Week 8 - L4 - Variants of Uncertain Significance in Clinical Genetics and Genetic Counseling

Definition and Nature of Variants of Uncertain Significance (VUS)

• Definition of Variant of Uncertain Significance (VUS):

  • A variation in a genetic sequence for which the direct association with disease risk remains unclear or unestablished.

  • Alternative terminology includes "variant of unknown significance" or "unclassified variant."

• Test Outcome Spectrum in Clinical Genetic Testing:

  • Positive Test Result: Occurs when an identified genetic variant is suspected or confirmed to be pathogenic (disease-causing).

  • Negative Test Result: Occurs when an identified genetic variant is suspected or confirmed to be benign (harmless).

  • Inconclusive Test Result (VUS): Occurs when a genetic test identifies a sequence variation whose clinical significance cannot be categorized as either clearly pathogenic or clearly benign, placing the result in an intermediate, uncertain classification.

• Clinical and Psychological Impact:

  • Ambiguity creates substantial uncertainty for both patients and healthcare providers.

  • Lacks clear clinical actionable data needed to guide definitive treatment decisions, surveillance strategies, or prophylactic interventions.

Clinical and Laboratory Methodologies for Evaluating VUS

• Segregation Analysis and Clinical Evaluation:

  • Conducting clinical evaluations on the index patient (proband) carrying the variant.

  • Extending genetic testing to biological family members, particularly those presenting similar clinical symptoms or disease phenotypes, to determine if the VUS co-segregates with the disease across generations.

• Aggregation of Literature and Population Databases:

  • Reviewing reported clinical cases across medical literature.

  • Querying publicly accessible centralized databases, primarily ClinVar and ClinVarMiner, to review global clinical submissions and variant entries.

• Assessment of Protein Structure and Function:

  • Computational (In Silico) Methods: Utilizing predictive algorithms and structural databases to evaluate how a nucleotide substitution alters amino acid sequence, protein folding, stability, or domain interactions.

  • Functional In Vitro Assays: Conducting or reviewing experimental studies that directly measure the physical and biochemical impact of specific single-nucleotide DNA changes on protein structure and cellular function.

Somatic Tissue Analysis and ClinVar Prevalence

• Somatic Tumour Analysis in Oncology:

  • Analyzing similar genetic variants with known functional impacts to infer the mechanism of an uncharacterized VUS.

  • Performing direct genomic sequencing on excised tumour tissue from cancer patients.

  • Conducting histological analysis and immunohistochemistry (IHC) using targeted antibodies to visualize spatial protein expression and cellular localization within tumour tissue.

• Epidemiological Prevalence of VUS:

  • Encountered in $10\% \text{ to } 31\%$ of all clinical cancer genetic tests.

• Infrastructure and Statistics of ClinVar:

  • ClinVar Definition: A freely accessible, public database archiving relationships between human genetic variations, health outcomes, and drug responses.

  • Aggregates submissions from clinical diagnostic laboratories, academic centers, and healthcare providers who assign clinical significance to identified variants.

  • Quantitative Overview from ClinVarMiner:

  • Nearly $2,000,000$ genetic variants in ClinVarMiner are currently classified as Variants of Uncertain Significance (VUS), forming a substantial proportion of total database entries.

  • Other primary ClinVar classification categories include benign, likely benign, likely pathogenic, and pathogenic.

Familial Pedigree Case Study: MSH6 Variant and Cancer Predisposition

• Familial Pedigree Overview:

  • Analysis of a complex, multi-generational family history demonstrating high cancer burden across multiple branches:

  • Branch 1: Documented diagnoses of lung cancer and pancreatic cancer.

  • Branch 2: Documented diagnoses of colon cancer and multiple individuals diagnosed with breast cancer across various ages of onset ($DX$).

• Proband Diagnostic Timeline:

  • Diagnosed with breast cancer at age $65$.

  • Diagnosed with pancreatic cancer at age $68$.

  • Deceased at age $69$ due to pancreatic cancer complications.

• Molecular Characterization and Testing History:

  • Initial genetic testing conducted post-breast cancer diagnosis (in $2018$ or $2019$) identified a Variant of Uncertain Significance (VUS).

  • Specific Gene Identification: The $MSH6$ gene.

  • Specific Nucleotide Alteration: A single-base substitution changing Adenine to Cytosine ($A \rightarrow C$).

  • Reclassification Over Time: Querying ClinVar reveals that the variant status evolved from VUS to a "conflicting classification of pathogenicity", with several laboratory submissions categorizing the alteration as "likely benign".

• Biological Function of MSH6:

  • The $MSH6$ gene encodes a crucial protein operating within the mismatch repair (MMR) pathway.

  • Functions to correct single-base mismatches and insertion/deletion loops occurring during DNA replication.

  • Loss or impairment of MMR protein function prevents DNA repair, causing an accumulation of uncorrected secondary genomic mutations.

• Multi-Generational Impact and Clinical Recommendations:

  • Family history includes two maternal aunts and a cousin diagnosed with breast cancer.

  • Genetic testing of affected living relatives showed negative results for known pathogenic variants, leaving open the possibility of an unidentified familial variant.

  • Surveillance Protocols for At-Risk Individuals:

  • Routine clinical follow-ups and heightened breast cancer vigilance.

  • Annual screening via Magnetic Resonance Imaging (MRI).

  • Universal Recommendation: All individuals with breast tissue should perform self-examinations and undergo regular general practitioner (GP) clinical examinations, as early detection represents the most effective prevention; immediate clinical evaluation is advised upon experiencing any abnormal symptoms.

Psychosocial and Equity Implications of Variant Reclassification

• Psychological Dynamics and Clinical Counselling Challenges:

  • Core Objective of Genetic Counselling: Calculating and effectively communicating individualized empirical and molecular disease risks to patients.

  • Interference of VUS: Inability to calculate precise risk metrics prevents counsellors from offering definitive guidance.

  • "Genetic Purgatory": A state of clinical and psychological uncertainty where patients lack a definitive diagnosis, leading to protracted anxiety and emotional distress while waiting for potential variant reclassifications.

  • Physical and Mental Self-Care: Managing family cancer histories and genetic uncertainty carries significant emotional trauma, necessitating comprehensive physical and psychological support.

• Ethnic Disparities and Database Representation:

  • Major source of healthcare disparities due to population bias in genomic research datasets.

  • Individuals of European descent are heavily over-represented in public genetic reference archives.

  • Individuals of non-European ancestry are under-represented, resulting in a disproportionate frequency of VUS results due to the lack of ethnicity-matched baseline population control data in ClinVar.

• Longitudinal Variant Reclassification Data ($2010$ vs. $2020$ Cohort Study):

  • Evaluation of variant cohort classifications analyzed in $2010$ and reclassified in $2020$:

  • $10$ variants originally designated as likely pathogenic were reclassified as likely benign by $2020$.

  • $77$ variants were categorized as Variants of Uncertain Significance (VUS) in $2020$.

  • The proportion of variants categorized as likely pathogenic substantially decreased between $2010$ and $2020$.

  • The total number of variants classified as pathogenic remained relatively stable.

  • Takeaway: Diagnostic and analytical methodologies continuously improve over time; reclassification updates reduce false-positive pathogenic classifications and inform patient risk management.

Clinical Reassessment and Case Studies on Inappropriate Interventions

• Case Study 1: Reassessment and Segregation Analysis in Breast Cancer

  • Clinical History: Patient diagnosed with breast cancer at age $30$ ($DX = 30$), with a strong family history involving a mother and sister both diagnosed with breast cancer.

  • Family Member Testing: Sister previously underwent genetic testing and was found to carry a VUS in the $BRCA2$ gene.

  • Inappropriate Surgical Referral: Based solely on the sister's $BRCA2$ VUS result, a gynecologist recommended that the index patient undergo a risk-reducing bilateral oophorectomy (surgical removal of both ovaries).

  • Genetic Counselling Re-evaluation: Genetic counselors tested the index patient and found she did not carry the sister's $BRCA2$ variant. The genetic counselling team concluded the prophylactic surgery was inappropriate, as a VUS that does not segregate with disease across affected family members is highly unlikely to be the underlying causative driver.

• Case Study 2: Invasive Surgical Consequences of Misclassified Variants

  • Clinical Scenario: Seven female family members tested positive for a $BRCA2$ variant initially classified as pathogenic.

  • Communicated Disease Risk: Patients were informed they possessed an $84\%$ cumulative lifetime risk of developing breast cancer and a $27\%$ risk of developing ovarian cancer by age $70$.

  • Surgical Interventions: Driven by this high risk, a majority of the seven women elected to undergo invasive risk-reducing surgical procedures (prophylactic mastectomies and oophorectomies).

  • Subsequent Variant Reclassification: Two years following the surgical procedures, updated genetic evidence led to the reclassification of the $BRCA2$ variant from pathogenic to a Variant of Uncertain Significance (VUS). Had this reclassification occurred prior to surgery, invasive procedures could have been avoided.

Systemic Frameworks, Ancestral Inequities, and Course Summary

• Ethical Frameworks for Non-Negligent Variant Reclassification:

  • Addressing clinical harms arising from variant reclassifications requires recognizing that such events typically stem from evolving scientific evidence rather than clinical negligence or diagnostic error.

  • Reclassification reflects the complex, dynamic nature of genomic data interpretation.

• Deepening Healthcare Inequities in Diverse Ancestry Groups:

  • Non-European populations face compounding negative impacts from variant reclassifications due to under-study rooted in:

  1. Inequitable access to specialized medical care.

  2. Deep-seated mistrust of healthcare and research institutions driven by historical unethical medical research.

  3. Persistent disparities in clinical care delivery.

  • Urgent Need for Action: Expanding genomic studies in racially and culturally diverse populations is essential to establish accurate, population-specific reference datasets, ensuring equitable diagnostic accuracy and preventing downstream clinical harm.

• Key Learning Outcomes of Genetic Counselling Integration:

  1. Explain the various types of clinical genetic tests and their specific diagnostic applications.

  2. Comprehend the core principles of genetic counselling and the role of genetic counsellors, emphasizing patient empowerment to support informed autonomous decision-making.

  3. Recognize and analyze the complex health, ethical, legal, and societal challenges linked to genomic medicine.

  4. Master domain terminology and concepts, including Variants of Uncertain Significance (VUS), segregation analysis, and longitudinal reclassification.

• Student Support and Educational Resources:

  • Moodle Discussion Forum: Available for submitting content-related questions for instructor response.

  • Student Consultations: Bookable 1-on-1 sessions with a trained Mental Health First Aider via Moodle links covering:

  • Academic and career path advising in genetics and genomics.

  • Mental health and psychosocial well-being support.