DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPE/KARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS

STUART SCHWARTZ, PhD

LABORATORY CORPORATION OF AMERICA, RESEARCH TRIANGLE PARK, NORTH CAROLINA

DISCLOSURE

I am an employee of LabCorp (Laboratory Corporation of America)

UNDERLYING QUESTIONS

What is a SNP How effective is the whole genome array analysis to detect chromosomal imbalances not seen with standard cytogenetic methods? How small of an alteration can be routinely detected easily and integrated into routine analysis What do these small alteration mean phenotypically

OVERVIEW

Introduction SNP Array - methodology SNP Array findings ~ 3000 abnormals Complexity Uniparental Disomy Consanguinity Prenatal Diagnosis and POCs Conclusions

OBJECTIVES

Describe the types of abnormalities detected by microarrays Review the implications of complexity
Translocations, markers, two hits

Review the impact of UPD and consanguinity Discuss the utilization of arrays for prenatal diagnosis and POC analysis

DETECTION OF GENOMIC CHANGES

Unbanded Chromosomes Chromosomes - 550 Band Level High Resolution Chromosomes FISH
DIRECTED ANALYSIS

20 Mb 10 Mb 3-5 Mb 150 kb

Array Analysis
NOT DIRECTED ANALYSIS

50 - 150 kb

GENOME ARRAY TECHNOLOGY

Gene number: ~25,000 functional genes Gene density: one per 45 kb

But very varied among chromosomes

Gene size: Average 20 kb

But enormous variation

Half of genes unknown function Interpretation of findings

Can detect abnormalities interpretation?

GENOME-WIDE AFFYMETRIX SNP ARRAY 6.0

More than 1.8 million markers across the entire genome for copy number analysis.
906,600 SNPs (Polymorphic probes for assessing genotype and copy number). 945,826 Structural Probes (Non-polymorphic probes for assessing copy number).

Marker spacing = Average 1.2 Kb Median 0.7 Kb (694 bases) 1.2 average

WHAT IS A SNP?

Single Nucleotide Polymorphism A SNP is a single base pair substitution of one nucleotide for another. This substitution must be found in the population at a frequency greater than 1.0%. E.g., one individual has a CAACCT sequence and another has a CAGCCT
LabCorp

SNP DESIGN
A 5 Genomic Sequence B T/G Patient DNA TAGCCATCGGTA N GTA C TCAATGATCAGCT ATCGGTAGCCAT A CAT G AGTTACTA PM Allele A 25mers ATCGGTAGCCAT C CAT G AGTTACTA PM Allele B SNP T/G 3 probe = 25 bases

SNP

NORMAL ALLELE DOSAGE

AA +1 AB 0 BB -1

Log 2

CN State

ALLELIC DIFFERENCE DELETION

ALLELIC DIFFERENCE - GAIN

ABNORMALITY - CRITERIA

Deletion
>200 kb in size less than 100.0% copy number variation (CNV) greater than 50 SNPs/CN probes within a 200 kb segment at least one OMIM annotated gene or within a region of clear clinical significance

Duplication
>500 kb in size at least one OMIM annotated gene

Known clinically significant gene region

Deletions and duplications are reported as small as 50 Kb

TYPES OF ABNORMALITIES

CATEGORIES OF ABERRATIONS

DELETED AND DUPLICATED SEGMENTS

Size <100kb 100-200kb 200 - 500kb 500kb 1Mb 1Mb 3Mb >3Mb % Deleted 2.1% 4.0% 27.1% 14.0% 28.6% 24.3% Size % Duplicated <100kb 0.2% 100-200kb 2.4% 200 - 500kb 15.1% 500kb 1Mb 39.9% 1Mb 3Mb 29.2% >3Mb 16.2%

INHERITANCE

GENES ARRAY [~3000 CASES]

Large changes multiple genes [619] Microdeletion/known pathogenic genes [367] Susceptibility genes [411] ?Susceptibility genes [284] Unknown function [1329]

EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS

15Q13.3 DELETION 17Q21.31 DELETION (MAPT) 1P36 DELETION 1Q21 MICRODELETION 1Q21 MICRODUPLICATION 22Q11.23 DELETION 3Q29 DELETION 9P DELETION 9P DUPLICATION 9Q34 DELETION ANGELMAN AUTISM BPES BRANCHIOOTORENAL CONGENITAL DIAPHRAGMATIC CRI-DU-CHAT CHRONIC GRANULOMATOUS DISEASE DUCHENNE MUSCULAR DYSTROPHY HOLOPROSENCEPHALY ICHTHYOSIS MICROPTHALMIA

MOWAT-WILSON MULTIPLE EXOSTOSES NEUROFIBROMATOSIS NOONAN PELIZAEUS-MERZBACHER DISEASE PSEUDOVAGINAL PERINEOSCROTAL HYPOSPADIAS PHELAN-MCDERMID POTOCKI-LUPSKI POTOCKI-SHAFFER PRADER-WILLI RENAL CYSTS AND DIABETES RETT SMITH-MAGENIS SOTOS SRY DELETION STICKLER VCF WARDENBURG-TYPE I WARDENBURG-TYPE IIA WILLIAMS WILLIAMS DUPLICATION WOLF-HIRSCHHORN

MICRODELETION SYNDROMES

Microdeletion syndromes well established

High resolution cytogenetics FISH

New microdeletion syndromes identified by arrays

17q21.31 deletion

More older microdeletion syndromes identified by array

Genotype first

SUSCEPTIBILITY GENES

Traditional view of genetics

Dominant, recessive, multigenic

Cytogenetics
Haploinsufficient; Over-expression

New Category
Susceptible
Important but not sufficient Parents with aberrations may be mildly affected or not affected

16p11.2 ABNORMALITIES

16p11.2 aberrations
Microdeletions Microduplications

Autism Parents with aberrations may be normal Important but not sufficient

1q21.1 ABNORMALITIES

1q21.1 aberrations
Microdeletions and microduplications

Patients with 1q21.1 aberrations show variable phenotype

Mild-moderate MR; microcephaly; cardiac anomalies; cataracts

Parents with aberrations may be mildly affected Demonstrates difficulties with new microdeletion/duplication syndromes

QUESTIONABLE SUSCEPTIBILITY

Precise effect of absence of loss or gain of genes questionable

Controversial at times Duplications
15q13.3; 16p13.11

Genes identified by GWAS; genes shown to have CNVs greater in autistic or other populations
PARK2; IMMP2L; 15q11.2 deletion

COMPLEXITY OF ARRAY RESULTS

Overall ~28% of samples show complexity

Structural abnormalities Two or more abnormalities in patient
Derivative chromosomes Recombinants Contiguous duplication/deletions TWO UNRELATED ABNORMALITIES

Will have an effect on phenotype

BALANCED REARRANGEMENTS

No loss or gain of genetic material

Inversions, translocations & insertions

Incidence: 1 in 500 live births

2-3 fold more common in mental retardation populations

De novo prenatal cases

A major diagnostic dilemma 8-10% risk of phenotypic abnormalities

CHROMOSOME 6 DELETION SECONDARY TO T(6;18)

18
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis

18q12.2 18q21.1 18q21.33 18q22.2-3

Del Ins(11) Del Ins(11)

RESULTS - REARRANGMENTS

100 de novo balanced rearrangements

56 with deletion/duplication of material
0.8 Mb to 15 Mb 15 to 70 genes deleted

117 copy number changes identified

16 of 17 studied without deletion - gene has been broken
1 neither broken or deleted

9 familial balanced rearrangement

0 with deletion of material 8 where a gene has broken
2 cases of an inheritance of familial disorder 6 cases where only the proband has the disease

RESULTS ABNORMALITIES

56% of de novo rearrangements with gain or loss of material

Considerable complexity
Only 29% demonstrated loss at one breakpoint 10% with deletions at 2 breakpoints 61% involved more than two chromosomes and one deletion

Only 57% of deletions/duplications were adjacent to the breakpoint

Many on same or other chromosome

80% of copy number changes deletions; 20% were duplications

MARKER - OVERVIEW

43 markers from 40 patients

SNP array analysis Cytogenetics and FISH

Multiple questions
Identification Proper characterization Phenotype correlation Mechanism of formation

INV DUP (15)

4 COPIES 3 COPIES 2 COPIES

ACENTRIC MARKER
Analphoid 2q Partial Trisomy der(2)(q32.3->q34)

Size:17,533 Kb SNP:1636 Genes: 30 (14 of 30 genes in OMIM)

TWO markers derived from ONE chromosome in an individual

Acentric G-band : 2p24.1-p24.3 Size : 6.6 Mb

Pericentromeric G-band : 2p11.2-q11.2 Size : 13.0 Mb

TWO markers derived from TWO chromosomes in an individual

G-band : 5p13.1 to 5q10 Size : 6.19 Mb

G-band : 15q10 to 15q13.3 Size : 10.77 Mb

MARKERS UNUSUAL CHARACTERISTICS

G-Band: 13Q31.3->QTER Size: 20.68 MB G-Band: 19 (9 SEGMENTS) Size: 6.89 MB

ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15

SUPERNUMERARY CHROMOSOME 8 AND UPD

Homozygosity Homozygosity Homo/Heterozygosity

Copy number state 4

DELINEATION OF TWO SIGNIFICANT ABNORMALITIES

A newborn was ascertained with a congenital heart defect and multiple congenital anomalies SNP array analysis revealed
A small deletion (1.37 Mb) in 7q11.23 consistent with Williams syndrome However, a second abnormality, a 1.39 Mb duplication in 22q11.21, was also detected The second abnormality would not have been detected with a directed FISH approach The second abnormality is likely to expand the phenotype of the proband

CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN

16p11.2 microdeletion
Log 2

7q11.23 microduplication
Log 2

1.97 Mb Duplication 611 kb Deletion

PWS/AS DELETION NOT DELETED ADDITIONAL DELETION

TWO HIT HYPOTHESIS

Girirjan et al (2010)
Using 16p12.1 as a model have suggested that many susceptibility genes may act as a two hit hypothesis Approximately 24% of cases had a second hit
Patients more severely affected than parents

Overall ~ 28% of our patients with two abnormalities

Those with known susceptibility genes ~15%

FAMILIAL DE NOVO

Overall fewer than expected abnormalities are de novo Type of abnormality parents studied
More susceptibility genes than originally thought More susceptibility genes parents are studied than known pathogenic deletions

Deletion and complex abnormalities more likely to be de novo

FREQUENCY - DE NOVO SIZE OF ABNORMALITIY

SIZE 100 200 kb 200 500 kb 500 kb 1 Mb 1 3 Mb > 3 Mb DELETION 25% 31% 11.3% 32.3% 79% DUPLICATION 3.7% 8.5% 15.7% 12.3% 63%

FAMILIAL DE NOVO TYPE OF ABNORMALITIY

TYPE ? Susceptibility Susceptibility Large Pathogenic Small % FAMILIAL 94.4 84.8 24.7 22.9 80.5 % DE NOVO 5.6 15.2 75.3 77.1 19.5

GENES ARRAY [~3000 CASES]

Large changes multiple genes [619] Microdeletion/ pathogenic genes [367] Susceptibility genes [411] ?Susceptibility genes [284] Unknown function [1329]
De novo [~311] Complex [372] Unknown [646 - ~21%]

Array Loss 341kb Array gain 234kb

Array gain 840kb Array loss 275kb Array loss 958kb Array loss 4.37Mb

IMPLICATIONS - I
Both

retrospective and prospective cases studied

~15.5% of cases studied prospectively shown not to be simple deletions or duplications complex ~35% of cases studied retrospectively complex Evidence for the need to study previously identified abnormalities with array analysis

IMPLICATIONS - II
The

majority of duplications (86%) are direct duplications, not inverted tandem Most deletions do not appear to be terminal (both retrospectively or prospectively ascertained) A higher than expected number of individuals have two or more abnormalities
Accounts for phenotypic abnormalities

IMPLICATIONS - III
Approximately

23.5% of abnormalities are facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are similar
Fewer overall duplications formed by LCRs
Phenotypically not ascertained Most

deletions are not facilitated by LCRs and are unique

IMPLICATIONS - IV
New

mechanisms responsible for abnormalities

Facilitated by repeats/but not LCRs Discontinuous duplications or deletions
Some facilitated by multiple sets of LCR

Duplication of chromosomal material from a non-adjacent region in the precise area where a deletion has occurred

IMPLICATIONS - IV
Multiple

mechanism for ring/marker formation

Breakpoint heterogeneity Formation by multiple chromosome Ring duplication rather than deletion Formation associated with UPD Facilitated by LCRs Pericentric heterochromatin involved, not alphasatellite DNA Formation involves non-continuous chromosomal segments

SNP ARRAY - IMPORTANCE

Can

detect extremely small abnormalities anywhere in the genome Will allow for good breakpoint delineation and determination of abnormalities
Importance in elucidation of mechanisms
Good

whole genome coverage

Terminal vs. interstitial abnormalities LCR involvement

COPY NEUTRAL HOMOZYGOSITY RUNS >1MB

AA AB BB

CN =2

Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients

40 35 30
% OF PATIENTS

25 20 15 10 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Mb BLOCKS

IDENTITY BY DESCENT

Chromosome 10: 97Mb Interval Total

IDENTITY BY DESCENT
1000

First Degree Consanguinity

900

800

Total Homozygosity >10 Mb

Denied Consanguinity
700

600

500

400

1st Cousins
300

2nd - 3rd Cousins

200

100

0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113

Patient #

PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH

All: Non-dysmorphic; IQ=70-90, but no significant genetic issues

5 5

Asperger syndrome, DD MLD Asperger syndrome Autism, DD, Speech Problems Proband IQ=60 Autism, DD, Speech Problems

TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD

Allelic Segregation

d15s217

d15s659

18.3 Mb

28.6 Mb

UPD 15

Copy Number State = 2.0

Red Brackets: Regions of homozygosity Light Blue Brackets: Regions of heterozygosity Dark Blue arrows: Recombination sites
-

MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE

Confirmed hetero-isoUPD 7mat: 29.9 and 8 Mb LCSH Intervals Detected in AF after CVS trisomy 7 mosaicism

EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)

A.

B.

F
.

90% DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13->QTER

D11S4463 D11S1383 D11S4463

Heterozygous Region (D11S1383)

Homozygous region (D11S4463)

Homozygous region (D11S4464)

BECKWITH-WEIDEMANN SYNDROME
Chromosome 11 SNP Array Results

AA AA/AB BB/AB BB

CN=2

CN=2

MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)

UPD RELATED RISK

1.

Imprinting syndromes Recessive allele disorders- relative to the length/site of the HZ run Occult trisomy- early gestational effects of mosaicism pre-rescue

2.

3.

CYTOGENETIC & ARRAY RESULTS - CULTURED CELLS

Cytogenetic Results Array Results Concordance

47,XX,+15 47,XY,+16 47,XX,+22 47,XX,+9 69,XXX 47,XY,+18 45,XX,der(13;14)(q10;q10) 46,XY 46,XY 46,XY 47,XX,+16[22]/46,XX[21]

XX,+15 XY,+16 XX,+22 XX,+9 XXX Triploid XY,+18 XX XY XY (60%) XY XX,+16 (60%)

+ + + + + + +* + + + +

TRISOMY 9 RESULT ALLELE DIFFERENCE

TRIPLOID RESULT

oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy

CYTOGENETIC VS. ARRAY COMPARISON OF DIRECT RAW TISSUE

CYTOGENETIC RESULT Aneuploidy/XX Aneuploidy/XX Complete Aneuploidy 46,XX (Fetal or MCC?) 46,XY 47,XY,+2[2]/46,XY ARRAY RESULT Pure Abnormal Mixed Abnormal Pure Abnormal Normal XX Normal XY Normal XY 16 3 3 7 2 1 1 2 1 # Cases

46,XX,t(3;8)[3]/46,XX[17] 48,XY,+21,+22 Tetraploid (XXYY) 46,XX (100% MCC) Normal Male Mole

MOLAR GENOTYPES
Normal 46,XX (one sperm x 2) 46,XY (two sperm) 69,XXX Normal 100% identity ~50% identity

Triploid normalization

ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES

DNA isolated from residual tissue in long term storage

Array results obtained in 33/34

NORMAL RESULTS = 17
NL XX = 5: 4 Pure and 1 with MCC NL XY = 12: 8 Pure and 4 with MCC

ABNORMAL RESULTS = 16 PURE TRISOMY or 45,X = 6 PURE TRIPLOID = 2 (XXX and XXY) PURE DELETION = 3 COMPLETE MOLE = 1 (XY, DISPERMY) TRISOMY with MCC = 4

PRENATAL DIAGNOSIS STUDIES

Validation of SNP array for prenatal in progress Utilization of Affymetrix 6.0 array
More conservative guidelines

Deletions 1MB; Duplications 2 Mb More restrictive definitive gene list

138 prenatal cases studied

clinically significant abnormalities detected (~7.7%)

Majority could not be detected by chromosomes

UPD 4 possible cases Consanguinity 6 cases

UTILITY OF SNP MICROARRAY ANALYSIS

High density coverage throughout entire genome

Both known and regions of potential clinical significance targeted Known regions targeted in high density More precise localization of abnormalities Ability to review archival data as new syndromes and genes identified Delineation of abnormalities in balanced rearrangements and markers

Routine detection of uniparental disomy Detection of identity by descent recessive allele risk

SNP ARRAY - LIMITATION

Involves extra work

Acquiring and using BACs FISH Problematic Where can these probes come from?

Variable phenotypic effects

1q21.1; 15q13.3 This is a major problem that everyone faces will only be resolved with research and good data collection

CONCLUSIONS

Have reviewed data of over 3,000 abnormalities detected by whole genome array Pathogenicity of genes can be delineated in ~80% of cases detected by array
All but 4% of 15,000 cases studied

Have delineated many new genes/regions that contribute to phenotype As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined lower unknown frequency

IMPLICATIONS - I
Both

retrospective and prospective cases studied

~15.5% of cases studied prospectively shown not to be simple deletions or duplications complex ~35% of cases studied retrospectively complex Evidence for the need to study previously identified abnormalities with array analysis

IMPLICATIONS - II
The

majority of duplications (86%) are direct duplications, not inverted tandem Most deletions do not appear to be terminal (both retrospectively or prospectively ascertained) A higher than expected number of individuals have two or more abnormalities
Accounts for phenotypic abnormalities

IMPLICATIONS - III
Approximately

23.5% of abnormalities are facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are similar
Fewer overall duplications formed by LCRs
Phenotypically not ascertained Most

deletions are not facilitated by LCRs and are unique

IMPLICATIONS - IV
New

mechanisms responsible for abnormalities

Facilitated by repeats/but not LCRs Discontinuous duplications or deletions
Some facilitated by multiple sets of LCR

Duplication of chromosomal material from a non-adjacent region in the precise area where a deletion has occurred

IMPLICATIONS - IV
Multiple

mechanism for ring/marker formation

Breakpoint heterogeneity Formation by multiple chromosome Ring duplication rather than deletion Formation associated with UPD Facilitated by LCRs Pericentric heterochromatin involved, not alphasatellite DNA Formation involves non-continuous chromosomal segments

SNP ARRAY - IMPORTANCE

Can

detect extremely small abnormalities anywhere in the genome Will allow for good breakpoint delineation and determination of abnormalities
Importance in elucidation of mechanisms
Good

whole genome coverage

Terminal vs. interstitial abnormalities LCR involvement

CONCLUSIONS
Much

more complexity to chromosomal aberrations than originally thought Structure of chromosomes examined and delineated
Fewer terminal deletions than previously believed Most duplications are tandem LCRs involvement in 23.5% of deletions and duplications do not count for the formation of the majority of abnormalities

CONCLUSIONS
New

mechanism of formation delineated

Only scratching the surface

Phenotypic

findings

Have always known considerable variability within cytogenetic syndromes Phenotypes may be altered by
Hidden complexity Additional abnormalities

VERY LAST THOUGHTS

Some abnormalities - difficult to interpret

Many factors need to consider Size doesnt always matter

Interpretation will only be possible with the acquisition of good clinical information and family follow-up
Parental phenotype and abnormality

Imperative for clinicians and laboratory personal to work together

ACKNOWLEDGEMENTS

LabCorp
Peter Papenhausen Jim Tepperberg Marcia Eisenberg Inder Gadi Rachel Burnside Vikram Jaswaney Hiba Risheg Romela Pasion

Affymetrix
Roger Schaller Richard Shippy

LabCorp
Brian Williford Carolyn Bullen Jessica Whaley-Davis Daniel Fuentes Renee Royster Josh Kesler

Referral physicians