Maria Franaszczyk

Does p.Q247X in TRIM63 Cause Human Hypertrophic Cardiomyopathy

Rationale: Variants in TRIM63, including a nonsense mutation (p.Q247X), have been suggested recently to cause hypertrophic cardiomyopathy. Objective: To verify pathogenicity of TRIM63 p.Q247X detected by whole-exome sequencing in a symptomless professional sports player seeking medical advice because of a prolonged QT interval found during a routine check-up. Methods and Results: Clinical studies were performed in the proband and his mother, who also carried TRIM63 p.Q247X. No evidence of hypertrophic cardiomyopathy was found in either person. Conclusions: The p.Q247X variant in TRIM63 is not likely to be a highly penetrant variant causing hypertrophic cardiomyopathy.

A study in Polish patients with cardiomyopathy emphasizes pathogenicity of phospholamban (PLN) mutations at amino acid position 9 and low penetrance of heterozygous null PLN mutations.

BackgroundIn humans mutations in the PLN gene, encoding phospholamban - a regulator of sarcoplasmic reticulum calcium ATPase (SERCA), cause cardiomyopathy with prevalence depending on the population. Our purpose was to identify PLN mutations in Polish cardiomyopathy patients.MethodsWe studied 161 unrelated subjects referred for genetic testing for cardiomyopathies: 135 with dilated cardiomyopathy, 22 with hypertrophic cardiomyopathy and 4 with other cardiomyopathies. In 23 subjects multiple genes were sequenced by next generation sequencing and in all subjects PLN exons were analyzed by Sanger sequencing. Control group included 200 healthy subjects matched with patients for ethnicity, sex and age. Large deletions/insertions were screened by real time polymerase chain reaction.ResultsWe detected three different heterozygous mutations in the PLN gene: a novel null c.9_10insA:(p.Val4Serfs*15) variant and two missense variants: c.25C > T:(p.Arg9Cys) and c.26G > T:(p.Arg9Leu). The (p.Val4Serfs*15) variant occurred in the patient with Wolff-Parkinson-White syndrome in whom the diagnosis of cardiomyopathy was not confirmed and his mother who had concentric left ventricular remodeling but normal left ventricular mass and function. We did not detect large deletions/insertions in PLN in cohort studied.ConclusionsIn Poland, similar to most populations, PLN mutations rarely cause cardiomyopathy. The 9thPLN residue is apparently a mutation hot spot whereas a single dose of c.9_10insA, and likely other null PLN mutations, cause the disease only with low penetrance or are not pathogenic.

Evidence for troponin C (TNNC1) as a gene for autosomal recessive restrictive cardiomyopathy with fatal outcome in infancy.

Restrictive cardiomyopathy is a rare form of pediatric cardiac disease, for which the known genes include MYH7, TNNT2, TNNI3, ACTC1, and DES. We describe a pediatric proband with fatal restrictive cardiomyopathy associated with septal hypertrophy and compound heterozygosity for TNNC1 mutations (NM_003280: p.A8V [c.C23T] and p.D145E [c.C435A]). This association between restrictive cardiomyopathy and TNNC1 mutations was strengthened by prospective observations on the second pregnancy in the family which revealed, in the presence of the same TNNC1 genotype, prenatally diagnosed hypertrophic cardiomyopathy which evolved into restrictive cardiomyopathy, heart failure and death at the age of 9 months. Contrary to previous reports, family and population analyses showed that each of the TNNC1 variants was not pathogenic when present alone. Our results (i) confirm that genetic backgrounds of hypertrophic cardiomyopathy and restrictive cardiomyopathy overlap and (ii) indicate that TNNC1 is a likely novel gene for autosomal recessive restrictive cardiomyopathy.

Titin Truncating Variants in Dilated Cardiomyopathy – Prevalence and Genotype-Phenotype Correlations

TTN gene truncating variants are common in dilated cardiomyopathy (DCM), although data on their clinical significance is still limited. We sought to examine the frequency of truncating variants in TTN in patients with DCM, including familial DCM (FDCM), and to look for genotype-phenotype correlations. Clinical cardiovascular data, family histories and blood samples were collected from 72 DCM probands, mean age of 34 years, 45.8% FDCM. DNA samples were examined by next generation sequencing (NGS) with a focus on the TTN gene. Truncating mutations were followed up by segregation study among family members. We identified 16 TTN truncating variants (TTN trunc) in 17 probands (23.6% of all cases, 30.3% of FDCM, 17.9% of sporadic DCM). During mean 63 months from diagnosis, there was no difference in adverse cardiac events between probands with and without TTN truncating mutations. Among relatives 29 mutation carriers were identified, nine were definitely affected (31%), eight probably affected (27.6%) one possibly affected (3.4%) and eleven were not affected (37.9%). When relatives with all affected statuses were combined, disease penetrance was still incomplete (62.1%) even after exclusion of unaffected relatives under 40 (82%) and was higher in males versus females. In all mutation carriers, during follow-up, 17.4% had major adverse cardiac events, and prognosis was significantly worse in men than in women. In conclusion, TTN truncating variants were observed in nearly one fourth of young DCM patient population, in vast majority without conduction system disease. Incomplete penetrance suggests possible influence of other genetic and/or environmental factors on the course of cardiotitinopathy. Counseling should take into account sex and incomplete penetrance.

The rs12526453 Polymorphism in an Intron of the PHACTR1 Gene and Its Association with 5-Year Mortality of Patients with Myocardial Infarction

Objective The rs12526453 (C/G) is a single nucleotide polymorphism in an intron of the PHACTR1 gene (phosphatase and actin regulator 1). The C allele is associated with increased risk of coronary artery disease in an unknown mechanism. We investigated its association with long-term overall mortality in patients with ST-elevation myocardial infarction (STEMI) treated invasively. Methods Two independent groups of patients with STEMI were analyzed: a derivation group (n= 638) and a validation one (n=348). Genotyping was performed with the TaqMan method. The analyzed end-point was total long term mortality. Additionally, transcriptomic analysis was performed in mononuclear blood leukocytes from rs12526453 CC monozygotes or G allele carriers. Results In the study group (mean age 62.3 ± 11.9 years; 24.9% of females, n=159), percentages of CC, CG, and GG genotypes were 45.3% (n=289), 44.7% (n=285), and 10% (n=64), respectively. In the 5-year follow-up 105 patients died (16.46%). CC homozygotes had significantly lower mortality compared to other genotypes: 13.1% (n=38) vs. 18.3% in G-allele carriers (n=67), (p=0.017, Cox`s F test). In the validation group 47 patients died within 3 years (13.5%). We confirmed lower mortality of CC homozygotes: 10.1 % (n=18) vs. 16.95% in G-allele carriers (n=29), (p=0.031, Cox`s F test). Transcriptomic analysis revealed a markedly higher expression of NLRP-2 in CC homozygotes. Conclusions The rs12526453 CC homozygotes (previously associated with increased risk of myocardial infarction) showed, in 2 independent samples, better long-term survival. The finding of such high effect size, after appropriate validation, could potentially be translated into clinical practice.

The 4q25, 1q21, and 16q22 polymorphisms and recurrence of atrial fibrillation after pulmonary vein isolation.

Introduction The efficacy of pulmonary vein isolation (PVI) in atrial fibrillation (AF) is well documented. Several single nucleotide polymorphisms (SNPs) are associated with AF, mainly in the 4q25 locus, but also in 16q22 and 1q21. The aim of our study was to test the association between those SNPs and short- and long-term results of PVI. Material and methods Patients with AF who underwent PVI between 2006 and 2009 were included in the study. Pulmonary vein isolation was performed using a 4-mm non-irrigated ablation catheter, circular mapping catheter, and the LocaLisa system. All patients were genotyped for the 4q25, 16q22, and 1q21 SNPs. Results Two-hundred and thirty-eight patients were included. The median follow-up was 45 months. Six-month efficacy was 59.7%. None of the polymorphisms was linked with the risk of AF recurrence after 6 months in univariate analysis. In multivariate analysis rs2200733 in the recessive model was linked significantly with AF recurrence (odds ratio 1.87, p = 0.008). None of the polymorphisms predicted AF recurrence in long-term follow-up. Conclusions There is a trend in the relationship between TT genotype of the rs2200733 polymorphism and increased rate of AF recurrence after PVI in short-term (6 months) follow-up. None of the tested SNPs 4q25, 16q22, and 1q21 correlated with the results of a single AF ablation in long-term follow-up.

HLA DQ2 HAPLOTYPE, EARLY ONSET OF GRAVES DISEASE, AND POSITIVE FAMILY HISTORY OF AUTOIMMUNE DISORDERS ARE RISK FACTORS FOR DEVELOPING CELIAC DISEASE IN PATIENTS WITH GRAVES DISEASE

OBJECTIVE The diagnosis of celiac disease (CD) in patients with different autoimmune diseases including Graves disease (GD) remains a challenge. The aims of our study were to: (1) assess the prevalence of CD in Polish patients with GD and (2) evaluate the prevalence of CD in the subgroups of patients with GD divided on the basis of clinical and human leukocyte antigen (HLA) typing criteria. METHODS The prospective study was conducted at an academic referral center. The study groups consisted of consecutive, euthyroid patients with GD (n = 232) and healthy volunteers without autoimmune thyroid diseases (n = 122). The diagnosis of CD was based on elevated immunoglobulin A autoantibodies to the enzyme tissue transglutaminase (IgA-TTG) and small intestine biopsy findings. RESULTS CD was diagnosed in 8 patients with GD (3.4%) and 1 healthy volunteer (0.8%). The development of CD in patients with GD was strongly associated with HLA-DQ2 haplotype (as predicted from linkage disequilibria, 14.6% vs. 1.5%, P = .009; odds ratio [OR] = 11.3; 95% confidence interval [CI] 1.3-252.7): 6 patients with CD carried HLA-DRB1(*)03, 1 carried an HLA-DRB1(*)04 allele, and 1 had an HLA-DRB1(*)07/(*)11 genotype. Multivariate analysis showed independent associations between CD and early GD onset (P = .014, OR = 9.6), autoimmunity in family (P = .029, OR = 6.3) and gastroenterologic symptoms (P = .031, OR = 8.1). CONCLUSIONS The results of our study suggest that serologic screening for CD may be considered in GD patients (1) with the HLA alleles typical for CD, (2) with an early onset of GD, or (3) a family history of autoimmunity. Moreover, the diagnosis of CD should be explored in euthyroid GD patients with nonspecific gastrointestinal symptoms.

Restrictive cardiomyopathy due to novel desmin gene mutation

Desminopathies are a heterogeneous group of diseases characterised by the presence of desmin-positive aggregates in muscle tissue and most commonly caused by desmin gene (DES) mutations. There are nearly 70 mutations known, and most of them are localised in coil 2B [Cepetanaki Y et al., Curr Opin Cell Biol. 2015; 32: 113–120]. Desminopathies manifest as cardiomyopathy and/or myopathy. In a patient with restrictive cardiomyopathy we found novel DES mutation (735+1G>T) predicted to cause altered splicing. This mutation has not been previously reported. The proband was a 62-year-old woman with progressive skeletal muscle weakness, who was wheel-chair bound at the time of diagnosis. First cardiological symptoms (syncopal episodes) led to VVI pacemaker implantation at the age of 46 years. With time she had progressive heart failure symptoms, New York Heart Association III, permanent atrial fibrillation, and episodes of sustained ventricular tachycardia, which prompted an upgrade of VVI to ICD-VR — at the time she was pacing-dependent. Neurological assessment showed severe myopathy of the inferior extremities. In 12-lead electrocardiogram: atrial fibrillation, ventricular pacing, rate 77 bpm. Two-dimensional echocardiographic study showed small well contracting left ventricle (left ventricular end-diastolic diameter: 35 mm, left ventricular ejection fraction: 55%) with markedly enlarged atria, restrictive pattern of mitral inflow, and severe tricuspid insufficiency (Figs. 1, 2). Laboratory assessment revealed increased level of N-terminal pro B-type natriuretic peptide (NT-proBNP; 1564; normal range: 0–125 pg/mL). Creatine kinase activity (maximal value: 423; normal range: 26–192 U/L) and troponin I levels (maximal value: 0.035; normal range: < 0.01 ng/mL) were increased. She died at the age of 65 years at home. Her son was diagnosed to have restrictive cardiomyopathy and died after heart transplantation at the age of 23 years. Her grandson was healthy at the age of 17 years. Only the proband was genetically tested. Genetic testing was performed using direct Sanger sequencing of the entire coding region of DES including splice sites (ABI 3130 Genetic Analyser). We identified novel splice variant IVS3+1G>T (735+1G>T) (Fig. 3). In the same nucleotide other splice variants have been described previously. The IVS3+1G>A variant disrupting exon 3 has been reported in a patient with unclassified cardiomyopathy, which evolved from hypertrophic to restrictive cardiomyopathy and later to dilated cardiomyopathy [Gudkova A et al., Pediatr Cardiol. 2013; 34(2): 467–470]. Another DES variant affecting the same splice site, IVS3+3A>G, has been previously reported in a patient with skeletal muscle weakness and complete atrioventricular block [Park KY et al., J Med Genet. 2000; 37(11): 851–857]. This particular case highlights the importance of genetic testing in rare familial cardiac diseases.

Novel truncating desmoplakin mutation as a potential cause of sudden cardiac death in a family.

704 available family members (mother and sister) and revealed no abnormalities. A genetic study was performed to gain insight into SCD in the family and identify family members at potential risk. DNA was extracted from peripheral blood by phenol extraction. Next-generation sequencing (NGS) in the proband was performed using the TruSight One (TSO, Illumina, San Diego, California, United States) sequencing panel. Selected genetic variants identified by NGS were followed up in the proband and relatives, using Sanger sequencing. We identified the frameshift deletion p.Thr2625fs (c.7871_7872delAC), annotated to transcript NM_004415.2 of the desmoplakin (DSP) gene, in the proband, but not in his healthy mother or sister (FIGURE 1F and 1G). Desmoplakin is a critical component of desmosome structures in the cardiac muscle, and the role of DSP mutations, including protein-truncating mutations, in the pathogenesis of cardiomyopathies is well established.1,2 The identified variant has not been described in the literature before or found in genomic databases (Phase 3 of 1000 Genomes, NHLBI GO Exome Sequencing Project [ESP] 6500 and Version 0.3 of ExAC). Although the recommendation of an implantable cardioverter defibrillator (ICD) as primary prevention remains controversial,3 it was necessary in this case. ICD was implemented owing to signs of biventricular involvement on CMR, the family history of SCD, and the result of genetic study in the patient with complex ventricular arrhythmia. A 2-year follow-up revealed that the patient remained asymptomatic, and cardiac function was stable with readings from the ICD memory Sudden cardiac death (SCD) of young, apparently healthy individuals raises questions about whether other family members are also at risk of SCD. A 30-year-old patient treated with bisoprolol (5 mg once daily) for ventricular arrhythmia for 6 months was admitted to our hospital for evaluation. A family history of SCD was identified: the patient’s brother died suddenly at the age of 39 years while working at the computer. A physical examination of our patient was unremarkable. A standard 12-lead electrocardiogram showed sinus bradycardia (47 bpm) and low voltage, fragmented QRS in limb leads (FIGURE 1A). A transthoracic 2-dimensional echocardiogram revealed a nondilated left ventricle with borderline ejection fraction; however, the right ventricle was dilated with mild global contractile dysfunction. Subsequent cardiac magnetic resonance (CMR) revealed a significantly enlarged right ventricle and a slightly enlarged left ventricle (right ventricular [RV] end-diastolic volume, 151 ml/m2, n <111, and left ventricular [LV] end-diastolic volume, 111 ml/m2, n <101) with slightly reduced LV and RV ejection fractions (49% and 46%, respectively) and global hypokinesis. Additionally, diffuse changes on late gadolinium enhancement (LGE) were found (FIGURE 1B–D). On 24-hour Holter monitoring, sinus bradycardia (without pauses), single premature ventricular contractions, ventricular couplets, and episodes of nonsustained ventricular tachycardia (2 polymorphic triplets) were observed. A family history revealed that the proband’s father died of heart failure at the age of 75 years, and a paternal cousin died suddenly (FIGURE 1E). Noninvasive clinical cardiac screening was performed in Correspondence to: Prof. Zofia T. Bilińska, MD, PhD, Ośrodek Badań Przesiewowych Dziedzicznych Chorób Układu Sercowo-Naczyniowego, Instytut Kardiologii, ul. Alpejska 42, 04-628 Warszawa, Poland, phone: +48 22 343 47 11, e-mail: zbilinska@ikard.pl (for clinical issues); Prof. Rafał Płoski, MD, PhD, Zakład Genetyki Medycznej, Warszawski Uniwersytet Medyczny, ul. Pawińskiego 3c, 02-106 Warszawa, Poland, phone: +48 22 572 06 06, e-mail: rploski@wp.pl (for genetic issues) Received: July 1, 2016. Revision accepted: August 29, 2016. Published online: September 27, 2016. Conflict of interests: none declared. Pol Arch Med Wewn. 2016; 126 (9): 704-707 doi:10.20452/pamw.3567 Copyright by Medycyna Praktyczna, Kraków 2016 CLINICAL IMAGE

Value of multilocus genetic risk score for atrial fibrillation in end-stage kidney disease patients in a Polish population

Genetic factors play a key role in the pathogenesis of atrial fibrillation (AF). We would like to establish an association between previously described single-nucleotide polymorphisms (SNPs) and AF in haemodialysed patients with end-stage kidney disease (ESKD-HD) as well as to assess the cumulative effect of all genotyped SNPs on AF risk. Sixteen SNPs were genotyped in 113 patients with AF-ESKD-HD and in 157 controls: without AF (NAF) and with ESKD-HD. The distribution of the risk alleles was compared in both groups and between different sub-phenotypes. The multilocus genetic risk score (GRS) was calculated to estimate the cumulative risk conferred by all SNPs. Several loci showed a trend toward an association with permanent AF (perm-AF): CAV1, Cx40 and PITX2. However, GRS was significantly higher in the AF and perm-AF groups, as compared to NAF. Three of the tested variables were independently associated with AF: male sex, history of myocardial infarction (MI) and GRS. The GRS, which combined 13 previously described SNPs, showed a significant and independent association with AF in a Polish population of patients with ESKD-HD and concomitant AF. Further studies on larger groups of patients are needed to confirm the associations.