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.

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.

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.

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.

Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2.

Phosphatases of regenerating liver (PRLs), the most oncogenic of all protein-tyrosine phosphatases (PTPs), play a critical role in metastatic progression of cancers. Recent findings established a new paradigm by uncovering that their association with magnesium transporters of the cyclin M (CNNM) family causes a rise in intracellular magnesium levels that promote oncogenic transformation. Recently, however, essential roles for regulation of the circadian rhythm and reproduction of the CNNM family have been highlighted. Here, we describe the crystal structure of PRL-1 in complex with the Bateman module of CNNM2 (CNNM2BAT), which consists of two cystathionine ß-synthase (CBS) domains (IPR000664) and represents an intracellular regulatory module of the transporter. The structure reveals a heterotetrameric association, consisting of a disc-like homodimer of CNNM2BAT bound to two independent PRL-1 molecules, each one located at opposite tips of the disc. The structure highlights the key role played by Asp-558 at the extended loop of the CBS2 motif of CNNM2 in maintaining the association between the two proteins and proves that the interaction between CNNM2 and PRL-1 occurs via the catalytic domain of the phosphatase. Our data shed new light on the structural basis underlying the interaction between PRL phosphatases and CNNM transporters and provides a hypothesis about the molecular mechanism by which PRL-1, upon binding to CNNM2, might increase the intracellular concentration of Mg2+ thereby contributing to tumor progression and metastasis. The availability of this structure sets the basis for the rational design of compounds modulating PRL-1 and CNNM2 activities.

Splicing defects caused by exonic mutations in PKD1 as a new mechanism of pathogenesis in autosomal dominant polycystic kidney disease.

The correct splicing of precursor-mRNA depends on the actual splice sites plus exonic and intronic regulatory elements recognized by the splicing machinery. Surprisingly, an increasing number of examples reveal that exonic mutations disrupt the binding of splicing factors to these sequences or generate new splice sites or regulatory elements, causing disease. This contradicts the general assumption that missense mutations disrupt protein function and that synonymous mutations are merely polymorphisms. Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder caused mainly by mutations in the PKD1 gene. Recently, we analyzed a substantial number of PKD1 missense or synonymous mutations to further characterize their consequences on pre-mRNA splicing. Our results showed that one missense and 2 synonymous mutations induce significant defects in pre-mRNA splicing. Thus, it appears that aberrant splicing as a result of exonic mutations is a previously unrecognized cause of ADPKD.

Severe manifestation of Bartter syndrome Type IV caused by a novel insertion mutation in the BSND gene.

Bartter syndrome Type IV is a rare subtype of the Bartter syndromes that leads to both severe renal salt wasting and sensorineural deafness. This autosomal recessive disease is caused by mutations in the gene encoding barttin, BSND, an essential subunit of the ClC-K chloride channels expressed in renal and inner ear epithelia. Patients differ in the severity of renal symptoms, which appears to depend on the modification of channel function by the mutant barttin. To date, only a few BSND mutations have been reported, most of which are missense or nonsense mutations. In this study, we report the identification of the first insertion mutation, p.W102Vfs*7, in the BSND gene of a newborn girl with acute clinical symptoms including early-onset chronic renal failure. The results support previous data indicating that mutations that are predicted to abolish barttin expression are associated with a severe phenotype and early onset renal failure.

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.

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.

RenalTube: a network tool for clinical and genetic diagnosis of primary tubulopathies.

UNLABELLED: The main purpose was to build a database while facilitating access to genotyping in order to improve the clinical and molecular knowledge of primary tubulopathies. Three tertiary referral centers of Spain collect clinical data through the site http://www.renaltube.com , while offering the analysis of 22 genes corresponding to 23 primary tubulopathies. There are three ways of collaboration: option 1 consists of adding patients to the database with clinical and biochemical information and requesting for genetic study, option 2 requires the payment of a fee for genetic analysis exclusively, and option 3 allows the enrollment of patients with a previously confirmed mutation. After 2 years of activity, RenalTube has collected data from 222 patients, the majority from Spain and Latin America (85.3 %). The most common tubulopathies are distal renal tubular acidosis (22.5 %) and classical Bartter syndrome (19.3 %) followed by familial hypomagnesemia with hypercalciuria and nephrocalcinosis (15.7 %) and Gitelman syndrome (15 %). Option 1 is the collaborating method preferred by doctors (62.3 %) followed by option 3 (36.3 %). CONCLUSION: RenalTube is a network-based registry that can be easily reached and filled out worldwide. A web-based approach with a multilateral collaboration scheme enhances the recruitment of data and promotes the understanding of underlying mechanisms of rare inherited diseases, defines more accurate diagnostic and follow-up criteria, develops new molecular techniques and will improve the overall care of the patients.

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.

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.

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).

Dent's disease: clinical features and molecular basis.

Dent's disease is an X-linked recessive renal tubulopathy characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, and progressive renal failure. LMWP is the most constant feature, while the other clinical manifestations show wide variability. Patients also present variable manifestations of proximal tubule dysfunctions, such as aminoaciduria, glucosuria, hyperphosphaturia, kaliuresis, and uricosuria, consistent with renal Fanconi syndrome. Dent's disease affects mainly male children, and female carriers are generally asymptomatic. In two-thirds of patients, the disease is caused by mutations in the CLCN5 gene, which encodes the electrogenic chloride/proton exchanger ClC-5. A few patients have mutations in OCRL1, the gene associated with the oculocerebrorenal syndrome of Lowe, which encodes a phosphatidylinositol-4,5-biphosphate-5-phosphatase (OCRL1). Both ClC-5 and OCRL1 are involved in the endocytic pathway for reabsorption of LMW proteins in the proximal tubule. This review will provide an overview of the important phenotypic characteristics of Dent's disease and summarize the molecular data that have significantly increased our comprehension of the mechanisms causing this disease.

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.

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.

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).

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.