Next-generation sequencing (NGS) of exomes and genomes has accelerated the identification of genes involved in Mendelian phenotypes. However, many NGS studies fall short of identifying causal variants, with estimates for success rates as low as 25% for uncovering the pathological variant underlying disease etiology. An important reason for such failures is familial locus heterogeneity, where within a single pedigree causal variants in two or more genes underlie Mendelian trait etiology. As examples of intra- and inter-sibship familial locus heterogeneity, we present 10 consanguineous Pakistani families segregating hearing impairment due to homozygous variants in two different hearing impairment genes and a European-American pedigree in which hearing impairment is caused by four variants in three different genes. We have identified 41 additional pedigrees with syndromic and nonsyndromic hearing impairment for which a single previously reported hearing impairment gene has been identified but only segregates with the phenotype in a subset of affected pedigree members. We estimate that locus heterogeneity occurs in 15.3% (95% confidence interval: 11.9%, 19.9%) of the families in our collection. We demonstrate novel approaches to apply linkage analysis and homozygosity mapping (for autosomal recessive consanguineous pedigrees), which can be used to detect locus heterogeneity using either NGS or SNP array data. Results from linkage analysis and homozygosity mapping can also be used to group sibships or individuals most likely to be segregating the same causal variants and thereby increase the success rate of gene identification.

Osteogenesis imperfecta (OI, MIM 610968) is a genetically and clinically heterogeneous disorder characterized by bone fragility. It is one of the rare forms of skeletal deformity caused by sequence variants in at least 14 different genes, including FKBP10 (MIM 607063) encoding protein FKBP65. Here we present three consanguineous families of Pakistani origin segregating OI in an autosomal-recessive pattern. Genotyping using either single-nucleotide polymorphism markers by Affymetrix GeneChip Human Mapping 250K Nsp array or polymorphic microsatellite markers revealed a homozygous region, containing a candidate gene FKBP10, among affected members on chromosome 17q21.2. Sequencing the FKBP10 gene revealed a homozygous novel nonsense variant (c.1490G>A, p.Trp497*) in the family A and two previously reported variants, including a missense (c.344G>A, p.Arg115Gln), in the family B and duplication of a nucleotide C (c.831dupC, p.Gly278ArgfsX295) in the family C. Our findings further extend the body of evidence that supports the importance of FKBP10 gene in the development of skeletal system.

Autosomal recessive hypotrichosis is a rare genetic irreversible hair loss disorder characterized by sparse scalp hair, sparse to absent eyebrows and eyelashes, and sparse axillary and body hair. The study, presented here, established genetic linkage in four families showing similar phenotypes to lysophosphatidic acid receptor 6 (LPAR6) gene on chromosome 13q14.11-q21.32. Subsequently, sequence analysis of the gene revealed two previously reported missense mutations including p.D63V in affected members of one and p.I188F in three other families. Molecular modeling and docking analysis was performed to investigate binding of a ligand oleoyl-L-alpha-lysophosphatidic acid (LPA) to modeled protein structures of normal and mutated (D63V, G146R, I188F, N248Y, S3T, L277P) LPAR6 receptors. The mutant receptors showed a complete shift in orientation of LPA at the binding site. In addition, hydropathy analysis revealed a significant change in the membrane spanning topology of LPAR6 helical segments. The present study further substantiated involvement of LPAR6-LPA signaling in the pathogenesis of hypotrichosis/woolly hair and provided additional insight into the molecular mechanism of hair development.

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