Clinical and Genetic Analysis of Human Hereditary Skeletal Disorders

Abstract

Skeletal dysplasia are a heterogenous group of disorders affecting specific skeletal elements or a generalized bone growth and development. They might be restricted to skeleton or involve other body systems to emerge as more complex anomalies. The genetic basis has been uncovered in a large number of congenital disorders that shows involvement of varied molecular pathways regulating skeletal patterning and growth. The advent of next generation sequencing has enabled a fast and more comprehensive genetic characterization of the observed phenotypes. The work presented in the dissertation describes clinical and genetic analysis of fourteen families (A-N) exhibiting skeletal disorders of different types. These families were recruited from different regions of the country. After clinical characterization, genetic analysis of these families was performed using microsatellite-based linkage analysis, Sanger sequencing, and exome sequencing. Five families (A, B, C, G, H) were tested for linkage to known genes/loci of the disorders using microsatellite markers. After establishing the linkage, mapped genes in the regions were sequenced following dideoxy chain termination method [Sanger Sequencing] to search for the disease-causing variants. Based on clinical diagnosis, three other families (E, J, L) were directly Sanger sequenced for the variants in the suspected culprit genes. Six families (D, F, I, K, M, N) were subjected to exome sequencing to search for the underlying causative genes. The study provided first time evidence of involvement of five genes (USP7, IGF2R, LRP6, PLCB1, MAGEB5) in developing specific skeletal disorders in human. The variant found in USP7 caused nonsyndromic postaxial polydactyly, in IGF2R nonsyndromic preaxial polydactyly and in LRP6 syndromic ectrodactyly (orofaciodigital syndrome). Variants found in two other genes PLCB1 and MAGEB5 segregated with a syndromic short stature (anemia and endocrine dysfunction). Four novel disease-causing variants were identified in four previously reported genes. ALX4 was found mutated in frontonasal dysplasia, TCTN3 in orofaciodigital syndrome IV, SCN9A in neuropathy short limb syndrome, and COL7A1 in ectodermal and skeletal phenotypes. Variants in SCN9A and COL7A1 were identified in the same family. VIII In addition, the present study revealed previously reported pathogenic variants in seven genes including IQCE in postaxial polydactyly, WNT1 in osteogenesis imperfecta, CTSK in pycnodysostosis, EXT2 in multiple hereditary exostosis, FGFR3 in achondroplasia, VPS13B and WRN in Cohen and Werner syndromes. The variants in the VPS13B and WRN were detected in the same family. In WNT1 gene, two different variants were identified in two separate families. In conclusion, the study of fourteen families, showing various forms of skeletal disorders, led to the identification of nine novel and eight previously reported disease causing variants in seventeen genes. Phenotypes in three families were the result of digenic inheritance. Disease-causing variants in five genes, causing various forms of skeletal disorders, have been reported for the first time in human. This study will support clinical diagnosis and genetic counselling in families segregating similar skeletal phenotypes in Pakistani and other populations. The work in this dissertation led to the publication of following articles: 1. Hayat A, Hussain S, Bilal M, Kausar M, Almuzzaini B, Abbas S, Tanveer A, Khan A, Siddiqi S, Foo JN, Ahmad F, Khan F, Khan B, Anees M, Mäkitie O, Alfadhel M, Ahmad W, Umair M (2020). Biallelic variants in four genes underlying recessive osteogenesis imperfecta. Eur J Med Genet 63:103954. [First three authors contributed equally to the study] 2. Hussain S, Umm-E-Kalsoom, Ullah I, Liaqat K, Nawaz S, Ahmad W (2020). A novel missense variant in the ALX4 gene underlies mild to severe frontonasal dysplasia in a consanguineous family. Genet Test Mol Biomarkers 24:217-223. 3. Hussain S, Nawaz S, Khan H, Acharya A, Schrauwen I, Ahmad W, Leal SM (2020). A splice site variant in TCTN3 underlies an atypical form of orofaciodigital syndrome IV. (In Preparation). 4. Hussain S, Liaqat K, Irfanullah, Nickerson DA, Bamshad MJ, University of Washington Center for Mendelian Genomics, Abdullah, Schrauwen I, Ahmad W, Leal SM (2020). LRP6 is a new gene for orofaciodigital syndrome. (In Preparation).