Clinical and Molecular Characterization of Human Hereditary Skeletal Deformities

Abstract

Research work, presented in the dissertation, described clinical and molecular analysis of twenty one families (A-U) segregating various types of skeletal deformities. Thirteen of these families (A-M) showed clinical features of dysostosis while eight others (N-U) represent condition of osteochondrodysplasia. After characterizing clinical spectrum in each family, genetic investigation, using various techniques, was conducted to search for the disease genes carrying the responsible sequence variants. Four families (A-D) presented features of split-hand/split-foot malformation (SHFM). Genotyping followed by Sanger sequencing or whole genome sequencing revealed four novel disease causing sequence variants in four different genes including WNT10B, DLX5, DLX6, and TP63. Five families (E-I) showed segregation of polydactyly in autosomal recessive pattern. In two of these families, E and F, whole genome and/or Sanger sequencing revealed two novel sequence variants (p.Arg113*, p.Leu506Glu) in the GLI1. This was the first report of involvement of GLI1 in causing skeletal disorder. In family G, SNPs based genotyping mapped the disease locus at chromosome 4p16.2-4p16.1 and 8q21.3- 8q21.3. However, exome sequencing failed to identify the variant segregating with the disease within the family. In another family (I), a novel locus for Polydactyly was mapped on chromosome 13q13.3–q21.2. Two families J and K presented features of isolated and syndromic form of brachydactyly, respectively. Direct Sanger sequencing revealed a deletion variant (p.Leu176Argfs*17) in the GDF5 in family J and a missense variant (p.Arg921Gln) in the TRPS1 in family K. Variable phenotypes of camptodactyly were found in two families (L and M). Genotyping using microsatellite markers established linkage in both the families at 17p13.3. However, sequence analysis revealed a novel homozygous indel variant (c.252_270delinsGCA; p.Phe85Glufs*108) only in family M. Eight families, presented here, showed various types of osteochondrodysplasia. Three of these families (N-P), segregated Bardet-Biedle Syndrome (BBS) in autosomal recessive pattern. The families N, O and P were mapped to BBS6 (20p12.2), BBS7 (4q27), and BBS8 (14q31.3), respectively. Sequence analysis revealed three novel homozygous mutations including p.Ala96Val in BBS6 in family N, p.Ala194del in BBS7 in family O, and p.Gln449His in TTC8 in family P. Two families Q and R showed frontonasal dysplasia segregating in autosomal recessive manner. SNPs micro-array followed by whole exome and Sanger sequencing revealed two novel disease causing variants including a non-sense (p.Gln202*) in the ALX3 in family Q and splice site (c.661-1G>C) in the ALX1 in family R. Two families, S and T, showed disproportionate short stature segregating in autosomal recessive pattern. Haplotype analysis established linkage of family S to the gene BMPR1B on chromosome 4q22.3. Sanger sequencing revealed a disease causing missense variant (p.Met397Arg) in the BMPR1B in the family. Microsatellite-based genotyping established linkage in the family T on chromosome 16q24.3. Sequence analysis detected a disease causing variant (p.Glu121Argfs*37) in the GALNS gene in family T. In family U, three affected individuals showed Waardenburg anophthalmia syndrome (Anophthalmia-syndactyly). SNPs based homozygosity mapping followed by Sanger sequencing revealed a homozygous missense variant (p.Cys271Tyr) in the SMOC1 gene in the family.