Germline structural variant as the cause of Lynch Syndrome in a family from Ecuador

Abstract

Colorectal cancer (CRC) is one of the most common cancers worldwide. Lynch Syndrome (LS) is the most common form of hereditary CRC and it is caused by germline defects in the DNA-mismatch repair (MMR) pathway. It is of extreme importance for affected LS patients and their relatives to identify the germline causative alteration to provide intensified surveillance to those at risk and allow early diagnosis and cancer prevention. Current approaches for LS molecular diagnosis typically involve screening of the MMR genes by targeted gene-panel sequencing and rearrangement screening. We report the identification and characterization of a novel germline structural variant encompassing 48.757 kb, involving the 3'-ends of the MLH1 and LRRFIP2 genes, as the cause of LS in a family of Ecuador. Whole-genome sequencing and transcriptomics allowed the identification of the genomic rearrangement and highlights the importance of the use of these additional approaches to achieve a comprehensive molecular diagnosis in some LS patients.

Fig. 1. Family tree and genetic testing of the index case for the MMR genes.

A Pedigree of the Ecuadorian family, where the index case is marked with an arrow. Black symbols represent individuals affected by either colorectal (upper right) or stomach cancer (lower left). Age of onset is also indicated. Female and male gender are circles and squares, respectively. Slashed individuals represent death. + symbol indicates carrier status for the rearrangement. - symbol indicates non-carrier status for the rearrangement. B MLPA results showing potential alterations in MLH1 exon 19 (probemix P003), and MLH1 exon 19 and exon LRRFIP2 exon 26 (probemix P248). C CGH array of index case indicative of a genomic alteration in the 3’ends of MLH1 and LRRFIP2 (red circle).

Fig. 2. Characterization of the complex rearrangement involving MLH1 and LRRFIP2.

A Visualization of WGS data with Integrative Genomics Viewer (IGV). Germline short reads from WGS allowed the detection of three SVs involving the 3’-ends of MLH1 and LRRFIP2. Representative paired reads with discordant pair orientation (RR, LL and RL) and aberrant mapping distance are depicted. A schematic map of the area of the rearrangement shows the three SVs and the six breakpoints, resulting in a five-segment map (A to E). The size of each segment in bp is indicated. The overlapped SVs are defined as a small inversion in MLH1 of 0.91 kb (fragments A and B, deep blue), a big inversion of 22.8 kb involving MLH1 and LRRFIP2 (fragments C and D, turquoise blue) and a tandem duplication of 37.88 kb in the LRRFIP2 gene (fragments D and E, green). B Characterization of the breakpoints of the SVs and proposed map of the rearrangement. IGV simplified coverage plot (IGV Count tool with an average read density window of 300 bp) allowed the characterization of the breakpoints and duplicated areas. The proposed map of the actual rearrangement fits the WGS coverage and the SV calling. Sanger sequencing profiles of the three PCRs (arrows) validated the breakpoints.