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)

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.

A novel point mutation in the DNA-binding domain (DBD) of the human glucocorticoid receptor causes primary generalized glucocorticoid resistance by disrupting the hydrophobic structure of its DBD.

CONTEXT: Primary generalized glucocorticoid resistance is a rare genetic condition characterized by partial end-organ insensitivity to glucocorticoids. Most affected subjects present with clinical manifestations of mineralocorticoid and androgen excess. The condition has been associated with inactivating mutations in the human glucocorticoid receptor (hGR) gene, which impair the molecular mechanisms of hGRα action, thereby reducing tissue sensitivity to glucocorticoids. OBJECTIVE: ΤHE aim of our study was to investigate the molecular mechanisms through which one previously described natural heterozygous V423A mutation, the second mutation detected in the DNA-binding domain (DBD) of the hGRα, affects glucocorticoid signal transduction. DESIGN AND RESULTS: Compared with the wild-type receptor, hGRαV423A demonstrated a 72% reduction in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone. The hGRαV423A receptor showed a significant reduction in its ability to bind to glucocorticoid-response elements of glucocorticoid-responsive genes, owing to structural alterations of the DBD confirmed by computer-based structural analysis. In addition, hGRαV423A demonstrated a 2.6-fold delay in nuclear translocation following exposure to the ligand, although it did not exert a dominant negative effect on the wild-type hGRα, had a similar affinity to the ligand with the wild-type receptor, and displayed a normal interaction with the GRIP1 coactivator in vitro. CONCLUSIONS: The natural mutant receptor hGRαV423A causes primary generalized glucocorticoid resistance by affecting multiple steps in the cascade of glucocorticoid receptor action, which primarily involve decreased ability to bind to target glucocorticoid response elements and delayed translocation into the nucleus.

Broad and unexpected phenotypic expression in Greek children with steroid-resistant nephrotic syndrome due to mutations in the Wilms' tumor 1 (WT1) gene.

Mutations in the Wilms' tumor suppressor gene 1 (WT1), most commonly within exons 8 or 9 or intron 9, are found in cases with the overlapping conditions of Denys-Drash and Frasier syndromes, as well as in patients with steroid-resistant nephrotic syndrome (SRNS). This study investigated the presence of WT1 gene mutations in cases with childhood SRNS, along with an evaluation of their clinical outcome. Twenty-seven Greek children with sporadic (19 cases) and familial (8 cases) SRNS were tested. Four phenotypically female patients with sporadic SRNS were found to carry de novo WT1 mutations, including two cases with p.R394W, and one case each with p.R366H, or n.1228+5G>A. Karyotype analysis found 46XX in three cases, but 46XY in one. No phenotype-genotype correlations were apparent in the WT1 gene positive cases since their clinical presentation varied broadly. Interestingly, one patient with a pathological WT1 nucleotide variation responded fully to combined therapy with cyclosporine A and corticosteroids. This study further illustrates that investigation of WT1 gene mutations is clinically useful to support definitive diagnosis in children presenting with SRNS in order to direct the most appropriate clinical management.

The Alport nephropathy: clinicopathological correlations.

The alleged dominance of diffuse attenuation of the glomerular basement membrane (GBM) in young children and females with Alport's Syndrome (AS) suggests that it might be the initial ultrastructural manifestation of type IV collagen defects. We carried out a 'blind' review of 130 renal biopsies obtained from 100 patients with AS, emphasizing the electron microscopy changes, and related the findings to the clinical presentation and outcome. The intracapillary distribution of (1) thickened, (2) attenuated and (3) normal GBM was assessed individually as: none (grade 0), <25% (grade 1), 25-50% (grade 2) and >50% (grade 3). Deafness was defined as persistent loss of > or =30 dBs. Proteinuria was measured as protein/creatinine ratios in early morning urine. Heavy proteinuria (> or =200 mg/mmol) correlated significantly with the presence of segmental and global glomerulosclerosis and foam cells. Comparing grades 0+1 vs. 3 GBM changes, using a 2x2 chi(2) test, there were significant correlations between grade 3 GBM thickening and male sex (P =0.005), heavy proteinuria (P =0.02) and deafness (P <0.001). GBM thickening did not correlate with age at the initial biopsy, but repeat biopsies demonstrated increasing thickening with age. The grades of GBM attenuation did not correlate with either age at biopsy or sex. In 11 biopsies with atypical lamina densa changes in thickened GBM segments, there were no differences in clinicopathological correlations compared with classical biopsies. Our data indicate that diffuse GBM attenuation can be an ultrastructural variant of the Alport nephropathy, but do not support the contention that it is the initial lesion.