MOLECULAR GENETIC ANALYSIS OF SELECTED NEUROLOGICAL INHERITED DISEASES

Abstract

This study was conducted to identify the loci and genes responsible to cause congenital neurological inherited diseases in selective Pashtoon families of Khyber Pakhtunkhwa region of Pakistan. For this purpose, five consanguineous/tribal endogamy families (A-E) suffering from oculocutaneous albinism, usher syndrome, primary microcephaly, and isolated clinical anophthalmia were selected and pedigrees were drawn. Blood samples were collected with informed consent from affected, as well as normal members of these families, and screened for disease associated mutations. These families were analyzed for linkage to all the known loci of oculocutaneous albinism, usher syndrome, primary microcephaly, and isolated clinical anophthalmia, using microsatellite STR markers. Direct sequencing was performed to find out disease associated mutations in the candidate genes. Molecular genetic analysis of family A with oculocutaneous albinism and golden red hair at birth was mapped to MC1R locus on chromosome 16q24.1. A novel mutation c.917G>A of MC1R gene was found to be consisting with OCA2 phenotype in family A. The identification of c.917G>A mutation in Pakistani family and its direct association with OCA2 phenotype is the first demonstration of a mutation of MC1R gene responsible for causing OCA2 phenotype in humans. By genetic linkage analysis, family B with diseased phenotype of Usher syndrome was mapped to USH1F locus on chromosome 10q21.22 (USH1F), which harbors PCDH15 gene. On sequencing of the PCDH15 gene, a novel homozygous c.1304 A>C transversion mutation was identified to be associated with the usher phenotype in the USH1F mapped family. This c.1304 A>C mutation predicts an amino-acid substitution of aspartic acid with an alanine at codon 435 (p.D435A) of PCDH15 protein product.Two families C and D with primary microcephaly were mapped to ASPM gene locus. On mutation screening of ASPM gene by PCR amplification and direct DNA sequencing, a common c.3978G>A transition, was identified in exon 17 of ASPM gene to be responsible for diseased phenotype in both the families. The identified mutation results into the substitution of an amino acid residue at position 1326 from tryptophan to a stop codon (i.e., p.Trp1326Stop). The family E with isolated clinical anophthalmia was mapped to SOX2 gene, which is located at chromosome 3q26.3-q27. On exonic and regulatory regions mutation screening of SOX2 gene, no disease-associated mutation was identified. It shows that another gene responsible for the development of eye might be present at chromosome 3q26.3-q27 and need to be identified and screened for disease- associated mutation in this family. It was concluded that the disease phenotypes of families with oculocutaneous albinism, usher syndrome, primary microcephaly, and isolated clinical anophthalmia were mapped by genetic linkage analysis. The candidate genes (MC1R, PCDH15, ASPM and SOX2) in the mapped regions were screened for disease associated mutations by PCR amplification and direct DNA sequencing. The novel disease- associated mutations were identified in MC1R and PCDH15. The disease associated mutation identified in ASPM gene was also reported in several other families of Pakistani origin with primary microcephaly. However, no disease associated mutation was identified in SOX2 gene, which indicates that possibly another gene might be present in the mapped region for disease phenotype.