The work presented in the dissertation includes genetic study of two of the several inherited disorders prevalent in Pakistani population.
Duchenne Muscular Dystrophy (DMD) and Becker Muscular dystrophy (BMD) are allelic X linked recessive disorders that cause progressive muscles degeneration. DMD is one of the most common and lethal inherited childhood muscle disorder of human worldwide. Clinically, DMD patients show delayed onset of walking, a progressive weakness with difficulty in running, climbing stairs and jumping, frequent falls, and marked calf hypertrophy. The progressive muscle degeneration results in the loss of ambulation at about age 10 and death usually before the end of the second decade due to respiratory or cardiac failure. The milder BMD variant shows a less severe but more variable phenotype, ranging from a slightly less severe DMD-like condition to a very mild condition.
DMD/BMD is caused by mutation in the dystrophin gene present at Xp21. The 2.6 MB dystrophin gene has 79 exons and is the largest known human gene. Dystrophin, the protein product of DMD/BMD locus is a 427 kDa rod shaped cytoskeletal protein that is located at the cytoplasmic face of muscle sarcolemma. Absence of dystrophin protein leads to sarcolemmal instability, muscle fragility, contraction induced damage and muscle fibers degeneration. In DMD muscle fibers, dystrophin is completely absent. The milder allelic disorder Becker muscle dystrophy is caused by mutations that result in reduced expression of dystrophin or expression of truncated partially functional forms of the protein.
About 60% of all DMD and BMD cases are due to gross gene deletions, 5% to duplications, and remaining 35% to point mutations. Gross gene deletions are the most frequent mutations in the dystrophin gene. Two deletion hotspots are localized to the proximal region of the gene extending from exon 1 to 19 and to the central region extending from exon 44 to 60. The frequency and pattern of exon deletions have been reported in different world populations. Not only is the distribution of intronic deletion breakpoints significantly different among the regional groups, their incidence also varies in different populations.
A total of 181 DMD and 4 BMD patients were recruited for this study. All patients were diagnosed as having DMD on the basis of the clinical course, high levels of serum creatine kinase, and histological findings of muscle biopsy specimens, electromyography, and known family histories. Deletions in the dystrophin gene were searched by Polymerase Chain Reaction (PCR)-based amplification of 16 exons from the proximal and distal hotspot regions of the gene. Intragenic deletions were detected in 42.16% of patients with 41.02% of deletions in the proximal hotspot and 58.97% in the distal hotspot region of the gene. The most frequently deleted exons were 50 and 47. The present analysis of dystrophin gene deletions in Pakistani populations, the first of its kind in this population, proposes a relatively low percentage of deletion mutations than that reported in most of other world populations.
Autosomal Recessive Primary Microcephaly (MCPH) is a neuro-developmental disorder characterized by severe underdevelopment of the brain cerebral cortex during fetal life. The brain is small but architecturally normal. Clinically MCPH is defined as congenital microcephaly with head circumference at least 3-4 standard deviations below age and sex mean accompanied by variable degree of mental retardation and no other neurological findings except presence of fits in rare cases.
MCPH demonstrate genetic heterogeneity and to date six loci (MCPHI-MCPH6) and four genes have been identified. These MCPH genes are believed to be involved in cell cycle regulation (Micro cephalin & CDK5RAP2), organization and orientation of mitotic spindle fibers (ASPM) and centrosomal production of microtubules (CENPJ) during neurogenesis.
For the present study, five families (A, B, C, D, E) showing primary microcephaly were ascertained from different regions of Pakistan. Patients were diagnosed on the basis of measurements of their head circumference, presence of variable degree of mental retardation and the absence of other neurological, metabolic or congenital defects. DNA samples of both affected and normal individuals from all the five families were tested for linkage to the known MCPH loci. Four of these families (A, B, C, D) showed linkage to the MCPH5 on chromosome lq31 and family E linked to MCPH2 on chromosome 1913.1-q13.2.
Sequence analysis of ASPM gene was performed in four families (A, B, C, D) that showed linkage to MCPH5 locus, however, pathogenic mutations were detected only in families B and D. In family B, a C to G transition was detected at nucleotide position 9557 (C9557G) in exon 23, leading to premature termination codon at amino acid 3186 (S3186X). In family D, sequence analysis of exon 21 of ASPM gene revealed an insertion of four nucleotides CATT at nucleotide position 9118 (9118insCAAT), leading to a frameshift and premature termination codon 5 bp downstream in the same exon. The mutation, S3186X, identified in family B was reported earlier in another Pakistani family by Bond et al. (2003). The second mutation, 9118insCAAT, detected in family D, however, represents a novel mutation. Failure to identify pathogenic mutations in families A and C, linked to MCPH5 locus, suggest that either the mutations are located in the regulatory sequences or that a second gene causing MCPH exists at this locus.
The family E, in the present study, showed linkage to MCPH2 locus on chromosome 19q13.1-q13.2. So far a candidate gene has not been identified at this locus. Haplotypes for micro satellite markers linked to MCPH2, generated in family E, failed to narrow down the reported defined minimum critical region for this locus.
Based on the research work, presented in the thesis, the following manuscripts are in preparation stage for submission to ‚€œMolecules and Cell‚€Ě and ‚€œJournal of Molecular Medicine‚€Ě for publications.
1. Revealing Intragenic Deletions in Dystrophin Gene: Screening 16 Exons of the Gene in 185 Pakistani Children with Duchenne/Becker Muscular Dystrophy
2. Novel Sequence Variants in ASPM Gene in Families with Autosomal Recessive Primary Microcephaly