Clinical and Molecular Characterization of Human Hereditary Skeletal Disorders in Consanguineous Families

Abstract

Human skeletal dysplasias are genetically heterogeneous group of disorders that results from errors in bone, cartilage and joint development. They are characterized by abnormalities in patterning, differentiation, linear growth and maintenance of the human skeleton. A complex series of signaling cascades, including FGF, BMP, TGF β, WNT, Notch, Hedgehog, are essential for proper development of skeleton. The study presented in the dissertation describes clinical and molecular analysis often consanguineous families (A-J) showing different types of skeletal disorders. These families were collected from different remote regions of Pakistan. Individuals with diseased phenotype in each family were clinically examined by expert medical officers at government hospitals. Subsequently the disorders were characterized at molecular level by testing linkage to known genes, human genome scan and DNA sequencing using both exome and cycle sequencing methods. In two families (A, B) after failing to establish linkage to known genes responsible for causing postaxial polydactyly, human genome scan using SNP markers was performed to map the causative genes. In family A, the locus was mapped to a 37.0 Mb region on chromosome 8q21.13-q24.12. Further, whole exome sequencing (WES) identified the first disease causing sequence varient (c.478C>T, p.Arg160*) in the gene FAM92A1, mapped on chromosome 8q21.13-q24.12. In the other family, B, genome scan revealed two regions of 37.0 Mb and 22.3 Mb on chromosome 2p14- q12.1, and 6p22.3-p21.1, respectively. Maximum possible multiple LOD score of 1.92 was obtained with SNP markers on both the chromosomes. In family C, segregating syndactyly, human genome scan mapped a disease locus, flanked by SNP rs2829950 and rs566038, on chromosome 21q21.3-q22.3. Maximum multipoint LOD score of 2.52 was obtained with the SNP rs761372. Using polymorphic microsatellite markers, linkage in three families (D, E, F), segregating split hand and foot malformation in autosomal recessive manner, was established to SHFM-6 on chromosome 12q11-q13. Subsequently sequence analysis of the WNT10B gene led to the identification of two novel sequence varients 4-bp deletion (c.1165_1168delAAGT) in family D and 7-bp duplication (c.300_306dupAGGGCGG) in family E. Three other families (G, H, I), segregating Ellis–van Creveld syndrome, were clinically characterized to record range of features observed in the affected members. Genotyping using microsatellite markers established linkage in two families (G, H) on chromosome 4p16. DNA sequence analysis revealed two novel sequence varients, one each in EVC and EVC2 gene. A nonsense sequence varient (p.Trp234*) was detected in EVC2 in the family G and 15-bp duplication (c.1932_1946dupAGCCCTCCGGAGGCT) in the EVC gene in the other family H. Affected individuals in the family J showed clinical features of acromesomelic dysplasia type Maroteaux (AMDM), segregated in an autosomal recessive manner. Genotyping using microsatellite markers established linkage in the family to NPR2 gene on chromosome 9p13-q12. However, DNA sequence analysis of the gene failed to identify any potential sequence variant, which could be responsible for generating AMDM phenotype in the family. Work presented in the dissertation resulted in publications of the following articles. 1. Aziz A, Muhammad Ansar, Regie Lyn P. Santos-Cortez,Kwanghyuk Lee, University of Washington Center for Mendelian Genomics, Deborah A Nickerson, Jay Shendure, Michael Bamshad, Wasim Ahmad, Suzanne M Leal (2014). Exome sequencing identified a non-sense mutation in the gene FAM92A1. (In Preparation) 2. Aziz A, Raza SI, Ali S, Ahmad W (2014). Novel homozygous mutations in the EVC and EVC2 genes in two consanguineous families segregating autosomal recessive Ellis–van Creveld syndrome. Clinical Dysmorphology (Submitted) 3. Aziz A , Irfanullah, Khan S, Zimri FK, Muhammad N, Rashid S, Ahmad W (2014). Novel homozygous mutations in the WNT10B gene underlying autosomal recessive split hand/foot malformation in three consanguineous families. Gene 534: 265–271