GENETIC MAPPING OF GENES INVOLVED IN AUTOSOMAL RECESSIVE MENTAL RETARDATION

Abstract

Mental Retardation (MR) or cognitive impairment is the most common and unsolved socio-economic problem in healthcare. MR is the condition with sub-average intellectual functioning, impairment in at least two of the adaptive skills (communication ability, self care, reading and writing ability etc) and onset before 18 year of age. Currently extensive knowledge exists regarding X-linked MR (217 loci and 98 genes) but only thirty loci and six genes for autosomal recessive NSMR have been discovered to date. In the current study five consanguineous Pakistani families were recruited for clinical and molecular analysis. Clinical investigations include the intelligence quotience (IQ) estimation (amended standard questionnaire), biometric data collection, computed tomography (CT) and magnetic resonance imaging scan (MRI) and biochemical testing. These investigations clearly indicate segregation of nonsyndromic mental retardation (NSMR) in these families except family A, which segregate autosomal recessive syndromic mental retardation. After clinical analysis, whole genome SNP genotyping with 500K Nsp array was carried out in the selected individuals of all families to perform homozygosity mapping followed by copy number analysis and microsatellite based genotyping. The clinical analysis of family A grossly showed myopathy (abnormal spectrum of EMG and elevated level of LDH enzyme), strabismus, neck webbing, facial deformities and poor developmental milestone. The family A was mapped to MRT5 locus, spanning over the region of 2.51 Mb [from 5,145,028 to 7,657,537 bp {May 2004 (NCBI35/hg17)}]. The subsequent mutation analysis of candidate genes identified a novel misssense (c.2100G>A) mutation in exon 19 of NSUN2 gene, which leads to substitution of Gly (GGA) with Arg (AGA) at amino acid position 679 (p.G679R). The subsequent expression studies of mutated NSUN2 encoded protein exhibited abnormal expression in cytoplasm indicating an important role of glycine residue, in protein localization and biogenesis. The family B with severe MR associated with speech disability and aggressive attitude was presented as segregating NSMR. Whole genome scan mapped this family to a 12.494 Mb region on chromosome 8. The identified HBD interval was flanked by rs6989820 and rs2237834 and harbors MCPH1 and TUSC3 genes, but sequencing of MCPH1 gene failed to reveal presence of pathogenic variant in the affected individuals of this family. The CNV analysis of the whole genome data identified a novel 170.63 Kb {Feb.2009 (GRCh37/hg19)} deletion in TUSC3 gene spanning entire gene except its promoter and first exon. Gross clinical spectrum (skeletal, microcephalic, neurologic symptoms) of family C was normal therefore it was treated as nonsyndromic MR family. This family was mapped to 2p25.3-p25.2 locus during exclusion mapping of known loci of ARNSMR. The identified region spans over the length of 6.57 Mb with a centromeric boundary defined by a recombination event involving markers D2S281 and D2S2166. This region has 21 protein coding genes but the sequence analysis of eight candidate genes in the linked interval did not revealed any pathogenic mutation. Molecular analysis of family D identified multiple homozygous regions on chromosomes 1, 3, 10, 20 and 22 (with positive hits on the basis of SNP data of four individuals) but the microsatellite genotyping in complete family ruled out loci on chromosome 1, 3 and 10. The linkage analysis, performed for all characterized homozygous loci, generated highest LOD score of 2.1039 at D20S602. Region of homozygosity on chromosome 20 was flanked by SNPs rs6140226 and rs6074396, delineating a minimum critical region of 4.998 Mb {Feb.2009 (GRCh37/hg19)}. This minimum critical region contains BTBD3 gene but the sequence analysis ruled out the presence of any pathogenic variant in the affected individuals of family D. So these findings either indicate involvement of other gene in this region or leading to the complex genetics by considering loci on chromosome 20 and 22 as responsible factors. The homozygosity mapping of family E revealed a single short HBD region on 2p12 from rs17020436 to rs11678145 which delineated a 1.1 Mb region. This short interval harbor one gene, four mRNAs and three spliced ESTs. The genetic player responsible for MR in this family can be identified by using next generation sequencing of the identified HBD to screen the pathogenic variants segregating with disease phenotype. The present work has shown that genetic players are associated with the significant number of Pakistani families with MR. The current study provides further support regarding genetic heterogeneity of MR in Pakistan, thus is a significant contribution towards the elucidation of common and emerging molecular pathways related to cognition/learning. Our study also signifies the importance of methylation in the development and functioning of the cognition and it is anticipated that additional players will be identified in future studies which have similar functions. The work presented in this thesis results in the following publications 1. Khan MA, Rafiq MA, Noor A, Ali N, Ali G, Vincent JB, Ansar M. A novel deletion mutation in the TUSC3 gene in a consanguineous Pakistani family with autosomal recessive nonsyndromic intellectual disability. BMC Med Genet 2011; 22;12(1):56. 2. Rafiq MA, Ansar M, Marshall CR, Noor A, Shaheen N, Mowjoodi A, Khan MA, Ali G, Amin-ud-Din M, Feuk L, Vincent JB, Scherer SW. Mapping of three novel loci for non-syndromic autosomal recessive mental retardation (NS-ARMR) in consanguineous families from Pakistan. Clin Genet 2010;78:478-83. 3. Khan MA, Rafiq MA, Noor A, Ansar M, Windpassinger C, Vincent JB. A novel gene, NSUN2, causes a new MR syndrome in a Pakistani origin family. Manuscript in preparation for submission to Nature Genetics.