Page 2V. Given the sequence or composi complementary strand. Be mindful of ifthe complementary strand is DNA or RNA \n of a single strand of DNA, predict the sequence or composi VI. Define the components and structure of a nucleosome and the role of histone H1. Describe the multiple levels of DNA packaging from the nucleosome to chromosome. Define heterochromatin and euchromatin. a Histones Hi: Wraps linker region of DNA H2A, H2B, H3, and H4: The “core” ™ mi mgone histones Very basic > binds with the acidic DNA 1. The positive charge interacts with the negatively charged phosphate backbone. “ewerome b. Nucleosome i. Components 1. Octameric core: 2 of each core histone = 8 histones 2. DNA (2tums around the core Nucteosome histones) 3. Linker DNA: Region between nucleosomes a. Wraps around a H1 histone & Levels of DNA packaging (tertiary structure) i. Wrapping around histones -> chromatin fiber 1.100 angstroms in diameter 6 nucleosomes - solenoid 1. Coil; 250 angstroms in diameter iii, > chromatin 1. Heterochromatin = Tightly packed chromatin that is poor in gene concentration Constitutive heterochromatin i. Never transcribed Highly repetitive Replicates late in S phase iv. Found near centromeres and at the ends b, Facultative heterochromatin i. Generally active, but is turned off or becomes genetically inert during some part of organism development e.g, the late replicating or inactive x in females 2. Euchromatin = Lightly packing form of chromatin that is rich in gene concentration a Sometimes a sign of active transcription b. Replicates early in S phase © Foundin both eukaryotes and prokaryotes iv. > chromosome TL Page 3VIL. Define and/or describe the following aspects of chromosome structures: homologs, sister chromatids, telomere, centromere vs. kinetochore, P-arm, Q-arm, euchromatin vs. heterochromatin. ‘a Homologs: A pair of chromosomes (one from the mother and one from the father) Homologs contain different alleles of the same gene Sex chromosomes are not homologs . Sister chromatids: Identical copies of a chromosome connected by a centromere i. Sister chromatids contain the same alleles of the same gene Telomere: TTAGGG repeats common to all human chromosomes found at thetips Implicated in aging There are also “subtelomeric repeats” just inside of those. Inside of those is the unique sequence, which is the target of FISH probes iv. Telomeric recombination is high 1. Only place where males have a higher rate than females 4d. Centromere: Primary constrictions of the chromosome and underlying DNA Interacts with microtubular proteins during cell division Surrounded by heterochromatin Variable in position kinetochore: The protein structure on either side of the centromere P-arm: Short arm Qarm: Longarm Euchromatin vs. Heterochromatin: See objective VI, above, Genomics (pp 19 - 22) 1. Define genome and recognize the approximate sizes of viral, bacterial, yeast, and human genomes. @ Genome: The total “distinct” DNA in a cell of a given organism b. Approximate sizes of haploid genomes (bp / mm) Viral: 7,600 / N/A Bacterial: 4,000,000 / 15 Yeast: 14,000,000 / 4.6 iv. Human: 2,900,000,000 / 990 |, Define the terms haploid and diploid and know how those terms correspond to human cells. a. Haploid: Cell with one copy of each chromosome i. 23in humans i.e. gametes, which contain only one member of each chromosome pair b. Diploid: Cell with two copies of each chromosome 46 in humans i.e. somatic cells, which contain chromosome pairs & Polyploidy: Abnormal cell with more than two copies of each chromosome TL Page 4Ill, Describe the informational content of genomes, including: coding regions, non-co DNA regions—including the various types of spacer DNA. a. Informational content The human genome has 6.4x10° base pairs per diploid human cell. 1. Thisis about 6 feet of DNA Only about 25% of the human genome encodes proteins. 1. There are 30,000 protein coding genes in the human genome Packing ratio of DNA is5,000 to 1 b. Coding Regions Protein coding genes: Encode for enzymes, structural proteins, etc RNA-coding genes: Encodes for rRNA, tRNA, ribozymes, et. Pseudogenes: Encodes for evolutionarily outdated (inert) genes that are still found in the genome Non-coding regions i. Regulatory sequences: These arethe sequences that are not transcribed or translated, but control the transcription of a gene or set of genes 1. Promoters, silencers, and enhancers d._ Spacer DNA regions — introns i. Unique sequence spacer DNA 11. Appears once in genome Repetitive DA: Can be repeated 1000s of times and make up “55% of the genome 1. Dispersed Repetitive DNA — Accounts for’"45% of the genome .Transposons — DNA sequences that jump into other locations in the genome, which can lead to dysfunction b, SINES (short interspersed elements): 90-500 bp long & LINES (long interspersed elements): < 7000 bp long 2. Satellite (a.k.a, tandem) Repetitive DNA — Accounts for “10% of the genome Repeated in tandem (next to each other) i. Microsatellite: 1-13 bp repeat unit; blocks of DNA 100s of bp long Minisatellite: 14-500 bp repeat unit; blocks of DNA 1000s of bp long a-satellite: 171 bp repeat unit; blocks of DNA millions of bp long 1. Located near centromeres iB regions, and spacer TL Page 5DNA Replication (pp 23 - 33) 1. Describe the roles of template and primer in DNA synthesis. a. Template: The basis upon which DNA is replicated Atemplate is always needed Replication is never de novo b. Primer: An RNA segment (usually) necessary for replication DNA polymerases require a short nucleotide sequence already in place 11. DNA polymerases must have a free 3-OH group available for attachment of incoming nucleotide RNA polymerases do not require a primer Il, Define semi-conservative replication. OG SOO B® SG SG a. Each parent strand is a template b. Each parent strand is conserved (not degraded) © See diagram > Rk 1g and lagging strands) arises from the 5’ G OGIO OK x to 3’ direction of polymerization. ‘a Because the strands are anti-parallel (one is to 3' left to right, and the other is’ to 3’ right to left) Therefore one strand (the leading strand) is made by continuous replication The other strand (the lagging strand) has to be laid down in fragments; Okazaki fragments 1. These fragments will ultimately be joined together IV. Recognize or draw diagrams representing bi-directional replication from specific origins for linear and circular genomes. Prokaryote Eukaryote V. Identify the primary events in the following stages of re (leading and lagging strands), termination, segregation. EXPLANATION IS|FORIE)COIi a. Pre-priming Regulated step — Once pre-priming occurs, replication must be carried out completely Recognition of origin and unwinding of starting site by helicase b. Priming/Initiation ‘Addition of primer to allow for elongation to occur ©. Elongation i. Leading strand 1. Continuous’ to ¥ 2. DNA polymerase Ill—two enzymes in one protein gstrand 1. Discontinuous —5' to 3’ direction /initiation, elongation TL Page 62. Primase a. Lagging strand needs to be primed often because of the discontinuous nature of lagging strand replication 3. DNA polymerase Il a. Synthesizes DNA by extension from the RNA primers 4. DNA polymerase — three enzymes in one protein DNA ligase — Joins fragments Details of lagging strand synthesis DNA poll synthesizes the new strand until it reaches an RNA primer b. DNA poll removes the RNA nucleotides (5’ to 3’ exonuclease) one at a time and attaches the correct deoxynucleotide (5’ to 3° polymerase} DNA poll has a3’ to5’ exonuclease then proofreads the new deoxynucleotide d._ DNA ligase seals the “nick” between the two fragments Replication Fork 1. Helicase 2. 958 3 Gyrase d. Termination i. Occurs when the two replication forks meet (in circular or polycistronic DNA) e. Segregation i. Done by topoisomerases VI. Define the roles and attributes of the following prot. Topoisomerase, gyrase, helicase, SSB. 5: primase, DNA polymerase, DNA ligase. ‘a. Primase - Makes an RNA primer anywhere near an origin of replication i. An RNA polymerase required for DNA synthesis 1 RNA polymerases do not need a free 3-OH . DNA polymerases ALWAYS requires a primer — needs free 3'-OH for attachment. No proofreading of RNA primer Must be removed later from both leading and lagging strands b. DNA polymerase TL Page 7i. DNA polymerase Il (leading strand) 1.5’ to 3’ DNA synthesis (DNA polymerase enzyme) ‘a. Makes the DNA polymer by extending from the 3-OH of the RNA primer 2. 3'to5! exonuclease a. The proofreading function — Removes an incorrectly incorporated nucleotide from the newly synthesized strand at the free 3-OH end i. Exonucleases work on the ends of the DNA DNA polymerase! (lagging strand) 1. 5’ to 3’ DNA synthesis — DNA polymerase enzyme 2. 3'to5! exonuclease — Proofreading function like DNA polymerase Il Removes mismatched nucleotides in 3 to 5’ direction 3. 5 to 3’ exonuclease —Snowplow functions Removes the RNA primers in theS’ to 3’ direction (on both strands) b. Not in DNA pol It © DNA ligase — Seals “nicks” between 2 fragments (on both strands) ‘Where the RNA primers were removed and replaced with deoxynucleotides d. Topoisomerase — Separates linked circles i. Accomplished by nicking and breaking either 1 or both strands of DNA 1. To separate two pieces of circular DNA, both are clipped & Gyrase— A helicase that unwinds in front of the fork Relieves the “over winding” by putting in “negative” unwinding fi, Drugtarget (ciprofloxacin) f. Helicase — Unwinds the DNA helix i. Separates parent strands g SSB —Single stranded DNA-binding protein i. Binds to the ssDNA strands to prevent re-annealing ~ Eukaryotic process © Packaging + DNA must be unpackaged from chromatin > replication > repack © Multiple origins + Each chromosome has multiple origins + Eacho in is initiated once during each cycle ‘+ CANNOT replicate DNA multiple times per cycle— lke E. coli © Eukaryotic DNA polymerases + Priming/initiating en2yme— DNA polymerase a ‘+ Part of complex that initiates DIVA synthesis ‘© Has primase activity — puts in RNA primer ‘+ DNA polymerase activity © Needs free 3-OH © Does not have a proofreading function + Major replicative enzyme —DNA polymerase 6 (acts like DNA pol Il) ‘© Does leading and lagging strands and 3' to5’ proofing TL Page 8+ DNA repair synthesis enzymes (6-7 other DNA polymerase) ‘+ Some involved in repair ‘+ Some involved in removing RNA primers from leading and lagging strands luding the role of 3’ to 5’ exonuclease and 5’ to 3’ ‘mechanisms of DNA polymerases, exonuclease activities. a. See objectives V and VI, above. VIII. Describe the problem encountered by lagging strand synthesis at the ends of linear DNA molecules. Identify the strategy (including the novel features) by which telomerase addresses that problem. Problem — There is no free 3'-OH at the end of the chromosome, so it cannot be primed RNA primer can start at the end, but once itis gone, DNA pol can’t bind b. Solution - Telomeres = Repeat sequences at the end of the chromosomes i. Telomerase (telomere synthetase) extends the 3' ends of the parent strands 1. Components of the enzyme a. Portable RNA template b. Reverse transcriptase activity vu. Implications 1. Cell aging— as the cell ages, telomerase activity decreases > cell death 2. Cancer cells — telomerase is always active, therefore cells do not die 1X. Identify the primary points at which the cell cycle is regulated. a G checkpoint - Start Determines if replication is going to occur (if started, it will complete) 1. Are conditions OK? 2. Does the DNA need repair first? b. Ga checkpoint Maaoase Is replication complete? (G2 checkpornl he chp cit Is the cell ready to divide? Metaphase checkpoint Areall the chromosomes condensed? : IF everything is OK, the cells divide % et (START) TL Page 9DNA Recombination, Mutation, and Repair (pp 34-44) DNA Recombination 38) 1. Define the terms syntenic genes, alleles, co-segregation, and recombination. ‘a. Syntenic genes - Genes on the same chromosome b. Alleles — Different forms of a gene present in a population i. Diploid organisms often have different alleles of a gene & Co-segregation —Syntenic alleles tend to stay together through generations 4d. Recombination — Occasionally co-segregation is disrupted resulting in a new combination of alleles on a chromosome Ml, Describe the le features and genetic implications of homologous recombination. ‘a. Exchange of DNA fragments between homologous chromosomes b. Occurs during crossing over (during meiosis) Homologous chromosomes align SDNA strands break and rejoin & Leads to additional genetic diversity i. Allows for experimentation — MAY result in increased fitness (as seen in evolution) Il, Describe how recombination frequencies among given sets of genes are used to indicate the relative distances between genes. a. The farther apart genes are, the higher the rate of recombination b. Experiments can be doneto determine likelihood of recombination and therefore the relative distance IV. Define the concept of transposition. Describe the genetic impact of transposon insertions and of homologous recombination between two transposons. a Transposition is the movement of transposons from one portion of a geneto another i. Made up “45% of DNA; About 3% actually carries protein-coding genes b. Potential effects (page 37 of the coursepack has diagrams) i. Insertional inactivation of a gene — The transposon gets inserted into a functional gene and disrupts the function ii. Insertional activation of a gene—The insertion of the transposon activates a gene that would not otherwise be expression — inappropriate expression, Deletion of a gene— Occurs if two transposons recombine cleaving the segment in between © Other biological impacts i. Bacterial transposons often carry antibiotic resistance genes Viruses insert their DNA like transposons and can cause the effects found above Allows for extensive variability the heavy and light chains of immunoglobulins DNA Mutation and Rey Et 44) 1. Describe the process by which physical damage to DNA becomes fixed as a permanent mutation. a. Sometimes mutant strands are used as templates > incorrect sequence on the daughter strands > leads to the exponential growth of incorrect genes Ml. Describe how domination, depurination, intercalating dyes and other chemicals, and radiation damage results in mutations. Spontaneous mutations — Replication errors (Base mismatches) TL Page 10b. Deamination — cytosine can spontaneously deaminate to uracil, which is replaced by a thymine (because uracil is not a DNA base} & Depurination — Clipping of a purine base (base is gone, but the backbones intact) > the body guesses what should be in the empty spot > mutation Transition mutation — Purine > Purine OR Pyrimidine > Pyrimidine Transversion mutations — Purine -> Pyrimidine OR Pyrimidine -> Purine Oxidative damage — Hydroxylation of carbons - shape change — mispairing Alkylation — Adding of methyl or ethyl groups > Improper hydrogen bonding Nucleotides (nucleoside) analogs — Used in drug therapies > mismatching > cell death Intercalating dyes — Inserts between bases and stretches a single strand New bases insert opposite the dye -> frame shift mutation i. Radiation damage i. UV irradiation — Leads to pyrimidine dimer formation 1. DNA structure is skewed > cannot base pair correctly -> mutations ii. lonizing radiation (X-rays, radioactivity) — Leads to dsDNA breaks-> chromosome translocations Il, Distinguish between cell-cycle arrest and apoptosis as cellular responses to DNA damage. a. Cell-eycle arrest — Stopping at a cell cycle checkpoint to assess damage and make repairs b. Apoptosis — When the cell is severely damaged/beyond repair > cell “suicide” IV. _ Describe the general features of the following DNA repair mechanisms, and human geneti associated with defects in these mechanisms. a. Direct repair of alkylation and UV photoproducts Dealkylation enzymes: Remove methyl and/or ethyl groups Photoreactivation of pyrimidine dimers (prokaryotes) — Use of visible light to fix dimers 1. Humans use nucleotide excision repair for pyrimidine dimers b. Base excision repair — Acts on single bases that are damaged i. DNA Ne glycosylase - Removes damaged base by cleaving N-glycosidic bond (leaves backbone) 1. Creates an AP site (Apurinic— missing a purine; Apyrimidic - missing a pyrimidine) ‘AP endonuclease — Cleaves the backbone (removes ribose-P) DNA polymerase/DNA ligase - Fills the gap; seals the “nick” ©. Nucleotide excision repair pathway — Repairs larger regions of damage i. Endonucleases and exonucleases “patrot” DNA > remove a short piece surrounding the damage > DNA polymerase fills in the gap -> DNA ligase seals the “nick” Patients with xeroderma pigmentosum cannot do excision repair (very sensitive to UV light) d. Mismatch repair i. fstrands are mismatched, the parent strand is deemed “correct” 1. Parent strand is methylated in various locations so it can be identified 2. Thereis a lagtime to allow for proofing before methylation ii. Hereditary nonpolyposis colorectal cancer (HNPCC) ~ Genetic defect in mismatch repair 1. Makes a person more susceptible to colon cancer e. dsDNA break repair Very error prone and mutagenic > chromosome translocations > incorrect reassembly BRCA1 and BRCA2 are key human genes that participate in recombination repair 1. Women with defects in either have >80% chance of developing breast cancer TL Page 11Mitosis and Meiosis (pp 45 - 58) 1. Compare and contrast the stages of mitosis and meio: Mitosi Meiosi Occurs in somatic cells ‘Occursin germ line cells (gonad) Takes about an hour Female begins at 4 months gestation, I* division is complete at ovulation, 2% division is complete at fertilization -Male beings at puberty, complete in 20 days Chromosomes do not pair Homologous chromosomes pair (prophase I) Usually no chiasmata or crossing over ‘Chiasmata and crossing over always occurs T cell division produces 2 daughter cells 2 cell divisions produce 4 daughter cells Wo change in chromosome number Chromosome number reduced to one of each pair (haploid set in each daughter cell) No changein gene content ‘All daughter cells may be genetically different, due to segregation oF chromosome pairs and crossing over between homologues Ml, Determine the “n” and “c” content of each state of mitosis and meiosis. nthe haploid number of unique chromosomes of an organism Determined by counting the number of centromeres For humans: 1n = 23 chromosomes b. ¢—the haploid number of copies of the unique DNA content Determined by counting the number of chromatids in the cell For humans: 1¢ = 23 copies IIL, List in correct sequence the stages of mitosis (somatic cell each stage (focus on the bold and/or italicized content). Interphase — Between divisions i. Atypical cell spends most of its life in interphase doing whatever it is programmed to do 1. Thisis called Go which is an indefinite Gy G, phase (gap 1) - 2n, 2c Interval between mitosis and replication 2. Cell carries on activities preparing to duplicate its DNA content ili, S phase (synthesis) - 2n, 3.4¢ 1. Cell replication occurs, duplicates its DNA iv. Gp phase (gap 2)- 2n, 4c Interval between thes phase and next mitosis 2. Some DNA repair occurs 3. Cell contains 2 identical copies of each of the 46 chromosomes ‘Identical chromosomes referred to as sister chromatids b._M phase — Mitosis (cell division) -> content returns to 2n, 2c i. Prophase 1. Chromosome condense 2. Mitotic spindle/centrioles begin to form 3. Each chromosome now contains two strands of dsDNA (sister chromatids), which lie parallel to one another and are connected together at one spot by the centromere ji, Prometaphase n) and the chromosomal events that occur in TL Page 121. Nuclear membrane disappears 2. Kinetochores (attached to the centromere) mature and attach to the spindle fibers to form kinetochore microtubules a. The remaining tubules in the spindle are polar microtubules b. The outsidetubules are astral microtubules Metaphase 1. Fully condensed chromosomes line up along the metaphase plate 2. Spindle fibers begin to contract 3. Chromosomes easily visible with LM iv. Anaphase 1. Centromeres divide as spindles begin to pull the sister chromatids toward opposite sides ofthe cell The sister chromatids are now referred to as individual chromosomes v. Telophase 1. Two nuclear membranes form while spindle fibers disappear 2. Chromosomes condense as the cell returns to interphase vi. Cytokinesis — splitting into two daughter cells > return to G, of interphase IV. List in correct sequence the stages of meiosis (germ cell division) and the chromosomal events that occur in each stage (focus on the bold and/or italicized content). a. Overview Meiosis 1 Reductive division (reduced cells from 2n to 1n) Meiosis il— Equatorial division (1n, 2c > 1n, 1¢) b. Interphase | i. Replication of chromosomal DNA occurs (similar to mitotic interphase) © Prophase Chromatin strands condense as the homologous pairs line up Synapsis occurs 1L._ The pairing of chromosomes where chiasmata (chiasma) form a. This is how crossing over (recombination) occurs in meiosis ili, 5 stages of prophase! 1. Leptotene— Chromosomes being to condense 2. Zygotene—_Homolog pairs, telomere first and zip down, synaptonemal complexes form 3. Pachvteié - crossing over occurs Homologs separate, remain attached at chiasmata 5. Diakinesis — Separation of homologs d. Metaphase! Complete formation of spindle and two centromeres of each bivalent (linked homologs) lie on the equatorial plane (they line up on the metaphase plate) e. Anaphase! i. Chiasmata disappear and the homologous chromosomes are pulled by the spindle fibers toward opposite poles of the cell f. Telophase! i. Begins when the chromosomes reach opposite sides of the cell TL Page 13,New nuclear membrane begins to form -> Cytokinesis (uneven in females > polar bodies) Each daughter cell contains the haploid number of chromosomes and each has two sister chromatids g. Interphase it Very brief 'No replication of DNA occurs Different from interphase | and mitotic interphase hh. Prophase I! The cell contains only the haploid number of chromosomes Chromosomes condense and the nuclear membrane disappears New spindle fibers are formed i, Metaphase it i. Chromosomes line up at the metaphase plate ‘Anaphase It Centromeres of sister chromatids split and migrate to the poles Sister chromatids may not be identical due to crossing over k. Telophase it Begins when sister chromatids reach opposite poles of the cell New nuclear membranes form —> Cytokinesis In males > 4 functional daughter cells iv. In females > 1 functional daughter cell and 2-3 polar bodies Female Meio: ‘Commences Early embryonic life (3°-4" week) Duration of Meiosi 10-50 years (only complete after fertilization) ‘Gametes/Meiosis 4 spermatids ‘Lovum and 2-3 polar bodies Gamete Production | 100-200 million per ejaculate ‘Lover per menstrual cycle V. Correlate chromosomal events in meiosis with the Mendelian concepts of independent assortment and segregation of alleles. a. Meiotic prophase |- Crossing over b. Meiotic anaphase I~ Segregation and Independent assortment VI. Describe the phenomenon of crossing-over (genetic recombination), segregation and independent assortment; explain how recombination affects segregations of alleles. ‘a. Crossing-over (recombination) Lining up of non-sister chromatids > formation of a bivalent (a tetrad of four chromatids) ‘When the bivalent separates, crossing over of genes can occur Occurs in the pachytene phase of meiotic prophase Segregation (Mendet’s 2” Law) No two allelic pairs are ever found in the same gamete Occurs in meiotic anaphase! Independent assortment (Mendel’s 3 Law) Members of a different gene pair sort into gametes independently Occurs in meiotic anaphase! TL Page 14Identify haploid and diploid stages of gametogenesis; describe each stage with respect to mitosis and meiosis. Spermatogonium Primary Secondary Spermatid Spermatozoan (46,XY) Spermatocyte Spermatocyte (46.XY) (23,X,0r23,Y) (23,X or 23,Y) Repeated mitoses sustain this production Meiosis 1 Meiosis I (40,XX) (46,XX) (23.X) (23.X) Oogonium Primary Secondary Oocyte Ovum & Oocyte (Prophase I) & 1% Polar body 2 polar bodies (@) 5 @) al} = (prenatal Meiosis! (Ovulation Meiosis II 2 pronuclei development) —_(atbirth) _ triggers) (metaphase) (fusion into [dictyotene stage] diploid zygote) TL Page 15Genetic Diversity and Disease (pp 59-67) Genetic jersity and Disease (pp 59 — 64) 1. Define and apply the following terms: allele, locus; homozygous and heterozygous; wild type and mutant alleles. a. Allele — different forms a gene may have in a population Diploid organisms have two alleles of each gene Locus — where a gene is found on a chromosome Homozygous - two identical alleles of a gene Heterozygous - two different alleles of a gene Wild type —the “normal” allele, or the version most often seen in a population Mutant alleles — are “different” alleles than normally seen Mutant alleles are not necessarily defective, sometimes they are improvements I, Recognize and provide examples of transitions, tranversions, insertions, deletions, as well as silent, missense, nonsense, and frameshift mutations. Transitions - Purine>Purine (e.g A>6) Transversions — Purine->Pyrimidine or Pyrimidine>Purine (e.g, CA) Insertions - Insertion of a base into the gene Deletions — Deletions of a base from the gene lent mutations — No effect Missense mut ns — Results in change of an amino acid to another amino acid Nonsense mutations — Results in change of an amino acid to a stop codon Frameshift mutations — An insertion or deletion that throws off the reading frame Ill, Distinguish between the effects of mutations in protein-coding regions and in regulatory regions of DNA. Describe specific examples of each kind of mutation represented by various forms of hemoglobinopathies. a. Mutation in protein coding regions Silent mutations > no change Missense mutations > possible protein dysfunction Nonsense mutations - possible protein dysfunction iv. Frameshift mutations ~ likely protein dysfunction n in regulatory regions Increased or decreased expression of the gene Incorrect response to expression signals Transcriptional regulatory signals - make more/less RNA iv. Translational regulatory signals — make more/less protein Hemoglobinopathies (| think these don't need to be memorized, just explain) Sickle-cell B-globin — Codon 6: GAG (Glu) to GUG (Val) HbC > Hemolytic anemia HbKansas > Reduced oxygen affinity iv. HbKempsey > Increased oxygen affinity v. HbMuskegon > No phenotype TL Page 16Describe and distinguish the biochemical and genetic effects of loss of function and gain of function mutations. Loss of function (LOF) alleles — More common because there are lots of ways to “break” a gene i. Typically recessive 1. Gene product is not expressed 2. Or, gene product is defective a Homozygous > pathology i. Lack of product Accumulation of intermediate b. Heterozygous > carriers with no pathology i. The wild type gene picks up the slack Occasionally dominant (there are exceptions) L._ Haplo-insufficiency — receiving half the amount of product is not sufficient a. eg. collagen; signaling molecules; receptors/ligands 2. Dominant negative alleles ~ mutant product interferes with wild type product eg leucine zipper proteins; PDH complex b. Gain of function (GOF} alleles — Protein does something new i. Typically dominant 1. Does the wrong job or does the right job under the wrong circumstances Phenotype is allele specific — depends on the mutation Gain of function often means loss of regulation Recognize or describe how mutations representing allelic heterogeneity and locus heterogeneity might affect ‘a metabolic pathway. a. Allelic heterogeneity — same/similar phenotypes may be caused by different mutant alleles 1. Many changes could occur in one enzyme 2. Could cause a problem in a pathway b. Locus heterogeneity i. Same/similar phenotypes caused by mutations at 2 (or more) different loc (different genes) 1 Enzymes working at different steps in the same pathway may be defective Recognize or describe the effects that regulatory or processing mutations might have on a metabolic pathway. a. Regulatory mutations — errors in silencers, enhancers, promoters, and transcription factors b. Processing errors — RNA processing (e.g. introns not property spliced) and protein processing sorders [Homework problems I Genetic Examples of Biochei Metabolic in each of the following categ 1. Recognize or name examples of specific genetic diseases wit enzymes, Enzyme cofactors, Hormones and receptors, Structural proteins, and Transport proteins Ml, Recognize or apply the principles of biochemical genetics (gain/loss of function, dominant/recessive, allelic/locus heterogeneity, haplo- insufficiency, enzymatic/regulatory/processing mutations) to diseases or pathways described in lecture, textbook, course pack, or in new situat ‘a. Review previous section “Biochemical Genetics (pp 65 - 67)" TL Page 17RNA Structure, Biosynthesis, and Processing (pp 68 - 82) RNA Structure (pp 68 ~ 71 |. Recognize and describe the structure of the four common bases and the sugar found in RNA. Nitrogenous Bases of RNA iva D-Ribose-5-phosphate Il. Describe the primary structure of RNA, and recognize the 5’ and 3’ ends of an RNA shown in a structure diagram. Il, Describe the participation of RNA in double-stranded nucleic acids, including G:U base pairs. GU pairing occurs in stem regions of dsRNA Aloop region follows the stem region IV. _ Describe the general functions, relative abundance, and complexities of the following classes of RNA: mRNA, FRNA, tRNA, hnRNA, snRNA. a, _- mRNA ~ messenger RNA — 35% of total RNA. Size: Highly variable Structure: 1. Monocistronic: Contains one open reading frame (eukaryotic) TL Page 182. Polycistronic: Contains multiple open reading frames (prokaryotic) ili, Abundance/Diversity: Low abundance/High diversity (10,000s of kinds) b. FRNA ribosomal RNA — 85% of total RNA Size: Depends on ifit is a prokaryotic or eukaryotic Structure: Very complicated; some mimic protein structure (and exhibit enzymatic activity) Abundance/Diversity: High abundance/Low diversity (1 kind) tRNA transfer RNA — 10-12% of total RNA Size: 75-90nt Structure: Clover leaf ‘Abundance/Diversity: Moderate abundance/Moderate diversity (35 kinds) d._ hnRNA heterogeneous nuclear RNA immature single stand of mRNA 1R— Amino eed tRNA molecule rcatourc Once it is processed and leaves tcp . the nucleus, itis considered to be Pious mature mRNA snRNA ~ small nuclear RNA toramalocuer i. Transcribed by RNA pol and RNA pol it Anticodon Splices introns from hnRNA Regulates transcription factors or RNAS: SE RNA pol Codon iv. Maintain telomeres thesis in E, coli {pp 7: 4) 1. List the essential components of a transcription rea a. DNA template bb. Ribonucleotide triphosphates (ATP, GTP, CTP, UTP) RNA polymerase 4d. Noprimer needed Il. Describe the subunit structure of E. coli core polymerase and holoenzyme. a. E.coli only has one RNA polymerase b. Core polymerase = a.BB' 4 subunits Catalytically active 1. Is unable to start dueto unknown start point Holoenzyme = a,BB' + sigma (0) factor i. factor directs the RNA polymerase where to start d._ RNA polymerase copies the bit of information needed for that cell at that moment ‘@. Reactions of RNA polymerase GTLInitiation (start) Elongation (go) Termination (stop) Ill, Define promoter, and describe the function of sigma factor. Promoter: Site were RNA polymerase binds to start transcription — Promoter sites: -35 box and -10 box b. factor directs the RNA polymerase where to start IV. Describe the steps that occur within each of the three stages of transcription (i termination). a. Initiation Binding to promoter sites by sigma factor and core polymerase Strand separation Formation of the first bond (first nucleotide is usually ATP, but sometimes GTP) iv. Sigma factor releases and RNA polymerase continues the reaction b. Elongation Direction: 5'to 3” Rate: 10° nt/sec (fast) No significant proofing iv. Unwinding/rewinding occurs before and after Termination Stop signal: Hairpin-loop and consecutive U's added to the end of the RNA RNA polymerase pauses RNA:DNA hybrid falls apart 1. RNAis no longer paired to DNA and “alls off” V. Recognize transcription as a site of action for antibiotics. a. Inhibition by DNA binding — blocks unwinding by RNA polymerase i. Actinomycin D = dactinomycin = cosmegen 1. Also a potent antitumor drug b. Inhibition by enzyme-binding— inhibits RNA polymerase enzyme activity i. Rifampicin — blocks initiation 1. Treatment for tuberculosis a. Resistant strains are mutated to not be sensitive to rifampicin tion, elongation, ithe: in Eukaryotes (pp 75 ~ 80) 1. Define the roles, relative activities, and locations of the three eukaryotic RNA polymerases. Enzyme Location Product Relative Activity RNA polymerase! | Nucleolus | Ribosomal RNA 50-70% RNA polymerase ll_| Nucleoplasm | Messenger RNA 29-40% RNA polymerase | Nucleoplasm_| Small RNAs (tRNAs) "10% Il, Describe the organization of rRNA genes, their location in the nucleus, and the processing of primary transcript into mature mRNA. a. Organization ‘Tandem arrays (500-700 genes on five human chromosomes), found close in 3D space All RIVA have identical promoter sequences TL Page 20One primary transcript codes for multiple proteins b. Location - Nucleolus © Processing Done by RNA polymerase | and accessory factors (TF FA, TF LB, and TFC) Site specific cleavage (e.g, 455 precursor -> 185 +5.85 + 285) Ill, Describe the general features of a promoter for RNA polymerase Il, in contrast to a prokaryotic promoter. ‘a. RNA pol Il synthesis of hnRNA (mRNA precursor), which is processed into mRNA b. Promoter has a-30 TATA box and a downstream promoter element (downstream of +1) RNA poll ‘Work is accomplished by the basal transcription factors 1. TFILD (TATA Binding Protein; TBP) binds TATA box and recruits T/sc initiation complex a Other TFIl's include A, B, D, €, F, H, and! d._ Regulatory structures for specific genes are also found upstream However due to the 3D conformation of the gene, -1000 and +1 may be close inguishing features of a promoter for RNA polymerase Il in contrast to a typical RNA pol II IV. Describe the di promoter. RNA pol Il tRNA, 5S rRNA, and small RNA b. RNA pol Il binds to internal promoter sequences (downstream from +1) © Enzyme i Accessories: TF IIIA, TF II-B, TF Il-C Stable transcription complex 1. TFIIEA and TF ill C are actually bound to the non-template strand V. Describe the events in processing of hnRNA into mRNA, including: a. The structure and role of the 5’ cap i. Modifications 1. Addition of GTP with 5’ —5’ linkage 2. Methylation of the N in the 7 position of guanine > 7-methy+G 3. Methylation of © on 2’ carbon of ribose a. On the first nucleotide; sometimes on the second as well Role 1L._Signal for subsequent processing — a mark for mRNA 2. Critical for efficient translation — ribosome recognition of mRNA b. The structure and role of the 3’ polyadenylate tail Modification — Addition of polyA residues to the 3’ end Role 1. Done because the termination of a transcript is not precise 2. Blocks binding of nucleases and tolerates a little degradation (loss of 100nt is OK) a. Endows mRNA with stability ii, Exception — Histone mRNAs are not polyadenylated iv. Use-Serves as a convenient tag on mRNA for microbiol ing, including. Define exon and intron 1. Exon — Coding sequence 2. Intron — Non-coding sequence TL Page 21a. “intervening sequences” Describe the general roles of snRNPs and the spliceosome 11. 5 snRNPs (small nuclear ribonucleoprotein particles) and'“300 proteins make up the machinery of the spliceosome a. The spliceosome spliced mRNA to remove introns UL Binds to thes’ splice site v2 Binds to the branch site U5 Bindsto the splice site U4-U6_| Assembles the spliceosome b, SnRNPS can also be antigens in auto-immune disorders Define what is meant by 5” and 3” (or donor and acceptor) splice sites, and describe the importance of the sequences at those sites 1.5’ (donor) site has the sequence AGU AG is part of the upstream exon b. GUis part of the intron 2. 3 (acceptor) site has the sequence CAGG CAG is part of the intron b. Gispart of the downstream exon 3. Aligase will join the two sites together > AGG VI. Describe the events in processing of eukaryotic tRNAs. a. 5’ and3! ends are trimmed b. Poly (CCA) is added to the 3’ end i. This is not coded for by the template Extensive modification of bases i. Over 40 modifications, methylations, oxidations, N to C switches Catalytic RNA (p 82} 1. Define what is meant by the phrase “catalytic RNA,” and give two examples. a RNA molecules that have catalytic (enzymatic) activity i. Self-splicing RNAs — Ribozymes 1. Have both endonuclease and ligase activity on themselves Spliceosomes — snRNAS do the cutting and ligation Ribosomes — rRNA is the catalyst for the peptidyl transferase reaction I, Recognize or describe the putative role of RNA as the primordial informational molecule. RNA can carry information b. RNA can copy information RNA can transfer/translate information d._ RNA can act on the information (can do the chemistry) 81] 1. Define the general concept of RNA-based gene silencing. a._RNA-based gene silencing is the body's mechanism to fight against RNA-based pathogens TL Page 22inguish between the origins of short interfering RNAs and microRNAs. a. siRNAs (short interfering RNAs) — Arise in response to an exogenous pathogen Copy RNA from the DNA genome Make a complimentary RNA Form dsRNA iv. Use dicer (an enzyme) to break it into 21nt pieces (this is siRNA) bb, _miRIAs (microRNAs) — Arise in responseto an endogenous pathogen RNA copied from the endogenous genome is self-complementary Use dicer to form a 21-22nt piece from the stem region of the stem-loop © Onestrand of the"2int dnRNA binds (through complementarity) to mRNA via RISC RISC = RNA-induced silencing complex Ill, Describe and distinguish between post-transcriptional and transcri Transcriptional Dicing of dsRNA prevents transcription Double-stranded regions of mRNA (by addition of siRNA and mRNA) interfere with polymerase activity b. Post-transcriptional i. Translation interference 1. No synthesis of the protein whose mRNA is bound with the siRNA/miRNA ji, mRNA degradation 1. The binding of the siRNNA/miRNA marks the mRNA for cleavage by nuclease IV. List or rearrange biological phenomena in which RNA silencing occurs. ‘a May be one mechanism to turn on/off genes quickly during early embryogenesis i. 2 daughter cells may be distinguished by shutting down genes in one cell, but not the other b. Defense against viruses (and other challenges) V. Describe the technological and potentially therapeutic application of RNA silencing. a. Targeted down-regulation of specific genes i. Deliver siRINAs to specific cells to shut off expression of a particular gene 1L._ Far superior to shutting down RNA polymerase in general ional effects of RNA silencing. TL Page 23,Protein Biosynthesis (pp 83 - 98) Translation of Genetic Information (pp 8! 6) 1. Describe the roles of mRNA, tRNA, rRNA, codons, and anticodons in the translation of genetic information from nucleic acid sequence to polypeptide sequence. a, mRNA is transcribed from the DNA template b, > rRNA subunits bind on the mRNA sequence © StRNA enters the sites on the rRNA leading to translation > protein Codons are the sequences found in the genes Anticodons are the complementary sequences found on the tRNA Il, Describe the “wobble” hypothesis; distinguish the concepts of “wobble” and “isoaccepting tRNAs”; recognize and/or construct codon:anticodon pairs, including base pairs involving U or |. ‘a. Wobble hypothesis — Some tRNAs can read morethan 1 codon b. Isoaccepting tRNAs A given type of amino acid may be carried by several different tRNAs The “Wobble” Rules of Codon-Anticodon Pairing. {Same information arranged in two ways) 5 Nucleotide of ¥ Nucleotide of ¥ Nucleotide of 5 Nucleotide of Anticodon Codon Codon Anticodon a U A TorU Cc GC c TorG G Corv GC Coru 1 U,G ora Uv 1G, ora U AorG Ill, Given a table of the genetic code, apply the rules of genetic coding {including wobble) to the following situations: a. Given a codon, identify the corresponding anticodon{s) and amino acid. b. Given an anticodon, identify the corresponding codon(s) and amino acid. identify the corresponding codon{s) and anticodon{s). . Given an amino aci Translational Machinery (pp 87) 1. Describe the ribosomes of bacteria and eukaryotes, including their size, subunits, and the RNAs and approximate protein composition within each subunit. a. Eukaryotic subunits are bigger i. Wang said we didn’t need to memorize the actual sizes b. The measurement of subunits (Svedbergs) is not additive Il. Describe the process of amino acid activation, including: a. The role and specificity of amino acid acyl-tRNA synthetases i. The amino acid acy-tRNA synthetases adds a charged amino acidto the tRNA Each amino acid has a distinct amino acid acy-tRNA synthetase 1. Has proofreading capability b. The position of the covalent bond formed with respect to both amino acid and tRNA i. This occurs between the O' of the amino acid carboxylic acid group and the 3’ CCA on tRNA TL Page 24¢. The role of ATP and the “cost” in high-energy bonds i. In the first step: amino acid + ATP > amino acid-AMP + PPi (PPi -> Pi + Pi) 1. From one ATP... the reaction requires the energy of two high-energy phosphate bonds Translation Mechanism (pp 88 1) 1. Compare and contrast the mechanisms used in E. coli and eukaryotes for identifying start codons. Identify the Positions of ribosome binding sites for each open reading frame in a sketch of a polygenic mRNA. a. Prokaryotes (polycistronic mRNA) — Ribosome recognition site (RRS) i. Before each open reading frame (ORF}, there is a sequence that can base pair with 165 rRNA 11. RRS consensus sequence = AGGAGGU ii, It then scans for the first AUG, which codes for formy-Methionine (fMet) b. Eukaryotes (monocistronic mRNA) ~ Scanning model 40s rRNA binds to the’ cap and scans for the first start codon 11. Translation CAN occur without 5’ cap, but with greatly reduced initiation rates The first AUG codes for Met (NOT fMet like in prokaryotes) Il, Describe the sequence of events leading to a translational initiation complex, including small and large subunits, mRNA, initiator tRNA, GTP and (unspecified) initiation factors. a, Small subunit (SSU) binds (as in the previous objective) b. Met-acyl tRNA pairs with the start codon Ina GTP requiring reaction, the large subunit (LSU) is locked into place i. With the Met-acyl tRIVA in the P-site of the initiation complex Il, Summarize the three principal steps in elongation, including the molecules that occupy the P and A sites within the ribosome and the role of GTP. Describe the formation of peptide bonds, including the implication for the direction of polypeptide chain growth. 1 706 ntatonconpin Delivery of aminoacyl-tRNA. Translocation 3 TL Page 25Delivery of aminoacyktRNA Met-acyl tRINA is in the P-site GTP is hydrolyzed to GDP to drive the delivery of the next amino acyHtRNA into the A-site b. Peptide bond forms i. Peptidyl transferase reaction (rRNA) 1L._ Charged amino acid in the P-site is attached to the amino acyHtRNA in the A-site 2. This leaves the tRNA in the P-site NOT bound to an amino acid © Translocation GTP is hydrolyzed to GDP to drive the translocation of the complex in the 3 direction 1. This is what causes the5’ to 3’ nature of protein synthesis, 2. The dipeptide acy-tRNA is now in the P-site and the A-site is empty to start over IV. Sketch or recognize and label a diagram representing polysomes. Growing pope ca a Polysomes (polyribosomes) are multiple ribosomes translating the same mRNA simultaneously b. The one on the 3’ end will have been on the longest > the longest translated protein chain v. NA FRNA pelymorese Qo a. In bacteria, transcription and translation are coupled i. This is possible because there is no nucleus 1. Therefore is it not possible in eukaryotes b. The complex closest to the 3’ end has been on the longest, as translation is 5’ to 3” TL Page 26VI. Briefly describe the mechanism by which stop codons are recognized and translation is terminated. a. The stop codons (UAA, UGA, UAG) bind termination factors and NOTtRNAS b. When the ribosomes reach the stop codon in the A-site they dissociate and the termination factors deave the peptide © One GTP is required to dissociate the ribosome VIL. Recognize or recall the steps in translation that may be sensitive to effects of at (actions of specific antibiotics need not be memorized at this time). Explain the potential effects that inhibitors of prokaryotic translation might have on mitochondrial translation. Sites of prokaryotic inhibition Inhibits initiation > misreading Binds 305 subunit 1. > misreading 2. > inhibition of amino acyltRNA binding ili, Inhibits peptidyl transferase activity of 50s iv. Binds to50 > inhibition of translocation b. Site shared by both prokaryotic and eukaryotic i. Acts as an analog of amino acyl tRNA > premature chain termination Sites of eukaryotic inhibition Inhibits peptidyl transferase reaction of 60S subunit Covalently modifies elongation factor by ADP-ribosylation Prokaryotic translation inhibitors should have similar effects on mitochondria, as they have similar internal machinery 1. ist the sequence of events (and cellular locations) proposed by the“: membrane-bound and secreted proteins. a. Secreted proteins (via the endomembrane pathway) i. Signal peptide (signal sequence) is translated 1. First “20 amino acids are hydrophobic Signal recognition particle (SRP) binds to the signal peptide as itis being synthesized, while still attached to the mRNA/ribosome and docks on the RER membrane Co-translational transport — As the ribosome continues translation, pushes the nascent polypeptide THROUGH the lipid bilayer of the ER on the basis of hydrophobicity TL Page 27 ignal hypothesis” for synthesis ofiv. Signal peptidase cleaves off the signal peptidase by proteolysis in the lumen of the ER v. > aprotein in the lumen of the ER > secreted protein b. Integral membrane proteins Signal peptide is made and the signal recognition particle (SRP) binds as it is being synthesized Peptidase cleaves off the signal protein Stop transfer signal leaves the protein spanning the membrane of the ER iv. > transmembrane protein (receptor) 1. The part of the protein in the lumen of the ER will face outside of the cell 2. The part of the protein in the cytoplasm will be the cytoplasmic domain 3. Thetrans membrane region > the transmembrane domain v. Thisis all kept in place by avery hydrophilic stretch of amino acids that will prevent it from going through the membrane popice Il, Define and describe the roles of signal peptide, signal recognition particle, ER, ribosome, signal peptidase, and stop-transfer sequence. ‘a. Signal peptide — Codes for import into the ER lumen A stretch of “15 hydrophobic amino acids ignal recognition particle — A ribonucleoprotein that serves as the receptor for signal peptide c ER Proteins insidethe ER lumen -> secretion Proteins spanning the ER membrane > spanning the cell membrane d._ Ribosome — Translates the protein & Signal peptidase — Cleaves the signal peptide from the polypeptide chain f.Stop-transfer sequence — Stops the transfer of the polypeptide into the ER lumen 92, 95 ~ 98) ranslational Proces: 1. Describe the forces that drive polypeptides to fold into specific secondary and tertiary structures. (Review) a. Thisis done by intermolecular forces, most notably hydrophobic forces. Ml, Define chaperonins and describe their function. a. Asset of proteins that assist others in folding (into higher order structures) TL Page 28Ill, Describe the role of proteolysis in maturation of proteins (as for insulin and digestive enzymes). a. As discussed in biochemistry, some molecules are secreted as zymogens Preproinsulin -> proinsulin > insulin + C-peptide ‘Trypsinogen -> Trypsin Pro-opiomelanocortin (POMC) > y-MSH, a-MSH, Endorphin, Enkephalin, and others Describe the structures and biological roles of the following chemical modification of amino acids, and give an w. ‘example of a protein so modi a. Phosphorylation Phosphate groups are added/removed to: Ser, Thr, Tyr, His, and Arg 1. Protein kinases put PO, groups on proteins 2. Protein phosphatases take them off ii. eg. glycogen phosphorylase ATP ADP b. Acetylation i. Addition of an acetyl group to a protein (from AcetyCoA) 1. _Acetyltransferases put acetyl groups on proteins 2. Deacetylases take them off e.g, histone acetylation > regulation of gene expression 1. Key mechanism in determining tightly packed euchromatin AcCoA CoA = = - & (Lens TT lueaniecon, Hhe 120 .Hydroxylation ‘Addition of O# groups e.g hydroxyproline and hydroxylysine for collagen structure osylation i. Enzymes transfer ADP-ribose groups from NAD" to His residues 1. Done by diphtheria toxin, cholera toxin, and pertussis toxin e.g, Translation elongation factors e.g, ion channels (ike cystic fibrosis CFTR) n (myristylation, palmitoylation) i. Covalent attachment of fatty acids to the plasma membrane 1. Attached to (processed) amino terminus or internal Cys residues Functions to anchor the protein to the membrane e.g, receptor and signal transduction proteins TL Page 29V. Compare and contrast O-linked and N-linked glycosylation with respect to: Ar Structure of oligosaccharides; Mechanism of sugar attachment. 10 acids that are modified; O-linked Nlinked ‘Amino Acids Modified “Mainly on Serine and Threonine “Asparagine Also on OH-Lys and OH-Pro ‘Structure of Oligosaccharides | -Appears to be a short saccharide chain ~"Core” is rich in mannose Mechanism of Sugar Attachment | -One at atime by a specific lycosyl ~The glycolipid dolicholphosphate transferase donates the sugars en bloc in the ER some units are cut out and terminal glycosylation occurs (in a one- by-one fashion) -Mostly in the Golgi Vi.__ Distinguish the nomenclature and structure of the oligosaccharides in the ABO blood group system. \dds GalNAca to the 3 of 2GalB1 of the non-reducing terminal of the oligosaccharide (dds Gala to the 3 of 2GalB1 of the non-reducing terminal of the oligosaccharide Blood Type | Type of Oligosaccharide | Antibody in Serum | Types of Serum that Cause Antigens Present on RBC ‘Agglutination of RBC 0 H ANtEA, AnteB None A A Ante 08 B B ‘Antica OA AB ‘AandB None OAB Vil. Describe the mechanisms used for targeting proteins to lysosomes, nucleus, and mitochondria. a. Lysosomes Targeting Signal — Lysosome targeting signal Chemical Nature - Mannose 6-phosphate Receptor for Signal - Mannose 6-phosphate receptor b. Into Nucleus Targeting Signal — Nuclear Localization Signal (NLS) Chemical Nature - Basic residues Receptor for Signal — importins (a and B) © Out of Nucleus i. Targeting Signal — Nuclear Export Signal (NES) Chemical Nature — Leucine-rich Receptor for Signal - Exportin-1 d. Mitochondria ‘Targeting Signal — Mitochondrial Signal Peptide (Matrix Targeting Signal) Chemical Nature — Amphipathic helix Receptor for Signal Involves receptor peptidases, ATP, and electrochemical gradient VIII. Describe the roles of secreted proteases and the lysosome in degrading extracellular proteins. ‘See Biochem notes i. Contain a variety of peptidases that break down the protein 1X. Describe the roles of ubiquitin and proteasomes in degrading intracellular proteins. ‘See Biochem notes i. Ubiquitin tagging (which requires ATP) > transport to proteasomes TL Page 30Regulation of Gene Expression (pp 99- 106) Strategies of Gene Regulat 100-102) 1. Explain the use of alternative sigma factors to regulate specific set of genes by recognition of specific promoter sequences. a. By using alternative sigma factors, the organism can trigger the transcription and translation of genes needed at that point in time b. Sigma Factors General (6) - Promoter for general purposes Heat shock (0) — Promoter for genes encoding heat shock proteins Nitrogen (0 Promoter for genes regulating nitrogen starvation List and define the following components of an operon: a. Promoter — Binding region that facilitates the transcription of genes b. Operator —A segment of DNA that a regulatory protein binds to Repressor — Protein that binds onto a DIVA segment and represses transcription q e Inducer — A molecule that promotes gene transcription Co-repressor — A protein that decreases gene expression by bindingto a transcription factor, which contains a DNA binding domain Unable to bind DNA by itself f.Polygenic mRNA — | assume it’s an mRNA coding for multiple genes Ill, Describe the effects of high or low concentrations of lactose and glucose on expression of the lac operon. a. Describe the activity of the lac repressor under these conditions AND Describe the activity of the CAP protein under these conditions Intracellular Proteins Bound to ‘Structural Genes [Glucose] [Lactose] | _Promoter Operator__| Transcription Rate a v None | Repressor bound v * + None None v v v CAP + cAMP_| Repressor bound v + + CAP + cAMP. None t “Figlucose] > extra sources of sugar do not need to be used > transcription is not promoted Vi glucose] > need to use lactose as a source of glucose > transcription is promoted lactose] + lactose needs to be digested > lactase transcription is not repressed iv. Ilactose] > lactose does NOT need to be digested ~ lactase transcription is repressed b. Apply the principles of a repressor or activator protein to a novel catabolic operon (i.e., predict how regulation would be affected by relevant metabolites) IV. Describe the effects of low or high concentrations of tryptophan on expression of the trp operon, and contrast the behavior of the Trp repressor with that of the Lac repressor. a. PI Trp] means there is no need for enzymes to make Trp i. Trp binds repressor protein > repressor binds to DNA ~ no transcription b. L{Trp] means that is need for enzymes to make Trp Repressor cannot bind to the operator -> RNA pol can bind > transcription & The Tip repressor, uses Trp as a co-repressor; the lac operon does not use glucose or lactose Tipis either on or repressed: Lac is either promoted, not regulated, or repressed TL Page 31Regulation of Eukaryotic Gene Expression (pp 10: |. Differentiate between polygenic mRNAs {from prokaryotic operons) and gene clusters in eukaryotes (e.g., histone, globin, rRNA genes). In eukaryotes, there are no operons i. > cannot make polygenic mRNA because of the scanning model of ribosome initiation of translation ji, AND, there are no multiple ORFs in eukaryotic mRNA b. Eukaryotes utilize gene clusters — related genes are near each other on the same chromosome Allows for: 1. Sequential unpacking of chromatin 2. Unpacking of a small region of chromatin opens up several related genes 3 s’ vs 8s SS 8 OY ° +> +> < , histone genes s.r > . 3 TT Fe erromosorne globin genes - os ns — > a EE [SLES ciroricme In the histone genes, the some mRNA is coded for from the sense strand and somes coded for from the antisense strand In the globin genes, sequences are found in proximity of each other Il, Describe how DNA methylation and chromatin condensation affect gene expression. heterochromatin. Describe the role of histone acetyltransferases in reducing chromatin packing. Describe the role of ATP-dependent remodeling enzymes in unpacking chromatin. a. DNA methylation Heterochromatin is heavily methylated (on adenine and guanine) ~ tighter packing Euchromatin contains much less methylation -> loose packing > expression b. Histone acetyl transferase i. Acetylates (by adding acetyl CoA) Lys of histones — reduction of positive charges on histones > weakens interactions with POs of DNA — reduction of packing of the nucleosome The acetylated histone may represent a new binding site for ATP-dependent “remodeling” enzymes (such as those that do demethylation) 1. Remodeling enzymes induce sliding of nucleosomes to increase gaps for RNA pol ll > TFIED can gain access to DNA & This all can lead to the expression, or lack thereof, of genes throughout development and life ‘This would be erased in germ cells during gametogenesis and new patterns are established for the next generation of cells prior to fertilization TL Page 32Ill, Define promoter, enhancer, and silencer with respect to gene expression. Distinguish between basal (or general) transcription factors and regulatory proteins (such as transcriptional activators). Describe the functions or activities associated with most regulatory proteins. Identify this as the “Receptor as a nn Factor” signaling pathway. a. Definitions i. Core promoter: TATA box + initiator site 1. Basal (general) transcription factor Enhancer AND silencers: Regulatory DNA sequences that are found at far distances away from the basal machinery, both upstream and downstream iptional regulatory proteins — the proteins that bind enhancers and silencers i. Has two parts: 1. DNA-binding domain (DBD) — binds to specific DNA-sequences of responsive elements Usually a helix-turn-helix, zinc finger, or leucine-zipper 2. Transcriptional regulatory domain (TRD) — interacts with the transcriptional machinery (basal complex) ‘a. Depend in the presence/absence of co-factors > activation or repression ji. Possible outcomes 1. Direct interactions with basal transcription factors 2. Recruit co-activators that modify chromatin a. ATP-dependent “remodeling” enzymes that induce “sliding” of nucleosomes along DNA increasing the gaps for RNA pol I, TFILD, etc. to gain access to DNA (eg Swi orSNF) 3. Covalent modification of histones — acetylation, methylation, phosphorylation, etc © Receptor as a Transcription Factor i. Steroid receptors are found in the cell, as steroids can readily cross the cell membrane 1. The receptors contain: a Hormone-binding domain b. DNA-binding domain (DBD) © Transcription regulatory domain (TRD) 2. Upon bindingto the steroid hormone a. The whole complex translocates into the nucleus b. The complex DBD binds to hormone-responsive elements on the DNA The complex TRD recruits the machine to activate hormone-responsive genes SREBP — Sterol Responsive Element-binding Protein 1. U{cholesterol] > protease activity on protein attached to cytoplasmic domain > cytoplasmic domain is free and translocates to the nucleus — activation of genes in cholesterol biosynthesis iii, NF-KB — Nuclear Factor (x-chain) 1. Anchored in cytoplasm by IB 2. Bacterial lipopolysaccharide (LPS) or viral infection (> cytokine induction) — ubiquitin- tagging and degradation of Ix-B > release of NF-KB to be translocated into the nucleus ~ activation of genes in inflammatory response TL Page 33,IV. List the structural motifs of three classes of DNA-binding proteins (helix-turn-helix, zinc-finger, and leucine tipper). a Helix-turn-helix Helix-turn-helix motif of protein Major groove of DNA A perfect fit Amino sequence forms aturn in ve z aoe Major groove b. Zinefinger Leucine zipper TL Page 34An Introduction to Dysmorphology (pp 109 - 118) |. Explain the scope of genetic disease in ordinary practice. ‘a. The vast majority of patients (71%) speak to their primary care physicians about genetic disorders jer how making a genetic diagnosis impacts upon individual and greater family uni a. Patient management Itis difficult to effectively treat the patient without knowing the underlying condition. al interventions Early recognition can allow for pre-emptive interventions Genetic counseling i. Allows family to: 1. Understand relative recurrence risks for a given diagnosis 2. Consider alternative options (i.e. adoption vs. child-birth) 3. Inform relatives of testing options 4. Can prevent unnecessary tests in other family members d._ Psychological impact Gives hope that the advancing field of genetics will find a solution Allows for participation in individual and family support groups Even with diagnosis “supportive” measures can be applied to the patient Ill, Understand the impact that Medical Genetics has in everyday general practice. a. There are >15,500 genetic disorders affecting more than 13 million Americans b. Dysmorphology affects 0.5% of all newborns and 7% of all stllborns & 3050% of post-neonatal deaths are due to congenital malformations d. 20-30% of all infant deaths are due to specific genetic disorders 185-50% are children with other congenital malformations 11-12% of all hospital admissions are for genetic causes (11.1% pediatric and 12% adult) 15% of all cancers have an inherited susceptibility 10% of all chronic diseases in adults have a genetic component At least 50% of mental retardation has a genetic component Iv. Know and contrast Genetic Disease presence and incidence in various patient populations. a. See other objectives V. Consider how birth defects and congenital malformations can lead one to suspect an underlying genetic diagnosis. ‘a. Knowingthe signs of certain conditions can help a physician determine possible underlying causes VI. Know the incidence of birth defects in newborn populations. ‘a. 6% of all births will entail a genetic malformation that will require medical attention by ageS b. Percentage of individuals with major malformations 0- 85% 1-13.4% 2-0.8% iv. 3-05% MW. Con TL Page 35VIL. Consider various etiologies of birth defects in newborn populations. ( er | newman | F] Bl retype ‘Sope ee. al cngealonenaes Forces end winoronarctes VIII. Consider various etiologies of birth defects, and their percentage of overall involvement in all that are either isolated or non-isolated. a. Isolated anomaly — Single structural defect, not associated with a syndrome i. Many have increased recurrence risks to future siblings or offspring b, Multiple congenital anomalies (or syndrome) — 2 or more defects in 2 of more systems Examples: Infant of Diabetic Mother Syndrome; Down Syndrome © Syndrome—A recurring pattern of multiple congenital anomalies (or physical signs) representing a known etiology i. Dueto either genetic and/or environmental causes 1. Examples: Down syndrome Trisomy 21 b. Williams syndrome —_Microdeletion of a chromosome © Prader-Willi syndrome— Chromosomal imprinting disorder 1X. know common genetic definitions and terminology. a. See other objectives X. Distinguish a syndrome from an association from a sequence. a. Syndrome: A recurring pattern of Multiple Congenital Anomalies (MCAs), representing a known etiology b, Sequence — single event/anomaly results in a cascade effect and multiple malformations Example: Pierre Robin Sequence: Micrognathia (small chin) > glossoptosis (displacement or retraction of the tongue) > U-shaped cleft palate TL Page 36& Association — A non-random occurrence of MCA’s NOT associate with a specific genetic etiology XI. Identify specific features of VACTERL association, Pierre-Robin sequence, and Stickler syndromes, noting ‘common features and distinguishing features. a. VACTERL association: i. Signs: 1. Vertebral anomalies 2. Anal anomalies 3, Tracheo-Esophageal fistula 4. Cardiac anomalies 5. Renal defects 6. Limb defects ii. Known to have increased incidence in: 1. Children of diabetic mothers 2. Children of mothers on statin (cholesterol lowering) drugs They may influence cholesterol dependent development pathways known to be associated with hydrocephalus and mutations in the FANCB gene on the X- chromosome b._ Stickler syndrome ‘Autosomal dominant Affects 1 in 7,500-9,000 newborns Distinctive facial appearance (malar hypoplasia no check bones) iv. Other symptoms: Hearing loss, early onset arthritis, skeletal abnormalities (hyper-extensibilty), ocular abnormalities v. Has oral cleft due to Pierre Robin sequence 1. > Pierre Robin can be an isolated sequence or part of a larger syndrome complex Xl. Understand what is dysmorphology and why is it important for syndrome identification. a. Dysmorphology ~ The knowledge of how abnormal embryonic and fetal development results in abnormal changes in body form i. tf we know the causes of different genetic abnormalities, we can work backwards to determine the underlying cause XII Understand what a major anomaly is; types of major anomalies; and, their importance relative to making a syndrome diagnosis. ‘Major anomaly - When a major organ system(s) is malformed early during embryogenesis b. Examples: spina bifida, anophthalmia (no eyes), cleft lip/palette, transposition (of the great arteries), renal agenesis (no kidneys), holoprosencephaly (single brain ventricle/hemisphere) & Less likely to identify a syndrome as the dysmorphias could have many causes XIV. Understand what a minor anomaly is; types of minor anomalies; relative to incidence of major anomalies; and their importance relative to making a syndrome diagnosis. ‘a. Minor anomaly - When a minor organ system/body feature is malformed late in embryogenesis b. Examples: Low set ears, hypospadias (incorrect urethral opening), bifid uvula, ASD, VSD, nail hypoplasia (small nails), post-axial polydactyly (extra digit “outside” of the pinky), 4"* metacarpal shortening, synophyris (unibrow), ear tag, clinodactyly (curved fingers), inward curved S* digit (form of clinodactyly) TL Page 37XV. know etiologies of genetic disease; Know relative frequency due to following causes: Approximate Etiologies Unknown Mendelian ti Chromosomal tw Teratogenic a a, “15% of all syndromes are caused by chromosomal aberrations b, “15-30% of severe mental retardations and 5-10% of mild retardations are due to detectable chromosome changes GTL Page38Basics of Cytogenetics Technology (pp 119 - 134) 1. Understand the basic cytogenetic techniques from specimen receiving to results reporti Specimen collection ‘Type of specimen — Depending on the goal of the test, samples are taken from different tissues 1. Constitutional chromosomes > blood lymphocytes 2. Tissues mosaicism (e.g, Palister-Killian syndrome, trisomy 20 mosaicism) > tissues from different germ layers (skin, gonad, etc.) 3. Acquired disorders associated with neoplasms > site of malignancy Appropriate containers — Sterile containers with appropriate medium 1. Bone marrow and peripheral blood —> anticoagulant (sodium heparin green-top tube) 2. Other solid tissues > collection/transport media or just kept moist by isotonic saline ili, Correct temperature 1. Refrigeration is helpful 2. Freezing can be damaging iv. Timely delivery 1. Some need to be received immediately (e.g, brain tumors) 2. Some can be delayed 1-3 days (e.g, blood) b. Specimen culturing i. Performed using strict aseptic techniques because microbes will cause culture failure ‘Types of culture 1. Suspension cultures ‘Cells are suspended in the growth medium and do not attach to the culture vessel b. eg, someneuroblastomas and most lymph nodes 2. Inssitu culture Attached monolayer cultures or anchorage-dependent cultures eg. chorionic villi, amniocytes, skin fibroblasts, etc Culture “environment” 1. _pH~7.25-7.40; controlled by a bicarbonate buffer in the media 2. Gas—5% COz 25% Oz and 93-95% Nz 3. Temperature — 37-375°C Growth curves fall off sharply over 38°C - cell death iv. Media 1. Medium is usually supplemented with serum for protein and growth factors Usually supplemented with fetal bovine serum (FBS) b. Alternatives to fetal bovine i. New-born calf— cheaper, but may betoxic Colostrum-free new-born calf less effective and expensive than FBS ¢ Other complements are antibiotics and L-glutamine v. Suitable cells 1. Lymphocytes in peripheral blood Peripheral blood is also useful for determination of constitutional karyotypes i. tis non-dividing so needs to be exposed to a mitogen: phytohemagglutinin (PHA for T cells) or pokeweed antigen (for B cells) TL Page 392. Fibroblasts from skin and tissue biopsies 3. Bone marrow cells 4, Fetal cells found in amniotic fluid or from chorionic villus biopsies Harvesting — Cells are processed to obtain metaphase for study purposes i. Step 1: Mitotic arrest 11. Usually with colcemid (or colchicine) a. Prevents formation of spindle fibers b. Causes chromosome condensation i. Condensation can be prevented by ethidium bromide, BrdU, etc. ii. Step 2: Hypotonic treatment 1. Usually with a0.75M KCI solution 2. Increases cell volume > chromosomes can spread out Active transport of K* and CI > water flow Step 3: Fixation 1. Usually 3:1 methanotacetic acid 2. Removes water from the cells, kills, and preserves them a. > hardened membranes and chromatin to allow for staining d._ Slide making i. Fixed cells are dropped onto glass slides and dried under conditions to optimize chromosome spreading and morphology Chromosomal banding (solid staining, banding, and selective staining) and what each represents. Which banding technology you should choose at certain conditions. Solid staining (a.k.a. conventional staining) i. Produces unbanded chromosomes 1. Allows grouping by size and shape a Only 1, 3, 9,16, and ¥ can be individually identified 2. Useful for: measurement of chromosome length, centromeric position, and arm ratio a. Also for: identifying satellite cells, secondary constrictions, dicentric or ring chromosomes, fragile sites, and breaks or gaps Uses Giemsa or aceto-orcein Not commonly used anymore b. Differential banding techniques i. Overview 1. Uses different dyes for banding 2. Standard patterns are set for different resolutions 3. Non-fluorescent procedures utilize the less expensive light microscope, are more permanent, and can produce better resolution ii, Qcbanding— Quinacrine staining 1. Common in Europe 2. Visualized with fluorescence The bright (glowing) Q bands (Q") correspond to dark G bands (G") 3. Useful in detecting heteromorphism (AT rich region) 4. Also useful for identifying Y chromosome as Yq12 glows very bright TL Page 40iii, G-banding~ Giemsa (or Wright or Leishman) staining 1. Common in the Us 2. Requires limited digestion by trypsin and done during prometaphase 3. Relatively permanent and sharp staining 4. Glight (6) and G-dark (G') reflect functional differences in the chromatin @ Euchromatin — Generally G- b. Constitutive heterochromatin — Generally G+ Facultative heterochromatin — G+ or G- 5. Visualized with light microscopy 6. An abnormality in G' is less harmful than an abnormality in G a Gis codingDNA iv. R-banding — Reverse staining 1. Banding pattern is opposite of G-banding Good for visualizing errors in the ends of chromosomes 2. Produced by incubation in a very hot phosphate buffer followed by Giemsa staining a. Splits AT rich segments, leaving GC rich DNA to stain 3. Coding DNA is R* & Selective staining techniques — adjunct procedures staining specific/specialized portions i. Frequently used procedures are: C-banding, silver staining, DAPI, Distamycin A staining, and Titerminal)-banding ii, Cbanding 1. Stains constitutive heterochromatin (termed h+, as in 9qh+) a. Acidic treatment > basic treatment > hot saline > Giemsa stain i. Results in sequential extraction of purine bases and selective brakeage of the phosphoribose backbone of DNA ii, Non-C-banding domains are eluted out b. Constitutive heterochromatin = inactive a. Classifying inherited size variants of 1, 9, and 16 b. Locatingthe position and number of centromere regions in a marker, ring, oF otherwise abnormal chromosome ¢ Identifying ¥qi2 in an unusual location d._ Crudely assessing the presence of active genes in small marker chromosomes Silver staining 1. Stains nucleolar organization regions (NORs) a. Reflects relative activity of the rRNA genes contains in the NORs b. Atheoretical maximum of 10 NORs can be visualized per cell 2. Failure of stain does not exclude the presence of NOR sequences 3. Commonly see: @ BigNOR b. Deletion NORS TL Page 414d. High resolution banding - Uses compounds which interfere with condensation — longer chromosomes & Dynamic staining techniques - Manipulate living cells for the purposes of study i. Frequently used in replication banding and sister chromatic exchange (SCE) Il, Recognize the common cytogenetic nomenclature used to des Chromosomal identification Sex chromosomes: X and Y ‘Autosomes: All non-sex chromosomes Groups: 1. A group: Chromosomes 1-3 2. B group: Chromosomes 4-5 3. C group: Chromosomes 6-12 and X 4. D group: Chromosomes 13-15 5 6 ibe normal and abnormal karyograms. E group: Chromosomes 16, 17, and 18 .F group: Chromosomes 19-20 7. G group: Chromosomes 21-22 with satellites and ¥ (without satellites) b. Position of the centromere ter P telomeres (s/sa) iF arn satelite centromere | stake 4 (stk) ater 4g Womees — Acrocentic Metacentric — Submetacentric P arm is always drawn up, by convention i. Metacentric— Centromere is near the middle 1. Two well defined arms with a length ratio varying from 1:1 to 25: Submetacentric - Centromere is nearer to one end Acrocentric— Centromere is near the end. 1. One arm is substantially shorter (ranges from 3:1 to 10:1) 2. Seen in chromosomes 13, 14, 15, 21, and 22 © Band nomenclature and idiogram (not ideogram) i. Band— Part of a chromosome distinguish from others by staining 1. Band 1 is closest to the centromere 2. Centromere is assigned bands p10 and qo Region — Portions of the chromosomes located by landmarks cen — Centromere iv. pter—Terminal of the P arm v. qter—Terminal of the Q arm Sub-bands - Portions of bands seen at high resolution Idiogram — Chromosome map stk - Stalk TL Page 42d. Terms s/sa Satellites Karyogram — Visual picture of the chromosomes Karyotype — Description of the karyogram (e.g. 47,XY +21 is Trisomy 21) del = Deletion der = derivative centric dup = Duplication fra =Fragile site h=heterochromatin inv =Inversion ish = Metaphase FISH mat/pat = maternal/paternal origin Robertsonian translocation ranslocation GTL Page 43,Common Chromosome Abnormalities (pp 135-148) Identify the various types of chromosome abnormalities (numerical, balance, and unbalanced structural] that are encountered in a clinical setting; understanding the description of common chromosome abnormalities using standard ISCN. Recognize the origins of certain chromosome abnorm Describe the risks associated with specific abnormal karyotypes, either for carriers or for their offspring. ities. Outline does not follow objectives, as the information makes little sense when separated Abnormalities of Chromosome Number a. Euploidy — Cell with a set or sets of n=23 chromosomes Haploi Diploid Triploid = 3n iv. Tetraploid =an b. Types of Triploidies i. Diandric- Type! 1. Occurs in 66% of triploidies 2. Extra set is paternal @2n sperm from failure of meiotic division b, 2normal sperm (dispermy) 3. Presents asa well-grown fetus with or without microcephaly and an abnormally large and cystic placenta usually classified as partial hydatidiform moles Digynic— Type! 1. Occurs in 34% of triploidies 2. Extra set is maternal a. 2n egg from failure of first meiotic division b. Retention of the second polar body 3. Presents as severe intrauterine growth retardation with relative macrocephaly and a small and noncystic placenta. 4, although there is no correlation between maternal age and triploidy, digyny can play a ‘major role in triploidy in the advanced maternal age group © Tetraploid i. Rarer than triploidy in spontaneous abortuses 1. Seen in 6-7% of abortuses with chromosome abnormalities 2. Rarely surviveto term Probable origin is chromosome duplication resulting from a failure of cytoplasmic cleavage during the first division 4d. Aneuploidy — Abnormal sets of chromosomes i. Monosomy ~ 1 copy of certain chromosomes 1. Tumer syndrome —45,X ‘Trisomy — 3 copies of certain chromosomes 1. Patau syndrome — Trisomy 13, GTL Page 442. Edward syndrome Trisomy 18 3. Down syndrome Trisomy 21 (47,XX,+21 or 47,XY,+21) 4. Klinefelter syndrome—47,XxY I Abnormalities of Chromosome Structure ‘a. Balanced chromosome rearrangements — No genetic material is added or lost > normal phenotype i. Inversion Single chromosome undergoes two breaks and is reformed in the inverted position 1. Paracentric— Inverted segment does not contain the centromere Crossing over with a paracentric inversion i. Usually results in non-viable (acentric or dicentric) daughter chromosomes - no risk of having offspring with abnormal phenotype 1. But, there is a50% chance of inheriting the same inversion 2. Pericentric— Inverted segment contains the centromere Crossing over with pericentric inversion i. Produce cells with a single centromere -> can produce gametes Recombination leads to chromosomes with either missing segments or with duplicate segments 1. Monosomy is less tolerated than trisomy 2. Leads to spontaneous abortuses or infertility 3. Risk No risk if passed on from parents b._ Slightly increased risk ifit is de novo Reciprocal Translocations - When two chromosomes exchange material 1. Results in a balanced karyotype 2 autosome with the lowest number is always specified first 3. Because of errors in chromosome segregation during anaphase I (and not crossing- over), carriers are at risk of producing offspring that are: a. Normal b. Normal containing balance set of translocated chromosomes Abnormal due to inheritance of an unbalanced set of chromosomes A derB a derA B A derA derB B TL Page 45iii, Robertsonian Translocations - Two acrocentric chromosomes fusing at the centromeres with loss of the satellites and short arms 1. Satellites and short arms contain genes for rRNA, which are found on other acrocentric. chromosomes (13, 14, 15, 21, and 22) Phenotype is normal, but problems occur during meiosis due to alternate segregation, as seen with reciprocal translocations 3, 21q21q Robertsonian translocation > trisomy 21 in all surviving progeny 4, Might lead to meiotic arrest > infertility in males iv. Insertions ~ Translocation, without reciprocity 1. Can lead to duplicate material and deleted material Material b. Unbalance rearrangements i. Deletions — Loss of chromosomal segment 1. Always results in unbalance karyotype 2. Types of deletions a Terminal i. Loss of end of chromosome b. Interstitial i. Loss of segment from within chromosome TL Page 46Duplications — Repetition of material within a chromosome 1. Can originate from unequal crossing-over 2. Will result in trisomy of the duplicate region (partial trisomy) 3. Clinical consequences depend on size and nature of the reg ili, Ring chromosomes n duplicated 1. Formed by deletions of both terminal ends (partial monosomy}and their subsequent fusion 2. Unstable during cell division and often lost a> may not be present in all cells (mosaic) iv. Isochromosomes— Chromosomes that are a mirror image about the centromere 1. Division of the centromere can result in: a Two copies of the same arm (normal) b. Mirror image around centromere & Centromeres in the wrong place d. Monosomy for 1 chromosome arm Trisomy forthe other arm ABC CBA Se Deletions and duplications OmmMs ood ammo. om> o> SE Oa OmmMd> Od Deletions and duplications GFED pEFG 2.q arms v. Extra Structurally Abnormal Chromosome (ESAC) ~ Abnormal chromosomes in addition to 46 1. Usually small and difficult to identify 2. Sometimes called marker chromosomes 3. May be benign or cause serious mental handicap TL Page 47‘Specific, Common Chromosomal Aberrations (pp 151 - 170) 1. Understand the incidence, etiology, and the dominant features of the following common chromosome syndromes: These are the only autosomal trisomies compatible with postnatal survival a. Down Syndrome - Trisomy 21 Occurs in 1/800 births (much higher incidence in children of mothers over 35) Most common genetic cause of moderate mental retardation Most common liveborn trisomy iv. 95% of Down Syndrome is from Trisomy 21 (due to non-disjunction at meiosis 1. “88% maternal origin 2. “86 paternal ori v. Recurrence risk is 1% in young mothers, but increases with age vi. Phenotype 1. Characteristic epicanthal folds and upstanting palpebral fissures Short neck and loose skin on the nape of the neck Flat nasal bridge Short, broad hands with a simian crease, and incurved" digit or clinodactyly Developmental delay, obvious by end of first year (1025-50) Musculo-skeletal problems a. Atlanto-axial instability b. Open mouth, microstomia (small mouth), and protruding tongue Short stature, brachycephaly (head wider than length) with flat occiput d. Flat face &Hypotonia (floppy baby) 7. Brain defects a. Holoprosencephaly — Single ventricle (lack of midline schism in development) 8 Cardiac defects 40% of newborns have congenital heart disease b. ASD, VSD, Pulmonary and/or Aortic atresia, and Hypoplastic left heart (lack of formation of left ventricle) & Endocardial cushion defects i. ASD, VSD, and abnormal mitral & tricuspid valve = Left to right shunt and tricuspid and mitral valve regurgitation ‘Amurmur may not be heard, so an echocardiogram is necessary 9. Gastrointestinal atresias (esophageal, duodenal, large intestinal, and anal) 10. Hematopoietic effects Higher incidence of Acute Myelogenous Leukemia (AML) i. Although it responds better to treatment than non-trisomy 21 AML 11. Endocrine: Hypothyroidism 12. Renal anomalies: Range from lack of kidneys to minor anomalies 13. CNS: Very high risk of developing early onset Alzheimer’s disease av ewn TL Page 48Translocation Down Syndrome 1. Occurs in 3-4% of Down Syndrome patients 2. Caused by unbalanced Robertsonian translocations of chromosome 21 and another acrocentric chromosome Usually a de novo event (should be confirmed by analysis of parents) 4, Abalanced translocation carrier mother has a 10-15% of Down Syndrome progeny, while a male carrier has a5% chance of affected progeny Mosaic Down Syndrome 1. Occurs in 2% of cases 2. Less than 100% of cells are abnormal > milder phenotype 3. Likely dueto non-disjunction event 21q21q Translocation 1. Involves 2 q arms 2. Denovo 3. All surviving progeny will have trisomy 21 b. Edwards Syndrome — Trisomy 18 i. Rare, but more severe than trisomy 21 1. Occurs in 1/6,000-1/8,000 live births 2. Usually live no morethan a few months (90% die by 1 year of age) a Generally, extreme life prolonging measures are not recommended, as death will be spontaneous Phenotype 1. Hypertonia, feeble activity 2. Failureto thrive 3, Microcephaly > severe mental retardation 4. Often severe malformation of the heart 5 6 7. Fists clenched, with second and fifth digits overlap the third and fourth Micrognathia 7. Short sternum, small nipples 8 Rocker-bottom feet ¢. Patau Syndrome - Trisomy 13 Occurs in 1/10,000 live births Phenotype 1. Failure of normal brain development (holoprosencephaly) > severe developmental retardation Micropthalmia—Small eyes Polydactyly Cardiac defects Hypoplasia of pelvis 6. Cleft lip, cleft palate, or both ili, “50% die within the first month ren Page 491. 18% survive first year iv. Generally, extreme life prolonging measures are not recommended 4d. Trisomy 16 = Fatal (no live births, most frequent trisomy in abortuses) I, Rationalize the basis for the spectrum of phenotypes that encompass a diagnosis of Down, Edward, and/or Patau Syndromes and consider how being mosaic for a chromosomal disorder further affects these clinical phenotypes. a. Early defects > wider range of defects i. Genetic defects from gametes are as early as can be b. Also, the lack of brain development in turn affects other body systems © Mosaicism will generally take the same symptoms and make them more mild i. Certain phenotypes will only be seen at sporadic locations in the body 1. For example, if a genetic defect caused skin discoloration, it would only occur in the areas with the affected cells Ill, Rationalize the statement that a syndrome does not necessarily confirm the underlying chromosomal causation, and relate this understanding to the chromosomal syndromes. a. Just because certain phenotypes are observed in a physical examination does not mean that the patient has a certain underlying cause b. For example, if a CT or MRI shows holoprosencephaly, it cannot be determined that the cause is trisomy 21 —Holoprosencephaly is found in trisomy 21 (Down syndrome) and trisomy 13 (Patau syndrome) IV. Understand dynamics of sex chromosome abnormal a. Understand the anatomy of the ¥ chromosome, and the importance of the | Peeudoautosomnal SRY region moe The Y chromosome is smaller than the X chromosome SRY (sex- determining region Y) — The region that triggers the events parm that converts the embryo into a male say 1. Females the “default” sex jons that contain genes that are arm inherited, much like the autosomal chromosomes (in females, both of PoeudSeutssomal b. Understand what Barr Bodies are, and under which circumstances they may region be noted Y chromosome Barr Bodies are condensed x Chromosomes 1. These used bodies of condensed chromatin used to be the method of determining sex 2. Number of Barr Bodies plus 1 is the number of X chromosomes in the cell a, Turner Syndrome females lack Barr Bodies The condensed chromosomes are the inactivated X chromosomes, as only one active X chromosome is needed 1. Lyonization is another term for X-inactivation TL Page 50©. Note how the Xist and Tsix are genes involved in X chromosome inactivation Xq13 = Inactivation center (Xie) Xist — X-inactive specific transcript 1. Expresses an RNA transcript that binds to and inactivates the x chromosome + inhibition of MOST genes 2. Only expressed on one copy of X Feeaon Tsix— Anti-sense gene of Xist 1. Expressed by the active x, asit antagonizes the function of the Xist gene on the inactivated chromosome iv. These genes do not affect the pseudo- autosomal regions 1. Thisis why Turner syndrome cases have a phenotype v. Ifthere are two copies of X, the damaged one is preferentially inactivated d. Understand what the pseudo-autosomal regions of the X and Y chromosomes represent, and how they impact upon sex chromosome syndromes They are regions that contain genes that are inherited, much like the autosomal chromosomes They persist in sex chromosome syndromes, as they are not inactivated by Xist e. Understand the consequences of having X-chromosomal material translocated onto autosomes, and why X-chromosome inactivation is important in these translocations In these cases the normal X chromosome is inactivated This allows for the expression of the translocated autosomal genes on the der X chromosome 1. However, if der X is passed on to progeny, it will be inactivated | aut = derX x \i der aut inactivated V. Know the incidence, etiology, and dominant phenotypic features of the following disorders so at to be able to identify them reat ical situations: i. Occurs is about 1/500 births 1. 1/400 males and 1/650 females TL Page 51b. Klinefelter syndrome i. Etiology: 47,XxY 1 Dominant featuré 1 Equal chance of extra chromosome being from mother or father ‘Appear normal before puberty Testes remain small (hypoorchidism) -> (testosterone production > underdeveloped secondary sexual characteristics 2. Can result in osteoporosis and gynecomastia (breast development) a Gynecomastia > ‘risk of breast cancer 3. Infertility 4, Moderately reduced 1a. a. 2/3have learning defects (mainly dyslexia) 5. Higher frequency of certain autoimmune disorders 6. Higher frequency of non-Hodgkins lymphoma and other lymphoid cancers ©. Turner Syndrome Etiology: 45,X Dominant features: 1. Lymphedema at birth 2. Short stature — Average height of 145cm (4°10") without growth hormone 3. Primary ovarian failure due to gonadal dysgenesis (streak ovary) 4. Very few are capable of having children (likely mosaics) 5. Lack of secondary sex characteristics 6. Elevated frequency of renal and cardiovascular anomalies a. Aortic stenosis b. Webbed neck & Shield-shaped chest with wide spaced nipples and underdeveloped breasts 7. Average or above average intelligence 8 Osteoporosis 9. Endocrine problems (including hyperthyroidism) 10-30% develop anti-thyroid antibodies > hypothyroidism VI. Compare and contrast paternal or maternal origins relative to type of chromosomal aneuploidy. Trisomy/Monosomy | % Parental | % Maternal 13-15, 10-12 87 16 0 100 18 45 5 2 57 93 XY 5 55 XXX 5 5 ‘Monosomy X 80 20 “75% of maternal meiosis errors are in meiosis j 25% are in meiosis I “*The X that is present in a45,X conceptus is the maternal X 80% of the time TL Page 52jer the ramifications of having a diagnosis of one of the different types of chromosomal abnormalities vi Con: relative to: a. Anaffect individual Some treatment may be available to alleviate symptoms b. Inthe parent of a diagnosed individual considering future children as well i. Depending on the genetic anomaly, the parents may consider other options (such as adoption) for future children Obtaining the diagnosis in an unborn child (ie: via amniocentesis) If it is known that a child has a certain anomaly, then precautions can betaken VIII. Understand why certain chromosomal aneuploidies are more likely to be present early in gestation rather than later in the gestation, and vice-versa. Different genes (and gene products) are necessary during different stages of development Mortality of Chromosomal Abnormalities Chromosome Abnormality | _% of abortuses that are ] Frequency among liveborns chromosomally abnormal 45K 2 15,000 (females) Triploidy 16 1/60,000 “Trisomy 16 16 0 Tetraploidy 6 0 Trisomy 21 5 17800 “Trisomy 22 5 0 Trisomy 18 3 178,000 TL Page 53Modes of Inheritance (pp 171 - 194) 1. Given a descriptive family history, draw a corresponding pedigree. ‘Common Pedigree symbols: Male: Fee: CQ) Gendertintnowm: <> vwsesat FZ rmpnms: D> sii Miscariage: <> “Termination: 22 ‘Other relationship lines: me tree OH ome OO “ao “oO Notre: By ebaice: Oyo Infesiiey Oyo For a relationship, males are typically to the left of females For sibship, children are placed in birth order, left to right Unaffected carrier: Represented by a dot in the shape Proband: First affected individual coming for medical attention Consultand: Unaffected individual seeking genetic counseling Pedigrees are simplified by grouping, represented with a number in the shape Unless otherwise specified, assume that affected individuals are heterozygous Il, Identify general pedigree characteristics that autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, and mitochondrial modes of inheritance. a. Autosomal dominant Phenotype expressed in heterozygotes Vertical transmission Males and females are equally affected 11. Equally likely to pass on mutation and equally likely to inherit it 2. Male-to-male transmission is possible iv. Unaffected individuals do not have affected children b. Autosomal recessive Phenotype is only expressed in homozygotes Males and females are equally affected ‘Trait may skip generations within a family, but are often only present in one sibshi spaoge TL Page 54iv. Affected individuals generally have unaffected parents (carriers), but an affected parent can also have an affected child (mate must be a carrier) 1. Obligate carrier: Someone that is assumed to be a carrier based on affected relatives v. Consanguinity (common bloodlines) is more frequently a factor 1. Represented on a pedigree by a double relationship line © X-linked dominant More females than males affected (generally 2:1, ignoring male infertility) Vertical transmission; disease can be seen in consecutive generations No male to male transmission (fathers do not pass geneto sons) iv. Males transmit to 100% of daughters (as they have to give their X to daughters) v. Males are more severely affected than females (dueto mosaicism) 1. Males express it in all of their cells 2. Females may only express it in some of their cells (see objective IX) a. About 50% of their cells would express the gene d. X-linked recessive i. Males are hemizygous and females are homozygous 1. Affected individuals are usually male 2. Typically, females are carriers but do not express the disease No maleto-male transmission “Skipping” of generations can be observed 1. Affected males pass gene on to unaffected daughters (carriers) 2. Carriers may then have affected sons e. Mitochondrial i. Mitochondrial mutation rate is 10 times higher than chromosomal 1. Dueto lack of mitochondrial DNA repair mechanisms Males and females are equally likely to inherit mutation Females transmit mutation to all offspring iv. Males do not transmit mutation to any offspring 1. Oocytes have mitochondria 2. Spermatocytes do not have mitochondria a. Affected males cannot affect their children v. Because cells contain several hundred mitochondria, only some may be mutated 1. Homoplasmy: All mitochondrial DNA are the same 2. Heteroplasmy: Some of the mitochondria are affected and some are normal vi. Clinical expression depends on: 1. Amount of heteroplasmy — The relative abundance of mutant mtDNAs 2. Tissue distribution of mutant mtDNAs 3. Threshold effect — The vulnerability of each tissue to impaired oxidative metabolism TL Page 55Ill, Recognize the modes of inheritance for each of the following genetic conditions or diseases... You should be able to recognize the clinical features of conditions that are presented in lecture. You will not be expected to know clinical features not discussed in lecture (course notes}: Listed in course objectives cystic fibrosis ~ Autosomal recessive Duchenne muscular dystrophy — X-linked recessive 1. Progressive neuromuscular disorder due to absence of, or minimal, dystrophin 2. Presents in early childhood with delayed milestones, waddling gait and difficulty climbing (due to proximal weakness), calf hypertrophy, and Gower maneuver Gower maneuver-> 3. Wheelchair bound by 12 years 4, Cardiomyopathy occurs after age 18 years 5. Reduced lifespan: usually teens-20s (due to respiratory and cardiac complications) Huntington disease — Autosomal dominant iv. Marfan syndrome — Autosomal dominant v. MELAS - Mitochondrial MELAS = Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes Encephalopathy with seizures and/or dementia Mitochondrial myopathy: lactic acidosis and/or ragged red fibers (RRF) on muscle biopsy Stroke like episodes, typically before age 40 years a Generally associated with seizures b. Gradually impair motor abilities, cognition, and vision 5. Onset in childhood (2-10 years): seizures, recurrent headaches, recurrent vomiting, limb ‘weakness, short stature, and hearing loss 6. Average life expectancy: 10-35 years 7. Heteroplasmy > Mother may only have minor features, or none at all Neurofibromatosis type 1 - Autosomal dominant Rett syndrome — X-linked dominant 1. MECP2 gene mutations 2. Females: Normal growth and development until 6-18 months of age b. Then, they enter a period of developmental stagnation then by rapid regression i. Progressive microcephaly (stunted head growth) Lose of purposeful hand movements, with development of stereotypies (repetitive or ritualistic movement, posture, or utterance) 1. Hand wringing, hand washing, etc. Progressive cognitive and developmental regression iv. Development of: seizures, ataxia, tremors, apnea, autism, growth failure PeNe TL Page 563. Males: a Severe neonatal encephalopathy, seizures, abnormal tone, breathing abnormalities b. Rarely survive infancy (possibly because males are hemizygous > no X inactivation) Vili, Sickle-cell anemia — Autosomal recessive 1, Most common condition in the African American population 2. 1/10-1/12 African Americans are carriers with 1/400-1/600 in the US affected b. Not listed in course objectives: Tay Sachs — Autosomal recessive Phenylketonuria — Autosomal recessive ili, Hemophilia A — X-linked recessive iv. Fragile X syndrome — X-linked recessive v. Achondroplasia - Autosomal dominant vi. Familial hypercholesterolemia - Autosomal dominant Frequency: 1/500 Heterozygotes have 2x normal cholesterol (can show incomplete dominance) Accelerated atherosclerosis Development of xanthomas 75% of men and 45% of women develop coronary disease 50% of men and 15% of women have fatal MI by age 60 Vil. Red-green color blindness — linked recessive 1, 8% of Caucasians (4-5% of Asians, 1-4% of African and Native American males) 2. 26 cannot perceive a primary color (usually red or green) at all 3. 6% can detect red and green, but have altered perception of shades of these colors IV. Given a pedigree, infer possible or probable modes of inheritance (or rule out unlikely modes of inheritance). a. See objective VI V. Identify obligate carriers in a pedigree. a. Obligate carrier: Someone that is assumed to be a carrier based on affected relatives b, In X-linked conditions: Female with an affected son + family history — obligate carrier 1. Son must get Y from father, and therefore cannot get X-linked from father Female with 2 affected sons -> obligate carrier Female with 1 affected son > MAY be an obligate carrier 1. This could result from ade novo mutation VI. Given a pedigree or family history information for any of the aforementioned modes of inheritance, predict the probability of affected offspring. Calculate the probability that any given individual has a specific genotype or phenotype. a. Salient features for determining mode: Vertical transmission > dominant inheritance Skipping generations — recessive inheritance Bias to affected males > X-linked recessive TL Page 57 ase eneiv. Bias to affected females = X-linked dominant v. Affected mother with all children affected > Mitochondrial Father to son transmission > NOT X-linked or mitochondrial Gender bias > LESS likely to be autosomal Male transmission > NOT mitochondrial ix. Unaffected mother with affected child OR affected mother with unaffected child > NOT homoplasmic mitochon dial b. See objective Vill for calculations VII. Given parental genotypes for a gene with two alleles construct and use a Punnett square to predict the number of offspring with various genotypes or phenotypes. A A A a a a AT aA AA AT AA aa AT ha Aa Ac aA AA A(_ AA Aa alfa Aa A A A a a a AT aA AA AA aa AT ha Aa a Aa ha al Aa aa al_aa aa A A A a a a Aa ha af fa aa af aa aa Aa ha al aa aa al_aa aa VIII. Describe the conditions for appropriate use of the sum rule and the product rule and apply those rules to simple questions of genetic predictions. ‘a Sum rule: Applied in “OR” conditions Either A or B will happen P(A) +P(B) b, Product rule: Applied in “AND” conditions ‘Aand B will happen P(A) x P(B) 1X. Understand the concept of X-inactivation and how it impacts X-linked inheritance. a. In females, one of the X chromosomes is turned off > both males and females have one active X i. Each cell establishes its own pattern (at the 16 to 64-cell stage) 1. Each daughter cell follows the same pattern 2. Basis of mosaicism b. I don’t think this has anything to do with the damaged X chromosome being preferentially inactivated Damaged chromosome ~ In an XX female, one altered X chromosome is not expressed 1. In order for females to express X-linked traits, they need to have the gene on both of their x chromosomes ii. This deals with damage and carrying a gene is not “damage” TL Page 58Factors That Complicate inheritance Patterns (pp 200 217) 1. PHENOTYPIC EXPRESSION a. Reduced penetrance Penetrance - Probability that an individual with a specific genotype will express a phenotype to any degree (expressed as a percentage) Complete penetrance — All people with the genotype express the phenotype (to some degree) 1. Allornothing Reduced penetrance — Not everyone with the genotype will express the phenotype 1. Can lead to “skipped” generations iv. Retinoblastoma - Malignant tumor of the retina 1. Autosomal dominant 2. Presents in early childhood 3. 60% sporadic: Unifocal a. Average age of dx: 24 months 4, 40% hereditary: May be bilateral or multifocal a. Average age of dx: 15 months 5. Penetrance is dependent on mutation; about 90% v. Chance of expression = Chance of inheritance x Penetrance b. Variable expressivity i. Variable phenotype amongst individuals with the same genotype 1. Found in the majority of genetic conditions What is expected: 1. Differences in what features are present and how strongly there are expressed 2. Difficult to predict phenotype in families As some relatives may not know they have the same genetic anomaly 3. Affected individuals may never seek medical attention due to mild symptoms Tuberous Sclerosis 11. Skin: Facial angiofibromas, hypomelanotic macules, shagreen patch (leathery skin), uungula, or periungual fibromas 2. CNS: Subependymal glial nodules, cortical tubers, seizures, MR 3. Renal and retinal hamartomas (neoplasms in the origin tissue) 4. Cardiac rhabdomyoma (rhabdomyoma = striated muscle tumor) ©. Pleiotropy A single gene produces diverse phenotypic effects (can affect multiple organ systems) cystic fibrosis 1. CFTR channel is involved in sodium and chloride transport in epithelial cells 2. Mutations cause salt imbalances, leading to thick, sticky mucus that causes obstructions 3. Causes symptoms in several areas of the body, including the exocrine pancreas, intestine, respiratory tract, male genital tract, hepatobiliary system, and exocrine sweat glands 4, Median age of survival: 30s Death usually due to end-stage lung disease TL Page 59d. Allelic heterogeneity i. Several different mutations of one gene that all cause the same disease 1L._Expression may vary depending on which alleles are present (variable expressivity) cystic fibro: 1. Over 1000 known mutations at the CFTR gene at locus 7431 with varying severity 2. 70% of mutations in the Caucasian populations are AFSO8 e. Locus heterogeneity Mutations at different loci (in different genes) > same genotype/phenotype Hearing loss - Several genes involved in non-syndromic hearing loss 1. Different genes > different inheritance patterns 2. Ifthere genes are recessive, two parents that are carriers for autosomal recessive genes from two different loci will not lead to expression in progeny f. Delayed age of onset Allinheritable conditions are present at birth (genotype), but not all are expressed (phenotype) Makes risk assessment for children difficult, as parent may not yet be presenting symptoms 1. Genetictesting is one possible solution Huntington disease 1. Progressive cognitive, psychiatric, and motor decline 2. Symptoms generally not seen until adulthood (average age of onset: 30s) Il, INTERACTION OF GENES a. Incomplete dominance Heterozygote (Aa) differs phenotypically to homozygous AA and homozygous aa 1. Heterozygotes are affected, but homozygotes have more sever effects (often fatal) ‘Achondroplasia 1. Most common form of inherited dwarfism 2. Short stature with disproportionately short arms and legs (rhizomelic shortening) 3. Large head and characteristic facial features (frontal bossing and mid-face hypoplasia) 4, Normal intelligence and life span A [a] 2% AA-Lethal ‘AA | Aa | 50% Aa—Achondroplasia a[ Aa | aa | 25% aa—Unaffected b. Codominance Different alleles have an ial effect on the phenotype ‘ABO Blood Groups Genotypes | Phenotypes | Antigens | Antibodies AA TypeA A ANteB A, TypeA A Ante B BB. TypeB B Antica BO Types B Antica (00 Type 0 None | AntrA, B AB TypeaB | AandB | None TL Page 60©. Epistasis Expression of one gene is prevented or modified by another gene (at a different locus) Hgene 1. TheH antigen needs to be present on the surface of RBCs for ABO blood types HH and Hh individuals > ABO blood typing occurs b. hh individuals > No blood types > Type 0 regardless of ABO genes d._Sex-influenced conditions Expressed in both genders, but with significantly different frequencies Male pattern baldness 1. Not an X-linked trait 2. Acts as an autosomal dominant trait in males and an autosomal recessive trait in females 3. Only one mutated allele required for males, but two required for females fe. Sex-limited conditions i. Exclusively expressed in only one gender Ill, MISCELLANEOUS/ENVIRONMENTAL a. Non-paternity i. Rate of non-paternity estimated to be 4-10% 1. Can be investigated by use of molecular techniques 2. May be revealed with DNA testing for genetic conditions May affect recurrence risk: 1L._ Siblings of an affected child may falsely be thought to be at an increased risk a. The child's paternal father most likely has different alleles prostate cancer) b. Phenocopies ‘An environmental influence mimicking a genetic disorder Examples: 1. Environmental infection/antibiotic) versus genetic hearing loss 2. Sporadic cancer in a hereditary cancer family ili, Issue: Patient may be thought to have a genetic condition that is actually not genetic 1. May be given higher than accurate recurrence risk 2. Affected individuals not at risk for their own offspring to be affected IV. DENOVO MUTATIONS/MOsAICISM a. De novo mutations Spontaneous (non-inherited) mutation occurring first in the proband Occurs in the egg or sperm cell - mutation is present in all cells Parents are not at an increased risk for a second affected child 1. However, proband has an increased risk for a passing mutation to progeny a. Risk would be equal to someone who inherited the mutation iv. Frequent in some diseases: 1. Usually autosomal dominant or X-linked, less common in autosomal recessive 2. Tuberous sclerosis: 67% 3. Duchenne muscular dystrophy: 33% TL Page 61New mutations need to occur in order to maintain the rate, as Duchenne muscular dystrophy is genetically lethal b. Somatic mosaicism Occurs after fertilization during mitosis > not all cells are affected 1. tf enough cells are affected > manifestation of disease Expression of disease may be less severe due to the presence of normal cells 1. Only some features may be expressed Difficult to determine risk of recurrence, as itis unknown if the germ cells will be affected iv. Examples: 1. Segmental neurofibromatosis 2. Neurofibromatosis: Café-au-lat spots, neurofibromas, axillary and inguinal freckling 3. Segmental neurofibromatosis: Findings are limited to a specific area of the body and do not cross midline Germline mosaicism Germ cells are a mixture of two different lines Can result in a portion of the germ cells having a mutation that the parent does not express Le Newer cae: Normal cots Gametes derived from this population have 50% chance of carrying mutant allele Population of germ cals in individual ili, Can lead to an unaffected parent having several affected children 1. Only a portion of the germ cells are affected iv. The majority of genetic conditions are thought to have a rate of <1% for germline mosaicism v. Ifthere is only one affected child in the family, you cannot determine if itis a denovo mutation or parental germline mosaicism 1. Denovo — Only child with mutation 2. Germline—Siblings may also have mutation vi. Duchenne muscular dystrophy: 15% risk of germline mosaicism in the mother when proband is only affected family member Osteogenesis imperfecta 1. 5-6% chance of germline mosaicism 2. Abnormality of collagen formation 3. Increased risk of fractures 4. Different types — spectrum of mild (few fractures) to lethal in the newborn period TL Page 62d. skewed X-inactivation i. One cell ine is preferentially activated (when it’s expected to beS0/S0 in a female) 1. Can be minimally skewed or as high as 100% a Dpercentage > Tlikelihood of effect 2. tf majority of cells with active X that carry the mutation a. >more severely affected, possibly lethal 3. If majority of cells with active X that does NOT carry mutation a > milder presentation Xlinked recessive: Female carriers may express phenotype 1. Depending on degree of skewing a female with the mutation in the majority of genes, the female may express some or all features of the condition 2. Examples: a. Brain in Fragile X (50% have some degree of MR) bb, Muscle in Duchenne muscular dystrophy (1/3 may have muscle weakness) ili, %linked dominant: Females with mutations may have milder expression than males 1. Females who carry mutation may not exhibit expected phenotype Normal X more often inactivated > More severely affected b, Mutated x more often inactivated > Milder than typical, possibly unaffected 2. Rett syndrome a. Afemale that doesn’t show symptoms may still be a carrier and have an affected child V. DETERMINING RECURRENCE RISKS WITH BAYESIAN ANALYSIS a. DO NOT NEED TO KNOW VI. OTHER INFORMATION a. Hereditary hemochromatosis — Increased iron absorption 1/9 Caucasians carry the severe mutation Often recognized in early stages 1. Thought to be preventable by reducing blood iron content Demonstrates: 1. Reduced penetrance — Not everyone with 2 mutations will develop HH 2. Variable expressivity — Individuals with HH may present very differently 3. Pleiotropy — Multiple organ systems affected 4. Locus heterogeneity — Usually due to HFE gene mutations, but three other genes are known 5. Allelic heterogeneity ~ 3 common mutations (C2822Y most severe) 6. Delayed age of onset Typically does not develop until adulthood 7. Phenocopies — May develop due to acquired conditions, such as alcoholic liver disease or hepatitis 8 Sex influenced — More common in males than females, protective effect of menstruation TL Page 63,‘Skeletal Skin arthritis ‘bronze or gray « joint pain dlscoloration \—— Heart = auhythnia . * failure liver s cirrhosis Rca Pancreas ele = diabetes mellitus Other symptoms include: * fatigue * weight loss * anemia * abdominal pain * impotence * menstrual iregularity carly menepause b. Disorder with reduced penetrance As previously stated, if parent with a disorder has a child who does not show signs of the disorder, it may be due to penetrance 1. The child could have the genotype, but is not expressing the phenotype Occurs in retinoblastoma Seeta Disorder with late age of onset i. Risk of transmission is modified depending on the age of the parent 1. The older a person is (and hasn’t presented with symptoms}, the less likely they are to be carryingthe gene Occurs in Huntington disease Seelf d._ Disorder with possible germline mosaicism If only one child carried a new disorder, itis possible that the effect is in the parental germ cells. Seelv-c TL Page 64Common Multiple Anomaly and Micro-Deletion Syndromes (pp 222 - 232) |. Explain the theorized causation of many micro-deletion syndromes. a Itis believed to be a recombination error leading to duplications and deletion of chromosomes During crossing over, there would be improper separation and two copies of a gene will end up on one chromosomes (duplication), leaving the other chromosome lacking the gene (deletion) Recognize the clinical features of several relatively well known and common micro-deletion syndromes. a. Wolf-Hirschborn Syndrome - del 4pter Profound mental retardation Severe grand mal and/or minor motor seizures Greek Helmet appearance 1. Ocular hypertelorism (eyes are far apart), Broad/beaked nose, Micrognathia (small chin) b. Cri-du-chat Syndrome — 5p. (missingSp; usually paternal) Newborns have weak high-pitched “cat-cry” Mental retardation, developmental delay, slow growth Micrognathia, microcephaly, hypertelorism, epicanthal folds, low-set ears ©. Velo-Cardial Facial aka DiGeorge Syndrome - 22q11 microdeletion Occurs in 1/4,000 births ‘Abnormalities primarily affect the 3" and 4" branchial arches during embryonic development 1.3" branch — Normal development of the parathyroid and thymus glands 2. 4" branch — Contributes to same structures, as well as cardiac development Parathyroid hypoplasia > hypocalcemia iv. Thymic aplasia > immunodeficiency v. Conotruncal heart defects (usually simple valvular involvement) Dysmorphic facial features Cleft tip, cleft palate, submucosal cleft (> hypernasal speech) Developmental delay Hypospadias (genital abnormalities) x. risk for schizophrenia (schizophrenia gene proposed to be at 22q11) 5% can be detected by gross chromosome analysis (95% need FISH probes) sms Syndrome — 7q11.23 microdeletion (there is an elastin gene at 7q11.23) i. Effin features: 1. Microcephaly 2. Periorbital puffiness/edema 3. Fulllips 4. Stellate iris 5 6 Malar hypoplasia (small cheekbones), full cheeks 3. Wide mouth, small mandible 7. High pitched, hoarse voice Cardiac involvement — supravalvular aortic stenosis Bladder diverticula - reflections of poor connective tissue strength iv. Hypercalcemia > stones, bones, and abdominal groans 1. Note that hypocalcemia results from DiGeorge v._ Proclivity towards music and/or musical fields and Loquacious (charming, engaging, and chatty) TL Page 65Ill, Identify the chromosomal location, and dominant phenotypic features of patients diagnosed with the following microdeletion syndromes: Wolf Hirschborn Syndrome, Cri-du-Chat Syndrome, Velo-Cardial Facial aka DiGeorge Syndrome, and Williams Syndrome. a See objective tt IV. Recognize the clinical features of several relatively common, single-gene gen: a. NF-1-Von Recklinghausen disease Occurs in 1/4,000 births ‘Autosomal dominant inheritance Half of all cases are due to a new mutation 1. Mutation rate is 1/10,000 iv. Exhibits: 1. Complete genetic penetrance — If you have the mutation you will manifest symptoms 2. Variable expressivity - Manifestation and/or age of onset will vary 3. Pleiotropy — Can have multiple phenotypic effects v. Clinical diagnosis — 2 or more of the following criteria 1. Six or more café au lait macules (25mm prepuberty and >15mm postpuberty) Skinfold freckling (axillary or inguinal - “where the sun don’t shine”) ‘Two or more neurofibromas, or one plexiform neurofibroma Optic glioma > tunnel vision and headache (needs annual screening) ‘Two or more Lisch nodules (benign hamartomas of the iris) Osseous lesion (sphen oid dysplasia) or thinning of long bone cortex with or without pseudoarthritis 7. First-degree relative with NEL vi. Those with NFI have an increased risk for cancer in general, as this is an overgrowth syndrome b. NF-2~Mutation of 22q12 (the “Merlin gene”) Occurs in 1/40,000 births Half of all cases of NF-2 are new mutations Bilateral eighth nerve vestibular schwannomas Confirms diagnosis 1L._ Symptoms of tinnitus (ringing), hearing loss, and balance dysfunction iv. If first degree relative has NF-2, then aSchwannoma or other cranial or peripheral nerves, meningiomas, or juvenile cataracts > diagnosis without bilateral vestibular schwannomas v. 2/3 of NF-2 patients develop spinal tumors, % develop meningiomas Exhibits: 1. Complete penetrance 2. Variable expressivity If the proband has a detectable mutation, there is a good role for DNA testing: 1. Primarily to detect at risk individuals early 2. Obviates need for annual MRIs commencing in teen years, and brain stem auditory evoked response testing to test for vestibular nerve involvement ay ewen TL Page 66© Marfan syndrome — Occurs in 1/5,000 (may be more frequent, just not being diagnosed) ‘Autosomal dominant inheritance Exhibits: complete penetrance and variable expressivity iv. Mutation in the FBN-1 gene (fibrilin) 1. Primarily missense, although nonsense/frameshift also described v. Deiiifahit Negative mutations result in more severe phenotypes, relative to individuals that have nonsense/frameshift mutations Fit Fibyitin protein Fibrilin oe monomers: polymers —— tate Normal: wt —— tO wt + —w FO —o—+ epee Nonsense: wt——» oe ae ad oe Se OH HD, Marfan: | wt > See misense es patient ™ ———r HED Vi. Clinical diagnosis is the presence of two or more of the following: 1. Skeletal manifestations — Arachnodactyly (long, thin fingers), Increased arm span, Abnormal upper to lower segment ratios, Hypermobility, Pectus excavatum, Stretch marks, Acetabulo protrusion 2. Cardiovascular: aortic aneurysm Preemptive B-blockers may slow down dilation b. Aggressive BP control mandatory & Screen with repeated echocardiograms with aortic root measurements d._ Preemptive aortic root replacement 3. Optic: Dislocated lens (upwardly shifted) and propensity to retinal tears 4. Dural ectasia- Widening of dural sac Confirmed by CT or MRI 5. Family — Confirmed diagnosis in a first-degree relative Vii. A gene testis available for detection of mutations in the fibrllin gene, but as with the other disorders, the results are only around 70% reliable V. Recognize the importance of diagnosing these conditions relative to: Implications. a See objectives il and iV ledical Implications and Genetic TL Page 67VI.__Understand need for adhering to clinical criteria when diagnosing several autosomal dominant, multiple anomaly syndromes: NF-1, NF-2, and Marfan Syndrome a See objective lv VII. Understand the concepts of complete genetic penetrance, variable expressivity, and locus heterogeneity. a. See objectives il and IV VIII. Rationalize how the concepts of complete genetic penetrance, variable expressivity, and pleiotropy influence and have impacted upon medical/genetic management of: NF-1, NF-2, and Marfan Syndrome a See objective lv TL Page 68Multifactorial inheritance (pp 233 - 243) 1. Understand the d \n of multifactorial diseases, and reflect on the distinct genetics of these diseases vs. those caused by single gene or chromosomal defects. ‘a. Multifactorial inheritance - Many clinical phenotypes are the result of shared genetic and environmental factors b. There difference between these diseases and those caused by a single gene or chromosomal defect is that multifactorial diseases require an outside stimulus i. Teratogens are one type of environmental factors that can contribute to congenital malformations that are multifactorial 1. Examples: certain medications, maternal conditions, alcohol 1, Explain how twin studies help to distinguish the effects of genes vs. environment. a. Dizygotictwins (aka DZ or fraternal twins) ~ share“50% of genes b. Monozygotictwins (aka MZ or identical twins) - share~“100% of genes i. By observing MZ twins exposed to different stimuli, you can determine if a phenotype is a result of genetics or environment Difficulties with twin studies: The environment for MZ twins may be more similar than for DZ twins —> skewed results Post-fertilization somatic mutations may make MZ twins not entirely identical In-utero environment may differ among pairs of MZ twins Ill, Understand how the incidence of a disease in Monozygotic vs. Dizygotic twins allows one to discern genetic influences on the respective disease. a. See objectives il and IV IV. Define concordance rates. a Concordance rates — how often individuals share the same trait/condition i. Traits among twins are determined to be concordant (shared) or discordant (not shared) b. When determined solely by genes: MZ twins have "100% concordance Dz twins have “50% concordance When determined solely by the environment: MZ twins and DZ twins havethe same concordance V. Define heritability. ‘Heritability — The proportion of the total variance of a train that is caused by genes ‘The difference between concordance rates: H = 2(Cuz— Coz) 1. Greater difference > higher heritability 2. Ranges from Oto 1 VI. Understand why high concordance rates do not necessarily reflect high heritability. a. Formeash MZ concordance = 0.95; DZ concordance = 0.87 > Heritability = 2(0.95 -0.87) =0.16 b. This occurs when some has a high rate of occurrence regardless of genes © Heritability does not tellus: How many genes can be involved How much each gene contributes to phenotype Mode of inheritance TL Page 69VII. Be able to recognize what condition(s) have a higher heritability based on concordance rate: and Dizygotic Twins. a. H=2(Cuz— Cox) Monozygotic © Quantitative Results ‘© Polygenictraits: Combined effects of multiple genes -> variation in phenotype © Multifactorial traits: Environmental and genetic influences -> variations in phenotype © Quantitative traits are multifactorial (i.e.: height, blood pressure) = Measured along a continuous scale as opposed to distinct and separate phenotypes + The phenotypes of multifactorial traits are often characterized by a bell-shaped distribution Vs Height One gene Muliple genes + environment VIIL. Consider and understand how both genetic and environmental factors contribute to the threshold of liability for a given medical condition. a. Example blood pressure: Genetic Formation of the heart, protein content of blood, baroreceptor reflex sensitivity Environmental Stress, diet, muscular tone (from exercise) 1X. Apply the threshold model of multifactorial diseases to several medical conditions, and understand how it, influences the genetic recurrence risk of these conditions. a. The threshold model applies to multifactorial diseases that exhibit a pattern of penetrance related to the number of predisposing factors i. The disease will manifest once the threshold is met/exceeded (i.e. cleft lip, cleft palate, omphaloecele, etc.) ii. This does not apply to diseases that are “present or absent” . Effect of gender on threshold — Threshold of liability may differ among genders for a specific disease Example: pyloric stenosis 1, Males (1/200) > Females (1/1,000) X. Understand and explain how differences in gender thresholds might affect recurrence risks in families affected by multifactorial diseases exhibiting gender threshold dependence. ‘a Recurrence — Risk that a relative will develop the same multifactorial disease (difficult to quantify) b. Factors leading to an increased recurrence risk in a family Affected family member is a close relative More family members are affected 1. VSD: 1 child = 3% risk; 2 children = 10% risk; 3 children = 25% risk (autosomal recessive) ili, The disease has more severe expression within that family iv. Affected individual is a member of the less commonly affected gender TL Page 70XI, Understand how recurrence risk can be affected by sex of the proband, and/or other clinical factors. a. ifthe proband belongs to the sex with the lower threshold, there is a lower risk of recurrence i. tfamalehas a lower threshold for disease X, he would have less genetic anomalies to pass onto Progeny. 1. However, male progeny have a higher risk. If a female has a higher threshold for the same disease, she would have more genetic anomalies to pass onto progeny. 1. However, female progeny have a lower risk Xl. Recognize the clinical scope of multi-factorial conditions covered in lecture. a. Pyloric stenosis Narrowing or obstruction of the pyloris Due to the same principal described in objective X! and that it is times more common in males: 1. fagirl has it, then a male sibling would have a higher likelihood of having tt 2. tfaboy has it, then a male sibling would have a lower likelihood of having it b. Neural tube defects Spina bifida and anencephaly — Failure of neural tube to close at “4 weeks gestations Occurs in 1/500 - 1/,000 ili, Recurrence risk = 3% for a first degree relative 1. Folic acid supplementation > decreased risk of recurrence a 0.4mgrecommended for all women, 4mg for history b. Taken one month prior to pregnancy and through first two months of gestation & 50-70% of ONTDs (open neural tube defects) may be avoided © Coronary artery disease 1. Causes 25% of deaths in the US eS aa Over a dozen genes have been ruttion identified ora Muttifactorial _— famiiv histo d. Thrombosis Da Formation of thrombosis 7 [apelipopiotein Risk increases with age—1/100,000 in . childhood up to 1/100 in the elderly 1. 1/10,000 in women under 40, ili, Genetic factors: Factor V Leiden, Prothrombin 20210A mutation, MTHFR mutation (methylenetetrahydrofolate reductase deficiency > '[homocysteinel), Protein S deficiency, Protein C deficiency, Antithrombin It deficiency iv. Environmental factors: Surgery/Trauma, Cancer, Chronic conditions (diabetes, obesity, hypertension, high cholesterol), Smoking, Prolonged inactivity, Hormonal (pregnancy, oral contraceptives, estrogen therapy) TL Page 71& Factor V Leiden (FVL) ~Specific mutation that increases the risk of VTE (venous thromboembolism} 3.8% of the general Caucasian population are heterozygous, 1/5,000 (0.02%) are homozygotes Risk of VTE 1°4-8 for Aa (<1% per year without risk factors) and 1'80x for aa Presence of other genetic factors may further increase risk 1. Prothrombin 202108 mutation affects 2-3% of the US Caucasian population a, 35 fold increased risk for VTE b. Prothrombin + FVL = 20-fold risk iv. Environmental factors: ‘Oral contraceptives: 4-fold risk Pregnancy:5-fold risk Oral contraceptives + FVL: 35-fold risk | Pregnancy + FVL: 20-25-fold risk Prothrombin + FVL + pregnancy: 100-fold risk (1% riskin women under 40) v._ Testing for inherited thrombophilia may include: 1. Genetictesting: FVL, prothrombin, MTHFR 2. Analyte: Protein C, protein S, antithrombin il, homocysteine vi. Who should be tested: 1. A first VTE before age50 years or unprovoked VTE at any age 2. Ahistory of recurrent VTE 3. VTE at unusual sites such as the cerebral, mesenteric, portal, or hepaticveins 4, VTE during pregnancy or postpardum, while on oral contraceptive or HRT (hormone replacement therapy) 5. Afirst VTE at any age in an individual with a first-degree family member with a VTE before age50 years or a strong family history Consider testing in: 11. Individuals with a known family history of a genetic predisposition to thrombosis 2. Women with unexplained fetal loss after ten weeks’ gestation Management 1. Avoid environmental risk factors (smoking, prolonged inactivity, oral contraceptive use) 2. Long-term anticoagulant therapy: Individuals homozygous for FVL b. Those with recurrent VTE 3. tf no history of VTE, long-term anticoagulant medication is not recommended a. Risk of a bleed from meds is greater than the risk of clotting in most individuals b. Consider short term treatment when at ‘risk (pregnancy, cancer, surgery) f. Cancer —5-10% of common cancers are thought to be due to a hereditary cancer syndrome i. Breast cancer 1. Genetic predisposing factors - BRCA1, BRCA2, and p53 gene mutations 2. Environmental factors — Related to estrogen and progesterone exposure a. Nulliparty (never pregnant) or Late age at first birth b. Early age of menses Late age at menopause 3. General population risk - 12-13% 4. One first degree relative (FDR) affected: 1.5-2x risk; 2 FDRs = 24x risk TL Page 72Colorectal cancer 1. Genetic predisposing factors — APC, MYH, and DNA repair gene mutations 2. Environmental risks — Low fiber diet and lack of exercise 3. General population risk — 6% 4. One first degree relative (FDR) affected: 2-3x risk; 2 FDRS = 3-4x risk Type | Diabetes (Insulin-Dependent Diabetes Mellitus) i. Strong association with several HLA class I! alleles (DR3 and DRA), and elevated risks for relatives of affected individuals 1. Over 20 other genes are being considered 35-40% concordance for MZ;5-10% concordance for DZ - heritability =05 to.0.7 Population risk: 1/500 iv. Risk for FDRs: 1/14 for siblings and 1/25 for offspring 1L._ Risk to siblings may increase to 18% if both HLA types shared, and to 20-25% if DR3/4 hh. Type ll Diabetes (Non-Insulin-Dependent Diabetes Mellitus) MZ concordance =59%; DZ concordance = 27% -> Heritability = 0.64 Some families affected by rare, autosomal dominant genes, most families exhibit multifactorial inheritance patterns ili, Obesity and lack of exercise are environmental factors iv. Population risk: 1. 6.2% for Caucasians 2. 10.2% for African Americans 3. 50% in certain Native American groups v. Risk for FDRs is 2x higher ic disorders i. Schizophrenia 1. MZ=47%; DZ 2. General population risk 3. Risk for siblings = 10% 4, Risk for offspring with one affected parent = 12.8%; two affected parents = 46.3% ii. Bipolar affective disorder 1. MZ=67% (22-93%); DZ = 20% (0-249) > 2. General population risk: 0.5-1% 3. Risk for FOR: 2.8-17.7% for bipolar and 7.0-22.4% for unipolar Xill, “Take home lesson” a Mendelian disorders, are more rare and, often have the largest impact on specific families b. Multifactorial disorders, are more common and, have largest impact on population as a whole 0.94 TL Page 73,Clinical Correlates of Genetic Anticipation (pp 244 — 253) 1. Recognize several potential clinical manifestations of a genetic disease that shows genetic anti family history. a. Genetic anticipation — Increased frequency of affected relatives, increased severity of phenotype, or earlier age of onset in successive generations b. Thisis observed Fragile X Syndrome Myotonic Dystrophy Huntington Disease iv. Friedrich’s Ataxia & TheSherman Paradox — a clinical description of Genetic Anticipation i. The effects of Fragile X seemed to worsen with each successive generation ii. Risk of expressing mental retardation could also be dependent on the position in the pedigree 1. Daughter of an unaffected male carrier was more likely to have affected offspringthan the mother of the unaffected male carrier ‘This showed that a premutation doesn’t cause severe symptoms, but as the gene was passed on, it progressed to a full mutation, especially when passed through a female. List the dominant features of Fragile X syndrome. ‘Incidence: 1/1,000 - 1/6,000; X-linked dominant Most common form of inherited mental retardation b. Clinical signs: Macrocephaly, large ears, macro- orchidism (large testicles), echolalia (parroting what someone says), perseverative speech (repetition of phrases), gaze avoidance 2.6% of individuals diagnosed with autism have Fragile X syndrome i. Fragile X isthe most common genetic cause of autism Ill, Understand the chromosomal and molecular mechanism underlying the Fragile X chromosome. a. The chromosomal change is the breakage of the X chromosome at the “Fragile site” i. Dueto trinucleotide expansion of a CGG repeat normally present at the’ end of the Frax gene 1L. Expansion to >200 repeats > methylation of rOROt@E -> inability to express the Frax MRNA/protein 2. Probability for insertions increases during meiosis and gamete production IV. Understand what trinucleotide repeats are, and what the ramifications of their expansion may be. a. Trinucleotide repeats are repeats of three nucleotides (such as CGG in Fragile X Syndrome) that are found in the chromosomes b. During replication, some of them will expand and some of them will contract Significant expansion => phenotype Premutation: Expansion of repeat, but not significant enough to cause phenotype i. Unstable; may lead to full mutation in next generation V. Consider how trinucleotide repeat expansions mechanistically cause the Sherman Paradox and Genetic Anti ‘a. With each passing generation, the mutation gets worse and worse b. Therefore, you can readily assume that someone who is on the verge of their permutation becoming a mutation, will have affected offspring TL Page 74Vi Con alleles. a. The pedigree to the right shows that pattern of 20 sina inheritance of Fragile X Syndrome jer how X-inactivation may contribute to phenotypic vari 1s of expanded Fragile X b. The other facet to consider is that even if a female has a mutant X chromosome, there is the possibly of Xinactivation i. This is never 100% for a mutant gene, so they may show more mild signs, or their phenotype may develop at a later age This could pose a problem when using an incomplete family history to determine if someone will pass on Fragile X VIL. Identify how sex of a carrier of a permutation allele might affect chance for trinucleotide expansion in subsequent offspring for: a. Fragile X Syndrome i. Xlinked > affect more males than females b. Myotonic Dystrophy i. Autosomal > equal sex distribution ©. Huntington Disease i. Autosomal > equal sex distribution jer broad clinical spectrum of several other trinucleotide repeat disorders. ‘a. FMR-1—Females with premutations have ‘risk of premature ovarian failure (POF aka menopause) i. However, females with full FMR-1 mutations are not affected with POF ‘This occurs because certain lengths of mutation pose the greatest risk, and once you exceed the range with the highest risk, the risk of POF begins to fall b. FXTAS — Fragile X-associated Tremor/Ataxia syndrome Progressively affects those older individuals carrying premutation expansions of the CGG repeat Mechanism is different from FraX mental retardation 1. Still expressing mRNA, but itis modified ations of exonic vs. non-exonic trinucleotide expansions on eventual RNAS or protein derived from an expansion allele. a. Exonic— Coding regions Expression is unchanged, but the mRNA codes for polyglutamine tracts ~ structural defects Effects of polyglutamine tracts 1. Affects particular subsets of neurons Causes protein aggregation in the nucleus Gain of function mutations Repeats tendo be smaller in size and variation Progressive neuronal dysfunction that begins in mid-life Larger expansion > earlier onset of symptoms (in general) ‘a. Known as anticipation VII, Con 1X. Define rami ay ewn TL Page 75ili, Examples: 1. Spinobulbar muscular atrophy (Kennedy disease) 2, Huntington's disease 3. Dentatorubrat-pallidoluysian atrophy (Haw-River syndrome) 4, Spinocerebellar ataxia type 1 5. Spinocerebellar ataxia type 2 6. Spinocerebellar ataxia type 3 (Machado-Joseph disease) 7. Spinocerebellar ataxia type 6 8 Spinocerebellar ataxia type 7 9. Spinocerebellar ataxia type 17 (recently recognized) b. Non-exonic—Non-coding regions Does not affect the mRNA, but affects the level of expression Examples: 1, Fragile X syndrome (FRAXA) 2. Fragile X“E” MR (FRAXE) 3, Frledigh’s/atakia (FRDA) — A quadranucleotide repeat 4, Myotonic dystrophy (DM) 5. Spinocerebellar ataxia type 8 (SCA8) 6. Spinocerebellar ataxia type 12 (SCA12) X. List the dominant features of Myotonic Dystrophy syndrome. 4 CTG expansion in 3’ untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene i. Codes for myotonin-protein kinase b. Occurs in 1/100,000 Repeat size: Normal: 5-37 Premuation: 38-49 DMPK protein levels are unaffected; problem appears to be with the processing of the expanded mRNA TG premutation repeat can expand after maternal transmission TG premutation repeat can contract after paternal transmission Symptoms depend on repeat size: 50-150 (mild) 1. Cataracts, mild myotonia (slow muscle relaxation), premature balding 2. Age of onset 60, with normal lifespan ii, 100-1000 (classic) 1. Weakness, myotonia, cardiac arrhythmias, pulmonary weakness with respiratory insufficiency 2. Age of onset 10-30 years, average age of death 48-55 years :1000-2000+ (congenital) 1. Most likely to occur after inheritance from maternal side (imprinting) a. If childis affected, mother is expected to have mild or classic form 2. Infantile hypotonia, respiratory insufficiency/failure, mental retardation 3. Onset at birth (0-10 years), lethal... but may be milder amon TL Page 76XI. Understand how anticipation and size of trinucleotide expansion/contraction influences phenotype of Myotonic Dystrophy. a See objective x Xil. List the dominant features of Huntington Disease. a. 3-7/100,000 (0.003-0.007%) b. Progressive disorder of motor, cognitive, and psychiatric disturbances i. 2/3 present with neurodegenerative changes 1. Spasticity, choreoiform movements, bradykinesia, hyperreflexia ii, 1/3 present with primarily psychiatric disturbances 1. Personality changes, psychosis (20-90%), schizophrenic psychosis (12%) © Cognitive decline over time, although speech is preserved XII Understand how anticipation and size of trinucleotide expansion/contraction influences phenotype of igton Disease. Increased expansion generally means earlier age of onset for typical neurodegenerative symptoms b. 10% of patients with onset before age 20 (60 CAG repeats) Expansion of CAG trinucleotide sequence in the HD (Huntingtin) gene -> production of abnormal Huntingtin > neuronal dysfunction CAG repeats in the HD gene Normal alleles: 10-26 or fewer CAG repeats, Intermediate alleles: 27. 5 CAG repeats -may have offspring with HD , but the individual is not at risk Mutant alleles: 36 or more CAG repeats -Reduced penetrance alleles: 36-~41 CAG repeats. -Fall penetrance alleles: 40 or more CAG repeats, XIV. Contrast genetic location of trinucleotide expansion in Huntington gene to that of Fragile X and Myotonic Dystrophy expansion locale, and how this impacts on protein functions of their respe a See objectives Vil and Ix XV. _ Consider how the phenomenon of trinucleotide expan expressivity, and locus heterogeneity known to occur Disease patients. a Genetic penetrance Generally, a longer repeat sequence would lead to a more severe phenotype However, in the case of POF, this is shown to be more of a guidelinethan a rule b. Variable expressivity i. Expressivity would be determined by the “severity” of the repeat Locus heterogeneity i. Not observed, as all three diseases occur at a specific locus genes. influences the genetic penetrance, variable Fragile X, Myotonic Dystrophy, and Huntington TL Page 77XVI. Friedrich’s Ataxia Occurs in 1/50,000 b, Autosomal recessive > progressive neurological deterioration commencing during puberty i. Trinucleotide expansion in an intron of the frataxin gene Results in gene silencing or lack of transcription of the frataxin mRNA. GAA triplet 1. Normally 7-22 repeats 2. >200in affected individuals © Cerebellar involvement Ataxia (gait disturbance) Dysarthria (speech problem) and subsequent dysphagia (swallowing problem) Titubation (excessive bobbing of the head or trunk to maintain its position in space) iv. Early signs could include scoliosis and pes cavus (high arched feet) d. Other symptoms i. Hearing loss Cardiac arrhythmia/hypertrophy Endocrine issues (including diabetes) iv. Low expression of Frataxin -> mitochondrial dysfunction due to iron accumulation 1. Normally, frataxin would localize in the mitochondrial membrane & Anticipation GAA expansion occurs with transmission from either parent Larger expansions occur as gene is passed from generation to generation but these are unstable, and can also contract (especially when paternal) Larger expansions + earlier onset and/or more severe clinical feature 1. Phenotype corresponds to the shorted of thetwo expansions in ratasin alleles TL Page 78Clinical Correlations of Genetic Imprinting (pp 254-263) 1. Understand that there are multiple levels of mammalian regulation of gene expression, and that imprint ‘one of those mechanisms. Genetic imprinting — Different expression of genetic material, at either the chromosomal or allelic level, depending on whether the genetic material is inherited from the male or female parent i. This is anon-Mendelian form of inheritance I, Rationalize how maternal or paternal inheritance of certain genes can and does influence human disease phenotypes. Although you inherit 2 copies of each gene (1 maternal, 1 paternal), a specific one or both of them may be necessary to express a certain phenotype b. Evidence shows that genetic imprinting is a predisposing/contributing mechanism for influencing the genetic penetrance, variable expressivity, and locus heterogeneity of several diseases Huntington disease — Earlier age of onset if inherited paternally Myotonic dystrophy Severe, early onset if inherited maternally Fragile X — Trinucleotide expansions more likely to occur if inherited maternally iv. Autosomal Dominant spinocerebellar ataxia — Severe, early onset if inherited paternally v. Neurofibromatosis - Increased severity with maternal transmission Il, Understand how genetic imprinting might be responsible for alterations in the genetic penetrance, variable expressivity, and/or locus heterogeneity of several medical conditions. a. These three concepts can all be applied to the examples in objective I IV. Understand the genetic composition of teratomas and how this confirms the existence of genetic imprinting in humans. a. fa fertilized egg has two pronuclei of maternal origin > ovarian teratomas or dermoid tumors ‘This is an example of unipaternal diploidy (46,Xx; all from mother) These tumors consist of diverse tissues like hair, teeth, bone, thyroid, etc. V. Understand the genetic composition of molar pregnancies and how this confirms the existence of genetic imprinting in humans. a. fa fertilized egg has two pronuclei of paternal origin -> hydatidiform mole (molar = grapes) i. Abnormal trophoblastic layer and little/no embryonic development VI. Compare and contrast the phenotypes of complete and partial molar pregnancies, and relate these differences to the underlying mechanism of genetic imprinting. a Completemole i. fa single sperm fertilizes an empty oocyte 1. 90% > the sperm derived genome duplicates 2. 10% > 2sperm fertilize the egg Can be 46,Xx or 46,XY Carries risk of malignancy to choriocarcinoma b. Partial mole Haploid ovum is fertilized or via dispermy-> 69,XXX or 69,XXY Fetal parts can be seen, no malignancy potential TL Page 79Vl. Compare and contrast ss and differences of Uniparental Diploidy and Uniparental Disomy. @.Uniparental diploidy is when you receive two haploid contributions from one parent (all chromosomes) b.Uniparental disomy is when a partial trisomy is when the body attempts to repair a partial trisomy, but kicks out the wrong chromosome (a single chromosome) i. For example, a47,Xx,+20 individual will have three copies of chromosome 20 (for example, 1 paternal and 2 maternal). Ideally, one of the two maternal chromosomes would be removed, but theres a 33% chance that the paternal chromosome is removed -> 46,Xx with uniparental disomy of chromosome 20 Of note, there have been no clinical symptoms reports for uniparental disomy of chromosomes 2Lor 22 VIII. Consider how differential methylation of a gene locus can act as a mechanism underlying genetic imprinting. ‘a, Methylation generally results in the inactivation of genes (by blocking transcription factors) Page 105 of the course pack talks about methylation of DNA ~ tightly packed heterochromatin b. Priorto fertilization an imprinting (methylation) pattern is established that persists throughout life i. Thisis erased in germ cells 1X. Understand the phenotype of the classical imprinting syndromes Prader-Willi and Angelman, as well as contrast the similarities and/or differences in their clinical presentations. a. Prader-Willi Syndrome — No paternal 15q11 (deleted or non-functional) Neonatal hypotonia Poor feeding, failure to thrive up to age 2 > @ 2years become voracious and steal food Short stature, small hands and feet iv. Micro-penis, hypogonadism v. Moderate to severe MR (accompanied by microcephaly) Obesity Lack of ability to regurgitate Great with puzzles Sometimes albinism b. Angel Syndrome—No maternal 15q11 (deleted or non-functional) Normal at birth, but manifest symptoms later in life Progressive microcephaly Severe MR, lack of speech onset iv. Gait ataxia, “happy puppet” syndrome v. Abnormal EEG, seizures can occur usually by age 3 Sometimes albinism Causes: 1, 5% of cases are mutations in the UBE3A gene, which is expressed in the brain 2. 25% of cases are DNA mutations that disrupt the imprinting center (-> improper methylation) of the Angelman/Prader-Will locus X. Explain how the following genetic mechanisms underlie the causation of the classical imprinting syndromes Prader-Willi and Angelman Syndromes: a. Inheritance of Genetic Deletions in the PWS/AS critical region PWS is dueto deleted or non-functional paternal 15q11 (60-70% of patients) AS is due to deleted or non-functional maternal 15q11 (70% of patients) TL Page 80b. Uniparental Disomy for the PWS/AS critical region 20-25% of PWS due to maternal uniparental disomy (no paternal chromosome 15) 2.4% of AS due to paternal uniparental disomy (no maternal chromosome 15) XI. As a conformation as to your understanding of the genetic mechanisms underlying Prader-Willi or Angelman ‘Syndromes, consider the hypothetical situation of either of these individuals having children (typically these indi luals are sterile) a. Consider if PWS or AS is due to deletion or uniparental disomy in the hypothetical parent Deletion — child will have PWS or AS, depending on parent with deletion Uniparental disomy > parent would be capable of having an unaffected child b. Understand how the sex of the parent would influence the outcomes relative to imprinting in these conditions. i. Deletion: Mother has deletion ~ child has Angelman syndrome 1. Father has deletion > child has Prader-Willi syndrome ii. Uniparental disomy: 1. Maternal disomy - child has Prader-Willi 2. Paternal disomy > child has Angelman XII. Understand how imprinting can impact upon several other diseases, including individuals affected by Beckwith-Weidemann Syndrome. ‘a. Congenital overgrowth syndrome occurring in 1/14,000 births b. Clinical features Macrosomia (big body), macroglossia (big tongue), hemi-hyperplasia (greater than normal asymmetry between the right and left halves of the body), visceromegaly (enlargement of the visceral organs) Posterior helical ear pits and creases Abdominal wall defects: omphalocele, diastesis rectii (vertical splitting of the abdominal wall), umbilical hernia © Clinical risks High risk for embryonal tumors, such as Wilm’s tumor of the kidney, and/or hepatoblastoma Neonatal hypoglycemia — Due to islet cell hyperplasia > hyperinsulinism d._Etiology/Imprinting 85% sporadic and 10-15% due to autosomal dominant inheritance 1.26 of cases involve detectable alterations in 1115 including duplications) 20% have paternal uniparental disomy at 11p15 > over-expression of insulin-like growth factor 2 (IGF2) Xill.Are all human chromosomes imprinted? a. No. The genes exhibiting Mendelian inheritance receive information from both chromosomes and therefore, cannot have those genes imprinted. XIV. Other examples of Uniparental Disomy Chromosome 7 > Russellsilver syndrome (IUGR (intrauterine growth restriction), post-natal short stature) and some rare instances of CF dueto uniparental inheritance of a mutant CF allele on chromosome 7 b. In Vitro Fertilization = increased rate of imprinting errors TL Page 81Population Genetics (pp 264 - 269) 1. Summarize the principle (and its mathematical basis) that describe the maintenance of low-frequency and recessive alleles within a population. a. Allele frequencies frequency of dominant allele (A) = frequency of recessive allele (a) ‘You must have one of the other, so total allele frequency: p+ q=1 Derivation of binomial equation (p+) (p+q)=(1)(1) (p+ q)?=1 p+ 2pq+q?=1 1. pt=Aa 2 2p 3 q’=aa I, Recognize or describe the conditions or assumptions under which the Hardy-Weinberg equilibrium principle holds. The binomial equation only holds true for 2 alleles b. Random mating (not selective mating) & Large population (principle does not work for small groups) d. No migration, no new mutations, no natural selection (same alleles are present) IL, Recognize or describe how each of the following conditions might affect prediction from the Hardy-Weinberg ‘equilibrium principle: a. Migrations — if a certain allele flows in or out of the population -> incorrect numbers b. New mutations — Are not accounted for by the equation and can -> incorrect numbers Natural selection — If certain traits lead to the death of a segment of the population, then those alleles ‘would slowly be removed from the population 4d. Nonrandom mating — if homozygous dominant only mated with homozygous dominant, then the recessive trait would eventually become extinct — Leads to increased retention of recessive traits f. Genetic drift -See migrations 8. Founder effects —Small group of population splits off and their traits would be “increased” as a percentage of the whole population (ie. if everyone with blue eyes moved to an island, then the alleles for blue eyes would be far more common) Bias of ascertainments — If the original numbers are wrong, then all calculations will be wrong Heterozygote advantage — In the case of sickle-cell, heterozygotes are resistant to malaria > advantage IV. Given the frequency in a population of a particular phenotype use the Hardy-Weinberg equation to calculate the corresponding allele frequencies and genotype frequencies. a See objective! V. Given the frequency of an allele for a particular trait (autosomal or X- the corresponding phenotype and genotype frequencies. a See objective | for autosomal b. For X-linked, remember that females are p?+ 2pq + q? and that males are p + q ced, recessive or dominant), calculate TL Page 82Recombinant DNA (pp 270-280) |. Describe the features of DNA sequences recognized by restriction endonucleases; sketch or recognize a DNA sequence that had dyad symmetry. Given a DNA sequence and a list of recognition and cleavage sites for restriction enzymes, identify the positions and number of DNA fragments that would be produced. a. Restriction endonucleases cut at specific sequences that show dyad symmetry b. These usually leave “sticky ends” (as shown in the examples below) as opposed to “blunt ends” © FoR! S'GAATIC’ > SG AATIC 3-CTTAAG 5" SCTTAA GS d BamHI S-cGATCC? > 5" GATCC:” 3-CCTAGGS’ B-CCTAG «GS! I, Describe in words or diagrams the general concept of recombinant DNA. Describe the function of DNA Describe the essential features of a cloning vector. List five types of cloning vectors and their distinguishing features. Basic concept Cut to DNA sources with the same restriction endonucleases Mix DNAs together Seal DAs together forming recombinant DNA b. Ligase—Takes a free 3-OH and a5'-PO, and bonds them together © Vector — The piece of DNA receiving the DNA insert to be studied The vector and the insert undergo transformation to enter the host Essential features of a vector: 1 Insertion site 2. Origin of replication 3. Selectable marker— to identify the cells that took up the insert Usually an antibiotic resistance gene ‘Types of vectors (do not memorize) Vector Type Host insert Special Features Organism | Size Range Plasmids E coli, yeast_| Upto 10 kbp | double-stranded circular —antibiotic resistances Xphage E coll 10-15 kbp | —double-stranded linear ~ efficient cloning of larger fragments Cosmids E coll Z-40Kbp___| — even larger fragments Bacterial Artificial | E. coli 50-200 kbp | — very large fragments for gene mapping Chromosomes (BACs) genome organization Yeast Artificial yeast 100-1,000 kbp | very large fragments for gene mapping, Chromosomes (YACs) genome organization = replication origin, centromere, telomeres TL Page 83,Il, Define genomic and cDNA inserts. Compare the informal and cDNA clones. Describe the synthesis of cDNA, including the role of reverse transcriptase. Explain using words or diagrams how expression vectors are used to direct the synthesis of a (human) protein in E, coli. List the unique features of an expression vector. Genomic insert - Takes full genome, digests it, and inserts the fragments into vectors i. Information content: 1. Full genome, including: a. Protein coding r b. introns © Regulatory information (promoters, enhancers, silencers) d._ Spacer DNA - repetitive DNA, “junk” DNA, ete. b. cDNA Takes just one coding region per insert i. How to make cDNA 1. Isolate all the mRNA that codes for a protein being actively made in the cell 2. Synthesize cDNAs using reverse transcriptase (GONA IS FFOH IRINA) a. Makes double stranded cDNAs of all the proteins actively made in the cell 3. These cDNAs are now the inserts to be processed and used to make recombinant DNA 4, Clone recombinant DNAs (with cDNA inserts) into a host organism Information content: 1. Hasthe same information as mRNAS ‘a. Exons, translation start codon, ORF, 5’ and 3' untranslated regions (UTRs), and the polyA tails 2. Will NOT have: ‘a. Introns, promoters, enhancers, intergenic DNA (spacer DNA - neither unique sequences nor repetitive DNA) & Expression clones — A special case of cDNA inserts Get host cell (bacterium) to synthesize protein encoded by cDNA 1. Use bacterial RNA polymerase and bacterial ribosometo make eukaryotic protein Require special vectors called expression vectors IV. Describe the concept of clone libraries and how such libraries are constructed. Distinguish between the concepts of selection and screening. Describe how molecular probes (DNA, RNA, oligonucleotide, antibody) can be used to identify specific recombinant DNA clones. Clone libraries are collections of clones, made simultaneously, from a given sample Genomic libraries — Use recombinant DNAs with genomic DNA inserts (DNA libraries — Use recombinant DNAs with cDNA inserts Expression libraries — Use recombinant DNAS with cDNA inserts ligated to expression vectors 1. Each colony makes the protein from whatever cDNA clone it received b. In order to find the correct clones, it is necessary to sort through the cells i. Selection — Kills all bacteria, except those with the vector 1. The vector contains a selective marker (usually an antibiotic resistance gene) 2. Tests can be run for the selective marker ‘Screening —Sort through remaining bacteria to find the one with the DNA insert of interest 1. Molecular probes are used to find sequences similar to the protein of interest nal content of genor ns (exons) TL Page 84Probes are made of DNA, RNA, synthetic oligonucleotides b, Must be labeled (radioactive label or fluorescent label) 2. Antibody (for expression libraries) Use Ab to detect protein of interest b, Make Ab ahead of time and label (with radioactive label or fluorescent label) & Ab binds at sites where protein being made V. Using words or diagrams, explain in concept (but not in technical detail) how recombinant DNA techniques can be used te a. Obtain a cloned gene fragment, given a protein sample or antibody Southern blot b. Obtain a specific protei Western blot VI. Identify the kinds of information revealed by Southern, Northern, and Western blots. Summarize these procedures, identifying the molecules being detected and the probes used. Southern blot — Detects specific DNA fragments Digest DNA Run electrophoresis to determine size of fragments ili, Denature DNA and transfer it to a filter iv. Hybridize probe to sequence of interest v. Visualize the fragment (usually via x-ray) b. Northern blot — Detects size (band position) and abundance (band intensity) of RNA species Isolate RNA Run electrophoresis to determine size of fragments Transfer to filter iv. Hybridize Western blot — Detects size (band position) and abundance (band intensity) of protein species Isolate protein Run electrophoresis to determine size of fragments Transfer to filter iv. US@aitibOUIES (does not hybridize) VIL. Describe the process and products of the polymerase chain reaction (PCR). a Gene specific primers Short ssDNA molecules REQUIRED — DNA polymerases need 3-OH end to attach incoming nucleotides b. The PCR cyde Denature dsDNA ‘Anneal primers DNA synthesis using DNA polymerase & Product is an exponential increase in copy number of DNA fragment of interest Each PCR cycle double the amount Each cycletakes “15 minutes (1 hour = billions of copies) d. Applications i. Sensitive— Can amplify very small amounts of DNA ‘given a gene fragment or probe TL Page 85Quantitative— starting quantity > Tproduct yield Productive The end product can be used in a wide array of tests GTL Page 86Diagnostic Applications of Molecular Analysis (pp 281 - 291) |. Define the following types of DNA polymorphisms: a Necessary terms: Polymorphism — A locus in which two or more alleles have gene frequencies of 1% of more Monomorphic— A locus in which 99% of a population has the same sequence Marker — Polymorphisms in a DNA sequence of which both sequence and position is known iv. Satellite DNA — Portion of DNA that contains highly repetitive DNA sequences 1. Microsatellites — Containing 1-13bp repeats in tandem; blocks of 100s of bp long 2. Minisatellites — Containing 14-5Obp repeats in tandem; blocks of 1000s of bp long, b. Restricted fragment length polymorphisms (RFLP) Variations in DNA sequences caused by the presence or absence of restriction sites Results in differing DNA fragment sizes between individuals’ DNA when digested with a particular (or set of) restriction endonucleases ©. Variable number of tandem repeats (VNTR) i. Used to detect variation between alleles based on the length of tandem repeats in DNA isatelites 1. Tandem repeats vary in size from a few to several dozen repeats, and are flanked by restriction sites 2. Aprobe specific for the repeated sequences is used 3. On aSouthern blot, the position of the visible probe indicates the size of the repeat 4, The size of the repeats can vary significantly within a population, and can be used to identify individuals d. Short/simple tandem repeats (STR) Smaller than minisatellites; only 2-13 base pairs length (microsatellites) May occur in several hundred repeats, also very variable within the population Generally, STRs are not flanked by restriction sites 1. Best isolated by PCR methods iv. Also usedto distinguish individuals v. Note: DNA fingerprinting uses both VNTRs andSTRs ple DNA sequence differences Analysis with ASO Probes (Allele-specific oligonucleotides) Can be made sensitive enough to detect a single nucleotide change Usually 10-20 base pairs long and all nucleotides need to pair for proper hybridization TL Page 87I Demonstrate the effects of the above polymorphisms using simple schematic diagrams or by Southern Blot or PCR results depicting these polymorphisms. allie “AI hash alle AL sills 2 AD” hastwo 5 s Lange | Frapmentsize small Heterozygote AIAZ —_Hemozygote Al Homoaygote A2 site “AU° asthe Sgestensies 5 digestion te Fragment se sal Herons AIA2 —— Homorygate AL Hormeaygnte A2 a. This also occurs with DNA markers and genes, but only if they remain syntenic; feeombination cam Occur “mi” allele AL “mut” allele AD x z aS allele Al MARKER, GENE MARKER GENE TL Page 88Il. Distinguish between informative and non-informative data for pedigree analysis. marker “m1” marker “m2” yama* I 1 mipx2* mint mifn2* ee ai 1 2° 3 4 2 mata" mnifna* sig 1 2 —_—= = = 2 - _ = --= = - - =- -=— - - - - Data for mi/m2 can be determined from the Southern blot. ‘As for which markers track for which allele, start with the homozygous recessives, knowing that they are both aa and m*m* From this data: 11-2 is unaffected, but carrier status is unknown 1. One m1 is from the other father and the other is from the mother; however, itis not possible to know which m1 was received from the mother ii, L-2is unaffected, and a carrier 1. Both parents have m2 syntenic with the gene, so the m2 must track with the diseased allele ili, 3 is unaffected, and not a carrier 1. Both parents have m1 does not track with the disease allele and these are both passed onto 3 TL Page 89IV. Use DNA polymorphisms as markers of distinct chromosomes and as alleles of specific loci for calculation recombination frequencies and for tracking chromosomes through pedigrees. From simple informative pedigree data for any two markers, estimate recombination frequency. a. See other objectives in this section V. Predict the phenotype and genotype for a. See other objectives in this section iduals in a pedigree based on informative polymorphism data. Other important information 11. Variable Number of Tandem Repeat Polymorphisms (VNTRs) Dien engi of iret VNTR alee DNA fom nts wih fret VNTR alles on Sather bl fofepeas 0 10 2 «3% 0 0 Allele Aiee A | = AleB | tee | — Altec aie [oe Alle — aie | - Aleee | —____ tee |__ - Aller = ater |= cE_AF BC DE cc Pact connt 1 0-2«3 4S atk 2. Analysis with ASO Probes a. Example: Mutant alleles for cystic fibrosis © = bybridization © = no hybridization 750 am> om [am> am] am] om[ am arsoe eo} o@ | ee] eo eo | eo GssiD 00 | 00 | 00] 00| ee] ec| eo Gsi2x 20| @0 | 00] e0/ ee| e0| eo Ni303K ° 20| ee| oo Patient T 2 3 4 567 Patient 2— Homozygous for AF508 > Affected Patient 3— Heterozygous for AF508 -> Unaffected; Carrier Patient 5 — Heterozygous for G551D and G542x > 72? 1. if mutations are on the same gene > Unaffected; Carrier 2. if mutations are on separate genes > Compound Heterozygote > Affected TL Page 90Applications of Molecular Analysis (pp 295 - 308) |. Understand how utilization of advanced cytogenetic techniques can detect specific submacroscopic chromosomal abnormalities not detectable by conventional karyotype. Convention karyotypes and banding stains only show chromosomes on a gross level b. To obtain information about the actual sequences contained, more advanced techniques are used. Il. Understand specific molecular technique of fluorescent in situ hybridization (FISH). ‘a. FISH probes are fluorescence labeled DNA probes b. They allow you detect for the presence of a specific piece of DNA S Centromeric Tolomeric _Chromosome-painting probe probe ‘probe —————— Ropetve-sequence probes Il. Understand limitations and/or benefits of using FISH to identi a. Single genes Pros: Can show the presence/absences of a single gene Cons: The sequence of the single gene must be known b. Centromeres i. Pros: Can show the presence of specific centromeres, which can then be used in conjunction with single gene FISH probes to test for sequences (as in testing for Williams on chromosome 7) Cons: Centromere staining alone provides little information other than the number of centromeres ©. Telomeres i. Pros: Allows you to visualize the telomeres of certain chromosomes d. Whole chromosome “paints” Pros: Allows the identification of chromosomes and identification of foreign material on a chromosome Cons: Does not provide information other than the presence/absence of a chromosome IV. Understand how PCR based methods can be used to facilitate diagnosis of a. Karyotype is only useful for large changes, therefore multi-plex PCR is used to detect smaller changes b. Agene segment is run through PCR to amplify it, then an array can show the results As shown on the right, sample 2 is missing gene fragments TL Page 91d._ Some other uses of PCR: i. Quantitative PCR will tell you if one or two alleles are present 1. For example, in the case of DMD, homozygous recessive > death. a. Mother can be heterozygous (two bands) or homozygous dominant (one band) ji. Methylation specific PCR can detect if imprinting occurs properly in cases of PWS/AS V. Understand the several limitations of FISH based and PCR based molecular techniques, such as false negative rates. PCR based techniques can detect 95% of deletions or duplications in the DMD gene. Therefore, a negative test does not mean the patient is negative for DMD b. One drawback of FISH probes is that they have to be specific for an amino acid sequences, so if the patient has a silent mutation, the results would appear the same as someone with missense mutation ‘This same concept applies to finding abnormalities, unless you know the sequence of the. abnormality, a FISH probe will not help you locate it VI. Understand how gonadal mosaicism can influence genetic counseling of diseases such as Duchenne Muscular Dystrophy, despite use of advanced molecular techniques. a. if the mutation occurs in the gonads, then testing the somatic cells would provide false reports i. Inthe example of the mother in the course notes, she may not be a carrier for the gene, but some of her eggs are due to gonadal mosaicism VIL. Understand how utilization of Comparative Genor detect numerous potential chromosomal aberrations using a single assay. CGH utilizes a series of cytogenetic tests and FISH probes to identify known chromosomal aberrations Even contains probes for some single gene disorders, gain/loss of large DNA sequences, as well as identifying normal chromosomes, trisomy, and monosomy. The test can even detect low levels (5%) of mosaicism b. These tests are also now able to detect copy number variations (CNVs) Duplications/insertions/deletions and complex variants of various gene regions CCNVs contribute to gene dosing and have been found in cancer, autism, schizophrenia, and learning disabilities There are about 1450 CNVs, covering about 12% of the genome VIII. Explain the several limitations of Comparative Genomic Hybridization methods, such as false negative rates, relative to type of mutations being analyzed. a Cannot detect balance translocations, or inversions (instances where there is no loss of genetic material) i. Therefore a karyotype should always be done before CGH b. Cannot detect point, missense, or small deletions 1X. Rationalize the utilization of Array based techniques to globally genotype individuals at numerous genetic Dueto levels of penetrance, expressivity, and age of onset, certain conditions may not be recognized by a physician until the disease has started to progress. b. By globally screening individuals, preventative/palliative measures can be taken to lessen the effects of the dysmorphology X. Define pharmacogenomics and toxicogenomics. a. Pharmacogenomics - Selecting drug treatments tailored to an individual's genotype TL Page 92i. Currently, testing is being done on cytochrome P450 in the liver to determine if there are variations in drug metabolism and/or efficacy based on genotype An allele for the enzyme cholesteryl ester transferase protein has been shows to affect clinical response to pravastin 11. 16% of the population is non-responsive to the medication 2. Thisis currently detected by coronary vessel intraluminal diameter, and not lipid levels b. Toxicogenomics — Identifies potential bad reactions to drugs or environmental contaminants based on an individuat’s genotype AHLA-B specific gene allele is highly associated with precipitation of Stevens-Johnson syndrome XI. Understand how advanced genetic techniques are being used to facilitate aspects of pharmacogenomics and toxicogenomics. a. See objective x XII. Define transcriptomics and its potential utility for clinical usage. a. Assessing the RNA expression pattern of all of the genes of a normal vs. mutant (tumor) tissue may reveal a pattern useful for understanding, as well as for prognostication and/or medical management b. Transcriptomic — Assessing global RNA expression levels of many genes at once for a given sample i. Tagged cDNA is made and then hybridized to a control sampleto test transcription rates XiIl.Consider how scrutinizing thousands of genes at once allows for greater diagnostic precision, as well as potential for improved therapy of diseases such as cancer. a. Proteomics — Assaying the presence or status of numerous proteins in a b. When it comes to cancer, sometimes ruling out what something isn’t is as important as determining what something is i. By screening for hundreds of known expression profiles, itis possible to narrow the scope of medical care, which can possibly lead to a better outcome. en sample simultaneously XIV. Other a. Mutation rate i =1/3()(1 +f) 1. 1=incidence 2. f=reproductive fitness For a disease like Duchenne Muscular Dystrophy, with f=0 (lethal) > Mutation = 1/3{!) 1. $0 1/3 of all cases are dueto a de novo mutation 2. 70-80% is from deletions/duplications on Xp21 TL Page 93,Clinical Case X: Hemochromatosis (pp 309 - 312) compounds in the body, and what are their function? a. Hemoglobin — Oxygen binding b, Myoglobin — Oxygen transfer from hemoglobin to muscle Heme containing enzymes cytochrome C d. Non-Heme containing enzymes |, How isiron absorbed and stored in the body? a. Absorption in duodenum and upper jejunum b, Mucosal uptake and intracellular transport Il, What proteins are involved in the transport and storage of iron? a. Transferin — Transport b. Hematin Ferritin -Storage IV. QUESTION: Which of these factor generally does not influence normal iron absorption Quantity of iron b. Composition of the diet ©. Presence of chronic hemolytic states 4d. Small bowel mucosal behavior & None of the above V. QUESTION: Which of the following statements is true? a. Ferritin’s only roleis to store iron. Also detoxifies iron b. Inheatthy individuals, ferritin levels remain constant with age. i. Start high, drop, then slowly rise. Nonheme iron is easily absorbed. Itis NOT easily absorbed Iron overload can occur in thalassemia major. The normal amount of iron in an adult male is 10-12g of iron. i. 65-150g VI. What are the relevant components of the patient’s medical and family history? a. Stiffness and pain in joints i. Radiographs show narrowing of metacarpophalangeal joints and chondrocalcinosis in wrist Fatigue and loss of libido Brother and uncle had liver related deaths i. As well as wife’s uncle d._Hepatomegaly and moderate ventricular enlargement Brown skin VI. What do the lab results tell you about the patient's status? a. Serum ferritin b. Transferrin saturation {Hemoglobin d._ Liver enzymes (Alanine aminotransferase and alkaline phosphatase) TL Page 94& 1 Glucose VII. What are the possible causes of either primary or secondary hemochromatosis? a Primary €282V mutations in HFE gene Other hemochromatoses b. Secondary Cirthosis ‘Anemia (some) Blood transfusion iv. Thalassemia v. Iron overload Liver disease 1X. QUESTION: The patient’s complain of decreased libido is most likely related to increased iron stores in: a. Testes b. Pituitary © Bonemarrow Liver Muscle X. QUESTION: Diagnosis of hemochromatosis can reasonably be made by all of the following methods except: ‘a. Measurement of serum transferring-iron saturation b, Measurement of serum ferritin levels ©. Determine of fibrosis on liver biopsy d._ Targeted mutation analysis of HFE & Sequence analysis of HFE XI. QUESTION: What is the chance that all three children will be carriers of hemochromatosis? a. 100% The father is homozygous recessive, so all of his children will carry the gene b. 67% 50% a 336 & Cannot tell Could be defended, if mother passes on an allele, they are not “carriers,” they have the disease In what ethnic group is hemochromatosis mutation €282Y found most often? a Irish/Celtic XIIl.What is the best approach to testing family members for hemochromatosis? a. Test wife, siblings, and then children (priority is oldest frst) XIV. Ifa mutation in HFE had not been found, what would have been you next step? Consider other hemochromatoses b. Consider alcohol XV. Given what you know about hemochromatosis, what treatment options and lifestyle changes would you discuss with this patient? Phlebotomy, No alcohol consumption, Liver transplant (not very affective), Decreased vitamin TL Page 95XVI. QUESTION: Which of the following best distingt hemochromatosis? a. The age of onset is earlier in HFE-related hemochromatosis. Earlier in TFR-2 b, TER-2related hemochromatosis is inherited as an autosomal dominant trait. i. Both recessive © Molecular testing is not available for TFR-2 hemochromatosis. Yes itis d. TER-2 mutations are more common than HFE mutat Penetrance is complete in TFR-2 hemochromatosis. i. Incomplete in both TFR-2 and HFE 1es HFE-related hemochromatosis from TFR-2 related GTL Page 96Prenatal Screening and Diagnosis (pp 315-327) 1. Understand the goal of screening and factors that determine if a screening testis offered. Recognize the differences between a screening and diagnostic test. a Goals of screening To identify individuals with an unrecognized disease To sort out persons who probably have the disease from those who probably do not b. Factors that determine if a screening testis offered Frequency of disease Cost to perform screen Ease of ability to screen iv. Ifthe condition you are screening for has an intervention/treatment & Screening test Atest for a particular disease given to patients who are asymptomatic Screening tests are generally cheap They are designed to be sensitive (detect lots of possible cases of the disease) but not as specific (accurately identify actual cases of disease) 1. However screening tests are seldom accurate (will not be 100% sensitive or specific) d. Diagnostictest i. Atest for specific diseases based on symptoms ‘To measure the progress or recovery from disease To confirm that a person is free from disease iv. Diagnostic tests are more accurate than screening tests Il, Understand the concepts of sensitivity and specificity. Understand how changing sensitivity and specificity “True Nes True Positive ! can affect a screening test. False Positive Critical value/cut-off— Chosen value that separates true positive and true negative Represented above by the dashed line Sensitivity — Proportion of affected individuals that test positive i. True positives/(True positives + false negatives) Specificity - Proportion of unaffected individuals that test negative True negatives/(True negatives + false positives) d._ Sensitivity and specificity are usually in opposition to each other i. PSensitivity > JSpecificity 1. Will detect all affected individuals, but also an increased number of false positives ii, TSpecficity > (Sensitivity 1. Will avoid all false positives, but you will miss a lot of the people who havethe disease TL Page 97Ill, Understand the effect of maternal age on chromosome abnormali a PMaternal age > Trisk of chromosomal abnormalities b. Accordingto the data, women above 35 are at an increased risk i. Ithas been determined that 35 is the age at which the risk of the test is greater than the risk of the disease being tested for IV. List the types of screens and tests performed in prenatal screening and diagnosis, and the benefits, limitations, and risks involved. a Screening i. Maternal serum screening 1. AFP (Alpha-fetoprotein) a 15-23 weeks b. Plevels > risk of open neural tube defects (ONTDs) and ventral wall defects © Wlevels > Trisk of Down syndrome i. Not recommend as a screen for Down syndrome, as better screening tests are available 2. Quadruple screening a. Tests levels of: AFP, hCG, Unconjugated estriol (uE3), and inhibin A b, Screens for: Down syndrome, Trisomy 18, and ONTDs i. May also detect rare conditions: Smith-Lemli-Optiz, X-linked ichthyosis, and ventral wall defects 3. First-trimester screening Blood tests for PAPP-A (pregnancy associated plasma protein A) and hCG b. Ultrasound for measurement of nuchal translucency (NT) © Screens for Down syndrome and Trisomy 18 i. Does not screen for ONTDs Ultrasound 1. Typically performed 20-22 weeks gestation 2. Evaluates anatomical structures of fetus, amniotic fluid levels, and placenta May detect ONTDs and other major malformations b. May detect soft signs (choroid plexus cysts, intracardiac echogenic foci) Integrated screening 1. Combination screening for PAPP-A, NT, and Quadruple screening 2. Blood samples a. First - 10-14 weeks gestation (first trimester) b. Second 15-23 weeks gestation (second trimester) 3. Ultrasound — 11-14 weeks gestation 4. Highest detection rate with the lowest screen positive rate a. In some areas, NTtest is not available (lack of trained sonographer) i. Can still have screening, but detection rate and (screen positive rate iv. ‘Screen positive rate 1. How many women will have a positive screen ‘a. Only indicates an increased risk for a condition (table just for illustration) TL Page 98‘Screening Method | Detection Rate for Down | Screen Positive Rate | Screen Positive Cutoff Syndrome (Sensitivity) | for Down syndrome _| for Down Syndrome ‘Quadruple Screening 80% 0% 1/270 First Trimester 80% 5% 1/260 Integrated (w/NT) 86% 1% 1/100 . Diagnosis i. Prenatal diagnosis 1. Usually offered only to those at an increased risk (when disease risk > testing risk) a. Advanced maternal age b, Abnormal serum screening Abnormal ultrasound findings d. Known genetic alleles in parents or affected sibling (need good family history) 2. Allwomen who come in before 20 weeks should have testing for aneuploidy Tests Ames tte eae ib ’\ contrtugation i . Fetus ened mee \ = sind | } Placenta Chorionic \\ through ines (So ae eee «| ae Ft Sere neta SN Boe sel ye Sov a Noa To Several’ | 0 0 weeks" karyotyping ja acer (8) Chorionevitus sampling (V8) 1. Amniocentesis ‘a Most common procedure b. Typically 15-22 weeks (can be done any time after 15 weeks) & Amniotic fluid contains fetal cells i. Can beusedto perform a variety of genetic disorders 1. FISH probes 2. AFP and acetylcholinesterase (AchE) levels for ONTDs d._ Risk for miscarriage — 1/200-1/300 (3.35%) 2. Early amniocentesis a Typically 11-14 weeks b. Cannot screen for ONTDs Risk i__Miscarriage—1% TL Page 99Club foot - 1-2% Adverse pulmonary development ~1-6% 3. Chorionic Villus Sampling a 10-12 weeks b. Done transcervically or transabdominally © Cannot screen for ONTDs d. May detect mosaicism in 1% of all sampling procedures i. Usually “confined placental mosaicism” 1. Cellline is present in the placenta, not in the fetus ji, Necessary to have amniocentesis in 2 trimester to determine between true mosaicism or confined placental mosaicism & Miscarriage risk— 1/100 Benefits of prenatal diagnosis 1. To prepare for the birth of a child with special needs a. Further testing b. Possible birth and surgical plans © Parental education 4d. Emotional preparation 2. Possible fetal treatment 3. Option of terminating an affected fetus iv. Fetal treatment 1. Only done (and available) when the risk of disorder > risk of the surgery 2. Fetal shunts for hydrocephalus or drainage of cystic hygromas 3. Open fetal surgery: spina bifida, teratoma, congenital cystic adenomatoid malformation of thelung v. Preimplantation Genetic Diagnosis 1. Common practice with in vitro fertilization a. Test for specific disorders, based on family history b. Test for inherited translocations, duplication/deletion syndromes, aneuploidy, and many single-gene disorders © Embryo biopsy i. Day3~1-2 blastomeres are removed at the 6-10 cell stage Day 4 —PCR and FISH testing Day5 — Unaffected embryos are implanted d._ Polar body diagnosis i. Performed on 1* and 2" polar body Used to test for transmission of carried gene or to screen for aneuploidy 1. Ovum’s composition can be inferred from polar body analysis Only looks at maternal contribution vi. Newborn Screening 1. Detects genetic diseases in the newborn population and prevents damage caused by the diseases (through presymptomatic treatment) 2. Approximately 4 million babies are screened each year (98% of al births) TL Page 1003000 affected infants detected b. Each state decides what tests they will include i. Michigan requires that all newborns are screened within 24-36 hours Non-invasive Prenatal Diagnosis 1. The “next generation” of prenatal testing (Already in use for Rh genotyping) 2. Uses cell free fetal DNA and RNA found in maternal blood V. Understand what screens or tests would be appropriate to offer in various ‘a. How far along a pregnant woman is/ What tests are available at various times during pregnancy Screening 1, 10-14 weeks: Blood test (10-14) — PAPP-A and hCG; Ultrasound (11-14) - Nuchal translucency 2. 15-23 weeks: AFP (Alpha-fetoprotein); Quadruple screening 3, 20-22 weeks: Ultrasound Diagnostic 1. 10-12 weeks: Chorionic villus sampling 2. 11-14 weeks: Early amniocentesis 3, >15 weeks: Amniocentesis (typically 15-22 weeks) 4, <20 weeks: Aneuploidy b. What information she wants to get out of testing i. Seetests in objective IV ¢. Her ethnic background/What carrier tests go with what ethnicity (do not memorize exact numbers) all 1. Qysticfibrosis, a. All couples who are planning pregnancy or currently pregnant should be offered | situations, such as: carrier screening for 23 of the most common mutations b, Most common in Caucasians EthnicGroup | Carrier Risk | Detection Rate | Residual Risk* Caucasian Vs 90% Va ‘Ashkenazi Jewish VB 7% 1/801 ‘African American 1/65 69% 1/207 Hispanic 1/46 57% 1/106 Asian’ 1/90 496 1/176 “The risk of beinga carrier after a negative test Ashkenazi Jewish ‘Condition Carrier Frequency (2pq) | Detection Rate Tay Sachs 1/30 3-986 ‘Cystic Fibrosis 1/29 98% 1/40 97% (Progressive nerve degeneration) Familial Dysautonomia 1/32 99% (Nerve development and survival) ii African American, Middle Eastern, Asian, and Mediterranean 1. Hemoglobin electrophoresis - Screening test for sickle cell and thalassemia TL Page 101Human Teratogens and Impact on Fetal Development (pp 328 - 337) 1. Understand what a teratogen is and the way a teratogen can manifest. a. Definitions Teratology — The study of environmentally-induced birth defects Teratogen — Any substance, agent, or process that interferes with normal development 1. These are potentially reversible b. Manifestations of a teratogen i. Death Growth retardation Malformation (structural abnormality or internal/external body parts) iv. Functional deficit (mental retardation, behavioral abnormalities) Ml, Recognize the various types of teratogens. a. Categories Infection agents: Toxoplasmosis, CMV, Herpes Physical agents: Hyperthermia, Radiation, Trauma, Operative procedures Chemical Agents/Drugs: Medications, Recreational drugs/alcohol, Herbal products, Occupational exposures iv. Maternal metabolic and genetic factors: Diabetes, Maternal PKU, Abnormal folate metabolism b. Characteristics i. Increased occurrence — Exposure during pregnancy is associated with increased occurrence of the phenotypic effect over the general population ii. Supportive animal model — Animal models should duplicate the human effect, 1. Ideally, this animal model should use the same route of exposure as the human exposure of concern Dose-response relationship — Likelihood and magnitude of response proportional to dose 1. Acertain threshold must be reached (to exceed compensatory mechanisms) ‘Varies from agent to agent iv. Biologic plausibility — Plausible biological explanation for mechanism of action © FDACode Category A— Controlled studies show no risk Category B— No evidence of human risk 1 Risk shown in animals Category C— Risk cannot be ruled out 1L._ Insufficient information 2. Benefits may justify the risk iv. Category D - Positive evidence of risk 1. Potential benefits may still outweigh the risk v. Category X — Contraindicated in pregnancy 1. Risk clearly outweighs benefits Ill. Understand the various factors affecting the response to a teratogen. Dose of the agent - Amount, route of administration, duration TL Page 102b, Timing of agent - Periods of greatest sensitivity, Does it make chronological sense? ‘Was the drug taken at the critical time in development to affect the target organ? ¢ Interactions with other environmental factors 4d. Host susceptibility ~ Individual differences in metabolism of teratogenic agents The mother’s genetics may affect how an agent/drug is metabolized & Period of exposure i. Each part, tissue, and organ of an embryo has a critical period during which its development may be disrupted IV. Understand critical periods of development and what effects you would expect to see based on the time of the medication exposure. Ss EE oe ‘Embryonic Period (in weeks) tal Period (in waeks) t z sa 5 zg 7 = ® TER Funcinel Defects on nor etna es] Moje Monel Knometis open! = Feriization Period Implantetion Period Ee Dating om Tn a Mtl Ped Gn wood | ‘Donte highly sonstive sagas in dovalopmert for that particular system HIE Less sinsitive toges for teratogons a. Allornone period 0-15 days post-conception The developing embryo is not susceptible to teratogens Will result in SAB or have no effect b. Organogenesis 3-8 weeks post-conception ‘When tissues and organs are forming 1. The embryo is most easily disrupted TL Page 1032. > Fetal death, Major malformations, Growth retardation, Impaired 1 il, Examples 1. Neural tube forms at 3.4 weeks 2. Heart formed by 8 weeks Later in pregnancy 1. Exposures can cause physiologic defects 1. Minor anomalies of the external ear, growth retardation, or functional disorders (MR) 8:12 weeks: Fetal death, vascular disruption, hemorrhage, and tissue loss 2.3" trimester: Stillbirth, Growth retardation, Impaired 10, Behavioral problems d. Multiple exposures (difficult to predict) ‘Additivity ~ Combined effect is the same as the sum of the individual effects ‘Antagonism — Combined agents counteract each other and have less of an effect Synergism — Combined effect is significantly greater than individual effects V. Know common teratogens presented and their effects. a. Medications (Chemical agents) i. Thalidomide 1. Used as a sedative or to treat pregnancy-related nausea a. Late 1950s— Early 1960s 2. Caused severe limb reduction defects (ie. phocomelia) in >10,000 babies worldwide ii. DES 1. Medication given to prevent pregnancy complications/loss a, 1938-1971 5-10 million pregnancies exposed 2. 1/3 of exposures + internal reproductive anomalies a. Vaginal adenosis, Cervical hoods, T-shaped uterus b. risk of vaginal cancer (clear cell carcinoma) in adult female children 1/1000 3. Increased risk of infertility 4. Effects may not be evident until years after the birth il, Angiotensin-Converting Enzyme (ACE) Inhibitors 1. Used to control hypertension 2. Mechanism: Reduced blood flow = {/placental perfusion and severe fetal hypotension 3. Causes fetal renal failure (renal tubular dysplasia) in 2° and 3" trimesters 4. Oligohydramnios + Fetal limb contractures, Craniofacial deformities, Hypoplastic lung development 5. Fetal skill ossification defects 6. Intrauterine Growth Restriction (IUGR) 7. Persistent patent ductus arteriosus b._ Drugs of abuse (chemical agents) Alcohol 1. Some manifest behavioral effects 2. Fetal alcohol syndrome ‘a. Only noted for regular users b. CDC— Major risk to fetus requires chronic, daily use iL Six or more drinks per day TL Page 104OR, 6+ drinks per occasion, with a monthly intake of at least 45 drinks 1. 40% of the above-described > FAS 2. 4 ormore drinks daily > 20% 3. 2ormore drinks daily > 10% & Categories of FAS i. Prenatal and postnatal growth restriction 1. Height, weight, head circumference (<10" percentile) Characteristic facial features 1. Narrow palpebral fissures, smooth and elongated phittrum with thin upper lip CNS abnormalities 1. Microcephaly, mental retardation, behavioral abnormalities Infections Cytomegalovirus (CMV) 11. 50-80% of women have been exposed to CMY prior to pregnancy . 0.7-4% of primary exposures occur during pregnancy i. "40% transmission to fetus ii. 10% of infected fetuses show symptoms of congenital CMV at birth CNS: Hydrocephaly, Microcephaly, Cerebral calcifications, Mental retardation Eye: Chorioretinitis, Blindness, Microphthalmia Other: Growth retardation, hearing loss, heart defects 5-15% of fetal infections have no symptoms are birth a. Delayed onset, b. Mental retardation, Hearing loss, Vision problems 6. Recurrent exposure during pregnancy: 1-14% a Only 0.2-2% produce congenital infection, with 1% of those showing symptoms at birth b, 5-10% may have longterm effects (hearing loss is most common) Congenital Rubella (German measles 1. Abnormalities very in frequency/severity/type based on month of gestation 2. Results in: Late fetal deaths (abortion), Growth retardation, Microcephaly, Cataracts, Deafness, Congenital heart defects a. Allorgans can be affected Herpes Simplex 1. HSV2 infections involvethe genital region ‘a. Have been associated with fetal and neonatal infections b. Usually acquired during the birth process 2. Risk estimates for neonatal HSV infection a. Primary infection: 33.50% b, Recurrent infection: 3% or less 3. Pregnancy complications include preterm delivery and intrauterine growth retardation 4, Clinical effects similar to CMV @ Microcephaly, Microphthalmia, Chorioretinitis veer TL Page 105d._ Physical agents Radiation 1L._ Increased risk associated with exposures of 10-20 rads or greater Diagnostic x-ray <5 rads is not known to be teratogenic b. Dose-response manner Radiation Estimated fetal dose {in milirads) Xray — Extremity 1 Xray — Chest 8 Mammogram 50 Xray — Lumbar Spine 25 Upper Gl Series 560 Barium Enema 800 CT of Abdomen 1000-5000 Radiation therapy — 1000 rads or more Very high risk teratogen 3. CNS: Microcephaly, Mental retardation, Seizures 4. Growth retardation 5. Minor anomalies of the eyes 6. Controversial increase in carcinogenesis Maternal conditions i. Hyperthermia 1. Body temperature over 101 degrees 2. Can be due to fever or exposuretto extreme heat (hot tubs, saunas, etc) 3. risk of neural tube defects if exposure is in first 4 weeks a. 3-4 weeks is the most critical time Diabetes Mellitus 1L._ Increased risk of: NTDs, Congenital heart defects, Skeletal defects (caudal regression syndrome), Kidney defects, Gastrointestinal defects, and Pulmonary hypoplasia 2. Poor control increases risk of malformations a, >20%6 risk has been reported in offspring of women with evidence of very poor control in the first trimester b, 610% is a general risk 3. Optimization of glycemic control before and during pregnancy has been associated with a significant risk reduction for major anomalies 4. Control is difficult as pregnancy is characterized by increased insulin resistance and reduced sensitivity to insulin action TL Page 106Cancer Genetics (pp 341 - 355) 1. Understand how genes play a role in cancer. Define and differentiate between tumor-suppressor genes and proto-oncogenes a Cancer is a genetic disease i. Mutation(s) occurringiin a specific cell, resulting from: 1. Spontaneous mutations 2. Induced mutations — Environmental agent (Carcinogen) 3. Inherited mutations b._ Definitions i. Oncogenes— A gene that turns into a cell into atumor cell 1. Generally involved in sporadic cancers 2. Rarely inherited 3. Act as dominant disease genes at the cellular level Only one mutated allele is needed 4, Proto-oncogenes— A normal gene that can become an oncogene a. Examples: i. Growth factors Growth factor receptors Signal transducers (protein kinases) iv. Transcription factors/proteins involved in DNA replication 5. Mutation — Proto-oncogenes become active oncogenes a. Increased expression of a proto-oncogene b. Change in structure/function of a proto-oncogene i. > Abnormal stimulation of cell division and proliferation ii. Tumor-suppressor genes 1. Most inherited cancers 2. Normal role: a Gatekeepers: i. Directly involved in regulation cell cycle growth inhi Mutations result in lack of growth inhibition b. Caretakers: i. DNA repair Mutations > cancer indirectly (Mutation accumulate) Sporadic cancer — Homozygous recessive 1. Requires mutations in both genes for cancer iv. Hereditary cancer — Heterozygous recessive 1. Requires a mutation in one gene for cancer & Cancer does not occur after a single genetic change Cancer-initiating gene mutation(s) Accumulation of additional mutations and genetic alterations 1. Normal cell > tumor/precancerous cells — cancer d. Oncogenes RET proto-oncogene TL Page 1071. Transmembrane tyrosine kinase receptor 2. Point mutation = constituently active tyrosine kinase > excessive activity 3. Multiple endocrine neoplasia (MEN2) Autosomal dominant inheritance of RET oncogene 4, People with RET mutations are at a 95-100% risk of Medullary thyroid carcinoma Typically develops by childhood early adulthood i. AgeS-25 for MEN2A Infancy in MEN2B, b. Recommended therapy: Prophylactic thyroidectomy i. By ageS for MEN2A By age 1 for MEN2B 5. Other features of RET proto-oncogene mutation ‘a. 50% - Pheochromocytomas b, 20-30% of MEN2A - Parathyroid adenoma/hyperplasia & MEN2B - Mucosal neuromas of the lips and tongue, enlarged lips, “Marfanoid” body habitus ii. Chromosome rearrangements activation oncogenes 1. These are typically sporadic cancers, and not inherited 2. Example: The Philadelphia Chromosome a. t(9;22)(q34:q11.2) i. Majority of CML (Chronic myelogenous leukemia) Frequently in ALL (Acute lymphoblastic leukemia) b. Breakpoints i. Chromosome 9 — ABL Chromosome 22— BCR © Results in a changeto the ABL gene function 3. Most chromosome rearrangements landing to oncogenes are hematopoietic diseases and lymphomas e Tumor-suppressor genes i. Most hereditary cancer syndromes are dueto inherited mutations in tumor-suppressor genes Define the multi-hit concept and two-hit hypothesis of cancer initiation. ‘exer 00 — 90 — §6-—¢8 ‘Two normal genes shit? 2 hie? Loss of one Loss of 2nd normal allele normal allele Cancer Hereditary Cancer One inherited 2 nit ‘mutation loss of normal allele Cancer (born with 1 hid, TL Page 108. Inhereditary cancer, the second “hit” is a sporadic/random event, which may not occur Ill, Identify features that are common in hereditary cancer syndromes. a. Increased risk — Higher chance of acquiring one hit (in any cell) vs. two hits (in the same cell) b. Earlier age of onset — Acquiring one hit vs. acquiring two hits (takes longer) & Bilateral/multifocal presentation — Increased chance of 2" hit occurring in more than one cell d. Multiple family members with related cancers & Rare or unusual cancers f. For most cancers, 5-10% of all diagnoses are hereditary IV. Recognize the clinical features and cancers that are associated with various hereditary cancer syndromes, as well as diagnostic criteria and medical management recommendations (for BRCA1/2, HNPCC, FAP). @. Hereditary Retinoblastoma 90% risk of cancer in heterozygotes Average age at diagnosis 1. Hereditary: 15 months 2. Sporadic: 24 months Bilateral or multifocal retinoblastoma is more common (very rare in sporadic) b. Hereditary Breast and Ovarian Cancer syndrome BRCA1 and BRCA2 genes: Accounts for 85% of hereditary breast/ovarian cancer families Caretaker genes: Involved in DNA repair, responds to double stranded DNA breaks Cancer BRCAI/2 Risks General Population Risk 56-87% lifetime Breast 10-20% by age 12% 30-50% by ageS0 Ovarian ZTAN Tes Male Breast BRCAL: 0.1% BRCA2: 6-7% Pancreatic 26% 1% Prostate 1633% T2156% ii, Testing recommendations 1. Breast Cancer a Increased screening i. Monthly self breast exam — Age 18 i, Clinical breast exam, twice a year— Age 25 fi, Annual mammogram — Age 25 b. Chemoprevention i. After age 35 ‘Tamoxifen for years Risk reduction: 50% © Prophylactic mastectomy i. Any age Risk reduction: 90% 2. Ovarian Cancer TL Page 109i, Risk reduction: 96% (Up to50% risk reduction for breast cancer) b. Increased screening i. Age 35 (A-12 and Transvaginal ultrasound For women who refuse/delay salpingo- oophorectomy 3. Males: Self-breast exam, annual clinical breast exams, consider mammogram iv. Ashkenazi Jewish v. Key features of BRCA1/2 families 1. Early onset breast cancer (<50 years old) 2. Bilateral breast cancer 3. Breast and ovarian cancer in same person/family 4, Male breast cancer vi Factors modifying inheritance 1. Reduced penetrance Variable expressivity Pleiotropy — Affects breast, ovary, pancreas, and prostate Allelic heterogeneity — Many different mutations in the ovarian cancer cluster region Locus heterogeneity — BRCA1 (17q21) and BRCA2 (13q12) Delayed age of onset Sex influenced disease Phenocopies — Family members with sporadic breast cancer may present 9, Multifactorial inheritance Vil. Key points in assessing a family history of breast cancer 1. Father's side is as important as the mother’s side 2. Distant relatives ARE important Especially those related to people dying at a young age 3. Unaffected relatives are also important a. Numbers and ages of death 4, Important to ask about other cancers in the family Colon Cancer ‘Approximately 5-10% of all colon cancer is thought to be hereditary Familial adenomatous polyposis (FAP) 1. Approximately 1% of all colon cancer 2. Autosomal dominant inheritance a APCgene b. 2/3 of cases are inherited; 1/3 due to new mutations 3. Characterized by 100s to 1000s of adenomas (polyps) liningthe colon and rectum 4. Average age of polyp onset: 16 May occur in young childhood PN OTE wN TL Page 1105. Cancer risks associated with FAP Site Risk of Cancer Colorectal "100% without colectomy (Average age: 39) ‘Small bowel (usually duodenum or periampulla) ‘412% (50-90% risk of polyps) Stomach (015% (50% risk of polyps) Pancreas "2b Thyroid (Papillary) "26 CNS (Medulloblastoma) 1% Liver (Hepatoblastoma) ‘1.6% (children 8 years of service, benefits will be granted without inquiry into disease’ existence at entry 2008 - Benefits cannot be denied unless the disability was noted at entry or compelling evidence or medical judgment exists that the disability incurred before enrollment f. NCAA andSickle Cell Testing i. 2006 death of Dale Lloyd it 1 Following football practice at Rice University a. Testing after death revealed sick cell status contributed to death b. Alawsuit was brought against the NCAA and Rice University i. June 2009 - NCAA agreed to recommend testing all athletes TL Page 114Life, Disability, Long-term Care Insurance Laws only apply to health in insurance Discrimination may still occur for other types of insurance 11. Life, disability, and long term care insurance companies have the right to ask about genetic testing and use the information to set premiums or determine coverage 2. Even if a person has not had genetic testing, they may be discriminated against based solely on family history of a condition 3. However, in some cases genetic testing helped show a negative status, despite family history of a genetic disorder Ill, Understand the benefits and limitations of genetic testing in an asymptomatic person. a. Benefits i. Medical management 1, Increased screening, risk reduction options ii. Life planning 1. Short-/long-term plans 2. Family planning 3. Financial planning Testing negative, or resolving uncertainty of risk b. Limitations Presymptomatic — Cannot determine when a disease will occur Predispositional — Cannot determine when or ifa disease will occur 1. May be detecting people who would have never developed condition Testing may not detect 100% of mutations related to condition 1. Example: Hereditary cancers IV. Understand the ethical issues involved in testing of minors (including prenatal testing) and under what conditions testing is recommended or not recommended. a Principles i. Patient autonomy — Respecting that the patient is in charge of his/her own health and has the right to make his/her own medical decisions L._ Right to know/right to not know Beneficence (doing good) ~ Promoting what is in the best interest of the patient (within THEIR morals and values, not yours) Nonmaleficence (do no harm) — Restrict behavior that may have a negative impact on someone iv. Genetic counselors do not make recommendations of who should and who should not have testing 1. We facilitate the decision making process for the patient 2. The patient should aways make their own decision (patient autonomy) a. However, the medical team is responsible for ensuring that there are no contraindications to testing, such as considering suicide, etc. (Nonmaleficence) b. Ethical issues i. Testing of Minors (Presymptomatic, Predispositional, and Carrier Testing) 1. Benefits of testing TL Page 115a. Reduces parental anxiety about the unknown b. To prepare for the future & To help in addressing risk when child is older 2. Harms of testing a. Taking away right for child not to know b. Potential damage to self-esteem and forming relationships © Potential harm to family relationships (child is treated differently) d._ Anxiety anticipating symptoms Possible discrimination 3. Conflicting principles ~ Beneficence for parents vs. Autonomy & Nonmaleficence for child a The benefits do not outweigh the harms for most conditions i. Testing of minors is recommended against by the American College of Medical Genetics, American Society of Human Genetics and National Society of Genetic Counselors 1. Except if the test results will affect the medical management of the patient from a young age (example: FAP) ii. Carrier testing in children 1. Assesses risk of them having affected children 2. Does not assess risk for child to develop condition 3, Same benefits and harms of testing apply Prenatal and pre-implantation genetic diagnosis of adult-onset disorders 1. Available — Primarily to allow for termination of an affected pregnancy 2. ifthe couple is planning to continue pregnancy even if affected, then testingiis not recommended iv. When testing on one person diagnoses another 1. Adult child wants to know genetic status, while at-risk parent does not want to know a, Same issue for identical twins (one wants to know; the other does not) 2. Conflicting principles ~ Beneficence and autonomy of patient vs. Nonmaleficence and autonomy of mother a. In most situations, the right of the patient to know their own medical information outweighs the other family members right to know V. Understand issues of impact of genetic testing on other family members who do not want information. a. Psychological Impact i. Anxiety 1. Worry about developing condition/symptoms Guilt 1. Parent feeling guilty for passing on mutation to child 2. Sibling feels guilty for not inheriting the mutation iii, Blame 1. Child blames parent for “giving them” the mutation iv. Depression 1. Inability to deal with abnormal result TL Page 1162. Difficulty coping with normal result Life decisions could have been made based on the thought of developing a disease (not goingto college, not having children, etc) v. Alteration of family relationships 11. Siblings with mutation may resent siblings without the mutation (or vice versa) 2. May impact marriage decisions/Childbearing decisions b. HDSA (Huntington Disease Society of America) i. Protocol 1L. Initial evaluations — Genetic counseling, Psychology, and Neurology 2. One month waiting time before blood is drawn 3. Test results — Always given in person a. Encourage individuals to bring a support person with them to appointment i. Preferably someone who is not at-risk of disease Reasons for Protocol 1. There are no risk reduction options for HD 2. Increased rate of adverse psychological reactions, including suicide for HD positive individuals VI. Understand duty to warn as it applies to genetic conditions. ‘a. Duty to warn — Information that affects the health of other individuals aside from your patient b. Conflicting principle— Beneficence of relatives versus autonomy/privacy of patient State Case Law Pate versus Threlkel 1. Woman with medullary thyroid cancer ‘a. Physician did not warn patient of potential risk to offspring b. Daughter developed medullary thyroid cancer three years later © Mother sued for doctor not warning the family, and won 2. Duty to warn family members is satisfied by telling the patient to tell their family ii, Safer versus Estate of Pack 1. Safer’s father had FAP when she was a child ‘a. Asan adult, Safer developed FAP and cancer (related to FAP) b. Daughter sued the father’s estate for failing to inform at risk family members & Safer lost, because she had a rectal screen as a child, which inferred notification of risk 2. Duty to warn is not satisfied by telling patient, but physician must take reasonable steps to warn immediate family members d._ Federal Legislation Itis permissibleto disclose health information without consent when the public interest is at risk (does not specifically refer to genetic disease) 1. Thereis serious or imminent threat to the health or safety of a person or the public 2. Thethreat constitutes an imminent, serious threat to an identifiable third party 3. The physician has the capacity to avert significant harm & US Bioethics Commission, American Society of Human Genetics and National Human Genome Institute TL Page 117