Functional Effects of the TMEM43 Ser358leu
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Rajkumar et al. BMC Medical Genetics 2012, 13:21 http://www.biomedcentral.com/1471-2350/13/21 RESEARCH ARTICLE Open Access Functional effects of the TMEM43 Ser358Leu mutation in the pathogenesis of arrhythmogenic right ventricular cardiomyopathy Revathi Rajkumar1, John C Sembrat1, Barbara McDonough2, Christine E Seidman2 and Ferhaan Ahmad1,3,4* Abstract Background: The Ser358Leu mutation in TMEM43, encoding an inner nuclear membrane protein, has been implicated in arrhythmogenic right ventricular cardiomyopathy (ARVC). The pathogenetic mechanisms of this mutation are poorly understood. Methods: To determine the frequency of TMEM43 mutations as a cause of ARVC, we screened 11 ARVC families for mutations in TMEM43 and five desmosomal genes previously implicated in the disease. Functional studies were performed in COS-7 cells transfected with wildtype, mutant, and 1:2 wildtype:mutant TMEM43 to determine the effect of the Ser358Leu mutation on the stability and cellular localization of TMEM43 and other nuclear envelope and desmosomal proteins, assessed by solubility assays and immunofluorescence imaging. mRNA expression was assessed of genes potentially affected by dysfunction of the nuclear lamina. Results: Three novel mutations in previously documented desmosomal genes, but no mutations in TMEM43, were identified. COS-7 cells transfected with mutant TMEM43 exhibited no change in desmosomal stability. Stability and nuclear membrane localization of mutant TMEM43 and of lamin B and emerin were normal. Mutant TMEM43 did not alter the expression of genes located on chromosome 13, previously implicated in nuclear envelope protein mutations leading to skeletal muscular dystrophies. Conclusions: Mutant TMEM43 exhibits normal cellular localization and does not disrupt integrity and localization of other nuclear envelope and desmosomal proteins. The pathogenetic role of TMEM43 mutations in ARVC remains uncertain. Background of cardiac desmosomes, namely, desmocollin-2 (DSC2), Arrhythmogenic right ventricular dysplasia/cardiomyo- desmoglein-2 (DSG2), desmoplakin (DSP), junctional pathy (ARVC) is an inherited disorder characterized by plakoglobin (JUP), and plakophilin-2 (PKP2)[3]. Muta- replacement of cardiomyocytes by adipose and fibrous tions can lead to instability of other desmosomal pro- tissue, primarily in the right ventricle (RV). This disrup- teins, resulting in their translocation from the cell tion can result in RV dysfunction, arrhythmias and sud- membrane to the cytoplasm [4,5]. den cardiac death. In the United States, 17% of sudden We first mapped by genetic linkage analysis a large death victims between the ages of 20 and 40 years had family with ARVC to a 9.3 cM region on chromosome ARVC. A large number of cases are unrecognized 3p23, known as locus ARVC5 [6]. Recently, Merner and because clinical tests are relatively insensitive to in vivo colleagues identified a c.1073 C > T mutation, leading detection of functional and structural changes in the RV to a p.Ser358Leu substitution, in a highly conserved [1,2]. In approximately 40% of patients, mutations have region of transmembrane protein 43 (TMEM43)atthis been identified in genes encoding constituent proteins locus in the same family [7]. TMEM43, also known as LUMA, is a highly conserved inner nuclear membrane * Correspondence: [email protected] (INM) protein. It has been suggested that TMEM43 can 1 UPMC Heart and Vascular Institute, Department of Medicine, University of cause pathological changes in the INM by affecting Pittsburgh, Pittsburgh, PA 15213, USA Full list of author information is available at the end of the article other protein complexes in it [8]. Furthermore, © 2012 Rajkumar et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rajkumar et al. BMC Medical Genetics 2012, 13:21 Page 2 of 9 http://www.biomedcentral.com/1471-2350/13/21 immunostaining of cardiac tissue from subjects with Primer3 utility http://frodo.wi.mit.edu/. Each identified TMEM43 mutations indicated reduced expression of mutation was screened in 150 unrelated and unaffected TMEM43 and plakoglobin, with TMEM43 showing control individuals to confirm its absence in normal localization at the sarcolemma [9]. However, little is population. Point mutations were detected by PCR knownaboutfunctionofTMEM43andthemechanism amplification of exons, followed by direct sequencing by which mutations cause ARVC. (ABI). Identified mutations were analyzed for pathogeni- The overall goal of this study was to investigate the city by using (1) protein prediction algorithms PolyPhen mechanisms by which mutations in TMEM43 may lead http://genetics.bwh.harvard.edu/pph/, SIFT http://sift. to ARVC. Given the novelty of the TMEM43 mutation jcvi.org/, MUpro http://www.ics.uci.edu/~baldig/muta- in the context of previously reported mutations that tion.html and PMut http://mmb2.pcb.ub.es:8080/PMut/, were primarily in genes encoding desmosomal proteins, (2) evolutionary conservation among species using Clus- we first sought to determine the prevalence of mutations talW analysis, and (3) localization within a functionally in TMEM43 relative to five desmosomal genes, namely, important domain. DSC2, DSG2, DSP, JUP, PKP2, in 11 ARVC probands. We found three novel mutations in different desmoso- Construction of wildtype and mutant TMEM43 expression mal genes, however, TMEM43 mutations were absent in vectors these probands. Next, since common mechanisms often Wildtype TMEM43 underlie each cardiomyopathy even in the setting of RNA was extracted from normal human heart tissue as genetic heterogeneity, we conducted in vitro studies to described elsewhere [11]. cDNA for TMEM43 was determine whether the functional abnormalities caused synthesized using SuperScript reverse transcriptase by the rare TMEM43 S358L parallel those found in the (Invitrogen). The sequence was verified by direct setting of desmosomal mutations. COS-7 cells were sequencing. The resulting fragment was cloned into the transfected with either wildtype or mutant TMEM43,or pcDNA3.1/NT-GFP-topo eukaryotic expression vector co-transfected with both. Our studies showed that (Invitrogen), which tags the expressed protein with GFP. mutant TMEM43 protein did not disrupt interactions Cloning and transformation into chemically competent among desmosomal and nuclear envelope proteins, and E. coli cells was performed according to the manufac- did not lead to altered cellular localization of itself or of turer’s instructions. Plasmid DNA was isolated using the desmosomal and nuclear envelope proteins. Further- QIAprep Spin Miniprep Kit (Qiagen) and quantified on more, mutant TMEM43 did not alter the expression of a UV spectrophotometer. The sequence was verified by genes as observed in the cardiomyopathy caused by direct sequencing. mutations in the nuclear membrane-associated protein, Ser358Leu mutant TMEM43 lamin A/C. Therefore, the role of the TMEM43 Ser358- The QuickChange XL Site-Directed Mutagenesis Kit Leu mutation in ARVC remains uncertain. (Stratagene) was used to introduce the Ser358Leu muta- tion into the wildtype TMEM43 expression vector. Methods Mutagenic primers were designed according to the man- Subjects ufacturer’s instructions. Plasmid DNA was isolated as A total of 11 families with ARVC were recruited by the described above and presence of the Ser358Leu muta- University of Pittsburgh Cardiovascular Genetics Center tion was verified by direct sequencing. and the Harvard Medical School Department of Genetics, in accordance with protocols approved by their respective Cell culture and transfection Institutional Review Boards. Informed consent was COS-7 cells were cultured in DMEM containing 10% obtained from all subjects. Clinical diagnosis of ARVC was fetal bovine serum (FBS), streptomycin, and penicillin G based on the recently modified criteria originally proposed at concentrations of 100 μg/ml each, and transfected by the International Task Force of the European Society of using Lipofectamine 2000 (Invitrogen). Briefly, cells were Cardiology and the International Society and Federation of grown overnight in supplemented DMEM medium and Cardiology [10]. Demographic and clinical data on pro- transfected in serum free medium with wildtype plasmid, bands and affected family members in whom a mutation mutant plasmid, or wildtype and mutant plasmid (at was identified are shown in Table 1. ratios of 1:1 and 1:2) for 6-8 hours. The medium in the plates was then replaced by supplemented DMEM. Cells Genetic mutation screening were harvested five days following transfection. TMEM43, DSC2, DSG2, DSP, JUP and PKP2 were screened for mutations in all probands, followed by con- Assessment of protein stability by solubility assays firmation of any potential mutation in other available Previous studies have suggested that disruption of cell- family members. Primers were designed using the cell adhesion because of desmosomal dysfunction is a Rajkumar et al. BMC Medical Genetics 2012, 13:21 Page 3 of 9 http://www.biomedcentral.com/1471-2350/13/21 Table 1 Clinical characteristics of ARVC subjects with mutations in desmosomal genes ARVC diagnostic Criteria Subject criteria fulfilled Family I.1 Family II.1 Family II.2 Family III.1 Family III.2 Family IV.1 Sex M M M F M M Age at diagnosis 41 20 17 35 18 16 (years) Global and