GENETICS

‘Numerical abnormalities of the sex chromosomes are better tolerated than numerical abnormalities of

the autosomes’. Discuss with specific examples. (2009 Resit)

Numerical abnormalities of the sex chromosomes are better tolerated and more compatible with life for

two reasons. Firstly, in males (XY), the Y chromosome carries relatively little genetic information.

Secondly, in females (XX), there is inactivation of certain gene loci of all but one X chromosomes. This

process is known as lyonisation, and the random inactivation of X chromosomes means females are

functionally hemizygous for X. This gene dosage compensation is important to ensure females do not

produce double the coded enzymes, and thus double the gene product, as males.

Turner’s Syndrome (45, X) is an example of a sex chromosome abnormality occurring once in every 2000

female live births. In this case, the single X chromosome undergoes lyonisation and as a result there is

halpoinsufficiency of certain genes leading to the clinical features of the disease. These include gonadal

dysgenesis, short stature, lymphoedema of the extremities, broad chest and widely spaced nipples.

Frequently encountered problems also include chronic heart disease, diabetes, hypothyroidism and

various autoimmune disorders. The SHOX gene is implicated in Turner’s Syndrome.

Klinefelters Syndrome (47, XXY) is another example occurring once in every thousand live male births. In

this case lyonisation of one of the X chromosomes still leaves a full X chromosome, leaving gene dosage

anomalies. Clinical features may not be apparent until later in life but include hypogonadism, tall

stature, sparse body hair and in 30% patients male breast development.

These two disorders, although they both confer infertility and various other health problems, have little

or no mental retardation associated with them. In contrast, autosomal numerical abnormalities are not

tolerated well and only three autosomal aneuploidies are compatible with life – Trisomies 13, 18, and 21

– common features of which are a myriad of congenital anomalies and growth and mental retardation.

Trisomy 21 is the most common, with 1/700 live births and is better known as Down’s Syndrome.

Usually it is due to an extra copy of chromosome 21, but can also be as a result of a Robertsonian

translocation (14, 21). It is a disorder of gene dosage, where the extra copy of the chromosome means

the production of extra gene product. Genes known to reside on Chromosome 21 (which are therefore

affected in DS) include APP, amyloid precursor protein, which is implicated in the pathology of early

Alzheimer’s disease in DS, and genes DYRK1A and DSCR1 which are involved in regulating the NFATc

signalling pathway required for vertebrate development and organogenesis. Clinical features of DS

include mental retardation, congenital anomalies, hypotonia, and the characteristic visible features of

epicanthic folds, flat facial palate, simian crease, abundant neck skin and ‘sandal toe’ gap.

In conclusion, numerical abnormalities of chromosomes can lead to a range of clinical syndromes, of

which abnormalities of the sex chromosomes fare better because of lyonisation of X chromosomes and

the small amount of information carried on the Y chromosome – therefore it is easier compensated for.

In contrast, abnormalities in autosomal chromosomes have a greater loss or gain of function, and are

therefore usually incompatible with life, or have greater mental, growth and congenital deficits.

Discuss the inheritance pattern, molecular basis and clinical features of Cystic Fibrosis. (2009 Resit)

Cystic Fibrosis is an Autosomal Recessive (AR) disorder affecting 1/2000 of Caucasians, with an increased

frequency of 1/1500 in the Irish population. 1/20 Irish people are carriers for the disease.

Autosomal Recessive disorders are only expressed when both alleles for the gene are mutated,

therefore only homozygotes are affected. This is because the product of the allele (eg. protein/enzyme)

is sufficient at less than 100% for normal function, and is normally involved in metabolic pathways. AR

disorders have a horizontal degree pattern, where the disease is normally seen in siblings and not

usually earlier generations and there is equal frequency and severity in both sexes.

Dd ---------------------------------Dd

DD Dd Dd dd

Unaffected Carrier Carrier Affected

Siblings of an affected individual have over 65% chance of being a carrier. Unaffected carrier parents of

an affected child have a 25% recurrence risk.

CF is caused by mutation on the CFTR (cystic fibrosis transmembrane receptor gene) on Ch7q. This codes

for an ion channel that regulated the liquid volume on epithelial surfaces through chloride secretion and

inhibition of sodium absorbtion.

In normal duct epithelia, Chloride is transported by Cl- channel that are opened by CAMP-regulated

phorphorlyation. In CF mutations cause defected CAMP-regulated Cl- transport. There are over 1100

mutations identified.

In airway epithelium, usually Cl- is secreted into the airway lumen, and Na+ is not reabsorbed, water

follows the osmotic gradient and the fluid is a watery secretion. In CF the defect causes problems in the

transporter, so Cl- is not secreted, and Na+ is absorbed, the water remains in epithelial cells which

causes the fluid in the airway to be more viscid.

In the sweat glands, the opposite is true – in the impaired transporter there is impaired reabsorption of

Cl-, which leads to increased levels of sweat chloride. This is the basis for the sweat test, used to help

diagnose CF.

In 70% Caucasians the mutation an inframe deletion of 3 base pairs (TTT), which codes for

Phenylalanine at position 508. This deletion disrupts normal processing of the protein, and leads to

degradation of the mutant protein – which means complete absence of CFTR from the cell membrane, a

severe form of CF with early onset. Other mutations can be less severe and permit relatively normal Cl-

transport. With so many possible mutations, molecular diagnosis is not 100% accurate because tests are

only available for the most common mutations.

Clinical features of the severe disease may present at birth, as meconium ileus, or later in infancy, when

there is decreased pancreatic enzyme secretion due pancreatic duct blockage. This leads to the

malabsorption of fats and presents clinically as a failure to thrive. Recurrent pulmonary infections are

common because the thick and heavy lung mucous is ideal environment for bacteria to thrive, including

Pseudomonas aeruginosa, burkholderia cepacia and Staphylococcus Aureus. Most males are sterile due

to absence of Vas deferens.

Treatment of CF is limited to merely treatment of symptoms – antibiotics for infections, organ

transplant, replacement of pancreatic enzymes and physiotherapy to manually clear mucous from the

respiratory tract. Advances are being made in the area of gene therapy but currently the life expectancy

is 30 years.

Unstable trinucleotide expansions cause genetic disease. Discuss with reference to specific examples.

(2008)

Discuss the inheritance pattern, molecular basis and clinical features of Huntington disease. (08/09

Resit)

Discuss the inheritance pattern, molecular basis and clinical features of Fragile X Syndrome. (2007)

Trinucleotide repeat disorders are a set of genetic disorders caused by the increase in the number of

trinucleotide repeats in certain genes exceeding the normal, stable threshold. They are unstable or

dynamic mutations that change as they are passed down generations. Trinucleotide repeat disorders

demonstrate genetic anticipation – a phenomenon whereby children may have phenotypic expression

of the disease before their parents, because of a greater number of trinucleotide repeats in their

genome.

Huntington’s Disease (HD) is an autosomally dominant neurodegenerative disorder characterised by

choreic movements and dementia. It is caused by expansion of a CAG trinucleotide repeat in the

huntingtin gene (HTT) located on chromosome 4p16.3. Repeat sequences of less than 35repeats are

considered normal and stable. Greater than 40 copies of the CAG sequence is diagnostic of HD, a disease

which has 100% penetrance. There can be up to 120 copies of the sequence, and the greater the

number of repeats, the earlier the onset of the disease - Juvenile HD occurs with greater than 70

repeats. The repeat sequence is meiotically unstable – and thus the increase in repeat sequence

(anticipation) is greater in spermatogenesis than oogenesis.

The huntingtin gene codes for huntingtin protein (HTT), which is ubiquitously expressed throughout the

body. It is very large and multifunctional, it interacts with over 200 cellular proteins involving numerous

cellular pathways. Although the precise function of HTT is unknown, it has been identified as a critical

protein in normal embryogenesis, and involved in transcription, cellular function and transport and

intracellular signaling.

In the disease pathogenesis of HD the mutant form of HTT, mHTT, increases the rate of decay of

neurons, in particular the Caudate nucleus of the basal ganglia. This area plays a key role in movement

and behaviour control, and death of neurones in this area explains the chorea associated with HD. The

exact mechanism of brain injury is still unknown, but the mutant HTT is a ‘gain of function’ mutation

that impairs normal HTT function. It is thought protein mis-folding plays a role in disease pathogenesis,

where HTT polyglutamine expansions are cleaved and translocated into the nucleus where they form

inclusion bodies and interfere with transcription factors, perturbing gene expression and initiating

neurodegeneration. The presence of mHTT causes cell loss by a combination of apoptosis and

impairment of normal energy metabolism.

Clinical features of HD usually begin with clumsiness, hyperreflexia, eye movement disturbances, and

gradually progress to choreic movement. Psychiatric features usually present as dementia, and affected

individuals invariably progress to a state of total dependence. The average age of onset is 40 years, but

with genetic anticipation can arise earlier. The disease has a low rate of new mutation, and it is rarely

seen in non-familial cases, affecting 4-7/100,000. The condition is not curable, but treatments to

decrease the severity of the symptoms are often employed – eg. drugs to reduce chorea and psychiatric

problems. Physical and speech therapy are also used in the early stages of disease progression.

Myotonic Dystrophy (Dystrophia Myotonica, DM1) is a common muscular dystrophy affecting adults,

affecting 1/8000. DM1 is an autosomal dominant disorder resulting from the expansion of a CTG

trinucleotide repeat in the dystrophia myotonica protein kinase gene (DMPK) on Ch19q13.2-q13.3.

Unaffected individuals have 5-37 copies, affected have >50 and congenital forms of the disease have

>1000 repeats. The extent of the expansion correlates with the severity of the disease. The repeat

sequence is most unstable during oogenesis, so the congenital form is usually passed from mother to

child.

The DMPK protein is predominantly localized at sites of neuromuscular and myotendinous junctions of

skeletal muscles. In patients with myotonic dystrophy the levels of detectable DMPK in skeletal and

cardiac muscle are much lower than in unaffected individuals. The CTG repeat sequence leads to a

disruption in RNA metabolism, expansion of the CUG RNA sequence, leading to misregulation of splicing

events of DMTK RNA. The expanded transcripts accumulate in the nucleus as ribonuclear inclusions. The

abnormal mRNA molecules produce altered levels of proteins- decreased muscle bind protein (usually

favoured in adults) and increased CTG-binding protein (usually present in foetus). Therefore the normal

foetal-adult splicing switch does not occur. Altered RNA processing events occur in the insulin receptors,

cardiac troponin T and chloride channels, correlating to the symptoms of DM1 – muscle myotonia

(hyperexcitability) and muscular wasting, insulin resistance and diabetes and cardiomyopathies. Other

associated symptoms include cataracts, testicular atrophy and frontal baldness.

DM1 has variable expression and anticipation.

DM2 also occurs as a repeat disorder, with expansion of the tetranucleotide CCTG, located in the ZNF9

gene on Ch3q. This is a DNA binding protein, and unrelated to DMTK, but the pathogenesis of DM1 and

DM2 are thought to be the same – repeat of RNA CCUG causes loss of the foetal-adult splicing switch,

leading to muscle degeneration. It is considered a milder form of dystrophy with no congenital form.

Fragile X Syndrome (FX) is a common, X-linked dominant disorder, affecting 1/4000 males and 1/8000

females. It is the most common form of mental retardation. Fragile X syndrome results from the

expansion of a CGG trinucleotide repeat in the FMR1 gene which encodes the FMRP protein on Xq27.3.

Normal number of repeats is 5-50 (most people have around 30). These people will have normal FMR

Protein levels and no clinical manifestations. The expansion of the mutation is a multistep process.

Premutation repeats of 50-200 leads to increased transcription and therefore increased expanded CGG

repeat mRNA. This abnormal mRNA causes cellular protein interactions leading to mRNA toxicity, with

decreased amount of FMRP produced. Depending on the size of the repeat sequence the individual can

have no noticeable symptoms, and be functionally unaffected, but a carrier of permutation.

Alternatively with greater number of repeats, it can lead to Premutation disorders including FX-

Associated Tremor/Ataxia Syndrome or Primary Ovarian Insufficiency, or Premutation-to-Full Mutation

spectrum disorders such as developmental delays, ADHD and Autism spectrum disorders.

Premutations tend to expand when maternally transmitted (during oogenesis). Spermatogenesis cannot

maintain the full mutation and as such it contracts the mutation to premutation size. Therefore,

mothers are more likely to pass on the full mutation to their sons, and fathers will not transmit the full

mutation to their daughters. (X linked – fathers cannot pass it on to sons).

Further expansion of the sequence greater than 200 repeats manifests as the full mutation, Fragile X

Syndrome. In this case, the gene sequence undergoes hypermethylation and gene silencing, so

reduced/absent transcription of sequence occurs. With reduced/absent mRNA there is reduced/absent

FMRP. The function of FMRP is not yet definitively known, but it is a cytoplasmic protein associated with

polyribosomes to control local protein synthesis by RNA binding. FX therefore dysregulates critical target

proteins involved in normal neuronal functions.

Fragile X Syndrome is more severe in males. Clinical features include moderate-severe mental

retardation, macro-orchidism with characteristic physical features of tall stature, large head, with long

narrow face and prominent ears. In females there are no distinguishing physical characteristics, 30%

carriers have mild mental retardation and behavioural phenotype shyness and poor eye contact is

sometimes the only indication of the disorder.

Explain what is meant by genomic imprinting and discuss why it is an important factor in some genetic

diseases. (2007)

Illustrate the relevance of genomic imprinting to genetic disease. (2009)

Genomic imprinting is a genetic phenomenon whereby certain genes are expressed exclusively (or

predominantly) by either maternal or paternal chromosomes. ‘Imprinted genes’ are these genes that

undergo allele specific gene expression depending on the parent of origin. These instructions are laid

down in the parental genome, before fertilisation occurs.

Generally paternally expressed genes (PEGS) are growth promoters, eg. IGF-2, and maternally expressed

genes (MEGS) are growth suppressors, eg. H19. This is the basis for Haig’s parental tug-of-war

hypothesis, which states fathers promote growth of big offspring for better survival, and mothers

promote smaller, more easily carried offspring.

Imprinting is evidenced in Angelman’s Syndrome (AS) and Prader-Willi Syndrome (PWS). These disorders

are homoalleic for 15q11-13, but have different phenotypes. In AS, the maternal chromosome 15 is

mutated and expressed (therefore, the paternal gene is ‘imprinted’ or inactivated). PWS is as a result of

mutation of the paternal Ch15. The gene deficiency may be as a result of chromosomal deletions,

uniparental disomy, point mutation or as a small deletion in the imprinting centres. Most occur from

cytogenic interstitial deletions on Ch15.

AS is characterised by profound intellectual disability; absent speech; microcephaly; ‘puppet-like’ gait

and facial features, paroxysmal laughter and seizures. The gene affected is UBE3A, which encodes a

protein in ubiquitin-mediated protein degration during brain development. The deletion removes the

single active copy of this gene. In PWS, the deficiency in SNRPN gene, which encodes a small nuclear

riboprotein in the brain, leads to dysmorphic mental retardation, hypogonadism, obesity and polyphagia

and a short stature. Usually the deletion is sporadic and has an occurance rate of 1/15,000.

Beckwith Wiedemann Syndrome (BWS) is also an imprinting disorder. Usually the maternal copy of

11p15.5 is imprinted (inactivated), and it is solely expressed by the paternal chromosome. It encodes

growth promoter IGF-2, an insulin growth factor gene. BWS can be caused by paternal trisomy of

11p15.5, paternal disomy of 11, paternal duplication of 11p15.5 region, translocations involving the

maternal 11p15.5 region, allowing it to escape imprinting, or loss of imprinting for any other reason.

Fundamentally the cause is double gene dosage of IGF-2 , leading to a range of clinical manifestations,

including pre- and post-natal overgrowth, visceromegaly, omphalocele and hypoglycaemia. Affected

individuals are at a 1000x increased risk of childhood tumours, and increased risk of colorectal cancer in

later life.

Explain how target DNA sequences are amplified in the polymerase chain reaction (PCR). Include in your

answer two clinical applications of PCR. (2008 Resit)

A young boy is suspected to have Duchenne muscular dystrophy. Describe the PCR reaction and how it

might be applied to confirm this diagnosis. (2007)

Describe what is meant by the term ‘microdeletion’, citing two specific examples in your answer. Briefly

describe how fluorescent in situ hybridisation technique is used to confirm the microdeletion in these

cases. (2008)

Polymerase chain reaction (PCR) is a technique used in molecular diagnostics to amplify a specific region

of ‘target’ DNA. It is used to produce many thousand copies of the same DNA sequence for cytogenic

analysis.

A solution is made of

1. DNA sequence

2. Primers - short single-stranded DNA sequences that are synthesized to correspond to the

beginning and ending of the DNA stretch to be copied

3. Taq polymerase enzyme, that moves along the segment of DNA, reading its code and assembling

a copy

4. Excess deoxynucleotide triphosphates to make the polymerase copy.

There are three steps in the PCR, which are repeated for 30-40 cycles. The cycles are done on an

automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture.

Each step - denatauration (alteration of structure), annealing (joining), and extension - takes place at a

different temperature:

1. Denaturation – 95C. The double stranded DNA helix unwinds and opens, leaving 2 single

stranded DNA sequences

2. Annealing – 60C. Primers bind to the single stranded templates.

3. Extension – 72C. The Taq polymerase binds to the primer-DNA pair and begins to form

complementary strands of DNA with the triphosphates, making a double stranded DNA

molecule.

As the cycles are repeated, more and more copies are generated and the number of copies of the

template is increased exponentially.

The DNA sequences obtained by PCR can then be used in a wide range of clinical tests including genetic

fingerprinting, paternity testing, detection of viral and genetic diseases, genotyping of specific

mutations, analysis of gene expression (RT-PCR) and gene cloning. PCR produces a lot of genetic material

from a small sample of DNA, which can be used further in diagnosis.

DNA electrophoresis separates DNA strands on the basis of size – DNA fragments, created by PCR, are

placed in a matrix of agarose/polyacrylamide, and the negatively charged phosphates in the DNA

backbone are attracted to the positively charged monitor and so move towards it. Smaller pieces of DNA

move faster than larger and are thus separated. The size/presence of strands are judged by running

them beside a standard ladder of known lengths. Deletions are detected when the band present in the

amplified normal (control) DNA is absent from the patient DNA.

A microdeletion syndrome is a syndrome caused by a chromosomal deletion spanning several genes that

is too small to be detected under the microscope using conventional cytogenic methods, ie.

Karyotyping. DiGeorge Syndrome (22q11.2) and Duchenne Muscular Dystrophy (Xp21.2) are two

examples.

Fluorescent in situ hybridisation is a molecular diagnostic technique used to detect and localise the

presence or absence of specific DNA sequences on chromosomes. The DNA material must be in the form

of a metaphase spread (where the chromosomes are actively dividing) or as interphase nuclei if

culturing is not possible (eg, amniotic cells). Labelled probes (eg. Labelled with digoxigenin), specific for

the area of interest allow multicolour analysis of the area. A detection system is also required (usually

fluorescent microscopy).

Three steps are involved in FISH:

1. Denaturation – heat denatures and opens the DNA helix

2. Hybridisation – the labelled probe (Cosmid/Alpha satellite/Painting probe) moves in and binds

to the target sequence.

3. Detection – visualised fluorescently using eg. Rhodamine anti-dig

In DiGeorge syndrome, there is a microdeletion in 22q11.2, which includes deletion of TUPLE1

sequence. A green probe targeted at this sequence, along with a red probe for 22q13 (as a control) are

mixed with the DNA material. Under fluorescent light, if there is no deletion, both red and green probes

should be visible, indicating they have hybridised DNA sequence. However, if there is a microdeletion at

22q11.2, no green light will be visible, because the DNA sequence is missing – it has been deleted. The

same technique is applied to DMD diagnosis, with a probe targeted at the Dystrophin gene on Xp21.2.

QFPCR stands for quantitative fluorescence polymerase chain reaction. Small sections (markers) of DNA

from the sample are amplified, labelled with fluorescent tags and the amounts measured by

electrophoresis. It relies on the amplification of short tandem repeats (STRs) – which are short

sequences of bases repeated a number of times – everybody has a different set (one from paternal and

one from maternal chromosomes). These repeats can be differentiated from each other on the basis of

length of repeat. It is used to detect trisomy (13,18,21) from amniotic DNA sample – if there are 3 copies

of 21 of different lengths, there will be 3 spikes on the graph instead of 2, which is diagnostic of trisomy.

Discuss the potential role of pharmacogenomics in the future development of efficient drug therapies.

(2009 Resit)

Describe the clinical relevance of mutations in the cytochrome p450 enzymes. Illustrate your answer

with specific examples. (2008)

Pharmacogenomics is the term used to describe the study of heredity as it relates to drug disposition

and action (ie. The way a person’s genome influences the pharmacokinetics and pharmacodynamics of

drugs in their body). They are polygenic traits, with discontinuous variation – there are variable allele

combinations at the same chromosomal locus.

Pharmacogenomics studies the variability in drug response in individuals and populations. Ideally, the

goal of therapeutic medicine is to provide the correct drug at the correct dose for every patient, every

time, maximising efficacy and minimising toxicity.

Many drugs entered the body must be metabolised into their active form by biotransformation. This

usually takes place in the hepatic endoplasmic reticulum (although it can also occur in other tissues – eg

the intestines by ‘first pass metabolism’), and in 75% cases metabolism is by Cytochrome P450 enzymes.

These phase 1 reactions are functionalisation reactions, to expose or introduce a functional group to the

compound, usually by oxidation reactions.

Polymorphisms in the p450 enzymes can lead to altered drug metabolism, either increasing or

decreasing the effect of the drug. The polymorphisms identified are:

Poor Metabolisers (PM), who lack the functional enzyme – homozygous for deficient allele

Intermediate Metabolisers (IM), who are heterozygous for 1 deficient allele

Extensive Metabolisers (EM), who have 2 normal alleles

Ultra Rapid Metabolisers (URM) who have multiple gene copies (increased gene dosage)

An alteration in a P450 enzyme is considered a pharmacokinetic polymorphism, because it changes the

effect the body has on the drug. Pharmacodynamic polymorphisms are changes in the drug target genes

– changes in drug receptors – and so changes the effect the drug has on the body.

CYP450 polymorphisms are clinically very important to ensure minimal toxicity and maximum efficacy

for the patient. A CYP2C9 polymorphism resulting in a poor metaboliser phenotype will experience

enhanced effect of Warfarin and Phenitoin. This is because the reduced metabolism of warfarin extends

the half life of the drug and therefore there is more active drug bioavailable in the plasma. Warfarin

inhibits VKORC1 enzyme to inhibit Vitamin K recycling to its active form. Because VitK is essential for the

synthesis of clotting factors, warfarin inhibits their synthesis and as such acts as an anticoagulant.

Increased plasma warfarin by either polymorphism in CYP2C9, or in the VKORC1 receptor will mean

increased plasma anticoagulants, which can cause catastrophic haemorrhage. Ideally for this patient, the

dosage should be reduced for therapeutic effect.

Patients should therefore be tested for both polymorphisms before being administered any drug with

documented P450 genetic variability. The goal for the future of pharmacogenomics is to identify the

genotype of the patient, to alter the dosage of drugs as required (ie. Decrease dosage to PMs, and

increase dosage to URMs) to achieve the maximum effect with minimal toxicity.

Describe the principles of multifactorial inheritance with reference to specific examples. Include in your

answer the methods used to disentangle the effects of genes and environment. (2009)

Multifactorial inheritance a form of inheritance where both genetic and environmental factors

determine a trait. It is not attributed to a single gene locus and is therefore considered polygenic. Skin

colour, height and weight are all normal multifactorial traits. These are quantitative traits, where most

people are centred around the mean but some are at extremes (eg. Very tall or very small)

The threshold model is a different form of MF inheritance. It shows an underlying distribution of

susceptibility in the population which follows a normal distribution with most people being unaffected.

Liability, in this case, refers to the sum of genetic and environmental influences that make an individual

more/less likely to express a trait, ie, predisposing alleles or exposure to relevant environmental factors.

Up to a certain point (threshold of liability) a person will not express a trait, but once the threshold is

passed, the trait will appear.

Affected individuals have a

combination of high susceptibility

alleles, combined with an

environmental triggering factor.

Multifactorial inheritance is demonstrated in many congenital and adult syndromes. Neural tube defects

result from defective closure of the developing NT in the first month of development. Defective anterior

neuropore closure results in anencephaly, where the absence of the midbrain, forebrain, membranes,

skull and skin exposes nervous tissue, usually with the child being stillborn. Defective closure in the

lumbar region leads to spina bifida, where the bony spinal arch fails to fuse and there is protrusion of

spinal tissue through the vertebral column. These congenital malformations of the NT have both

environmental and genetic risk factors. Environmentally, folic acid is required for homocysteine

metabolism, and insufficient maternal folic acid will lead to a toxic build up of homocysteine, affecting

the foetus. Genetically, a polymorphism in methylenetetrahydrofolate reductase (MTHFR) resulting in

decreased enzyme activity, will decrease folate levels and increase levels of homocysteine in the blood.

To prevent NTDs, folic acid dietary supplements are taken pre-conception, which has decreased the risk

in NTD-prone families from 4-1% and has decreased the incidence overall by 75%.

To differentiate between the genetic factors and environmental factors in MF inheritance, many studies

are performed on twins – both monozygotic and dizygotic. Monozygotic twins have an identical

genome, so any variability in MF traits is most likely environmental. Of particular importance are studies

on MZ twins that were separated at birth, and were hence exposed to different environments, as this

can help identify specific risk factors for specific diseases.

Other studies use adopted children to differentiate between genetic and environmental factors. Since

children adopted into a family are not genetically related, but have been exposed to the same

environment, it indicates any MF traits are likely to be predominantly caused by genetic factors.

Discuss the current strategies in gene therapy. Illustrate in your answer how different mutations

influence the approach that might be adopted. (2008 Resit). (2009)

Gene therapy is a technique that uses genes to treat or prevent a disease. Somatic gene therapy is

currently the only feasible gene therapy and involves replacing the defective gene in somatic cells only.

The disease is therefore still inherited in future generations, but the disease phenotype is less/not

expressed in the treated individual. Germline gene therapy involves modifying the genome of the sex

cells so the genetic modification is passed on to offspring. This therapy is not currently in use because of

ethical and technical barriers.

There are four gene therapy strategies.

Gene Augmentation therapy is used for diseases caused by loss of function, where the gene product ahs

less or no function. This form of therapy introduces extra copies of the normal gene into somatic cells to

increase gene product to a level where normal phenotype is restored. Research and testing is currently

underway for the use of this therapy in treating many autosomal recessive disorders, including CF.

Suicide Gene therapy is where the gene is directed to target cells and expressed to cause killing of the

cells. It is generally applied to cancer cells. The gene expressed either produces a lethal toxin, or it

synthesises a drug metabolising enzyme which when treated with a nontoxic produg converts the

prodrug to the active form. For example, HSV thymidine Kinase converts ganciclovir from an inactive

prodrug to a cytotoxic agent by phosphorylation.

Targeted Mutation Correction is used in disorders that have a dominant negative mutation (ie where the

mutated product inhibits/interferes with the normal product produced by the normal allele). Because

the mutant product inhibits the action of the normal product, gene augmentation is unlikely to work.

TMC works to correct the mutation or inhibit its expression. This form of therapy could be potentially

useful in the treatment of Marfan Syndrome, to prevent the expression of mutant fibrillin-1.

Targeted Inhibition of Gene Expression is similar to TMC, and is used to prevent the expression of

deleterious gene in gain of function mutations. This is where the mutation causes increased effect of the

gene product, or the gene product gains a new and abnormal function. This therapy seeks to block

expression of the gene at DNA, RNA or protein level, thus blocking the pathogenic gene product.

Write a short note on ectopic pregnancy, including the predisposing factors, clinical presentation,

diagnosis and management. (2007)

An ectopic pregnancy is one where the fertilised ovum is implanted at any site other than the normal

uterine location. It can be implanted anywhere along the reproductive tract; most commonly (90%)

implantation occurs in the fallopian tube, called a tubal pregnancy. Other sites of implantation are in the

ovaries, and rarely the abdominal cavity.

Any factor that stops the passage of the ovum to the uterus is considered a predisposing factor for an

ectopic pregnancy. This includes pelvic inflammatory disease, peritubal adhesions, endometriosis and

tubal ligation. However 50% of tubal pregnancies occur in normal tubules with no anatomical cause.

Early development of the embryo is relatively normal with the formation of placental tissue and

amniotic sac. Eventually the placental tissue invades the wall of the tube and causes total rupture, which

can lead to an intratubal haematoma or intraperitoneal haemorrhage. An ectopic pregnancy presents

clinically as severe abdominal pain, approximately 6 weeks after the last menstrual period.

Haemorrhage can cause hypotension and cardiogenic shock, and therefore ectopic pregnancies are

considered a medical emergency.

Diagnosis is by serum betaHCG (levels will be lower in an ectopic pregnancy than a normal pregnancy).

An ultrasound will be performed to determine the location, and a laparotomy/laparoscopy performed if

required. The ectopic embryo will be surgically removed, and medical treatment of Methotrexate can

also be administered.

Ectopic pregnancies are always non-viable pregnancies.

‘The stage of development of a human embryo determines its susceptibility to teratogens’. Discuss.

(2008 Resit)

Teratogens are environmental agents which may cause developmental disruptions following maternal

exposure to them. Examples are drugs, radiation, alcohol and teratogens are responsible for 7-10%

congenital abnormalities.

The mechanisms of teratogenesis are obscure and incompletely understood. Three important principles

are identified – the dose of teratogen, the genotype of the embryo and the stage in development at

which the mother is exposed.

The stage of development of a human embryo is an important factor in teratogenesis, but it is not the

sole factor that determines the foetal susceptibility. The most critical periods in human development are

when there is cell division, cell differentiation and morphogenesis taking place. Exposure to a teratogen

in the first two weeks post-fertilisation leads to disturbances in zygote cleavage and implantation. Either

the disruptive effects are compensated for by powerful regulators, or it results in spontaneous abortion.

During the embryonic period (weeks 3-8), tissues and organs are forming and if development is

disrupted at this stage, it will result in major congenital abnormalities in that organ/tissue.

Between weeks 9 and 38, the foetal period, exposure to teratogens may cause physiologic defects,

minor morphological anomalies or functional disturbances (eg mental retardation) in the foetus.

It is evident that early exposure to teratogens causes more serious defects. It is important to note that

there is a dose-response relationship for teratogens – a small amount of exposure may not cause as

great a defect. There are also genetic differences in response to a teratogen, involving the foetal

genotype. This is demonstrated by exposure to Phenytoin, an anticonvulsant. 5-10% exposed foetuses

develop hydantoin syndrome, 33% have some congenital anomalies but over 50% are unaffected. This

indicates that the mere exposure to a teratogen does not necessarily mean all foetuses will be affected

in the same way.

What features characterise pre-eclamptic toxaemia? Briefly discuss the pathogenesis, clinical features

and complications of the condition. (2008 Resit)

Pre-eclamptic toxaemia or pre-eclampsia is a complication of pregnancy, characterised by hypertension,

proteinuria and oedema. It occurs most commonly in the last trimester of pregnancy, often in

primiparous women and occurs in approximately 6% of pregnancies.

The aetiology is not yet known, however, it is thought that an intrinsic defect in the trophoblast causes

altered vascular flow. This leads to placental ischaemia and decreased uteroplacental perfusion. This

stimulates vasoconstrictor substances and inhibits vasodilator molecules (causing the hypertension) and

generalised endothelial dysfunction occurs. PET can progress to eclampsia – a very serious condition

involving seizures and convulsions – if left untreated.

PET usually initially presents as oedema of the ankles and feet, followed by persistent hypertension (at

or above 140/90mmHg) and proteinuria. Patients may also present with headaches, visual disturbances,

and rarely, in severe cases with disseminated intravascular coagulation (DIC) and/or organ damage.

Treatment for mild-moderate forms of PET is bed rest and a suitable antihypertensive agent. For

established PET or eclampsia, the pregnancy must be induced and the baby delivered, because both

mother and baby are at high risk.

Complications that may arise are eclampsia, intrauterine growth reduction (lack of perfusion to foetus),

haemolysis, elevated liver enzymes and low platelets.

Name one congenital infection that may not be apparent at birth. Discuss any measures that are

available to prevent this infection. (2008)

Cytomegalovirus is a herpes virus that infects 40% of the population in Ireland. It is spread through body

fluids and can cross the placenta. In primary infection of a pregnant woman, there is a 1% chance of the

child being infected. 10% of those infected will develop a serious apparent-at-birth infection. Of the

remaining 90% that are asymptomatic at birth, 85% will be normal, and 15% will develop symptoms

later.

[In Ireland, there are around 70,000 births per year, therefore 700 babies infected. 70 will be

symptomatic at birth, 630 non-symptomatic. 90 babies will develop problems later – 70+90=160 babies

infected with CMV per year in Ireland].

Symptoms of infection that are apparent at birth include IUGR, microcephaly, petechiae and eye

defects, as chorioretinitis. Inapparent-at-birth infections may present later in postnatal check-ups, as

sensorineural deafness and /or cognitive impairment and developmental delay.

There is no specific treatment for CMV. In many mothers, the virus is not apparent and non-

symptomatic so they may not even realise they have been infected. Because the virus is quite

ubiquitous, the only prevention is maintaining hygiene. Early detection of affected babies is important to

prevent complications.

[Or]

Rubella is a viral infection that can cross the placenta. If the primary infection occurs in the mother

during the first trimester, the Rubella ‘triad’ of major defects is apparent at birth. This includes cardiac

anomalies, eye defects (cataracts) and ear defects (deafness). The baby is sometimes decribed as a

‘blueberry muffin baby’ because of the extensive petechiae and hepatosplenomegaly.

If the infection occurs in the second or third trimester, the effects are somewhat less serious. Usually

deafness is noticed at postnatal check-ups, and intellectual disability and behavioural problems can also

be attributed to the congenital infection.

Prevention of congenital Rubella is by pre-pregnancy vaccination. The vaccination must be administered

before the pregnancy because it is a live virus. Antenatal testing for maternal serum markers for rubella

IgG and IgM are taken to compare with post-natal samples, if required.

List the risk factors for prematurity and briefly discuss the conditions affecting premature infants. (2008)

(2009 Resit)

Prematurity refers to the physiological state of a newborn that is born pre-term – with less than 37

weeks gestation. It is a major cause of neonatal mortality, accounting for 10% of worldwide neonatal

mortality, and 25% in the US. Premature birth is relatively common, with one in every eight babies born

premature in the USA.

Risk factors for prematurity are

Pre-eclampsia

Hypertension

Age – older than 35 years or younger than 18

Short cervix or uterine malformations that may not be able to carry a full term baby

History of previous premature birth

Maternal diabetes

Infection – UTI, group B Strep, STIs and the TORCH complex

Multiple pregnancies (56% twin pregnancies are born pre-term)

Maternal use of tobacco, alcohol and drugs

Pre-term birth puts infants at an increased risk of complications in numerous organ systems. This is

because the organs need 37 weeks or more in utero to develop properly. If born before they are fully

developed they can be immature and may not function as well.

Neurological complications incurred are mostly as a result of hypoxia. Intracranial haemorrhage can

cause mental retardation and Retinopathy of Prematurity (ROP) can cause scarring and retinal

detachment. Both of these conditions are due to the underdeveloped and weak blood vessels, which are

very sensitive to hypoxia and leak causing haemorrhage into tissues. Hypoxic Ischaemic Encephalopathy

causes stroke and necrosis in the brain leading to developmental delay or profound mental retardation,

depending on the extent of damage. Apnoea of Prematurity and developmental delay are other

neurological complications.

In the cardiovascular system, the ductus arteriousus is a vessel that connects the pulmonary artery to

the aorta to bypass the lungs in intrauterine life. At first breath, the lungs expand and gas exchange in

the lungs decreases pulmonary vascular resistance, so blood flows to the lungs through the pulmonary

artery. The ductus arteriousus usually closes fully within 7-10 days after birth. In premature babies, the

ductus arteriosus remains open, which is known as a Patent Ductus Arteriosus. Failure of the ductus to

close means the pressure will be higher in the systemic circulation which will reverse bloodflow. This will

eventually lead to right sided heart failure.

Respiratory Distress Syndrome is the leading cause of morbidity and mortality in premature babies,

affecting 50% of those born at 24 weeks, and 20% of those born after 32 weeks. Surfactant lines the

alveoli in the lungs and acts to decrease surface tension and therefore keep alveoli open. It is secreted

by type II Pneumocytes, which only differentiate from type I pneumocytes until late pregnancy. With

insufficient surfactant, the alveoli collapse on exhalation and resist expansion on second breath. The

energy required to overcome the cell-cell affinity in alveoli requires considerable effort, which damages

the alveolar lining. This causes an outpouring of fluid and blood into the alveolar lining and airspaces.

Plasma constituents also bind to remaining surfactant which further impairs function. Although the

alveoli are adequately perfused, they are not ventilated, which leads to hypoxia. Hypoxia, as mentioned

previously, is the main cause of neurological damage, and it can also exacerbate a patent ductus

arteriousus by causing a right-left shunt.

Treatment for RDS is by CPAP ventilator (continuous positive airway pressure). These deliver high

quantities of O2 to the newborn. However, the high O2 tensions can lead to O2 toxicity, which is known

as Bronchopulmonary Dysplasia.

Gastrointestinal and Metabolic pathways are also affected in prematurity – problems include

hypoglycaemia, hypocalcaemia, feeding difficulties, rickets of prematurity and inguinal hernia.

Necrotising Enterocolitis is the most common acquired GI emergence encountered in Neonatal ICU.

Ischaemia of the intestinal mucosal (due to hypoxia) leads to bacterial colonisation, usually by

Clostridium Difficile. The lesions vary from pseudomembranous collitus to bowel perforation and

gangrene. It is treated as a medical emergency.

Finally, pre-term babies often experience haematologic complications. Anaemia of prematurity is

encountered because iron stores are usually only built up in late pregnancy, and this can lead to feeding

difficulties and failure to thrive. Jaundice is one of the most common features of premature infants, and

it is due to the liver being immature and deficient in enzyme glucuranyl transferase, which conjugates

bilirubin for excretion. Bilirubin is derived from the destruction of red blood cells and the catabolism of

released haeme, and is toxic in its unconjugated form. Without treatment, hyperbilirubinaemia will lead

to Kernicterus, a condition due to unconjugated bilirubin toxicity in the brain, which leads to lethargy

and death in 75% cases. Fortunately, it can be treated with phototherapy, where UV light converts

bilirubin to less toxic isomers which can be excreted.

List the micro-organisms that may cause an erythematous maculopapular rash. Discuss one of these

with regard to clinical presentation, complications, treatment and prevention. (2009)

A maculopapular rash is an area of redness characterised by macules (spots) and papules (bumps). It can

be caused by a number of viruses; measles, rubella, human parvovirus B19, herpes 6. It can also be

caused by SPE toxin of Streptococcus Pyogenes and as a result of vasculitis in Kawasaki’s disease.

Rubella is a moderately infectious virus which uncommon in the present day due to vaccination. Many

cases can be subclinical, but symptomatic infections present as a mild fever, lymphadenopathy in the

head and neck and as arthralgia in older patients. The maculopapular rash develops within 2-3 days and

fades within 2-5 days of onset. The incubation period for the virus is 17-20 days.

Complications arising from rubella infection include thrombocytopaenia purpura and encephalitis, both

of which are rare. The main complication is infection with rubella during pregnancy, leading to

congenital rubella infection of the foetus. If the infection is incurred in the first trimester, it is most

serious and the child will have the Rubella ‘triad’ of cardiac anomalies, hearing deficit and eye problems

including cataracts. Usually microcephaly and mental retardation also result, and the baby born has the

characteristic ‘blueberry muffin baby’ appearance of hepatosplenomegaly and extensive petechiae.

Infections in the second and third trimester result in less serious defects, most commonly deafness and

developmental delay.

There is no treatment for rubella as it is a viral infection. Prevention in the form of immunisation is

available in the MMR vaccine, which is part of the childhood vaccination schedule in Ireland, and has

greatly reduced the incidence of rubella since its introduction in 1988.