High proportion of genetic cases in patients with advanced cardiomyopathy including a novel homozygous Plakophilin 2-gene mutation

Cardiomyopathies might lead to end-stage heart disease with the requirement of drastic treatments like bridging up to transplant or heart transplantation. A not precisely known proportion of these diseases are genetically determined. We genotyped 43 index-patients (30 DCM, 10 ARVC, 3 RCM) with advanced or end stage cardiomyopathy using a gene panel which covered 46 known cardiomyopathy disease genes. Fifty-three variants with possible impact on disease in 33 patients were identified. Of these 27 (51%) were classified as likely pathogenic or pathogenic in the MYH7, MYL2, MYL3, NEXN, TNNC1, TNNI3, DES, LMNA, PKP2, PLN, RBM20, TTN, and CRYAB genes. Fifty-six percent (n = 24) of index-patients carried a likely pathogenic or pathogenic mutation. Of these 75% (n = 18) were familial and 25% (n = 6) sporadic cases. However, severe cardiomyopathy seemed to be not characterized by a specific mutation profile. Remarkably, we identified a novel homozygous PKP2-missense variant in a large consanguineous family with sudden death in early childhood and several members with heart transplantation in adolescent age

In vitro functional analyses of arrhythmogenic right ventricular cardiomyopathy-associated desmoglein-2-missense variations

Gaertner A, Klauke B, Stork I, Niehaus K, Niemann G. In vitro functional analyses of arrhythmogenic right ventricular cardiomyopathy-associated desmoglein-2-missense variations. PloS one. 2012;7(10): e47097.BACKGROUND: Although numerous sequence variants in desmoglein-2 (DSG2) have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), the functional impact of new sequence variations is difficult to estimate. METHODOLOGY/PRINCIPAL FINDINGS: To test the functional consequences of DSG2-variants, we established an expression system for the extracellular domain and the full-length DSG2 using the human cell line HT1080. We established new tools to investigate ARVC-associated DSG2 variations and compared wild-type proteins and proteins with one of the five selected variations (DSG2-p.R46Q, -p.D154E, -p.D187G, -p.K294E, -p.V392I) with respect to prodomain cleavage, adhesion properties and cellular localisation. CONCLUSIONS/SIGNIFICANCE: The ARVC-associated DSG2-p.R46Q variation was predicted to be probably damaging by bioinformatics tools and to concern a conserved proprotein convertase cleavage site. In this study an impaired prodomain cleavage and an influence on the DSG2-properties could be demonstrated for the R46Q-variant leading to the classification of the variant as a potential gain-of-function mutant. In contrast, the variants DSG2-p.K294E and -p.V392I, which have an arguable impact on ARVC pathogenesis and are predicted to be benign, did not show functional differences to the wild-type protein in our study. Notably, the variants DSG2-p.D154E and -p.D187G, which were predicted to be damaging by bioinformatics tools, had no detectable effects on the DSG2 protein properties in our study

Flow cytometry-based assay for rECD-binding.

<p><b>A</b> Representative histograms of FITC-fluorescence for rECD-wt binding with 5 mM CaCl₂ (1) or with 2 mM EGTA (2). HT1080 cells were incubated with (black line) or without (negative control, grey filled area) rECD-wt. Bound rECD-wt was detected with anti-HisFITC. <b>B</b> Column plots representing the ratios of rECD-wt-binding (ratio_rECD-bound; for calculation see Supporting Information) indicated as mean±SEM of 3 independent measurements as detected by flow cytometry. The ratio_rECD-bound was significantly (p<0.01) decreased from 1.21±0.03 for samples incubated in 5 mM CaCl₂ (1) to 1.05±0.03 for samples incubated with 2 mM EGTA (2) showing that rECD-wt has a Ca²⁺-dependent binding to HT1080 cells. Statistical analysis was performed with unpaired student’s t-test (GraphPad Prism 5.01).</p

In Vitro Functional Analyses of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmoglein-2-Missense Variations - A

<p><b>Schematic view of the rECD with analysed ARVC-associated variations.</b> The dotted line shows the predicted PC cleavage site. SS = signal sequence, Pro = prodomain, EC1-EC4 = DSG2 extracellular cadherin subdomains 1-4. <b>B</b> Recombinantly expressed proteins were identified as DSG2-ECD with anti-DSG2-10G11 by Western blot analysis. The calculated apparent molecular weights were 67.5±1.5, 72.5±3.5, 70.0±3.0, 70.0±3.0, 70.5±2.5, and 69.0±4.0 (mean±SEM; n = 2) for the proteins in the traces in 1, 2, 3, 4, 5 and 6, respectively. <b>C</b> Coomassie-R-250 staining revealed the purity of the proteins. 1 = rECD-wt, 2-6 = rECDs as labelled in <b>A</b>.</p

Adhesion properties of rECDs.

<p><b>A+B</b> Flow cytometry-based assay for the binding of 0.8 µM rECD-wt or -variants to HT1080. <b>A</b> Representative histograms of FITC- fluorescence for binding of rECD-wt- and rECD-R46Q (as indicated). Bound rECD was detected with anti-HisFITC. As a negative control, HT1080 cells were incubated with only anti-HisFITC (grey filled area). <b>B</b> Column plots representing the ratio of rECD-binding related to the negative control (ratio_rECD-bound) as detected by flow cytometry. Ratios_rECD-bound are indicated as mean± SEM of 7 independent measurements for rECD-variants and 9 independent measurements for rECD-wt with rECDs from at least 3 different purifications. Statistical analysis was performed by one-way ANOVA with Dunnett’s posttest using rECD-wt as a control (GraphPad Prism 5.01). rECD-R46Q-binding to HT1080 is increased 1.8-fold as compared to rECD-wt. Other ARVC-associated variants have no influence on rECD-binding to HT1080. <b>C</b> Representative Western blot (with anti-DSG2-10G11) of rECDs crosslinked in a 5 mM CaCl₂ containing buffer with BS³ (+) or of controls (-) reveals that rECD wild-type and variants exist in solution as monomers (m), dimers (d), and oligomers (o).</p

Evaluation of CD data.

<p>Results of the deconvolution with DichroWeb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Whitmore1" target="_blank">[67]</a> using the CONTINLL-algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Provencher1" target="_blank">[68]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-vanStokkum1" target="_blank">[69]</a> and the CRYST175 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Evans1" target="_blank">[70]</a> reference data set. The results are presented as means±SEM [%] for three independent measurements. α-helical content (<b>A</b>) was 5.9±0.8 and 5.9±0.5 without Ca²⁺ (-Ca²⁺) and 3.2±1.4 and 3.2±0.2 with 5 mM CaCl₂ for rECD-wt-nat and rECD-wt-denat, respectively. β-strand content (<b>B</b>) was 38.2±0.3 and 36.6±1.3 without Ca²⁺ and 40.5±0.8 and 40.7±0.6 with 5 mM CaCl₂. Analysis of the secondary structure with two-way ANOVA showed that the α-helix content (<b>A</b>) was significantly decreased (p<0.05) while the β-strand content (<b>B</b>) was significantly increased (p<0.01) by the addition of 5 mM CaCl₂ (+Ca²⁺). However, as shown by two-way ANOVA, purification conditions had no significant effect on the rECD secondary structure.</p

Comparison of rECD fragment ion peaks generated by MALDI-ISD using QuPE [<b>71</b>].

<p><b>A</b> The location of <i>ECD</i> and <i>Pro-ECD</i> and the positions of the corresponding c-ions are illustrated on the schematic view of the rECD. <b>B+C</b> Each row shows extracts of the MALDI-ISD spectra for the particular rECDs. Peaks in one column represent the ions of the m/z ratios indicated below the negative control. The corresponding c-ions are indicated for each peak. The calculated monoisotopic masses [M+H]⁺ are shown at the bottom of each column. <b>B</b> Fragment ion peaks representing the <i>ECD</i>-c-ions; only rECD-R46Q shows no fragment ions corresponding to <i>ECD </i><b>C</b> Fragment ion peaks representing the <i>Pro-ECD-</i>c-ions; only rECD-R46Q shows fragment ions corresponding to <i>Pro-ECD</i>.</p

Investigated DSG2-variants.

<p>All data were obtained from the ARVC database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-vanderZwaag1" target="_blank">[64]</a> and the corresponding references. TFC = task force criteria, EC = extracellular cadherin domain, DCM = dilatative cardiomyopathy.</p>a<p>The prevalence in controls for the DSG2-V392I was adapted to the results in our research group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Klauke1" target="_blank">[35]</a>.</p>b<p>Grantham, R. (1974). “Amino acid difference formula to help explain protein evolution.” Science 185(4154):862–864.; Li, W. H., C. I. Wu, et al. (1984). “Nonrandomness of point mutation as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications.” J Mol Evol 21(1): 58–71.</p>c<p>Kumar, P., S. Henikoff, et al. (2009). “Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.” Nat Protoc 4(7): 1073–1081.</p>d<p>Ramensky, V., P. Bork, et al. (2002). “Human non-synonymous SNPs: server and survey.” Nucleic Acids Res 30(17): 3894–3900.</p

Representative images for detecting the localisation of wild-type and variants of full-length-DSG2-EYFP in HT1080 for three independent transfection experiments:

<p><b>DSC2b-HT1080 cells were transfected with full-length(fl)-DSG2-pEYFP; live cells were analysed with a fluorescence miscroscope one day after transfection.</b> R46Q, D154E, D187G, K294E and V392I indicate the sequence variant in fl-DSG2-EYFP, wt fl-DSG2-wt-pEYFP, and C the LFA mock transfected control. Chimeric DSG2-proteins localised preferentially to the cell borders. ARVC-associated variations had no detectable influence on the localisation of fl-DSG2-EYFP in DSC2b-HT1080. Images were acquired through YFP and phase-contrast filters. Scale (red bar) = 10 µm.</p

Compound Heterozygous FKTN Variants in a Patient with Dilated Cardiomyopathy Led to an Aberrant &alpha;-Dystroglycan Pattern

Fukutin encoded by FKTN is a ribitol 5-phosphate transferase involved in glycosylation of &alpha;-dystroglycan. It is known that mutations in FKTN affect the glycosylation of &alpha;-dystroglycan, leading to a dystroglycanopathy. Dystroglycanopathies are a group of syndromes with a broad clinical spectrum including dilated cardiomyopathy and muscular dystrophy. In this study, we reported the case of a patient with muscular dystrophy, early onset dilated cardiomyopathy, and elevated creatine kinase levels who was a carrier of the compound heterozygous variants p.Ser299Arg and p.Asn442Ser in FKTN. Our work showed that compound heterozygous mutations in FKTN lead to a loss of fully glycosylated &alpha;-dystroglycan and result in cardiomyopathy and end-stage heart failure at a young age