Genetic analysis of skeletal disorders in a Pakistani population through whole exome sequencing

Introduction Genetic skeletal disorders are an inherited and heterogenous group of rare bone conditions with a prevalence rate of 1/5000 live births. In Pakistan, approximately 55% of genetic skeletal disorders are characterized by cases polydactyly, syndactyly, synpolydactyly, split hand and foot malformation. Over 19 different genetic skeletal disorders resulting from mutations in 43 genes have so far been reported in the Pakistani population. Whole exome sequencing (WES) is technique used to test the protein coding regions (exons) of a gene to identify disease causing variants. The technique is very useful when an underlying genetic disorder is suspected, but the genetic cause cannot be narrowed down to a finite list of candidate genes. The aim of this study is to identify pathogenic variants that define the diagnosis and etiology of various types of skeletal disorders in the Pakistani population. Methods 35 families with various skeletal disorders were recruited from different regions of Pakistan. The following data was collected: demographic data (age, sex, and ethnicity), clinical phenotype, age at diagnosis, radiological data, pedigree, and previous genetic tests. Blood samples affected and healthy individuals of the families were collected and DNA extracted by the phenol/chloroform extraction method and stored at -20ºC. Whole Exome Sequencing (WES) was performed (3 trios, 2 quad and 1 proband) at Macrogen Inc. – Korea (Library Truseq DNA library and TruSeq DNA Exome, Platform Novaseq). Sanger Sequencing was performed to confirm candidate gene variants and facilitate segregation analysis. Results So far, WES was performed in 6 selected families and a pathogenic homozygous mutation found in 1 consanguineous family (16.7%) with 3 siblings affected by a spondlylar-epi-metaphyseal dysplasia. We identified a known pathogenic missense variant (c.697G>A, p.Asp233Asn) in the GALNS gene which causes MPS type IV. Sanger sequencing revealed the 2 other affected siblings were homozygous for this variant, while an unaffected sibling and the parents were carriers. We have not been able to determine a genetic cause in 3 families and remaining families are under analysis. Conclusion We have identified the genetic cause of unexplained short stature in a consanguineous Pakistani family. Unaffected siblings have a 2/3s chance of being a carrier and genetic testing can assist in precisely determining carrier status. MPS IV is an autosomal recessive condition and a couple who are carriers have a 25% chance of a pregnancy affected by MPS IV. Enzyme replacement treatment is now available for this condition, but the cost is prohibitive and treatment is not available to all affected individuals in the world.