Analysis and diagnosis of targeted medical and genetic disorders in human population of Khyber Pakhtunkhwa, Pakistan

Abstract

Genes are essential elements of our genetic material “the DNA”. Normal function of each gene is important for the daily works of our body. Any alteration in the structure or function of genes is termed as “genetics” which may lead to structural or functional abnormalities of our body and organs. Alterations that mainly affect DNA can be transferred from parents to offspring’s and have to the potential to segregate in the subsequent generations. Thus, a DNA change may be persistent and has the potential to affect many generations continuously allowed to segregate. Genetic diseases that affect less than 1/2,000 individuals in Western countries are referred to “rare genetic diseases”. Pakistan in general, and Khyber Pakhtunkhwa in specific, constitutes ofmany subgroups i.e. socio-economic, ethnic or culturally isolated populations. In majority of cases, these people prefer to marry their closer relatives for example first degree cousins, ethnically matched subgroups or with whom residing for centuries in a same locality. These inbreeding brings DNA similarity among the upcoming generations and have very little chance to be genetically different from each other. This way once a person has got DNA alteration it may prevail generations-after-generations and in case of disease variants may lead to abnormalities that could not mend in the new-borns. These genetic alterations are screened by DNA sequencing and compared with the wild type DNA sequences to identify the causative variants. The field enormously emended in the very recent past by using Sanger sequencing and next generation sequencing tools and a basic tool for mutation detection in the disease carrying patients. Sanger sequencing is implied to targeted sequencing for small range screening analysing a few candidate genes, while whole exome sequencing and whole genome sequencing is used for large scale, economic and time saving experiments. The present dissertation describes clinical and molecular analysis of a consanguineous family from a remote village of Khyber Pakhtunkhwa, Pakistan. There were two ` xiv phenotypes a. palmoplantar keratoderma and b. intellectual disability presented by the family in a fashion that either the affected individuals carried both or only one. Palmoplantar keratoderma (PPK) phenotype was presented in three generations with autosomal dominant mode of inheritance. The patients were characterized the thickness of skin over the palms andsoles.They also had hyperkeratotic lesions restricted only to the pressure regions of palms or extend longitudinally along the fingers. Previously fourteen genes had been assigned to PPK phenotype. Whole exome sequencing followed by Sanger sequencing andin-silico bioinformatics analyses we found a novel heterozygous variant in COL20A1 gene (NM_020882.2, c. 392C > G; p.Ser131Cys) in all the affected individuals of the three generations. The alteration lied in the loop region close to fibronectin type III-1 domain of the collagen 20 α1 protein. This mutation was not found in the 219 unaffected healthy controls of Pakhtun ethnicity collected from various districts of Khyber Pakhtunkhwa. The variant was assigned “pathogenic” by in-silico prediction tools. To the best of our knowledge, this gene had not been associated with any human genetic disorders previously. Thus, we identified COL20A1 gene for the first time as a potential candidate to segregate with the disease phenotype of PPK in Pakistani family. The intellectual disability of segregated in autosomal recessive mode; however, no causative variant was assigned in the whole exome sequencing data. The causative variant might lie in deep intronic region for which we recommend whole genome sequencing. Next generation sequencing is becoming a powerful tool in molecular diagnostics and helping families with genetic disorders on-time and accurate results. Most the developed countries have already started WES analysis for newborn screening and genetic counselling programs. WES has the potential to be used as one of the basic genetic testing tool in the near future.