Abstract:
The main objective of the present investigation is to understand the molecular genetics of
autosomal recessive genetic disorders in the Pakistani population by studying primary
microcephaly and certain eye disorders i-e primary congenital aphakia, primary
congenital glaucoma and retinitis pigmentosa.
Autosomal recessive primary microcephaly (MCPH) is a neuro-developmental congenital
disorder in which the affected individuals have significantly reduced brain size
(occipitofrontal head circumference at least -4SD) accompanied with mild to moderate
intellectual impairment. Though MCPH has a single clinical phenotype, it is a genetically
heterogeneous disorder with seven loci reported to date (MCPH1 through MCPH7).
Twenty MCPH families (designated as MCP) were ascertained from various regions of
Pakistan. Linkage analysis was performed for all the families as a result of which five
families established linkage to MCPH2, one family to MCPH4, eight families were found
linked to MCPH5, two families to MCPH6, four families were found unlinked to any of
the reported locus, one of which was later found linked to the seventh locus MCPH7.
Since the underlying genes have not yet been identified for both MCPH2 and MCPH4,
candidate gene approach was used to find the gene responsible. Six potential candidate
genes (MAG, SIRT2, ZNF302, ZNF599, CHST8 and SNX26) were sequenced for MCPH2
linked families and nine genes (BUB1B, CHAC1, CHP, COPS2, FGF7, NUSAP1, PAK6,
RHOV and TYRO3) were selected and sequenced for the MCPH4 locus, which did not
reveal any pathogenic mutation. Since sequencing all the candidate genes for both these
loci was beyond the scope of this study, the high-throughput sequencing facility was then
used to sequence the candidate regions coupled with DNA capture experiments to capture
only the regions of interest rather than sequencing the entire genome.
XIIIEight families linked to MCPH5 which is the most common locus responsible for MCPH.
Mutation screening of the underlying gene ASPM in these families revealed 4 novel and 3
known mutations. The novel mutations identified in the present study are,
c.9677_9678insG, c.2938C>T, c.9595A>T and c.7894C>T, all leading to a premature
stop codon. Four families linked to this locus shared three known mutations
(c.8508_8509delGA, c.3978G>A and c.9730C>T) which have already been reported.
Two families (MCP10 and MCP26) established linkage to MCPH6, whose gene is
CENPJ. Extensive bi-directional sequencing of all the coding exons as well as exon-
intron boundaries did not revealed any pathogenic mutation.
Four MCPH families (MCP12, MCP23, MCP30 and MCP33) were initially found
unlinked to any of the known loci, and subjected to homozygosity mapping by using SNP
6.0 array. Large homozygous regions were identified across the genome which will be
further narrowed down to obtain the minimum critical regions. By the discovery of the
seventh locus (MPCH7), MCP12 was found to have linkage to the MCPH7 locus with the
underlying gene STIL. Bi-directional sequencing of all the coding exons of STIL has not
revealed any mutation.
Molecular genetic analyses of autosomal recessive eye disorders comprise the second
part of this dissertation. Eye disorders could either present as isolated entity or combined
with other phenotypes to have a syndromic manifestation. Inherited eye disorders are
genetically heterogeneous as they have a wide range of phenotypic outcome.
Family CT-1 was diagnosed to have primary congenital aphakia wih complete absence of
lens. Initially, screening for all thirteen autosomal recessive cataract loci was done, to
rule out surgical cataract removal, but the family was found linked to 1p34.3-p32.2
XIVharboring the gene FOXE3 which is responsible for causing aphakia. Sequence analysis
revealed a nonsense mutation c.720C>A, changing cysteine 240 to a stop codon
(p.Cys240X). The mutation was counter confirmed using a restriction enzyme DdeI.
Since this mutation has already been reported in a family from Madagascar, haplotype
analysis was done for both families which ruled out the ancestral origin of the mutation.
Family RP5 had the clinical diagnosis of primary congenital glaucoma with secondary
cataract. CYP1B1 is the candidate gene for this phenotype, whose sequence analysis
revealed a novel frameshift mutation 862insdelG>CC (p.A288fsX326). This frameshift
mutation leads to a premature stop codon truncation 38 amino acids downstream.
Families RP6 and RP7, initially diagnosed as early-onset blindness, were subjected to
SNP 6.0 array for homozygosity mapping to identify homozygous regions. Analysis of
RP6 family is still underway. However in family RP7, by combining the SNP 6.0 data
and marker analysis, a reported nonsense mutation (p.W278X) was found in the gene
AIPL1, known to be responsible for causing Leber Congenital Amaurosis.
The work presented in this thesis has been published in the following articles:
1. Aujum I, Eiberg H, Baig SM, Tommerup N, Hansen L (2010). A mutation in the
FOXE3 gene causes congenital primary aphakia in an autosomal recessive
consanguineous Pakistani family. Mol Vis 16:549-555.
2. Muhammad F, Mahmood Baig SM, Hansen L, Hussain MS, Anjum I, Aslam M,
Qureshi JA, Toilat M, Kirst E, Wajid M, Nurnberg P, Eiberg H, Tommerup N,
and Kjaer KW (2009). Compound heterozygous ASPM mutations in Pakistani
MCPH families. Am J Med Genet A 149A (5): 926-30.