Muhammad Arshad Rafiq

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

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Publications (26)119.19 Total impact

  • [show abstract] [hide abstract]
    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: 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: 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
  • AJHG. 01/2011;
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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
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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
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    ABSTRACT: We have identified a consanguineous Pakistani family where oligodontia is inherited along with short stature in an autosomal-recessive fashion. Increased bone density was present in the spine and at the base of the skull. Using high-density single-nucleotide polymorphism microarrays for homozygosity mapping, we identified a 28 Mb homozygous stretch shared between affected individuals on chromosome 11q13. Screening selected candidate genes within this region, we identified a homozygous nonsense mutation, Y774X, within LTBP3, the gene for the latent TGF-beta binding protein 3, an extracellular matrix protein believed to be required for osteoclast function.
    The American Journal of Human Genetics 05/2009; 84(4):519-23. · 11.20 Impact Factor
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    ABSTRACT: Deletions of single or multiple exonic regions within the dystrophin gene can be detected using current molecular methods in approximately 65% of the patients with X-linked recessive neuromuscular disorder, Duchenne/Becker muscular dystrophy (DMD/BMD). Population-based variations in frequency and distribution of dystrophin gene deletions have been reported in DMD/BMD patients. In the present study, the first in the Pakistani population, frequency and distribution of deletions of 18 exons clustered in two hot spots within the dystrophin gene in 211 unrelated DMD patients were analyzed. A total of 211 patients suffering from DMD were ascertained, and intragenic deletions within the dystrophin gene were detected on polymerase chain reaction amplification of the genomic DNA using 18 primer sets clustered within two major deletion hot spots. lovd v.1.1.0 software from the Leiden Muscular Dystrophy website has been used to predict in-frame and out-of-frame deletions. Intragenic deletions were detected in 86 patients (40.75%): 35 patients (40.69%) had deletions within the proximal hot spot, and 51 patients (59.30%) had deletions confined to the distal deletion hot spot of the dystrophin gene. The most frequently deleted exons were 50, 6, 47, 13 and 52 with deletion frequencies of 15.11%, 12.79%, 10.46%, 8.13%, and 4.65%, respectively. lovd v.1.1.0 predicted out-of-frame deletions in 67 DMD patients and in-frame deletions in 19 DMD patients. The observed proportion of intragenic deletions in the Pakistani population is relatively low, which is comparable with most of the Asian data. Also, deletions in 67 patients (77.9%) are in agreement with the frame-shift rule.
    Pediatrics International 05/2008; 50(2):162-6. · 0.88 Impact Factor
  • British Journal of Dermatology 04/2008; 158(3):621-3. · 3.76 Impact Factor
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    ABSTRACT: Localized autosomal recessive hypotrichosis (LAH) is rare disorder affecting the scalp, trunk and extremities and largely sparing the facial, pubic and axillary hair. Mutations in desmoglein 4 (DSG4) gene are responsible for LAH which maps to human chromosome 18q12. In this study a recurrent intragenic deletion mutation (Ex5_8del) was identified in DSG4 gene in two Pakistani families of Balochi and Sindhi origins. Manifestation of identical intragenic deletion mutation in eight Pakistani families, six reported earlier and two here, is exceptionally evocative of the dispersion of ancestral chromosome in different ethnic groups through common ancestors.
    Archives for Dermatological Research 09/2006; 298(3):135-7. · 2.71 Impact Factor
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    ABSTRACT: Hereditary hearing impairment (HI) displays extensive genetic heterogeneity. Autosomal recessive (AR) forms of prelingual HI account for approximately 75% of cases with a genetic etiology. A novel AR non-syndromic HI locus (DFNB47) was mapped to chromosome 2p25.1-p24.3, in two distantly related Pakistani kindreds. Genome scan and fine mapping were carried out using microsatellite markers. Multipoint linkage analysis resulted in a maximum LOD score of 4.7 at markers D2S1400 and D2S262. The three-unit support interval was bounded by D2S330 and D2S131. The region of homozygosity was found within the three-unit support interval and flanked by markers D2S2952 and D2S131, which corresponds to 13.2 cM according to the Rutgers combined linkage-physical map. This region contains 5.3 Mb according to the sequence-based physical map. Three candidate genes, KCNF1, ID2 and ATP6V1C2 were sequenced, and were found to be negative for functional sequence variants.
    Human Genetics 02/2006; 118(5):605-10. · 4.63 Impact Factor
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    ABSTRACT: Atrichia with papular lesions (APL) is a rare autosomal recessive form of total alopecia, characterized by hair loss soon after birth and the development of papular lesions of keratin-filled cysts over extensive areas of the body. Mutations in the hairless (hr) gene, a putative single zinc finger transcription factor, have been implicated in the pathogenesis of this disorder. In the present study, we describe two novel deletion mutations in exons 2 and 8 of the human hairless gene leading to frameshift and downstream premature termination codons in two consanguineous Pakistani families affected with atrichia.
    Archives for Dermatological Research 12/2005; 297(5):226-30. · 2.71 Impact Factor
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    ABSTRACT: Ectodermal dysplasia (ED) represents a heterogeneous group of genetic disorders characterized by the absence or deformity in two or more of the ectodermal appendages. We have studied an autosomal recessive form of ED in 13 individuals over six generations from an inbred Pakistani family. The clinical features of the affected individuals include highly dystrophic nails and thin hair on scalp, fine eyebrows and eyelashes, and thin body hair. Genome-wide linkage analysis of 390 microsatellite markers mapped the ED gene to the 3.92 cM interval flanked by markers D10S1710 and D10S1741 on chromosome 10q24.32-q25.1. Multipoint linkage analysis generated a maximum logarithm of odds ratio score of 4.79 in the interval D10S1239-D10S1264, which corresponds to 6.35 Mb.
    Journal of Investigative Dermatology 03/2005; 124(2):338-42. · 6.19 Impact Factor

Publication Stats

273 Citations
119.19 Total Impact Points

Institutions

  • 2011–2012
    • 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
  • 2004–2008
    • Quaid-i-Azam University
      • Department of Biochemistry
      Islāmābād, Islamabad Capital Territory, Pakistan