[Show abstract][Hide abstract] ABSTRACT: In recent years, the translation of genomic discoveries into mainstream medical practice and public health has gained momentum, facilitated by the advent of new technologies. However, there are often major discrepancies in the pace of implementation of genomic medicine between developed and developing/resource-limited countries. The main reason does not only lie in the limitation of resources but also in the slow pace of adoption of the new findings and the poor understanding of the potential that this new discipline offers to rationalize medical diagnosis and treatment. Here, we present and critically discuss examples from the successful implementation of genomic medicine in resource-limited countries, focusing on pharmacogenomics, genome informatics, and public health genomics, emphasizing in the latter case genomic education, stakeholder analysis, and economics in pharmacogenomics. These examples can be considered as model cases and be readily replicated for the wide implementation of pharmacogenomics and genomic medicine in other resource-limited environments.
Human genomics 06/2015; 9(1):11. DOI:10.1186/s40246-015-0033-3
[Show abstract][Hide abstract] ABSTRACT: Intellectual disability (ID) is a major public health burden on most societies with significant socioeconomic costs. It has been shown that genetic mutations in numerous genes are responsible for a proportion of hereditary forms of ID. NOP2/Sun transfer RNA (tRNA) methyltransferase family member 2 encoded by NSUN2 gene is a highly conserved protein and has been shown to cause autosomal recessive ID type 5 (MRT5). In this study, we recruited an Emirati consanguineous family with a patient diagnosed with ID. Whole-exome sequencing revealed a homozygous variant c.1020delA in NSUN2 gene. The variants segregated in an autosomal recessive mode of inheritance in the family. This variant is novel and causes a frameshift and premature stop codon. At the messenger RNA (mRNA) level, relative expression analysis showed a decreased level of NSUN2 mRNA in the affected child compared to a healthy individual. Mutation prediction analysis and clinical investigation confirmed the pathogenic nature of the identified variant. We therefore conclude that c.1020delA mutation in NSUN2 is most likely the cause of ID in our patient.
Journal of Molecular Neuroscience 06/2015; DOI:10.1007/s12031-015-0592-8 · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Muscle, skeletal, receptor tyrosine kinase (MuSK) is a key organizer at the postsynaptic membrane and critical for proper development and maintenance of the neuromuscular junction. Mutations in MUSK result in congenital myasthenic syndrome (CMS). We hypothesized that the CMS-causing missense mutation (P344R), found within the cysteine rich domain of the protein, will affect its conformational tertiary structure. Consequently, the protein willmisfold, get retained in the endoplasmic reticulum (ER)leading to the loss of its biological function through degradation by the highly conserved ER Associated protein Degradation (ERAD)machinery. We report how P344R-MuSK mutant is trafficking-deficient when expressed at 37 °C inHeLa, COS-7 and HEK293cell lines. It colocalized with the ER marker calnexinin contrast to the MuSKwild-type whichlocalized to the plasma membrane. The N-glycosylation status of the P344R mutant is that of an immature and not properly post-translationally modified protein. Inhibition of protein synthesis showed that the P344R mutant'shalf-life is shorter than MuSKwild-type protein. Proteasomal inhibition resulted in the stabilization of the mutant protein.The mutant protein ishighly ubiquitinatedcompared to wild type suggesting its targeting for proteasomaldegradation.P344R-MuSK mutant'strafficking defect is correctable by culturing the expressing cells at 27 °C and is able to autophosphorylate.Moreover, chemical compounds namely 2.5% glycerol,1% dimethyl sulfoxide, 10 μMthapsigargin and 1 μM curcuminimproved thematuration and exit of the mutant protein from the ER.These findings open perspectives for potential therapeutic interventionfor patients with CMSharboring the P344R-MuSK mutation.
The International Journal of Biochemistry & Cell Biology 01/2015; 60. DOI:10.1016/j.biocel.2014.12.015 · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies have implicated the WW domain-containing oxidoreductase encoding gene (WWOX) in a severe form of autosomal recessive neurological disorder. This condition showed an overlapping spectrum of clinical features including spinocerebellar ataxia associated with generalized seizures and delayed psychomotor development to growth retardation, spasticity, and microcephaly. We evaluated a child from a consanguineous Emirati family that presented at birth with growth retardation, microcephaly, epileptic seizures, and later developed spasticity and delayed psychomotor development. Screening for deletions and duplications using whole-chromosomal microarray analysis identified a novel homozygous microdeletion encompassing exon 5 of the WWOX gene. Analysis of parental DNA indicated that this deletion was inherited from both parents and lies within a large region of homozygosity. Sanger sequencing of the cDNA showed that the deletion resulted in exon 5 skipping leading to a frame-shift and creating a premature stop codon at amino acid position 212. Quantification of mRNA revealed striking low level of WWOX expression in the child and moderate level of expression in the mother compared to a healthy control. To the best of our knowledge, this is the first homozygous germline structural variation in WWOX gene resulting in truncated transcripts that were presumably subject to NMD pathway. Our findings extend the clinical and genetic spectrum of WWOX mutations and support a crucial role of this gene in neurological development.
[Show abstract][Hide abstract] ABSTRACT: Deficiency of Asparagine Synthetase (ASNSD, MIM 615574) is a very rare autosomal recessive disorder presenting with some brain abnormalities. Affected individuals have congenital microcephaly and progressive encephalopathy associated with severe intellectual disability and intractable seizures. The loss of function of the asparagine synthetase (ASNS, EC 188.8.131.52), particularly in the brain, is the major cause of this particular congenital microcephaly. In this study, we clinically evaluated an affected child from a consanguineous Emirati family presenting with congenital microcephaly and epileptic encephalopathy. In addition, whole-exome sequencing revealed a novel homozygous substitution mutation (c.1193A > C) in the ASNS gene. This mutation resulted in the substitution of highly conserved tyrosine residue by cysteine (p.Y398C). Molecular modeling analysis predicts hypomorphic and damaging effects of this mutation on the protein structure and altering its enzymatic activity. Therefore, we conclude that the loss of ASNS function is most likely the cause of this condition in the studied family. This report brings the number of reported families with this very rare disorder to five and the number of pathogenic mutations in the ASNS gene to four. This finding extends the ASNS pathogenic mutations spectrum and highlights the utility of whole-exome sequencing in elucidation the causes of rare recessive disorders that are heterogeneous and/or overlap with other conditions.
[Show abstract][Hide abstract] ABSTRACT: Dysequilibrium syndrome (DES, OMIM 224050) is a genetically heterogeneous condition that combines autosomal recessive non-progressive cerebellar ataxia with mental retardation. The subclass dysequilibrium syndrome type 1 (CAMRQ1) has been attributed to mutations in the VLDLR gene encoding the Very Low Density Lipoprotein Receptor (VLDLR). This receptor is involved in the reelin signalling pathway that guides neuronal migration in the cerebral cortex and cerebellum. Three missense mutations (c.1459G>T; p.D487Y, c.1561G>C; p.D521H and c.2117G>T; p.C706F) have been previously identified in VLDLR gene in patients with DES. However, the functional implications of those mutations are not known and therefore we undertook detailed functional analysis to elucidate the cellular mechanisms underlying their pathogenicity. The mutations have been generated by site-directed mutagenesis and then expressed in cultured cell lines. Confocal microscopy and biochemical analysis have been employed to examine the subcellular localization and activities of the mutated proteins relative to wild type. Our results indicate that the three missense mutations lead to defective intracellular trafficking and ER retention of the mutant VLDLR protein. This trafficking impairment prevents the mutants from reaching the plasma membrane and binding exogenous Reelin, the initiating event in Reelin signalling. Collectively, our results provide evidence that ER quality control is involved in the functional inactivation and underlying pathogenicity of these DES-associated mutations in the VLDLR.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 08/2014; 1843(12). DOI:10.1016/j.bbamcr.2014.08.013 · 5.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Congenital myasthenic syndromes with endplate acetylcholinesterase deficiency are very rare autosomal recessive disorders characterized by onset of the disease in early childhood, general weakness exacerbated by exertion, ophthalmoplegia and refractoriness to anticholinesterase drugs. To date, all reported cases have been attributed to mutations in 18 genes including the COLQ gene which encodes for a specific collagen that anchors acetylcholinesterase at the basal lamina of the neuromuscular junction. We identified an inbred Syrian family with two children of consanguineous parents from two branches affected with congenital myasthenic syndrome with endplate acetylcholinesterase deficiency.
The absence of acetylcholinesterase antibodies was demonstrated biochemically in the patients. Consequently, all the coding regions, exon-intron boundaries and the 5’ and 3’ untranslated regions of the COLQ gene were amplified and sequenced using the Sanger sequencing method.
We observed that the severity of the phenotype in the two affected children was variable. One patient had mild symptoms that included difficulties in gait and feeding with mild respiratory insufficiency. Her sibling died in the first months of life due to severe respiratory failure. The second patient had severe symptoms from birth and has been mechanically ventilated. DNA sequencing revealed a novel homozygous single nucleotide substitution mutation (c.1010T>C) in the COLQ gene in both patients. This substitution leads to a missense amino acid substitution at position 337 of the protein (p.Ile337Thr). This mutation is likely to impair the COLQ’s trimeric organization and therefore it’s anchoring within the synaptic basal lamina.
We identified the molecular cause underlying CMS in two patients. The marked phenotypic variation suggests that other factors including modifier genes may affect the severity of this disease.
[Show abstract][Hide abstract] ABSTRACT: Purpose Fifteen missense mutations in the frizzled family receptor 4 (FZD4) reported to cause familial exudative vitreoretinopathy (FEVR), were evaluated to establish the pathological cellular mechanism of disease and to explore novel therapeutic strategies. Methods The mutations were generated by site-directed mutagenesis and expressed in HeLa and Cos-7 cell lines. Confocal fluorescence microscopy (CFM) and N-glycosylation profiling were used to observe the subcellular localization of the mutant proteins relative to wild type. Polyubiquitination studies were used to establish the involvement of the proteasome. Culturing at reduced temperatures and incubation in the presence of chemical compounds were used to enhance mutant protein processing and exit out of the ER. Results CFM of the mutants showed three distinct subcellular localizations: a plasma membrane pattern, an ER pattern and a mixed pattern to both compartments. CFM and N-glycosylation profiling established the predominant ER localization of P33S, G36N, H69Y, M105T, M105V, C181R, C204R, C204Y and G488D mutants. Co-expression of these mutants with wild-type FZD4 showed the inability of the mutants to trap wild-type FZD4. Culturing the expressing cells at reduced temperatures or in the presence of chemical agents directed at ameliorating protein misfolding resulted in partial rescue of the trafficking defects observed for M105T and C204Y mutants. Conclusion Defective trafficking resulting in haploinsufficiency is a major cellular mechanism for several missense FEVR-causing FZD4 mutants. Our findings indicate that this trafficking defect might be correctable for some mutants, which may offer opportunities for the development of novel therapeutics for this condition.
[Show abstract][Hide abstract] ABSTRACT: The rare autosomal genetic disorder, Spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL), is reported to be caused by missense or splice site mutations in the human discoidin domain receptor 2 (DDR2) gene. Previously our group has established that trafficking defects and loss of ligand binding are the underlying cellular mechanisms of several SMED-SL causing mutations. Here we report the clinical characteristics of two siblings of consanguineous marriage with suspected SMED-SL and identification of a novel disease causing mutation in the DDR2 gene.
Clinical evaluation and radiography were performed to evaluate the patients. All the coding exons and splice sites of the DDR2 gene were sequenced by Sanger sequencing. Subcellular localization of the mutated DDR2 protein was determined by confocal microscopy, deglycosylation assay and Western blotting. DDR2 activity was measured by collagen activation and Western analysis.
In addition to the typical features of SMED-SL, one of the patients has an eye phenotype including visual impairment due to optic atrophy. DNA sequencing revealed a novel homozygous dinucleotide deletion mutation (c.2468_2469DelCT) on exon 18 of the DDR2 gene in both patients. The mutation resulted in a frameshift leading to an amino acid change at position S823 and a predicted premature termination of translation (p.S823Cfs*2). Subcellular localization of the mutant protein was analyzed in mammalian cell lines, and it was found to be largely retained in the endoplasmic reticulum (ER), which was further supported by its N-glycosylation profile. In keeping with its cellular mis-localization, the mutant protein was found to be deficient in collagen-induced receptor activation, suggesting protein trafficking defects as the major cellular mechanism underlying the loss of DDR2 function in our patients.
Our results indicate that the novel mutation results in defective trafficking of the DDR2 protein leading to loss of function and disease. This confirms our previous findings that DDR2 missense mutations occurring at the kinase domain result in retention of the mutant protein in the ER.
BMC Medical Genetics 04/2014; 15(1):42. DOI:10.1186/1471-2350-15-42 · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Whereas many genes associated with intellectual disability (ID) encode synaptic proteins, transcriptional defects leading to intellectual disability are less well understood. We studied a large, consanguineous pedigree of Arab origin with 7 members affected with ID and mild dysmorphic features. Homozygosity mapping and linkage analysis identified a candidate region on chromosome 17 with a maximum multipoint LOD score of 6.01. Targeted high-throughput sequencing of the exons in the candidate region identified a homozygous 4-base pair deletion (c.169_172delCACT) in the METTL23 (methyltransferase like 23) gene, which is predicted to result in a frameshift (p.His57Valfs*11). Overexpressed METTL23 protein localized to both nucleus and cytoplasm, and physically interacted with GABPA (GA binding protein transcription factor, alpha subunit). GABP, of which GABPA is a component, is known to regulate the expression of genes such as THPO (thrombopoietin) and ATP5B (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide), and is implicated in a wide variety of important cellular functions. Overexpression of METTL23 resulted in increased transcriptional activity at the THPO promoter, while knockdown of METTL23 with siRNA resulted in decreased expression of ATP5B, thus revealing the importance of METTL23 as a regulator of GABPA function. The METTL23 mutation highlights a new transcriptional pathway underlying human intellectual function.
Human Molecular Genetics 02/2014; 23(13). DOI:10.1093/hmg/ddu054 · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Germline heterozygous mutations in the tumor suppresser NF1 gene cause a cancer predisposition syndrome known as neurofibromatosis type 1 (NF1). This disease is one of the most common multisystem disorders with an estimated incidence of 1 in 3,000 to 1 in 4,000 births. Clinically, NF1 patients are prone to develop "café au lait" spots, neurofibromas, Lisch nodules, freckling of the axillary, or inguinal region and optic nerve gliomas.
In the present study, we report clinical and molecular findings of five unrelated patients and seven cases from four families with NF1 from UAE. To reveal the genetic defects underlying NF1 in our cohort of patients, we screened the whole coding and splice site regions of the NF1 gene. In addition, MLPA or CGH array has been used to screen for structural variations including deletions, indels, and complex rearrangements.
This resulted in the identification of five distinct novel mutations and two previously reported ones. These variations included three missense and one nonsense mutations, one single base, one dinucleotide, and one large deletion.
Four mutations were inherited, and the remaining were absent from both parents and therefore are "de novo" mutations. This analysis represents the spectrum of NF1 mutations in UAE and supports the premise of absence of hotspot mutations in the NF1 gene. Moreover, no obvious genotype-phenotype correlations were observed in our patients.
Child s Nervous System 01/2014; 30(7). DOI:10.1007/s00381-013-2352-9 · 1.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Variants in the head and tail domains of the MYO7A gene, encoding myosin VIIA, cause Usher syndrome type 1B (USH1B) and nonsyndromic deafness (DFNB2, DFNA11). In order to identify the genetic defect(s) underling profound deafness in two consanguineous Arab families living in UAE, we have sequenced a panel of 19 genes involved in Usher syndrome and nonsyndromic deafness in the index cases of the two families. This analysis revealed a novel homozygous insertion of AG (c.1952_1953insAG/p.C652fsX11) in exon 17 of the MYO7A gene in an Iraqi family, and a homozygous point mutation (c.5660C>T/p.P1887L) in exon 41 affecting the same gene in a large Palestinian family. Moreover, some individuals from the Palestinian family also harbored a novel heterozygous truncating variant (c.1267C>T/p.R423X) in the DFNB31 gene, which is involved in autosomal recessive nonsyndromic deafness type DFNB31 and Usher syndrome type II. Assuming an autosomal recessive mode of inheritance in the two inbred families, we conclude that the homozygous variants in the MYO7A gene are the disease-causing mutations in these families. Furthermore, given the absence of retinal disease in all affected patients examined, particularly a 28 year old patient, suggests that at least one family may segregate a DFNB2 presentation rather than USH1B. This finding further supports the premise that the MYO7A gene is responsible for two distinct diseases and gives evidence that the p.P1887L mutation in a homozygous state may be responsible for nonsyndromic hearing loss.
[Show abstract][Hide abstract] ABSTRACT: The chemokine receptors components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5∆32) confer high degree of resistance to HIV infection, Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5∆32 variant is present in 10-15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians).
The prevalence of CCR5 gene variants including CCR5∆32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5∆32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing.
Among Emiratis, the allele frequency of the CCR5∆32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5∆32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans.
We conclude that the allele frequency of the most critical CCR5 ploymorphism (∆32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.
[Show abstract][Hide abstract] ABSTRACT: Background
Inherited intellectual disability (ID) conditions are a group of genetically heterogeneous disorders that lead to variable degrees of cognition deficits. It has been shown that inherited ID can be caused by mutations in over 100 different genes and there is evidence for the presence of as yet unidentified genes in a significant proportion of patients. We aimed at identifying the defective gene underlying an autosomal recessive ID in two sibs of an Emirati family.
A combined approach involving homozygosity mapping and whole-exome sequencing was used to identify the causative mutation. RNA analysis was performed to gain further insight into the pathogenic effect of the detected mutation.
We have identified a homozygous splicing mutation (c.1219_1222+1delAAAGG) in the LINS gene in the affected children. LINS is the human homologue of the Drosophila segment polarity gene lin that encodes an essential regulator of the wingless/Wnt signaling. The identified mutation alters the first consensus nucleotide of the 5' donor splice junction of intron 5 and the 3' end of exon 5. Transcript analysis revealed that this change leads to an exon skipping event resulting in direct splicing of exon 4 to exon 6. Another mutation in LINS has been described very briefly in an Iranian family with autosomal recessive ID and microcephaly.
Our study confirms that LINS, a modulator of the WNT pathway, is an indispensable gene to human cognition and this finding sheds further light on the importance of WNT signaling in human brain development and/or function.