Defective claudin-10 causes a novel variation of HELIX syndrome through compromised tight junction strand assembly.

Formation of claudin-10 based tight junctions (TJs) is paramount to paracellular Na+ transport in multiple epithelia. Sequence variants in CLDN10 have been linked to HELIX syndrome, a salt-losing tubulopathy with altered handling of divalent cations accompanied by dysfunctional salivary, sweat, and lacrimal glands. Here, we investigate molecular basis and phenotypic consequences of a newly identified homozygous CLDN10 variant that translates into a single amino acid substitution within the fourth transmembrane helix of claudin-10. In addition to hypohidrosis (H), electrolyte (E) imbalance with impaired urine concentrating ability, and hypolacrimia (L), phenotypic findings include altered salivary electrolyte composition and amelogenesis imperfecta but neither ichthyosis (I) nor xerostomia (X). Employing cellular TJ reconstitution assays, we demonstrate perturbation of cis- and trans-interactions between mutant claudin-10 proteins. Ultrastructures of reconstituted TJ strands show disturbed continuity and reduced abundance in the mutant case. Throughout, both major isoforms, claudin-10a and claudin-10b, are differentially affected with claudin-10b showing more severe molecular alterations. However, expression of the mutant in renal epithelial cells with endogenous TJs results in wild-type-like ion selectivity and conductivity, indicating that aberrant claudin-10 is generally capable of forming functional paracellular channels. Thus, mutant proteins prove pathogenic by compromising claudin-10 TJ strand assembly. Additional ex vivo investigations indicate their insertion into TJs to occur in a tissue-specific manner.

Matching clinical and genetic diagnoses in autosomal dominant polycystic kidney disease reveals novel phenocopies and potential candidate genes.

PURPOSE: Autosomal dominant polycystic kidney disease (ADPKD) represents the most common hereditary nephropathy. Despite growing evidence for genetic heterogeneity, ADPKD diagnosis is still primarily based upon clinical imaging criteria established before discovery of additional PKD genes. This study aimed at assessing the diagnostic value of genetic verification in clinical ADPKD. METHODS: In this prospective, diagnostic trial, 100 families with clinically diagnosed ADPKD were analyzed by PKD gene panel and multiplex ligation-dependent probe amplification (MLPA); exome sequencing (ES) was performed in panel/MLPA-negative families. RESULTS: Diagnostic PKD1/2 variants were identified in 81 families (81%), 70 of which in PKD1 and 11 in PKD2. PKD1 variants of unknown significance were detected in another 9 families (9%). Renal survival was significantly worse upon PKD1 truncation versus nontruncation and PKD2 alteration. Ten percent of the cohort were PKD1/2-negative, revealing alternative genetic diagnoses such as autosomal recessive PKD, Birt-Hogg-Dubé syndrome, and ALG9-associated PKD. In addition, among unsolved cases, ES yielded potential novel PKD candidates. CONCLUSION: By illustrating vast genetic heterogeneity, this study demonstrates the value of genetic testing in a real-world PKD cohort by diagnostic verification, falsification, and disease prediction. In the era of specific treatment for fast progressive ADPKD, genetic confirmation should form the basis of personalized patient care.

Evaluating pathogenicity of SLC34A3-Ser192Leu, a frequent European missense variant in disorders of renal phosphate wasting.

Loss-of-function mutations of SLC34A3 represent an established cause of a distinct renal phosphate wasting disorder termed hereditary hypophosphatemic rickets with hypercalciuria (HHRH). SLC34A3 encodes the renal phosphate transporter NaPi2c expressed at the apical brush border of proximal renal tubules. Substitution of p.Ser192Leu is one of the most frequent genetic changes among HHRH patients in Europe, but has never been systematically evaluated, clinically or on a cellular level. Identification of a 32-year-old female with a homozgyous c.575C>T, p.Ser192Leu substitution enabled a more comprehensive assessment of the impact of this missense variant. Clinically, the patient showed renal phosphate wasting and nephrocalcinosis without any bone abnormalities. Heterozygous carriers of deleterious SLC34A3 variants were previously described to harbor an increased risk of kidney stone formation and renal calcification. We hence examined the frequency of p.Ser192Leu variants in our adult kidney stone cohort and compared the results to clinical findings of previously published cases of both mono- and biallelic p.Ser192Leu changes. On a cellular level, p.Ser192Leu-mutated transporters localize to the plasma membrane in different cellular systems, but lead to significantly reduced transport activity of inorganic phosphate upon overexpression in Xenopus oocytes. Despite the reduced function in ectopic cellular systems, the clinical consequences of p.Ser192Leu may appear relatively mild, at least in our index patient, and can potentially be missed in clinical practice.

Mutations in DZIP1L, which encodes a ciliary-transition-zone protein, cause autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1, has been associated with ciliary dysfunction. Here, we describe mutations in DZIP1L, which encodes DAZ interacting protein 1-like, in patients with ARPKD. We further validated these findings through loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and to the distal ends of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. In agreement with a defect in the diffusion barrier, we found that the ciliary-membrane translocation of the PKD proteins polycystin-1 and polycystin-2 is compromised in DZIP1L-mutant cells. Together, these data provide what is, to our knowledge, the first conclusive evidence that ARPKD is not a homogeneous disorder and further establish DZIP1L as a second gene involved in ARPKD pathogenesis.

An efficient and comprehensive strategy for genetic diagnostics of polycystic kidney disease.

Renal cysts are clinically and genetically heterogeneous conditions. Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent life-threatening genetic disease and mainly caused by mutations in PKD1. The presence of six PKD1 pseudogenes and tremendous allelic heterogeneity make molecular genetic testing challenging requiring laborious locus-specific amplification. Increasing evidence suggests a major role for PKD1 in early and severe cases of ADPKD and some patients with a recessive form. Furthermore it is becoming obvious that clinical manifestations can be mimicked by mutations in a number of other genes with the necessity for broader genetic testing. We established and validated a sequence capture based NGS testing approach for all genes known for cystic and polycystic kidney disease including PKD1. Thereby, we demonstrate that the applied standard mapping algorithm specifically aligns reads to the PKD1 locus and overcomes the complication of unspecific capture of pseudogenes. Employing careful and experienced assessment of NGS data, the method is shown to be very specific and equally sensitive as established methods. An additional advantage over conventional Sanger sequencing is the detection of copy number variations (CNVs). Sophisticated bioinformatic read simulation increased the high analytical depth of the validation study and further demonstrated the strength of the approach. We further raise some awareness of limitations and pitfalls of common NGS workflows when applied in complex regions like PKD1 demonstrating that quality of NGS needs more than high coverage of the target region. By this, we propose a time- and cost-efficient diagnostic strategy for comprehensive molecular genetic testing of polycystic kidney disease which is highly automatable and will be of particular value when therapeutic options for PKD emerge and genetic testing is needed for larger numbers of patients.

c-Src mediated tyrosine phosphorylation of plakophilin 3 as a new mechanism to control desmosome composition in cells exposed to oxidative stress.

Plakophilins (PKP1 to PKP3) are essential for the structure and function of desmosomal junctions as demonstrated by the severe skin defects observed as a result of loss-of-function mutations in mice and men. PKPs play additional roles in cell signaling processes, such as those controlling the cellular stress response and cell proliferation. A key post-translational process controlling PKP function is phosphorylation. We have discovered that reactive oxygen species (ROS) trigger the c-Src kinase-mediated tyrosine (Tyr)-195 phosphorylation of PKP3. This modification is associated with a change in the subcellular distribution of the protein. Specifically, PKP3 bearing phospho-Tyr-195 is released from the desmosomes, suggesting that phospho-Tyr-195 is relevant for the control of desmosome disassembly and function, at least in cells exposed to ROS. Tyr-195 phosphorylation is transient under normal physiological conditions and seems to be strictly regulated, as the activation of particular growth factor receptors results in a modification at this site only when tyrosine phosphatases are inactivated by pervanadate. We have identified Tyr-195 of PKP3 as a phosphorylation target of epidermal growth factor receptor signaling. Interestingly, this PKP3 phosphorylation also occurs in certain poorly differentiated adenocarcinomas of the prostate, suggesting a possible role in tumor progression. Our study thus identifies a new mechanism controlling PKP3 and hence desmosome function in epithelial cells.

The desmosomal plaque proteins of the plakophilin family.

Three related proteins of the plakophilin family (PKP1_3) have been identified as junctional proteins that are essential for the formation and stabilization of desmosomal cell contacts. Failure of PKP expression can have fatal effects on desmosomal adhesion, leading to abnormal tissue and organ development. Thus, loss of functional PKP 1 in humans leads to ectodermal dysplasia/skin fragility (EDSF) syndrome, a genodermatosis with severe blistering of the epidermis as well as abnormal keratinocytes differentiation. Mutations in the human PKP 2 gene have been linked to severe heart abnormalities that lead to arrhythmogenic right ventricular cardiomyopathy (ARVC). In the past few years it has been shown that junctional adhesion is not the only function of PKPs. These proteins have been implicated in cell signaling, organization of the cytoskeleton, and control of protein biosynthesis under specific cellular circumstances. Clearly, PKPs are more than just cell adhesion proteins. In this paper we will give an overview of our current knowledge on the very distinct roles of plakophilins in the cell.