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The oculocerebrorenal syndrome of Lowe is a rare X-linked disease characterized by congenital cataracts, proximal renal tubulopathy, muscular hypotonia and mental impairment. This disease is caused by mutations in the OCRL gene encoding membrane bound inositol polyphosphate 5-phosphatase OCRL1. Here, we examined the OCRL gene of two Lowe syndrome patients and report two new missense mutations that affect the ASH domain involved in protein-protein interactions. Genomic DNA was extracted from peripheral blood of two non-related patients and their relatives. Exons and flanking intronic regions of OCRL were analyzed by direct sequencing. Several bioinformatics tools were used to assess the pathogenicity of the variants. The three-dimensional structure of wild-type and mutant ASH domains was modeled using the online server SWISS-MODEL. Clinical features suggesting the diagnosis of Lowe syndrome were observed in both patients. Genetic analysis revealed two novel missense variants, c.1907T>A (p.V636E) and c.1979A>C (p.H660P) in exon 18 of the OCRL gene confirming the clinical diagnosis in both cases. Variant c.1907T>A (p.V636E) was inherited from the patient's mother, while variant c.1979A>C (p.H660P) seems to have originated de novo. Analysis with bioinformatics tools indicated that both variants are pathogenic. Both amino acid changes affect the structure of the OCRL1 ASH domain. In conclusion, the identification of two novel missense mutations located in the OCRL1 ASH domain may shed more light on the functional importance of this domain. We suggest that p.V636E and p.H660P cause Lowe syndrome by disrupting the interaction of OCRL1 with other proteins or by impairing protein stability.

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAM, Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations., Hay una corrección posterior a este artículo: http://hdl.handle.net/10486/703679, Background X-linked hypophosphatemia (XLH) is a hereditary rare disease caused by loss-of-function mutations in PHEX gene leading tohypophosphatemia and high renal loss of phosphate. Rickets and growth retardation are the major manifestations of XLH in children, but there is a broad phenotypic variability. Few publications have reported large series of patients. Current data on the clinical spectrum of the disease, the correlation with the underlying gene mutations, and the long-term outcome of patients on conventional treatment are needed, particularly because of the recent availability of new specific medications to treat XLH. Results The RenalTube database was used to retrospectively analyze 48 Spanish patients (15 men) from 39 different families, ranging from 3 months to 8 years and 2 months of age at the time of diagnosis (median age of 2.0 years), and with XLH confirmed by genetic analysis. Bone deformities, radiological signs of active rickets and growth retardation were the most common findings at diagnosis. Mean (± SEM) height was − 1.89 ± 0.19 SDS and 55% (22/40) of patients had height SDS below—2. All cases had hypophosphatemia, serum phosphate being − 2.81 ± 0.11 SDS. Clinical manifestations and severity of the disease were similar in both genders. No genotype—phenotype correlation was found. Conventional treatment did not attenuate growth retardation after a median follow up of 7.42 years (IQR = 11.26; n = 26 patients) and failed to normalize serum concentrations of phosphate. Eleven patients had mild hyperparathyroidism and 8 patients nephrocalcinosis. Conclusions This study shows that growth retardation and rickets were the most prevalent clinical manifestations at diagnosis in a large series of Spanish pediatric patients with XLH confirmed by mutations in the PHEX gene. Traditional treatment with phosphate and vitamin D supplements did not improve height or corrected hypophosphatemia and was associated with a risk of hyperparathyroidism and nephrocalcinosis. The severity of the disease was similar in males and females, This research has been partially funded by Kyowa Kirin Farmacéutica S.L.U., project PI17/01745 from Instituto de Salud Carlos III, Acción Estratégica en Salud 2017-2020 and FEDER funds, Fondo de Investigaciones Sanitarias (FIS), Fundación Nutrición y Crecimiento (FUNDNYC), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA) and Fundación para la Investigación y la Innovación Biosanitaria del Principado de Asturias (FINBA)

To find out if cardiovascular alterations are present in pediatric patients with X-linked hypophosphatemia (XLH). Multicentre prospective clinical study on pediatric patients included in the RenalTube database ( www.renaltube.com ) with genetically confirmed diagnosis of XLH by mutations in the PHEX gene. The study’s protocol consisted of biochemical work-up, 24-h ambulatory blood pressure monitoring (ABPM), carotid ultrasonography, and echocardiogram. All patients were on chronic treatment with phosphate supplements and 1-hydroxy vitamin D metabolites. Twenty-four patients (17 females, from 1 to 17 years of age) were studied. Serum concentrations (X ± SD) of phosphate and intact parathyroid hormone were 2.66 ± 0.60 mg/dl and 58.3 ± 26.8 pg/ml, respectively. Serum fibroblast growth factor 23 (FGF23) concentration was 278.18 ± 294.45 pg/ml (normal

The maintenance of magnesium (Mg2+) homeostasis is essential for human life. The Cystathionine-beta-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) have been described to be involved in maintaining Mg2+ homeostasis. Among these CNNMs, CNNM2 is expressed in the basolateral membrane of the kidney tubules where it is involved in Mg2+ reabsorption. A total of four patients, two of them with a suspected disorder of calcium metabolism, and two patients with a clinical diagnosis of primary tubulopathy were screened for mutations by Next-Generation Sequencing (NGS). We found one novel likely pathogenic variant in the heterozygous state (c.2384C>A; p.(Ser795*)) in theCNNM2gene in a family with a suspected disorder of calcium metabolism. In this family, hypomagnesemia was indirectly discovered. Moreover, we observed three novel variants of uncertain significance in heterozygous state in the other three patients (c.557G>C; p.(Ser186Thr), c.778A>T; p.(Ile260Phe), and c.1003G>A; p.(Asp335Asn)). Our study shows the utility of Next-Generation Sequencing in unravelling the genetic origin of rare diseases. In clinical practice, serum Mg2+ should be determined in calcium and PTH-related disorders. This study was supported by three grants from the Department of Health (2017111014, 2018111097 and 2019111052) and one grant from the Department of Education (IT1281-19) of the Basque Government. This work is generated within the Endocrine European Reference Network (Project ID number of Endo-ERN: 739527). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Hyperinsulinemic hypoglycemia (HI) and congenital polycystic kidney disease (PKD) are rare, genetically heterogeneous disorders. The co-occurrence of these disorders (HIPKD) in 17 children from 11 unrelated families suggested an unrecognized genetic disorder. Whole-genome linkage analysis in five informative families identified a single significant locus on chromosome 16p13.2 (logarithm of odds score 6.5). Sequencing of the coding regions of all linked genes failed to identify biallelic mutations. Instead, we found in all patients a promoter mutation (c.-167G>T) in the phosphomannomutase 2 gene (PMM2), either homozygous or in trans with PMM2 coding mutations. PMM2 encodes a key enzyme in N-glycosylation. Abnormal glycosylation has been associated with PKD, and we found that deglycosylation in cultured pancreatic β cells altered insulin secretion. Recessive coding mutations in PMM2 cause congenital disorder of glycosylation type 1a (CDG1A), a devastating multisystem disorder with prominent neurologic involvement. Yet our patients did not exhibit the typical clinical or diagnostic features of CDG1A. In vitro, the PMM2 promoter mutation associated with decreased transcriptional activity in patient kidney cells and impaired binding of the transcription factor ZNF143. In silico analysis suggested an important role of ZNF143 for the formation of a chromatin loop including PMM2. We propose that the PMM2 promoter mutation alters tissue-specific chromatin loop formation, with consequent organ-specific deficiency of PMM2 leading to the restricted phenotype of HIPKD. Our findings extend the spectrum of genetic causes for both HI and PKD and provide insights into gene regulation and PMM2 pleiotropy.

Dent's disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and eventual renal failure. Various types of mutations in the renal chloride channel gene, CLCN5, have been identified in patients with this disease. We studied a Spanish patient with Dent's disease and found, by polymerase chain reaction amplification of the CLCN5 exons, an abnormally large exon 11. Sequencing analysis revealed that this was attributable to the insertion in codon 650 of an Alu element of the "young" Ya5 subfamily. The Alu element was inserted with the same orientation as the CLCN5 gene and arose de novo on the maternal chromosome. Polymorphism analysis indicated that the insertion occurred in the germline of the maternal grandfather. The presence of a long poly(A) tract and evidence for a 16-bp target-site duplication implied that the Alu element was integrated by retrotransposition. This mutation predicts a truncated ClC-5 protein that lacks part of the carboxy-terminus and is likely to result in loss of function of the chloride channel. Insertions of Alu sequences, which are rarely found in coding regions, have occasionally been reported to cause other genetic diseases. However, this is the first report of a retrotransposon insertion in the CLCN5 gene associated with Dent's disease.

Background. Dent’s disease is characterized by lowmolecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets and eventual renal failure. The disease is caused by mutations in the Xlinked chloride channel CLCN5 gene, which encodes a 746-amino-acid protein expressed in renal tubules. These mutations have been reported in unrelated families from the UK, USA, Japan and other countries. We were interested in identifying additional mutations in the CLCN5 coding region of Spanish patients with Dent’s disease. Methods. Five patients from three unrelated Spanish families were studied. Leukocyte genomic DNA from patients and their relatives was used with CLCN5-specific primers for polymerase chain reaction amplification of the coding region and exon–intron boundaries. Amplified products were analysed by single-strand conformational polymorphism analysis, DNA sequencing and restriction enzyme analysis. Results. Low-molecular-weight proteinuria and hypercalciuria were detected in all the patients, nephrocalcinosis in two patients, and rickets or osteopenia in three patients. We identified three new CLCN5 mutations consisting of two nonsense mutations, Leu433Stop and Arg718Stop, and an insertional frameshift mutation, 65insT, which results in a stop at codon 98. These three mutations predict truncated ClC-5 proteins that, respectively, lack 314, 649 and 28 amino acids at the carboxy terminus, and are likely to result in loss of function. These mutations were shown to co-segregate with the disease in each of the three families. Conclusions. Our study is the first to characterize mutations in the CLCN5 gene in Spanish patients with Dent’s disease and expands the spectrum of CLCN5 mutations associated with this disease.

Journal Article; Research Support, Non-U.S. Gov't; Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal recessive tubular disorder characterized by excessive renal magnesium and calcium excretion and chronic kidney failure. This rare disease is caused by mutations in the CLDN16 and CLDN19 genes. These genes encode the tight junction proteins claudin-16 and claudin-19, respectively, which regulate the paracellular ion reabsorption in the kidney. Patients with mutations in the CLDN19 gene also present severe visual impairment. Our goals in this study were to examine the clinical characteristics of a large cohort of Spanish patients with this disorder and to identify the disease causing mutations. We included a total of 31 patients belonging to 27 unrelated families and studied renal and ocular manifestations. We then analyzed by direct DNA sequencing the coding regions of CLDN16 and CLDN19 genes in these patients. Bioinformatic tools were used to predict the consequences of mutations. Clinical evaluation showed ocular defects in 87% of patients, including mainly myopia, nystagmus and macular colobomata. Twenty two percent of patients underwent renal transplantation and impaired renal function was observed in another 61% of patients. Results of the genetic analysis revealed CLDN19 mutations in all patients confirming the clinical diagnosis. The majority of patients exhibited the previously described p.G20D mutation. Haplotype analysis using three microsatellite markers showed a founder effect for this recurrent mutation in our cohort. We also identified four new pathogenic mutations in CLDN19, p.G122R, p.I41T, p.G75C and p.G75S. A strategy based on microsequencing was designed to facilitate the genetic diagnosis of this disease. Our data indicate that patients with CLDN19 mutations have a high risk of progression to chronic renal disease. Yes

Alu sequences are short, interspersed elements that have generated more than one million copies in the human genome. They propagate by transcription followed by reverse transcription and integration, causing mutations, recombination, and changes in pre-mRNA splicing. We have recently identified a 345-bp long Alu Ya5 element inserted in codon 650 within exon 11 of the chloride channel ClC-5 gene (CLCN5) of a patient with Dent’s disease. A microsatellite pedigree analysis indicated that the insertion occurred in the germline of the maternal grandfather. Dent’s disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and nephrocalcinosis. Here, we found, by RT-PCR amplification of RNA extracted from the patient’s blood and subsequent DNA sequencing, that the Alu insertion led to an aberrant splicing of the CLCN5 pre-mRNA that skipped exon 11. Using the ESE finder and RESCUE-ESE Web interfaces, we identified two high-score exonic splicing enhancer (ESE) sequences in the site of insertion. The functional significance of these ESE motifs is suggested by our observation that these sequences are highly conserved among mammal CLCN5 genes. Therefore, we suggest that the Alu insertion causes exon skipping by interfering with splicing regulatory elements. The altered splicing would predict a truncated ClC-5 protein that lacks critical domains for sorting and chloride channel function.

Sirs, We have read with great interest the brief report of Boussemart et al. [1] published online first and strongly agree with their recommendation of strict vigilance of the fluid status in young patients with nephrogenic diabetes insipidus (NDI) who begin treatment. In fact, we would also recommend that not only are water balance and frequent clinical assessment, including hemodynamical status when necessary, crucial, but blood and urine sodium determination as well. The authors attribute their patient’s water intoxication to a strong enhanced sodium reabsorption due to the additive effect of hydrochlorothiazide and indomethacin combination therapy together with the lack of any physiological control of water intake secondary to gastric feeding. However, we experienced a water intoxication case in a 2-month-old male infant who only received initial hydrochlorothiazide treatment and was only bottle fed. Our patient was admitted to the hospital for evaluation of prolonged fever. Hypernatremia (Na 158 mEq/l) was revealed on admission, and a repeated medical history from their parents revealed that the infant had experienced polyuria–polydipsia since the first days of life, with a daily oral water intake of 500 ml added to his formula bottles, which they had considered normal. A dehydration test with subsequent 1-desamino-8-D-arginine vasopressin administration confirmed the diagnosis of NDI, and sequence analyses revealed mutation p.Trp200GlyfsX12 in exon 4 of the AVPR2 gene. Hydrochlorothiazide treatment was initiated at 2 mg/kg per day orally twice a day. Six hours after the first dose had been administered, the infant developed transient profuse sweating, paleness and hypothermia. The serum sodium concentration was 141 mEq/l at that time (pretreatment control 153 mEq/l), indicating water intoxication. Consequently, further hydrochlorothiazide treatment was stopped. The infant clinically recovered in the following hours. The treatment was reinitiated the following day at a lower dose, 1 mg/kg per day orally twice a day, which was well tolerated; no serum sodium control was obtained. The third day of treatment, 5 h after receiving the morning dose of hydrochlorothiazide, the patient's clinical condition worsened once again, with sweating, paleness, hypothermia, weakness, lethargy and gasping breathing. The serum sodium level was checked and the patient admitted to the pediatric intensive care unit (PICU). As the serum sodium concentration was 137 mEq/l, we were unable to demonstrate hyponatremia, but we hypothesized that there had probably been a significant serum sodium and subsequent osmolar shift secondary to water intoxication that had led to cerebral swelling. A cranial computerized scan showed no findings at that moment, and the patient recovered spontaneously during the following hours. Polyuria–polydipsia and hypernatremia continued the following days after the withdrawal of treatment. Therefore, 2 days later treatment was once again reinitiated at a lower dose (0.5 mg/kg per day twice a day). This treatment was well tolerated and was progressively increased during the following days (maximum 4 mg/kg per day) until neutral water balances were be achieved. Central venous catheter Pediatr Nephrol (2009) 24:2277–2278 DOI 10.1007/s00467-009-1236-4