DSG2 mutations contribute to arrhythmogenic right ventricular dysplasia/cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a disorder characterized by fibrofatty replacement of cardiac myocytes that typically manifests in the right ventricle. It is inherited as an autosomal dominant disease with reduced penetrance, although autosomal recessive forms of the disease also occur. We identified four probands with ARVD/C caused by mutations in DSG2, which encodes desmoglein-2, a component of the cardiac desmosome. No association between mutations in this gene and human disease has been reported elsewhere. One of these probands has compound-heterozygous mutations in DSG2, and the remaining three have isolated heterozygous missense mutations, each disrupting known functional components of desmoglein-2. We report that mutations in DSG2 contribute to the development of ARVD/C.     

Geographical distribution of plakophilin-2 mutation prevalence in patients with arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy (AC) is characterised by myocardial fibrofatty tissue infiltration and presents with palpitations, ventricular arrhythmias, syncope and sudden cardiac death. AC is associated with mutations in genes encoding the desmosomal proteins plakophilin-2 (PKP2), desmoplakin (DSP), desmoglein-2 (DSG2), desmocollin-2 (DSC2) and junctional plakoglobin (JUP). In the present study we compared 28 studies (2004-2011) on the prevalence of mutations in desmosomal protein encoding genes in relation to geographic distribution of the study population. In most populations, mutations in PKP2 showed the highest prevalence. Mutation prevalence in DSP, DSG2 and DSC2 varied among the different geographic regions. Mutations in JUP were rarely found, except in Denmark and the Greece/Cyprus region.     

Crosstalk between desmoglein-2/desmocollin-2/Src kinase and coxsackie and adenovirus receptor/ZO-1 protein complexes,regulates blood-testis barrier dynamics

Morphological studies in the testis reported the presence of ‘desmosome-like’ junctions between Sertoli cells at the blood-testis barrier, whose function is also constituted by tight junctions and basal ectoplasmic specializations. Unfortunately, little is known about the role of desmosomes in blood-testis barrier dynamics. This study aims to fill this gap with the functional investigation of two desmosomal cadherins, desmoglein-2 and desmocollin-2, by their specific knockdown in Sertoli cells cultured in vitro. Reminiscent of the blood-testis barrier in vivo, desmosome-like structures were visible by electron microscopy when Sertoli cells were cultured at high density, thereby forming a polarized epithelium with functional cell junctions. At this point, we opted to focus our efforts on desmoglein-2 and desmocollin-2 based on results which illustrated desmosomal mRNAs to be expressed by Sertoli and germ cells, as well as on results which illustrated desmoglein-2 to co-immunoprecipitate with plakoglobin, c-Src and desmocollin-2. Simultaneous knockdown of desmoglein-2 and desmocollin-2 not only led to a reduction in and mislocalization of zonula occludens-1, but also perturbed the localization of c-Src and coxsackie and adenovirus receptor at the cell–cell interface, resulting in disruption of tight junction permeability barrier. We hereby propose a novel regulatory protein complex composed of desmoglein-2, desmocollin-2, c-Src, coxsackie and adenovirus receptor and zonula occludens-1 at the blood-testis barrier.     

Germline mutations in BMPR1A/ALK3 cause a subset of cases of juvenile polyposis syndrome and of Cowden and Bannayan-Riley-Ruvalcaba syndromes

Juvenile polyposis syndrome (JPS) is an inherited hamartomatous-polyposis syndrome with a risk for colon cancer. JPS is a clinical diagnosis by exclusion, and, before susceptibility genes were identified, JPS could easily be confused with other inherited hamartoma syndromes, such as Bannayan-Riley-Ruvalcaba syndrome (BRRS) and Cowden syndrome (CS). Germline mutations of MADH4 (SMAD4) have been described in a variable number of probands with JPS. A series of familial and isolated European probands without MADH4 mutations were analyzed for germline mutations in BMPR1A, a member of the transforming growth-factor beta-receptor superfamily, upstream from the SMAD pathway. Overall, 10 (38%) probands were found to have germline BMPR1A mutations, 8 of which resulted in truncated receptors and 2 of which resulted in missense alterations (C124R and C376Y). Almost all available component tumors from mutation-positive cases showed loss of heterozygosity (LOH) in the BMPR1A region, whereas those from mutation-negative cases did not. One proband with CS/CS-like phenotype was also found to have a germline BMPR1A missense mutation (A338D). Thus, germline BMPR1A mutations cause a significant proportion of cases of JPS and might define a small subset of cases of CS/BRRS with specific colonic phenotype.     

Desmoglein-2: a novel regulator of apoptosis in the intestinal epithelium

Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated down-regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation.     

Mutations in KIF11 cause autosomal-dominant microcephaly variably associated with congenital lymphedema and chorioretinopathy

We have identified KIF11 mutations in individuals with syndromic autosomal-dominant microcephaly associated with lymphedema and/or chorioretinopathy. Initial whole-exome sequencing revealed heterozygous KIF11 mutations in three individuals with a combination of microcephaly and lymphedema from a microcephaly-lymphedema-chorioretinal-dysplasia cohort. Subsequent Sanger sequencing of KIF11 in a further 15 unrelated microcephalic probands with lymphedema and/or chorioretinopathy identified additional heterozygous mutations in 12 of them. KIF11 encodes EG5, a homotetramer kinesin motor. The variety of mutations we have found (two nonsense, two splice site, four missense, and six indels causing frameshifts) are all predicted to have an impact on protein function. EG5 has previously been shown to play a role in spindle assembly and function, and these findings highlight the critical role of proteins necessary for spindle formation in CNS development. Moreover, identification of KIF11 mutations in patients with chorioretinopathy and lymphedema suggests that EG5 is involved in the development and maintenance of retinal and lymphatic structures.     

Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis

Proteolytic modification of components of the extracellular milieu by metalloproteinases plays important roles in the regulation of multiple cellular and physiological processes and pathological conditions. ADAMTS1 is a secreted enzyme of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases, which is related to angiogenesis and inflammation processes. Here, we describe a proteomic screening for putative ADAMTS1 substrates by analyzing the protein profiles obtained from cultures of transfected cells overexpressing the protease as compared to parental cells. Conditioned medium proteins of cultures of the two cell lines have been quantitatively compared by DIGE. Proteins showing differential levels have been identified by MS techniques leading to the finding of five potential new substrates of ADAMTS1: the basement membrane proteins nidogen-1 and -2, the desmosomal protein desmocollin-3, and the extracellular glycoproteins dystroglycan 1 and Mac-2-binding protein. Nidogen-1 and -2 have been further validated as substrates by immunochemical analysis. Our results demonstrate the utility of the DIGE proteomic technique for the discovery of specific substrates of matrix proteases.     

A mutation in COL9A1 causes multiple epiphyseal dysplasia: further evidence for locus heterogeneity

Multiple epiphyseal dysplasia (MED) is an autosomal dominantly inherited chondrodysplasia. It is clinically highly heterogeneous, partially because of its complex genetic background. Mutations in four genes, COL9A2, COL9A3, COMP, and MATR3, all coding for cartilage extracellular matrix components (i.e., the alpha2 and alpha 3 chains of collagen IX, cartilage oligomeric matrix protein, and matrilin-3), have been identified in this disease so far, but no mutations have yet been reported in the third collagen IX gene, COL9A1, which codes for the alpha1(IX) chain. MED with apparently recessive inheritance has been reported in some families. A homozygous R279W mutation was recently found in the diastrophic dysplasia sulfate transporter gene, DTDST, in a patient with MED who had a club foot and double-layered patella. The series consisted of 41 probands with MED, 16 of whom were familial and on 4 of whom linkage analyses were performed. Recombination was observed between COL9A1, COL9A2, COL9A3, and COMP and the MED phenotype in two of the families, and between COL9A2, COL9A3, and COMP and the phenotype in the other two families. Screening of COL9A1 for mutations in the two probands from the families in which this gene was not involved in the recombinations failed to identify any disease-causing mutations. The remaining 37 probands were screened for mutations in all three collagen IX genes and in the COMP gene. The probands with talipes deformities or multipartite patella were also screened for the R279W mutation in DTDST. The analysis resulted in identification of three mutations in COMP and one in COL9A1, but none in the other two collagen IX genes. Two of the probands with a multipartite patella had the homozygous DTDST mutation. The results show that mutations in COL9A1 can cause MED, but they also suggest that mutations in COL9A1, COL9A2, COL9A3, COMP, and DTDST are not the major causes of MED and that there exists at least one additional locus.     

Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ～65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113-1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) -mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC(50) 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.     

The binding of the RyR2 calcium channel to its gating protein FKBP12.6 is oppositely affected by ARVD2 and VTSIP mutations

Arrhythmogenic right ventricular dysplasia/cardiomyopathy type 2 (ARVD2, OMIM 600996) and stress-induced polymorphic ventricular tachycardia (VTSIP, OMIM 604772) are two cardiac diseases causing juvenile sudden death, both associated with mutations in the RyR2 calcium channel. By using a quantitative yeast two-hybrid system, we show that VTSIP- and ARVD2-associated point mutations influence positively and negatively, respectively, the binding of RyR2 to its gating protein FKBP12.6. These findings suggest that ARVD2 mutations increase RyR2-mediated calcium release to cytoplasm, while VTSIP mutations do not affect significantly cytosolic calcium levels, thereby explaining the clinical differences between the two diseases. The present two-hybrid system appears to be an efficient molecular tool to assay the binding of FKBP12s proteins to both cardiac RyR2 and skeletal muscle RyR1 isoforms, circumventing the full-length expression of this class of giant channels. We also provide evidence of the suitability of this system to test new drugs that target RyRs-FKBP12s interactions and do not affect yeast growth.     

Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements

Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.     

Abnormal connexin43 in arrhythmogenic right ventricular cardiomyopathy caused by plakophilin-2 mutations

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disorder of cardiomyocyte intercalated disk proteins causing sudden death. Heterozygous mutations of the desmosomal protein plakophilin-2 (PKP-2) are the commonest genetic cause of ARVC. Abnormal gap junction connexin43 expression has been reported in autosomal dominant forms of ARVC (Naxos and Carvajal disease) caused by homozygous mutations of desmosomal plakoglobin and desmoplakin. In tissue culture, suppression of PKP-2 results in decreased expression of connexin43. We sought to characterize the expression and localization of connexin43 in patients with ARVC secondary to heterozygous PKP-2 mutations. Complete PKP-2 gene sequencing of 27 ARVC patients was utilized to identify mutant genotypes. Endomyocardial biopsies of identified carriers were then assessed by immunofluorescence to visualize intercalated disk proteins. N-cadherin was targeted to highlight intercalated disks, followed by counterstaining for PKP-2 or connexin43 using confocal double immunofluorescence microscopy. Immunofluorescence was quantified using an AdobeA Photoshop protocol, and colocalization coefficients were determined. PKP-2 siRNA experiments were performed in mouse cardiomyocyte (HL1) cell culture with Western blot analysis to assess connexin43 expression following PKP-2 suppression. Missense and frameshift mutations of the PKP-2 gene were found in four patients with biopsy material available for analysis. Immunofluorescent studies showed PKP-2 localization to the intercalated disk despite mutations, but associated with decreased connexin43 expression and abnormal colocalization. PKP-2 siRNA in HL1 culture confirmed decreased connexin43 expression. Reduced connexin43 expression and localization to the intercalated disk occurs in heterozygous human PKP-2 mutations, potentially explaining the delayed conduction and propensity to develop arrhythmias seen in this disease.     

Arrhythmogenic cardiomyopathy: diagnosis,genetic background,and risk management

Arrhythmogenic cardiomyopathy (AC), also known as arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), is a hereditary disease characterised by ventricular arrhythmias, right ventricular and/or left ventricular dysfunction, and fibrofatty replacement of cardiomyocytes. Patients with AC typically present between the second and the fourth decade of life with ventricular tachycardias. However, sudden cardiac death (SCD) may be the first manifestation, often at young age in the concealed stage of disease. AC is diagnosed by a set of clinically applicable criteria defined by an international Task Force. The current Task Force Criteria are the essential standard for a correct diagnosis in individuals suspected of AC. The genetic substrate for AC is predominantly identified in genes encoding desmosomal proteins. In a minority of patients a non-desmosomal mutation predisposes to the phenotype. Risk stratification in AC is imperfect at present. Genotype-phenotype correlation analysis may provide more insight into risk profiles of index patients and family members. In addition to symptomatic treatment, prevention of SCD is the most important therapeutic goal in AC. Therapeutic options in symptomatic patients include antiarrhythmic drugs, catheter ablation, and ICD implantation. Furthermore, patients with AC and also all pathogenic mutation carriers should be advised against practising competitive and endurance sports.     

Clinical and molecular findings in osteoporosis-pseudoglioma syndrome

Mutations in the low-density lipoprotein receptor-related protein 5 gene (LRP5) cause autosomal recessive osteoporosis-pseudoglioma syndrome (OPPG). We sequenced the coding exons of LRP5 in 37 probands suspected of having OPPG on the basis of the co-occurrence of severe congenital or childhood-onset visual impairment with bone fragility or osteoporosis recognized by young adulthood. We found two putative mutant alleles in 26 probands, only one mutant allele in 4 probands, and no mutant alleles in 7 probands. Looking for digenic inheritance, we sequenced the genes encoding the functionally related receptor LRP6, an LRP5 coreceptor FZD4, and an LRP5 ligand, NDP, in the four probands with one mutant allele, and, looking for locus heterogeneity, we sequenced FZD4 and NDP in the seven probands with no mutations, but we found no additional mutations. When we compared clinical features between probands with and without LRP5 mutations, we found no difference in the severity of skeletal disease, prevalence of cognitive impairment, or family history of consanguinity. However, four of the seven probands without detectable mutations had eye pathology that differed from pathology previously described for OPPG. Since many LRP5 mutations are missense changes, to differentiate between a disease-causing mutation and a benign variant, we measured the ability of wild-type and mutant LRP5 to transduce Wnt and Norrin signal ex vivo. Each of the seven OPPG mutations tested, had reduced signal transduction compared with wild-type mutations. These results indicate that early bilateral vitreoretinal eye pathology coupled with skeletal fragility is a strong predictor of LRP5 mutation and that mutations in LRP5 cause OPPG by impairing Wnt and Norrin signal transduction.     

Clinical characterisation and risk stratification of patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy ≥50 years of age

Purpose

With the increased use of genetic testing for arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), this disease is being increasingly recognised among elderly patients. However, elderly ARVD/C patients were underrepresented in prior cohorts. We aimed to describe the phenotypical characteristics and outcomes among ARVD/C patients surviving ≥50 years.

Methods

We assessed detailed phenotypical data of 29 patients who (1) presented at ≥50 years of age; and (2) fulfilled 2010 Task Force Criteria (TFC) for ARVD/C by last follow-up. Primary outcome was the occurrence of a major ventricular arrhythmia (sudden cardiac death, resuscitated sudden cardiac arrest or sustained ventricular tachycardia).

Results

The majority (55?%) of elderly ARVD/C subjects were male, with a mean age of 59.0 ± 5.8 years at presentation. Study participants fulfilled a median of six (IQR 5–8) TFC criteria by last follow-up, of which arrhythmia criteria were most frequent (97?%), followed by structural criteria (83?%), depolarisation criteria (72?%) and repolarisation criteria (69?%). By last follow-up, 15 (52?%) patients had experienced major ventricular arrhythmias. Most patients (n = 12) presented with this arrhythmia, while three experienced the event during 5.4 ± 3.2 years of follow-up. Compared with patients without an arrhythmic event, patients with major arrhythmias were more likely to be proband (p < 0.001) and male (p = 0.042). Likewise, survival free from sustained ventricular arrhythmia was lower among probands and males.

Conclusion

Phenotypic characteristics of elderly ARVD/C patients are characterised by depolarisation abnormalities and structural cardiac changes. Ventricular arrhythmias in this elderly cohort are associated with male gender and proband status.

     

TMEM43-S358L mutation enhances NF-κBTGFβ signal cascade in arrhythmogenic right ventricular dysplasia/cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a genetic cardiac muscle disease that accounts for approximately 30% sudden cardiac death in young adults. The Ser358Leu mutation of transmembrane protein 43 (TMEM43) was commonly identified in the patients of highly lethal and fully penetrant ARVD subtype, ARVD5. Here, we generated TMEM43 S358L mouse to explore the underlying mechanism. This mouse strain showed the classic pathologies of ARVD patients, including structural abnormalities and cardiac fibrofatty. TMEM43 S358L mutation led to hyper-activated nuclear factor κB (NF-κB) activation in heart tissues and primary cardiomyocyte cells. Importantly, this hyper activation of NF-κB directly drove the expression of pro-fibrotic gene, transforming growth factor beta (TGFβ1), and enhanced downstream signal, indicating that TMEM43 S358L mutation up-regulates NF-κB-TGFβ signal cascade during ARVD cardiac fibrosis. Our study partially reveals the regulatory mechanism of ARVD development.     

Regulation of intestinal epithelial intercellular adhesion and barrier function by desmosomal cadherin desmocollin-2

The role of desmosomal cadherin desmocollin-2 (Dsc2) in regulating barrier function in intestinal epithelial cells (IECs) is not well understood. Here, we report the consequences of silencing Dsc2 on IEC barrier function in vivo using mice with inducible intestinal–epithelial-specific Dsc2 knockdown (KD) (Dsc2^ERΔIEC). While the small intestinal gross architecture was maintained, loss of epithelial Dsc2 influenced desmosomal plaque structure, which was smaller in size and had increased intermembrane space between adjacent epithelial cells. Functional analysis revealed that loss of Dsc2 increased intestinal permeability in vivo, supporting a role for Dsc2 in the regulation of intestinal epithelial barrier function. These results were corroborated in model human IECs in which Dsc2 KD resulted in decreased cell–cell adhesion and impaired barrier function. It is noteworthy that Dsc2 KD cells exhibited delayed recruitment of desmoglein-2 (Dsg2) to the plasma membrane after calcium switch-induced intercellular junction reassembly, while E-cadherin accumulation was unaffected. Mechanistically, loss of Dsc2 increased desmoplakin (DP I/II) protein expression and promoted intermediate filament interaction with DP I/II and was associated with enhanced tension on desmosomes as measured by a Dsg2-tension sensor. In conclusion, we provide new insights on Dsc2 regulation of mechanical tension, adhesion, and barrier function in IECs.