Medical Genetics Practise Questions

A 28-year-old woman (Individual III-2) presents with progressive muscle weakness and exercise intolerance. Her mother (II-2) experienced similar but milder symptoms starting at age 45. Her maternal grandmother (I-1) was unaffected. Two of her maternal aunts show varying degrees of muscle fatigue, while her maternal uncle is completely unaffected. The patient's father and his family have no history of muscle disorders.

Muscle biopsy from III-2 reveals ragged red fibers, and genetic testing identifies a pathogenic mutation in mitochondrial DNA (MT-TL1 gene). Heteroplasmy analysis shows:

• III-2 (patient): 85% mutant mtDNA in muscle, 45% in blood

• II-2 (mother): 60% mutant mtDNA in muscle, 30% in blood

• II-3 (affected aunt): 50% mutant mtDNA in muscle, 25% in blood

• II-4 (mildly affected aunt): 35% mutant mtDNA in muscle, 20% in blood

Discuss the following aspects of this case:

• Explain the inheritance pattern and why only maternal relatives are affected

• Why do different individuals and different tissues show different levels of mutant mtDNA?

• How does heteroplasmy level correlate with disease severity in this family?

• If III-2 has children, what is the likelihood they will be affected, and can we predict severity?

• What role does the threshold effect play in determining who shows symptoms?

Inheritance pattern:

This is a mitochondrial inheritance pattern. Only individuals on the maternal lineage (I-1 → II-2, II-3, II-4 → III-2) are affected. It could be the case that people on the father’s side could be affected but he wouldn’t pass it on. 

Heteroplasmy variation:

Heteroplasmy refers to the coexistence of both wild-type and mutant mtDNA within an individual. The variation occurs through several mechanisms:

• Maternal bottleneck: During oogenesis, the mtDNA population undergoes a dramatic reduction followed by amplification, leading to random drift in mutant load between offspring.

• Tissue-specific distribution: Different tissues have different energetic demands and different replication rates. Muscle tissue (high energy demand, post-mitotic) accumulates mutant mtDNA more readily than blood (proliferating cells may have selection against severely dysfunctional mitochondria).

• Somatic drift: Over time, random partitioning during cell division leads to different heteroplasmy levels in different tissues and different individuals.

Correlation with severity:

There is a clear correlation between heteroplasmy level and disease severity:

• III-2 with 85% mutant mtDNA in muscle has severe, early-onset symptoms (age 28)

• II-2 with 60% mutant mtDNA has moderate symptoms starting later (age 45)

• II-3 and II-4 with 50% and 35% respectively show progressively milder phenotypes

This demonstrates the threshold effect: below ~30-40% mutant mtDNA, cellular respiration is sufficient and symptoms may not manifest. Above this threshold, insufficient wild-type mtDNA leads to energy deficits and symptoms emerge.

Risk to III-2's children:

All of III-2's children will inherit mitochondria exclusively from her, so all will inherit some proportion of mutant mtDNA. However, the exact heteroplasmy level in each child is unpredictable due to the maternal bottleneck effect. With maternal heteroplasmy at 85%, her children are at high risk of inheriting substantial mutant loads, but levels could range from ~40% to near 100%. Severity cannot be precisely predicted but statistical risk is high given the mother's high heteroplasmy.

Prenatal testing of heteroplasmy in fetal tissue or preimplantation genetic diagnosis could provide some information, though heteroplasmy levels may shift during development.

Threshold effect:

The threshold model explains why individuals with similar heteroplasmy can have different outcomes and why tissue-specific manifestations occur. Tissues with high energy demands (muscle, brain, heart) have lower thresholds—they need more functional mitochondria to maintain ATP production. This is why muscle biopsy shows the highest mutant loads and correlates best with symptoms.

A 42-year-old woman of European ancestry (Ms. K) undergoes direct-to-consumer genetic testing and receives a polygenic risk score (PRS) for type 2 diabetes (T2D). Her PRS places her in the 88th percentile compared to the reference population, with an estimated odds ratio of 1.8 for developing T2D compared to the population average.

Background information:

• Her mother developed T2D at age 58

• Her maternal grandmother developed T2D at age 65

• No paternal history of T2D

• Ms. K is overweight (BMI 28), sedentary, and has a diet high in processed foods

• Her HbA1c is currently 5.9% (pre-diabetic range: 5.7-6.4%). HbA1c is a marker of long term glucose control, the higher the less controlled your glucose levels are.

GWAS information provided with her PRS:

• Her PRS is calculated from 500 SNPs identified through GWAS meta-analyses. The variants include:

  • TCF7L2 rs7903146 (OR = 1.37 per T allele; Ms. K is CT)

  • FTO rs9939609 (OR = 1.18 per A allele; Ms. K is AA)

  • PPARG rs1801282 (OR = 0.86 per G allele; Ms. K is CC, no protective alleles)

  • 497 other SNPs with individual ORs ranging from 1.02 to 1.15

• Ms. K wants to understand: "Does this mean I will get diabetes? Should I tell my sister to get tested?"

Discuss the following:

• Explain what a polygenic risk score represents and how it differs from monogenic disease risk

• Calculate Ms. K's approximate relative genetic risk from the three major SNPs listed (show your working hint - when we assume indiepnende, we just multiply the odds ratios together)

• How do environmental factors modify her genetic risk, and what is her absolute risk?

• What are the limitations of applying this PRS to Ms. K?

• What advice would you give Ms. K about prevention and screening?

What PRS represents:
A polygenic risk score aggregates the effects of many common genetic variants (SNPs), each contributing a small effect to disease risk. Unlike monogenic diseases where a single mutation causes disease (e.g., cystic fibrosis: CFTR mutation → disease), T2D is polygenic (many genes) and multifactorial (genes + environment).

Key differences:

• Monogenic: Genotype ≈ phenotype (predictable, high penetrance)

• Polygenic: Genotype → probability of phenotype (uncertain, requires environmental triggers)

Ms. K's PRS of 88th percentile means she carries more T2D risk alleles than 88% of the reference population. This shifts her position on the liability curve toward the disease threshold, but doesn't guarantee disease. Her actual outcome depends on whether her combined genetic + environmental load exceeds the threshold.

Relative genetic risk calculation:

For the three major SNPs:

• TCF7L2 rs7903146: Ms. K is CT so has 1 copy of T allele, so OR = 1.37

• FTO rs9939609: Ms. K is AA so 2 copies of A allele so OR = 1.18² = 1.39

• PPARG rs1801282: Ms. K is CC so 0 protective G alleles so OR = 1.00 (no effect)

Assuming SNPs act independently (multiplicative model):

Combined OR = 1.37 1.39 1.00 = 1.90

This approximates the overall PRS-derived OR of 1.8, suggesting these three SNPs contribute substantially to her genetic risk, with the remaining 497 SNPs adding smaller cumulative effects.

Environmental factors and absolute risk:

Ms. K's absolute risk combines genetic and environmental contributions:

1. Baseline population risk: ~10% of European women develop T2D by age 70

2. Genetic risk: OR = 1.8 increases risk to approximately 10% × 1.8 = 18% genetic component

3. Environmental risk factors:

• Overweight (BMI 28): OR ~2.0

• Sedentary lifestyle: OR ~1.3

• Poor diet: OR ~1.2

• Pre-diabetic HbA1c (5.9%): Indicates already impaired glucose regulation

(All this is from the literature and not expected BTW)

If we just used a simple multiplicative model the combined risk would be 18% 2.0 1.3 * 1.2 = ~56%

This is a rough estimate and actual risk calculation would use more sophisticated models accounting for interactions and age, etc. However, it illustrates that Ms. K's environmental factors amplify her genetic predisposition substantially. Someone with the same PRS but healthy lifestyle might have only ~20-25% lifetime risk.

Limitations of this PRS:

1. Population-specific: GWAS and PRS are derived primarily from European ancestry populations. Linkage disequilibrium (LD) patterns differ between ancestries, so the same SNPs may have different predictive power in African, Asian, or admixed populations. Ms. K is European, so this PRS is appropriate, but her sister should confirm her ancestry before using the same test.

2. Incomplete heritability: Twin studies estimate T2D heritability at ~70%, but GWAS-derived PRS typically explains only ~10-20% of variance. This "missing heritability" reflects:

• Rare variants not captured by SNP arrays

• Gene-gene and gene-environment interactions

• Epigenetic factors

• Structural variants

3. Probabilistic, not deterministic: An OR of 1.8 means 1.8× average risk, but individual outcomes vary enormously. Some in the 88th percentile never develop T2D; some in lower percentiles do.

4. Environment dominates: Unlike high-penetrance monogenic diseases, T2D is highly preventable through lifestyle modification. Genetic risk can be largely offset by maintaining healthy weight, diet, and exercise.

5. Does not replace clinical assessment: Her HbA1c of 5.9% is a stronger near-term predictor than PRS. Clinical factors (age, BMI, family history, glycemic control) should guide management.

Advice for Ms. K:

• Prevention: Ms. K's genetic risk is modifiable. Evidence shows that lifestyle intervention (weight loss, exercise, dietary changes) reduces T2D incidence by ~58%, even in high-risk individuals. Genetic risk does not equate to inevitability.

• Specific actions:

  • Weight loss goal: Even 5-7% body weight reduction significantly lowers risk

  • Exercise: 150 minutes/week moderate activity (e.g., brisk walking)

  • Dietary changes: Reduce processed foods, increase fiber, control portions

  • Her HbA1c is pre-diabetic → reversible with intervention

• Monitoring: Given her PRS, pre-diabetic HbA1c, and family history, she should have HbA1c checked annually. Earlier detection allows earlier intervention.

• Family communication: Ms. K's sister shares ~50% of genetic variants. The sister likely has elevated genetic risk, but PRS is not perfectly heritable (depends on which parental alleles were inherited). Encourage the sister to:

  • Consider testing if interested

  • Focus on modifiable risk factors (which benefit everyone regardless of genetics)

  • Discuss family history with her physician

• Psychological impact: Reassure Ms. K that this is risk information, not a diagnosis. Many people with high PRS never develop T2D. Emphasize empowerment: she has actionable steps to reduce risk substantially.

• Clinical integration: PRS should complement, not replace, standard clinical risk assessment (family history, BMI, HbA1c, blood pressure). Her physician can integrate PRS into a comprehensive prevention plan.

A clinical geneticist evaluates a 35-year-old man (Individual II-1) presenting with multiple café-au-lait spots limited to his right arm and trunk on the right side of his body. No other features of neurofibromatosis type 1 (NF1) are present. His 8-year-old daughter (III-1) has been diagnosed with classical NF1, presenting with multiple café-au-lait spots distributed across her entire body, axillary freckling, and several cutaneous neurofibromas.

Genetic testing results:

• II-1 (father): Blood DNA sequencing shows no NF1 mutation. Deep sequencing of DNA from a skin biopsy from affected skin (right arm) reveals a pathogenic NF1 frameshift mutation present in ~18% of reads. Unaffected skin (left arm) shows <1% mutant reads (technical background).

• III-1 (daughter): Blood DNA shows the same NF1 mutation as found in her father's affected skin, present in ~50% of reads consistent with heterozygosity in all cells.

Discuss the following:

• Explain why Individual II-1 has localised features while his daughter has generalised disease

• At what developmental stage did the mutation likely arise in II-1? Use the distribution of features to support your answer

• How does mosaicism explain the apparent "incomplete penetrance" in the father?

• Calculate the approximate percentage of II-1's germline cells that carry the mutation, based on transmission to III-1

• Why is genetic counseling different for II-1 compared to someone with constitutional (non-mosaic) NF1?

Localised vs generalised disease:
Individual II-1 has somatic mosaicism—the NF1 mutation arose post-zygotically during early development, affecting only a subset of cells. The localised distribution (right arm and trunk) suggests the mutation occurred in a progenitor cell that gave rise to tissues on that side of the body. Because only ~18% of cells in affected skin carry the mutation, the disease burden is insufficient to cause neurofibromas or systemic features—just café-au-lait spots in that region.

In contrast, III-1 inherited the mutation constitutionally (present in the fertilizing sperm), so every cell in her body carries the mutation (~50% of reads reflects heterozygosity). This leads to generalised, classical NF1 with features across her entire body.

Developmental timing:
The localised, unilateral distribution suggests the mutation occurred during early embryogenesis, likely during gastrulation or early organogenesis (approximately weeks 2-4 of development). At this stage:

• Cells are still migrating and establishing body axes

• A mutation in a single progenitor cell would affect all descendants of that cell

• The right-sided distribution indicates the mutation occurred in a cell contributing to the right side of the embryo before left-right patterning was complete

• If it occurred earlier (at the 2-4 cell stage), distribution would be more extensive; if later (after organogenesis), the patch would be smaller and more localised

The relatively large affected area (~25% of body surface, estimated) is consistent with a mutation arising when there were ~100-1000 cells in the embryo..

Mosaicism and "incomplete penetrance":
Individual II-1 would appear to have incomplete penetrance if we only considered family history without molecular analysis:

• His daughter is affected with classical NF1

• He appears "unaffected" or only mildly affected

• This would suggest he carries the mutation but doesn't express it (reduced penetrance)

However, molecular testing reveals he is a carrier, but only in a mosaic pattern. The mutation is present but below the threshold needed for full clinical manifestation. This illustrates how mosaicism can masquerade as reduced penetrance—once we identify the mosaic genotype, we understand the parent does carry the mutation, just not in all cells.

Germline mosaicism calculation:
Since III-1 inherited the mutation, at least some of II-1's germline cells must be mutant. With 50% of reads in III-1's blood (heterozygous), we know she inherited one mutant allele. If we assume:

• Transmission follows Mendelian ratios from mosaic germline

• The mutation is present in a proportion p of II-1's sperm

The risk of transmission ≈ p × 50% (if sperm is mutant, 50% chance of transmission).

We only have one affected child, which limits statistical power. However, empirical data on gonadal mosaicism in NF1 suggests that when somatic mosaicism is detected, germline mosaicism levels can range from 1% to >50%. Given that II-1 successfully transmitted the mutation, a rough estimate would be 20-40% of germline cells carry the mutation (though this is uncertain without testing multiple offspring or sperm samples).

More precisely: if we detected 18% mosaicism in somatic tissue (skin), germline levels could be similar, but are often different due to independent clonal expansion during gametogenesis.

Genetic counseling implications:
Counseling for II-1 is substantially different from constitutional NF1:

1. Recurrence risk: Unlike constitutional NF1 (50% transmission), II-1's risk depends on germline mosaicism level. Based on one affected child, empirical risk for subsequent children is approximately 20-30% (lower than 50%, but not negligible).

2. Personal health: II-1's personal risk for NF1 complications (optic gliomas, malignant peripheral nerve sheath tumors) is much lower than classical NF1 because most of his body lacks the mutation. However, he may develop complications in affected tissues.

3. Future children: Each pregnancy carries independent risk. Unlike constitutional NF1 (where risk is constant 50%), II-1's risk depends on germline composition, which might evolve (clonal expansion of mutant germ cells could potentially increase risk over time, though this is theoretical).

4. Prenatal testing considerations: If II-1 and his partner wish to pursue prenatal testing for future pregnancies, the recurrence risk is meaningful enough to justify testing, unlike truly sporadic de novo cases.

5. Other family members: II-1's parents and siblings are not at increased risk (his mutation is de novo and mosaic). This differs from someone with constitutional NF1, whose siblings would have 50% risk if the parent were affected.

1. A single-nucleotide substitution in the LRRK2 gene replaces glycine with serine at amino-acid position 2019 (LRRK2 G2019S). The mutation increases the kinase activity of LRRK2 and is associated with autosomal dominant Parkinson’s disease. You would like to generate a Drosophila model to investigate how this mutation affects dopaminergic neuron function.

1. Design a strategy to produce a Drosophila model in which the effect of the LRRK2 G2019S mutation can be examined specifically in dopaminergic neurons.

Because the G2019S substitution increases LRRK2 kinase activity, a suitable strategy is to generate a transgenic Drosophila line expressing the mutant LRRK2 gene (human or fly version).To restrict expression to dopaminergic neurons, the transgene should be placed under the control of regulatory elements that are active specifically in these cells. In Drosophila, the regulatory region of the tyrosine hydroxylase gene (TH) is commonly used because it drives expression in dopaminergic neurons.

An alternative is to use the GAL4/UAS binary expression system, which is widely used in Drosophila. The mutant LRRK2 gene can be cloned downstream of UAS, and this UAS‑LRRK2 G2019S line can then be crossed to a TH‑GAL4 driver line. Progeny carrying both transgenes will express the mutant protein specifically in dopaminergic neurons. This approach also allows the same UAS‑LRRK2 G2019S line to be combined with other GAL4 drivers to test effects in different tissues or developmental stages, and avoids any issues with reduced viability or reproductive capability of flies expressing the mutant LRRK2 gene. 

To generate the transgenic TH-LRRK2 G2019S or UAS‑LRRK2 G2019S line, the construct can be integrated into the fly genome via P-element-mediated integration. (not covered how exactly this works in the lectures, but look it up if you are interested!). 

1. Flies generated using your strategy show impaired climbing ability compared with wild-type flies. You decide to test a panel of candidate drugs to see whether any improve this locomotor phenotype. Discuss the advantages and disadvantages of using your Drosophila model for this type of drug-testing application compared with using a mouse model.

Using the Drosophila model for drug testing offers several advantages, but also important limitations when compared with a mouse model. Fly disease models can be generated quickly and easily, are inexpensive to maintain, and breed rapidly, allowing large numbers of animals to be tested. This makes it feasible to examine many compounds or multiple doses in a short period of time. The locomotor phenotype can be measured using the negative geotaxis (climbing) assay, which is simple, rapid, and scalable. The short lifespan of flies also allows drug effects on age‑related phenotypes to be assessed within days or weeks rather than months, and ethical considerations and costs are substantially lower than for vertebrate models.

However, there are important disadvantages. Mouse models more closely replicate human dopaminergic neuron biology, brain circuitry, and drug metabolism, meaning that results obtained in mice are generally more translatable to human disease. Mice also allow assessment of more complex behavioural and pathological features of Parkinson’s disease than is possible in flies.

Because of these strengths and limitations, a practical strategy is to use Drosophila for initial large‑scale screening of candidate compounds, and then test promising hits in a mouse disease model to evaluate their relevance in a mammalian system

1. AMT-130 is an in vivo gene therapy for Huntington’s disease that uses an AAV5 vector to deliver a DNA construct encoding an artificial microRNA designed to reduce expression of the HTT gene. After neurosurgical injection into the striatum, the vector transduces neurons and drives long-term production of the miRNA, which then lowers both mutant and wild-type huntingtin mRNA levels.

Evaluate whether an ex vivo gene therapy approach would have been feasible.

An ex vivo gene therapy strategy for Huntington’s disease would not be feasible because the disease affects post-mitotic neurons deep within the brain. These cells cannot be harvested without causing significant damage, and even if this was possible, transplanted genetically modified cells would be unlikely to make correct connections with other cells. Modifying a small number of neurons would probable not provide a sufficient therapeutic effect.

1. Discuss the strategic design choices made (including choice of delivery vector, delivery of DNA construct instead of RNA delivery, miRNA instead of siRNA, injection into striatum). 

AAV5 was chosen as the delivery vector because it efficiently transduces neurons and supports long‑term, stable expression from episomal DNA. Among AAV serotypes, AAV5 shows strong tropism for striatal neurons and spreads well within brain tissue after stereotactic injection. Although non‑viral vectors generally pose fewer safety risks, they have extremely poor transfection efficiency in neurons and cannot provide durable expression, making them unsuitable for a therapy that must suppress HTT for years.

Direct injection into the striatum is required because AAV5 does not cross the blood–brain barrier. Systemically deliverable vectors such as AAV9 can cross the BBB, but they distribute widely throughout the CNS and peripheral tissues, which would cause off‑target huntingtin suppression and increase toxicity. Stereotactic delivery ensures high local vector concentration in the most affected brain region while limiting systemic exposure.

Delivering a DNA construct rather than RNA enables long‑term production of the therapeutic microRNA. RNA‑based therapies would require repeated administration, which is impractical and risky given the need for neurosurgical delivery. A DNA cassette allows sustained expression from a single procedure.

A miRNA scaffold was chosen instead of siRNA because siRNAs expressed from DNA can overwhelm the RNAi machinery and cause cellular toxicity. Artificial miRNAs are processed through endogenous miRNA pathways, improving tolerability and allowing stable expression. The miRNA used in AMT‑130 is engineered to selectively target HTT mRNA.

AMT‑130 reduces both mutant and wild‑type huntingtin. Although loss of wild‑type HTT carries risks due to its roles in vesicle trafficking and neuronal survival, designing allele‑specific therapies is challenging because the mutant and wild‑type transcripts differ only by CAG repeat length. 

(The developers of AMT-130 have not stated publicly which regulatory elements are used for the construct. Enhancers/promoters that either drive ubiquitous expression or neuron-specific expression would be suitable).

1. In some patients with Duchenne Muscular Dystrophy, the causative mutation is a nonsense mutation in exon 23 of the DMD gene. You want to generate a mouse model that reproduces this mutation so that you can test mutation-specific gene therapy approaches.

1. To generate a mouse model carrying the equivalent mutation in the mouse DMD gene, you use the ‘tag-and-exchange’ gene targeting by homologous recombination method. This involves the consecutive use of two targeting vectors to modify the genome of embryonic stem cells.

The first vector looks like this: 

Selection after the first step occurs with Geneticin (G418). Design a second vector that can be used to achieve the desired genetic change (selection after the use of the second vector would be with Ganciclovir). 

1. Is random integration of vector 1 or vector 2 an issue using this strategy?

If random integration occurs in step 2, cells will be killed by Ganciclovir treatment.

However, if random integration occurs in step 1, and homologous recombination between the randomly integrated vector 1 and vector 2 in step 2, then these cells would survive both the positive selection after step 1, and the negative selection after step 2. So random integration of vector 1 can be problematic. 

1. Suggest how you could test if ES cells carry the correct modification. 

You could carry out PCR of a region that includes the mutation and a region outside of the region of homology and then sequence the PCR product to test for the presence of the mutation. This would confirm that the mutation is in the correct genomic location. 

You would like to test an in vivo gene therapy approach that leads to the excision of exon 23 using CRISPR-Cas9, which should restore the reading frame of the DMD gene. Which viral vector would be suitable for delivery to muscle, and what expression cassette(s) would it need to carry?

AAV9 is a suitable viral vector because it efficiently transduces skeletal and cardiac muscle in vivo in the mouse. To excise exon 23 with CRISPR–Cas9, the vector must deliver a Cas9 nuclease together with two guide RNAs that cut in the introns flanking exon 23. Because AAV has a limited packaging capacity, a smaller Cas9 such as SaCas9 can be used so that Cas9 and both sgRNAs fit into a single vector. Cas9 should be expressed from a muscle‑specific promoter to restrict nuclease activity to muscle tissue, while each guide RNA is driven by a Pol III promoter such as U6. This combination allows CRISPR‑mediated deletion of exon 23.