Sequence variation in PPP 1R13L results in a novel form of cardio‐cutaneous syndrome
2017; Springer Nature; Volume: 9; Issue: 3 Linguagem: Inglês
10.15252/emmm.201606523
ISSN1757-4684
AutoresTzipora C. Falik‐Zaccai, Yiftah Barsheshet, Hanna Mandel, Meital Segev, Avraham Lorber, Shachaf Gelberg, Limor Kalfon, Shani Ben Haroush, Adel Shalata, Liat Gelernter‐Yaniv, Sarah Chaim, Dorith Raviv Shay, Morad Khayat, Michal Werbner, Inbar Levi, Yishay Shoval, Galit Tal, Stavit A. Shalev, Eli Reuveni, Emily Avitan‐Hersh, Eugene Vlodavsky, Liat Appl‐Sarid, Dorit Goldsher, Richard N. Bergman, Zvi Segal, Ora Bitterman‐Deutsch, Orly Avni,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoResearch Article9 January 2017Open Access Source DataTransparent process Sequence variation in PPP1R13L results in a novel form of cardio-cutaneous syndrome Tzipora C Falik-Zaccai Corresponding Author Tzipora C Falik-Zaccai [email protected] orcid.org/0000-0003-0065-1922 Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Yiftah Barsheshet Yiftah Barsheshet orcid.org/0000-0003-0883-8425 Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Hanna Mandel Hanna Mandel Metabolic Disease Unit, Rambam Health Care Campus, Haifa, Israel Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Search for more papers by this author Meital Segev Meital Segev Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Avraham Lorber Avraham Lorber Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Department of Pediatric Cardiology, Rambam Health Care Campus, Haifa, Israel Search for more papers by this author Shachaf Gelberg Shachaf Gelberg Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Limor Kalfon Limor Kalfon Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Shani Ben Haroush Shani Ben Haroush Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Adel Shalata Adel Shalata orcid.org/0000-0001-5209-2072 The Winter Genetic Institute, Bnei Zion Medical Center, Haifa, Israel Search for more papers by this author Liat Gelernter-Yaniv Liat Gelernter-Yaniv Pediatric Cardiology Clinic, Bnei Zion Medical Center, Haifa, Israel Search for more papers by this author Sarah Chaim Sarah Chaim Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Dorith Raviv Shay Dorith Raviv Shay Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Morad Khayat Morad Khayat The Genetic Institute, Ha'emek Medical Center, Afula, Israel Search for more papers by this author Michal Werbner Michal Werbner Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Inbar Levi Inbar Levi Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Yishay Shoval Yishay Shoval Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Galit Tal Galit Tal Metabolic Disease Unit, Rambam Health Care Campus, Haifa, Israel Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Search for more papers by this author Stavit Shalev Stavit Shalev Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel The Genetic Institute, Ha'emek Medical Center, Afula, Israel Search for more papers by this author Eli Reuveni Eli Reuveni Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Emily Avitan-Hersh Emily Avitan-Hersh Department of Dermatology, Rambam Health Care Campus, Haifa, Israel Search for more papers by this author Eugene Vlodavsky Eugene Vlodavsky Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Department of Pathology, Rambam Health Care Campus, Haifa, Israel Search for more papers by this author Liat Appl-Sarid Liat Appl-Sarid Department of Pathology, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Dorit Goldsher Dorit Goldsher Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Department of Diagnostic Imaging, Rambam Health Care Campus, Haifa, Israel Search for more papers by this author Reuven Bergman Reuven Bergman Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Department of Dermatology, Rambam Health Care Campus, Haifa, Israel Search for more papers by this author Zvi Segal Zvi Segal Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Department of Ophthalmology, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Ora Bitterman-Deutsch Ora Bitterman-Deutsch Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Dermatology Clinic, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Orly Avni Corresponding Author Orly Avni [email protected] orcid.org/0000-0002-9270-9564 Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Tzipora C Falik-Zaccai Corresponding Author Tzipora C Falik-Zaccai [email protected] orcid.org/0000-0003-0065-1922 Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Yiftah Barsheshet Yiftah Barsheshet orcid.org/0000-0003-0883-8425 Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Hanna Mandel Hanna Mandel Metabolic Disease Unit, Rambam Health Care Campus, Haifa, Israel Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Search for more papers by this author Meital Segev Meital Segev Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Avraham Lorber Avraham Lorber Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Department of Pediatric Cardiology, Rambam Health Care Campus, Haifa, Israel Search for more papers by this author Shachaf Gelberg Shachaf Gelberg Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Limor Kalfon Limor Kalfon Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Shani Ben Haroush Shani Ben Haroush Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Adel Shalata Adel Shalata orcid.org/0000-0001-5209-2072 The Winter Genetic Institute, Bnei Zion Medical Center, Haifa, Israel Search for more papers by this author Liat Gelernter-Yaniv Liat Gelernter-Yaniv Pediatric Cardiology Clinic, Bnei Zion Medical Center, Haifa, Israel Search for more papers by this author Sarah Chaim Sarah Chaim Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Dorith Raviv Shay Dorith Raviv Shay Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Morad Khayat Morad Khayat The Genetic Institute, Ha'emek Medical Center, Afula, Israel Search for more papers by this author Michal Werbner Michal Werbner Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Inbar Levi Inbar Levi Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Yishay Shoval Yishay Shoval Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Galit Tal Galit Tal Metabolic Disease Unit, Rambam Health Care Campus, Haifa, Israel Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Search for more papers by this author Stavit Shalev Stavit Shalev Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel The Genetic Institute, Ha'emek Medical Center, Afula, Israel Search for more papers by this author Eli Reuveni Eli Reuveni Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Emily Avitan-Hersh Emily Avitan-Hersh Department of Dermatology, Rambam Health Care Campus, Haifa, Israel Search for more papers by this author Eugene Vlodavsky Eugene Vlodavsky Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Department of Pathology, Rambam Health Care Campus, Haifa, Israel Search for more papers by this author Liat Appl-Sarid Liat Appl-Sarid Department of Pathology, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Dorit Goldsher Dorit Goldsher Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Department of Diagnostic Imaging, Rambam Health Care Campus, Haifa, Israel Search for more papers by this author Reuven Bergman Reuven Bergman Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel Department of Dermatology, Rambam Health Care Campus, Haifa, Israel Search for more papers by this author Zvi Segal Zvi Segal Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Department of Ophthalmology, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Ora Bitterman-Deutsch Ora Bitterman-Deutsch Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Dermatology Clinic, Galilee Medical Center, Nahariya, Israel Search for more papers by this author Orly Avni Corresponding Author Orly Avni [email protected] orcid.org/0000-0002-9270-9564 Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Search for more papers by this author Author Information Tzipora C Falik-Zaccai *,1,2, Yiftah Barsheshet2,‡, Hanna Mandel3,4,‡, Meital Segev2, Avraham Lorber4,5, Shachaf Gelberg2, Limor Kalfon1, Shani Ben Haroush1, Adel Shalata6, Liat Gelernter-Yaniv7, Sarah Chaim1, Dorith Raviv Shay1, Morad Khayat8, Michal Werbner2, Inbar Levi1, Yishay Shoval1, Galit Tal3,4, Stavit Shalev4,8, Eli Reuveni2, Emily Avitan-Hersh9, Eugene Vlodavsky4,10, Liat Appl-Sarid11, Dorit Goldsher4,12, Reuven Bergman4,9, Zvi Segal2,13, Ora Bitterman-Deutsch2,14 and Orly Avni *,2 1Institute of Human Genetics, Galilee Medical Center, Nahariya, Israel 2Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel 3Metabolic Disease Unit, Rambam Health Care Campus, Haifa, Israel 4Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel 5Department of Pediatric Cardiology, Rambam Health Care Campus, Haifa, Israel 6The Winter Genetic Institute, Bnei Zion Medical Center, Haifa, Israel 7Pediatric Cardiology Clinic, Bnei Zion Medical Center, Haifa, Israel 8The Genetic Institute, Ha'emek Medical Center, Afula, Israel 9Department of Dermatology, Rambam Health Care Campus, Haifa, Israel 10Department of Pathology, Rambam Health Care Campus, Haifa, Israel 11Department of Pathology, Galilee Medical Center, Nahariya, Israel 12Department of Diagnostic Imaging, Rambam Health Care Campus, Haifa, Israel 13Department of Ophthalmology, Galilee Medical Center, Nahariya, Israel 14Dermatology Clinic, Galilee Medical Center, Nahariya, Israel ‡These authors contributed equally to this work *Corresponding author. Tel: +972 4 9107493; E-mail: [email protected] *Corresponding author. Tel: +972 72 2644921; E-mail: [email protected] EMBO Mol Med (2017)9:319-336https://doi.org/10.15252/emmm.201606523 Correction(s) for this article Sequence variation in PPP1R13L results in a novel form of cardio-cutaneous syndrome04 September 2017 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Dilated cardiomyopathy (DCM) is a life-threatening disorder whose genetic basis is heterogeneous and mostly unknown. Five Arab Christian infants, aged 4–30 months from four families, were diagnosed with DCM associated with mild skin, teeth, and hair abnormalities. All passed away before age 3. A homozygous sequence variation creating a premature stop codon at PPP1R13L encoding the iASPP protein was identified in three infants and in the mother of the other two. Patients’ fibroblasts and PPP1R13L-knocked down human fibroblasts presented higher expression levels of pro-inflammatory cytokine genes in response to lipopolysaccharide, as well as Ppp1r13l-knocked down murine cardiomyocytes and hearts of Ppp1r13l-deficient mice. The hypersensitivity to lipopolysaccharide was NF-κB-dependent, and its inducible binding activity to promoters of pro-inflammatory cytokine genes was elevated in patients’ fibroblasts. RNA sequencing of Ppp1r13l-knocked down murine cardiomyocytes and of hearts derived from different stages of DCM development in Ppp1r13l-deficient mice revealed the crucial role of iASPP in dampening cardiac inflammatory response. Our results determined PPP1R13L as the gene underlying a novel autosomal-recessive cardio-cutaneous syndrome in humans and strongly suggest that the fatal DCM during infancy is a consequence of failure to regulate transcriptional pathways necessary for tuning cardiac threshold response to common inflammatory stressors. Synopsis A new cardio-cutaneous syndrome associated with fatal dilated cardiomyopathy is discovered in children with sequence variations in PPP1R13L, which encodes for iASPP. Patient skin-derived fibroblasts are hypersensitive to inflammatory stimuli, responding with NF-κB-dependent high expression levels of pro-inflammatory cytokines. In a murine model, the affected hearts are associated with pro-inflammatory transcriptional programs starting early after birth. Further abnormal increase happens with age and in response to inflammatory triggers. Sublethal doses of LPS are fatal in the murine model. Introduction Dilated cardiomyopathy (DCM) is characterized by impaired contraction of heart ventricles leading to overt heart failure. It results in ~10,000 deaths per year in the United States and is the primary indication for cardiac transplantation. Most patients present symptoms during adulthood, but DCM may also occur in childhood. Genetically inherited DCM is highly diverse, caused by over 40 genes (Kloos et al, 2012) and mostly inherited in an autosomal-dominant manner. Numerous syndromes, many with genetic etiologies, have been reported in association with DCM. Syndromic DCM may involve skin, CNS, and musculo-skeletal systems. Inherited cardio-cutaneous syndromes (CCSs) such as Naxos and Carvajal are two examples, caused by genes encoding desmosomal proteins. These CCSs are clinically characterized by woolly hair, palmoplantar keratoderma, and skin fragility, all of which appear in early childhood, and cardiac abnormality manifested from young adulthood (Bolling & Jonkman, 2009; Jiang et al, 2011; Rickelt & Pieperhoff, 2012). iASPP (the inhibitor of apoptosis-stimulating protein of p53) is an evolutionarily conserved member from worm to human of the ASPP (ankyrin repeat, SH3 domain, and proline-rich region containing protein) family (Yang et al, 1999; Bergamaschi et al, 2003; Herron et al, 2005; Minekawa et al, 2007; Chikh et al, 2011; Toonen et al, 2012). iASPP is encoded by PPP1R13L and expressed mainly in epithelial cells of the skin, testes, heart, and stomach, but also in other tissues. iASPP is associated with multiple proteins including the transcription factors NF-κB (Yang et al, 1999; Herron et al, 2005) and p53 (Bergamaschi et al, 2003, 2006). Functionally, iASPP inhibits transcriptional activity of NF-κB and p53, though the underlying mechanism is unclear. We report for the first time a novel human CCS, associated with a premature stop codon in PPP1R13L and ablation of iASPP expression in three Arab Christian infants and in the mother of two additional infants. Our data show how the loss of iASPP lowers the heart's threshold to inflammatory response and strongly suggests that this hypersensitivity underlies the severe DCM seen in our patients. Results Patients and families Clinical, dermatologic, and cardiologic details of five Arab Christian patients (Fig 1A) aged 4–30 months, diagnosed with autosomal-recessive (AR) CCS (Fig 1B and C) following an inter-current viral infection, are summarized in Table 1. Parents of all patients are consanguineous and reside in the same village. Three patients presented with unusually sparse and woolly hair and two had wedged teeth and dry skin (Fig 1B). Dermatologic evaluation was unavailable for patients VI12 and VI13. Patient VI4 presented with bilateral cloudy cornea and congenital corneal cyst, and no behavioral visual response. Figure 1. Clinical characterization of the CCS patients Pedigree of the extended family. Filled symbols indicate affected members. Arrow indicates the proband. Circles, females; squares, males; slant, deceased; and asterisk, no DNA available for molecular diagnosis. Phenotype of the patients as indicated. (a) Sparse woolly hair. (b) Protrusion of upper lip due to deformity of teeth. (c) Ichthyosis-like fine scale and erythema. Images of the hearts of the patients as indicated. (a) M-mode echocardiography taken from the parasternal long-axis view of the left ventricle (LV) illustrating poor LV function with severe involvement of the interventricular septum (IVS) and reduced motion of the left ventricular posterior wall (LVPW). (b) Bi-dimensional apical four-chamber view with swirling of SMOG heart chambers as a result of poor myocardial function. The SMOG is enhanced in the right atrium. (c) Bi-dimensional apical four-chamber view with free tricuspid valve regurgitation (TR) due to lack of cooptation of the tricuspid valve leaflets during systole (marked by a red bar) as a result of critical reduction of right ventricular function and right ventricular enlargement. (d) Right and left ventricular thrombi seen in the short-axis apical parasternal view. T1, large right ventricular thrombus; T2 and T3, left ventricular thrombi. (e) The color-flow Doppler picture: TR. Free regurgitation of the tricuspid valve shown in modified four chambers apical view. RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; and TR, tricuspid regurgitation color-flow Doppler. (f) M-mode from long-axis view of the left ventricle; moderately reduced LV function with fractional shortening of 26%, enlarged RV. (g) Modified four-chamber view: severe right atrium enlargement, no coaptation of the tricuspid valve leaflets during systole, leading to severe TR. (h) Modified four-chamber view: severe right atrium enlargement, severe tricuspid regurgitation. Photomicrograph demonstrating fibrosis and interstitial inflammation in the heart of patient VI10. (a, c) Post-mortem heart sections (2.7 Y). (a) Prominent fibrosis and lymphocytic interstitial inflammation (hematoxylin & eosin ×50). (c) Subepicardial inflammation and mild interstitial “myocarditis” (hematoxylin & eosin ×100). (b, d) Fetal heart (VI7) homozygous for p.Tyr747Ter. No inflammatory infiltrate or other histological abnormalities are seen. (b) Normal arrayed myocardium (hematoxylin & eosin ×50). (d) Subepicardial region with no histopathological changes (hematoxylin & eosin ×100). Sanger sequencing confirming that the patient VI10 is homozygous for the causative SV c.2241C > G, p.Tyr747Ter in PPP1R13L. Segregation of c.2241C > G, p.Y747X in PPP1R13L in the studied family. The affected girl VI10 is homozygous for the causative SV p.Y747X, as are the four fetuses that were aborted. The parents are obligate carriers as shown, and, of the two healthy sisters, one carries the wild-type allele only, and the other is heterozygous for the causative SV. Source data are available online for this figure. Source Data for Figure 1 [emmm201606523-sup-0005-SDataFig1.pptx] Download figure Download PowerPoint Table 1. Clinical characteristics of the five patients reported in this study with a novel CCS Patient M.A (VI10) Patient A.J (VII1) Patient Y.M (VI4) R (VI12) older sister R (VI13) DCM Age of presentation/diagnosis (months) 6 12 30 4 10 Consanguinity + + + + + Age at death 2.8 years 1.5 years 2.8 years 9 months 1.5 years Cardiac Severe DCM + + + + + Mitral valve regurgitation ++ ++ + Severe tricuspid valve regurgitation + + + Small pericardial effusion + ++ On EKG: multiple bi-geminis, multiple VPB'sa + + Sinus tachycardia + Disarray and vacuolization of cardiomyocytes + NA NA NA NA Bicuspid aortic valve + Dominant RV − − + Severely enlarged RV with a severe reduction in RV contraction Severely enlarged right atrium Moderately reduced LV function − − Dominant LV + Left ventricular enlargement with reduced systolic function with marked involvement of the interventricular septum followed by biventricular dysfunction and akinetic RV and tricuspid regurgitation + Left ventricular hypertrophy and dilation with marked reduction of LV systolic function followed by biventricular dysfunction with RV failure and tricuspid regurgitation − + VSD, marked reduced LV function enlarged heart shadow on chest X-ray + VSD, marked reduced LV function enlarged heart shadow on chest X-ray Skin Impaired thermoregulation − − − NA NA Sparse, dry, wooly hair + + + NA NA Ichthyosis-like fine scale and erythema + − + NA NA Pruritus − − + NA NA Fibroma − − + NA NA Palmo-plantar keratoderma − − − NA NA Dystrophic nails + − − NA NA Wedge-shaped teeth + + + NA NA Orthopedic Intoeing bilateral + + + NA NA Facial cysmorphism High forehead hairline + + + NA NA Border/low and depressed nasal bridge + + + NA NA Cleft lip and palate − − − + + Ophthalmological abnormalities − − Bilateral cloudy cornea and congenital corneal cyst No behavioral visual response − − Failure to thrive + + − + + Motor development Delayed Delayed Delayed NA NA Cognitive development Normal Normal Normal NA NA Fetal brain U/S Normal Normal Normal Brain MRI NA NA Distortion of the eyeball with sand clock appearance, thin optic nerves, cystic protrusion of the eyeball LT> RT Decreased brain volume secondary ventriculomegaly. Decreased white matter maturity for age. NA NA Brain CT Middle cerebral artery infarct VSD, ventricular septal defect; LV, left ventricle; RV, right ventricle. Data were collected regarding medical history, metabolic measurements, imaging, electrophysiological studies, and muscle biopsy. Complete physical, dermatological, and cardiological examinations were performed on the five patients. The disease phenotype in all patients is similar. a Ventricular Premature Beats. Metabolic analyses and skeletal muscle biopsy were normal. All patients passed away before reaching 3 years of age. Post-mortem histology of one heart (patient VI10) revealed myocarditis (Fig 1D). The parents of the other patients refused post-mortem analysis. Couple V3 and V4 electively aborted four fetuses between weeks 21 and 31 of gestation with midline brain, eye, and facial defects (patients VI5–8). In the extended family, two more affected girls (VI12, VI13) passed away before initiation of this study and no DNA/fibroblasts were available from them. No pathologic sequence variations (SeVa) were found in the five main genes involved in CCS (TP63 (MIM 603273), EDAR (MIM 604095), DSP (MIM 125647), PKP2 (MIM 604536), and JUP (MIM 173325)) sequenced in VI10. Homozygosity mapping, haplotype analysis, and whole exome deep sequencing (WES) Homozygosity SNP analysis revealed a large homozygous region on chromosome 19 (chr19: 44615969-55644865). WES of VI10, validated by Sanger sequencing, identified a homozygous SeVa, which creates a premature stop codon in exon 11 (c.2241C> G, p.Tyr747Ter) of PPP1R13L (Fig 1E). PPP1R13L is located on chromosome 19q13.1 (chr19: 45897911-45909607; MIM 607463) and encodes the iASPP protein. Segregation analyses (Fig 1F) confirmed homozygosity of this SeVa in three patients and carrier state in their parents. The mother (V6) of the affected sisters VI12 and VI13 (from whom DNA was not available) was found to be a carrier (Fig 1A), as well as the maternal grandmother (IV8). The father-V5 presented with sudden death and was unavailable for testing. Four fetuses (VI5–8) were also homozygous for this SeVa. Most likely, in this highly inbred family, the two different disorders, CCS and facial/brain malformations of the fetuses, were caused by mutations in distinct genes. Alternatively, the facial/brain malformations might have an incomplete penetrance with this CCS. Haplotype analysis using DNA markers closely linked to PPP1R13L D19S903, D19S902 support a common founder haplotype in all patients (data not shown). The c.2241C>G in PPP1R13L is not present in dbSNP or in the 1000 Genomes project databases. To identify the SeVa frequency among the Arab Christian community who reside in the same village and among the Arab Christian community in northern Israel, one hundred individuals from each group were studied. Eleven heterozygous carriers were identified in the first group with no healthy individuals found to be homozygous to this SeVa. One heterozygous carrier was identified in the second group, thus indicating existence of a genetic isolate for this novel CCS. PPP1R13L SeVa and expression PPP1R13L mRNA level was lower in the VI4, VI10, and VII1 patient skin-derived fibroblasts (PDFs) than in control age-matched skin-derived fibroblasts (CFs; Fig 2A). The iASPP protein was completely absent in PDFs of VI4, VI10, and VII1 (Fig 2B and C). PPP1R13L knockdown confirmed that the band appearing specifically in the CFs and not in the PDFs was iASPP (Fig 2D). Figure 2. PPP1R13L SeVa and expression A. PPP1R13L mRNA expression level is lower in VI10 PDFs than in CFs (set as 1). The qPCR results are the mean values of three independent experiments ± SD. B, C. Western blot (WB) analyses demonstrated the absence of iASPP in VI10, VI4, and VII1 PDFs. D. WB analysis demonstrated that PPP1R13L-shRNA downregulated the putative iASPP band. Source data are available online for this figure. Source Data for Figure 2 [emmm201606523-sup-0006-SDataFig2.pptx] Download figure Download PowerPoint NF-κB-dependent pro-inflammatory response Considering that iASPP interacts and represses transcriptional activity of NF-κB (Yang et al, 1999; Takada et al, 2002; Herron et al, 2005; Sullivan & Lu, 2007) and that dysregulation of NF-κB is involved in the development of heart failure (Gordon et al, 2011), we hypothesized that absence of iASPP can unleash NF-κB to increase transcription of pro-inflammatory mediators and, as a consequence, induce prolonged inflammatory processes and eventually DCM. In accordance with our hypothesis, PDFs from either one of the two patients VI10 and VII1 expressed higher mRNA levels of the inflammatory cytokines IL1B, TNFA, IL6, and of the chemokine IL8 than CFs following stimulation with LPS (Fig 3A and B). The mRNA expression level of another known NF-κB-target gene, the chemokine MCP1, increased similarly in PDFs and CFs. Two other putative NF-κB-target genes MMP9 and IKB were induced neither in the CFs nor in PDFs. These results may suggest a restricted effect of iASPP on expression of selected NF-κB-target genes. In VI4-PDFs, we determined increased expression of IL1B and TNFA but not of IL6 and IL8 (data not shown). This may reflect heterogeneity in human fibroblasts. To further dissect the effect of iASPP deficiency on the expression of pro-inflammatory mediators, we knocked PPP1R13L down in CFs (Fig 3C). The mRNA levels of the inflammatory mediators were higher in response to LPS stimulation in the PPP1R13L-silent CFs than in control CFs, confirming the role of iASPP in the regulation of the inflammatory response. Figure 3. Upregulation of pro-inflammatory mediators in PDFs A, B. Higher induction of inflammatory cytokine mRNA levels in LPS-stimulated VI10 PDFs (A) or VII1 PDFs (B) than in CFs. The expression level in resting conditions of the PDFs was set as 1. The qPCR results are the mean values of at least three independent experiments ± SD. Differences between knockdown and control with P-values ≤ 0.05 (Student's t-test) are indicated with an asterisk. C. Knockdown of PPP1R13L in CFs resulted in increased mRNA expression levels of inflammatory mediators. The qPCR results are the mean values of three independent experiments ± SD. Differences between knockdown and control with P-values (Student's t-test) are indicated with asterisk in the graph. Download figure Download PowerPoint Amount of nuclear NF-κB subunit p65 was higher and its nuclear duration longer following LPS stimulation in VI10-PDFs than in CFs (Fig 4A). ChIP assay demonstrated that p65 associated more strongly with IL1B promoter 1 h following LPS stimulation in PDFs than in CFs, where binding activity was almost below detection (Fig 4B). Similar results were obtained using primers amplifying the TNFA promoter. In contrast, binding activity of p65 was induced neither at the p53-target gene p21 promoter in both PDFs and CFs nor at the IKB promoter, whose expression was unresponsive to the presence of LPS or iASPP under these experimental conditions. To finally confirm involvement of NF-κB downstream to iASPP in the regulation of inflammatory genes, VI10-PDFs and CFs were stimulated in the presence or absence of the pharmacologic inhibitor of NF-κB activity BOT-64. As seen in Fig 4C, the in
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