Abstract
Lynch syndrome is the most common type of hereditary colorectal cancer and is characterized by a germline mutation in one of the DNA mismatch repair (MMR) genes (PMS2, MSH2, MSH6 or MLH1). Identification of Lynch syndrome patients and their relatives enables initiation of interventions that can significantly increase prognosis and facilitate early cancer detection. Established methods for MMR mutation detection sometimes fail to identify a pathogenic mutation confirming the diagnosis suspected based on clinical manifestations. In this study, haplotype analysis was performed to assess indications of a hereditary germline mutation in MMR genes in suspected Lynch syndrome families with inconclusive genetic test results. Family members affected by a hereditary disease inherit a copy of a mutated gene from a progenitor and share a chromosomal segment that is identical by descent around the disease locus. Haplotype analysis can identify genes involved in hereditary disorders by exploiting the cosegregation of disease phenotype and haplotype markers. Marker panels comprised of SNP and microsatellite loci were constructed for the four MMR genes and genotyped by Sanger sequencing and multiplex fragment analysis by capillary electrophoresis, respectively. Haplotype configurations were reconstructed based on genotype data and pedigree structures. Disease-segregating haplotypes were identified in several of the families, and thus supported the hypothesis of an undetected MMR germline mutation harbored by affected family members. The study revealed conditions acting as determinants for the obtention of informative MMR haplotype pedigrees, including pedigree structure and size. Including microsatellites in the marker panels offered a higher resolution of haplotypes, which is especially advantageous in pedigree analysis. However, insufficient marker variance posed a challenge for informative haplotype-phenotype associations and deduction of haplotype lineages within the families. The established haplotype analysis methodology provides a starting point for the development of a supplementary tool for identification of Lynch syndrome patients.