Detection and characterization of mutation in mismatch repair genes in a filipino family with HNPCC (Hereditary Nonpolyposis Colorectal Cancer)

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal syndrome largely caused by germline mutations in any of the six mismatch repair genes. Eighty per cent of the reported cases are attributed to mutations in two MMR genes: hMLH1 and hMSH2. Mutation carriers exhibit a characteristic phenotype called microsatellite instability. The objective of this study is to determine the MSI and MMR status of members of an HNPCC family who satisfied the Amsterdam Criteria. This family is composed of 118 individuals, 26 of whom are included in the study. A non-HNPCC family of 10 members was used as the control group. DNA was extracted from peripheral blood samples and analyzed for the presence of microsatellite instability (MSI) using single strand conformation polymorphism (SSCP). Mutation in the hMSH2 and hMLH1 mismatch repair genes was determined using denaturing high performance liquid chromatography (dHPLC). The type of mutation was ultimately characterized by DNA sequencing. MSI screening was performed using the Bethesda Panel of Markers, where instability in two or more of the five markers translates into high MSI. Results showed that 92.3% of the HNPCC Family exhibited high MSI while 60% of the control family exhibited the same status of instability. MMR gene mutation analysis revealed that 4 symptomatic (diagnosed with cancer) members and 10 asymptomatic HNPCC members have mutations in exon 14 of the hMSH2 gene. In addition, six symptomatic and 2 asymptomatic members showed mutations in exon 4 of the hMLH1 gene, and two symptomatic members were found positive for mutation in exon 3 of the same gene. On the other hand, three members of the non-HNPCC family showed mutations in exon 14 of hMSH2. DNA sequencing of samples from three members of the HNPCC family were found positive for frameshift mutation and substitution in exon 14 of the hMSH2 gene. Two consecutive substitutions were also detected in the exon 4 of the hMLH1 gene while another substitution was identified in the exon 3 of the same gene. On the other hand, one member of the HNPCC family and two members of the Non-HNPCC family did not show any nucleotide change in DNA sequencing despite having positive result in dHPLC screening. The potential of using peripheral blood in screening for MSI and MMR gene mutation is shown in this study