Mutations Screening of Selected Genes Involved in Familial Cases of Skeletal Disorders from Khyber Pakhtunkhwa
INTRODUCTION: Human skeletal disorders result from abnormalities in the development of bone, cartilage, and joint. A chain of intricate signaling pathways (FGF, TGFβ, BMP, and WNT etc ;) are crucial for appropriate skeletogenesis. The human skeletal dysplasias result from abnormal regulation in the primary or secondary stage of these pathways as reviewed recently. Pathogenic variants of genes involved in metabolism within the cell, extracellular matrix, or regulation of transcription can result in dysregulation of cell-cell and cell-matrix signaling thereby altering cell division, differentiation, apoptosis and tissue patterning. The clinical phenotypes point to the major action of particular constituents of these pathways, although they work together to regulate the formation of bone, cartilage, and joint. OBJECTIVE: Mutation screening of potential candidate genes in familial disorders is well established through Sanger sequencing. In the present study, three families of skeletal disorders including two families of post axial polydactyly segregating disease phenotype in autosomal recessive fashion and a family with brachydactyly type D phenotype were investigated for the molecular pathogenicity. Mutation screening of HOXD13 in skeletal disorders was performed through Sanger sequencing. METHODOLOGY: After getting informed written consent and approved from the Institutional Review Board (IRB), KMU Peshawar, clinical Investigations of the selected families were performed in the Hayatabad Medical Complex, Peshawar. Blood Sampling was done in EDTA tubes, DNA extruded using Organic Phenol Chloroform Method. Polymerase Chain Reaction was performed in TPersonal thermo-cycler. Primers were designed using Primers 3 Software and DNA Sequencing done using Bi-Directional DNA sequencing kits (ABI) and Automated DNA analyzer ABI 3730. Sequencing was performed in the Core Facility Laboratory of Cincinnati Children Hospital Ohio, USA. BioEdit Software and ClustalW were used for Sequence Alignment. RESULTS: Mutation analysis of the pathogenic variant in families demonstrating autosomal recessive non-syndromic postaxial polydactyly and autosomal dominant non-syndromic brachydactyly phenotypes was performed by direct sequencing of HOXD13 gene as a potential candidate. Bidirectional sequencing of HOXD13 gene including all coding exons and intron-exons boundaries in an affected and an un affected individuals of each family was performed by automated sequencer ABI 3730XL DNA Analyzer (Life Technologies, USA). However, sequencing analysis of the affected individuals in each family did not reveal any pathogenic variant. CONCLUSION: Mutations screening, for molecular pathogenicity of HOXD13 gene in affected individuals of families with skeletal disorders including postaxial polydactyly and brachydactyly type D phenotypes were investigated and did not reveal pathogenic variant. KEY WORDS: Skeletogenesis, Pathogenic variants, Phenotype, Autosomal recessive, Non-syndromic