Prevalence of the mitochondrial A 1555G mutation in Moroccan patients with non-syndromic hearing loss.

UNLABELLED: Mutations in mitochondrial DNA (mtDNA), especially the A1555G transition in the 12S rRNA gene, are one of the causes of both aminoglycoside-induced and non-syndromic sensorineural hearing loss. OBJECTIVE: The aim of this study was to determine the prevalence of the A1555G mitochondrial mutation in Moroccan patients. METHODS: We performed molecular characterization by PCR-RFLP and direct sequencing of one hundred and sixty four patients (84 unrelated familial and 80 sporadic cases) with a congenital sensorineural non-syndromic hearing loss and one hundred normal hearing controls for the occurrence of the A1555G mutation. RESULTS: Mutational analysis of the mtDNA showed the presence of the homoplasmic A1555G mutation in three families, leading to a frequency of 3.6% similar to that reported for European-populations. No A1555G mutation was detected in sporadic and controls cases. However, we detected in twenty normal hearing controls a novel polymorphism A1557C, which was not found in patient samples. We further evidenced the presence of the A1438G mitochondrial polymorphism in four patients with sensorineural hearing loss and in five controls. CONCLUSION: Our results show that the occurrence of the A1555G mutation in hearing impaired patient's accounts for 3.6% in a Moroccan patients and those novel mtDNA polymorphisms might contribute to a novel sub-haplogroup specific of the Magrheb.

Assessment of phylogenetic structure of Berber-speaking population of Azrou using 15 STRs of Identifiler kit.

Allele frequencies for 15 STR autosomal loci of Identifiler kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) in the Moroccan population of Berber-speaking of Azrou, were assessed from a sample of 201 unrelated individuals. Markers D18S51, D2S1338, FGA and D21S11 present the highest power of discrimination (PD) values while D21S11 was the most polymorphic locus in the studied population. The phylogenetic tree established among worldwide populations, shows that Berber-speaking population of Azrou was so close to the Berber-speaking population of Asni but also to the Arab-speaking population of southern Morocco. Nevertheless, a significant distance was observed between populations of Azrou and Bouhria even they share the same dialect (Amazigh) and belong to the same geographical area (Morocco). The 15 STR loci studied appear to be highly discriminating, thus providing a powerful tool for forensic applications, paternity investigation, individual identification and anthropological studies.

Allele frequencies of 15 autosomal STR loci in the southern Morocco population with phylogenetic structure among worldwide populations.

Polymerase chain reaction (PCR) amplification using the AmpFl STR Identifiler kit was performed in a random sample of 204 unrelated individuals from the Arabic-speaking population of the southern Morocco. Allele frequencies of 15 STRs loci (D13S317, D16S539, D2S1338, vWA, TPOX, D18S51, D5S818, FGA, D8S1179, D21S11, D7S820, D19S433, CSF1PO, TH01 and D3S1358) have been reported in this population. Markers D18S51, FGA, D2S1338 and D21S11 had the highest power of discrimination (PD) values while TH01 was the most informative locus in the studied population. The phylogenetic tree established among worldwide populations and genetic distance values show a great affinity between the Southern Moroccan population, Saudian, Moroccan of Asni and Andalusian. Our data is useful for anthropological and other comparative studies of populations and is powerful for forensic and paternity testing in the Arabic-speaking population of the Southern Morocco.

Carrier frequencies of mutations/polymorphisms in the connexin 26 gene (GJB2) in the Moroccan population.

Mutations in the Connexin 26 gene (GJB2/Cx26) are responsible for more than half of all cases of prelingual nonsyndromic recessive deafness in Caucasians. The carrier frequency of the 35delG-GJB2 mutation was found to be as high as 2-4% in the Mediterranean populations. Different GJB2 mutations were reported in the Moroccan patients with autosomal recessive nonsyndromic hearing loss; however, rare studies were carried out on the carrier frequencies of these mutations in the healthy populations. The aim of this study was to estimate the carrier frequencies of the GJB2 mutations in the Moroccan population. The molecular analysis of the 35delG mutation and other GJB2 sequence variations was performed in 386 healthy unrelated Moroccan individuals with no known hearing loss. Five GJB2 sequence variations at heterozygous state were found: two mutations, 35delG and 109G > A (V37I), and three polymorphisms, 79G > A (V27I), 341G > A (E114G), and 457G > A (V153I). The carrier frequency of the 35delG mutation was the highest with 2.07% [95% confidence interval (0.90-4.04%)], followed by that of the V37I mutation with 1.43% (0.06-5.39). The carrier frequency of V27I, E114G, and V153I changes was estimated to be 0.71% (0.01-4.34). This finding shows that the 35delG carrier frequency found here is similar to the one observed in Mediterranean populations. It provides new information about GJB2 carrier rates facilitating the diagnosis and the genetic counseling in the Moroccan population.

The analysis of three markers flanking GJB2 gene suggests a single origin of the most common 35delG mutation in the Moroccan population.

In Caucasian populations a single mutation, 35delG, accounts for the majority of GJB2 gene mediated hearing loss, with carrier frequencies estimated between 2-4%, possibly resulting from a founder effect rather than from a mutational hot spot. In Moroccan population, the 35delG mutation accounts for 90.8% of all GJB2 mutated alleles in deaf patients with a carrier frequency of 2.65%. The aim of this study was to evaluate whether the 35delG mutation has derived from a single origin in the Moroccan population. We enrolled 30 unrelated deaf patients homozygous for the 35delG mutation and 165 unrelated control individuals negative for this mutation, and genotyped three microsatellite markers flanking the GJB2 region: D13S141, D13S175 and D13S143. Data analysis revealed that the 35delG mutation is associated with particular alleles of these markers, with significant linkage disequilibrium for the 125 and 105 nucleotide long alleles of D13S141 and D13S175, and that a single specific haplotype accounts for 68% of the chromosomes carrying the 35delG mutation. The estimate age of 35delG mutation is 135 generations or approximately 2700 years old. Like in other Mediterranean populations, our results suggest that in the Moroccan population the 35delG mutation has derived from a single origin in a common founder process.

Absence of GJB3 and GJB6 mutations in Moroccan familial and sporadic patients with autosomal recessive non-syndromic deafness.

UNLABELLED: Deafness is an etiologically heterogeneous trait with a wide variety of genetic and environmental causes. It is generally considered that genetic factors account for at least half of all cases of profound congenital deafness, which can be classified in two categories - dominant or recessive - according to the mode of inheritance and in two types - syndromic or non-syndromic - according to the presence or absence of some other specific clinical features. Mutations in the gene GJB2, encoding the gap junction protein connexion 26, are considered to be responsible for up to 50% of familial cases of autosomal recessive non-syndromic hearing loss and for up to 15-30% of the sporadic cases. It has also been reported that mutations in the GJB6 and GJB3 genes contribute to autosomal recessive and autosomal dominant hearing defects in many populations. OBJECTIVE: The aim of this study was to screen mutations in GJB6 and GJB3 genes in Moroccan patients with autosomal non-syndromic hearing loss. METHODS: We carried out 95 patients with non-syndromic hearing loss. The patients, who were negative for homozygous mutations in GJB2 gene and GJB6-D13S1830 deletion, were tested for the coding regions of GJB6 and GJB3 genes by direct sequencing. RESULTS: No deleterious mutation in GJB6 and GJB3 genes was detected in all deaf patients tested. Only a C357T silent transition in heterozygous state was found in the GJB3 gene in one patient. CONCLUSION: The present data demonstrated that mutations in the GJB6 and GJB3 genes are an infrequent cause of non-syndromic deafness in Morocco.

No association between T222P/LGR8 mutation and cryptorchidism in the Moroccan population.

BACKGROUND: Cryptorchidism is the most common genital anomaly in men. The INSL3/LGR8 system is involved in testicular descent via gubernacular development. INSL3 binds with high affinity to its receptor LGR8 and receptor activation is associated with cAMP signaling. Analysis of human INSL3 and LGR8 mutations confirms that some cases of cryptorchidism are caused by mutations in these genes. The T222P mutation is the only one within the LGR8 gene associated with the cryptorchidism phenotype. A strong association of the T222P mutation with cryptorchidism was found in an Italian population. Due to the same mutation being found in patients within the Mediterranean area, a possible founder effect of this mutation is supposed. METHODS: We screened 109 patients with cryptorchidism and 250 controls in a Moroccan population. RESULTS: We found that 3 of the 109 patients tested carry the T222P mutation and 4 individuals in the control group also carry the mutation. CONCLUSIONS: Our results show in fact that the same mutation is present in the Moroccan population, but an association between cryptorchidism and the T222P mutation was not found.

Genotype determination in Moroccan hepatitis B chronic carriers.

BACKGROUND: In Morocco, chronic liver disease related to hepatitis B virus (HBV) is a public health burden. Treatment of chronic hepatitis B is often complicated by the appearance of escape mutants after treatment with nucleoside analogs, especially with genotypes responsible for the more severe form of the disease. OBJECTIVES: In the present study we investigate the prevalence of the different HBV genotypes in Morocco since no previous careful study has been attempted. METHODS: Epidemiological data from 91 chronically infected patients (45 women and 46 men) were collected prospectively. Sera were tested for anti-HBc IgG, HBeAg, anti-HBe antibody and liver enzymes. Restriction Fragment Length Polymorphism (RFLP) analysis was confirmed by subsequent sequencing of the pre-S and S region of the viral genome in order to determine which HBV genotypes were prevalent among Moroccan patients. RESULTS: The mean age was 41+/-12.4 years. Ten patients (11%) were positive for hepatitis B e antigen (HBeAg) and 81 (89%) were positive for anti-HBe antibodies. By the RLFP method, genotype D, pattern D2, was found in the 77 cases where HBV was successfully amplified. Phylogenetic analysis based on pre-S/S sequences revealed that genotype D in Morocco differed from others D strains subgenotypes (D1, D2, D3 and D4). In addition, the pre-core mutant defined as HBeAg-negative/anti-HBe-positive and HBV DNA positive was detected in 86% of cases. CONCLUSIONS: Our results clearly show that genotype D and pre-core mutant are highly prevalent in Morocco.

Prevalence of common HFE and SERPINA1 mutations in patients with hepatocellular carcinoma in a Moroccan population.

BACKGROUND: Hereditary hemochromatosis and SERPINA1 mutation were reported to affect liver functions. Our objective was to estimate the prevalence of HFE and SERPINA1 (formerly known as alpha1-antitrypsin, AAT) mutations and assess their influence on hepatocellular carcinoma development. METHODS: This study included 222 controls and 96 cases with hepatocellular carcinoma. PCR-RFLP was used to characterize S and Z alleles in SERPINA1, as well as C282Y/H63D alleles of HFE. RESULTS: In healthy subjects and hepatocellular carcinoma patients as well, no homozygotes for the C282Y mutation were found. In controls, heterozygosity and homozygosity for the H63D mutation were 27 and 0.9%, respectively. Among patients, homozygosity for the H63D mutation was 3.1%, whereas heterozygosity for C282Y and H63D was 2.1 and 35.4%, respectively. Interestingly, albeit it does not reach significance (p=0.062), H63D was more prevalent in hepatocellular carcinoma patients than in controls (38.5 vs. 27.9%, respectively). The association was stronger when considering only male patients with hepatocellular carcinoma (47.1 vs. 23.6, p=0.001). Allele frequencies of S and Z in controls were 0.45% (95% CI=0.2-1.07) and 0.22% (95% CI=0.2-0.6), respectively, and 1 for S and 0% for Z in HCC. No significant difference was found between cases and controls. CONCLUSIONS: We provide a novel appraisal of HFE and SERPINA1 mutations prevalence in the Moroccan population. Results are consistent with the worldwide spread of the H63D and S mutation and the north European restriction of the C282Y and Z. Our results show that H63D carriage is increased among hepatocellular carcinoma patients, suggesting that it may confer an increased susceptibility to hepatocellular carcinoma even in a heterozygous state. On the contrary, HFE C282Y and SERPINA1 mutations do not contribute to hepatocellular carcinoma development.

Genetic polymorphism in the manganese superoxide dismutase gene is associated with an increased risk for hepatocellular carcinoma in HCV-infected Moroccan patients.

Hepatocellular carcinoma (HCC) ranks among the 10 most common cancers worldwide. The main risk factors for its development are hepatitis B and C virus infections. Hepatitis B and C viruses induce chronic inflammation and oxidative stress that could predispose a cell to mutagenesis and proliferation. Manganese superoxide dismutase (MnSOD) catalyses the detoxification of free radicals, thus playing a crucial role in the protection against damage. A valine (Val) to alanine (Ala) substitution at amino acid 9, mapping within the mitochondrion-targeting sequence of the MnSOD gene, has been associated with an increased cancer risk. The aim of our study was to investigate a possible association of the Val/Ala-MnSOD polymorphism and HCC development in Moroccan patients. Genotypes were determined by means of PCR and RFLP analysis in 96 patients with HCC and 222 control subjects matched for age, sex, and ethnicity. Homozygous Ala/Ala carriers were 31% in the cases and 18% in the controls, which corresponds to an odds ratio (OR) of 2.89, with a 95% confidence interval (CI) of 1.47-5.68. Stratification into subgroups based on HCV infection status revealed an even more increased risk for homozygous Ala/Ala carriers with hepatitis C infection (38.2% in the cases versus 14.8% in the control subjects OR, 5.09; 95% CI, 1.76-14.66). Our findings provide further evidence of an association between the Ala-9Val MnSOD polymorphism and HCC occurrence in hepatitis C virus-infected Moroccan patients.

Analysis of MYO7A in a Moroccan family with Usher syndrome type 1B: novel loss-of-function mutation and non-pathogenicity of p.Y1719C.

PURPOSE: Mutations in the MYO7A gene are responsible for Usher syndrome type 1B (USH1B), the most common USH1 subtype, which accounts for the largest proportion of USH1 cases in most populations. Molecular genetic diagnosis in Usher syndrome is well established and identification of the underlying mutations in Usher patients is important for confirmation of the clinical diagnosis and genetic counseling. METHODS: We analyzed a large consanguineous USH1 family from Morocco and linked the disease in this family to the MYO7A/USH1B locus. RESULTS: We identified the frequently described missense change p.Y1719C. In addition, we found the homozygous c.1687G>A mutation in the last nucleotide of exon 14, which is predicted to result in aberrant splicing and may lead to loss of MYO7A transcript. We further showed that p.Y1719C is not disease-causing but does represent a frequent polymorphism in the Moroccan population, with an estimated carrier frequency of 0.07. CONCLUSIONS: This finding has an important impact for molecular diagnosis and genetic counseling in USH1B families.

AZF microdeletions and partial deletions of AZFc region on the Y chromosome in Moroccan men.

AIM: To evaluate for the first time the frequency of Y chromosome microdeletions and the occurrence of the partial deletions of AZFc region in Moroccan men, and to discuss the clinical significance of AZF deletions. METHODS: We screened Y chromosome microdeletions and partial deletions of the AZFc region of a consecutive group of infertile men (n = 149) and controls (100 fertile men, 76 normospermic men). AZFa, AZFb, AZFc and partial deletions of the AZFc region were analyzed by polymerase chain reaction (PCR) according to established protocols. RESULTS: Among the 127 infertile men screened for microdeletion, four subjects were found to have microdeletions: two AZFc deletions and two AZFb+AZFc deletions. All the deletions were found only in azoospermic subjects (4/48, 8.33%). The overall AZFc deletion frequency was low (4/127, 3.15%). AZF microdeletions were not observed in either oligoasthenoteratozoospermia (OATS) or the control. Partial deletions of AZFc (gr/gr) were observed in a total of 7 of the 149 infertile men (4.70%) and 7 partial AZFc deletions (gr/gr) were found in the control group (7/176, 3.98%). In addition, two b2/b3 deletions were identified in two azoospermic subjects (2/149, 1.34%) but not in the control group. CONCLUSION: Our results suggest that the frequency of Y chromosome AZF microdeletions is elevated in individuals with severe spermatogenic failure and that gr/gr deletions are not associated with spermatogenic failure.

The Pro variant of the p53 codon 72 polymorphism is associated with hepatocellular carcinoma in Moroccan population.

AIM: Codon 72 polymorphism of the p53 gene has been implicated in cancer risk, and it has been suggested that it may have an impact on the clinical outcome of the disease. Our objective was to evaluate the association between p53 polymorphism at codon 72 and hepatocellular carcinoma (HCC) in the Moroccan population. METHODS: Genomic DNA was extracted from peripheral blood cells of 96 patients with HCC and 222 controls without HCC matched for age, gender and ethnicity. Codon 72 polymorphism of p53 was identified by PCR-restriction fragment length polymorphism, confirmed by sequencing. RESULTS: Patients with HCC had higher frequencies of Pro/Pro (13.5% vs. 6.3%, P < 0.02) than controls and consequently a 2.3-fold increased risk of liver cancer development (odds ratio [OR], 2.304; 95% confidence interval [CI], 1.014-5.234). In addition, we found a significant association between the p53Arg72Pro polymorphism and the female gender in HCC. Men with Pro/Pro genotype had a 1.57-fold increased risk for HCC, whereas the corresponding genotype in women had a 4.4-fold increased risk of HCC (OR, 4.4; 95% CI, 1.18-16.42). No correlation between the polymorphism and HCC risk was found when comparing the hepatitis C virus (HCV)-positive cases to HCV-positive controls. However, HCV-negative subjects and Pro/Pro genotype had a 3.31-fold increased risk for HCC. CONCLUSION: These results provide evidence that p53 polymorphism at codon 72 is a modifier of hepatocarcinogenesis, especially in women and HCV-negative subjects.

GJB2 (connexin 26) gene mutations in Moroccan patients with autosomal recessive non-syndromic hearing loss and carrier frequency of the common GJB2-35delG mutation.

OBJECTIVE: Mutations in the connexin 26 gene (GJB2), which encodes a gap-junction protein expressed in the inner ear, have been shown to be responsible for a major part of autosomal recessive non-syndromic hearing loss in Caucasians. The aim of our study was to determine the prevalence and spectrum of GJB2 mutations, including the (GJB6-D13S1830) deletion, in Moroccan patients and estimate the carrier frequency of the 35delG mutation in the general population. METHODS: Genomic DNA was isolated from 81 unrelated Moroccan familial cases with moderate to profound autosomal recessive non-syndromic hearing loss and 113 Moroccan control individuals. Molecular studies were performed using PCR-Mediated Site Directed Mutagenesis assay, PCR and direct sequencing to screen for GJB2, 35delG and del(GJB6-D13S1830) mutations. RESULTS: GJB2 mutations were found in 43.20% of the deaf patients. Among these patients 35.80% were 35delG/35delG homozygous, 2.47% were 35delG/wt heterozygous, 3.70% were V37I/wt heterozygous, and 1 patient was E47X/35delG compound heterozygous. None of the patients with one or no GJB2 mutation displayed the common (GJB6-D13S1830) deletion. We found also that the carrier frequency of GJB2-35delG in the normal Moroccan population is 2.65%. CONCLUSIONS: These findings indicate that the GJB2-35delG mutation is the major cause of autosomal recessive non-syndromic hearing loss in Moroccan population. Two other mutations were also detected (V37I and E47X), in agreement with similar studies in other populations showing heterogeneity in the frequencies and types of mutation in connexin 26 gene.

Novel mutations involving the INSL3 gene associated with cryptorchidism.

PURPOSE: Cryptorchidism affects 1% to 9% of full-term male neonates. Hypospadias is the second most frequent congenital anomaly seen in newborn males. These pathological conditions are part of the testicular dysgenesis syndrome. Insulin-like factor 3 and LGR8 (leucine-rich repeat-containing G protein-coupled receptor 8), acting as a hormone and a receptor, respectively, are involved in control of the first phase of testicular descent via gubernacular development. MATERIALS AND METHODS: The study group consisted of 184 patients, of whom 52 presented with unilateral cryptorchidism, 37 presented with bilateral cryptorchidism, 19 presented with cryptorchidism and hypospadias, 1 presented with bilateral cryptorchidism and micropenis, and 75 presented with isolated hypospadias. A control panel consisted of 270 controls, including 127 fertile, and 143 fertile noncryptorchid males. Insulin-like factor 3 mutations were analyzed by direct sequencing and restriction enzyme digestion. We analyzed the ability of the mutant insulin-like factor 3 peptides identified in this study to activate LGR8 receptor in an ex vivo assays. RESULTS: We identified 3 novel insulin-like factor 3 variants, including C-19G, V18M and R105H, in 3 of the 109 patients (2.75%) but in none of the 270 controls. The V18M mutation in the insulin-like factor 3 signal peptide had a significant deleterious effect in activating LGR8 receptor in ex vivo studies (p<0.05). To our knowledge we report the first variant in the promoter region of the insulin-like factor 3 gene in a patient with cryptorchidism in association with micropenis. CONCLUSIONS: Mutations involving the insulin-like factor 3 gene may contribute to other anomalies of male genital development, such as micropenis.

Male sterility thermal thresholds in Drosophila: D. simulans appears more cold-adapted than its sibling D. melanogaster.

Numerous different criteria may be used for analysing species thermal adaptation. We compared male sterility thresholds in the two most investigated cosmopolitan siblings, D. melanogaster and D. simulans. A survey of various populations from Europe and North Africa evidenced consistent differences between the two species, and a detailed analysis was made on flies from Marrakech. Sharp sterility thresholds were observed in both species but at different temperatures: D. simulans appeared more tolerant to cold than its sibling (difference 1 degrees C) but more sensitive to heat (difference 1.5 degrees C). When transferred to an optimum temperature of 21 degrees C, D. simulans males, sterilized by a low temperature, recovered more rapidly than males of D. melanogaster; the reverse was true on the high temperature side. The analysis of progeny number also revealed the better tolerance of D. simulans males to cold but a lesser tolerance to heat. From these observations, we might expect that D. simulans should be more successful in cold temperate countries than its sibling, while ecological observations point to the contrary. Our data clearly show the difficulty of comparing ecophysiological data to field observations, and also the need of extensive comparative life history studies in closely related species.