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
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Ka-Kiu Claire Fung
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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 andmolecular 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 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.
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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.
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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.
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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
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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.
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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 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
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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
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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
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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
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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
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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
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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
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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
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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. 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.
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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.