SSM 2012 2013 Medicine MBChB Year 1

Molecular Genetics
SSM1 Literature Review:

Introduction to Molecular Biology in Medicine

and Prenatal Diagnosis of Holoprosencephaly

Candidate Number: 1081 Convenor Name: Word Court: Professor P S Rudland 3,088

Ka-Kiu Claire Fung

Abstract
Holoprosencephaly (HPE; 1 in 16,000 live births1; 1 in 250 foetuses2) is a brain disorder resulting from incomplete separation of the prosencephalon during the third and fourth weeks of gestation, causing a wide spectrum of craniofacial symptoms. Clinical phenotypes range from single cerebral hemisphere and cyclopia to unaffected carriers in autosomal dominant HPE families. 3 This disorder is genetically heterogeneous, but there are also environmental causes that contribute to HPE. The main genes that are found to be causative agents of HPE phenotypes are SHH, ZIC2, SIX3, and TGIF, although there are at least 10 HPE loci found. Currently, various imaging methods such as foetal ultrasound are used to establish a diagnosis. DNA screening is also offered to HPE families to monitor the development of the foetus. These processes include multicolour FISH to detect for deletions and quantitative PCR to confirm diagnoses made using the former method. There are no known treatments, although there are ways to manage the clinical manifestations of the disease to a limited extent. This paper reviews the gene mutations that leads to HPE, and compares and contrasts the methods of molecular diagnosis that can be used to establish a diagnosis, the subtype and its severity.

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1. Introduction
Holoprosencephaly (HPE) is the most common form of malformation in the forebrain in humans1. It has three clinical subtypes4. The most severe is alobar HPE, where the brain has not divided at all. An alobar HPE patient will have fused cerebral hemispheres and midline grey matter structures; the corpus callosum and third ventricle are typically absent. The moderate case is semi-lobar HPE, where the brain has somewhat divided. It includes a fusion of frontal lobes with the presence of interhemispheric fissure posteriorly; part of the corpus callosum is present. The mildest is lobar HPE, where there is considerable evidence of separate brain hemispheres. The brain of a lobar HPE patient will clearly show two lobes, but will have misshapen ventricles as a result of the lack of septum pellucidum. Milder craniofacial characteristics of HPE include microcephaly, hypotelorism, flat (or absent) nasal bridge and single maxillary central incisor. Around 80% of severe HPE patients have characteristic facial dysmorphisms. Their severity range from median cleft lip and/or palate to cyclopia, occasionally coupled with an overriding proboscis. Other microforms of HPE exist, resulting in sharp and narrow nasal bridge5, developmental delay 6, and more. Aside from physical features unique to this disorder, all forms of HPE also involve similar clinical manifestations7, including seizures and pituitary dysfunction. There is a common misconception that children with HPE do not survive beyond early infancy. However, many with milder cases (as well as some who are severely affected) can live beyond 12 months. To date, results from various studies of the cause of HPE can be summarized as follows: 15% were related to environmental causes; 45% patients are chromosomal HPE. 25% of the remaining isolated (nonchromosonal and nonsyndromic) HPE are caused by microdeletions and molecular anomalies, leaving 75% with unidentified aetiology. 8
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The most common non-genetic cause of human HPE is maternal diabetes mellitus, which increases the chance for infants having any form of HPE by 1% -- a 200-fold increase from the normal incidence9. More recently, an association between cholesterol-lowering agents with HPE has been discovered, but its causal relationship is also not yet proven 10.

2. Aim
This paper aims to look at the genetic determinants of HPE and to compare and contrast the methods in which foetal cells can be analysed in order to establish a diagnosis, the subtype and its severity. The paper will then go on to suggest any potential developments in treatment. To do so, the molecular genetics of HPE and the current hypotheses regarding its aetiology is first discussed.

3. Methodology
Databases such as Google Scholar and Pubmed were the main resources for gathering information for this paper. Another inevitable resource is the University Library resource application that, with a student ID, allows full access to various journals. Initially, molecular genetics AND holoprosencephaly was searched. Others had AND holoprosencephaly after a key word, which included role of SHH, molecular screening, molecular diagnosis, microdeletions, mutations and prenatal gene therapy. The first search resulted in 5,600 results, and Mercier S et als Genetic this search. Counseling and Molecular Prenatal
19

paper on of

Diagnosis

Holoprosencephaly (HPE) is an example of an article that was found under

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4. Molecular Genetics of HPE

Four genes have so far been linked to the majority of HPE cases: Sonic Hedgehog (SHH), ZIC2, SIX3 and TGIF. Other genes that contribute to the craniofacial abnormalities are known, and these are summarized in the table below. Table 111 Genes and its loci that contribute to HPE Human gene SIX3 SHH TGIF ZIC2 PTCH1 GLI2 DISP1 NODAL Human locus HPE1 HPE2 HPE3 HPE4 HPE5 HPE6 HPE7 HPE8 HPE9 HPE10 21q22.3 2p21 7q36 18p11.3 13q32 2q37.1-q37.3 9q22.3 14q13 2q14 1q42 10q (unknown) Forebrain and eye development Ventral VNS patterning Transcriptional repressor including retinoids Axis formation and dorsal brain development (unknown) Receptor for hedgehog ligands (unknown) Transcription factor mediating hedgehog signalling (unknown) Release of hedgehog ligands TGF-like ligand involved in midline and laterality establishment FOXH1 8q24.3 Transcription factor for NODAL signalling
Table 1 is taken directly from the article cited.

Chromosome

Molecular Function

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Close examination of mutations in the genes mentioned showed that they would result in proteins with reduced or absent biological function
11

However, there is still a substantial number of HPE cases that do not have any apparent mutations, which leads to the belief that there must be more genes that contribute to HPE. The large number of genes that have an associative link with HPE also describes the large phenotypic spectrum, as not all responsible genes are structurally altered or lost simultaneously. 11 4.1 Sonic Hedgehog signalling and HPE

Studies confirmed that a common cause of characteristic HPE phenotypes is SHH signalling dysfunction. SHH receptor PTCH1, ligand transporter DISP1 and transcription factor GLI2 are three additional genes in the SHH signalling pathway, and lesions in any of them would contribute to formation of HPElike phenotypes. 11 SHH is considered the main gene to cause HPE phenotypes because the removal of SHH signals or insensitivity to them directly causes cyclopia.

Figure 2 is taken directly from article cited for table 1.

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Colour Orange Black Green Black arrows Light blue strip (posterior to ANR) Blue area, centre of yellow circle Yellow circle Green arrows Red line Red dot Red arrows

Representation Hindbrain Anterior neural ridge (ANR) Midbrain-hindbrain boundary (MHB) Fibroblast Growth Factor 8 (FGF8) Telencephalon Eye field Mesencephalon WNT proteins Notochord Prechordal plate SHH protein (Axial midline)

Figure 2 represents a typical flat neural plate prior to neurolation stage, viewed from above. The anterior is at the top and hindbrain and spinal cord are at the bottom. The ANR and MHB secrete FGF8 that promote growth and expansion of the telencephalon. Prior to neurolation, the eye field within the mesencephalon is continuous in the midline, caudal to the telencephalon. 11 MHB produces WNT proteins that are initially inhibited by rostral inhibitors. The paraxial mesoderm (caudal to MHB) secretes retinoic acid, but an enzyme removes this as it enters the MHB. The axial midline secretes SHH protein that separates the eye field, as shown from the 1A 1A progression.11 Diagram 1B (in Figure 2) shows a defective SHH secretion system from the axial midline, causing failure of the eye field to separate, leading to the most characteristic cycloptic phenotype of severe HPE (1B). If there is a reduced SHH secretion (as in milder forms of HPE), the patient would result with hypotelorism as the eye field only partially separates. 11

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4.2

Effects of ZIC2, SIX3, TGIF on SHH signalling and HPE

Mutations in the ZIC2 gene are the second most common detectable alteration in HPE patients
12

, with lesions in SIX3 being the third, and

mutations in TGIF, fourth. All mutations contribute to the formation or deletion of restriction sites, which can be detected by various DNA sequencing methods that will aid in the establishment of a diagnosis (This is discussed in detail in Section 5. Establishing a Diagnosis). ZIC2 targets the transcription factors that mediate SHH protein signals; therefore, a lack thereof would effectively have the same phenotypic effects on HPE patients as those who have diminished SHH signalling pathways.
13

SIX3 has multiple roles, but its main role is to regulate SHH protein secretion in the ventral forebrain, which, again, lends itself into the same developmental pathway. 13 TGIF is thought to code for a transcription factor that competitively inhibits the binding of retinoic acid to its receptor13. Thus, depleted TGIF levels will indirectly cause an increase of retinoic acid levels that exceeds the enzymatic ability to degrade it in the MHB. In an experiment in 2005, by targeting the deletions of exons 2 and 3, which encode 98% of amino acids, mice lacking TGIF were generated. Western blotting proved that these mice had no detectable TGIF protein, and that were both viable and fertile with no HPE symptoms in the forebrain. This suggests the possible functional redundancy of TGIF14. 4.3 Effects of GLI2 on SHH signalling and HPE

Three GLI genes have implications on SHH signals. GLI2 acts as the central transcriptional activator, and recently, it has been discovered that the aminoterminal transcriptional repressor domain of the gene plays a pivotal role in the dominant-negative activity resulting from mutations 15.

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Mutations have reported to cause HPE-like phenotypes with pituitary anomalies. Cleft lip and/or palate are common phenotypes of patients with GLI2 mutations.15 Contrastingly, ZIC2 mutations often result in the absence of typical HPE facial abnormalities.

4.4

The multiple-hit hypothesis

This is perhaps the most widely accepted hypothesis explaining HPE. First described in 200216, this theory suggests the interplay of genetic and environmental factors, for example, the role of cholesterol in HPE 2. In order for full and accurate activity, SHH molecules must be covalently modified by cholesterol. Furthermore, an adequate supply of cholesterol in cells that are receiving the SHH signal is also required for appropriate responsiveness of said cells. Aside from dividing the eye field, SHH signalling pathway is also involved with an enormous diversity of molecular developmental stages, for example, survival of migrating cranial neural crest cells into facial primordia. 21 The relationship between SHH activity and cholesterol regulation remains obscure, but there is a significant association between the perturbation of cholesterol metabolism in early embryonic development and its effects on SHH mechanism. 21

4.5

Mutations in the SHH gene

SHH is on the 7th chromosome. Various molecular screening techniques revealed a total of 17 mutations of SHH, including three nonsense mutations, three deletions and eleven missense mutations. The 17 loci where mutations take place are as follows:

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Figure 3 shows the schematic representation of SHH gene and mutations. It is taken directly from article cited for section 4.5.

Of the nonsense mutations found, the first was c.72C>A in exon 1 that causes premature termination of SHH translation. The effects of the other two, c.388G>T and c.474C>G in exon 2, are unclear17, but it is noted to be unique to semilobar HPE. A deletion of six bases at the 316th nucleotide position results in the absence of two amino acids in the SHH protein, which leads to alobar HPE, and the deletion of nine bases at the 526th nucleotide (c.526_534del GAGTCCAAG) results in microcephaly and absence of part of the corpus callosum. The c.211delG mutation causes semilobar HPE, and, again, is inherited. 17 The remaining missense mutations can cause a variety of HPE cases some sporadic and some inherited. Missense mutations may alter various restriction sites, for example, a c.329C>A transversion destroys the EaeI restriction site, and a c.449C>G mutation creates a SexAI restriction site, rendering the SHH protein malfunctional. 17

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4.6

Mutations in the ZIC2 gene

Figure 4

13

shows the schematic representation of ZIC2 gene and mutations.

Most of the nine mutations of the ZIC2 gene cause a premature termination during transcription. A c.172G>T transversion or c.107A>C transversion creates two different restriction sites, and an insertion of 17 base pairs was also found in the terminus of the first exon that can cause alobar HPE. 17

4.7

Mutations in SIX3 gene

Figure 5

13

shows the schematic representation of SIX3 gene and mutations.

Of the eight mutations noted in the SIX3 gene, one is a GG insertion in c.556_557 causes a frameshift that leads to a nonsense mutation in the homeodomain, one 35-basepair duplication that creates a stop codon in the homeodomain; and there are six missense mutations four in SIX domain and two in homeodomain. Each of the missense mutations led to a creation of a different restriction site on the gene.17

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4.8

Mutations in TGIF gene

Figure 6

13

shows the schematic representation of TGIF gene (A) and mutations in the

protein (B).

Of the two mutations detected in TGIF, a c.177C>G transversion creates a restriction site, and the c.320A>T mutation causes microcephaly, cleft lip and palate and mild mental retardation. 13 Mutations were determined using PCR and denaturing high-performance liquid chromatography analysis (DHPLC). 8.5% of HPE patients presented with SHH mutations13, whereas TGIF mutations are detected in only 1.6% of HPE cases. This, again, suggests that mutations in SHH are the principle cause of HPE. 13

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5.

Establishing a Diagnosis

Traditionally, a CT scan or prenatal ultrasound is sufficient to confirm the diagnosis of HPE, define the clinical subtype, and identify any associated abnormalities of the central nervous system (CNS)18. These methods can detect CNS and facial abnormalities of severe HPE as early as the first trimester. However, they are less effective in detecting milder forms of HPE. Although foetal MRI provides better characterization of brain malformations, it is only successful in the third trimester of the pregnancy 4. This means that there is a possibility that HPE may be undiagnosed until birth, or symptoms could be misdiagnosed as being isolated, such as isolated cleft lip or palate15. Thus, the parents would not have had the chance to decide if they wanted to continue with the pregnancy or not. The identification of the four main genes and the lesions within them that are responsible for HPE allows prenatal screening of any mutations through obtaining foetal cells from chorionic villus samples or from the amniotic fluid in the mothers uterus via amniocentesis. Genetic counselling is based on clinical evaluation exploring family history, environmental and associated factors. This is a process where patients or relatives at risk of transmitting the disorder are advised of the consequences and nature of HPE, the probability of transmitting it, the options open to them and family planning. A standard karyotype can diagnose 24 45% of all cases as it allows visualization of large deletions or duplications within HPE genes19. However, if HPE is isolated or nonsyndromic, further tests are needed. Results The tests that are currently used to screen for HPE microdeletions that lead to the formation or deletion of a restriction site include DHPLC, DNA sequencing20, quantitative multiplex PCR for short fluorescent fragments20
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(QMPSF), (MLPA).

multi-coloured
21

fluorescent

in

situ

hybridization21

(FISH),
8

quantitative PCR

, and multiplex ligation probe-dependent amplification

As mentioned before, DHPLC can be used to analyse the genes for any mutations17. QMPSF, introduced in 2002, is a process used for rapid determination of HPE genes20. Oligonucleotide primer pairs for amplification corresponding to the four main genes are used to construct a multiplex PCR. It is then used to construct one multiplex PCR that generates ten PCR fragments including two to three products for each HPE gene. Multiplex PCR is performed in this reaction mixture, The data obtained from these tests accurately diagnoses any microdeletions.
Figure 7

Figure 7

20

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Figure 7 shows the detection of HPE gene deletion by QMPSF. In each panel, the electrophoregram of the patient is in red, and it is superimposed upon a control (blue). a on figure 7 shows the deletion of the entire SHH gene and b shows the deletion of ZIC2. 20 Another method, multicolour FISH, is used to detect submicroscopic rearrangements 21. DNA is PCR amplified first, and three bacterial artificial chromosome probes are labelled with one fluorescent dye each. Standard FISH mapping confirms the correct chromosomal location of each probe, and they can be identified based on its unique colour and its chromosomal location. Used alongside M-FISH, qPCR allows analysis of smaller sequences, detecting deletions of individual exons confirm findings from M-FISH.
21

. It is often used to

Figure 8

21

is taken directly from its source, showing the chromosomal localization of the six

FISH probes,

Colour Light blue Yellow Pink Green Red Dark blue

Gene DISP1 SIX3 SHH ZIC2 TGIF FOXA2

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In 2007, MLPA was introduced8. It is PCR-based, and allows simultaneous testing of all subtelomeres. A capillary analyser also allows visualization, normalization and comparison of electrophoretic profiles based on size standard and signal strength.

Figure 9

Figure 9 shows two charts of MLPA results from female patients. The first represents normality, where all subtelomeric probes have a ratio close to 1.0 when compared to normal results, and the second shows a 7q deletion (where ratio is now 0.5), and is associated with a gain in a 7p telomere (where the ratio is 1.5). 8

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Discussion Although DHPLC is the most commonly used procedure, studies carried out by Dubourg and Lazaro found that the sensitivity and specificity of DHPLC exceeds 96%, but it cannot test for any other genes that may be responsible for HPE, which could cause a misdiagnosis. 17 QMPSF allows an accurate analysis of electrophoregrams of different samples, not by comparing the different peak intensities, but through normalization of different samples. Therefore, it can detect heterozygous deletions as well as duplications accurately and cost effectively as it uses less DNA templates and reagents than the following methods. Until 2004, M-FISH was considered the best method 8, as it allows the detection of somatic chromosomal mosaicism 8. However, the average probe size used in the experiment carried out by Bendavid C et al. 21 was between 100 150kb, which is slightly larger than HPE genes, which could potentially lead to false negative results, causing misdiagnoses. It is also time consuming and requires fresh specimens, making it costly. MLPA is a process that has a low false-positive rate confirmation processes such as qPCR have proved that it is 83% accurate, as recorded by recorded by Bendavid C et al 8. MLPA requires small quantities of genomic DNA, which makes it easier and more cost efficient, and consequently, it is used more regularly now than M-FISH.
8

The results

obtained for MLPA are also more reliable, as a larger number of procedures can be carried out than for M-FISH, due to the fact that the latter requires fresh samples of DNA, which is not readily available in large quantities processes such as QMPSF
20 21

. M-

FISH also requires a larger sample of DNA template and reagents than for , therefore making it more costly and a less ideal method used for routine diagnosis of HPE. Another process which may be added to regular HPE screening is array comparative genomic hybridization (a-CGH). It can help identify unbalanced
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subtelomeric anomalies as MLPA does, but can also determine the breakpoints simultaneously, making it more suitable to help clinicians diagnose obscure HPE anomalies. 8 Overall, molecular prenatal diagnosis of HPE demands a more

encompassing approach, incorporating primarily foetal imaging, and especially allows more reassurance if the known mutation in an index case is absent in the foetus before MRI imaging.

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6. Final Conclusions and Future Works

This paper covers the molecular genetics of holoprosencephaly, focussing mainly on the effects of Sonic Hedgehog signalling pathways in the brain and how it causes HPE phenotypes. Current comprehension of the disorder is far from complete:

Understanding of the underlying problem There remains a large percentage of HPE patients whose genotypic anomalies are unidentified and therefore cannot be diagnosed. It has been proved that HPE has a strong inheritance association, however it is still not fully understood how sporadic HPE-linked genetic mutations come about. Secondly, of the genetic mutations that have been identified, only some have been confirmed to be directly responsible for HPE phenotypes. There are some that are noted are unique to HPE genotype but have not found any phenotypic correlation. Their significance to the disorder has yet to be understood. The studies that have been reviewed in this paper have not shown any conflict between findings of responsible genetic mutations. Some have debated the significance of SHH pathway in causing of HPE phenotypes, but others have mentioned that SHH is the main responsible gene. The lack of disagreement in findings could be because HPE is still a relatively young field, and the research today focuses on identifying and suggesting all responsible mutations rather than critiquing the current information.

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Potential treatment Currently, there is no standard course of treatment for this disorder, but there are ways to manage the symptoms of HPE to a limited extent, including hormone replacement therapy for pituitary dysfunction and antiepileptic drugs for seizures, etc. In the future, in utero gene therapy may provide a cure for HPE. Stilldividing stem cells that are inaccessible later in life could also be a target the developing foetus may also be more compliant to the uptake and permanent integration of DNA. One suggestion as to why in utero gene therapy may succeed is that the foetal immune system is functionally immature, which may permit the induction of immunological tolerance to the vector and its transgene, and aid in postnatal repeat vector administration if necessary. With the current imaging technology, a minimally invasive procedure can be carried out in order to deliver the transgene to the foetus. 22

The aforementioned areas represent the pinnacle of the many issues that surrounds the field of embryonic development. Our knowledge of this disorder has far-reaching implications in allowing full understanding of the most common cause of malformation of the brain.

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References
1. Orioli IM, Castilla EE, Ming JE, Nazer J, Aguiar M, Llerena JC, Muenke M. Birth Prevalence of Holoprosencephaly Genes in a South American (ECLAMC) Population. Human Genetics. 2001; 109: 16. 2. Edison R, Muenke M. The Interplay of Genetic and Environmental Factors in Craniofacial Morphogenesis: Holoprosencephaly and the Role of Cholesterol. Congenital Anomalies (Kyoto). 2003; 43: 121. 3. Muenke M, Cohen MM. Genetic Approaches to Understanding Brain Development: Holoprosencephaly as a Model. Medical Retardation and Developmental Disabilities Research Reviews. 2000; 6: 1521. 4. Hahn JS, Barnes PD. Neuroimaging Advances in Holoprosencephaly: Refining the Spectrum of the Midline malformation. American Journal of Medical Genetics. Part C, Seminars in Medical Genetics. 2010; 154C: 12032 5. Solomon BD, Lacbawan F, Jain M, Domen S, Roessler E, Moore C, et al. A novel SIX3 Mutation Segregates with Holoprosencephaly in a Large Family. American Journal of Medical Genetics. 2009; 149A: 91925. 6. Heussler HS, Suri M, Young ID, Muenke M. Extreme Variability of Expression of a Sonic Hedgehog Mutation: Attention difficulties and holoprosencephaly. Archives of Disease in Childhood. 2002; 86: 293 6 7. Solomon BD, Mercier S, Vlez JI, Pineda-Alvarez DE, Wyllie A, Zhou N, et al. Analysis of Genotype Phenotype Correlations in Human Holoprosencephaly. American Journal of Medical Genetics. Part C, Seminars in Medical Genetics. 2010; 154C: 13341.
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8. Bendavid C, Dubourg C, Pasquier L, Gicquel I, Le Gallou S, Mottier S, et al. MLPA Screening Reveals Novel Subtelomeric Rearrangements in Holoprosencephaly. Human Mutations. 2007; 28(12): 1189 1197 9. Barr M Jr, Hanson JW, Currey K, Sharp S, Toriello H, Schmickel RD, Wilson GN. Holoprosencephaly in infants of diabetic mothers. Journal of Paediatrics. 1983; 102: 5658. 10. Edison RJ, Muenke M. Central nervous system and limb anomalies in case reports of first trimester statin exposures. New England Journal of Medicine. 2004a; 350: 157982. 11. Roessler E, Muenke M. The Molecular Genetics of

Holorprosencephaly. American Journal of Medical Genetics. Part C, Seminars in Medical Genetics. 2010, 154C: 52 61 12. Roessler E, Lacbawan F, Dubourgh C, Paulussen A, Herbergs J, Hehr U, et al. The full spectrum of holoprosencephaly-associated mutations within ZIC2 gene in humans predict loss-of-function as the predominant disease mechanism. Human Mutation. 2009; 30: E541 E544. 13. Aguilella C, Dubourg C, Attia-Sobool J, Vigneron J, Blayau M, Pasquier L, et al. Molecular screening of TGIF gene in holoprosencephaly identification of two novel mutations. Human Genetics. 2003; 112: 131 134 14. Shen J, Walsh CA. Targeted disruption of TGIF, the Mouse Ortholog of Human Holoprosencephaly Gene, Does Not Result in Holoprosencephaly in Mice. Molecular and Cellular Biology 2005; 25(9): 3639 3647 15. Berolacini CDP, Ribeiro-Bicudo LA, Petrin A, Richieri-Costa A, Murray JC. Clinical Findings in Patients with GLI2 Mutations Phenotypic Variability. Clinical Geneicst 2012; 80: 70-75
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16. Ming J, Muenke M. Multiple Hits during Early Embryonic Development: Digenic Diseases and Holoprosencephaly. American Journal of Medical Genetics. 2002; 71: 1017 1032 17. Dubourg C, Lazaro L, Pasquier L, Bendavid C, Blayau M, Le Duff F, Durou MR, et al. Molecular Screening of SHH, ZIC2, SIX3 and TGIF genes in Patients with Features of Holoprosencephaly Spectrum: Mutation Review and Genotype Phenotype Correlations. Human Mutations. 2004; 24: 43 51 18. Barkovich AJ, Maroldo TV. Magnetic Resonance Imaging of Normal and Abnormal Brain Development. Topics in Magnetic Resonance Imaging. 1993; 5: 96122. 19. Mercier S, Dubourg C, Belleguic M, Pasquier L, Loget PLucas J, et al. Genetic Counseling and Molecular Prenatal Diagnosis of Holoprosencephaly (HPE). American Journal of Medical Genetics 2010; 154C: 191 196 20. Bendavid C, Dubourg C, Gicquel I, Pasquier L, Saugier-Veber P, Durou MR, et al. Molecular Evaluation of Foetuses with Holoprosencephaly shows high incidence of Microdeletions in the HPE Genes. Human Genetics. 2006; 119: 1 8 21. Bendavid C, Haddad BR, Friffin A, Huizing M, Dubourg C, Gicquel I, et al. Multicoloured FISH and quantitative PCR can detect Submicroscopic deletions in Holoprosencephaly Patients with a Normal Karyotype. Journal of Medical Genetics. 2006; 43: 496 500 22. David AL, Waddington SN. Candidate Diseases for Prenatal Gene Therapy. Methods in Molecular Biology. 2012; 891: 9 39

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Appendix
Date 15/01/2013 Introduction to the SSM structure by Prof. Rudland. The seminar included information about what is required in the paper. We were put into groups of three and present on the use of molecular biology in the diagnosis of a disease for 16/02/2013. 16/01/2013 Presentation on the uses of molecular biology in the diagnosis of a disease. Presentations from other groups included the treatment of disease and in agriculture. 17/01/2013 Practical 1 two experiments were carried out. The experiment included an unknown sample of genetic material that required the use of TLC plate and chromatography to determine its nature. Our sample was DNA and our experimental value for its concentration was 0.26mg/mL, which was accurate. 18/01/2013 In a group of 7, we were given a set of data to deduce the identities of 21 people and to create a pedigree chart to show the three family trees. The data provided included results from Southern blotting, genetic profiling and PCR analysis. We had to deduce which individual were carriers of diseases, which individuals were not carriers, and which were affected. 21/01/2013 A seminar on the introduction of nucleotides, nucleosides and DNA genetic code was given by Prof. Rudland. 22/01/2013 A seminar on the introduction to manipulation of the genetic code and its involvement in biochemistry in Medicine.

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24/01/2013

Practical 2 Few experiments were carried out to analyse a sample of an unknown polynucleotide using enzyme hydrolysis and paper chromatography. A map of the unknown DNA molecule was sketched using data obtained from the experiments.

25/01/2013

Individual presentations were given on our SSM topics. Each presentation was 10 12 minutes. My topic of choice was What is holoprosencephaly and how is it diagnosed?

28/01/2013

A seminar on molecular biology in the cloning of DNA was given by Prof. Rudland.

29/01/2013

A seminar on the production of recombinant proteins was given by Prof. Rudland.

01/02/2013

In groups of 7, we used data given to suggest links between the different cancers and the changes in environment and habits. The data provided included a table showing cancer incidence annually from 1950 to 1990, results from AMES tests, DNA sequencing of RAS gene and RFLP of fragments detected after Southern blotting.

04/02/2013

The final seminar on the engineering of proteins was given by Prof. Rudland.

Alongside these mandatory sessions with our convenor, I carried out my research on my topic (as mentioned in section 3. Methodology), completed my paper and submitted it on 06/02/2013.

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