G2691A and C2491T mutations of factor V gene and pre-disposition to myocardial infarction in Morocco.

Coagulation factor Leiden mutation has been described as a common genetic risk factor for venous thrombosis; however, this mutation was reported to be practically absent in an African population. Recently, a novel non-sense mutation in the gene encoding factor V has been associated with the risk of occurrence of cardio-cerebrovascular diseases such as stroke and venous thrombosis. The aim of the present study was to investigate whether the factor V Leiden (FVL) and C2491T non-sense mutations are associated with the risk of developing myocardial infarction. Genotyping of FVL and C2491T FV was performed using the polymerase chain reaction restriction fragment length polymorphism method on a sample of 100 patients with myocardial infarction as well as 211 controls. In the study population, the frequency of the FVL mutation was practically zero. However, with regard to the C2491T mutation, the TT genotype was associated with an increased risk of myocardial infarction [odds ratio (OR)=3.16, 95% confidence interval (CI): 1.29-7.71, P=0.03]. A significant association between the C2491T FV mutation and the risk of myocardial infarction was identified using recessive (OR=2.74, 95% CI: 1.14-6.58, P=0.04), dominant (OR=1.85, 95% CI: 1.13-3.04, P=0.02) and additive (OR=1.88, 95% CI: 1.25-2.80, P=0.004) models. Furthermore, a positive correlation was found between the presence of the C2491T FV mutation and hypertension (P=0.02), which is associated with myocardial infarction. In conclusion, the results of the present study suggested that the C2491T non-sense mutation of the FV gene may be a risk factor for myocardial infarction in a Moroccan population.

Genetic polymorphisms of T-1131C APOA5 and ALOX5AP SG13S114 with the susceptibility of ischaemic stroke in Morocco.

Ischaemic stroke is a multifactorial disease. Genetic polymorphisms involved in lipid, inflammatory and thrombotic metabolisms play an important role in the development of ischaemic stroke. The present study aimed to assess the relationship between T1131C APOA5 and SG13S114 ALOX5AP polymorphisms and the risk of ischemic stroke in 175 cases and 201 controls. Genotyping was performed by high resolution melting and polymerase chain reaction restriction fragment length polymorphism methods. In the case of T-1131C APOA5, a modest risk of ischaemic stroke was noticed with CC (OR: 2.86; 95% CI = 1.24-6.58; Pc = 0.039) and C allele (OR: 1.54; 95% CI = 1.01-2.33; Pc = 0.014). For SG13S114 ALOX5AP, a significant association was observed among subjects with TT (OR: 2.57; 95% CI =1.49-4.83; Pc = 0.009) and T allele (OR: 1.59; 95% CI = 1.16-2.19; Pc = 0.008). According to the risk factors of ischaemic stroke, a positive correlation was observed only between SG13S114 variant of ALOX5AP gene and hypertension (Pc = 0.026). Despite lower sample size, T-1131C APOA5 and SG13S114 variants could be considered an independent genetic risk factor of ischaemic stroke in Moroccan population.

Screening of exon 11 of BRCA1 gene using the high resolution melting approach for diagnosis in Moroccan breast cancer patients.

BACKGROUND: Identification of specific mutations in cancer patients may lead to the discovery of genes, which can affect susceptibility and/or prognosis. It has previously been reported that mutations in BRCA1 and BRCA2 genes are linked to breast cancer. Here, we evaluated the use of the High Resolution Melting (HRM) approach to screen for mutations in exon 11 of BRCA1 gene in Moroccan patients. METHODS: HRM analysis was used to screen exon 11 from 71 breast cancer patients in order to detect different variants. Conventional Sanger sequencing was used to confirm the presence of possible mutations. Distribution of different SNPs was determined by SNaPshot analysis software. RESULTS: In order to assess the efficacy of the HRM approach to screen for mutations, especially in diagnosis, we first used two samples with previously known mutations, "2924delA and 3398delC". Indeed, these previously known sequence variants were detected by the HRM approach and yielded melting curves with atypical shape relative to wild-type control sequences. We then analyzed, 69 samples from breast cancer patients using the HRM method, and were able to detect two samples with atypical curves. Sequencing of the two samples, using the conventional Sanger approach, confirmed the presence of the same SNP (c.2612C > T) in both samples. CONCLUSIONS: Our results strongly suggest that the HRM approach represents a reliable and highly sensitive method for mutation scanning, especially in diagnosis.

Novel mutations in the amyloid precursor protein gene within Moroccan patients with Alzheimer's disease.

In Morocco, Alzheimer's disease (AD) affects almost 30,000 individuals, and this number could increase to 75,000 by 2020. To our knowledge, the genes predisposing individuals to AD and predicting disease incidence remain elusive. In this study, we aimed to evaluate the genetic contribution of mutations in the amyloid precursor protein (APP) gene exons 16 and 17 to familial and sporadic AD cases. Seventeen sporadic cases and eight family cases were seen at the memory clinic of the University of Casablanca Neurology Department. These patients underwent standard somatic neurological examination, cognitive function assessment, brain imaging, and laboratory tests. Direct sequencing of exons 16 and 17 of the APP gene was performed on genomic DNA of AD patients. In this original Moroccan study, we identified seven novel frameshift mutations in exons 16 and 17 of the APP gene. Interestingly, only one novel splice mutation was detected in a family case. There is a strong correlation between clinical symptoms and genetic factors in Moroccan patients with a family history of AD. Therefore, mutations in APP gene exons 16 and 17 may eventually become genetic markers for AD predisposition.

Prenatal diagnosis of BMD in Morocco: evolution and limits.

The muscular dystrophy is a group of inherited disorders characterized in the most of cases by progressive muscle weakness. The best known are X-linked disorder Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). BMD is a milder form of the disease with a later age of onset and a slower clinical progression. The DMD gene, located on Xp21, is the largest human gene in the human genome (2.3 Mb). DMD gene consists of 79 exons and codes for dystrophin protein. A 9-year-old boy, who experienced symptoms of the disease, was admitted to the Casablanca University Children's Hospital. The patient, with no known family history of significant muscle disease, was first examined at 4 years of age because of walking difficulties and a limited hands force. Blood tests revealed elevated serum levels of creatine kinase (7.60 U/L). The electromyogram showed myopathic changes, consisting of polyphasic potentials, and the muscular biopsy revealed dystrophic aspect. Analysis of the dystrophin-encoding gene by PCR deletion analysis of the dystrophin gene was performed by multiplex PCR primer sets of Chamberlain and Beggs. The analysis showed a deletion of exons 45 to 49. Mother genetic testing showed the heterozygosis deletion.

G894T endothelial nitric oxide synthase polymorphism and ischemic stroke in Morocco.

Nitric oxide plays a major role in the regulation of cerebral blood flow and loss of its function leads to alteration of the vascular relaxation given its central role in the physiology of the vascular system. G894T eNOS polymorphism could have adverse effects on the expression and activity of endothelial nitric oxide synthase, which can result in functional impairment of the endothelium and contribute to the development of ischemic stroke in the different models of transmission. In this study, genotyping with PCR-RFLP and HRM (high resolution melting) methods were conducted on 165 ischemic stroke patients as well as 182 controls. The goal here was to compare genotyping with PCR-RLFP primer sequences of eNOS gene (size < 300 bp) to HRM. Our data suggests a statistically significant association between G894T eNOS polymorphism and ischemic stroke in recessive, dominant and additive models with P < 0.05 and odds ratio of 2.68 (1.08-6.70), 1.78 (1.16-2.73), and 1.71 (1.21-2.43) respectively. In sum, although the sample size is relatively small, it suggests that G894T eNOS polymorphism could be a potentially important genetic marker of ischemic stroke in the Moroccan population. Future studies should be conducted in this direction taking into consideration the functional activity of eNOS.

Methylenetetrahydrofolate reductase C677T variant in Moroccan patients with inflammatory bowel disease.

BACKGROUND: The association of genetic polymorphisms related to metabolism of homocysteine and folate with inflammatory bowel disease has been evidenced. Several studies have identified genetic variants of MTHFR as significant susceptibility loci for Crohn's disease (CD) and ulcerative colitis (UC). The C677T genetic polymorphism in the MTHFR gene is found to be associated with a thermolabile variant enzyme that shows a reduced activity. Therefore, we investigated whether the C677T variant confers genetic susceptibility to CD or UC and evaluated the genotype-phenotype associations in the Moroccan population. METHODS: The present study included 96 inflammatory bowel disease patients (68 patients with CD and 28 with UC) and 182 healthy controls. DNA samples were genotyped for the MTHFR (C677T) mutation by the PCR-RFLP method. Statistical analyzes were performed using MedCalc software, Chi square test and Fisher test. RESULTS: The respective odds ratio for CD, UC and control group were, 1.55 (CI 95%: 0.53-4.53, P=0.52); 0.50 (CI 95%: 0.06-4.15, P=0.52) and 0.50 (CI 95%: 0.06-4.15, P=0.52). Thus, no statistically significant association with the disease was observed in frequency of the TT variant in comparison to healthy controls. Stratification of IBD patients on the basis of CD or UC showed that individuals carrying at least one T allele are not protected against Crohn's disease. Furthermore, clinical features of the disease did not show any significant association. CONCLUSION: In conclusion, the present study indicates that the genetic risk for IBD is not modulated by MTHFR C677T polymorphism in Moroccan population.

Evolution of molecular diagnosis of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the commonest of the muscular dystrophies. The DMD gene (DMD) is the biggest human gene and the most common molecular defect in the DMD gene, accounting for approximately 65 % of cases of DMD, is the deletion of one or more exons. The most basic method still in regular use involves multiplex PCR of the exons, known to be most commonly deleted. The multiplex is relatively simple. Quantitative analysis of all exons of the gene and multiplex ligation-dependent probe amplification have brought about an improvement in mutation detection rate, as they will detect all exon scale deletions as well as duplications, widely used to detect exonic and intronic mutations. As a sensitive and discriminative tool, MLPA can be used for prenatal testing. A more recent development in quantitative analysis is the use of oligonucleotide-based array comparative genomic hybridization.

Sickle cell disease with double stroke in a Moroccan family.

The sickle-cell disease is a group of chronic hemolytic diseases which associates three types of injuries: severe anemia, severe infections, and ischemic vaso-occlusive crisis that are secondary to conflicts between small vessels and red blood cells too deformable. Thus, organic various complications may arise. Its prevalence in Europe is estimated to be about 1/150 and reaches 15 % in the Mediterranean areas. Clinical manifestations vary widely from one person to another and from one moment to another. In addition to anemia and bacterial infections, vaso-occlusive crisis may manifest by focal ischemia. In the long term, the VOC may compromise the function of a particular tissue or organ. The transmission is autosomal recessive. The sickle-cell diseases are determined by combinations of two abnormal alleles of beta globin gene including at least one which carries the mutation beta 6 glu-val (Hb S). We report the case of a girl aged 11 years, who presented two strokes in the interval of 8 months, which manifested by a complete right hemiplegia and aphasia confirmed by head CT scan; the electrophoresis of the hemoglobin and the molecular test had confirmed the diagnosis of sickle-cell disease, and we were allowed to spread better reflection on the prevention of stroke, which remains a frequent and serious complication of sickle-cell disease.

Large-scale meta-analysis of genetic studies in ischemic stroke: Five genes involving 152,797 individuals.

BACKGROUND: Ischemic stroke descent has a genetic basis. Stroke represents a complex trait, which is assumed to be polygenic. On this topic, the role of a wide number of candidate genes has been investigated in stroke through association studies. MATERIALS AND METHODS: We performed a literature-based systematic review of genetic association studies in stroke abound several populations. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined for each gene-disease association. Following a review of 300 manuscripts, five candidate gene variants were analyzed among 152,797 individuals (45,433 cases and 107,364 controls). RESULTS: For these five candidate genes studied, the prothrombin OR is 1,57 (1,23-2,89), the factor V Leiden OR is 1,43 (0,67-6,24), the mean OR of angiotensin I converting enzyme (ACE) insertion/deletion (I/D) polymorphism is 1,11 (1,02-1,25), the summary OR for the C677T variant of 5,10-methylenetetrahydrofolate reductase (MTHFR) is 1,23 (0,61-1,47) and the pooled OR for the apolipoprotein E (APOE) gene is 0,95 (0,77-1,14) . CONCLUSION: These data suggest the genetic associations of some genes with ischemic stroke and it is necessary to compete with other genes. Our findings could represent an epidemiological base and a useful tool to address further molecular investigations and to realize more detailed meta-analyses.

Prevalence of angiotensin-converting enzyme, methylenetetrahydrofolate reductase, Factor V Leiden, prothrombin and apolipoprotein E gene polymorphisms in Morocco.

BACKGROUND: Evidence of the influence of genetic risk factors on cardiovascular diseases is more or less established. These genetic factors are involved in several pathways affecting blood pressure regulation, blood coagulation, homocysteine and lipid metabolisms. AIM: We evaluated frequencies of five genetic polymorphisms to assess their informativeness as markers for prospective clinical studies. SUBJECTS AND METHODS: 182 healthy Moroccan subjects were genotyped for ACE I/D by amplification alone and by amplification followed by enzymatic digestion for other polymorphisms. RESULTS: Allele frequencies of ACE ID, MTHFR C677T were 76.6%, 26.9% for D and T alleles, respectively. APOE polymorphism showed 11.3%, 78.6% and 10.2% for the alleles E2, E3 and E4, respectively. The frequency for FII G20210A polymorphism was around 2.7% for A allele. Our data showed an absence of FVL mutation. Using allele frequencies, genetic distances between Moroccan and other populations revealed an independent variability of these polymorphisms. CONCLUSION: These values appear to be influenced by findings in European and African peoples, and may be considered in assessing the clinical significance of a predisposition to cardiovascular disease.

A rapid polymerase chain reaction-based test for screening Steinert's disease (DM1).

Myotonic dystrophy (DM) is a multisystemic neuromuscular disorder caused by a dynamic mutation of (CTG) trinucleotide repeats in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). The aim of the present study was to establish the use of polymerase chain reaction (PCR)-based simple and rapid method for initial sample screening. Only a minority of samples were tested positive with the above method and need to be detected by tri primer (TP)-PCR and Southern blotting which is more time consuming and involves use of radioactive material. This study concerned 24 patients from nine families with a clinical diagnosis of the DM1. DNA extracted from the blood was used for amplification of the triplet repeat sequences at the DMPK loci. We obtained two bands for the normal subjects and one band for patients corresponding to normal DMPK allele, confirmed by the TP-PCR and the Southern blot. This rapid test for initial screening of samples for the presence of DMPK mutations is economical and reliable method. This method reduces the number of samples needing TP-PCR and Southern blotting.

Prader-Willi syndrome: Methylation study or fluorescence in situ hybridization first?

Prader-Willi syndrome (PWS) is neurogenetic disorder involving the imprinting mechanism at 15q11-13 region. We report a 4-year-old girl who was referred to our laboratory to be investigated for clinical obesity, mental deficiency and respiratory problems. The patient was born for non-consanguineous and healthy biological parents. After normal pregnancy, the patient was delivered by cesarean section at full term, with a birth weight of 2500 g, and the height and head circumference were unknown. In neonatal stage, she presented severe hypotonia with feeding problems. Her developmental progress was delayed. She walked and developed speech at the age of 3 years. Since the age of 3 years, she presented severe dental problems. Methylation study had confirmed the diagnosis, and for detecting etiology, fluorescence in situ hybridization using probes for small nuclear ribonucleoprotein polypeptide N (SNRPN), which map inside the chromosomal region 15q11-15q13, was necessary to confirm the 15q11-15q13 deletion of paternal chromosome 15, which is the predominant genetic defect in PWS. In conclusion, we report this case with an objective to reinforce the necessity of analysis of DNA methylation within the 15q11-13 region, which is an important tool for the correct diagnosis among children presenting with neonatal hypotonia, mental deficiency and obesity.

Duchenne and Becker muscular dystrophy: contribution of a molecular and immunohistochemical analysis in diagnosis in Morocco.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders caused by mutations of the DMD gene located at Xp21. In DMD patients, dystrophin is virtually absent; whereas BMD patients have 10% to 40% of the normal amount. Deletions in the dystrophin gene represent 65% of mutations in DMD/BMD patients. To explain the contribution of immunohistochemical and genetic analysis in the diagnosis of these dystrophies, we present 10 cases of DMD/BMD with particular features. We have analyzed the patients with immunohistochemical staining and PCR multiplex to screen for exons deletions. Determination of the quantity and distribution of dystrophin by immunohistochemical staining can confirm the presence of dystrophinopathy and allows differentiation between DMD and BMD, but dystrophin staining is not always conclusive in BMD. Therefore, only identification involved mutation by genetic analysis can establish a correct diagnosis.