Barrett’s oesophagus has previously been reported in familial cohorts implying underlying genetic origin and inheritance[1, 2], however no causative genetic variants have been identified. As next generation sequencing (NGS) now allows the identification of all genetic variants in an individual and has previously identified causative mutations in mendelian disorders, we propose to use NGS to identify genetic variants inherited by affected individuals from families with familial Barrett’s oesophagus.
We have identified a number of families with autosomal dominant familial Barrett’s oesophagus. Clinical data, oesophageal biopsies, and blood samples were gathered from members of these families. Initially, we selected 12 affected and 1 unaffected individual across 3 highly penetrant families for genomic analysis. DNA was isolated and used for whole exome capture followed by NGS using the Illumina® HiSeq 2000 sequencer. Variants causing a downstream change to protein sequence were identified by comparison with the human reference genome. Variants were prioritised for segregation analysis based on a heterozygous genotype, and a low frequency in the general population (when compared to publically accessible genomic data). Validation and segregation analysis was carried out by conventional Sanger sequencing.
NGS produced an average read depth of 74 per base and on-target capture rate of 93.2%. After quality control filtering, an average of 6478 variants per family was detected. By applying algorithms to identify rare genetic variants likely to induce a change in protein function, we reduced candidate variants to around 900 per family. Of these, 178 were novel (not in dbSNP and 1000 genome databases). Around 10 heterozygous variants per family were predicted to cause a significant change to protein function. Prior validation of a subset of variants by Sanger sequencing confirmed our filtering methods. Segregation analysis in the same family and comparison of identified genes across different families will be used to further refine the list of candidate variants for future biological validation.
NGS is a validated and robust method for discovering variants, and its combined use in families with inherited Barrett’s oesophagus has identified candidate genetic variants. Ongoing work will refine our candidate list with the aim of ultimately identifying the underlying molecular basis for the inheritance of Barrett’s oesophagus in these families.