Genotypic variability in patients with clinical diagnosis of Bartter syndrome type 3.

Bartter syndrome (BS) is a salt-losing hereditary tubulopathy characterized by hypokalemic metabolic alkalosis with secondary hyperaldosteronism. Confirmatory molecular diagnosis may be difficult due to genetic heterogeneity and overlapping of clinical symptoms. The aim of our study was to describe the different molecular findings in patients with a clinical diagnosis of classic BS. We included 27 patients (26 families) with no identified pathogenic variants in CLCNKB. We used a customized Ion AmpliSeq Next-Generation Sequencing panel including 44 genes related to renal tubulopathies. We detected pathogenic or likely pathogenic variants in 12 patients (44%), reaching a conclusive genetic diagnosis. Variants in SLC12A3 were found in 6 (Gitelman syndrome). Median age at diagnosis was 14.6 years (range 0.1-31), with no history of prematurity or polyhydramnios. Serum magnesium level was low in 2 patients (33%) but urinary calcium excretion was normal or low in all, with no nephrocalcinosis. Variants in SLC12A1 were found in 3 (BS type 1); and in KCNJ1 in 1 (BS type 2). These patients had a history of polyhydramnios in 3 (75%), and the mean gestational age was 34.2 weeks (SD 1.7). The median age at diagnosis was 1.8 years (range 0.1-6). Chronic kidney disease and nephrocalcinosis were present in 1 (25%) and 3 (75%) patients, respectively. A variant in CLCN5 was found in one patient (Dent disease), and in NR3C2 in another patient (Geller syndrome). Genetic diagnosis of BS is heterogeneous as different tubulopathies can present with a similar clinical picture. The use of gene panels in these diseases becomes more efficient than the study gene by gene with Sanger sequencing.

Molecular aspects and long-term outcome of patients with primary distal renal tubular acidosis.

BACKGROUND: Primary distal renal tubular acidosis (dRTA) is a rare genetic disorder caused by impaired distal mechanisms of urinary acidification. Most cases are secondary to pathogenic variants in ATP6V0A4, ATP6V1B1, and SLC4A1 genes, which encode transporters regulating acid-base balance in the collecting duct. METHODS: Retrospective study of molecular and clinical data from diagnosis and long-term follow-up (10, 20, and 40±10 years) of 16 patients with primary dRTA diagnosed in childhood. RESULTS: Molecular analyses revealed nine patients had ATP6V0A4 pathogenic variants, five in ATP6V1B1, and two in SLC4A1. A novel intragenic deletion and a common ATP6V0A4 gene variant (c.1691 + 2dupT) in ATP6V0A4 occurred in two-thirds of these patients, suggesting a founder effect. Median age at diagnosis was 3.25 months (IQR 1, 13.5), which was higher in the SLC4A1 group. Median SDS height at diagnosis was -1.02 (IQR -1.79, 0.14). Delayed clinical diagnosis was significantly related to growth failure (P = 0.01). Median SDS height at 20 years follow-up was -1.23 (IQR -1.71, -0.48), and did not significantly improve from diagnosis (P = 0.76). Kidney function declined over time: at last follow-up, 43% had moderate to severe chronic kidney disease (CKD). Adequate metabolic control was not related to CKD development. Incidence of sensorineural hearing loss (SNHL) was high in ATP6V1B1 patients, though not universal. Patients harboring ATP6V0A4 variants also developed SNHL at a high rate (80%) over time. CONCLUSIONS: Patients with dRTA can develop moderate to severe CKD over time with a high frequency despite adequate metabolic control. Early diagnosis ameliorates long-term height prognosis.

Hereditary distal renal tubular acidosis: Genotypic correlation, evolution to long term, and new therapeutic perspectives.

Distal renal tubular acidosis (DRTA) is a rare disease resulting from a failure in the normal urine acidification process at the distal tubule and collecting duct level. It is characterised by persistent hyperchloremic metabolic acidosis, with a normal anion gap in plasma, in the presence of high urinary pH and low urinary excretion of ammonium. To date, 5 genes whose mutations give rise to primary DRTA have been described. Alterations in the ATP6V1B1 and ATP6V0A4 genes are inherited recessively and are associated with forms of early onset and, in many cases, with neurosensorial deafness. Pathogenic variants in the SLC4A1 gene are habitually inherited dominantly and give rise to milder symptoms, with a later diagnosis and milder electrolytic alterations. Nonetheless, evolution to nephrocalcinosis and lithiasis, and the development of chronic kidney disease in the medium to long term has been described in a similar manner in all 3 groups. Lastly, recessive forms of DTRA associated to mutations in the FOXI1 and WDR72 genes have also been described. The clinical management of DTRA is based on bicarbonate or citrate salts, which do not succeed in correcting all cases of the metabolic alterations described and, thus, the consequences associated with them. Recently, a new treatment based on slow-release bicarbonate and citrate salts has received the designation of orphan drug in Europe for the treatment of DTRA.

Characterization of two novel mutations in the claudin-16 and claudin-19 genes that cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal-recessive renal tubular disorder characterized by excessive urinary losses of magnesium and calcium, bilateral nephrocalcinosis and progressive chronic renal failure in childhood or adolescence. The disease is caused by mutations in the tight-junction proteins claudin-16 and claudin-19 that are encoded by the CLDN16 and CLDN19 genes, respectively. Patients with CLDN19 mutations also are affected with severe ocular abnormalities. The aim of our study was to identify and characterize the molecular defects causing this disease in a Georgian girl and two Spanish siblings. Clinical and biochemical parameters were studied. The CLDN16 and CLDN19 genes were analyzed by DNA sequencing. The functional consequences of the identified mutations on pre-mRNA splicing were investigated using a minigene assay. Sequence analysis revealed that the patient from Georgia was homozygous for a novel mutation, c.602G > A; p.(G201E), in exon 4 of the CLDN16 gene. The two Spanish siblings were homozygous for a new CLDN19 mutation, c.388G > T; p.(G130C), located in exon 2, and both parents were heterozygous carriers of the mutation. Bioinformatics analysis predicted that the amino acid substitutions generated by these mutations were pathogenic. Functional studies showed that mutation c.388G > T also results in partial skipping of CLDN19 exon 2, which would imply significant alterations in the claudin-19 protein structure. Conversely, CLDN16 mutation c.602G > A had no effect on pre-mRNA splicing. Our study expands the genotypic classification of this rare disease and provides the first report of a CLDN19 mutation affecting splicing.

Poor phenotype-genotype association in a large series of patients with Type III Bartter syndrome.

INTRODUCTION: Type III Bartter syndrome (BS) is an autosomal recessive renal tubule disorder caused by loss-of-function mutations in the CLCNKB gene, which encodes the chloride channel protein ClC-Kb. In this study, we carried out a complete clinical and genetic characterization in a cohort of 30 patients, one of the largest series described. By comparing with other published populations, and considering that 80% of our patients presented the p.Ala204Thr Spanish founder mutation presumably associated with a common phenotype, we aimed to test the hypothesis that allelic differences could explain the wide phenotypic variability observed in patients with type III BS. METHODS: Clinical data were retrieved from the referral centers. The exon regions and flanking intronic sequences of the CLCNKB gene were screened for mutations by polymerase chain reaction (PCR) followed by direct Sanger sequencing. Presence of gross deletions or duplications in the region was checked for by MLPA and QMPSF analyses. RESULTS: Polyuria, polydipsia and dehydration were the main common symptoms. Metabolic alkalosis and hypokalemia of renal origin were detected in all patients at diagnosis. Calciuria levels were variable: hypercalciuria was detected in 31% of patients, while 23% had hypocalciuria. Nephrocalcinosis was diagnosed in 20% of the cohort. Two novel CLCNKB mutations were identified: a small homozygous deletion (c.753delG) in one patient and a small deletion (c.1026delC) in another. The latter was present in compound heterozygosis with the already previously described p.Glu442Gly mutation. No phenotypic association was obtained regarding the genotype. CONCLUSION: A poor correlation was found between a specific type of mutation in the CLCNKB gene and type III BS phenotype. Importantly, two CLCNKB mutations not previously described were found in our cohort.

Novel mutations associated with nephrogenic diabetes insipidus. A clinical-genetic study.

UNLABELLED: Molecular diagnosis is a useful diagnostic tool in primary nephrogenic diabetes insipidus (NDI), an inherited disease characterized by renal inability to concentrate urine. The AVPR2 and AQP2 genes were screened for mutations in a cohort of 25 patients with clinical diagnosis of NDI. Patients presented with dehydration, polyuria-polydipsia, failure to thrive (mean ± SD; Z-height -1.9 ± 2.1 and Z-weight -2.4 ± 1.7), severe hypernatremia (mean ± SD; Na 150 ± 10 mEq/L), increased plasma osmolality (mean ± SD; 311 ± 18 mOsm/Kg), but normal glomerular filtration rate. Genetic diagnosis revealed that 24 male patients were hemizygous for 17 different putative disease-causing mutations in the AVPR2 gene (each one in a different family). Of those, nine had not been previously reported, and eight were recurrent. Moreover, we found those same AVPR2 changes in 12 relatives who were heterozygous carriers. Further, in one female patient, AVPR2 gene study turned out to be negative and she was found to be homozygous for the novel AQP2 p.Ala86Val alteration. CONCLUSION: Genetic analysis presumably confirmed the diagnosis of nephrogenic diabetes insipidus in every patient of the studied cohort. We emphasize that we detected a high presence (50 %) of heterozygous females with clinical NDI symptoms. WHAT IS KNOWN: • In most cases (90 %), inherited nephrogenic diabetes insipidus (NDI) is an X-linked disease, caused by mutations in the AVPR2 gene. • In rare occasions (10 %), it is caused by mutations in the AQP2 gene. What is new: • In this study, we report 10 novel mutations associated with NDI. • We have detected a high presence (50 %) of heterozygous carriers with clinical NDI symptoms.

Genetics of type III Bartter syndrome in Spain, proposed diagnostic algorithm.

The p.Ala204Thr mutation (exon 7) of the CLCNKB gene is a "founder" mutation that causes most of type III Bartter syndrome cases in Spain. We performed genetic analysis of the CLCNKB gene, which encodes for the chloride channel protein ClC-Kb, in a cohort of 26 affected patients from 23 families. The diagnostic algorithm was: first, detection of the p.Ala204Thr mutation; second, detecting large deletions or duplications by Multiplex Ligation-dependent Probe Amplification and Quantitative Multiplex PCR of Short Fluorescent Fragments; and third, sequencing of the coding and flanking regions of the whole CLCNKB gene. In our genetic diagnosis, 20 families presented with the p.Ala204Thr mutation. Of those, 15 patients (15 families) were homozygous (57.7% of overall patients). Another 8 patients (5 families) were compound heterozygous for the founder mutation together with a second one. Thus, 3 patients (2 siblings) presented with the c. -19-?_2053+? del deletion (comprising the entire gene); one patient carried the p.Val170Met mutation (exon 6); and 4 patients (3 siblings) presented with the novel p.Glu442Gly mutation (exon 14). On the other hand, another two patients carried two novel mutations in compound heterozygosis: one presented the p.Ile398_Thr401del mutation (exon 12) associated with the c. -19-?_2053+? del deletion, and the other one carried the c.1756+1G>A splice-site mutation (exon 16) as well as the already described p.Ala210Val change (exon 7). One case turned out to be negative in our genetic screening. In addition, 51 relatives were found to be heterozygous carriers of the described CLCNKB mutations. In conclusion, different mutations cause type III Bartter syndrome in Spain. The high prevalence of the p.Ala204Thr in Spanish families thus justifies an initial screen for this mutation. However, should it not be detected further investigation of the CLCNKB gene is warranted in clinically diagnosed families.

Eculizumab in the treatment of atypical hemolytic uremic syndrome in infants.

A 28-day-old male newborn weighing 3.6 kg was given a diagnosis of atypical hemolytic-uremic syndrome, new-onset thrombotic microangiopathy (TMA; hemoglobin, 7.7 g/dL; schistocytes, 9%), thrombocytopenia (platelets, 49 × 10(3)/µL [49 × 10(9)/L]), and acute kidney failure (serum creatinine, 1.13 mg/dL [99.8 µmol/L], corresponding to estimated glomerular filtration rate [eGFR] of 15 mL/min/1.73 m(2) [0.25 mL/s/1.73 m(2)]). Repeated high-volume plasma infusions were ineffective. Plasma exchange was attempted, but not tolerated. The patient required mechanical ventilation and continuous renal replacement therapy. He developed multiple intestinal perforations and leg skin necrosis due to systemic TMA. A low C3 level (36 mg/dL) suggested complement activation. Eculizumab, 300 mg, was administered, and within 48 hours the patient recovered from acute kidney failure, with complete hematologic remission 2 weeks later. The infant, 14 months old at the time of writing, continues to receive eculizumab, 300 mg, every 3 weeks; he is free of disease activity and has a normal creatinine level of 0.2 mg/dL (17.68 µmol/L; corresponding to eGFR of 110 mL/min/1.73 m(2) [1.83 mL/s/1.73 m(2)]), but mild proteinuria (urinary protein-creatine ratio, 1 mg/g). Results of additional studies, including probing for cobalamin anomalies and measuring levels of ADAMTS13, complement factor H (CFH), factor I (CFI), and membrane cofactor protein (MCP), were unremarkable. Antibodies to CFH were undetectable, and mutation testing of the genes for CFH, CFI, and MCP gave negative results. Treatment with eculizumab was life saving, and with continued treatment, the patient showed sustained freedom from clinical TMA complications.

Methylation cycle, arginine-creatine pathway and asymmetric dimethylarginine in paediatric renal transplant.

BACKGROUND: Hyperhomocysteinaemia represents an important cause of morbidity in recipients of renal transplants, but few investigations have been carried out to evaluate the status of the methylation cycle and its relation with levels of new cardiovascular biomarkers, such as asymmetric dimethylarginine (ADMA). METHODS: Twenty-six children and adolescents aged 7-18 years (17 male, 9 female) with stable renal transplants were recruited for the study. None had received treatment with folate, vitamin B(12) or statins. Levels of ADMA in plasma and of components of the methylation cycle and arginine (Arg)-creatine pathway in plasma and urine were analysed by specific analytical methods. Results were compared to those obtained by us with identical methods in healthy children of similar age. RESULTS: Concentrations of homocysteine (Hcys), S-adenosylhomocysteine (SAH) and ADMA were significantly higher, while S-adenosylmethionine (SAM)/SAH and Arg/ADMA ratios were significantly lower than controls. Arg/ADMA ratio correlated with plasma guanidinoacetate. The components of the methylation cycle, Hcys and SAH correlated with renal function. CONCLUSIONS: Children with renal transplant showed low methylation power (SAM/SAH) mainly due to increased levels of SAH which acts as a cardiovascular biomarker. Elevated values of ADMA and low Arg/ADMA coefficients also represent a novel finding because it inhibits nitric oxide synthesis contributing to endothelial dysfunction and cardiovascular risk in such patients.

The arginine-creatine pathway is disturbed in children and adolescents with renal transplants.

Cardiovascular disease is an important cause of morbidity in recipients of renal transplants. The aim of the present study was to analyze the status of the arginine-creatine pathway in such patients, given the relationship between the arginine metabolism and both renal function and the methionine-homocysteine cycle. Twenty-nine children and adolescents (median age 13, range 6-18 years), who had received a renal allograft 14.5-82.0 months before, were recruited for the study. On immunosuppressive therapy, all patients evidenced an adequate level of renal function. Plasma concentrations of homocysteine and glycine were significantly higher, whereas urinary excretions of guanidinoacetate and creatine were significantly lower than controls. Urinary excretions of guanidinoacetate and creatine correlated positively with creatinine clearance. Urinary excretion of creatine was negatively correlated with plasma concentration of homocysteine. The demonstration of disturbances in the arginine-creatine pathway in patients with well-functioning renal transplants and in absence of chronic renal failure represents a novel finding. We speculate that the low urinary excretion of guanidinoacetate and creatine is probably related to the nephrotoxic effect of immunosuppressive therapy and to defective methylation associated with the presence of hyperhomocysteinemia.

Essential fatty acid deficiency profile in patients with nephrotic-range proteinuria.

Plasma free fatty acids are bound to albumin, filtered through the glomeruli, and reabsorbed at the proximal nephron. The aim of the present investigation was to determine if urinary loss of fatty acids results in essential fatty acid (EFA) deficiency in patients with nephrotic-range proteinuria. We studied 12 patients aged 9 months to 23 years (eight male, four female) four suffering from congenital nephrotic syndrome (NS) and eight from different renal diseases. Six patients were studied postrenal transplantation. Proteinuria ranged between 41 and 829 mg/m2/h. Results were compared with data obtained in 83 healthy children. The patients had significantly lower values for plasma arachidonic acid content and EFA index (omega3 + omega6/omega7 + omega9). Deficiency in polyunsaturated fatty acids (PUFA) was especially manifest in infants with congenital NS. Plasma content of arachidonic and docosahexaenoic acids related negatively with the degree of proteinuria. In the lineal regression model, the degree of proteinuria explained 60% of the variability of plasma values of those fatty acids. We conclude that plasma fatty acid status should be regularly monitored in patients with nephrotic-range proteinuria, especially in young infants with congenital NS, who represent a population at special risk with regard to neurological development.

Bone mineral density and bone turnover in patients with Bartter syndrome.

The aim of this investigation was to evaluate bone mineral density (BMD), by use of DXA, and bone turnover, in patients with Bartter syndrome (BS). Ten patients (2 with BS type II and 8 with BS type III) were included in the procedure. Age at study varied between 2 and 30 years. During the studies usual treatment with indomethacin, spironolactone, and potassium chloride was maintained. Results were compared with those obtained in the 20 asymptomatic parents. Height of the patients at the time of the study did not differ from reference values (Z-score -1.2 to +0.8). Three patients (1 with BS type II and 2 with BS type III) presented reduced lumbar spine BMD or overt osteopenia (BMD Z-scores: -2.3, -1.3, and -1.1). BMD did not correlate significantly with age. Paternal and maternal femoral neck BMD values correlated significantly with lumbar spine BMD of the patients (r=0.65, P<0.05, and r=0.80, P<0.01). Lumbar spine BMD Z-scores correlated negatively with urinary Ca excretion when values both from patients and parents were jointly analyzed (r=-0.43, P<0.05). Plasma calcium concentration was significantly higher (P<0.001) and plasma phosphate Z-score was significantly lower (P<0.05) in the patients than in the parents. However, no significant differences were observed in values for intact PTH, 1,25 (OH)(2)D(3) and 25 (OH)D(3). Intact PTH values correlated positively with BMD Z-scores at lumbar spine (r=0.45, P<0.05) and at femoral neck (r=0.63, P<0.01). Age-corrected biochemical markers of bone formation (plasma alkaline phosphatase and osteocalcin concentrations) were normal whereas age-corrected markers of bone reabsorption (urinary PYD and DPD excretion) were significantly higher than parental values (P<0.01 and <0.05, respectively). We conclude that: (1) reduced BMD is not an exclusive feature of neonatal BS and it can be also observed in classic BS; (2) the loss of bone mineral is not progressive, probably because of the hypocalciuric effect of indomethacin therapy; and (3) this study did not determine whether loss of bone mass is the cause or the consequence of hypercalciuria although the beneficial effect of indomethacin therapy implies the latter.

Long-term renal follow-up of extremely low birth weight infants.

There is evidence that low birth weight caused by intrauterine growth retardation adversely affects normal renal development. Very little information on this issue is available on children born very prematurely. This investigation examined clinical and functional renal parameters in 40 children (23 boys, 17 girls) ranging in age between 6.1 and 12.4 years and weighing less than 1000 g at birth. Results were compared to those obtained in 43 healthy children of similar age and gender. Study subjects were significantly smaller and thinner than control subjects (mean height SDS: -0.36 vs. +0.70; and mean BMI SDS: -0.56 vs. +1.18). Systolic, diastolic, and mean blood pressures did not differ from those of controls. Renal sonography revealed no abnormality, and mean percentiles for renal length and volume appeared normal. In comparison with controls, plasma creatinine concentration (0.62+/-0.1 vs. 0.53+/-0.1 mg/dl) and estimated creatinine clearance (117+/-17 vs. 131+/-17 ml min(-1) 1.73 m(-2)) differed significantly. No significant differences were observed in microalbuminuria values, but five study subjects (12.5%) presented values above the upper limit of normality. A defect in tubular phosphate transport was also evident: TmP/GFR (3.6+/-0.4 vs. 4.2+/-0.8 mg/dl) and TRP (83+/-5% vs. 90+/-4%) were significantly lower, and urinary P excretion, estimated by the ratio UP/UCr, was significantly higher (1.2+/-0.4 vs. 0.9+/-0.4 mg/mg) than controls. Urinary calcium excretion, estimated by the UCa/UCr ratio, was also significantly higher (0.15+/-0.07 vs. 0.12+/-0.09 mg/mg). These data clearly demonstrate that both GFR and tubular phosphate transport are significantly diminished in school-age children born with extreme prematurity, probably as a consequence of impaired postnatal nephrogenesis.

Genetic and metabolic determinants of increased plasma plasminogen activator inhibitor-1 activity in children with renal transplants.

Recent studies have shown that activity of plasminogen activator inhibitor-1 (PAI-1), a prothrombotic protein, may be increased in transplanted patients. The aim of the present investigation was to determine PAI-1 activity in pediatric recipients of renal transplants and to establish the relative contribution of both genetic and metabolic factors. In 29 children and adolescents with stable renal transplants, we related plasma PAI-1 activity to an indicator of inflammatory status [plasma concentration of C-reactive protein (CRP)] and to elements of the insulin resistance syndrome [body mass index (BMI), fasting insulinemia, HOMA index and plasma triglyceride, HDL-cholesterol, apolipoproteins A-1 and B concentrations]. Polymorphisms of PAI-1, apolipoprotein E (apoE) and angiotensin-converting enzyme (ACE) genes were also investigated. In all patients the study was repeated 1 year later. PAI-1 activity remained constantly elevated (23.4+/-22.8 and 18.6+/-7.8 U/ml in the first and second study, respectively, P=NS). Plasma PAI-1 activity correlated positively with CRP ( P=0.001), BMI z score ( P=0.02), fasting insulinemia ( P=0.009), and HOMA index ( P=0.006). No significant correlations were found in this population between plasma PAI-1 activity and age, gender, time elapsed after transplantation and plasma homocysteine, total cholesterol, LDL-cholesterol, HDL-cholesterol, apolipoprotein B, and apolipoprotein A-1. Plasma PAI-1 activity was not related to the cumulative dose of prednisone, cyclosporin A, or tacrolimus. Plasma PAI-1 activity was significantly higher in 5 children with apoE3/apoE4 genotype. No apparent influences of the PAI-1 4G/4G and ACE I/D genotypes were observed. In a multiple stepwise regression model, fasting insulinemia and apoE3/apoE4 genotype accounted for 45% of the observed plasma PAI-1 variability. We conclude that increased PAI-1 activity in children with stable renal transplants is determined both by genetic factors and by metabolic factors, the latter mainly linked to the insulin resistance syndrome.