CLDN16 Research Articles

#6286 NEPHROCALCINOSIS IN 3 GENERATIONS OF FEMALE PATIENTS DUE TO A PATHOGENIC MEN1 MUTATION

Abstract Background and Aims Multiple Endocrince Neoplasia type 1 (MEN1) syndrome, has an autosomal dominant transmission pattern, characterized by hyperparathyroidism, benign or malignant tumors in pancreas islets, pituitary tumors, thymic carcinomas, adrenal cortex adenomas and angio-fibromas of the skin. We are describing 2 cases of MEN1 with first and cardinal manifestations within the kidney. Method Medical records review, whole exome sequencing (WES), bioinformatics analysis by Ingenuity Pathway Analysis, QIAGEN Results Patient 1. A 28 year old female presented with horseshoe kidney, multiple pyelonephritis episodes, primary hyperparathyroidism, hypercalcemia, magnesiuria, reduced bone mass, and nephrocalcinosis. Her mother and grandmother suffered from hyperparathyroidism. WES analysis demonstrated a 12-base deletion between intron 9 and exon 10 in MEN1 gene (c.1351-3_1359delCAGGTGCGGCAG) with an apparent pathogenicity [1]. An accompanying alteration of CLDN16 gene (c.324+13C>G, rs369250510) was also detected. Till now this alteration is considered benign. Patient 2. A 48 year old female, with primary hyperparathyroidism in the last 15 years (the mother of Patient 1), with nephrocalcinosis since 1997 (calcium oxalate stones), preserved renal function, hypercalcemia, magnesiuria, reduced bone mass, hypertelorism with concurrent diplopia. In 2013, parathyroeidectomy resulted in stabilization of nephrocalcinosis. WES revealed the same pathogenic deletion as in her daughter. A research for epigastric pain revealed a cystic pancreatic mass, with accompanying increase in serum gastrin and chromogranin. A pituitary adenoma is manifested with a marginally increased serum prolactin. Her mother suffers from primary hyperparathyroidism with nephrocalcinosis. Conclusion In familial nephrocalcinosis/lithiasis/hyperparathyroidism genetic counselling and screening defines individualized treatment and may prevent extra renal disease manifestations.

Furosemide rescues hypercalciuria in familial hypomagnesaemia with hypercalciuria and nephrocalcinosis model

Aim: Impaired calcium homeostasis limits life expectancy and quality in familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC). This rare disease is caused by loss-of-function mutations in CLDN16 or CLDN19 genes leading to impaired paracellular reabsorption of divalent cations along the cortical thick ascending limb (cTAL). The ensuing late distal nephron and collecting duct system partially compensate for the defect in cTAL by increased transcellular Ca 2+ reabsorption via the luminal transient receptor potential channel V5 (TRPV5), as well as basolateral plasma membrane calcium ATPase (PMCA) and sodium-potassium exchanger (NCX1). The loop diuretic furosemide induces compensatory activation in these distal segments due to blockade of NaCl and divalent cation reabsorption in the preceeding TAL. Although furosemide enhances urinary calcium excretion via inhibition of the aforementioned cTAL in general population, this effect is not expected in FHHNC patients with already severely impaired Ca 2+ transport in the cTAL. The present study follows the hypothesis that furosemide may alleviate hypercalciuria in this disease by activation of the distal transcellular Ca 2+ transport proteins. Methods: Cldn16-deficient mice (Cldn16-/- ) served as a FHHNC model. Wild-type (WT) and Cldn16-/- mice were treated with furosemide (7 days of 40 mg/kg bw) or vehicle. We assessed renal electrolyte handling (metabolic cages) and key divalent transport proteins. Results: Cldn16-/- mice show increased Ca 2+ excretion than WT and compensatory stimulation of Cldn2 in the proximal tubule, as well as of TRPV5, and NCX1 in the distal nephron at baseline. Furosemide reduced hypercalciuria in Cldn16-/- mice and enhanced TRPV5 and PMCA levels in Cldn16-/- but not in WT mice. Conclusions: Furosemide alleviated hypercalciuria in a mouse FHHNC model, likely via stimulation of transcellular luminal and basolateral Ca 2+ transport systems in the distal nephron and collecting duct. These results may have a clinical implication in FHHNC patients. No conflict of interests This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Furosemide rescues hypercalciuria in familial hypomagnesaemia with hypercalciuria and nephrocalcinosis model.

Perturbed calcium homeostasis limits life expectancy in familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC). This rare disease occurs by loss-of-function mutations in CLDN16 or CLDN19 genes, causing impaired paracellular reabsorption of divalent cations along the cortical thick ascending limb (cTAL). Only partial compensation takes place in the ensuing late distal convoluted tubule, connecting tubule, and collecting duct, where the luminal transient receptor potential channel V5 (TRPV5), as well as basolateral plasma membrane calcium ATPase (PMCA) and sodium-potassium exchanger (NCX1) mediate transcellular Ca2+ reabsorption. The loop diuretic furosemide induces compensatory activation in these distal segments. Normally, furosemide enhances urinary calcium excretion via inhibition of the aforementioned cTAL. As Ca2+ reabsorption in the cTAL is already severely impaired in FHHNC patients, furosemide may alleviate hypercalciuria in this disease by activation of the distal transcellular Ca2+ transport proteins. Cldn16-deficient mice (Cldn16-/- ) served as a FHHNC model. Wild-type (WT) and Cldn16-/- mice were treated with furosemide (7 days of 40 mg/kg bw) or vehicle. We assessed renal electrolyte handling (metabolic cages) and key divalent transport proteins. Cldn16-/- mice show higher Ca2+ excretion than WT and compensatory stimulation of Cldn2, TRPV5, and NCX1 at baseline. Furosemide reduced hypercalciuria in Cldn16-/- mice and enhanced TRPV5 and PMCA levels in Cldn16-/- but not in WT mice. Furosemide significantly reduces hypercalciuria, likely via upregulation of luminal and basolateral Ca2+ transport systems in the distal nephron and collecting duct in this model for FHHNC.

P-211 Claudin-16, its clinical and prognostic value in colorectal cancer

Claudin-16, also known as CLDN-16 or paracellin-1, plays a major role in tight junction-specific occlusion of the intercellular space, through calcium-independent cell-adhesion activity, and Involved in paracellular magnesium reabsorption. The CLDN16 gene is a protein coding gene, mutations of which are associated with diseases such as Hypomagnesemia 3, renal and primary hypomagnesemia. Among its related pathways are cell junction organization and Blood-Brain Barrier and immune cell transmigration. Tight junctions represent one mode of cell-to-cell adhesion in epithelial or endothelial cell sheets, forming continuous seals around cells and serving as a physical barrier to prevent solutes and water from passing freely through the paracellular space. Evidence has now identified tight junction as key barriers to metastatic disease and cancer progression. We have previously shown that CL16 is associated with survival in breast cancer. The present study explored the expression and relevance of Claudin-16 in human colorectal cancer. Claudin-16 expression levels in human colorectal tumour and normal tissues were quantitatively analysed using gene transcript analyses. Expression was determined against the clinical and pathological parameters and clinical outcome of the patients including recurrence, metastatic disease and deaths related to colorectal cancer. The Mann Whitney U statistical test was utilised for comparisons and logistic regression, with Kaplan-Meier used for survival analyses. Expression of Claudin-16 was significantly reduced in tumour specimens in this colorectal cohort (p=0.0016, tumour n=83; normal n=41). Paired tumour/normal tissues also indicated a significantly reduces expression in tumour samples (p= 0.0287). There was a progressive reduction in Claudin-16 expression with increasing TNM status (p < 0.08). Intriguingly, expression of Claudin-16 was significantly lower in tumours originating in right colon and the rectum (p < 0.034) and expression was reduced in tumours that were both invasive/non-invasive (p < 0.026). When evaluating disease outcome, patients that remained alive and well had higher levels of Claudin-16 when compared to those with local recurrence, metastatic disease and had dies from cancer (p < 0.0238). Kaplan-Meier curves indicated that Clausin-16 was associated with recurrence of disease and death from colorectal cancer (p < 0.042). Claudin-16 has a highly differential expression in colorectal cancer and exhibits properties that in future may enable its use as a possible prognostic indicator.

Association study of CLDN14 variations in patients with kidney stones.

Claudin-14 protein plays an essential role in regulating calcium ions in the kidney and ear. Two phenotypes, hearing loss and kidney stones, were reportedly associated with variations in the CLDN14 gene. This study aimed to understand CLDN14 mutations’ contribution to hearing loss and renal stone formation in a Pakistani cohort. We analyzed CLDN14 sequence variations in 100 patients, along with healthy individuals, to assess whether specific polymorphisms were associated with the disease. Also, we performed an in silico analysis using a mutation database and protein annotation. The rs219779’s genotype CT (p = 0.0020) and rs219780’s genotype AG (p = 0.0012) were significantly associated with kidney stones. We also found that a novel haplotype, “TA” associated with kidney stone formation, has moderate linkage disequilibrium. The TA haplotype was significantly correlated with a kidney stone risk formation of 3.76-fold (OR (CI 95%) = 3.76 (1.83–7.72)) and p = 0.0016 compared to other haplotypes. In silico analysis revealed that mutations associated with hearing loss were not correlated with renal stone formation but affected claudin-14 protein stability. We structurally mapped a novel TA haplotype of CLDN14 that, based on our analysis, likely contributes to the pathogenesis of renal stones.

Abstract TP179: Candidate Genes For Small Vessel Ischemic Stroke: A Gwas Pilot Study

Background: The genetic basis of small vessel stroke (SVS) is poorly understood. Few genetic loci have been identified that may provide insights into SVS pathogenesis. We sought to identify genetic associations with incident SVS. Methods: A GWAS analysis was performed on a prospective cohort of patients enrolled in the American Stroke Association- Bugher Small Vessel Intracranial Disease Whole Genome Association Study which included patients from 4 hospitals in North Carolina recruited between December 2007-August 2012. Patients with SVS in the prior 2 years confirmed clinically and on neuroimaging were compared to a control group who had no previous history of stroke. A logistic regression model with SVS as the outcome and single nucleotide polymorphisms (SNPs) as the main predictor was performed and adjusted for baseline demographics and clinical characteristics, including age, race/ethnicity, sex, tobacco use, history of hypertension, diabetes, hyperlipidemia, atrial fibrillation, history of stroke/TIA, and principal components. Because of the relatively small sample size, SNPs with p-values &lt;1E-4 were considered significant. Results: The cohort included 139 patients who had SVS and 64 controls. Among this cohort, 50% were men and 38% African-American. The mean age of the study group was 63-years. Sixteen SNPs in 9 genes met the statistical cutoff for association with SVS. These were involved in extracellular matrix integrity (CHD23, CLDN14, SPTBN1), neuronal differentiation (DSCAML1), cholesterol transport (GRAMD1B), transcriptional regulation (JARID2, LINC02111, LINC01993) and voltage-gated potassium channel (KCND2). Conclusion: In this pilot GWAS study, several loci variations were associated with SVS. Although this study is hypothesis generating, previous studies have also shown an association of SVS with genes involved in extracellular matrix integrity. In addition, we found it interesting that SNPs in both CLDN14 and CDH23 genes, which have been linked to non-syndromic sensorineural deafness, are also more common in SVS. Some forms of sensorineural deafness have been associated with cognitive impairment and stroke, and if a genetic association is confirmed, it would suggest a common pathophysiology may link the two conditions.

Activation of the calcium sensing receptor increases claudin-14 expression via a PLC -p38-Sp1 pathway.

Activation of the basolateral calcium sensing receptor (CaSR) in the renal tubular thick ascending limb (TAL) increases claudin‐14 expression, which reduces paracellular calcium (Ca2+) permeability, thus increasing urinary Ca2+ excretion. However, the upstream signaling pathway contributing to altered CLDN14 gene expression is unknown. To delineate this pathway, we identified and then cloned the CaSR responsive region including the promoter of mouse Cldn14 into a luciferase reporter vector. This 1500 bp sequence upstream of the 5′ UTR of Cldn14 variant 1, conferred increased reporter activity in the presence of high extracellular Ca2+ (5 mM) relative to a lower (0.5 mM) concentration. Assessment of Cldn14 reporter activity in response to increased extracellular Ca2+ in the presence or absence of specific inhibitors confirmed signaling through PLC and p38, but not JNK. Overexpression of SP1 attenuated Cldn14 reporter activity in response to CasR signaling. SP1 is expressed in the TAL and phosphorylation was attenuated by CaSR signaling. Finally, activating mutations in the CaSR increased Cldn14 reporter activity while a dominant negative mutation in the CaSR inhibited it. Together, these studies suggest that basolateral activation of the CASR leads to increased Cldn14 expression via a PLC‐ stimulated p38 pathway that prevents Sp1 mediated repression.

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

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare tubulopathy caused by mutations in the CLDN16 or CLDN19 genes. Patients usuallydevelop 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 functionimpairment, especially in patients with CLDN19 mutations, have not been addressed. Retrospective multicenter study of 30 genetically confirmed FHHNC Spanish patients. We analyzed kidney function impairmentconsidering as outcomeschronic kidney disease (CKD) stage3 and annual estimated glomerular filtration rate (eGFR) decline, to identify factors associated with thedifferent phenotypes. 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 ± 4.30years. No differences in CKD stage3-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 stablekidney function, despite similar initial eGFR. Approximately 60% of CLDN19 patients presented ocular abnormalities. Furthermore, we confirmed high phenotypic intrafamilial variability. In a contemporary cohort of FHHNC patients with CLDN19 mutations, females seemed to progress to CKD-stage3 faster than males. Increased PTH levels at baseline may indicate amore severe renal course. There was high phenotype variability among patients with CLDN19 mutations and kidney function impairment differedeven betweensiblings.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC; OMIM 248250) is a rare autosomal recessive kidney disease caused by mutations in the CLDN16 or CLDN19 genes encoding the proteins claudin-16 and claudin-19, respectively. These are involved in paracellular magnesium and calcium transport in the thick ascending limb of Henle's loop and account for most of the magnesium reabsorption in the tubules. FHHNC is characterized by hypomagnesaemia, hypercalciuria, and nephrocalcinosis, and progresses to kidney failure, requiring dialysis and kidney transplantation mainly during the second to third decades of life. Patients carrying CLDN19 mutations frequently exhibit associated congenital ocular defects leading to variable visual impairment. Despite this severe clinical course, phenotype variability even among siblings has been described in this disease, suggesting unidentified epigenetic mechanisms or other genetic or environmental modifiers. Currently, there is no specific therapy for FHHNC. Supportive treatment with high fluid intake and dietary restrictions, as well as magnesium salts, thiazides, and citrate, are commonly used in an attempt to retard the progression of kidney failure. A kidney transplant remains the only curative option for kidney failure in these patients. In this review, we summarize the current knowledge about FHHNC and discuss the remaining open questions about this disorder.

Hypomagnesemia with Hypercalciuria Leading to Nephrocalcinosis, Amelogenesis Imperfecta, and Short Stature in a Child Carrying a Homozygous Deletion in the CLDN16 Gene

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disease caused by mutations in the CLDN16 or CLDN19 gene; however, few cases develop classical amelogenesis imperfecta. Herein, we report the case of a boy with early clinical renal manifestations that started at 1year of age and presenting with dental hypoplasia and growth delay. The patient presented with vomiting, polyuria, and polydipsia. Apart from recurrent sterile leukocyturia, erroneously treated as infectious, he was normal, except for short stature and amelogenesis imperfecta with gradually discolored teeth. Laboratory tests revealed hyperparathyroidism, hypomagnesemia, severe hypercalciuria, and hypermagnesuria on 24-h urine testing. Helical computed tomography confirmed nephrocalcinosis. We performed whole-exome sequencing (WES) to test the hypothesis of FHHNC and oligogenic inheritance of amelogenesis. Analysis of the WES binary sequence alignment/map file revealed the presence of exon 1 of the CLDN16 and absence of the other exons [c.325_c918*? (E2_E5del)]. We confirmed a CLDN16 E2_E5 homozygous deletion by multiplex ligation-dependent probe amplification and polymerase chain reaction assays. Although most mutations causing FHHNC are missense and nonsense mutations in the CLDN16 or CLDN19 gene, large deletions occur and may be misled by WES, which is generally used for genetic screening of oligogenic disorders. The patient received cholecalciferol, magnesium oxide and potassium citrate. Later, the combination with hydrochlorothiazide plus amiloride was prescribed, with a good response during follow-up. Our report broadens the phenotype of FHHNC, including severe early-onset amelogenesis and short stature, and reinforces the phenotype-genotype correlation of the large deletion found in CLDN16.

In-Depth Bioinformatic Study of the CLDN16 Gene and Protein: Prediction of Subcellular Localization to Mitochondria.

Background and Objectives: The defects in the CLDN16 gene are a cause of primary hypomagnesemia (FHHNC), which is characterized by massive renal magnesium wasting, resulting in nephrocalcinosis and renal failure. The mutations occur throughout the gene’s coding region and can impact on intracellular trafficking of the protein or its paracellular pore forming function. To gain more understanding about the mechanisms by which CLDN16 mutations can induce FHHNC, we performed an in-depth computational analysis of the CLDN16 gene and protein, focusing specifically on the prediction of the latter’s subcellular localization. Materials and Methods: The complete nucleotide or amino acid sequence of CLDN16 in FASTA format was entered and processed in 14 databases. Results: One CpG island was identified. Twenty five promoters/enhancers were predicted. The CLDN16 interactome was found to consist of 20 genes, mainly involved in kidney diseases. No signal peptide cleavage site was identified. A probability of export to mitochondria equal to 0.9740 and a cleavable mitochondrial localization signal in the N terminal of the CLDN16 protein were predicted. The secondary structure prediction was visualized. Νo phosphorylation sites were identified within the CLDN16 protein region by applying DISPHOS to the functional class of transport. The KnotProt database did not predict any knot or slipknot in the protein structure of CLDN16. Seven putative miRNA binding sites within the 3’-UTR region of CLDN16 were identified. Conclusions: This is the first study to identify mitochondria as a probable cytoplasmic compartment for CLDN16 localization, thus providing new insights into the protein’s intracellular transport. The results relative to the CLDN16 interactome underline its role in renal pathophysiology and highlight the functional dependence of CLDNs-10, 14, 16, 19. The predictions pertaining to the miRNAs, promoters/enhancers and CpG islands of the CLDN16 gene indicate a strict regulation of its expression both transcriptionally and post-transcriptionally.

Atypical presentation of familial hypomagnesemia with hypercalciuria and nephrocalcinosis in a patient with a new claudin-16 gene mutation

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an autosomal recessive tubular disorder caused by mutations in genes that encode renal tight junction proteins claudin-16 or claudin-19, which are responsible for magnesium and calcium paracellular reabsorption in the thick ascending limb of Henle’s loop. Progressive renal failure is frequently present, and most of the patients require renal replacement therapy still during adolescence. In this case report, we describe a new homozygous missense mutation on CLDN16 gene (c.592G>C, Gly198Arg) in a 24-year-old male patient diagnosed with FHHNC after clinical investigation due to incidental detection of altered routine laboratorial tests, who was firstly misdiagnosed with primary hyperparathyroidism. In addition, it illustrates an atypical presentation of this disease, with late onset of chronic kidney disease, improving the phenotype-genotype knowledge of patients with FHHNC.

The expression level of the CLDN14 gene in patients with urolithiasis - a preliminary report

Summary&#x0D; Aim: An attempt to assess the expression of the CLDN14 gene at the mRNA level in patients diagnosed with urolithiasis in relation to healthy persons constituting a comparative group.&#x0D; Material and methods: The material for examinations was peripheral blood from 23 patients, including 12 women and 11 men with diagnosed kidney stones disease and 6 donors (control group), including 2 women and 4 men who did not have kidney stones.&#x0D; Results and conclusions: The studies showed differences in the level of CLDN14 gene expression in both the examined and the comparative group.&#x0D; &#x0D; Key words: CLDN14, gene expression, urolithiasis

The expression level of the CLDN14 gene in patients with urolithiasis - a preliminary report

The expression level of the CLDN14 gene in patients with urolithiasis - a preliminary report

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.

Genetics of common complex kidney stone disease: insights from genome-wide association studies.

Kidney stone disease is a common disorder in Western countries that is associated with significant suffering, morbidity, and cost for the healthcare system. Numerous studies have demonstrated familial aggregation of nephrolithiasis and a twin study estimated the heritability to be 56%. Over the past decade, genome-wide association studies have uncovered several sequence variants that confer increased risk of common complex kidney stone disease. The firstreported variants were observed at the CLDN14 locus in the Icelandic population. This finding hassince beenreplicated in other populations. The CLDN14 gene is expressed in tight junctions of the thick ascending limb of the loop of Henle, where the protein is believed to play a role in regulation of calcium transport. More recent studies have uncovered variants at the ALPL, SLC34A1, CASR, and TRPV5 loci, the first two genes playing a role in renal handling of phosphate, while the latter two are involved in calcium homeostasis. Although genetic data have provided insights into the molecular basis of kidney stone disease, muchremains to be learned about the contribution of genetic factors to stone formation. Nevertheless, the progress made in recent years indicates that exciting times lie ahead in genetic research on kidney stone disease.

Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods

BackgroundMolecular and cellular pathophysiological events occurring in the majority of rare kidney diseases remain to be elucidated. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disorder caused by mutations in either CLDN16 or CLDN19 genes. This disease is characterized by massive urinary wasting of magnesium and calcium, osmosis deregulation and polyuria. Patients with p.G20D homozygous mutation in CLDN19 gene exhibit different progression to kidney failure suggesting that beyond the pathogenic mutation itself, other molecular events are favoring disease progression. Due to the fact that biopsy is not clinically indicated in these patients, urinary exosome-like vesicles (uEVs) can be envisioned as a valuable non-invasive source of information of events occurring in the kidney. Exosome research has increased notably to identify novel disease biomarkers but there is no consensus standardized protocols for uEVs isolation in patients with polyuria. For this reason, this work was aimed to evaluate and refine different uEVs isolation methods based on differential centrifugation, the gold standard method.ResultsCharacterization by NTA, cryo-TEM and immunoblotting techniques identified the most appropriate protocol to obtain the highest yield and purest uEVs enriched fraction possible from urine control samples and FHHNC patients. Moreover, we tested five different RNA extraction methods and evaluated the miRNA expression pattern by qRT-PCR.ConclusionsIn summary, we have standardized the conditions to proceed with the identification of differentially expressed miRNAs in uEVs of FHHNC patients, or other renal diseases characterized by polyuria.

Genetic analysis of CLDN14 in the Chinese population affected with non-syndromic hearing loss

ObjectiveThe CLDN14 gene, encoding the tight junction protein Claudin-14, has been proposed as a candidate causative gene affecting autosomal recessive non-syndromic hearing loss (ARNSHL). Genetic analysis of nonsynonymous single-nucleotide variations (nsSNVs) in CLDN14 has been performed in different populations. The role of CLDN14 nsSNVs in contributing to hearing loss in Chinese populations would be investigated in this study. MethodsTarget screening for CLDN14 variations were conducted in 500 unrelated patients diagnosed with non-syndromic hearing loss (NSHL). ResultsNo reported pathogenic CLDN14 nsSNVs in heterozygote or homozygote were detected in this study, however, we identified 4 heterozygous nsSNVs [c.11C > T, p.(Thr4Met); c.16G > A, p.(Val6Met); c.68T > C, p.(Ile23Thr); c.367A > C, p.(Thr123Pro)] in CLDN14. The 4 nsSNVs are located at claudin-14 transmembrane domains, but assessed to be poorly conservative and non-pathogenic via multiple in silico algorithms. The structure-based analysis also suggested that the 4 nsSNVs had less structural and functional impact on claudin-14. ConclusionOur findings indicated that CLDN14 might not be a major causative gene for NSHL in Chinese populations, which would contribute to fully understanding the genetic cause of NSHL in the East Asian populations.

Mutations in OTOF, CLDN14 & SLC26A4 genes as major causes of hearing impairment in Dhadkai village, Jammu & Kashmir, India.

Background & objectives:A high incidence of hearing impairment is reported from the village of Dhadkai in the State of Jammu and Kashmir, India. Prevalence of endogamy in this community suggested a common genetic basis for the disorder. A genetic study was undertaken to ascertain the basis for the high incidence of hearing impairment in this region.Methods:In a two-step approach to identify the causative mutation/s, a whole-genome-based linkage analysis of an extended family of 45 members was carried out, which included 23 affected and 22 unaffected members. Mutational analysis for the candidate deafness genes helped reveal causative mutations in the family. In addition, seven deafness-causing genes, Cx26, SLC26A4, CLDN14, TMPRSS3, TMC1, TMIE and USH1C, were analyzed in smaller families with hearing impairment.Results:In the 45-member extended family, the critical chromosomal region mapped to 2p24-p22. The c.2122C>T (p.R708X) mutation in OTOF in 2p24-p22was identified as being the causal change. Linkage to 2p24-p22 locus was not observed in a particular branch of this extended family. Analysis of seven known deafness-causing genes in this branch revealed a mutation, c.254T>A (p.V85D), in CLDN14. Among seven small families unrelated to the 45-member extended family, hearing loss was attributable to p.R708X in OTOF in three families and to p.V85D in CLDN14 in one family; a new mutation c.1668T>A (p.Y556X) SLC26A4 was identified in two families and the causative change could not be identified in one family.Interpretation & conclusions:This study suggested considerable genetic heterogeneity in the causation of hearing loss in Dhadkai. Recessive mutations were observed in at least three genes causing hearing loss: OTOF (p.R708X), SLC26A4 (p.Y556X) and CLDN14 (p.V85D). Mutation p.R708X appeared to be the major cause of hearing impairment in Dhadkai.

Diagnostic strategy for inherited hypomagnesemia

Hereditary hypomagnesemia is difficult to diagnose accurately because of its rarity and the variety of causative genes. We established a flowchart for identifying responsible genes for hypomagnesemia, and we confirmed its diagnostic efficacy in patients with suspected inherited hypomagnesemia. We established a flowchart and applied it to five index cases with suspected inherited hypomagnesemia. Direct sequence analysis was used to detect the causative gene variants in four cases, and targeted sequencing analysis using next-generation sequencing (NGS) of all causative genes for hypomagnesemia was used in one. Expected pathogenic variants were detected in the HNF1B, TRPM6, CLDN16, CASR, or SLC12A3 gene in all five cases. The results of all genetic analyses were consistent with the clinical diagnostic results using the flowchart. Accurate genetic diagnosis is crucial for estimating the prognosis, detecting complications in organs other than the kidneys, and for directing genetic counseling. The developed flowchart for identifying responsible genes for hypomagnesemia was useful for diagnosing inherited hypomagnesemia. In addition, NGS analysis will help to resolve clinical difficulties in making an accurate diagnosis and thus improve the diagnostic strategy for inherited hypomagnesemia.