Clinical and Genetic Characterization of Hereditary Skeletal Disorders in Families of Pakistani Origin

Abstract

Genetic disorders of the bones and joints, interrupting the bone development, leads to abnormal shape and size of the skeleton. In a vast variety of congenital disorders, genetic basis has been discovered, revealing the direct involvement of various molecular pathways that regulate limb patterning and development. The study, displayed in the dissertation, demonstrated clinical and genetic investigation of fifteen families exhibiting a variety of skeletal abnormalities. The study was carried out in stages, which included a visit to rural areas of Pakistan to construct a pedigree of the family, collection of the blood samples, clinical and radiological examination, linkage analysis, Exome-sequencing, Sanger sequencing, and in silico analysis. In five families, linkage to the candidate genes was established by typing polymorphic microsatellite markers. This was followed by Sanger sequencing to search for the disease-causing variants. In nine other families hunt for the sequence variants was carried out using exome sequencing of DNA of affected members. The selected variants in each family were tested by Sanger sequencing for their segregation in the families. Various bioinformatics tools were then used to test pathogenicity of the variants. In a few cases protein modelling was performed to study effect of the variants on protein structure and function. Analysis of the data, presented here, provided first evidence of involvement of five genes (IGFBP3, LRP6, STKLD1, EFCAB7, NIM1K) in developing distinct bone disorders. The variant found in IGFBP3 caused non-syndromic split hand/split foot malformation (SHFM), in LRP6 syndromic form of SHFM, in STKLD1 nonsyndromic pre-axial polydactyly, and in EFCAB7 and NIM1K non-syndromic postaxial polydactyly. In addition, eight novel disease-causing variants were identified in seven previously reported genes. This included WNT10B mutated in SHFM, KIAA0825 in post-axial polydactyly, LRP4 in cenani-lenz syndactyly, MEGF8 in carpenter syndrome, GJA1 in non-syndromic syndactyly, MKKS in Bardet Biedl syndrome (BBS), and SP7 in osteogenesis imperfecta. In three families, previously reported sequence variants were identified in WNT10B, and IQCE genes. The discovery of novel genes and disease-causing variants has not only broadened the mutation spectrum, but it will also facilitate genetic counselling in the present and other families segregating various forms of skeletal abnormalities. Such studies will particularly help various ethnic communities in Pakistan where cousin marriages are strictly followed. The research work, presented in the dissertation, led to the publication of following articles is as follows: