4-Phenylbutyric Acid Treatment Reduces Low-Molecular-Weight Proteinuria in a Clcn5 Knock-in Mouse Model for Dent Disease-1.

Dent disease-1 (DD-1) is a rare X-linked tubular disorder characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrolithiasis and nephrocalcinosis. This disease is caused by inactivating mutations in the CLCN5 gene which encodes the voltage-gated ClC-5 chloride/proton antiporter. Currently, the treatment of DD-1 is only supportive and focused on delaying the progression of the disease. Here, we generated and characterized a Clcn5 knock-in mouse model that carries a pathogenic CLCN5 variant, c. 1566_1568delTGT; p.Val523del, which has been previously detected in several DD-1 unrelated patients, and presents the main clinical manifestations of DD-1 such as high levels of urinary b2-microglobulin, phosphate and calcium. Mutation p.Val523del causes partial ClC-5 retention in the endoplasmic reticulum. Additionally, we assessed the ability of sodium 4-phenylbutyrate, a small chemical chaperone, to ameliorate DD-1 symptoms in this mouse model. The proposed model would be of significant value in the investigation of the fundamental pathological processes underlying DD-1 and in the development of effective therapeutic strategies for this rare condition.

Enfermedades renales que cursan con hipomagnesemia. Comentarios acerca de una nueva tubulopatía hipomagnesémica de origen genético / Renal diseases that course with hypomagnesemia. Comments on a new hereditary hypomagnesemic tubulopathy

Las enfermedades renales que cursan con hipomagnesemia son un grupo complejo y variopinto de tubulopatías producidas por mutaciones en genes que codifican proteínas que se expresan en la rama gruesa ascendente del asa de Henle y en el túbulo contorneado distal. En el presente artículo revisamos la descripción inicial, la expresividad clínica y la etiología de cuatro de las primeras causas de tubulopatías hipomagnesémicas que se describieron: las enfermedades de Bartter tipo 3 y Gitelman, la hipomagnesemia con hipocalcemia secundaria autosómica recesiva y la hipomagnesemia familiar con hipercalciuria y nefrocalcinosis. A continuación, se describen los patrones bioquímicos básicos que se observan en las hipomagnesemias tubulares renales y las modalidades de transporte e interacción que concurren entre los transportadores implicados en la reabsorción de magnesio en el túbulo contorneado distal. Finalmente, se comunica la reciente descripción de una nueva tubulopatía hipomagnesémica, la hipomagnesemia con hipocalcemia secundaria tipo 2 causada por una reducción de la actividad del canal TRPM7 (AU)

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 (AU)

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.

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).

Concordance between two intrapersonal psychological resilience scales: how should we be measuring resilience?

BACKGROUND: While resilience has generated a lot of interest in mental health, operationalizing the construct of resilience remains an important challenge. This study aims to evaluate the concordance of two resilience scales that evaluate intrapersonal aspects of resilience in adolescents. METHODS: Cross-sectional evaluation of internal consistency, concordance, and correlation of the Individual Protective Factors Index Questionnaire (IPFI) and the Adolescent Resilience Scale (ARS) in sixth grade students of three low-income public schools in Colombia. RESULTS: 325 adolescents (41.5% female) participated in the study (72.5% response rate). Mean age was 12.1 years (standard deviation [SD]: 1.04). Of a possible score from 1-4, the mean adjusted IPFI score was 3.3 (SD: 0.3; Cronbach's alpha: 0.87). Of a possible score from 21-105, the total ARS score was 76.4 (SD 13.0; Cronbach's alpha: 0.82); both distributions were non-normal and left-skewed. The Lin's concordance correlation coefficient was 0.34 and the Spearman correlation coefficient was 0.52 (p-value < 0.0001 for both). Notably, 10 adolescents (3.1% of the sample) had a score in the lowest quartile in one of the two instruments, and a score in the highest quartile in the other instrument. CONCLUSIONS: There was low concordance between the scales, with notable lack of overlap in who was identified as having "low" levels of resilience. To better elucidate and operationalize the construct of resilience, studies using resilience scales should consider greater focus in understanding what aspects of the construct are being measured and how they relate to meaningful variables (well-being, risk of illness, etc.).

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

La gota es una artritis inflamatoria recurrente provocada por el depósito de cristales de urato monosódico en las articulaciones. Entre los factores de riesgo que predisponen a padecer gota se encuentran aquellos no modificables como sexo, edad, raza y genética y los modificables como dieta y estilo de vida. Se ha indicado que la heredabilidad de los niveles de ácido úrico en sangre es superior al 30%, lo que indica que la genética tiene un papel clave en dichos niveles.La hiperuricemia es a menudo una consecuencia de la reducción de la excreción renal de urato, ya que más del 70% se excreta por el riñón, principalmente, por el túbulo proximal.Los mecanismos que explican que la hiperuricemia asociada a la reducción de la excreción renal de urato es, en gran medida, una tubulopatía proximal, se han empezado a conocer al saberse la existencia de dos genes que codifican los transportadores URAT1 y GLUT9 que, cuando son portadores de mutaciones de pérdida de función, explican las dos variantes conocidas de hipouricemia tubular renal.Algunos polimorfismos presentes en esos genes pueden tener un efecto contrario de ganancia de función, con la consecuencia de un incremento en la reabsorción de urato. A la inversa, polimorfismos de pérdida de función en otros genes que codifican trasportadores implicados en la excreción de urato (ABCG2, ABCC4) favorecen la hiperuricemia.Los métodos de asociación genómica amplia (GWAS) han permitido localizar nuevos locus relacionados con gota asociada a reducción de la excreción renal de urato (NIPAL1, FAM35A). (AU)

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). (AU)

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).

Bioactive Metabolites from the Endophytic Fungus Aspergillus sp. SPH2.

In the current study, an ethyl acetate extract from the endophytic fungus Aspergillus sp. SPH2 isolated from the stem parts of the endemic plant Bethencourtia palmensis was screened for its biocontrol properties against plant pathogens (Fusarium moniliforme, Alternaria alternata, and Botrytis cinerea), insect pests (Spodoptera littoralis, Myzus persicae, Rhopalosiphum padi), plant parasites (Meloidogyne javanica), and ticks (Hyalomma lusitanicum). SPH2 gave extracts with strong fungicidal and ixodicidal effects at different fermentation times. The bioguided isolation of these extracts gave compounds 1-3. Mellein (1) showed strong ixodicidal effects and was also fungicidal. This is the first report on the ixodicidal effects of 1. Neoaspergillic acid (2) showed potent antifungal effects. Compound 2 appeared during the exponential phase of the fungal growth while neohydroxyaspergillic acid (3) appeared during the stationary phase, suggesting that 2 is the biosynthetic precursor of 3. The mycotoxin ochratoxin A was not detected under the fermentation conditions used in this work. Therefore, SPH2 could be a potential biotechnological tool for the production of ixodicidal extracts rich in mellein.

Non-Heterosexual Medical Students Are Critically Vulnerable to Mental Health Risks: The Need to Account for Sexual Diversity in Wellness Initiatives.

Phenomenon: Mental health problems among medical students are a worrisome issue; recent studies have shown that one-third may be suffering major depressive disorder and one out of ten had suicidal ideation. Few studies have evaluated the association of medical students' mental health and their sexual orientation. This study aimed to evaluate differences in mental health indicators among medical students with diverse sexual orientations at a South American medical school. Approach: This study is a secondary analysis of cross-sectional data obtained through an electronic survey. The survey assessed demographics, academic variables, and several mental health scales and indexes, including: World Health Organization Well-being Index, Satisfaction With Life Scale, Family APGAR (Adaptability, Partnership, Growth, Affection, and Resolve), Self-Reporting Questionnaire, Athens Insomnia Scale, Eating Attitudes Test, and Alcohol Use Disorders Identification Test. Sexual orientation was assessed using self-identification and responses dichotomized as heterosexual and non-heterosexual. Findings: 554 students completed the survey (response rate: 70%). Mean age was 20.6 years, and the sample was 58.7% women. Eighty-two participants (14.8%) self-identified as non-heterosexual; this group comprised mostly males, fewer of whom lived with their family, and more of whom used loans or scholarships to pay university tuition fees. After adjustment for sex and tuition fee payment, non-heterosexual orientation was significantly associated (adjusted Odds Ratios [aOR] above 3.00) with rating mental health as bad, self-perceiving a need for mental health evaluation/treatment, and reporting last-year use of psychiatric medication. Lastly, non-heterosexual respondents reported more frequent psychiatric symptoms (depression/anxiety scores, suicidal ideation, eating disorder symptoms and substance use) with an aOR between 2.17 and 2.51. Insights: This study suggests that self-identified non-heterosexual medical students exhibit worse mental health outcomes evaluated through validated self-report scales and subjective perception of mental health status. This report specifically indicates that non-heterosexual medical students report family dysfunction more often and have less social support, which serve as additional risk factors. Future studies must assess social support, clarify the impact of family and peer support in mental health problems, and explore students' views on their sexual identity and the burden imposed by experiences of discrimination.

Short report: cross sectional comparison of anxiety and depression symptoms in medical students and the general population in Colombia.

There are not many direct comparisons of anxiety and depression symptoms between medicine students and the general population. This study aimed to determine the type of anxiety and depression symptoms at a medical school in Bogota (Colombia), and to compare the findings with population data. All students enrolled at a medical school were invited to participate in 2017. The Self-Reporting Questionnaire was applied to measure anxious and depressive symptoms. These findings were compared with data from the National Survey of Mental Health 2015 that were obtained with the same instrument in the same age range (18-25 years). A total of 554 students participated in the survey (70% response rate); 477 were between 18 and 25 years old. Anxiety symptoms (≥5) were reported by 44.9% of the medical students and 6.8% of the general population, and depression symptoms (≥7) were reported by 33.9% and 2.6%, respectively. The most prevalent symptoms reported by the medical students were fatigue, lack of sleep, and feeling tired all the time. In turn, the general population reported headaches, fear, nervousness, tension, and uneasiness. All comparisons showed p-values < 0.01. As prevalence is higher and symptom profile is different, specific policies are needed to reduce risk throughout medical degree programmes.

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.

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.

Consideraciones acerca de las bases moleculares de algunas tubulopatías en relación con la endogamia y los desplazamientos poblacionales / Considerations about the molecular basis of some kidney tubule disorders in relation to inbreeding and population displacement

No disponible

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.

Splicing Analysis of Exonic OCRL Mutations Causing Lowe Syndrome or Dent-2 Disease.

Mutations in the OCRL gene are associated with both Lowe syndrome and Dent-2 disease. Patients with Lowe syndrome present congenital cataracts, mental disabilities and a renal proximal tubulopathy, whereas patients with Dent-2 disease exhibit similar proximal tubule dysfunction but only mild, or no additional clinical defects. It is not yet understood why some OCRL mutations cause the phenotype of Lowe syndrome, while others develop the milder phenotype of Dent-2 disease. Our goal was to gain new insights into the consequences of OCRL exonic mutations on pre-mRNA splicing. Using predictive bioinformatics tools, we selected thirteen missense mutations and one synonymous mutation based on their potential effects on splicing regulatory elements or splice sites. These mutations were analyzed in a minigene splicing assay. Results of the RNA analysis showed that three presumed missense mutations caused alterations in pre-mRNA splicing. Mutation c.741G>T; p.(Trp247Cys) generated splicing silencer sequences and disrupted splicing enhancer motifs that resulted in skipping of exon 9, while mutations c.2581G>A; p.(Ala861Thr) and c.2581G>C; p.(Ala861Pro) abolished a 5' splice site leading to skipping of exon 23. Mutation c.741G>T represents the first OCRL exonic variant outside the conserved splice site dinucleotides that results in alteration of pre-mRNA splicing. Our results highlight the importance of evaluating the effects of OCRL exonic mutations at the mRNA level.