Hypomagnesaemia with varying degrees of extrarenal symptoms as a consequence of heterozygous CNNM2 variants.

Variants in the CNNM2 gene are causative for hypomagnesaemia, seizures and intellectual disability, although the phenotypes can be variable. This study aims to understand the genotype-phenotype relationship in affected individuals with CNNM2 variants by phenotypic, functional and structural analysis of new as well as previously reported variants. This results in the identification of seven variants that significantly affect CNNM2-mediated Mg2+ transport. Pathogenicity of these variants is further supported by structural modelling, which predicts CNNM2 structure to be affected by all of them. Strikingly, seizures and intellectual disability are absent in 4 out of 7 cases, indicating these phenotypes are caused either by specific CNNM2 variant only or by additional risk factors. Moreover, in line with sporadic observations from previous reports, CNNM2 variants might be associated with disturbances in parathyroid hormone and Ca2+ homeostasis.

Renal diseases that course with hypomagnesemia. Comments on a new hereditary hypomagnesemic tubulopathy.

Renal diseases associated with hypomagnesemia are a complex and diverse group of tubulopathies caused by mutations in genes encoding proteins that are expressed in the thick ascending limb of the loop of Henle and in the distal convoluted tubule. In this paper, we review the initial description, the clinical expressiveness and etiology of four of the first hypomagnesemic tubulopathies described: type 3 Bartter and Gitelman diseases, Autosomal recessive hypomagnesemia with secondary hypocalcemia and Familial hypomagnesemia with hypercalciuria and nephrocalcinosis. The basic biochemical patterns observed in renal tubular hypomagnesemias and the modalities of transport and interaction that occur between the transporters involved in the reabsorption of magnesium in the distal convoluted tubule are described below. Finally, the recent report of a new renal disease with hypomagnesemia, type 2 hypomagnesemia with secondary hypocalcemia caused by reduced TRPM7 channel activity is described.

Characterization of pre-mRNA Splicing Defects Caused by CLCN5 and OCRL Mutations and Identification of Novel Variants Associated with Dent Disease.

Dent disease (DD) is an X-linked renal tubulopathy characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis and progressive renal failure. Two-thirds of cases are associated with inactivating variants in the CLCN5 gene (Dent disease 1, DD1) and a few present variants in the OCRL gene (Dent disease 2, DD2). The aim of the present study was to test the effect on the pre-mRNA splicing process of DD variants, described here or in the literature, and describe the clinical and genotypic features of thirteen unrelated patients with suspected DD. All patients presented tubular proteinuria, ten presented hypercalciuria and five had nephrolithiasis or nephrocalcinosis. CLCN5 and OCRL genes were analyzed by Sanger sequencing. Nine patients showed variants in CLCN5 and four in OCRL; eight of these were new. Bioinformatics tools were used to select fifteen variants with a potential effect on pre-mRNA splicing from our patients' group and from the literature, and were experimentally tested using minigene assays. Results showed that three exonic missense mutations and two intronic variants affect the mRNA splicing process. Our findings widen the genotypic spectrum of DD and provide insight into the impact of variants causing DD.

New SLC22A12 (URAT1) Variant Associated with Renal Hypouricemia Identified by Whole-Exome Sequencing Analysis and Bioinformatics Predictions.

Renal hypouricemia (RHUC) is a rare hereditary disorder caused by loss-of-function mutations in the SLC22A12 (RHUC type 1) or SLC2A9 (RHUC type 2) genes, encoding urate transporters URAT1 and GLUT9, respectively, that reabsorb urate in the renal proximal tubule. The characteristics of this disorder are low serum urate levels, high renal fractional excretion of urate, and occasional severe complications such as nephrolithiasis and exercise-induced acute renal failure. In this study, we report two Spanish (Caucasian) siblings and a Pakistani boy with clinical characteristics compatible with RHUC. Whole-exome sequencing (WES) analysis identified two homozygous variants: a novel pathogenic SLC22A12 variant, c.1523G>A; p.(S508N), in the two Caucasian siblings and a previously reported SLC2A9 variant, c.646G>A; p.(G216R), in the Pakistani boy. Our findings suggest that these two mutations cause RHUC through loss of urate reabsorption and extend the SLC22A12 mutation spectrum. In addition, this work further emphasizes the importance of WES analysis in clinical settings.

Pathogenic Variants of SLC22A12 (URAT1) and SLC2A9 (GLUT9) in Spanish Patients with Renal Hypouricemia: Founder Effect of SLC2A9 Variant c.374C>T; p.(T125M).

Renal hypouricemia (RHUC) is a rare inherited disorder characterized by impaired urate reabsorption in the proximal tubule resulting in low urate serum levels and increased urate excretion. Some patients may present severe complications such as exercise-induced acute renal failure and nephrolithiasis. RHUC is caused by inactivating mutations in the SLC22A12 (RHUC type 1) or SLC2A9 (RHUC type 2) genes, which encode urate transporters URAT1 and GLUT9, respectively. In this study, our goal was to identify mutations associated with twenty-one new cases with RHUC through direct sequencing of SLC22A12 and SLC2A9 coding exons. Additionally, we carried out an SNPs-haplotype analysis to determine whether the rare SLC2A9 variant c.374C>T; p.(T125M), which is recurrent in Spanish families with RHUC type 2, had a common-linked haplotype. Six intragenic informative SNPs were analyzed using PCR amplification from genomic DNA and direct sequencing. Our results showed that ten patients carried the SLC22A12 mutation c.1400C>T; p.(T467M), ten presented the SLC2A9 mutation c.374C>T, and one carried a new SLC2A9 heterozygous mutation, c.593G>A; p.(R198H). Patients carrying the SLC2A9 mutation c.374C>T share a common-linked haplotype, confirming that it emerged due to a founder effect.

Possible role for rare TRPM7 variants in patients with hypomagnesaemia with secondary hypocalcaemia.

BACKGROUND: Hypomagnesaemia with secondary hypocal-caemia (HSH) is a rare autosomal recessive disorder caused by pathogenic variants in TRPM6, encoding the channel-kinase transient receptor potential melastatin type 6. Patients have very low serum magnesium (Mg2+) levels and suffer from muscle cramps and seizures. Despite genetic testing, a subgroup of HSH patients remains without a diagnosis. METHODS: In this study, two families with an HSH phenotype but negative for TRPM6 pathogenic variants were subjected to whole exome sequencing. Using a complementary combination of biochemical and functional analyses in overexpression systems and patient-derived fibroblasts, the effect of the TRPM7-identified variants on Mg2+ transport was examined. RESULTS: For the first time, variants in TRPM7 were identified in two families as a potential cause for hereditary HSH. Patients suffer from seizures and muscle cramps due to magnesium deficiency and episodes of hypocalcaemia. In the first family, a splice site variant caused the incorporation of intron 1 sequences into the TRPM7 messenger RNA and generated a premature stop codon. As a consequence, patient-derived fibroblasts exhibit decreased cell growth. In the second family, a heterozygous missense variant in the pore domain resulted in decreased TRPM7 channel activity. CONCLUSIONS: We establish TRPM7 as a prime candidate gene for autosomal dominant hypomagnesaemia and secondary hypocalcaemia. Screening of unresolved patients with hypocalcaemia and secondary hypocalcaemia may further establish TRPM7 pathogenic variants as a novel Mendelian disorder.

Gout associated with reduced renal excretion of uric acid. Renal tubular disorder that nephrologists do not treat.

Gout is recurrent inflammatory arthritis caused by the deposition of monosodium urate crystals in the joints. The risk factors that predispose to suffering from gout include non-modifiable factors such as gender, age, ethnicity and genetics, and modifiable factors such as diet and lifestyle. It has been shown that the heritability of uric acid levels in the blood is greater than 30%, which indicates that genetics play a key role in these levels. Hyperuricaemia is often a consequence of reduced renal urate excretion since more than 70% is excreted by the kidneys, mainly through the proximal tubule. The mechanisms that explain that hyperuricaemia associated with reduced renal urate excretion is, to a large extent, a proximal renal tubular disorder, have begun to be understood following the identification of two genes that encode the URAT1 and GLUT9 transporters. When they are carriers of loss-of-function mutations, they explain the two known variants of renal tubular hypouricaemia. Some polymorphisms in these genes may have an opposite gain-of-function effect, with a consequent increase in urate reabsorption. Conversely, loss-of-function polymorphisms in other genes that encode transporters involved in urate excretion (ABCG2, ABCC4) can lead to hyperuricaemia. Genome-wide association study (GWAS) methods have made it possible to locate new gout-related loci associated with reduced renal urate excretion (NIPAL1, FAM35A).

Hereditary kidney diseases associated with hypomagnesemia.

In the kidney, a set of proteins expressed in the epithelial cells of the thick ascending loop of Henle and the distal convoluted tubule directly or indirectly play important roles in the regulation of serum magnesium levels. Magnesium reabsorption in the thick ascending loop of Henle occurs through a passive paracellular pathway, while in the distal convoluted tubule, the final magnesium concentration is established through an active transcellular pathway. The players involved in magnesium reabsorption include proteins with diverse functions including tight junction proteins, cation and anion channels, sodium chloride cotransporter, calcium-sensing receptor, epidermal growth factor, cyclin M2, sodium potassium adenosine triphosphatase subunits, transcription factors, a serine protease, and proteins involved in mitochondrial function. Mutations in the genes that encode these proteins impair their function and cause different rare diseases associated with hypomagnesemia, which may lead to muscle cramps, fatigue, epileptic seizures, intellectual disability, cardiac arrhythmias, and chronic kidney disease. The purpose of this review is to describe the clinical and genetic characteristics of these hereditary kidney diseases and the current research findings on the pathophysiological basis of these diseases.

Gout associated with reduced renal excretion of uric acid. Renal tubular disorder that nephrologists do not treat. / La gota asociada a reducción de la excreción renal de ácido úrico. Esa tubulopatía que no tratamos los nefrólogos.

Gout is recurrent inflammatory arthritis caused by the deposition of monosodium urate crystals in the joints. The risk factors that predispose to suffering from gout include non-modifiable factors such as gender, age, ethnicity and genetics, and modifiable factors such as diet and lifestyle. It has been shown that the heritability of uric acid levels in the blood is greater than 30%, which indicates that genetics play a key role in these levels. Hyperuricaemia is often a consequence of reduced renal urate excretion since more than 70% is excreted by the kidneys, mainly through the proximal tubule. The mechanisms that explain that hyperuricaemia associated with reduced renal urate excretion is, to a large extent, a proximal renal tubular disorder, have begun to be understood following the identification of two genes that encode the URAT1 and GLUT9 transporters. When they are carriers of loss-of-function mutations, they explain the two known variants of renal tubular hypouricaemia. Some polymorphisms in these genes may have an opposite gain-of-function effect, with a consequent increase in urate reabsorption. Conversely, loss-of-function polymorphisms in other genes that encode transporters involved in urate excretion (ABCG2, ABCC4) can lead to hyperuricaemia. Genome-wide association study (GWAS) methods have made it possible to locate new gout-related loci associated with reduced renal urate excretion (NIPAL1, FAM35A).

Heterogeneity is a common ground in familial hypomagnesemia with hypercalciuria and nephrocalcinosis caused by CLDN19 gene mutations.

BACKGROUND: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare tubulopathy caused by mutations in the CLDN16 or CLDN19 genes. Patients usually develop hypomagnesemia, hypercalciuria, nephrocalcinosis and renal failure early in life. Patients with CLDN19 mutations may also have ocular abnormalities. Despite clinical variability, factors associated with kidney function impairment, especially in patients with CLDN19 mutations, have not been addressed. METHODS: Retrospective multicenter study of 30 genetically confirmed FHHNC Spanish patients. We analyzed kidney function impairment considering as outcomes chronic kidney disease (CKD) stage 3 and annual estimated glomerular filtration rate (eGFR) decline, to identify factors associated with the different phenotypes. RESULTS: Of thirty patients, 27 had mutations in the CLDN19 gene (20 homozygous for the p.G20D mutation) and 3 in the CLDN16. Age at diagnosis was 1.71 (0.67-6.04) years and follow-up time was 8.34 ± 4.30 years. No differences in CKD stage 3-free survival based on CLDN19 mutation (p.G20D homozygous vs. other mutations) or gender were found, although females seemed to progress faster than males. Patients with more pronounced eGFR decline had higher PTH levels at diagnosis than those with stable kidney function, despite similar initial eGFR. Approximately 60% of CLDN19 patients presented ocular abnormalities. Furthermore, we confirmed high phenotypic intrafamilial variability. CONCLUSIONS: In a contemporary cohort of FHHNC patients with CLDN19 mutations, females seemed to progress to CKD-stage 3 faster than males. Increased PTH levels at baseline may indicate a more severe renal course. There was high phenotype variability among patients with CLDN19 mutations and kidney function impairment  differed even between siblings.

The phenotypic and genetic spectrum of patients with heterozygous mutations in cyclin M2 (CNNM2).

Hypomagnesemia, seizures, and intellectual disability (HSMR) syndrome is a rare disorder caused by mutations in the cyclin M2 (CNNM2) gene. Due to the limited number of cases, extensive phenotype analyses of these patients have not been performed, hindering early recognition of patients. In this study, we established the largest cohort of HSMR to date, aiming to improve recognition and diagnosis of this complex disorder. Eleven novel variants in CNNM2 were identified in nine single sporadic cases and in two families with suspected HSMR syndrome. 25 Mg2+ uptake assays demonstrated loss-of-function in seven out of nine variants in CNNM2. Interestingly, the pathogenic mutations resulted in decreased plasma membrane expression. The phenotype of those affected by pathogenic CNNM2 mutations was compared with five previously reported cases of HSMR. All patients suffered from hypomagnesemia (0.44-0.72 mmol/L), which could not be fully corrected by Mg2+ supplementation. The majority of patients (77%) experienced generalized seizures and exhibited mild to moderate intellectual disability and speech delay. Moreover, severe obesity was present in most patients (89%). Our data establish hypomagnesemia, seizures, intellectual disability, and obesity as hallmarks of HSMR syndrome. The assessment of these major features offers a straightforward tool for the clinical diagnosis of HSMR.

Two new missense mutations in the protein interaction ASH domain of OCRL1 identified in patients with Lowe syndrome.

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.

Nail-Patella syndrome with early onset end-stage renal disease in a child with a novel heterozygous missense mutation in the LMX1B homeodomain: A case report.

Nail-Patella syndrome (NPS) is an inherited disease characterized by nail and skeletal anomalies, nephropathy and glaucoma. The diagnosis of NPS is based on clinical findings, including hypoplastic or absent patella, dystrophic nails, dysplasia of the elbows and iliac horns. However, the main determinant of NPS prognosis is nephropathy, which may range from asymptomatic proteinuria to end-stage renal disease. NPS is caused by heterozygous loss-of-function mutations in the LMX1B gene, which encodes the LIM homeodomain transcription factor LMX1B. LMX1B serves an essential role in the physiological development of dorsal-ventral limb structures, morphogenesis and function of podocytes, as well as in development of the anterior segments of the eyes, and in certain types of neurons. The present study aimed to identify the disease-causing mutation in a 2-year old girl with nephrotic syndrome that evolved rapidly to end-stage renal disease. The patient showed classical symptoms of NPS including dystrophic nails and an absence of the patellae. DNA sequence analysis identified a novel missense variant in exon 4 of LMX1B (c.709T>C, p.S237P); this substitution affected a conserved serine residue in the homeodomain of LMX1B and was predicted to be pathogenic. In silico modeling of the homeodomain revealed that the p.S237P mutation converted the A236-S237-F238 segment of α-helix 1 into a strand. It was hypothesized that this mutation affected binding of the transcription factor to its target DNA, thus abrogating transcription activation, which would explain the phenotype that manifested in the patient.

Novel compound heterozygous mutations of CLDN16 in a patient with familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

BACKGROUND: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an autosomal recessive tubulopathy characterized by excessive urinary wasting of magnesium and calcium, bilateral nephrocalcinosis, and progressive chronic renal failure in childhood or adolescence. FHHNC is caused by mutations in CLDN16 and CLDN19, which encode the tight-junction proteins claudin-16 and claudin-19, respectively. Most of these mutations are missense mutations and large deletions are rare. METHODS: We examined the clinical and biochemical features of a Spanish boy with early onset of FHHNC symptoms. Exons and flanking intronic segments of CLDN16 and CLDN19 were analyzed by direct sequencing. We developed a new assay based on Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) to investigate large CLDN16 deletions. RESULTS: Genetic analysis revealed two novel compound heterozygous mutations of CLDN16, comprising a missense mutation, c.277G>A; p.(Ala93Thr), in one allele, and a gross deletion that lacked exons 4 and 5,c.(840+25_?)del, in the other allele. The patient inherited these variants from his mother and father, respectively. CONCLUSIONS: Using direct sequencing and our QMPSF assay, we identified the genetic cause of FHHNC in our patient. This QMPSF assay should facilitate the genetic diagnosis of FHHNC. Our study provided additional data on the genotypic spectrum of the CLDN16 gene.

Novel missense mutation affecting the LIM-A domain of LMX1B in a family with Nail-Patella syndrome.

Nail-patella syndrome (NPS) is a rare autosomal dominant disease characterized by developmental defects of dorsal limb structures, the kidney, and the eye, that manifest as dysplastic nails, hypoplastic or absent patella, elbow dysplasia, iliac horns, glomerulopathy, and adult-onset glaucoma, respectively. This disorder is inherited in an autosomal dominant mode and is caused by heterozygous loss-of-function mutations in the LMX1B gene, which encodes the LIM homeodomain transcription factor LMX1B. In this study, we report the clinical findings of a Spanish family, from the Canary Islands, with three affected members who displayed varying phenotypes. DNA sequence analysis identified a novel heterozygous missense mutation in LMX1B, c.305A>G, p.(Y102C), that segregated with the disease. The tyrosine residue affected by the mutation is highly conserved in evolution, and is located in the LIM-A domain, next to one of the cysteine residues involved in zinc binding, suggesting that p.(Y102C) affects LMX1B function by disturbing its interactions with other proteins. Our results expand the mutation spectrum of LMX1B and provide insight into the molecular mechanisms of NPS pathology.

Hipouricemia renal hereditaria tipo 1 y 2 en tres niños españoles. Revisión de casos pediátricos publicados.

Hereditary renal hypouricemia is a rare autosomal recessive genetic disorder that involves an isolated defect in uric acid reabsorption at the renal tubules. Patients present with serum uric acid concentrations of less than 2mg/dl (119 micromol/L) with increased fractional excretion above 10%. Most of the patients are asymptomatic and are detected incidentally. However, complications such us nephrolithiasis, hematuria, acute renal failure exercise-induced or after dehydration for acute gastroenteritis, or posterior reversible encephalopaty syndrome (PRES) may develop. Hereditary renal hypouricemia is confirmed by molecular genetic analysis of the two genes which codify the uric acid transport in the kidney tubules. The renal hypouricemia type 1 (OMIM 220150) is characterized by loss-of-function mutations in the SLC22A12 gene which encodes URAT 1 transporter, and the hypouricemia type 2 (OMIM 612076) is caused by defects in the SLC2A9 gene. Homozygous mutations of SLC2A9 cause the most severe forms of the disease. Most mutations have been identified in Japanese adults, and only a few in children. We describe three asyntomatic pediatric Spanish patients with renal hypouricemia, with genetic confirmation, and we make a revision of all of the pediatric cases with genetic study published in the literature.

Exonic CLDN16 mutations associated with familial hypomagnesemia with hypercalciuria and nephrocalcinosis can induce deleterious mRNA alterations.

BACKGROUND: Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis type 1 is an autosomal recessive disease characterized by excessive renal magnesium and calcium excretion, bilateral nephrocalcinosis, and progressive chronic renal failure. This rare disease is caused by mutations in CLDN16 that encodes claudin-16, a tight-junction protein involved in paracellular reabsorption of magnesium and calcium in the renal tubule. Most of these variants are located in exons and have been classified as missense mutations. The functional consequences of some of these claudin-16 mutant proteins have been analysed after heterologous expression showing indeed a significant loss of function compared to the wild-type claudin-16. We hypothesize that a number of CLDN16 exonic mutations can be responsible for the disease phenotype by disrupting the pre-mRNA splicing process. METHODS: We selected 12 previously described presumed CLDN16 missense mutations and analysed their potential effect on pre-mRNA splicing using a minigene assay. RESULTS: Our results indicate that five of these mutations induce significant splicing alterations. Mutations c.453G > T and c.446G > T seem to inactivate exonic splicing enhancers and promote the use of an internal cryptic acceptor splice site resulting in inclusion of a truncated exon 3 in the mature mRNA. Mutation c.571G > A affects an exonic splicing enhancer resulting in partial skipping of exon 3. Mutations c.593G > C and c.593G > A disturb the acceptor splice site of intron 3 and cause complete exon 4 skipping. CONCLUSIONS: To our knowledge, this is the first report of CLDN16 exonic mutations producing alterations in splicing. We suggest that in the absence of patients RNA samples, splicing functional assays with minigenes could be valuable for evaluating the effect of exonic CLDN16 mutations on pre-mRNA splicing.

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.