Muhammad Arshad Rafiq

Centre for Addiction and Mental Health, Toronto, Ontario, Canada

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Publications (50)211.25 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Background Autosomal recessive intellectual disability (ID) is genetically heterogeneous and most of the genes causing it remain undiscovered. Objective We have ascertained 11 consanguineous families multiplex for IDs in order to identify new loci for autosomal recessive genes for non-syndromic ID, or to aid pinpointing mutations in known causative gene/loci. Methodology Microarray genotyping (Affymatrix 250K) was performed to identify homozygosity-by-descent (HBD) in all affected families. Results Analysis of genotypes revealed 45 potential HBD regions across the families, although these may be rationalised down to 39. Two families share an overlapping HBD region on 7q11.21. In one family, X-linkage also looks plausible, and a new ID gene near the centromere may be a likely cause. In one family, no HBD region was found, and thus we exclude autosomal recessive mutation as the likely cause in this family. Copy-number variation (CNV) was also performed and revealed no CNVs, homozygous or heterozygous, segregating with the phenotype. Conclusion The homozygous loci identified in this study might harbour candidate genes for ID in these studied families. Therefore, we are proceeding with next-generation sequencing analysis of the families, using whole-exome approaches, and anticipate that this will identify the causative gene/mutation within the identified HBD regions for many of the families studied here.
    Acta Neuropsychiatrica 12/2014; · 0.61 Impact Factor
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    ABSTRACT: Intellectual disability (ID) is a genetically heterogeneous disorder with more than 50 mutated genes to date. ID is characterized by deficits in memory skills and language development with difficulty in learning and problem solving. It affects ~2% of population with difference in severity based on the Intelligence quotient (IQ) scores from mild forms with IQ range from 50 to 70, to profound forms with IQ below 20-25. For detection of disease-causing mutation in such a heterogeneous disorder, exome sequencing is a powerful tool as almost all known gene can be assessed simultaneously in a high-throughput manner. In this study, we performed exome-sequencing for several families with non-syndromic autosomal recessive ID (NS-ARID). After genotyping and exome sequencing, several interesting genes were detected. For example, a homozygous missense mutation was detected in LRP2 gene in a Pakistani patient with mild ID from a marriage between second cousins. The variation was absent in 400 ethnically matched healthy control chromosomes and is not listed in SNP databases. The LRP2 mutation identified here is located in one of the LDL-receptor class Adomains which is a cysteine-rich repeat that plays a central role in mammalian cholesterol metabolism suggesting that alteration of cholesterol processing pathway can lead to intellectual disability. In another Pakistani family, a homozygous missense mutation in NUP205 gene was detected in a female patient with mild ID. Sanger sequencing analysis showed complete segregation within the family. Functional studies are ongoing to verify the pathogenicity of variants detected. Here we describe the use of autozygosity mapping and whole exome sequencing to identify new genes for NS-ARID in several families. Due to the widely use of exome sequencing where all the genes are assessed at once, more genes for ID are identified leading to identification of underlying pathways.
    American Society of Human Genetics, San Diego; 10/2014
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    ABSTRACT: Dendritic spines represent the major site of neuronal activity in the brain, serving as the receiving point for neurotransmitters, and undergoing rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next generation sequencing in two consanguineous families segregating non-syndromic autosomal recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein in the formin family of actin cytoskeleton nucleation factors, highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites, and that germline inactivation of mouse Fmn2, previously demonstrated to have a conditioned fear-learning defect, showed decreased spine density. Furthermore, patient Induced Pluripotent Stem Cell (IPSC)-derived neural cells showed correlated decreased synaptic density. Thus FMN2 mutations link intellectual disability either directly or indirectly to actin-mediated synaptic spine density regulation.
    The American Journal of Human Genetics 10/2014; · 11.20 Impact Factor
  • Clinical Genetics 07/2014; · 4.25 Impact Factor
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    ABSTRACT: In this study, we have performed autozygosity mapping on a large consanguineous Pakistani family segregating with intellectual disability. We identified two large regions of homozygosity-by-descent (HBD) on 16q12.2-q21 and 16q24.1-q24.3. Whole exome sequencing (WES) was performed on an affected individual from the family, but initially, no obvious mutation was detected. However, three genes within the HBD regions that were not fully captured during the WES were Sanger sequenced and we identified a five base pair deletion (actually six base pairs deleted plus one base pair inserted) in exon 7 of the gene FBXO31. The variant segregated completely in the family, in recessive fashion giving a LOD score of 3.95. This variant leads to a frameshift and a premature stop codon and truncation of the FBXO31 protein, p.(Cys283Asnfs*81). Quantification of mRNA and protein expression suggests that nonsense-mediated mRNA decay also contributes to the loss of FBXO31 protein in affected individuals. FBXO31 functions as a centrosomal E3 ubiquitin ligase, in association with SKP1 and Cullin-1, involved in ubiquitination of proteins targeted for degradation. The FBXO31/SKP1/Cullin1 complex is important for neuronal morphogenesis and axonal identity. FBXO31 also plays a role in dendrite growth and neuronal migration in developing cerebellar cortex. Our finding adds further evidence of the involvement of disruption of the protein ubiquitination pathway in intellectual disability.
    Human Genetics 03/2014; · 4.63 Impact Factor
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    ABSTRACT: Mirror movements (MRMV) are involuntary movements on one side of the body that mirror voluntary movements on the opposite side. Congenital mirror movement disorder is a rare, typically autosomal-dominant disorder, although it has been suspected that some sporadic cases may be due to recessive inheritance. Using a linkage analysis and a candidate gene approach, two genes have been implicated in congenital MRMV disorder to date: DCC on 18q21.2 (MRMV1), which encodes a netrin receptor, and RAD51 on 15q15.1 (MRMV2), which is involved in the maintenance of genomic integrity. Here, we describe a large consanguineous Pakistani family with 11 cases of congenital MRMV disorder reported across five generations, with autosomal recessive inheritance likely. Sanger sequencing of DCC and RAD51 did not identify a mutation. We then employed microarray genotyping and autozygosity mapping to identify a shared region of homozygosity-by-descent among the affected individuals. We identified a large autozygous region of ~3.3 Mb on chromosome 22q13.1 (Chr22:36605976-39904648). We used Sanger sequencing to exclude several candidate genes within this region, including DMC1 and NPTXR. Whole exome sequencing was employed, and identified a splice site mutation in the dynein axonemal light chain 4 gene, DNAL4. This splice site change leads to skipping of exon 3, and omission of 28 amino acids from DNAL4 protein. Linkage analysis using Simwalk2 gives a maximum Lod score of 6.197 at this locus. Whether or how DNAL4 function may relate to the function of DCC or RAD51 is not known. Also, there is no suggestion of primary ciliary dyskinesis, situs inversus, or defective sperm in affected family members, which might be anticipated given a putative role for DNAL4 in axonemal-based dynein complexes. We suggest that DNAL4 plays a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, and in commissural neurons of the corpus callosum in particular. This, in turn, could lead to faulty cross-brain wiring, resulting in MRMV.
    Human Genetics 01/2014; · 4.63 Impact Factor
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    ABSTRACT: Autosomal recessive causes of intellectual disability (ARID) have, until very recently, been under-researched due to the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. We have ascertained more than 165 multiplex, consanguineous ARID families from Pakistan. These families are selected for lack of obvious syndromic features. Our strategy includes genotyping family members on genome-wide single nucleotide polymorphism microarrays, looking for large regions of shared homozygosity (and haploidentity) between affected individuals (homozygosity-by-descent, or autozygosity). We also screen for potential disease-related CNVs- either as a shared homozygous genotype, or heterozygous as a potential cause of phenocopy. We firstly exclude any known ARID genes in HBD regions, then either select candidates from within the HBD region for mutation screening by Sanger sequencing, or we embark on whole exome sequencing to identify disease mutations. Our successes include a number of new genes for apparent non-syndromic ARID, such as MAN1B1, TRAPPC9, NSUN2, as well as new genes for syndromic forms or ARID, such as Joubert syndrome (CC2D2A and TCTN2), and many known ARID genes (TUSC3, TPO, VPS13B, PEX1, PSPH, PMM2). Here we describe the use of autozygosity mapping and whole exome sequencing to identify an additional 6 new genes for NS-ARID. As more and more genes for ID are identified, using these and other strategies, we are building a picture of the biological pathways that, when perturbed, may lead to intellectual disability.
    American Association of Human Genetics, Boston; 10/2013
  • World Congress of Psychitric Genetics, Boston; 10/2013
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    ABSTRACT: Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.
    The American Journal of Human Genetics 04/2012; 90(5):856-63. · 11.20 Impact Factor
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    ABSTRACT: Intellectual disability (ID), or mental retardation (MR), is a neurodevelopmental disorder that has a huge impact on the health care system. Here we report a study on non-syndromic autosomal recessive intellectual disability (NS-ARID) in a consanguineous Pakistani family. Two affected and one unaffected members of a family were genotyped using Affymetrix 500K single-nucleotide polymorphism (SNP) microarrays. Analysis of microarray data identified three genomic regions (11q24.1-q25, 14q11.2 and 17q24.2-q24.3) that were homozygous-by-descent (HBD) in both affected individuals. All family members (2 affected and 6 unaffected) were genotyped by using highly polymorphic microsatellite markers present in three regions of HBD discovered by microarray analysis. By using this approach we were able to exclude HBD for the 14q11.2 and 17q24.2-q24.3 regions. Using whole exome sequence capture followed by next generation sequencing on the SOLiD 4, we identified a homozygous splice donor site G>A mutation in both affected individuals in a gene on 11q24.2. Sequencing of cDNA from both affected individuals revealed a new splice site 44 nucleotide downstream in the intronic region. Consequently, 15 extra amino acids are added to the protein, and are predicted to disrupt crucial protein function. Studies to measure the effect of mutation on protein activity are being conducted.
    American Association of Human Genetics annual meeting; 01/2012
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    ABSTRACT: We have used genome-wide genotyping to identify an overlapping homozygosity-by-descent locus on chromosome 9q34.3 (MRT15) in four consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability (NS-ARID) and one in which the patients show additional clinical features. Four of the families are from Pakistan, and one is from Iran. Using a combination of next-generation sequencing and Sanger sequencing, we have identified mutations in the gene MAN1B1, encoding a mannosyl oligosaccharide, alpha 1,2-mannosidase. In one Pakistani family, MR43, a homozygous nonsense mutation (RefSeq number NM_016219.3: c.1418G>A [p.Trp473*]), segregated with intellectual disability and additional dysmorphic features. We also identified the missense mutation c. 1189G>A (p.Glu397Lys; RefSeq number NM_016219.3), which segregates with NS-ARID in three families who come from the same village and probably have shared inheritance. In the Iranian family, the missense mutation c.1000C>T (p.Arg334Cys; RefSeq number NM_016219.3) also segregates with NS-ARID. Both missense mutations are at amino acid residues that are conserved across the animal kingdom, and they either reduce k(cat) by ∼1300-fold or disrupt stable protein expression in mammalian cells. MAN1B1 is one of the few NS-ARID genes with an elevated mutation frequency in patients with NS-ARID from different populations.
    The American Journal of Human Genetics 07/2011; 89(1):176-82. · 11.20 Impact Factor
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    ABSTRACT: We present measurements of the excitation spectra from the 2p53p [5/2]3,2 levels in neon using two-step laser excitation and ionization in conjunction with an optogalvanic detection in dc and rf discharge cells. The 2p53p [5/2]3,2 intermediate levels have been approached via the collisionally populated 2p53s [3/2]2 metastable level. The Rydberg series 2p5(2P3/2)nd [7/2]4 (12
    Physical Review A 07/2011; 84. · 3.04 Impact Factor
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    ABSTRACT: Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.
    Cell 05/2011; 145(4):513-28. · 31.96 Impact Factor
  • Cell 05/2011; 145(4):513. · 31.96 Impact Factor
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    ABSTRACT: We have used genome-wide genotyping to identify a homozygosity-by-descent locus in a consanguineous Pakistani family with three intellectual disability (ID) plus distal myopathy to a 2.6Mb region on 5p15.32-p15.31, and have identified a missense mutation, Gly679Arg, at a conserved residue within the gene NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA), as well as functioning in spindle assembly during mitosis as well as chromosome segregation. The Gly679 residue in orthologous proteins is highly conserved across the animal kingdom. Analysis of the Gly679Arg mutation through Myc-tagged constructs carrying the mutation and overexpressed in HeLa, COS7 and HCC1954 breast cancer cells indicates that the effect of the mutation at the cellular level appears to be the prevention of NSUN2 localizing within the nucleolus, and occasionally the nucleus also. Thus it is likely that the role of NSUN2 within the nucleolus is crucial to normal neurodevelopmental processes.
    American Association of Human Genetics annual meeting; 01/2011
  • AJHG. 01/2011;
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    ABSTRACT: Intellectual disability (ID), or mental retardation (MR), is a neurodevelopmental disorder that has a devastating impact on the affected individuals and their families, as well as on health and social system. Here we report a study on non-syndromic autosomal recessive intellectual disability (NS-ARID) in consanguineous Pakistani families. Seven families (MR28, MR62, MR64, MR70, MR72, MR74 and PK11) were genotyped using Affymetrix 500K single-nucleotide polymorphism (SNP) microarrays. This approach allowed us to identify seven homozygous-by-descent (HBD) loci: 18q22.3-q23, 7p14.1-q11.22, 8q23.1-q24.21, 6q13-q16.2, 16q12.2-q21, 8p11.21-q13.2 and 11p11.2-q13.4.Three out of the seven loci mapped (8q23.1-q24.21, 6q13-q16.2 and 11p11.2-q13.4) overlap with previously reported studies and reduce the critical HBD region. Three other loci (18q22.3-q23, 16q12.2-q21 and 8p11.21-q13.2) have not been reported previously. We have sequenced DNA from four families (MR70, MR72, MR74 and PK11) by whole exome sequencing and exome data is being analyzed to discover disease causing mutations. Genes within the HBD region for family MR64 on 8q23.1-q24.21 have been sequenced and no causative mutation found so far. Sequencing of all genes within the HBD region discovered in family MR28 did not expose any exonic or splice junction changes. The remaining locus of MR62 (7p14.1-q11.22) family already mapped in patients with phosphoserine phosphatase (PSPH) deficiency, which is responsible for mental retardation and additional clinical phenotypes. Compound heterozygous mutations in the PSPH gene have been reported in patients with PSPH deficiency. We sequenced PSPH and found a homozygous missense mutation, Ala35Thr, in all affected members. This is first report of a homozygous change in PSPH. Studies to measure the effect of mutation on enzymatic activity reveal that the enzyme with the Ala35Thr, mutation has about a 5-fold reduced catalytic activity. The side chain of Ala35 is in a small hydrophobic pocket near the catalytic site. Threonine may make the side chain too bulky and polar to fit in the small pocket.
    American Association of Human Genetics annual meeting; 01/2011
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    ABSTRACT: Intellectual disability (ID) is a serious disorder of the central nervous system with a prevalence of 1-3% in a general population. In the past decades, the research focus has been predominantly on X-linked ID (68 loci and 19 genes for non syndromic X linked ID) while for autosomal recessive nonsyndromic ID (NSID) only 30 loci and 6 genes have been reported to date. Genome-wide homozygosity mapping with 500 K Nsp1 array (Affymetrix), CNV analysis, PCR based breakpoint mapping and DNA sequencing was performed to explore the genetic basis of autosomal recessive nonsyndromic ID in a large Pakistani family. Data analysis showed linkage at 8p23 locus with common homozygous region between SNPs rs6989820 and rs2237834, spanning a region of 12.494 Mb. The subsequent CNV analysis of the data revealed a homozygous deletion of 170.673 Kb which encompassed the TUSC3 gene. We report a novel deletion mutation in TUSC3 gene which is the second gene after TRAPPC9 in which mutation has been identified in more than one family with autosomal recessive NSID. The study will aid in exploring the molecular pathway of cognition.
    BMC Medical Genetics 01/2011; 12:56. · 2.54 Impact Factor
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    ABSTRACT: To date, of 13 loci with linkage to non-syndromic autosomal recessive mental retardation (NS-ARMR), only six genes have been established with associated mutations. Here we present our study on NS-ARMR among the Pakistani population, where people are traditionally bound to marry within the family or the wider clan. In an exceptional, far-reaching genetic survey we have collected more than 50 consanguineous families exhibiting clinical symptoms/phenotypes of NS-ARMR. In the first step, nine families (MR2-9 and MR11) with multiple affected individuals were selected for molecular genetic studies. Two families (MR3, MR4) showed linkage to already know NS-ARMR loci. Fifteen affected and 10 unaffected individuals from six (MR2, MR6, MR7, MR8, MR9 and MR11) families were genotyped by using Affymetrix 5.0 or 6.0 single-nucleotide polymorphism (SNP) microarrays. SNP microarray data was visually inspected by dChip and genome-wide homozygosity analysis was performed by HomozygosityMapper. Additional mapping was performed (to exclude false-positive regions of homozygosity called by HomozygosityMapper and dChip) on all available affected and unaffected members in seven NS-ARMR families, using microsatellite markers. In this manner we were able to map three novel loci in seven different families originating from different areas of Pakistan. Two families (MR2, MR5) showed linkage on chromosome 2p25.3-p25.2. Three families (MR7, MR8, and MR9) that have been collected from the same village and belong to the same clan were mapped on chromosome 9q34.3. MR11 maps to a locus on 9p23-p13.3. Analysis of MR6 showed two positive loci, on chromosome 1q23.2-q23.3 and 8q24.21-q24.23. Genotyping in additional family members has so far narrowed, but not excluded the 1q locus. In summary, through this study we have identified three new loci for NS-ARMR, namely MRT14, 15 and 16.
    Clinical Genetics 02/2010; 78(5):478-83. · 4.25 Impact Factor
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    ABSTRACT: Mental retardation/intellectual disability is a devastating neurodevelopmental disorder with serious impact on affected individuals and their families, as well as on health and social services. It occurs with a prevalence of approximately 2%, is an etiologically heterogeneous condition, and is frequently the result of genetic aberrations. Autosomal-recessive forms of nonsyndromic MR (NS-ARMR) are believed to be common, yet only five genes have been identified. We have used homozygosity mapping to search for the gene responsible for NS-ARMR in a large Pakistani pedigree. Using Affymetrix 5.0 single nucleotide polymorphism (SNP) microarrays, we identified a 3.2 Mb region on 8q24 with a continuous run of 606 homozygous SNPs shared among all affected members of the family. Additional genotype data from microsatellite markers verified this, allowing us to calculate a two-point LOD score of 5.18. Within this region, we identified a truncating homozygous mutation, R475X, in exon 7 of the gene TRAPPC9. In a second large NS-ARMR/ID family, previously linked to 8q24 in a study of Iranian families, we identified a 4 bp deletion within exon 14 of TRAPPC9, also segregating with the phenotype and truncating the protein. This gene encodes NIK- and IKK-beta-binding protein (NIBP), which is involved in the NF-kappaB signaling pathway and directly interacts with IKK-beta and MAP3K14. Brain magnetic resonance imaging of affected individuals indicates the presence of mild cerebral white matter hypoplasia. Microcephaly is present in some but not all affected individuals. Thus, to our knowledge, this is the sixth gene for NS-ARMR to be discovered.
    The American Journal of Human Genetics 12/2009; 85(6):909-15. · 11.20 Impact Factor

Publication Stats

475 Citations
211.25 Total Impact Points

Institutions

  • 2011–2014
    • Centre for Addiction and Mental Health
      • Molecular Neuropsychiatry and Development Laboratory
      Toronto, Ontario, Canada
  • 2010
    • SickKids
      Toronto, Ontario, Canada
  • 2006–2009
    • COMSATS Institute of Information Technology
      • Department of Biosciences
      Islāmābād, Islāmābād, Pakistan
  • 2001–2009
    • Quaid-i-Azam University
      • • Department of Physics
      • • Department of Biochemistry
      Islāmābād, Islamabad Capital Territory, Pakistan