Prof. of Medical Genetics, UQU-Faculty of Medicine, Department of Medical Genetics
Skills and Expertise
Molecular GeneticsGenomicsBiotechnologyNext Generation SequencingGenotypingAutism Spectrum DisordersMutationReal-Time PCRClinical TeachingSingle Nucleotide PolymorphismSNP GenotypingHuman Molecular GeneticsMedical GeneticsDementiaSplicingGene AmplificationDNA Mutational AnalysisCapillary Electrophoresis ABI SequencingDuchenne Muscular DystrophyDNA MicroarrayMultiplex PCRSkin DiseasesGenotype-PhenotypeGenetic DiseasePCR-RFLPMale InfertilityMultiplex AssaysMicrosatellite RepeatsHaplotypesARMS-PCRTransglutaminasesPhenylketonuriasMTHFR genetic polymorphismsFrameshift MutationMultifactorial InheritancePhenylalanine Hydroxylase
Feb 2009 - Feb 2011
- Medical genetics
- Mecca, Makkah, Saudi Arabia
- Principal Investigator
- Project: funded by KACST.. Mutations spectrum map of the whole phenylalanine hydroxylase gene in the phenylketonuric Saudi patients
Detection of the genetic variants for nephrotic syndrome (a genetically heterogeneous disease) within the whole human genome using NGS strategy.
Research Item (56)
Conflicting results have arisen among different ethnic populations with regard to the ability of tumor necrosis factor (TNF) to control the development of bronchial asthma. We examined common TNF polymorphisms (TNFA -1031C>T, TNFA -308G>A, and TNFB +252A>G) to develop a model of the associations between these genetic markers and the development of the disease in Egypt. Amplified DNA from buccal mucosa was genotyped for 240 children using polymerase chain reaction-restriction fragment length polymorphism. Skin prick test, total serum immunoglobulin E levels, and assessment of pulmonary functions were investigated. The onset age for one-third of the asthma patients in our study was between 7 and 10 years. The TNFA -1031C>T and TNFA -308G>A polymorphisms were strongly associated with the risk of asthma (p = 0.007, and p = 0.000, respectively), but the TNFB +252A>G polymorphism was not (p = 0.6). We detected a significant linkage between the +252A>G and -1031C>T, and another between the +252A>G and the -308G>A (p < 0.0001 for both). The -1031C>T and -308G>A polymorphisms were not linked (p = 0.14). The -308A/A genotype was absent, and the -308A allele was expressed only in patients with -308G/A heterozygosity (13%). All but the +252G/A genotype were also strongly associated with the severity of disease. Environmental factors, as genetic variations, clearly influence susceptibility, the onset, progression, and severity of bronchial asthma. More information is needed to develop genetic models of susceptibility for different ethnic populations.
Background: In individuals with Duchenne muscular dystrophy (DMD), exon skipping treatment to restore a wild-type phenotype or correct the frame shift of the mRNA transcript of the dystrophin (DMD) gene are mutation-specific. To explore the molecular characterization of DMD rearrangements and predict the reading frame, we simultaneously screened all 79 DMD gene exons of 45 unrelated male DMD patients using a multiplex ligation-dependent probe amplification (MLPA) assay for deletion/duplication patterns. Multiplex PCR was used to confirm single deletions detected by the MLPA. Results: There was an obvious diagnostic delay, with an extremely statistically significant difference between theage at initial symptoms and the age of clinical evaluation of DMD cases (t value, 10.3; 95% confidence interval 5.95–8.80,P< 0.0001); the mean difference between the two groups was 7.4 years. Overall, we identified 147 intragenic rearrangements: 46.3% deletions and 53.7% duplications. Most of the deletions (92.5%) were between exons 44 and 56, with exon 50 being the most frequently involved (19.1%). Eight new rearrangements, including a mixed deletion/duplication and double duplications, were linked to seven cases with DMD. Of all the cases, 17.8% had duplications with no hot spots. In addition, confirmation of the reading frame hypothesis helped account for new DMD rearrangements in this study. We found that 81% of our Saudi patients would potentially benefit from exon skipping, of which 42.9% had a mutation amenable to skipping of exon 51.Conclusions:Our study could generate considerable data on mutational rearrangements that may promote future experimental therapies in Saudi Arabia.
Project - Genetic Variations in Urinary Bladder Cancer Among Saudi Patients
The Research Team has recently published the first article (https://doi.org10.1155/2017/1474560) including 52 Saudi patients with urothelial bladder carcinoma (UBC) for genotyping of SNP variants within the GSTT1, GSTM1, Cytochrome P450, TP53, MTHFR, and MTRR gene.
We evaluated the associations between seven single nucleotide polymorphisms and susceptibility to urothelial bladder carcinoma (UBC) in a Saudi population. Genomic DNA was taken from buccal cells of 52 patients with UBC and 104 controls for genotyping of GSTT1, GSTM1, rs4646903, rs1048943, TP53 rs1042522, rs1801133, and rs1801394 using PCR and TaqMan assays.The rs1801133 and rs1801394 variants showed strong associations with UBC (OR = 2.3, 𝑃 = 0.0002; OR = 2.6, 𝑃 = 0.0001, resp.). Homozygosity of Pro72 conferred a significant double risk in cases compared with controls (30.8% versus 15.4%), but the homozygote Arg/Arg had no effect on risk. Genotypic combinations of GSTM1/GSTT1, rs4646903/rs1048943, and rs1801133/rs1801394 exhibited significant linkage with the disease (𝜒2 = 10.3, 𝑃 = 0.006; 𝜒2 = 13.9, 𝑃 = 0.003; and 𝜒2 = 20.4, 𝑃 = 0.0004, resp.). The GSTM1 and rs1042522Arg and rs1801394G variant alleles were more frequent in current smokers with UBC (52.4%, 52.5%, and 64.3%, resp.) than were the corresponding wild-types. Despite some variants having only a slight effect on UBC risk, the interaction effect of combined genetic biomarkers—or even the presence of one copy of a variant allele—is potentially much greater. Perhaps more studies regarding next-generation genetic sequencing and its utility can add to the risk of UBC.
Question - What is the best DNA extraction method for genomics applications?
I am wondering you are going to use your isolated DNA templates in NGS or micro-array methods, but you could not have a budget for DNA isolation kit.
Objectives: Single nucleotide polymorphisms (SNPs) have been reported in different autistic populations. Here we present the first association study investigating SNPs of some genes; serotonin receptor (HTR2A IVS2A>G rs7997012; HTR2C 68G>C rs6318), serotonin transporter (SLC6A4 rs3813034), ankyrin repeat and kinase domain containing 1 (ANKK1 rs1800497), methylenetetrahydrofolate reductase (MTHFR rs1891394), and BDNF rs6265 in Saudi autistic children. Epidemiologic, clinical and psychometric aspects were used to examine the possible risk factors of autism. Methods: We used TaqMan SNP genotyping to examine 68 Saudi children (48 Males and 20 females) diagnosed with autism according to DSM-IV criteria & ICD-10 criteria, including deficits in reciprocal social interaction, impaired verbal, and non-verbal communication as well as restricted, repetitive, and stereotyped patterns of behaviors. Healthy controls (n= 78) with no history of mental illnesses, behavioral disorders or substance abuse were used. The severity of behavioral symptoms in cases was assessed at admission using the Childhood Autism Rating Scale (CARS). Hardy-Weinberg equilibria of the genetic variants were assessed using online software (http://www.oege.org/software/hwe-mrcalc.shtml). Chi-square tests were used to compare sociodemographic and clinical characteristics. Odds ratios and confidence intervals were calculated. Results: Overall, our data provide strong evidence of associations between these two SNPs and risk of autism in this population. Compared to healthy subjects, children with autism showed significant overexpression of the mutant alleles in the SNPs rs7997012, rs6318, rs3813034, rs1800497, and rs1891394, but not in the rs6265 SNP. Data on linkages or associations between these genetic loci and the disease vary among different ethnicities. Conclusion: Our findings presented associations between autism and some genetic variants under investigation, and showed that the potential influence of just one copy of the mutant alleles in the Saudi patients. Replicating our findings in other ethnic population may support our data before making any firm generalizations. Ongoing analyses of genetic variants associated with autism are being extended using next-generation sequencing and some fruitful data are going to be published.
Limited research has assessed associations between schizophrenia and genetic variants of the ankyrin repeat and kinase domain containing 1 (ANKK1) and lymphotoxin-alpha (LTA) genes among individuals of Middle Eastern ancestry. Here we present the first association study investigating the ANKK1 rs1800497 (T>C) and LTA rs909253 (A>G) single-nucleotide polymorphisms in an Egyptian population. Among 120 patients with DSM-IV and PANSS (Positive and Negative Syndrome Scale) assessments of schizophrenia and 100 healthy controls, we determined the genotypes for the polymorphisms using endonuclease digestion of amplified genomic DNA. Results confirmed previous findings from different ethnic populations, in that the rs1800497 and rs909253 polymorphisms were both associated with risk of schizophrenia. Differences between the genotypes of cases and controls were strongly significant (P = 0.0005 for rs1800497 and P = 0.001 for rs909253). The relative risk to schizophrenia was 1.2 (P = 0.01) for the C allele and 0.8 (P = 0.04) for the G allele. The CC, GG, and combined CC/AA genotypes were all more frequent in cases than in controls. These results support an association between ANKK1 and LTA genetic markers and vulnerability to schizophrenia and show the potential influence of just one copy of the mutant C or G allele in the Egyptian population.
Question - Do you know any available kit for DNA extraction from stool. I am working on cryptosporidium?
Dear Dr. Malky
You can easily isolate purified DNA from stool using MagaZorb® DNA Mini-Prep Kit Protocol (Promega Cor.). The Agency in SA is Abdallah Fouad Co.
Introduction and Objectives Methylenetetrahydrofolate reductase (MTHFR) 677CT marker influences the risk and severity of Alzheimer's disease (AD) and whether AD is associated with homocysteine, vitamin B12, and cholesterol levels in Egypt. Aims The aim of this study was to determine the genotype and allele frequencies of the rs1801133 SNP and to evaluate the influence of genotype on risk and severity of disease in Egyptian patients with AD. Methods Forty-three Alzheimer's cases and 32 non-AD controls were genotyped for the 677C>T polymorphism. Clinical characteristics and levels of homocysteine, vitamin B12, and cholesterol were assessed. Results No significant differences in the frequencies of the MTHFR alleles or genotypes between AD cases and controls (P= 0.14) were identified. The 677T mutant allele was significantly overrepresented in AD cases compared to controls (OR = 2.22; P= 0.03). The 677T/T frequency was three times higher in AD patients than in controls, which could increase plasma homocysteine levels. Severe cases of AD were the most frequent in patients with the T/T genotype (11.6%). The effect of the MTHFR polymorphism on the risk of AD may be independent of homocysteine, vitamin B12, or even cholesterol levels. Conclusions The MTHFR 677C>T polymorphism—especially the presence of one copy of the T allele—appears to confer a potential risk for the development of AD. The T/T genotype may contribute to hypercysteinemia as a sensitive marker.
We evaluated whether TAP1-rs1135216 (p.637D>G) and PSMB9-rs17587 (p.60R>H) were significantly associated with the risk and severity of vitiligo among Saudi patients. One hundred seventy-two subjects were genotyped for the TAP1-rs1135216 and PSMB9-rs17587 variants using endonuclease digestions of amplified genomic DNA. The TAP1-rs1135216 and PSMB9-rs17587 mutant alleles were strongly associated with vitiligo, with odds ratios showing five fold and two fold risks ( P < 0.0001 and P = 0.007 , resp.). In TAP1-rs1135216, the 637G mutant allele was more frequent in cases (74%) than in healthy controls. In cases, the 60H mutant allele PSMB9-rs17587 was less frequent (42%) than the wild-type 60R allele (58%). Vitiligo vulgaris was the most common type of disease, associated with the DG (55%) and GG (46%) genotypes for rs1135216 and with the RH genotype (59%) for rs17587. The heterozygous 637DG and 60RH genotypes were each linked with active phenotypes in 64% of cases. In conclusion, the TAP1-rs1135216 and PSMB9-rs17587 variants are significantly associated with vitiligo, and even one copy of these mutant alleles can influence the risk among Saudis. Vitiligo vulgaris is associated with genotypes containing the mutant G and H alleles.
Screening of Yq has become one of the most frequently performed postnatal molecular genetic tests in Egypt. A survey sponsored by WHO estimated the prevalence of infertility among Egyptian couples to be 12% (4.3% for primary infertility and 7.7% for secondary infertility) . The 10-Mb AZF region on the q-arm of the Y chromosome is frequently deleted in men with unexplained spermatogenic failure. Microdeletions are linked to AZF loci in 20-30% of patients with non-obstructive azoospermia and in 3-7% of patients with severe idiopathic oligospermia . AZF microdeletions are associated with varied testicular histology, ranging from Sertoli-Cell-Only (SCO) syndrome to hypospermatogenesis to maturation arrest. We aim to determine the tag sequence-tagged sites (STSs) in the AZF-region of Yq associated with azoospermia and severe oligospermia in infertile Egyptian men.
Question - What is the best DNA extraction method for genomics applications?
I used to work with Qiagen-Spin column kits for direct sequencing, microarray Chips, or NGS. The ready-spin Column protocol is recommended by the above-mentioned strategies.
Objective: We evaluated whether the methylenetetrahydrofolate reductase (MTHFR) 677C>T marker influences the risk and severity of Alzheimer's disease (AD) and whether AD is associated with homocysteine, vitamin B12, and cholesterol levels in Egypt. Methods: Forty-three Alzheimer's cases and 32 non-AD controls were genotyped for the 677C>T polymorphism. Clinical characteristics and levels of homocysteine, vitamin B12, and cholesterol were assessed. Results: No significant differences in the frequencies of the MTHFR alleles or genotypes between AD cases and controls (P = 0.14) were identified. The 677T mutant allele was significantly overrepresented in AD cases compared to controls (OR = 2.22; P = 0.03). The 677T/T frequency was three times higher in AD patients than in controls, which could increase plasma homocysteine levels. Severe cases of AD were the most frequent in patients with the T/T genotype (11.6%). The effect of the MTHFR polymorphism on the risk of AD may be independent of homocysteine, vitamin B12, or even cholesterol levels. Conclusions: The MTHFR 677C>T polymorphism--especially the presence of one copy of the T allele--appears to confer a potential risk for the development of AD. The T/T genotype may contribute to hypercysteinemia as a sensitive marker.
Bronchopulmonary dysplasia (BPD) remains as a major and increasing burden in Egypt. To determine whether alleles of TNFα-238G > A affect the risk of BPD or the severity of BPD in preterm infants in Egypt. We prospectively genotyped 220 premature neonates (birth weight <1,500 g and gestational age 26–32 weeks) for the −238 polymorphism, and assessed the clinical risk factors for BPD in our study populations. Infants with BPD were mechanically ventilated. Infants who developed BPD (n = 120) had a younger gestational age (31.0 ± 2.1 weeks vs. 34.3 ± 1.5 weeks) and lower birth weight (1,490 ± 360 g vs. 1,880 ± 520 g) than infants who did not develop BPD (n = 100). Results of antenatal steroid supplementation, surfactant therapy, or sepsis might affect the genetic modulation of BPD. The −238G > A polymorphism was associated with a twofold risk of BPD (OR = 2.86; 95% confidence interval, 1.35–3.83). Despite the dominance of the G allele in the Egyptian population, the −238A allele was more common among infants with BPD (23%) than among infants without BPD (15%). The A allele occurred less often in infants with mild BPD (9%) than in infants with severe (39%) or moderate (52%). The AA genotype occurred in 15% of cases but in none of the controls. The TNFα −238G > A polymorphism—particularly the presence of an A allele—should be evaluated as a biomarker to predict the clinical outcome of preterm infants with BPD in Egypt. Even the presence of one copy of this mutant allele appears to be sufficient to influence the severity of disease. Pediatr Pulmonol. 2013; 48:699–706.
Abstract Reported to date, strong evidence exists in multiple studies for genetic predisposing in the development of diabetic nephropathy, and no studies addressed this issue among Egyptian population. The results of angiotensin converting enzyme gene (ACE) in the susceptibility to nephropathy in type 1 diabetes with nephropathy are conflicting. We aim to identify the associations of two ACE gene polymorphisms (PstI, A > G substitution and a 287-bp insertion/deletion) with nephropathy in type 1 diabetes in Egyptian children/adolescents. Our case-control study contained 140 diabetic individuals; 80 diabetic with nephropathy as cases, and 60 diabetic subjects without nephropathy as control group. Amplified DNA from peripheral leucocytes/buccal mucosa was genotyped for using polymerase chain reaction and enzymatic assay. We found no significant differences in the distribution of ACE insertion/deletion and PstI genotypes or allele frequencies were observed between the examined groups. Frequencies of PstI–indel haplotypes were similar in all of our study groups. In both cases and control subjects, ACE activity and microalbuminuria were highest among D/D homozygotes and lowest in I/I homozygotes, while a dissimilar result was seen in PstI polymorphism. Our findings in Egyptian population strongly conclude that there is no association between the ACE gene I/D and PstI polymorphisms with nephropathy in type 1 diabetes.
There is strong evidence that supports the role of tumour necrosis factors (TNF-alpha/beta) as common genetic factors, located on 6p21.1-6p21.3 loci, in the pathogenesis of asthma disease. In this study, we extended our research work on TNFA to include the genotyping of Saudi asthmatic children as regards to TNFB gene (namely as lymphotoxin-α, LTA). We examined 60 asthmatic Saudi children compared to 56 healthy non-asthmatics using the PCR-RFLP analyses to identify the polymorphism +252A>G in intron 1 in lymphotoxin-α gene. We identified 55% of the allele A of the LTA∗NcoI polymorphism in subjects with asthma disease, and 45% of the allele G. In this study, the frequency of the LTA∗NcoI-A/A genotype was 40% preferably to the LTA∗NcoI-G/A and LTA∗NcoI-G/G genotypes. In addition, the severe persistent asthmatic cases were associated with the LTA∗NcoI-AA genotype at a frequency of 80%, while the genotype LTA∗NcoI-GG are associated with the mildest form of the disease. Consequently, one could predict the severity of asthma and hence the polymorphism of the LTA∗NcoI. Herein, we stated that more than 93% of Saudi children under investigation lived in the randomized areas of western regions of Saudi Arabia. In conclusion, genotype frequencies for the LTA+252 polymorphisms were significantly different from the controls. These findings may have implications for future early intervention studies by helping to identify infants at increased risk for wheezing and childhood asthma.
We have updated the dataset of the molecular spectrum of the β-thalassemia (β-thal) in Upper Egypt. Buccal swabs were analyzed from 94 unrelated patients with β-thal major (β-TM) using reverse dot-blot and multiplex ampliﬁcation refractory mutation system-polymerase chain reaction ARMS-PCR). The most frequent mutation was IVS-I-110 (G>A) (57%). The IVS-I-110, IVS-I-6 (T>C) and IVS-I-1 (G>A) mutations accounted for 87% of the β-thal anomalies. The codon 39(C>T) and frameshift codon (FSC) 6 (–A) ( GAG >–GG) mutations were only detected in Al-Minya and Qina, respectively. We did not observe the IVS-II-745 (C>G) or –101 (C>T) mutations. Forty-three percent of Upper Egyptians were homozygotes. Our efforts were an important step to complete the mutation map of β-thal in Egypt restricted to Cairo and the Nile Delta regions. This study wil help to develop preventative programs for Upper Egyptians. It addressed the genetic drift of the β-thal gene mutations in Africa, Asia, and Europe. Keywords β-Thalassemia (β-thal), Mutation spectrum, Upper Egyptians, Genetic drift
Screening of Yq has become one of the most frequently performed postnatal molecular genetic tests in Egypt. Our purpose was to determine the tag sequence-tagged sites (STSs) in the AZF -region of Yq associated with azoospermia and severe oligospermia in infertile Egyptian men. We analyzed blood samples from 49 infertile men (28 with azoospermia and 21 with severe oligospermia) using multiplex PCR for six common AZFa, AZFb, and AZFc STS markers,, as recommended by the European Academy of Andrology. Twenty-four (37%) microdeletions with five separate deletions were identified. We found 66.7% of the deletions in the AZFb locus, 20.8% in the AZFa locus, and 12.5% in the AZFc locus. Some common haplotypes (7 of 10) were identified in our sample population. Haplotypes H3 (corresponding to sY127) and H4 (corresponding to sY134) were the most common. We suggest that screening with a minimum of three STSs-sY86, sY127, and sY134-would provide the highest level of clinical sensitivity in genetic testing among infertile Egyptian men. Moreover, separate microdeletions were localized in infertile Y-chromosome patients.
The aim of this study is to assess the prevalence of six common mutations in the Mediterranean basin and Turkey among a large group of Egyptian PKU cases Phenylketonuria (PKU) is one of the most common inborn errors of amino acid metabolism that is caused by deficiency of hepatic phenylalanine hydroxylase (PAH). This deficiency is attributed to more than 528 mutations in the PAH gene. Ninety unrelated patients with PKU (180 alleles) were screened for six mutations (IVS10-11G>A, R261Q, R252W, Y277D, E221G and G272S) using polymerase chain reaction-restriction fragment length polymorphism. The IVS10-11G>A mutation was found in thirty alleles (17%), the R261Q in twelve (7%) and R252W in three (1.6%), while Y277D, E221G and G272S were not found in this patient group. Screening for six Mediterranean mutations identified a heterogeneous pattern among Egyptian PKU patients with a high frequency of IVS10-11 G>A (17%) (Tab. 2, Ref. 31). Full Text (Free, PDF) www.bmj.sk.
Background. In spite of a large body of scientific evidence that genetic factors contribute strongly to autism, linkage studies have failed to implicate any specific region as a susceptibility locus. Therefore, instead of searching for a single major gene, minor functional deficits in several genes causing a small change in gene activity might be implicated. Of these functional polymorphisms, the HoxA1 and HoxB1 genes responsible for hindbrain development appeared to be implicated in the development of autism at least in some studies. Methods. We performed a case-control analysis of 25 controls and family based analysis of 41 simplex families forming complete trios, in search of a specific preponderance of an allelic variant of the HoxA1 and HoxB1 genes in relation to the development and severity of autism as detected by the childhood autism rating scale (CARS). To detect a substitution variant at the first exon of HoxA1 gene and an insertion variant at the first exon of HoxB1 gene, genomic DNA was amplified using polymerase chain reaction (PCR) followed by digestion with restriction endonucleases. Results. Our results showed a statistically significant preponderance of males (p< 0.05) and a highly significant association with mental retardation (p< 0.005), 58.5% of the sample had moderate to severe autism. The most prevalent genotypes were A/A genotype and +/+ for the HoxA1 and HoxB1 gene loci in the Egyptian sample group. No statistically significant association was found between the HoxA1 or HoxB1 gene variants and the occurrence of autism. similarly when matching the probands to paternal and maternal genes separately, no statistically significant relationship was detected (p<0.05). Finally, we could not find an association between the gene variants and the severity of autism. Conclusion. Association studies of HoxA1 and HoxB1 with autism have been carried out in various populations, but no such study is available on the Egyptian population. Additional studies are needed to address further the possible association of these neurodevelopmental genes and autism.
Objectives: Genetic typing of the intronic (CA)n loci within the dystrophin gene was exploited for linkage analysis and diagnosis of carrier status. Design and methods: IP-RP-HPLC was developed for genotyping of 98 chromosomes. Results: The typing of 2-bp STRs with mean SD of +/- 0.43 bp ensured an allele's identity with 99.7% confidence. Conclusion: The automation enabled the accurate sizing of unlabeled alleles with an excellent reproducibility of 100% with significant labor saving and reduced reagent costs.
Vitiligo is a very common, often heritable acquired disorder characterized by wellcircumscribed milky white cutaneous macules devoid of identifiable melanocytes. Vitiligo appears to be more commonly observed in parts of the body exposed to the sun and in darker skin types and may develop at any age. Our study contained 47 unrelated Egyptian young-aged vitiligo patients (age range 2-18 y) and 14 healthy volunteers of matched age range. All patients experienced active vitiligo lesions with no signs of other autoimmune disorders. To genotype the TAP1 (C>T intron 7) and LMP7 (G>T intron 6), we amplified the genomic DNA using polymerase chain reaction (PCR) using genomic DNA followed by enzymatic digestion. Our results showed no significant difference between TAP1 C>T (intron 7) and LMP7 G>T (intron 6) alleles and healthy controls (p= 0.3 or 0.5, respectively). Even so, the odd ratios (ORs) for the genotypes of the TAP1 (C>T) were 1.78 (0.4 to 7.0), 0.8 (0.5 to 1.2), and 1.19 (0.2 to 4.9) for the TAP1-CC, CT, TT-genotypes, respectively. The ORs of LMP7 (intron G>T) genotypes were 1.4 (0.3 to 6.0), 0.8 (0.5 to 1.3), 1.19 (0.3 to 3.6) for GG, GT, and TT, respectively. However, a major contribution of both TAP1 and LMP7 polymorphisms to vitiligo susceptibility cannot be excluded. Further studies of other alleles within the TAP and LMP gene regions in Egyptian patients is recommended to demonstrate a possible role for MHC class I antigen processing and/or presentation pathway in the antimelanocyte autoimmune response in vitiligo pathogenesis
Phenylketonuria is the most common inborn error of amino acid metabolism in Egypt with a relatively higher incidence of 1:7,500. Unrelated fifty-one PKU probands were selected from the database-records at the Medical Genetics Center, AinShams University-Cairo. We analyzed the DNA samples using polymerase chain reaction (PCR) combined with restriction enzyme assays, or allele specific oligonucleotide (ASO) testing and direct sequencing to detect 10 PAH gene mutations in exons 2, 3, 6, 7 and 11. We interestingly identified a novel missense CpG site R243P mutation. Moreover, three new known mutations L48S, delEX3 and Y277D unreeled in the Egyptian population. The ten detected mutations covered 58% representing 47 positive chromosomes. The most common mutation was represented by IVS10nt546 (10.8%), while the total missense mutations in our sample group account for the majority of mutations 40%. The high heterozygosity of the mutant PAH locus in Egypt suggests that multiple founder events would explain the presence of hyperphenylalaninemia in Egypt. Further studies will however be necessary to fully exploit the potential of PAH gene analysis to reconstruct the genetic history of PKU in Egypt in context with migrations among European and Mediterranean populations.
Duchenne and Becker muscular dystrophy (D/BMD) are X-linked recessive disorders resulting from mutations in the DMD gene. Since there is no cure or effective treatment for progressive muscular dystrophy, prevention of the disease is important and strongly depends on carrier status in-formation. Two-thirds of DMD/BMD cases are familial, thus female relatives are candidates for carrier-risk assessment. Segregation analysis of polymorphic short tandem (CA)n repeats [STR-(CA)n] was used to establish and compare the haplotypes of DMD patients with those of their at-risk relatives in order to determine the carrier status. However, 59 D/BMD index families and 35 of their at-risk female relatives were analyzed using the ion-pair reversed phase high performance liquid chromatography (IP-RP-HPLC) method. Comparison between the re-sults of CPK of the carriers and linkage analysis revealed that values higher than the normal level were compatible in 100% of the cases with the carrier status. On the other hand, normal values do not distinguish between the healthy and carrier populations. In conclusion, the unlabeled IP-RP-HPLC-STR assay represents an excellent molecular tool for carrierstatus identification and consequently the genetic counseling for the early prevention of such diseases.
Tumor necrosis factor (TNF) is a proinflammatory cytokine that is eminently important in the pathogenesis of bronchial asthma. Bronchial asthma is a frequent respiratory disease characterized by variable airflow obstruction, inflammation of the airways, and bronchial hyper-responsiveness (BHR). In an effort to find out the polymorphism(s) in genes whose variant(s) have been implicated in asthma phenotypes, we examined the genetic effects of TNF (TNFA and TNFB) polymorphisms on the Egyptian asthmatic children. In this study, skin prick test (SPT), total IgE level, pulmonary functions including FVC, PEF, FEV1, and FEF25−75, and bronchial asthma hyper-responsiveness (BHR), were investigated. Sixty asthmatic subjects, as defined by standard MRC respiratory questionnaire, plus 40 healthy controls were genotyped for two common single-nucleotide polymorphisms (SNP) using enzymatic digestion of polymerase chain reaction (PCR). Asthma was significantly more common in subjects with TNFA−1031C>T (P= 0.007). Although the SNP containing TNFB+252A>G polymorphism might seem to be excluded in our sample as a cause of the disease (P= 0.6), it was in a very strong linkage disequilibrium with TNFA−1031C>T (P= 0.000002). All the TNFA−1031C>T genotypes were in a strong association with the severity of the asthma. Incidentally, the LTαNcoI-AA (80%) was the most predominant genotype with the severe form. However, someone might predict the severity of asthma and consequently the phenotype of an asthmatic individual by knowing the polymorphism of either the TNFA−1031C>T or even the LTαNcoI. These findings may have implications for future early intervention studies by helping to identify infants at increased risk forchildhood asthma.
Genetic typing of the intronic (CA)n loci 44, 45, 49, and 50 within the dystrophin (DMD) gene is widely used to determine carrier status for Duchenne and Becker muscular dystrophies. This is typically accomplished by electrophoresis-based methods follo-wing PCR amplification with fluoresceently labeled primers. We have used the unlabeled ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) to develop a simple and rapid method for rapid identification of female carriers of the dystrophin gene. DNA molecular size markers were efficiently used to type the 2-bp short tandem repeat (STR). In our Egyptian sample of 98 chromosomes, the most common alleles within the DMD gene were 200, 176, 245, and 243 bp due to STR44, 45, 49, and 50 markers, respectively. The true heterozygosity values of these markers range from 73.5 to 90.9%, and observed allele numbers range from 8 to 17 in 98 chromosomes revealing high polymorphism of the four (CA)n system of the DMD gene.
Pediatric asthma is considered a complex multifactorial disease, with an obvious genetic predisposition and the possible involvement of noxious environmental factors. Glutathione S-transferase genes are known as risk factors predisposing to some environ-mentally induced diseases. This study has examined the hypothesis that glutathione S-transferase (GSTM1) genotype may play a role in asthma and wheezing occurrence among those exposed to tobacco smoke. Genomic DNA samples isolated from 35 asthmatic children and 35 healthy children were amplified using the flanking GSTM1 primer set premixed with the internal set. Asthmatic children showed a significant high prevalence of the GSTM1-null genotype (odds ratio [OR] 2.2, 95% confidence interval [CI] 1.4-3.4). Among GSTM1 null children, in utero smoke exposure was associated with increased prevalence of asthma (OR 3.7, 95% CI 1.9- 7.3). The intermediate electrophilic metabolites, arising in the first phase of detoxification of tobacco smoke, are not utilized by GST enzyme in asthmatic children. These intermediate metabolites may therefore attack cells and provoke oxidative stress, which contribute to the pathogenesis of asthma. Our findings indicate that there are important longterm effects of in utero smoke exposure in a genetically suscptible group of children (genetic-environmental interaction).
Partial gene deletion is the major type of mutation leading to Duchenne muscular dystrophy (DMD) and its mild allelic form, Becker muscular dystrophy (BMD). Amplification of the genomic DNAs of 152 unrelated dystrophin patients using multiple primers detected 78 (51.3%) probands with deletion mutations. We predicted the translational reading frame for all the deletions in Egyptian dystrophin males. The frameshift rule was confirmed positively ranging for 50 to 67% of the cases depending on the type of disease. We discuss ways of accounting for some exceptions from the frameshift hypothesis in the central and proximal regions. These explanations may help in developing procedures for reducing the severity of dystrophin phenotypes to restore the correct frame by disrupting the translational fidelity. Great efforts have been put into the development of effective 'gene correction' procedures via such intrinsic mechanisms. In addition, we mapped regional difference in deletion mutation frequencies within the DMD gene locus between the different Egyptian governorates. There were no double deletions in the Egyptian dystrophin males.
Autosomal recessive congenital ichthyosis (ARCI) is a rare heterogeneous keratinization disorder of the skin. It is clinically divided into 2 subtypes, lamellar ichthyosis (LI) and congenital ichthyosiformis erythroderma (CIE). We investigated forty-three ARCI Egyptian individuals in 16 severe LI, and 10 CIE families. We identified 5 alleles in two Egyptian families as having intron-5/exon-6 splice acceptor mutation recognized by the MspI restriction endonuclease. This promoted to a frequency of 9.6% for this mutation (5 splice-mutation alleles/52 alleles tested). We extended our previous dataset to update the detection of R142H mutation in 4 CIE Egyptian families and one LI phenotype (frequency of 28.8%; 15/52), whereas we still had no R141H among our Egyptian population. There was no correlation between phenotype and genotype in our study. Surprisingly, the mutant alleles detected in intron-5 acceptor splice-site were associated with the other extreme of CIE phenotypes rather than the severe LI form. We clearly demonstrated that the ARCI Egyptian families in Upper Egypt was ethnically pure and had a tendency not to be a hybrid with other populations in Lower Egypt, Delta zone and Cairo city.
Beta-thalassemia major is an inherited blood disorder leading to life-threatening anemia and requiring regular blood transfusion and iron-chelating therapy throughout life from early childhood. This study aimed at antenatal diagnosis of beta-thalassemia using both DNA from fetal cells in maternal circulation versus amniocentesis in attempt to establish a non-invasive and easy prenatal diagnostic strategy. Identification of the prevalent beta-thalassemia mutant alleles of fetal DNA in maternal circulation using the recent technology of denaturing high performance liquid chromatography (DHPLC) after ARMS-PCR amplifications of all the DNA samples were identified. Genomic DNA was prepared from 42 peripheral blood samples (14 probands, 14 fathers to test for paternal mutant alleles and 14 mothers to test both maternal DNA as well as the circulating fetal DNA in their blood) together with 12 amniotic fluid samples taken at early amniocentesis. The most prevalent genotypes among thalassemic probands of the study were b+IVS1nt110/bIVS1nt1 b+IVS1nt110/ bCD39 and bIVS1nt1/b+IVS1nt6 with 14.28% frequency for each. Using DHPLC-WAVE system (Transgenomic Inc., Crewe, UK), amplification of fetal DNA from maternal blood may provide a prenatal diagnostic test for beta-thalassemia with the potential of identifying 85.7% of the affected pregnancies in this study that represent the com-pound heterozygote conditions. Invasive testing may be limited to only 14.3% of b-thalassemia pregnancies (homozygote conditions). The comparative study between the DHPLC and the classical approaches of mutation screening revealed that the DHPLC allows the application of a non-invasive, rapid, highly sensitive and cost-effective strategy for prenatal diagnosis especially when testing large number of samples.
- Jan 2003
Autosomal recessive congenital ichthyosis (ARCI) is a rare heterogeneous keratinization disorder of the skin. It is clinically divided into 2 subtypes, lamellar ichthyosis (LI) and congenital ichthyosiformis erythroderma (Cl E). We investigated forty-three ARCI Egyptian individuals in 16 severe LI, and 10 CIE families. We identified 5 alleles in two Egyptian families as having intron-5/exon-6 splice acceptor mutation recognized by the Mspl restriction endonuclease. This promoted to a frequency of 9.6% for this mutation (5 splice-mutation alleles/52 alleles tested). We extended our previous dataset to update the detection of R142H mutation in 4 CIE Egyptian families and one LI phenotype (frequency of 28.8%; 15/52), whereas we still had no R141 H among our Egyptian population. There was an observation concerning the correlation between phenotype and genotype in our study. Surprisingly, the mutant alleles detected in intron-5 acceptor splicesite were associated with the other extreme of CIE phenotypes rather than the severe LI form. We clearly demonstrated that the ARCI Egyptian families in Upper Egypt was ethnically pure and had a tendency not to be a hybrid with other populations in Lower Egypt, Delta zone and Cairo.