molecular genetics and prenatal diagnosis of holoprosencephaly

7/29/2019 Molecular Genetics and Prenatal Diagnosis of Holoprosencephaly

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SSM 2012 – 2013

Medicine

MBChB Year 1

SSM1 Literature Review:

Introduction to

Molecular Biology

in Medicine

Molecular Genetics

and Prenatal

Diagnosis of

Holoprosencephaly

Candidate Number: 1081Convenor Name: Professor P S Rudland

Word Court: 3,088



Ka-Kiu Claire Fung

Page | 2

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.



Ka-Kiu Claire Fung

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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 andmolecular anomalies, leaving 75% with unidentified aetiology. 8



Ka-Kiu Claire Fung

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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 al’s 19 paper on

Genetic Counseling and ‘Molecular’ Prenatal Diagnosis of

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

this search.



Ka-Kiu Claire Fung

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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 1 – Genes and its loci that contribute to HPE

Human

gene

Human

locus

Chromosome Molecular Function

– HPE1 21q22.3 (unknown)

SIX3 HPE2 2p21 Forebrain and eye

development

SHH HPE3 7q36 Ventral VNS patterning

TGIF HPE4 18p11.3 Transcriptional repressor

including retinoids

ZIC2 HPE5 13q32 Axis formation and dorsal

brain development

– HPE6 2q37.1-q37.3 (unknown)

PTCH1 HPE7 9q22.3 Receptor for hedgehog

ligands

– HPE8 14q13 (unknown)

GLI2 HPE9 2q14 Transcription factor mediating

hedgehog signalling

– HPE10 – (unknown)

DISP1 – 1q42 Release of hedgehog ligands

NODAL – 10q TGFβ-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.



Ka-Kiu Claire Fung

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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 HPE-

like 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.



Ka-Kiu Claire Fung

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Colour Representation

Orange Hindbrain

Black Anterior neural ridge (ANR)

Green Midbrain-hindbrain boundary (MHB)Black arrows Fibroblast Growth Factor 8 (FGF8)

Light blue strip (posterior to ANR) Telencephalon

Blue area, centre of yellow circle Eye field

Yellow circle Mesencephalon

Green arrows WNT proteins

Red line Notochord(Axial midline)

Red dot Prechordal plate

Red arrows SHH protein

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



Ka-Kiu Claire Fung

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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 patients12

, 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 receptor 13. 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 amino-

terminal transcriptional repressor domain of the gene plays a pivotal role in

the dominant-negative activity resulting from mutations 15.



Ka-Kiu Claire Fung

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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:



Ka-Kiu Claire Fung

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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 thatcauses premature termination of SHH translation. The effects of the other

two, c.388G>T and c.474C>G in exon 2, are unclear 17

, 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 526

th

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 Sex AI restriction site,

rendering the SHH protein malfunctional. 17



Ka-Kiu Claire Fung

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4.6 Mutations in the ZIC2 gene

Figure 413

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



Ka-Kiu Claire Fung

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4.8 Mutations in TGIF gene

Figure 613

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



Ka-Kiu Claire Fung

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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 mother’s 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, DNAsequencing20, quantitative multiplex PCR for short fluorescent fragments20



Ka-Kiu Claire Fung

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(QMPSF), multi-coloured fluorescent in situ hybridization21

(FISH),

quantitative PCR 21, and multiplex ligation probe-dependent amplification 8

(MLPA).

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

20

Figure 7



Ka-Kiu Claire Fung

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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

rearrangements21

. 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 21. It is often used to

confirm findings from M-FISH.

Figure 821

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

FISH probes,

Colour Gene

Light blue DISP1

Yellow SIX3

Pink SHH

Green ZIC2

Red TGIF

Dark blue FOXA2



Ka-Kiu Claire Fung

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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 98

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



Ka-Kiu Claire Fung

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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 quantities21

. M-

FISH also requires a larger sample of DNA template and reagents than for

processes such as QMPSF 20, 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 arraycomparative genomic hybridization (a-CGH). It can help identify unbalanced



Ka-Kiu Claire Fung

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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.



Ka-Kiu Claire Fung

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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.



Ka-Kiu Claire Fung

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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. Still-

dividing 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.



Ka-Kiu Claire Fung

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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: 1–6.

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: 1–21.

3. Muenke M, Cohen MM. Genetic Approaches to Understanding Brain

Development: Holoprosencephaly as a Model. Medical Retardation

and Developmental Disabilities Research Reviews. 2000; 6: 15–21.

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: 120–32

5. Solomon BD, Lacbawan F, Jain M, Domené 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:

919–25.

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, Vélez 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: 133–41.



Ka-Kiu Claire Fung

Page | 22

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: 565–8.

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: 1579–82.

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 – PhenotypicVariability. Clinical Geneicst 2012; 80: 70-75



Ka-Kiu Claire Fung

Page | 23

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: 96–122.

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.



Ka-Kiu Claire Fung

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.

molecular genetics and prenatal diagnosis of holoprosencephaly

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