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Functional evaluation of tumour-specific variants of p16INK4a/CDKN2A: correlation with protein structure information
M Ruas1, S Brookes, N Q McDonald
1Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
Abstract:
Inherited mutations in the CDKN2A/INK4a/MTS1 tumour suppressor gene on chromosome 9p21 are associated with familial predisposition to melanoma and other tumour types. Nonsense and missense mutations are also found in a variety of sporadic cancers, and over 140 sequence variants have already been recorded in the literature. In assessing the relevance of these variants and for counselling members of affected families, it is important to distinguish inactivating mutations from harmless polymorphisms. Existing functional assays have frequently reached conflicting conclusions and no single test appears adequate. Here we evaluate a number of alternatives including a novel assay based on retroviral delivery of p16INK4a cDNAs into human diploid fibroblasts. Among the 17 sequence variants analysed, three distinct categories can be distinguished: those that abrogate the binding of p16INK4a to CDK4 and CDK6, those that alter the properties of the protein without preventing it from interacting with CDKs, and those that have no discernible effect on protein function. These distinctions can be rationalized by considering the impact of the amino acid changes on the three-dimensional structure of the protein.
Insights
Distinguishing inactivating mutations from polymorphisms in the CDKN2A tumor suppressor gene is crucial for cancer risk assessment. A novel assay categorizes variants based on their impact on p16INK4a protein function and interactions.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Germline mutations in the CDKN2A (also known as INK4a/MTS1) tumor suppressor gene are linked to hereditary cancer syndromes, particularly melanoma.
- Numerous sequence variants within CDKN2A have been identified in both familial and sporadic cancers, necessitating accurate functional assessment.
Purpose of the Study:
- To develop and evaluate a reliable functional assay for classifying CDKN2A variants.
- To differentiate between pathogenic inactivating mutations and benign polymorphisms for improved genetic counseling and risk assessment.
Main Methods:
- Evaluation of multiple functional assays for assessing CDKN2A variants.
- Development of a novel assay utilizing retroviral delivery of p16INK4a cDNAs into human diploid fibroblasts.
- Analysis of 17 distinct CDKN2A sequence variants.
Main Results:
- Three categories of variants were identified: those disrupting p16INK4a binding to CDK4/CDK6, those altering protein properties without affecting CDK interaction, and those with no functional impact.
- The novel retroviral assay demonstrated utility in categorizing variants based on their functional consequences.
- Structural considerations of amino acid changes correlate with observed functional distinctions.
Conclusions:
- A robust method is needed to accurately assess the pathogenicity of CDKN2A variants.
- The developed assay provides a means to classify variants based on their functional impact on p16INK4a.
- Understanding the structural basis of variant effects aids in predicting functional outcomes.