Identification of Rare Noncoding Variants in Familial Nonmedullary Thyroid Carcinoma

Familial nonmedullary thyroid carcinoma (FNMTC) occurs when three or more family members are affected by usually papillary thyroid carcinoma (PTC), the most common form of NMTC. While the heritability to NMTC is among the highest of all cancers, the genetic determinants among NMTC families are not well understood. Here, we aim to understand the contribution of rare noncoding germline variants in the etiology of FNMTC.

We previously reported whole-genome sequencing (WGS) and linkage analysis in 17 PTC families and reported on 41 protein-coding variants in 40 genes that cosegregated with PTC in 11 of the families. Herein, we further leveraged our WGS data to include noncoding variants in our analysis for all 17 families. We hypothesized that most of the pathogenic noncoding variants would be located in theoretical or empirically determined regulatory regions that demonstrate at a minimum, basal thyroid expression, a positive family linkage score, and co-segregation among PTC-affected individuals. To test this hypothesis, we adopted a unique filtering strategy to identify variants that occurred in known DNA elements and transcription factor binding sites, near regions known to impact on gene expression or splicing in thyroid tissue, and/or in characterized thyroid enhancers. We annotated variants using two analyses (ENCODE and transcription factor binding site) within the BasePlayer software. We separately analyzed (1) expression quantitative trait loci, (2) splicing quantitative trait loci, and (3) thyroid enhancers. We then ranked variants according to predicted pathogenicity and performed Sanger sequencing in all individuals of each family.

In total, 121 variants were selected based on in-silico prediction and our custom ranking analysis in each pedigree. Of these, 56 variants showed cosegregation among all PTC-affected individuals and were absent from unaffected individuals. This included candidate variants from five of the six PTC families for whom no protein-coding variants were previously found.

Our data suggest that noncoding variants are important in the etiology of FNMTC and provide a framework for identifying noncoding germline variants using a novel approach. Further studies are needed to functionally characterize these variants to better understand the molecular mechanism of their pathogenicity.