Clinical and Genetics Charactrization of human Hereditary Skeletal Deformities in Consanguineous Families

Abstract

Genetic skeletal disorders (GSDs) constitute a rare, heterogeneous, unique group of bone growth disorders affecting the homeostasis, development of bones, and resulting in anomalous size and shape of the skeleton. Syndromic and non-syndromic skeletal disorders epitomize a public health problem that affects 1/4000 individuals worldwide, thus leading to high health cost and poor quality of life. Detailed information about pathophysiologic mechanisms and disease-causing genetic defects is prerequisite in order to provide proper clinical intervention for different GSDs. With this inkling in mind, the present study was planned to investigate sixteen families manifested with GSDs from different populations at clinical and molecular levels. A total of sixteen families (A-P) segregating different forms of skeletal disorders were genetically and clinically characterized in the present study. The present study was performed in different steps, which included visit to remote areas in Pakistan to construct family pedigree, collection of blood samples, clinical (radiological) examination of at least two affected individuals in each family, genetic linkage analysis using STS microsatellite markers/SNPs microarray, whole exome sequencing (WES) and Sanger sequencing. The identified pathogenic variants were also analyzed for their pathogenicity using in-silico and in-vitro approaches. In the four families (A-D), after failing to establish linkage to the known genes/loci responsible for pre-axial and post axial polydactyly, WES was performed to identify the candidate causing gene. In family A, WES identified a homozygous splice acceptor site variant (c.395-1G>A) in intron 5 of IQCE gene on chromosome 7p22.3. In-vitro analysis using mini-gene splice assay in the family revealed a frameshift variant (p.Gly132Valfs*22). In family B, WES revealed a homozygous missense variant (c.223G>A; p.Asp75Asn) in a potential novel gene GLT8D1 (3p21.1). In family C, a nonsense mutation (c.84C>A: p.Tyr28*) was identified in the C9orf96 (STKLD1) gene mapped on chromosome 9q34.2. The C9orf96 is the first candidate gene identified to cause autosomal recessive non-syndomic pre-axial polydactyly. In family D with autosomal recessive uni-lateral pre-axial polydactyly, WES identified a Abstract XXVI novel biallelic deletion of ZNF468 and ZNF28 genes located on chromosome 19q13.41. In three families, segregating osteogenesis imperfecta, scanning human genome using SNP markers, mapped the causative homozygous region on chromosome 17q21.1- q21.31 in family E and F. Sequence analysis of the previously reported gene FKPB10 on 17q21.1-q21.31 led to the identification of a novel nonsense mutation (c.1490G>A; p. Trp497*) in family E and a previously reported missense variant (c.344G>A; p.Arg115Gln) in family F. In family G, using WES a homozygous splice acceptor site variant (c.359-3C>G) in the intron 2 of the WNT1 gene was identified, located on chromosome 12q13.12. Affected individuals in family H and I were diagnosed with acromesomelic dysplasia type Grebe (AMDG) phenotypes, and those in family J with acromesomelic dysplasia type maroteaux (AMDM). Linkage in families (H and I) was established to the GDF5 locus on chromosome 20q11.22, and in family J to the NPR2 locus on chromosome 9p13-q12. Subsequently, Sanger sequencing of GDF5 gene identified two novel homozygous variants, (c.157_158dupC and c.872G>A) in the family H and I. In family J, an already known homozygous splice donor site variant (c.2986+2T>G) was detected in intron 20 of the NPR2 gene. Three families (K-M), segregating split hand/foot malformation (SHFM) were investigated in the present study as well. Family K and L were subjected to whole genome SNP array analysis. In family K, SNP microarray identified two potential homozygous regions including a 35Mb on chromosome 12 and 11Mb on chromosome 4. This family will be subjected to WES upon availability of funds. Whole genome SNP array following WES in family L revealed a novel frameshift variant (c.409delA; p.Ser137Alafs*19) in the EPS15L1 gene located on chromosome 19p13.11. After establishing linkage in family M to WNT10B on chromosome 12q13.12, Sanger sequencing identified previously reported 7bp duplication (c.300_306dupAGGGCGG) in the WNT10B gene. Clinical and radiological examination of family N and family O showed typical Ellisvan Creveld syndrome (EvC) phenotypes. In family N, WES revealed two homozygous variants in the EVC2 (c.30dupC; p.Thr11Hisfs*45) and TMC1 gene Abstract XXVII (c.1696-1G>A). In family O, WES identified novel compound heterozygous variants (c.919T>C; p.Ser307Pro, c.2894+3A>G) in exon 7 and 20 of the EVC gene. Finally, clinical examination of affected members in family P displayed major features of Bardet-Biedl syndrome (BBS). DNA from an affected individual of the family was subjected to WES. A novel nonsense variant (c.119C>G; p.Ser40*) was identified in exon 1 of MKKS (BBS6) gene located on chromosome 20p12.2. In conclusion, the study, presented here, identified four novel candidate genes, novel and recurrent mutations in few previously reported genes causing different skeletal deformities. The identification of novel skeletal related genes not only improves the overall understanding of skeletal development system but also helps in creating new research dimensions such as understanding different pathways. GSDs results from mutations in different genes that encode different transcription factors (TFs), extracellular matrix proteins, signal transducers (channel proteins, receptors, ligands), RNA processing molecules, tumor suppressors, cellular transporters, enzymes (chaperones), binding proteins, cilia proteins, and numerous proteins having unknown function. The novel gene identified here, such as the IQCE share a common Hedgehog signaling pathway including the EVC/EVC2, thus helping in further understanding there important role in limb patterning, and skeletal developmental. The novel and recurrent mutations might help in the proper genotype–phenotype correlation which will help in prenatal testing and genetic counseling of the affected families. Further functional characterization of the genes, discovered here, is required to elucidate their roles in skeletal development and pathophysiology of myriad skeletal disorders.