Identification of High-Risk Single Nucleotide Polymorphisms in the Human CYB5R3 Gene Responsible for Recessive Congenital Methemoglobinemia: A Computational Approach.

Introduction: NADH-cytochrome b5 reductase deficiency due to pathogenic variants in the CYB5R3 gene causes recessive congenital methemoglobinemia (RCM) type I or type II. In type I, cyanosis from birth is the only major symptom, and the enzyme deficiency is restricted only to erythrocytes. Whereas in type II, cyanosis is associated with severe neurological manifestations, and the enzyme deficiency is generalized to all tissues. Methods: In this study, several computational methods (SIFT, Polyphen-2, PROVEAN, Mutation Assessor, Panther, Phd-SNP, SNPs&GO, SNAP2, Align, GVGD, MutPred2, I-Mutant 2.0, MUpro, Duet, ConSurf and Netsurf-2.0 tools) were used to find the most deleterious nsSNPs in the CYB5R3 gene. Furthermore, structural analysis by Swiss-PDB viewer, protein-ligand docking using FTSite, and protein-protein interaction using STRING were carried out to evaluate the impact of these nsSNPs on the protein structure and function. Results: Our in silico analysis suggested that out of 339 nsSNPs of the CYB5R3 gene, 17 (L47H, L47P, R61P, L73R G76D, G76C, P96H, G104C, S128P, G144D, P145S, L149P, Y151H, M177T, I178T, I216N, and G251V), are the most deleterious. Among them, two (P96H and S128P) were reported to be associated with the severe form RCM type II, six are related to RCM type I (G104C, G144D, P145S, L149P, M177T, and I178T), and the remaining nine high-risk nsSNPs have not yet been reported in RCM patients. Discussion: This study highlighted the potential pathogenic nsSNPs of the CYB5R3 gene. To comprehend how these most harmful nsSNPs contribute to disease, it is crucial to experimentally validate their functional effects.

First Observation of HbM-Saskatoon at the Origin of Neonatal Cyanosis in a Tunisian Baby.

Several causes are known to be at the origin of neonatal cyanosis among them methemoglobinemia is by inheritance of an hemoglobin (Hb) M variant. This is a rare condition never been reported in Tunisia so far. Here, we report a Tunisian newborn with refractory cyanosis since birth. As cardiac and respiratory diseases were ruled out, methemoglobinemia was suspected. Hematological parameters, concentration of methemoglobin, capillary electrophoresis, and amplification sequencing of the HBB gene were performed. Computational analysis was achieved by different in silico tools to investigate the mutation effect. The diagnosis was established by a raised MetHb, confirmed by the presence HbM-Saskatoon [Beta63 (E7) His>Tyr] by capillary electrophoresis and molecular analysis. The identified mutation occurred as a de novo mutation. In silico analysis confirmed the pathogenicity of the mutation. To our knowledge, this is the first time that this mutation has been reported in the Tunisian population. In view of its low incidence rate, clinicians might misdiagnose cyanosis caused by HbM, which can lead to inappropriate treatment and clinical complications. An up-to-date literature review of HbM disease is presented in this study.

Clinical and genetic investigation of catecholaminergic polymorphic ventricular tachycardia in a consanguineous Tunisian family.

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare disease presenting with syncopal events and sudden cardiac death at a young age in the absence of structural heart disease. Two major genes have been shown to be responsible for CPVT: RYR2 and CASQ2 genes involved in calcium homeostasis.Methods: We report here clinical and molecular investigation of a consanguineous Tunisian family including three affected members. Involvement of RYR2 and CASQ2 genes was investigated.Results: Mutation screening for RYR2 gene showed that no mutation were detected in the coding sequence. A novel variation c.572C/T was identified in CASQ2 gene leading to a p.Pro191Leu.Conclusion: To our knowledge, this is the first clinical and genetic investigation of CPVT in North Africa.

Molecular investigation of distal renal tubular acidosis in Tunisia, evidence for founder mutations.

BACKGROUND: Distal renal tubular acidosis (dRTA) is a rare genetic disease caused by mutations in different genes involved in the secretion of H+ ions in the intercalated cells of the collecting duct. Both autosomal dominant and recessive forms have been described; the latter is also associated with sensorineural hearing loss. METHODS: Twenty-two Tunisian families were analyzed for mutations in the ATP6V1B1 and ATP6V0A4 genes by direct sequencing. Dating of the founder mutations was performed. RESULTS: Two founder mutations in the ATP6V1B1 gene were found in 16/27 dRTA cases. The p.Ile386Hisfs*56 founder mutation was estimated to be older than 2400 years and no correlations were found with deafness. For the remaining patients, two mutations in the ATP6V0A4 gene, one of them being novel, were found in three Tunisian cases. The presence of a heterozygous missense mutation p.T30I, of the ATP6V1B1 gene, was identified in six patients, while no mutations of the second gene were detected. No deleterious mutations of either ATP6V1B1 or ATP6V0A were found for the two probands. CONCLUSION: Our study gives evidence of phenotypic and genotypic heterogeneity of dRTA in the Tunisian population. Five different mutations were found, two of them were due to a founder effect, and screening of these mutations could provide a rapid and valuable tool for diagnosis of dRTA.

Clinical and genetic investigation of a large Tunisian family with complete achromatopsia: identification of a new nonsense mutation in GNAT2 gene.

Complete achromatopsia is a rare autosomal recessive disease associated with CNGA3, CNGB3, GNAT2 and PDE6C mutations. This retinal disorder is characterized by complete loss of color discrimination due to the absence or alteration of the cones function. The purpose of the present study was the clinical and the genetic characterization of achromatopsia in a large consanguineous Tunisian family. Ophthalmic evaluation included a full clinical examination, color vision testing and electroretinography. Linkage analysis using microsatellite markers flanking CNGA3, CNGB3, GNAT2 and PDE6C genes was performed. Mutations were screened by direct sequencing. A total of 12 individuals were diagnosed with congenital complete achromatopsia. They are members of six nuclear consanguineous families belonging to the same large consanguineous family. Linkage analysis revealed linkage to GNAT2. Mutational screening of GNAT2 revealed three intronic variations c.119-69G>C, c.161+66A>T and c.875-31G>C that co-segregated with a novel mutation p.R313X. An identical GNAT2 haplotype segregating with this mutation was identified, indicating a founder mutation. All patients were homozygous for the p.R313X mutation. This is the first report of the clinical and genetic investigation of complete achromatopsia in North Africa and the largest family with recessive achromatopsia involving GNAT2; thus, providing a unique opportunity for genotype-phenotype correlation for this extremely rare condition.

Clinical and genetic investigation of pediatric cases of Wolff-Parkinson-White syndrome in Tunisian families.

BACKGROUND: Wolff-Parkinson-White (WPW) syndrome is an autosomal-dominant heart disease characterized by an accessory pathway that arises from an aberrant conduction from the atria to the ventricles. Several mutations within the PRKAG2 gene were shown to be responsible for WPW. This gene encodes the γ2 regulatory subunit of adenosine monophosphate (AMP)-activated protein kinase, which functions as a metabolic sensor in cells, responding to cellular energy demands. METHODS: This first study of WPW in a North African population comprises the clinical and genetic investigation of 3 Tunisian families, including 11 affected members. The involvement of the PRKAG2 and NKX2-5 genes was investigated. RESULTS: Mutation screening showed that with the exception of two already reported single-nucleotide polymorphisms, no mutations were detected within the coding region of PRKAG2 or in the NKX2-5 gene. CONCLUSIONS: This study provides further evidence of the genetic heterogeneity of WPW.

Lack of association between renin-angiotensin system (RAS) polymorphisms and hypertension in Tunisian type 2 diabetics.

BACKGROUND: The genes encoding renin-angiotensin system (RAS) components are potent candidate genes in both hypertension and diabetes namely ACE encoding the angiotensin converting enzyme and AGT encoding angiotensinogen. It has been suggested that the insertion/deletion (I/D) polymorphism in intron 16 of ACE gene is associated with ACE levels, and M235T gene polymorphism is associated with plasma AGT levels. AIM: We examined in this report the association between ACE I/D and AGT M235T polymorphisms with hypertension status in Tunisian type 2 diabetic subjects. METHODS: Thirty nine hypertensive and 22 normotensive type 2 diabetic Tunisian patients were recruited for this study. The I/D polymorphism of ACE gene was analysed with nested PCR in order to avoid mistyping heterozygous individuals and the M235T polymorphism of AGT gene was analysed using PCR and allele specific restriction. RESULTS: The distribution of DD, ID and II genotypes did not significantly differ between type 2 diabetic patients with or without hypertension (DD: 49%; ID: 41%; II: 10% vs DD: 36%; ID: 55%; II: 9%, respectively) (chi2=1.06, p=0.58). There was also no significant statistical difference between these two groups for the M235T polymorphism (TT: 20%; MT: 54%; MM: 26% vs TT: 27%; MT: 41%; MM: 32%, respectively) (chi2=0.95, p=0.62). CONCLUSION: RAS polymorphisms do not seem to play a role in the development of hypertension in the studied Tunisian type 2 diabetic subjects.

Clinical and genetic investigation of isolated microspherophakia in a consanguineous Tunisian family.

Microspherophakia seems to be the most specific feature of the Weill-Marchesani Syndrome, which could be due to mutations within the ADAMTS10 gene. As the locus responsible for isolated microspherophakia is still unknown, because the reported cases are rare, we checked whether the ADAMTS10 gene is involved in isolated microspherophakia in a Tunisian family. A consanguineous family (MSP-M), including six family members and two patients, presented with decreased vision secondary to bilateral isolated microspherophakia. A linkage analysis was carried out using microsatellite markers flanking the ADAMTS10 candidate gene. In the MSP-M family, isolated microspherophakia is likely inherited as an autosomal-recessive disease. Using a homozygosity-mapping strategy, haplotypic analysis using four STRs showed an exclusion of linkage between the ADAMTS10 gene and the disease locus in this family. Our study suggests that isolated microspherophakia and the Weill-Marchesani Syndrome are not allelic to the ADAMTS10 gene.

Clinical and genetic investigation of atrial septal defect with atrioventricular conduction defect in a large consanguineous Tunisian family.

BACKGROUND: Atrial septal defect (ASD) is an autosomal dominant disease characterized by left-to-right shunting and increased right ventricular output. Approximately 5-10% of congenital heart diseases (CHD) are due to ASD, which is one of the most frequent CHD found in adults. The gene responsible for ASD was mapped to chromosome 5q35 encoding the transcription factor NKX2-5 that plays an important role for the regulation of septation during cardiac morphogenesis. METHODS: A Tunisian family including four affected members was investigated. Individuals were genotyped using the polymorphic microsatellite markers D5S394 and D5S2069 overlapping the NKX2-5 gene. RESULTS: We report here clinical and molecular investigation of a Tunisian consanguineous family with four affected members. Two presented with ASD associated with prolonged PR interval, whereas the other two presented only a prolonged PR interval. We also identified five asymptomatic individuals in the same family with ventricular preexcitation. Although the patients were products of a consanguineous marriage, no other abnormalities were observed in this family. Genotyping and linkage analysis showed exclusion of linkage between the gene responsible for ASD in this family and NKX2.5 gene. CONCLUSIONS: Our results further confirm the genetic heterogeneity of ASD.

Lack of association between the angiotensin-converting enzyme gene (I/D) polymorphism and diabetic nephropathy in Tunisian type 2 diabetic patients.

OBJECTIVE: The aim of the present study was to investigate whether the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism is associated with diabetic nephropathy and type 2 diabetes in the Tunisian population. DESIGN: A case-control study was conducted among 141 unrelated type 2 diabetic patients with (90 patients) or without nephropathy (51 patients) and 103 non-diabetic controls with normal fasting blood glucose. Genotyping was performed using a nested polymerase chain reaction amplification in order to identify correctly heterozygous individuals. RESULTS: The distribution of DD, ID and II genotypes did not significantly differ between type 2 diabetic patients with or without nephropathy (DD: 44%; ID: 46%; II: 10% vs. DD: 41%; ID: 47 %; II: 12%, respectively). There was also no significant statistical difference between the genotype distribution and allele frequencies of the (I/D) polymorphism in all type 2 diabetic subjects compared to non-diabetic controls with normal fasting blood glucose (DD: 43%; ID: 46%; II: 11% vs. DD: 37%; ID: 48%; II: 15%, respectively). CONCLUSIONS: In the present preliminary study, the (I/D) polymorphism within the ACE gene is likely not associated with diabetic nephropathy nor with type 2 diabetes in the Tunisian studied population.