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Home > Identification of Genes and Mutations Involved in Primary Microcephaly and Inherited Limb Disorders in Pakistani Families

Identification of Genes and Mutations Involved in Primary Microcephaly and Inherited Limb Disorders in Pakistani Families

Thesis Info

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Author

Farooq, Muhammad

Program

PhD

Institute

Quaid-I-Azam University

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2010

Thesis Completion Status

Completed

Subject

Natural Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/handle/123456789/719

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726395389

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Numerous genetic conditions have been described clinically but the molecular etiology for most of them is still unknown. With the advancement in the field of molecular biology powerful techniques have been developed to localize these conditions in the human genome and subsequent identification of causative genes. Functional analysis of causative genes leads to the discovery and understanding of novel genetic processes and pathways underlying disease conditions including normal developmental pathways. Linkage analysis studies in Mendelian disorders to identify the causative genes and mutations are possible using large pedigrees with multiple affected individuals. Analysis of alleles using microsatellite markers and genome wide SNPs lead to the discovery of novel genes and loci for specific disorders. The main aim of this thesis was to analyze families with autosomal recessive primary microcephaly and families with inherited limb disorders particularly polydactyl, syndactyly and brachydactyly to identify the causative mutations or chromosomal loci. Autosomal recessive primary microcephaly (MCPH) is a neurogenic disorder characterized by reduced head circumference (≤4 SD) and variable degree of mental retardation without any other neurological manifestations. The normal brain architecture is preserved despite the fact that brain size is reduced to three folds. In the first part of this study, genetic analysis of eleven primary microcephaly families was carried out. Linkage analysis using highly polymorphic microsatellite markers confirmed linkage in six families to ASPM (MCPH5), two CENPJ (MCPH6), one MCPH2 locus and haplotype analysis in two families demonstrated compound heterozygosity for ASPM. Sequencing of ASPM in six potentially linked families (MCP3, MCP6, MCP7, MCP9, MCP11 and MCP17) revealed six homozygous mutations in the affected subjects (A1160fs1181X, Y2245fs2258X, R3233X, Y3164X, S3186X, and R3244X respectively) and two possible compound heterozygous families (MCP35 and MCP18) demonstrated compound heterozygous mutations (W1326X/R3107X and R1019X/Q2632X, respectively). Compound heterozygous patients (W1326X & R3107X) also have additional clinical symptoms of seizures. Two families linked to MCPH6 locus (MCP21, MCP22) demonstrate 1bp deletion mutation c.17_18delC (T6fsX3) in exon 2 of CENPJ leading to premature termination of protein. This mutation was previously reported in two Northern Pakistani families. XVIFamily MCP15 established linkage to MCPH2 locus on chromosome 19q (19q13.1-q13.2). MCPH2 locus was defined by markers D19S416 and D19S420 which was about 7.6 cM in two consanguineous families from Northern Pakistan. However, the region is significantly reduced in MCP15 which is defined by markers D19S416 and D19S47. This substantially decreases the minimum critical interval from 7.6 cM to about 4.4 cM containing 162 genes. Family MCP36 has only single affected child. Molecular analysis using microsatellite markers revealed that affected individual is homozygous for the MCPH1 locus. By sequencing I have identified a novel nonsense mutation in exon 4 of MCPH1/microcephalin. The mutant protein lacks both of the C-terminal BRCT domains required for the normal functioning of protein during cell cycle progression and DNA repair mechanism. The second part of thesis comprised of genetic analysis of inherited limb disorders. Inherited limb malformations are genetically heterogeneous group of conditions with wide range of phenotypic manifestations. Inherited limb disorders occur as an isolated entity or syndromic form and are of clinical significance due to their severity and overall frequency. Limb development is a cascade of complex pathways involving patterning, growth and differentiation. Molecular characterization of inherited limb disorders may lead to the identification of novel genes and signalling pathways important for normal limb development during organogenesis. Family PD1 with preaxial polydactyly and triphalangeal thumb revealed autosomal dominant inheritance. Linkage analysis using microsatellite markers D7S550, D7S559 and D7S2423 was performed and maximum multipoint LOD score of 1.93 at recombination frequency θ= 0.1 was obtained. This region spans SHH and its cis-acting regulatory element (ZRS), which is well conserved among various species lying in intron 5 of LMBR1. Direct Sequencing of ZRS identified a novel point mutation (T>G) in ZRS element at base position 4976 in intron 5 of LMBR1. Many point mutations have been identified in ZRS leading to disruption of SHH expression during limb development leading to preaxial duplication in upper limbs. Electrophoretical mobility shift assay (EMSA) demonstrated a marked difference between wild and the mutant probe which uniquely bound a specific subset of nuclear transcription factors extracted from Caco-2 cells. It is suggested that altered transcription factor affinity may be important for our understanding of how single nucleotide substitutions in long distance regulatory elements changes cis-regulation of its target gene. XVIIGreig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder which affects limb and craniofacial development. GCPS was mapped to chromosome 7p13. Mutations in GLI3 had been described in GCPS patients. In the present study four novel GLI3 mutations in four distinct families have been identified. In family PD2, a single nucleotide substitution mutation [c.1702A>T (p.R568X)] leading to immediate stop codon in exon 13 is identified. Two base pairs deletion mutation [c.1853_1854delAC (p.Y618fs)] leading to frameshift and premature terminated protein product of 673 amino acids is identified in family PD316 in a family from Denmark. Both the mutations R568X and Y618fs lie in zinc finger domain in the first third of GLI3 producing truncated protein product which may affect the DNA binding property of zinc finger domain leading to possibly haploinsufficiency of GLI3. In family PD7, a novel C to T substitution at coding nucleotide 4574 (p.P1525L) in exon 15 of GLI3 is identified. The third mutation which is a missense (c.4574C>T (p.P1525L) lies in the last third of GLI3. Missense mutation P1525L lies in the C terminal region of GLI3 protein in the transactivation domain. In family PD16, at coding nucleotide position 3557, C to T substitution leading to missense incorporation of amino acid (p.P1186L) is identified. The variability in phenotype with respect to mutation in the affected family members may help to understand the phenotypic spectrum of GLI3 mutation. Brachydactyly is a rare and genetically heterogeneous disorder. In the present study a novel locus in a large consanguineous family with recessive form of brachydactyly type E is localized on chromosome 6p22.3 by homozygosity mapping using 10K SNP analysis. The physical linkage interval lies between 15,837,143 to 16,579,402 bp which is about 742 Kb. Maximum two point LOD score (Zmax) of 5.00 at recombination fraction (θ=0.0) was calculated at marker locus D6S18xAG. This region spans only seven genes including four pseudogenes. Sequencing of protein coding genes which include MYLIP, GMPR and ATXN1 did not reveal any mutation. Analysis using SNP6 array also did not identify any homozygous deletion or duplication in the region. However, smaller deletions or duplication (≤30 kb) cannot be excluded. Family PD14A shows cutaneous syndactyly of 3 rd and 4 th digit in hands. After exclusion using microsatellite markers on chromosome 2q34-q36 (syndactyly type I), 3p21.31 (zygodactyly), 2q31 (SPD1 locus, HOXD13), 6q22.31 (GJA1, syndactyly type III), 22q13.31 (SPD2), 14q11.2- q12 (SPD3) and 17p13.3 (syndactyly type IX) genome wide 10K SNP analysis was performed. XVIIIAfter fine mapping using microsatellite markers a single homozygous region on chromosome 9 flanked by markers, SNP_A-1518820 and D9S21AT (marker not available in Marshfield genetic map) was identified. The physical positions of flanking markers are 12018387 bp to 15340449 bp on chromosome 9 with maximum LOD score (Zmax) of 2.35 for given locus (θ=0.00). The region spans only 22 genes. A novel single nucleotide G to A substitution at coding nucleotide position 1289 (c.1289G>A) in exon eight leading to missense incorporation of glutamine instead of arginine at amino acid position 430 of Frem1 (p.R430Q) is identified. Arginine at 430 amino acid position of Frem1 is not only conserved among different vertebrate species but also conserved among Frem family of genes. However, possibility of missense mutation in Frem1 producing a defect in digit separation requires more families to study in addition to the functional studies in experimental models to prove the pathogenic nature of this mutation." xml:lang="en_US
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المبحث الثاني: عن المطر والإحساس

المبحث الثاني: عن المطر والإحساس

قصيدة (علی وقع المطر) ([1])لنازک الملائكة

أمطري، لا ترحمي طیفي في عمقِ الظلام
أمطري، صبّي علیّ السیل، یا روح الغمام
لا تُبالي([2]) أن تعیدیني علی الأرض حطام
وأحیلیني([3]) ، إذا شئت، جلیداً أو رُخام

أترکي ریح المساء الممطر الداجي([4]) تجنّ
ودعي الأطیار، تحت المطر القاسي، تئنّ
أغرقي الأشجار بالماءِ ولا یحزنکِ غصن
زمجري([5])، دويّ([6])، فلن أشکو، لن یأتیک لحن

أمطري فوقي، کماشئت، علی وجھي الحزین
لا تبالي جسدي الراعش، في کفّ الدجون([7])
أمطري، سیلي علی وجھي، أو غشيّ عیوني
بلّلي ما شئت کفيّ وشعري وجبیني

أغرقي، في ظلمۃ اللیل، القبور البالیہ([8])
وألطمي، ما شئتِ أبواب القُصور العالیہ
أمطري، في الجبل الناءي([9])، وفوق الھاویۃ([10])
أطفءي النیران، لا تُبقي لحيّ باقیہ


آہ ما أرھبکِ الآن، وقد ساد السکون
غیر صوتِ الرّیح، في الأعماق([11]) تدوي في جنون
لم تزل تھمي([12])، من الأمطار، في الأرض، عیون
لم یَزٖل، قلبي حزیناً، تحت أمواج الدُجون


أیّھا الأمطارُ، قد ناداکِ قلبي البشريُّ
ذلک المفرق في الأشواق، ذاک الشاعريّ
إغسلیہ، أم تری الحزنُ حماہ([13]) الأبديّ
أبداً ، مثلک یا أمطارُ، دفاقٌ نقيُّ

أبداً یسمعُ، تحت اللیل، وقعَ القطرات
ساھماً یحلم بالماضي وألغاز الممات
یسأل الأمطار: ما أنتِ؟ وما سرّ الحیاۃ؟
وأنا، فیم وجودي؟ فیم دمعي وشکاتي([14]

ما أنا؟ ما أنتِ یا أمطار؟ ما ذاک الخضمُّ(

Economic Policies of Pakistan During Military Rules an Analytical Study in Islamic Perspective

In the history of Pakistan, three military governments ruled for a quite long period. Nonetheless, they made no stable policies for the economic development of Pakistan. As a result, Pakistan could not make progress on strong foundation. Economic policies were meant for a shorter interval. Therefore, they only resulted in cosmetic change in the economy. The results of these policies were restricted to a certain class and the public suffered from the negative impact of these policies. Public welfare got secondary importance which resulted in economic inequality. Wealth circulated within a few families. In all the three reigns, no strategy was formed for the paying of external debt. On the one hand, the salvation of Pakistan was put at stake by increasing external debt and aid while on other hand, masses suffered added direct and indirect taxes. In these conditions, common man could not fulfill his basic necessities of life. Pakistan could make considerable progress, if any of the governments might have considered Islamic principles of Islam.

Genetic Basis of Water Stress Tolerance in Triticum Aestivum L.

Water stress is a key constraint in grain production of bread wheat. Using genomic and advanced breeding tools, genetic improvement in wheat and other crops is possible under water stress conditions. Fifty wheat genotypes were screened against water stress using traits like relative water contents and excised leaf water loss. Two water stress tolerant (Chakawal-50 and mairaj-08) and two susceptible (9436 and Millat-11) genotypes were identified and crossed to develop F1, F2 and backcross generations. Genetic analysis was conducted for relative water contents, excised leaf water loss, cell membrane thermo-stability, photosynthetic rate, stomatal conductance, CO2 assimilation, chlorophyll contents, plant height, flag leaf area, specific flag leaf area, peduncle length, spike length, number of tillers per plant, grain weight per spike, number of grains per spikes, 1000-grain weight, grain yield per plant and harvest index. Presence of additive, dominance and epistatic components were identified. Gene action was found complex showed that selection should be done in later generations for improvement of traits. Medium to high broad sense heritability, low to medium narrow sense heritability and low to high genetic advance was observed. Analysis under normal and water stress conditions revealed heterosis for relative water contents, excised leaf water loss, chlorophyll contents and plant height. Correlation analysis showed that relative water contents, cell membrane thermo-stability, photosynthetic rate and grain yield per plant were positively correlated with each other. Excised leaf water loss was negatively correlated with cell membrane thermo-stability, photosynthetic rate, relative water contents and grain yield per plant. A total of 30 ISSR markers were used to survey F2 population developed by a cross of Chakawal-50 (tolerance) and 9436 (susceptible) revealing a lots of DNA polymorphism. DNA polymorphism was used to construct genetic linkage map. In linkage analysis 73 bands produced from 4 ISSR markers were mapped on chromosomes. One QTL for photosynthetic rate on chromosome 3A, one for cell membrane thermo-stability on chromosome 2B and one for relative water contents on chromosome 4D was identified. These QTLs could be used to mold wheat genome into water stress tolerant wheat genome.