GC238 Rare Disease Genetic Testing For Precision Medicine

At the end of this lecture, you will be able to:

1. Identify the key challenges in rare disease diagnosis.
2. Use case studies to demonstrate the importance of diagnosis on management for rare diseases.
3. Explain rare disease screening and the implications.

• Rare disease questions tend to appear as pattern recognition MCQs (e.g., skin findings + seizures → tuberous sclerosis) or as "first investigation" and "genetic counselling" SAQs.
• Understanding the diagnostic odyssey concept is tested in clinical scenarios where a patient has seen multiple doctors without diagnosis.
• Newborn screening in Hong Kong is a high-yield list that can appear in paediatrics and public health contexts.
• Links directly to pharmacogenomics lectures (precision medicine), inherited cardiac conditions (genetic testing), immunodeficiencies (SCID screening), and cancer genetics.

A rare disease is any condition with a prevalence of less than 1 in 2,000. ^[1]

This is the internationally accepted threshold used by the EU (Orphan Drug Regulation) and adopted by many countries. Why 1 in 2,000? Because below this prevalence, commercial pharmaceutical development is not financially viable without special incentives (orphan drug legislation), and clinical expertise becomes scarce.

Although individually rare, they are collectively common with global rare disease point prevalence estimated at 3.5–5.9%. ^[1]

This is the key conceptual shift: while each rare disease affects few people, the aggregate burden is enormous.

Why is the HK prevalence lower than global? The HK figure (1.5%) comes from hospital administrative data using ORPHAcodes in ICD-10 and likely underestimates true prevalence due to undiagnosed cases and coding limitations. The global estimate (3.5–5.9%) uses the Orphanet database which includes all catalogued rare diseases.

Rare diseases can be found in every area of medicine; they present across all ages and demographics; however, their individually low prevalence means they are not an educational priority. ^[1]

Serial reports indicate that inadequate knowledge and misconceptions about the prevalence of rare diseases result in suboptimal patient care, compounded by insufficient formal medical education. ^[1]

Why does this matter clinically?

• Doctors in any specialty will encounter rare disease patients — they are not confined to genetics clinics.
• Lack of awareness → misdiagnosis, inappropriate treatment, delay.
• Up to 80% of rare conditions are estimated to be caused by an underlying genetic susceptibility. ^[1]
• Most rare conditions manifest in childhood. ^[1]

This is one of the most important concepts in the lecture and is very testable:

The extended journey to achieve an accurate diagnosis is referred to as the "diagnostic odyssey." ^[1]

Why does the diagnostic odyssey occur?

1. Low individual prevalence → most doctors have never seen the condition
2. Phenotypic overlap with common diseases → misdiagnosis
3. Lack of formal education → not considered in differential
4. Fragmented healthcare → seeing many specialists without coordination
5. Limited availability of specialized tests

Impact:

• Significant distress to patients and families ^[1]
• Costly to the healthcare system (in the UK: excess NHS cost > £3.4 billion while undiagnosed) ^[1]
• Significant burden of disease and/or disability ^[1]

Red Flags: ^[1]

• Unusual symptoms
• Non-response to standard treatment
• Family history of similar or unexplained illnesses

Stepwise Approach: ^[1]

1. Detailed history (medical, familial, environmental)
2. Comprehensive physical exam
3. Review of prior investigations and treatments

Why these red flags matter from first principles:

• "Unusual symptoms" → symptoms that don't fit a recognizable pattern suggest an uncommon cause
• "Non-response to standard treatment" → if the treatment for the presumed diagnosis isn't working, the diagnosis may be wrong; also, some rare diseases have specific pathophysiology that standard therapies don't address
• "Family history" → 80% of rare diseases are genetic; a family pattern of unexplained illness is a strong clue

The stepwise approach explained:

• History must include a three-generation pedigree (as emphasized in clinical genetics ^[2]) — consanguinity, ethnic background, neonatal history, developmental milestones
• Physical exam must include dysmorphic examination — subtle facial features, skin findings, skeletal anomalies can point to syndromic diagnoses ^[3]
• Reviewing prior investigations avoids repeating expensive tests and may reveal patterns missed when results were interpreted in isolation

Genetic Testing: ^[1]

• MLPA (Multiplex Ligation-dependent Probe Amplification) — for copy number variation (exon deletion or duplication)
• Targeted gene panels — for specific phenotypes
• Whole exome sequencing (WES)
• Whole genome sequencing (WGS)

Biochemical Tests: ^[1]

• Enzyme assays (e.g., lysosomal storage disorders)
• Metabolic screening (e.g., plasma amino acids)
• Auto-antibodies panel (e.g., immune-mediated encephalitis panels)

Imaging: ^[1]

• MRI/CT for structural anomalies
• Functional imaging (e.g., PET for metabolic disorders)

Biopsy and Histopathology: ^[1]

• E.g., rare cancers or inflammatory conditions or musculoskeletal conditions

From senior notes ^[2]: Types of WES/WGS:

• Singleton: proband only
• Duo: proband + either parent
• Trio: proband + both parents (most informative — can identify de novo variants)
• Quad: proband + both parents + sibling

Why trio WES/WGS is preferred: By comparing the child's genome against both parents, you can:

1. Identify de novo variants (present in child but not parents) — strong evidence for pathogenicity
2. Determine phase (which allele came from which parent) for autosomal recessive conditions
3. Filter out benign familial variants, dramatically reducing the list of candidate variants

Positive outcomes: ^[1]

• Validation of Symptoms: Patients feel heard and understood
• Access to Targeted Care: Opportunity for specific treatments or interventions, or participation in clinical trials
• Emotional Relief: Reduction in uncertainty and anxiety about the unknown
• Support Networks: Connection with patient advocacy groups and communities

Challenges Post-Diagnosis: ^[1]

• Treatment Limitations: Many rare diseases have no specific therapy
• Ongoing Care Needs: Long-term multidisciplinary care often required
• Psychosocial Adjustments: Coping with chronic illness or prognosis
• Financial Strain: Costs of specialised care and medications
• Family planning decisions: Genetic counselling

Why genetic counselling is essential ^[2]:

• Should ALWAYS be done before and after genetic tests
• Pre-test: explain purpose, limitations, possible outcomes, implications for family members
• Post-test: interpret results, discuss prognosis, reproductive options, cascade testing of at-risk relatives
• Refer to appropriate resources: other specialties, HCA, patient support groups

Preimplantation genetic testing (PGT) is recommended when couples risk transmitting a known genetic abnormality to their children. ^[1]

How PGT works:

1. IVF is performed to create embryos
2. At blastocyst stage (day 5), a few trophectoderm cells are biopsied
3. Cells are analyzed (by NGS, array CGH, or targeted PCR depending on type)
4. Only unaffected embryos are transferred to the uterus

NIPT is most often used to look for chromosomal disorders, related to presence of an extra or missing copy of a chromosome (aneuploidy). ^[1]

NIPT can look for: ^[1]

• Down syndrome (trisomy 21)
• Trisomy 18 (Edwards syndrome)
• Trisomy 13 (Patau syndrome)
• Extra or missing copies of X and Y chromosomes (sex chromosome aneuploidies)

How NIPT works:

• Cell-free fetal DNA (cffDNA) circulates in maternal blood from ~10 weeks gestation (originates from placental trophoblast cells)
• NIPT sequences this cffDNA and uses statistical algorithms to detect over-representation of specific chromosomes
• It is a screening test (not diagnostic) — positive results require confirmation by amniocentesis or CVS with karyotyping/array CGH
• Sensitivity > 99% for trisomy 21; lower for trisomy 18/13 and sex chromosome aneuploidies

Newborn screening panel in HK: ^[1]

1. Neonatal hearing screening
2. G-6PD deficiency
3. Congenital hypothyroidism
4. Severe combined immunodeficiency (SCID)
5. Inborn error of metabolism
6. Spinal muscular atrophy

The list is expected to be expanding with the availability of new and/or effective treatment. ^[1]

Early detection allows early treatment and better clinical outcome. ^[1]

Why each condition is screened:

Wilson & Jungner criteria for screening (classical framework — know this for exams):

1. Important health problem
2. Accepted treatment available
3. Facilities for diagnosis and treatment available
4. Recognizable latent/early symptomatic stage
5. Suitable test available
6. Test acceptable to population
7. Natural history adequately understood
8. Agreed policy on whom to treat
9. Cost of case-finding economically balanced
10. Case-finding should be a continuing process

SMA NBS perfectly fulfills these criteria now that effective treatments exist (nusinersen approved for pre-symptomatic SMA in HK in May 2022 ^[1]).

The clinical pharmacology lecture discusses how genetic testing guides drug selection — the same principle applies here. In rare diseases, identifying the molecular defect guides not just drug choice but the entire management strategy. For example:

• CYP2C19 poor metabolizers → switch from clopidogrel to ticagrelor ^[6]
• HLA-B*15:02 → avoid carbamazepine (Stevens-Johnson syndrome risk) ^[7]
• TSC1/TSC2 mutation → use mTOR inhibitor (everolimus) ^[1]

Genetic testing is equally critical in cardiomyopathies ^[8]:

• Familial DCM: TTN, LMNA mutations → guides ICD implantation decisions
• ARVC: desmosomal gene mutations → activity restriction + screening of relatives
• HCM: sarcomeric gene mutations → cascade family screening

SCID is on the HK NBS panel ^[1]. The TREC assay detects absent T-cell receptor excision circles (indicating failed T-cell development). Without screening, SCID infants present with recurrent severe infections and die within the first year. With early HSCT (ideally before 3.5 months), survival exceeds 90%. ^[9]

Germline genetic testing (e.g., BRCA1/2, Lynch syndrome genes) requires pre- and post-test genetic counselling ^[4], just as discussed in this lecture for rare diseases. This distinguishes germline testing from somatic mutation profiling (e.g., EGFR in lung cancer), which does not typically require formal genetic counselling ^[4].

1. MCQ: Photo of facial angiofibromas + ash leaf spots in a child with seizures → "What is the most likely diagnosis?" → TSC
2. MCQ: What investigation confirms TSC diagnosis? → Genetic testing for TSC1/TSC2
3. SAQ: "List the Hong Kong newborn screening panel" → 6 items
4. SAQ: "Explain the concept of diagnostic odyssey" → definition + impact (time, cost, distress, misdiagnosis rate)
5. MCQ: "Which screening test specifically detects chromosomal aneuploidy non-invasively?" → NIPT
6. MCQ: "A test for a rare inherited metabolic disorder has very high specificity but moderate sensitivity. Best clinical use?" → Confirmatory testing after positive screen (2025 MCQ Q6)

Stem: "Disease diagnosis often requires ancillary investigations such as laboratory testing. A laboratory test for detecting a rare inherited metabolic disorder has a very high specificity but moderate sensitivity. Which of the following clinical scenarios BEST suits this test?"

Options: A. Confirmatory testing after a positive screening test B. Initial screening for asymptomatic population C. Population-wide epidemiological studies D. Testing in emergency setting for rapid diagnosis

Answer: A

Rationale: High specificity = very few false positives = when it's positive, you can be confident it's a true positive → ideal for confirmatory testing. Screening tests need high sensitivity (to catch all cases, even at the cost of some false positives). This question directly relates to the lecture's discussion of rare disease screening followed by confirmatory testing. Option B is wrong because screening needs high sensitivity, not specificity.

Stem: "A 63-year-old man with metastatic lung adenocarcinoma has been on targeted therapy against epidermal growth factor receptor (EGFR) for 2 years. Chest X-ray shows suspected malignant pleural effusion but no malignant cells are detected on cytological examination of the pleural fluid. Which of the following tests on the pleural tap sample is MOST APPROPRIATE to evaluate the doctor's suspicion of metastatic carcinoma?"

Options: A. Biochemical test on the cell-free component of the sample for glucose and protein contents B. Genetic testing of the cell-free component of the sample for EGFR mutation C. Genetic testing of the cellular component of the sample for EGFR mutation D. Rapid tumour cell culture of the cellular component of the sample

Answer: B

Rationale: No malignant cells on cytology → the cellular component is uninformative. Cell-free DNA (cfDNA) in the pleural fluid may contain tumour-derived DNA with EGFR mutations. This is the principle of liquid biopsy applied to pleural fluid. Testing cell-free component for EGFR mutation can confirm malignant origin even when cytology is negative. This connects to the lecture's theme of genetic testing enabling diagnosis where conventional methods fail.

Stem: "Molecular genetic tests are now commonly used to assist diagnostic pathology particularly in challenging cases. Which of the following gene mutation tests is applied to assist histopathological diagnosis of ovarian Sertoli-Leydig cell tumour, a sex cord stromal tumour?"

Options: A. BRCA B. DICER C. EGFR D. KRAS

Answer: B (DICER)

Rationale: DICER1 somatic mutations are characteristic of Sertoli-Leydig cell tumours and help confirm the diagnosis histopathologically. BRCA is for hereditary breast/ovarian cancer (high-grade serous carcinoma). EGFR is for lung adenocarcinoma. KRAS is for colorectal/pancreatic cancer. This tests knowledge of which genetic test matches which rare tumour — a precision medicine application.