Using Sequence Data to Localise Developmental Mutations

Transverse Hemimelia (TH) is a developmental limb malformation in which the limb is normally developed proximally but distal structures are lost and appear to be amputated. The phenotype can involve unilateral or bilateral limb loss. In human, most cases are sporadic but some families have affected relatives, suggesting a complex genetic etiology. In Mediterranean Italian River Buffalo, TH manifests as the absence of a variable portion of one or both distal limbs and appears to be inherited as an autosomal recessive. In 2008, the prevalence of TH cases was between 2 and 5% in Campania suggesting an allele frequency as high as 22%. We have developed an analytical pipeline for the detection of autosomal recessive disorders that involves the whole genome sequencing of cases and comparing the variants detected within the genome of cases to those found within controls. When the genome sequence of a single case is compared to those of 16 controls we find, on average, 827 homozygous variants in coding genes that are not found in the controls and when 2 cases are compared to 16 controls we find, on average, 36 candidate mutations. To identify the mutation responsible for TH, we sequenced 4 distantly related cases to an average depth of 12X coverage and obtained sequences on 14 control animals (5X-10X coverage) from the Buffalo Genome Sequencing Consortium. All sequences were aligned to the UMD3.1 bovine reference assembly and variants were called against bovine. Results will be presented at the Workshop.