Claudin-10b N48K alters electrophysiological properties in MDCK-C7 cell layers.

<p>(A) Dilution potentials (and thus the permeability ratio for Na⁺ and Cl^-; P_Na/P_Cl) of cell layers expressing claudin-10b WT (blue) and claudin-10b N48K (red) are significantly higher than those of control cell layers (black; empty vector). Cell layers transfected with claudin-10b WT maintain these properties over several weeks whereas cell layers transfected with claudin-10b N48K lose these properties over a time-course of three weeks. n = 1–28, means +/- SEM. (B) Expression of claudin-10b WT (blue symbols) cause reduced TER (x-axis) and increased dilution potentials (y-axis) compared to vector transfected controls (black symbols). The effects when expressing claudin-10b N48K (red symbols) were less pronounced. n = 8–40, means +/- SEM. (C) Permeability for monovalent cations (P_X) relative to P_Na. Expression of claudin-10b WT altered Eisenman-sequence for monovalent cations from Eisenman-sequence IV in control layers to Eisenman-sequence VIII to X, depending on transfection strength. Expression of claudin-10b N48K resulted in Eisenman-sequences between III and X. n = 2–13, means +/- SEM.</p

The <i>CLDN10b</i> specific gene variant segregates anhidrosis and mild kidney failure.

<p>(A) Pedigree of the consanguineous Pakistani family segregating anhidrosis and heat intolerance, alacrima, xerostomia, mild kidney failure and Mg²⁺ retention (black filled symbols). All affected individuals are homozygous for chromosome 13q32 marker alleles flanking the <i>CLDN10</i> gene with the missense variant c.144C>G (black bars). (B) Body temperature measurements at rest over 25 min when exposed to 45°C and 45% humidity in affected (n = 2; black line) and age-matched controls (n = 3; grey line). The patients interrupted the study after 20 min due to distress. (C) The N48K variant is located within the 1^st extracellular loop (ECL1) and results in the introduction of the positively charged Lysine in the claudin consensus sequence W- G/NLW-C-C (filled amino acids). The first 73 amino acids (black circles) are specific for the claudin-10b isoform encoded by exon 1b. Grey circles correspond to amino acids shared with the claudin-10a isoform. +/- indicate charged residues in the extracellular loops. ECL–extracellular loop. TM–Transmembrane segment. (D-E) Immunostaining of the secretory portion of sweat glands from a healthy control (D) and from the affected ind. 15 (E). In the control, claudin-10 (green) is predominantly found in the canaliculi, the cell peripheries and in membranes lining the lumen. Strong occludin staining (red) is found lining the main lumen of the sweat gland. In the patient, claudin-10 signal (green) is observed in membranes facing the lumen but shows a more even intracellular distribution without accumulation in canaliculi but in spots resembling vesicles. A strong occludin signal (red) is observed both in the canaliculi and in membranes lining the main lumen. Scale bars: 20μm.</p

Reduced size of cysts formed by MDCK-C7 cells transfected by claudin-10b N48K.

<p>(A) Histogram of the size distribution of cyst lumen diameter (grey, untransfected control, n = 151 cysts [10 different z-stacks]; red, claudin-10b WT transfected cells: clone #3, n = 140 [9], clone #39, n = 187 [11]; blue claudin-10b N48K transfected cells: clone #5, n = 167 [9]; clone # 21, n = 113 [7]). (B) Normal distribution of the data shown in (A) to visualize the shift in lumen size in the presence of claudin-10b. (C) Representative images of cysts (red, actin-staining by phalloidin-Alexa 594; blue, nuclei stained with DAPI), (D) TJ localization of N48K and WT claudin-10b in 2D culture. (E) Presence of WT and N48K claudin-10b (green) in 3D culture (co-staining as in D).</p

Homology modeling predicts structural alterations of claudin-10b p.N48K.

<p>(A) Homology model of the tertiary structure of claudin-10b based on crystal structure of murine claudin-15 (PBD ID: 47P9) as template. Backbone is shown as cartoon in green and residues of the claudin consensus motif (W-G/NLW-C-C), T27, D28 and K51 are shown as sticks in violet. (B) Region of claudin-10b model around N48 (green stick) in detail highlighting likely electrostatic interactions (dotted lines) between N48 and backbone of surrounding residues (T27, L49, W50) as well as potential electrostatic interaction between residues K51 and D28. (C) In the claudin-15 structure (cyan), the corresponding residue N47 participates in similar interactions (dotted lines) whereas residues Y50 (corresponding to K51) and S27 (corresponding to D28) do not interact. (D) The p.N48K substitution (blue) prevents the interactions mediated by N48 and may also disturb a D28-K51 interaction. O atoms, red; N atoms, dark blue; S yellow.</p

The claudin-10b N48K increases FRET efficiency in cis.

<p>(A, B) FRET efficiencies for YFP-claudin-10b WT/CFP-claudin-10b WT interaction (blue), YFP-claudin-10b N48K/CFP-claudin-10b N48K (red) and YFP-claudin-10b N48K/CFP-claudin-10b WT (black) constructs overexpressed in HEK293 cells (A) and MDCK-C7 cells (B) plotted against the YFP/CFP ratio. Only values above the critical YFP/CFP ratio as defined by Milatz et al. (2015) are shown. (C) In HEK293 (** p<0.01) and in MDCK-C7 (# p<0.05) cells average FRET efficiency is significantly higher for YFP-claudin-10b N48K/CFP-claudin-10b N48K interaction than for YFP-claudin-10b WT/CFP-claudin-10b WT interaction. Interaction between claudin-10b N48K/claudin-10b WT is significantly lower than for both claudin-10b N48K/claudin-10b N48K and claudin-10b WT/claudin-10b WT (** p<0.01, only tested in HEK cells). Statistical analysis: MDCK-C7, t-test, n = 19 (WT) and 21(N48K); HEK293, t-test + Bonferroni-Holm, n = 83 (WT), 52 (N48K) and 15 (WT-N48K). Error bars are presented with ± standard error (SEM).</p

Perturbed formation of tight junctions and inhibition of claudin-10b trans-interactions by the N48K mutation.

<p>(A-D) Freeze fracture electron microscopy of HEK293 cells expressing YFP-claudin-10b. (A) Stable expression of wt claudin-10b led to formation of complex mesh-works of branched tight junction strands. Numerous continuous strands (arrow) were detected on the protoplasmic face (P-face, PF) and as particle-free grooves (arrowhead) on the exoplasmic face (E-face, EF) of the plasma membrane. (B) Stable expression of claudin-10b N48K resulted in few tight junction strands and less complex meshworks. Strands were detected on the P-face as rows of rather separated intramembranous particles (black arrow) and two- dimensional particle arrays (white arrow). (C) Similar to the stable expression, transient expression of wt claudin-10b led to mesh-works of continuous tight junction strands detected on the P-face (arrow) and E-face (arrowhead). (D) For transient expression of claudin-10b N48K, tight junction mesh-works were infrequent. Particle-type strands were detected as noncontinuous rows of separated, beaded intramembranous particles (black arrow) on the P-face. Scale bars, 200 nm. (E-H) Inhibition of YFP-claudin-10b trans-interactions by the p.N48K mutation. (E) Transiently expressed claudin-10b WT (green) is enriched at contacts (arrows) between claudin-expressing HEK293 cells suggesting trans-interaction. Arrowheads indicate plasma membrane outside of contacts between two claudin-expressing cells. (F) Transient expression of claudin-10b N48K (green) in HEK293 cells does not show strong enrichment at contacts (arrows) between cells suggesting lack of trans-interaction. (G) For transient expression in HEK293 cells, the enrichment factor (Enr. F) was significantly lower for claudin-10b N48K compared to claudin-10b WT. n = 27–44; *, p < 0.01. (H) For stable expression in HEK293 cells, the product of enrichment factor and length of enrichment (Enr. F x Length) was significantly lower for claudin-10b N48K compared to claudin-10b WT. n = 27–42; *, p < 0.01. Means +/- SEM. Size bars: 5μm.</p

Results from urine (spot) and serum analysis in six affected family members.

<p>Results from urine (spot) and serum analysis in six affected family members.</p

Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

<div><p>Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of <i>EYS</i> revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in <i>RP1</i>, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.</p></div

Causative mutations and putatively pathogenic variants identified in this study.

<p>Causative alleles are being listed as “allele 1” and “allele 2” in resolved cases. Additional alleles are shown if the minor allele frequency is below 3% and if <i>in silico</i> prediction suggests putative pathogenicity. The inheritance pattern was largely delineated from pedigree informations. In patients 22, 23, 77, 100, 116 and 119, the true mode of inheritance had not been evident from the pedigree information and was finally deduced from the genotype. a, this study. References for studies cited in this table can be found in the Supplementary Material (References S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078496#pone.0078496.s001" target="_blank">File S1</a>). n.d., not defined; f, female; m, male; ar, autosomal recessive; ad, autosomal dominant; s, sporadic. Xl, X-linked. Cau, Caucasian; Ger, Germany; Tur, Turkey; KSA, Kingdom of Saudi Arabia; Pol, Poland; Au, Austria; Syr, Syria; Pak, Pakistan; DRC, Democratic Republic of the Congo; Mor, Morocco; UAE, United Arab Emirates; E-Eur, East Europe; SE-Eur, Southeast Europe.</p

Hemizygosity of a <i>CRX</i> mutation in a recessive consanguineous LCA family.

<p><b>A.</b> Compound-heterozygosity for a potentially protein-extending no-stop mutation (c.899A>G/p.(*300Trpext*118); here designated as Ext) abrogating the natural termination codon in exon 4 and a deletion of the same exon (delE4) <i>in trans</i> in patient 110 and her brother. <b>B.</b> Graphical view of the LOD score calculation from genomewide SNP mapping for this family previous to NGS testing: Genomewide homozygosity mapping prior to NGS did not identify a clear candidate locus. The combined maximum parametric LOD score of 2.4 was not obtained. <b>C.</b> Scheme of the <i>CRX</i> gene and coverage plots for CNV analysis from NGS data (Illumina MiSeq), indicating a heterozygous deletion of exon 4 (upper panel, absolute coverage based on read count; lower panel, SeqNext CNV analysis). See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078496#pone-0078496-g002" target="_blank">Figure 2C</a>. <b>D.</b> Schematic representation of the mapped sequencing reads for the no-stop mutation (Integrative Genomics Viewer). The mutation (arrow) was present in all 65 reads covering this region of the gene and therefore appeared homozygous. <b>E.</b> Electropherograms from Sanger sequencing of the no-stop mutation with hemizygosity in patient 110 (upper panel) and heterozygosity in her mother (lower panel). <b>F.</b> Summary of the disease-causing genetic constellation in patient 110 and her brother (superimposition on parental alleles).</p