Genetic association study between T-786C NOS3 polymorphism and essential hypertension in an Algerian population of the Oran city.

BACKGROUND: Essential hypertension is an important risk factor for the development of cardiovascular disease. Important candidate genes such as NOS3 gene have been widely studied and reported to be associated with essential hypertension (HTN) in human populations. AIM: We aim in this study to analyze the relationship between NOS3 -786T/C, a common genetic variant and HTN in a sample of the Algerian population of the Oran city. METHODS: A case-control study has been performed in 154 subjects including 77 hypertensives and 77 normotensives. The recruitment of these subjects was done in local Health Centers of the city of Oran, West Algeria. HTN was defined as elevated systolic blood pressure SBD≥140  mmHg and or sustained diastolic blood pressure DBP≥90  mmHg, measured using an Omron® Automatic BP Monitor - M-3W machine. Consents were obtained from all participants. Polymerase chain reaction (PCR) combined with restrictive fragment length polymorphism (RFLP) was used to genotype the NOS -786T/C variant. RESULTS: The distribution of the allelic frequencies did not differ between cases and controls (OR = 1.48; 95%CI [0.94-2.32], P = 0.09). However, after adjustment with the age, sex, and body mass index, we observed significant association between NOS -786C allele and HTN status (OR = 2.08; 95%CI [1.18-3.66], P = 0.01). CONCLUSION: Our results indicate that the C allele of the NOS3 gene is associated with increased risk of essential hypertension in this sample of the Algerian population of the Oran city. Further validation in larger samples is needed to confirm this finding.

Prevalence of Novel MAGED2 Mutations in Antenatal Bartter Syndrome.

BACKGROUND AND OBJECTIVES: Mutations in the MAGED2 gene, located on the X chromosome, have been recently detected in males with a transient form of antenatal Bartter syndrome or with idiopathic polyhydramnios. The aim of this study is to analyze the proportion of the population with mutations in this gene in a French cohort of patients with antenatal Bartter syndrome. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The French cohort of patients with antenatal Bartter syndrome encompasses 171 families. Mutations in genes responsible for types 1-4 have been detected in 75% of cases. In patients without identified genetic cause (n=42), transient antenatal Bartter syndrome was reported in 12 cases. We analyzed the MAGED2 gene in the entire cohort of negative cases by Sanger sequencing and retrospectively collected clinical data regarding pregnancy as well as the postnatal outcome for positive cases. RESULTS: We detected mutations in MAGED2 in 17 patients, including the 12 with transient antenatal Bartter syndrome, from 16 families. Fifteen different mutations were detected (one whole deletion, three frameshift, three splicing, three nonsense, two inframe deletions, and three missense); 13 of these mutations had not been previously described. Interestingly, two patients are females; in one of these patients our data are consistent with selective inactivation of chromosome X explaining the severity. The phenotypic presentation in our patients was variable and less severe than that of the originally described cases. CONCLUSIONS: MAGED2 mutations explained 9% of cases of antenatal Bartter syndrome in a French cohort, and accounted for 38% of patients without other characterized mutations and for 44% of male probands of negative cases. Our study confirmed previously published data and showed that females can be affected. As a result, this gene must be included in the screening of the most severe clinical form of Bartter syndrome.

A new SLC12A3 founder mutation (p. Val647Met) in Gitelman's syndrome patients of Roma ancestry / Una nueva mutación fundadora en SLC12A3 (p. Val647Met) en pacientes con síndrome de Gitelman de etnia gitana

Background: Gitelman's syndrome (GS) is an autosomal recessive disorder caused by mutations in the SLC12A3 gene. GS is characterized by hypokalaemic metabolic alkalosis, hypomagnesemia and hypocalciuria. Most of the reported patients of Roma ancestry are homozygous for an SLC12A3 intron 9 frameshifting mutation (c.1180+1G>T). Some forms of Bartter's syndrome result from mutations in the CLNCKB gene and clinically overlap with GS. Objectives: To characterize a second SLC12A3 mutation in Roma patients negative for the intron 9 variant. Methods: SLC12A3 and CLNCKB genes were analyzed by next-generation sequencing in two Spanish and Greek gypsy patients who were negative for the intron 9 splicing mutation. Sanger sequencing was performed to confirm the putative mutations in patients and family members. Results: We identified a missense variant (p.Val647Met, c.1939G>A) in both cases, and both were homozygous for Met. This mutation was also found in three additional patients; two homozygous and one heterozygous compound with the intron 9 splicing mutation. This new SLC12A3 mutation seems to be characteristic of gipsy GS patients and was linked to the same haplotype in all cases, supporting a founder origin. All the patients showed biochemical features characteristic of GS. Conclusion: We report a second founder mutation among GS patients of Roma ethnic background. The direct screening of this mutation would facilitate the characterization of patients who are negative for the more common intron 9 +1G>T mutation (AU)

Antecedentes: El síndrome de Gitelman (SG) es un trastorno autosómico recesivo causado por las mutaciones en el gen SLC12A3.El SG se caracteriza por una alcalosis metabólica hipopotasémica, hipomagnesemia e hipocalciuria. La mayoría de los pacientes de etnia gitana notificados son homocigotos para la mutación con desplazamiento del marco de lectura del intrón 9 de SLC12A3 (c.1180+1G>T). Algunas formas del síndrome de Bartter proceden de las mutaciones del gen CLNCKB y se solapan clínicamente con el SG. Objetivos: Determinar las características de una segunda mutación en SLC12A3 en pacientes de etnia gitana con resultados negativos en la variante intrón 9. Métodos: Se analizaron los genes SLC12A3 y CLNCKB mediante secuenciación de nueva generación en 2 pacientes -uno español y otro griego- de etnia gitana con resultados negativos en la mutación de empalme del intrón 9. Se llevó a cabo una secuenciación de Sanger para confirmar las supuestas mutaciones en los pacientes y sus familiares. Resultados: Se identificó una variante con cambio de sentido (p.Val647Met, c.1939G>A) en ambos casos, y ambos eran homocigotos con respecto a Met. También se observó esta mutación en 3 pacientes adicionales, 2 homocigotos y uno heterocigoto compuesto con la mutación del intrón 9. Esta nueva mutación del SLC12A3 parece ser característica de los pacientes con SG de etnia gitana y se relacionó con el mismo haplotipo en todos los casos, lo que indica un origen fundador. Todos los pacientes presentaron rasgos bioquímicos propios del SG. Conclusión: Informamos de una segunda mutación fundadora en los pacientes con SG de etnia gitana. El cribado genético directo de esta mutación facilitará la determinación de las características de los pacientes con resultados negativos en la mutación del intrón 9+1G>T, que es más frecuente (AU)

Signification of distal urinary acidification defects in hypocitraturic patients.

BACKGROUND AND OBJECTIVES: Hypocitraturia has been associated with metabolic acidosis and mineral disorders. The aim of this study was to investigate the occurrence of urinary acidification defects underlying hypocitraturia. MATERIALS AND METHODS: This retrospective observational study included 67 patients (32 men), aged 40.7±15.1 years with hypocitraturia (<1.67 mmol/24-h) and nephrolithiasis, nephrocalcinosis, and/or bone demineralization, referred to our center from 2000 to 2015. We aimed to assess renal distal acidification capacity, prevalence and mechanisms of urinary acidification defects. Patients with low baseline plasma HCO3- (<22 mmol/L) were studied by bicarbonate loading or furosemide/fludrocortisone tests. Patients with normal baseline plasma HCO3- had an ammonium-chloride challenge test. A normal response was a decrease in urinary pH <5.3 and an increase in urinary NH4+ ≥33 µmol/min and defined idiopathic hypocitraturia. RESULTS: Eleven patients (16.4%) had low HCO3- and overt distal acidification defect. Three had a mutation in the gene encoding AE1, 4 had Gougerot-Sjögren syndrome and no cause was found in the remaining 4 cases. Fifty-six patients (83.6%) had normal HCO3-; of those, 33 (58.9%) had idiopathic hypocitraturia. Among the 23 (41%) remaining patients, 12 were unable to increase urinary NH4+ excretion (among them, 8 were able to decrease urinary pH and 4 were not) whereas 11 were able to increase urinary NH4+ excretion but unable to decrease urinary pH. These 11 patients had higher fasting urinary calcium, reflecting bone resorption, than the other 12 patients: median 0.41 [0.24-0.47] vs. 0.22 [0.08-0.37] mmol/mmol creatinine (P = 0.04). CONCLUSIONS: Patients with hypocitraturia and normal plasma HCO3- frequently show a latent acidification defect that can be further dissected into one of several subtypes based on urinary pH and NH4+ response to the acid load. Those patients with impaired urine acidification capacity but preserved NH4+ excretion exhibit particularly high calciuria and should be identified to optimize nephrolithiasis prevention.

A new SLC12A3 founder mutation (p.Val647Met) in Gitelman's syndrome patients of Roma ancestry.

BACKGROUND: Gitelman's syndrome (GS) is an autosomal recessive disorder caused by mutations in the SLC12A3 gene. GS is characterized by hypokalaemic metabolic alkalosis, hypomagnesemia and hypocalciuria. Most of the reported patients of Roma ancestry are homozygous for an SLC12A3 intron 9 frameshifting mutation (c.1180+1G>T). Some forms of Bartter's syndrome result from mutations in the CLNCKB gene and clinically overlap with GS. OBJECTIVES: To characterize a second SLC12A3 mutation in Roma patients negative for the intron 9 variant. METHODS: SLC12A3 and CLNCKB genes were analyzed by next-generation sequencing in two Spanish and Greek gypsy patients who were negative for the intron 9 splicing mutation. Sanger sequencing was performed to confirm the putative mutations in patients and family members. RESULTS: We identified a missense variant (p.Val647Met, c.1939G>A) in both cases, and both were homozygous for Met. This mutation was also found in three additional patients; two homozygous and one heterozygous compound with the intron 9 splicing mutation. This new SLC12A3 mutation seems to be characteristic of gipsy GS patients and was linked to the same haplotype in all cases, supporting a founder origin. All the patients showed biochemical features characteristic of GS. CONCLUSION: We report a second founder mutation among GS patients of Roma ethnic background. The direct screening of this mutation would facilitate the characterization of patients who are negative for the more common intron 9 +1G>T mutation.

The relationship between MTHFR C677T gene polymorphism and essential hypertension in a sample of an Algerian population of Oran city.

BACKGROUND: Many studies have investigated the role of 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism in essential hypertension (EH), but with conflicting results. AIM: To determine the eventual association between 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and hypertension in a sample of Algerian population from the Oran city. METHODS: A case-control study has been performed in 154 subjects including 82 hypertensives defined as subjects with elevated systolic blood pressure SBD≥140mmHg and or sustained diastolic blood pressure DBP≥90mmHg, and 72 normotensive subjects. Polymerase chain reaction (PCR) combined with restrictive fragment length polymorphism (RFLP) was used to detect the MTHFR C677T variant. RESULTS: We observe no significant differences between allelic and genotypic frequencies between cases and controls for C677T polymorphism (OR=1.51, 95% CI=0.89-2.56, P=0.13). Analyses adjusted for age, sex and body mass index improved the association level, though the association was still not significant (30% vs. 22%, OR=1.75, 95% CI=0.95-3.24, P=0.07). CONCLUSION: This work showed that genetic polymorphism related to the MTHFR gene (C677T) is not associated with the risk of hypertension in this sample of Algerian population. Larger case-control samples are required to clearly assess the role of this genetic variant in EH.

PHACTR1 Is a Genetic Susceptibility Locus for Fibromuscular Dysplasia Supporting Its Complex Genetic Pattern of Inheritance.

Fibromuscular dysplasia (FMD) is a nonatherosclerotic vascular disease leading to stenosis, dissection and aneurysm affecting mainly the renal and cerebrovascular arteries. FMD is often an underdiagnosed cause of hypertension and stroke, has higher prevalence in females (~80%) but its pathophysiology is unclear. We analyzed ~26K common variants (MAF>0.05) generated by exome-chip arrays in 249 FMD patients and 689 controls. We replicated 13 loci (P<10-4) in 402 cases and 2,537 controls and confirmed an association between FMD and a variant in the phosphatase and actin regulator 1 gene (PHACTR1). Three additional case control cohorts including 512 cases and 669 replicated this result and overall reached the genomic level of significance (OR = 1.39, P = 7.4×10-10, 1,154 cases and 3,895 controls). The top variant, rs9349379, is intronic to PHACTR1, a risk locus for coronary artery disease, migraine, and cervical artery dissection. The analyses of geometrical parameters of carotids from ~2,500 healthy volunteers indicate higher intima media thickness (P = 1.97×10-4) and wall to lumen ratio (P = 0.002) in rs9349379-A carriers, suggesting indices of carotid hypertrophy previously described in carotids of FMD patients. Immunohistochemistry detected PHACTR1 in endothelium and smooth muscle cells of FMD and normal human carotids. The expression of PHACTR1 by genotypes in primary human fibroblasts showed higher expression in rs9349379-A carriers (N = 86, P = 0.003). Phactr1 knockdown in zebrafish resulted in dilated vessels indicating subtle impaired vascular development. We report the first susceptibility locus for FMD and provide evidence for a complex genetic pattern of inheritance and indices of shared pathophysiology between FMD and other cardiovascular and neurovascular diseases.

Functionomics of NCC mutations in Gitelman syndrome using a novel mammalian cell-based activity assay.

Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was, therefore, to analyze the functional consequences of NCC mutations in a mammalian cell-based assay, followed by analysis of mutated NCC protein expression as well as glycosylation and phosphorylation profiles using human embryonic kidney (HEK) 293 cells. NCC activity was assessed with a novel assay based on thiazide-sensitive iodide uptake in HEK293 cells expressing wild-type or mutant NCC (N59I, R83W, I360T, C421Y, G463R, G731R, L859P, or R861C). All mutations caused a significantly lower NCC activity. Immunoblot analysis of the HEK293 cells revealed that 1) all NCC mutants have decreased NCC protein expression; 2) mutant N59I, R83W, I360T, C421Y, G463R, and L859P have decreased NCC abundance at the plasma membrane; 3) mutants C421Y and L859P display impaired NCC glycosylation; and 4) mutants N59I, R83W, C421Y, C731R, and L859P show affected NCC phosphorylation. In conclusion, we developed a mammalian cell-based assay in which NCC activity assessment together with a profiling of mutated protein processing aid our understanding of the pathogenic mechanism of the NCC mutations.

Observations of a large Dent disease cohort.

Dent disease classically combines low-molecular-weight proteinuria, hypercalciuria with nephrocalcinosis, and renal failure. Nephrotic range proteinuria, normal calciuria, and hypokalemia have been rarely reported. It is unknown whether the changes in phenotype observed over time are explained by a decrease in glomerular filtration rate (GFR) or whether there is any phenotype-genotype relationship. To answer this we retrospectively analyzed data from 109 male patients with CLCN5 mutations (Dent-1) and 9 patients with mutation of the OCRL gene (Dent-2). In Dent-1 disease, the estimated GFR decreased with age, by 1.0 to 1.6 ml/min per 1.73 m(2)/yr in the absence and presence of nephrocalcinosis, respectively, with no significant difference. Median values of low-molecular-weight proteinuria were in the nephrotic range and remained at the same level even in late renal disease. End-stage renal disease occurred in 12 patients, at a median age of 40 years. Hypercalciuria decreased with glomerular filtration and was absent in 40% of the patients under 30 and 85% of those over the age of 30. Hypophosphatemia did not resolve with age and calcitriol concentrations were in the upper normal range. Kalemia decreased with age, with half of the patients over the age of 18 presenting with hypokalemia. Thus, no phenotype/genotype correlation was observed in this cohort of patients with Dent disease.

Mutations in ATP6V1B1 and ATP6V0A4 genes cause recessive distal renal tubular acidosis in Mexican families.

BACKGROUND: Autosomal recessive distal renal tubular acidosis (dRTA) is a rare disease characterized by a hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitraturia, nephrocalcinosis, and conserved glomerular filtration rate. In some cases, neurosensorial deafness is associated. dRTA is developed during the first months of life and the main manifestations are failure to thrive, vomiting, dehydration, and anorexia. METHODS: Nine unrelated families were studied: seven children, a teenager, and an adult with dRTA. Hearing was preserved in four children. Coding regions of the genes responsible for recessive dRTA were analysed by Sanger sequencing. RESULTS: Molecular defects were found in the genes ATP6V1B1 and ATP6V0A4. We identified three homozygous variants in ATP6V1B: a frameshift mutation (p.Ile386Hisfs*56), a nucleotide substitution in exon 10 (p.Pro346Arg), and a new splicing mutation in intron 5. Three patients were homozygous for one novel (p.Arg743Trp) and one known (p.Asp411Tyr) missense mutations in the ATP6V0A4 gene. Three patients were compound heterozygous: one proband displayed two novel mutations, the frameshift mutation p.Val52Metfs*25, and a large deletion of exons 18-21; two probands showed the missense mutation p.Asp411Tyr and as a second mutation, p.Arg194Ter and c.1691+2dup, respectively. CONCLUSION: ATP6V0A4 and ATP6V1B1 genes were involved in recessive dRTA of Mexican families. All ATP6V1B1 mutations detected were homozygous and all patients developed sensorineural hearing loss (SNHL) early in infancy. ATP6V0A4 mutations were found in one infant and three children without SNHL, and in one teenager and one adult with SNHL confirming the phenotypic variability in this trait. The mutation p.Asp411Tyr detected in four Mexican families was due to a founder effect. Screening of these mutations could provide a rapid and valuable tool for diagnosis of dRTA in this population.

Familial Hypocalciuric Hypercalcemia Types 1 and 3 and Primary Hyperparathyroidism: Similarities and Differences.

CONTEXT: Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogeneous condition resembling primary hyperparathyroidism (PHPT) but not curable by surgery; FHH types 1, 2, and 3 are due to loss-of-function mutations of the CASR, GNA11, or AP2S1 genes, respectively. OBJECTIVE: This study aimed to compare the phenotypes of patients with genetically proven FHH types 1 or 3 or PHPT. DESIGN, SETTING, AND PATIENTS: This was a mutation analysis in a large cohort, a cross-sectional comparison of 52 patients with FHH type 1, 22 patients with FHH type 3, 60 with PHPT, and 24 normal adults. INTERVENTION: There were no interventions. MAIN OUTCOME MEASURES: Abnormalities of the CASR, GNA11, and AP2S1 genes, blood calcium, phosphate, and PTH concentrations, urinary calcium excretion were measured. RESULTS: In 133 families, we detected 101 mutations in the CASR gene, 68 of which were previously unknown, and in 19 families, the three recurrent AP2S1 mutations. No mutation was detected in the GNA11 gene. Patients with FHH type 3 had higher plasma calcium concentrations than patients with FHH type 1, despite having similar PTH concentrations and urinary calcium excretion. Renal tubular calcium reabsorption levels were higher in patients with FHH type 3 than in those with FHH type 1. Plasma calcium concentration was higher whereas PTH concentration and urinary calcium excretion were lower in FHH patients than in PHPT patients. In patients with FHH or PHPT, all data groups partially overlapped. CONCLUSION: In our population, AP2S1 mutations affect calcium homeostasis more severely than CASR mutations. Due to overlap, the risk of confusion between FHH and PHPT is high.

Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1.

Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.