Functional testing strategy for coding genetic variants of unclear significance in MLH1 in Lynch syndrome diagnosis.
Carcinogenesis
; 36(2): 202-11, 2015 Feb.
Article
en En
| MEDLINE
| ID: mdl-25477341
ABSTRACT
Lynch syndrome is caused by inactivating mutations in the MLH1 gene, but genetic variants of unclear significance frequently preclude diagnosis. Functional testing can reveal variant-conferred defects in gene or protein function. Based on functional defect frequencies and clinical applicability of test systems, we developed a functional testing strategy aimed at efficiently detecting pathogenic defects in coding MLH1 variants. In this strategy, tests of repair activity and expression are prioritized over analyses of subcellular protein localization and messenger RNA (mRNA) formation. This strategy was used for four unclear coding MLH1 variants (p.Asp41His, p.Leu507Phe, p.Gln689Arg, p.Glu605del + p.Val716Met). Expression was analyzed using a transfection system, mismatch repair (MMR) activity by complementation in vitro, mRNA formation by reverse transcriptase-PCR in carrier lymphocyte mRNA, and subcellular localization with dye-labeled fusion constructs. All tests included clinically meaningful controls. The strategy enabled efficient identification of defects in two unclear variants the p.Asp41His variant showed loss of MMR activity, whereas the compound variant p.Glu605del + p.Val716Met had a defect of expression. This expression defect was significantly stronger than the pathogenic expression reference variant analyzed in parallel, therefore the defect of the compound variant is also pathogenic. Interestingly, the expression defect was caused additively by both of the compound variants, at least one of which is non-pathogenic when occurring by itself. Tests were neutral for p.Leu507Phe and p.Gln689Arg, and the results were consistent with available clinical data. We finally discuss the improved sensitivity and efficiency of the applied strategy and its limitations in analyzing unclear coding MLH1 variants.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Nucleares
/
Neoplasias Colorrectales Hereditarias sin Poliposis
/
Adenosina Trifosfatasas
/
Enzimas Reparadoras del ADN
/
Proteínas Adaptadoras Transductoras de Señales
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Proteínas de Unión al ADN
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Reparación de la Incompatibilidad de ADN
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Adult
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Female
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Humans
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Male
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Middle aged
Idioma:
En
Revista:
Carcinogenesis
Año:
2015
Tipo del documento:
Article