L1 Genetics Genetic Tests - Clinical applications

Overview

This lecture sets out when and how genetic tests are used in the clinic, and what can be done with the result. It begins with the sheer scale of human genetic variation and the problem this creates for interpretation (the significance spectrum, and the variant of uncertain significance in particular), then compares the available testing modalities by scope and cost, and sets out the three pillars of clinical utility: diagnosis and prognosis, reproductive decisions, and therapeutic intervention. Inherited retinal disorders, principally retinitis pigmentosa, are used as the anchor case throughout, tracked from the microarray era of the 2009 Auckland audit, through next-generation sequencing panels tailored to the New Zealand population in 2017, to Luxturna gene therapy. The closing synthesis frames genetic testing as a four-phase end-to-end clinical workflow rather than a standalone laboratory service.

Module and tutorial context

The genetics vertical module (MBChB Year 3) runs as follows:

• L1 14th April, Genetic Tests: Clinical applications (Prof Hughes)
• L2 17th April, Genetics of Autism (Prof Robertson)
• Tutorial 8, Genetic testing in an eye clinic
• L3 22nd April, Heritability in disorders of metabolism (Prof Hughes)
• L4 24th April, Genetics of Diabetes and Obesity (Prof Hughes)
• L5 1st May, Direct to consumer genetic tests (Prof Hughes)
• L6 11th May, Genome damage and repair: radiation, free radicals (Prof Bicknell)
• Tutorial 9, Genetics in Obesity and Diabetes
• Tutorial 10, Whole genome analysis
• Tutorial 11, Polycystic Kidney Disease and Direct to Consumer Genetic Testing

Core objectives in clinical genetics (the three aims of this lecture):

1. Indications and risks: understanding when to deploy genetic tests, weighing the clinical benefits against the complexities of identifying variations.
2. Diagnostic modalities: outlining the characteristics, scope, and limitations of modern sequencing tests used for inherited conditions.
3. Applied interventions: translating molecular diagnoses into actionable clinical utility, using inherited retinal disorders as the primary anchor case.

Tutorial 8, Genetic Testing: Types and Clinical Uses. Intended to revise the principles of the genetics of monogenic disease, with later teaching relating to complex genetic disorders; the cases chosen relate to the nervous system module and to ongoing developments in genetic diagnosis and treatment. Outcomes: outline the types of indications, risks and benefits of testing for specific variations, and outline the characteristics of tests used for genetic conditions. Preparation is to read Pradhan et al. (2009), “An audit of genetic testing in diagnosis of inherited retinal disorders: a prerequisite for gene-specific intervention”, Clinical and Experimental Ophthalmology 37: 703-711. An MCQ pretest on Moodle precedes all genetics tutorials in ELM3. [tutorial handout screenshot is truncated at the lower edge; any content below the MCQ paragraph is not visible]

The scale of human variation

The problem genetic testing has to solve is one of filtering: a very large number of variants per person, of which only a handful are clinically relevant. The lecture presents this as an inverted funnel, narrowing tier by tier:

• ~20,000 protein-coding genes, roughly 1% of the genome
• 25,000 to 50,000 rare variants per individual, mostly variants of uncertain significance
• ~10,000 non-synonymous variations across 5,000 genes per individual
• 50 to 100 disease-associated variants, 3 to 7 CNVs, and ~75 de novo variations

Allele frequency versus effect size. Variants can be placed on a plot of allele frequency (very rare, rare 0.001, low frequency 0.005, common 0.05) against effect size (low 1.1, modest 1.5, intermediate 3.0, high 50.0). The occupied territory runs along a descending diagonal:

• Rare alleles causing Mendelian disease: low frequency, high effect size.
• Low-frequency variants of intermediate effect: the middle of the diagonal.
• Common variants implicated in common disease by genome-wide association: high frequency, low effect size.
• Common variants of high effect (top right) barely exist; few examples are known.
• Rare variants of small effect (bottom left) are very hard to identify by genetic means.

Categorising the code: the significance spectrum

Variants are classified along a five-band spectrum, in order:

Benign → Likely benign → Variant of Uncertain Significance (VUS) → Likely pathogenic → Pathogenic

The Variant of Uncertain Significance is the primary diagnostic hurdle: a variant is identified, but its exact impact on disease risk remains clinically undefined.

Key classifications of variation:

• SNPs (single nucleotide polymorphisms): present at greater than 1% population frequency.
• CNVs (copy number variations): larger insertions and deletions.

Worked database examples (all in CFTR, illustrating three points on the spectrum):

Variant	Condition listed	Classification
NM_000492.3(CFTR):c.2424T>C (p.Tyr808=), Chr7:117232645 (GRCh37)	not specified	Likely benign
NM_000492.3(CFTR):c.2374C>G (p.Arg792Gly), Chr7:117232595 (GRCh37)	Cystic fibrosis, not specified	Uncertain significance (Apr 13, 2016)
NM_000482.3(CFTR):c.830G>A (p.Trp277Ter), Chr7:117176688 (GRCh37)	Cystic fibrosis	Pathogenic (Oct 1, 2013)

Note the pattern: the synonymous change (p.Tyr808=) is likely benign, the missense change (p.Arg792Gly) sits as a VUS, and the nonsense change (p.Trp277Ter) is pathogenic.

The genetic diagnostic toolkit

Test modality	Scope	Estimated cost	Ideal clinical use case
Single gene testing	Specific pathogenic variation	~NZ$100	Known specific conditions (e.g. haemochromatosis)
Targeted gene panel	Panel of variations linked to a disease phenotype	~NZ$300-400	Phenotype with known genetic heterogeneity (e.g. Retinitis pigmentosa)
Whole exome sequencing (WES)	Resequencing the ~2% of the genome coding for proteins	~NZ$400-500	Unidentified variations in an affected child
Whole genome sequencing (WGS)	High-resolution comprehensive analysis	~NZ$1,000	Complex, unsolved diagnostic challenges

The targeted gene panel row is highlighted on the slide as the modality of interest for the retinal-disorder case used later in the lecture.

Adjunct tools:

• Chromosome microarray analysis (aCGH) for CNVs
• Genotyping for risk association
• RNA analysis for oncology

The three pillars of clinical utility

1. Diagnosis and prognosis: establishing a clear genotype/phenotype relationship, to understand disease trajectory and to resolve unusual clinical cases.
2. Reproductive decisions: Pre-implantation genetic diagnosis (PGD) during IVF, and prenatal diagnosis (PND) in post-conception embryos. In PGD, a cell is biopsied from the embryo, the embryo is classified as affected or unaffected on that result, and an unaffected embryo is the one selected for transfer.
3. Therapeutic intervention: the ultimate goal, enabling traditional targeted gene therapies and emerging CRISPR applications.

The economics of clarity

• Specialised genetic testing: mean cost ~NZ$400forthetest,plusNZ$200 for DNA extraction and international courier logistics. Historic turnaround times measured in weeks. [a grey rectangle partially obscures the slide text near “turnaround times measured ___ in weeks”; a word may be hidden]
• Standard radiological imaging: a routine CT or MRI of the brain or orbits ranges from NZ$250toNZ$575, depending on the use of contrast.

Conclusion: selective genetic testing is a highly opportune and cost-effective investigation relative to standard specialised diagnostics, and acts as an essential prerequisite for gene-specific interventions.

The clinical anchor: inherited retinal disorders

• The phenotype: Retinitis pigmentosa (RP) causes severe progressive peripheral vision loss. The paired street-scene photographs contrast a normal full field of view with the RP view, which retains only a small central island of vision with the periphery black, that is, tunnel vision.
• The genotype: extreme genetic and allelic heterogeneity. Small pedigrees often prevent narrowing down the causative gene by inheritance pattern alone.
• Convergence of inheritance modes: autosomal dominant, autosomal recessive, X-linked and mitochondrial (MIDD) inheritance can all produce the same RP phenotype.

The eye cross-section is labelled with sclera, choroid, retina, cornea, pupil, lens, iris, ciliary body and optic nerve, with a magnified inset of the retinal cell layers.

Background from Pradhan et al. (2009)

The lecture reproduces the paper by Monika Pradhan, Ian Hayes and Andrea Vincent (Department of Ophthalmology, University of Auckland; Eye Department and Northern Regional Genetic Service, Auckland District Health Board). A/P Andrea Vincent (Ocular Genetic Clinic, Greenlane, Retina Specialists) leads the following week’s tutorial.

• Background: an exponential increase in genes implicated in inherited retinal disease over the preceding decade, but genetic and phenotypic heterogeneity limited mutation detection. High sequencing cost and long turnaround times meant gene testing was not a viable option, particularly in New Zealand. Advances including microarray-based mutation analysis and not-for-profit laboratories made testing affordable and time-efficient, enabling genetic diagnostics to become an integral component of the work-up for inherited retinal disease.
• Scale of the field: 144 retinal disease-causing genes identified to date, with a further 48 loci or genes mapped (RetNet). For RP specifically, at least 15 causative dominant genes, 15 recessive genes and two X-linked genes have been identified, yet a large number of affected individuals remain without molecular characterisation. For some genes, sequencing is available only in research laboratories, often with relatively low yield. [the BACKGROUND column of the screenshot is cut off mid-sentence at “in addition, only in 50%”; the remainder is not visible]
• Methods: genetic testing for inherited retinal disorders was initiated via the Ocular Genetic Clinic in Auckland two years before the audit; a retrospective audit of that period was carried out.
• Results: 35 probands underwent genetic testing, covering X-linked retinoschisis, Leber congenital amaurosis, retinitis pigmentosa, albinism, achromatopsia, Usher syndrome, Stargardt disease and mitochondrial disease. 54% of tests (19/35) showed a rare variant or pathogenic mutation. Three couples proceeded to investigate the options of prenatal diagnosis and/or pre-implantation genetic diagnosis.
• Conclusion: the introduction of genetic testing, largely via disease arrays, was highly successful at clarifying disease genotype in the cohort. It is a timely and cost-effective investigation that should be elemental to the assessment of inherited retinal disease, and testing in an opportune fashion permits genetic counselling, enables families to make reproductive choices, and might allow the possibility of gene therapy interventions.

The 2009 reality check: microarray limitations

Yield: 19 informative and 16 uninformative tests out of 35, a 54% diagnostic yield.

Clinical diagnosis	Positive	Uninformative	Total
LCA	3	2	5
Usher	3	0	3
MIDD	1	4	5
ADRP	3	3	6
OCA	1	1	2
XL retinoschisis	2	0	2
Stargardt	2	1	3
ADOA	1	1	2
ARRP	0	3	3
CSNB	1	0	1
Achromatopsia	1	0	1
XLRP	0	1	1
CPEO	1	0	1
Total	19	16	35

Why the yield was limited:

• 66% of tests used early microarray analysis.
• These chips only tested for known mutations in known genes.
• Uninformative results stemmed from inaccurate phenotyping, limited screening methods, or undiscovered genes.
• DNA was analysed in 7 different laboratories internationally, with up to 20 weeks for results.

The slide's doughnut chart labels "16 Uninformative Tests" twice (top right and bottom right) while the left segment is labelled "19 Informative Tests"; this appears to be a slide labelling error and is transcribed as shown.

The next-generation sequencing breakthrough (2017)

Vincent et al. (2017), “Next-generation sequencing targeted disease panel in rod-cone retinal dystrophies in Māori and Polynesian reveals novel changes and a common founder mutation”, published 09 May 2017 (doi:10.1111/ceo.12983).

• The problem: standard international gene panels missed variations specific to the New Zealand demographic.
• The cohort: NGS of a targeted retinal gene panel deployed in 16 patients of Māori and Polynesian ancestry.
• The clinical yield: 10 entirely novel variations discovered.
• The breakthrough: identification of a common founder mutation responsible for 16% of autosomal recessive retinal disease within the Māori population.

Bridging to treatment: Luxturna gene therapy

Luxturna was approved by NZ MedSafe in 2024 and has been commercially available in the USA since 2022.

Mechanism of action, in three steps:

1. Vector encapsulation: the functional RPE65 gene is encapsulated in an adeno-associated virus (AAV) vector.
2. Subretinal injection: the replication-defective viral vector is injected directly into the retina.
3. Genome integration and vision restoration: AAV does not integrate into the genome; the RPE65 gene derived from the AAV vector is expressed in retinal cells to restore vision.

Synthesis: the modern genomic diagnostic pathway

Phase 1 → Phase 2 → Phase 3 → Phase 4, with Phase 4 as the endpoint:

1. Clinical phenotype: recognising phenotypic markers and genetic heterogeneity (e.g. tunnel vision in retinitis pigmentosa).
2. Investigation: deploying the cost-effective toolkit, that is targeted panels, whole exome sequencing, or whole genome sequencing.
3. Variant resolution: navigating the significance spectrum from ambiguous VUS to definitive pathogenic variations.
4. Clinical action: enabling precise prognosis, pre-implantation genetic diagnosis, and targeted viral-vector interventions such as Luxturna.

Genetic testing is not merely a laboratory service; it is an end-to-end clinical workflow connecting molecular code to tangible medical clarity.

Self-test

1. List the four tiers of the human variation funnel, with the figure given for each.
2. Describe where Mendelian disease alleles and common GWA-identified variants sit on a plot of allele frequency against effect size, and explain why the top-right and bottom-left regions are sparse or intractable.
3. List the five categories of the variant significance spectrum in order.
4. Define a variant of uncertain significance and explain why it is described as the primary diagnostic hurdle.
5. Distinguish a SNP from a CNV as the lecture defines them.
6. List the four main test modalities of the diagnostic toolkit with the scope, approximate NZ cost, and ideal use case of each.
7. Name the three adjunct tools listed alongside the main modalities and what each is used for.
8. Describe the three pillars of clinical utility of a genetic diagnosis.
9. Describe the steps of pre-implantation genetic diagnosis as the slide presents it.
10. Compare the cost of specialised genetic testing with routine CT or MRI of the brain or orbits, and state the conclusion drawn from that comparison.
11. Describe the phenotype of retinitis pigmentosa and explain why its genotype makes inheritance-pattern-based gene identification difficult.
12. List the four inheritance modes shown converging on the retinitis pigmentosa phenotype.
13. State the diagnostic yield of the 2009 Auckland audit, with the numerator and denominator, and list the three stated causes of uninformative results.
14. Explain why early microarray-based testing had a ceiling on its diagnostic yield.
15. Describe the problem, cohort, and two main findings of the 2017 next-generation sequencing study in Māori and Polynesian patients.
16. Describe the three-step mechanism of action of Luxturna, and state what happens to the AAV vector genome after delivery.
17. List the four phases of the modern genomic diagnostic pathway.
18. A patient presents with progressive loss of peripheral vision and a small pedigree with no clear inheritance pattern. Explain which test modality the lecture would favour and why, and what clinical actions a positive result could enable.
19. Explain how the 2009 audit and the 2017 study together illustrate the relationship between test technology and diagnostic yield.

Answers

Reveal answers

1. ~20,000 protein-coding genes (~1% of the genome); 25,000 to 50,000 rare variants, mostly VUS; ~10,000 non-synonymous variations across 5,000 genes; and 50 to 100 disease-associated variants, 3 to 7 CNVs and ~75 de novo variations.
2. Rare alleles causing Mendelian disease sit at low allele frequency with high effect size; common variants implicated in common disease by genome-wide association sit at high frequency with low effect size, with low-frequency intermediate-effect variants between them along the diagonal. Common variants of high effect (top right) are almost absent, with few known examples; rare variants of small effect (bottom left) are very hard to identify by genetic means.
3. Benign, likely benign, variant of uncertain significance, likely pathogenic, pathogenic.
4. A VUS is a variant that has been identified but whose exact impact on disease risk remains clinically undefined. It is the primary diagnostic hurdle because the test has produced a finding that cannot be translated into a diagnosis or a risk statement.
5. A SNP is a single nucleotide polymorphism present at greater than 1% population frequency; a CNV is a copy number variation, that is a larger insertion or deletion.
6. Single gene testing, scope a specific pathogenic variation, ~NZ$100,forknownspecificconditionssuchashaemochromatosis.Targetedgenepanel,scopeapanelofvariationslinkedtoadiseasephenotype,NZ$300-400, for a phenotype with known genetic heterogeneity such as retinitis pigmentosa. Whole exome sequencing, resequencing the ~2% of the genome coding for proteins, ~NZ$400-500,forunidentifiedvariationsinanaffectedchild.Wholegenomesequencing,high-resolutioncomprehensiveanalysis,NZ$1,000, for complex unsolved diagnostic challenges.
7. Chromosome microarray analysis (aCGH) for CNVs, genotyping for risk association, and RNA analysis for oncology.
8. Diagnosis and prognosis, establishing a genotype/phenotype relationship to understand disease trajectory and resolve unusual cases; reproductive decisions, via pre-implantation genetic diagnosis during IVF and prenatal diagnosis in post-conception embryos; and therapeutic intervention, enabling targeted gene therapies and emerging CRISPR applications.
9. A cell is biopsied from the embryo, the embryo is classified as affected or unaffected on the basis of that cell’s result, and an unaffected embryo is selected.
10. Genetic testing costs a mean of ~NZ$400plusNZ$200 for DNA extraction and international courier logistics, with historic turnaround times of weeks; routine CT or MRI of the brain or orbits costs NZ$250toNZ$575 depending on contrast. The conclusion is that selective genetic testing is a highly opportune and cost-effective investigation relative to standard specialised diagnostics, and an essential prerequisite for gene-specific interventions.
11. RP causes severe progressive peripheral vision loss, leaving a small central island of vision (tunnel vision). Its genotype shows extreme genetic and allelic heterogeneity, and small pedigrees often prevent narrowing down the gene by inheritance pattern alone.
12. Autosomal dominant, autosomal recessive, X-linked, and mitochondrial (MIDD).
13. 54%, that is 19 informative of 35 tests (16 uninformative). Uninformative results stemmed from inaccurate phenotyping, limited screening methods, or undiscovered genes.
14. 66% of tests used early microarray chips, which only tested for known mutations in known genes; anything caused by an undiscovered gene or an unlisted mutation could not be detected. Testing was also fragmented across 7 international laboratories with up to 20 weeks for results.
15. The problem was that standard international gene panels missed variations specific to the New Zealand demographic. A targeted retinal NGS panel was deployed in 16 patients of Māori and Polynesian ancestry. It discovered 10 entirely novel variations and identified a common founder mutation responsible for 16% of autosomal recessive retinal disease in the Māori population.
16. Step 1, the functional RPE65 gene is encapsulated in an AAV vector; step 2, the replication-defective vector is injected subretinally, directly into the retina; step 3, the RPE65 gene derived from the vector is expressed in retinal cells to restore vision. AAV does not integrate into the genome.
17. Phase 1 clinical phenotype, phase 2 investigation, phase 3 variant resolution, phase 4 clinical action.
18. The picture fits retinitis pigmentosa, a phenotype with known genetic heterogeneity, and the small pedigree removes inheritance pattern as a way of narrowing the candidate gene, so a targeted gene panel (~NZ$300-400) is the modality the lecture highlights for exactly this situation. A positive result enables precise diagnosis and prognosis through the genotype/phenotype relationship, genetic counselling and reproductive choices via PGD or prenatal diagnosis, and potentially a gene-specific therapy such as Luxturna if the causative gene is RPE65.
19. The 2009 audit used largely microarray-based testing limited to known mutations in known genes and achieved a 54% yield with up to 20 week turnaround. The 2017 study used next-generation sequencing of a targeted panel, which was able to detect entirely novel variations and a population-specific founder mutation that international panels had missed. Yield is therefore constrained by what the technology is capable of interrogating, not only by the biology of the patient.