Molecular autopsy and subsequent functional analysis reveal de novo DSG2 mutation as cause of sudden death

Sudden cardiac death (SCD) is a common cause of death in young adults. In up to 80% of cases a genetic cause is suspected. Next-generation sequencing of candidate genes can reveal the cause of SCD, provide prognostic management, and facilitate pre-symptomatic testing and prevention in relatives. Here we present a proband who experienced SCD in his sleep for which molecular autopsy was performed.We performed a post-mortem genetic analysis of a 49-year-old male who died during sleep after competitive kayaking, using a Cardiomyopathy and Primary Arrhythmia next-generation sequencing panel, each containing 51 candidate genes. Autopsy was not performed.Genetic testing of the proband resulted in missense variants in KCNQ1 (c.1449C > A; p.(Asn483Lys)) and DSG2 (c.2979G > T; p.(Gln993His)), both absent from the gnomAD database. Familial segregation analysis showed de novo occurrence of the DSG2 variant and presence of the KCNQ1 variant in the proband's mother and daughter. KCNQ1 p.(Asn483Lys) was predicted to be pathogenic by MutationTaster. However, none of the KCNQ1 variant carrying family members showed long QTc on ECG or Holter. We further functionally analysed this variant using patch-clamp in a heterologous expression system (Chinese Hamster Ovary (CHO) cells) expressing the KCNQ1 mutant in combination with KCNE1 wild type protein and showed no significant changes in electrophysiological function of Kv7.1.Based on the above evidence, we concluded that the DSG2 p.(Gln993His) variant is the most likely cause of SCD in the presented case, and that there is insufficient evidence that the identified KCNQ1 p.(Asn483Lys) variant would confer risk for SCD in his mother and daughter. Fortunately, the DSG2 variant was not inherited by the proband's two children. This case report indicates the added value of molecular autopsy and the importance of subsequent functional study of variants to inform patients and family members about the risk of variants they might carry.     

A compound heterozygosity of Tecrl gene confirmed in a catecholaminergic polymorphic ventricular tachycardia family

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is one of the most common causes of sudden cardiac death (SCD) during childhood and in adolescence. Trans-2, 3-enoyl-CoA reductase-like (Tecrl) gene mutations (Arg196Gln and c.331+1G > A splice site mutation) were first reported in CPVT. Tecrl homozygous c.331+1G > A splice site mutation in iPSCs revealed a definite correlation between Tecrl and Ca²⁺ transport in cardiomyocytes. However, no other researchers have confirmed Tecrl mutations in CPVT with literature review. In this study, a case of compound heterozygosity in the Tecrl gene (Arg196Gln and c.918+3T > G splice site mutation) was first identified in a 13-year-old boy with CPVT by whole-exome sequencing (WES) and confirmed by Sanger sequence. Support vector machine and neural network analysis predicted that Arg196Gln mutation could decrease the stability of Tecrl structure, the confidence scores were ?0.8929 and ?0.9930. A STRUM server also confirmed that Arg196Gln mutation may decrease the binding capacity of the substrate and cause an amino acid substitution immediately upstream of the 3-oxo-5-alpha steroid 4-dehydrogenase domain. According to the “human splicing finder” indication and Alamut Visual Splicing Prediction, the c.918 + 3T > G mutation could influence Tecrl variable splicing. Thus, we confirmed that Tecrl as a new gene which is associated with CPVT.     

Physiological consequences of the P2328S mutation in the ryanodine receptor (RyR2) gene in genetically modified murine hearts

Aim: To explore the physiological consequences of the ryanodine receptor (RyR2)‐P2328S mutation associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). Methods: We generated heterozygotic (RyR2^p/s) and homozygotic (RyR2^s/s) transgenic mice and studied Ca²⁺ signals from regularly stimulated, Fluo‐3‐loaded, cardiac myocytes. Results were compared with monophasic action potentials (MAPs) in Langendorff‐perfused hearts under both regular and programmed electrical stimulation (PES). Results: Evoked Ca²⁺ transients from wild‐type (WT), heterozygote (RyR2^p/s) and homozygote (RyR2^s/s) myocytes had indistinguishable peak amplitudes with RyR2^s/s showing subsidiary events. Adding 100 nm isoproterenol produced both ectopic peaks and subsidiary events in WT but not RyR2^p/s and ectopic peaks and reduced amplitudes of evoked peaks in RyR2^s/s. Regularly stimulated WT, RyR2^p/s and RyR2^s/s hearts showed indistinguishable MAP durations and refractory periods. RyR2^p/s hearts showed non‐sustained ventricular tachycardias (nsVTs) only with PES. Both nsVTs and sustained VTs (sVTs) occurred with regular stimuli and PES with isoproterenol treatment. RyR2^s/s hearts showed higher incidences of nsVTs before but mainly sVTs after introduction of isoproterenol with both regular stimuli and PES, particularly at higher pacing frequencies. Additionally, intrinsically beating RyR2^s/s showed extrasystolic events often followed by spontaneous sVT. Conclusion: The RyR2‐P2328S mutation results in marked alterations in cellular Ca²⁺ homeostasis and arrhythmogenic properties resembling CPVT with greater effects in the homozygote than the heterozygote demonstrating an important gene dosage effect.     

Identification of a PKP2 gene deletion in a family with arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disease characterized by progressive myocardial loss, with fibro-fatty replacement, and high frequency of ventricular arrhythmias that can lead to sudden cardiac death. ARVC is a genetically determined disorder, usually caused by point mutations in components of the cardiac desmosome. Conventional mutation screening of ARVC genes fails to detect causative mutations in about 50% of index cases, suggesting a further genetic heterogeneity. We performed a genome-wide linkage study and a copy number variations (CNVs) analysis, using high−density SNP arrays, in an ARVC family showing no mutations in any of the desmosomal genes. The CNVs analysis identified a heterozygous deletion of about 122 kb on chromosome 12p11.21, including the entire plakophilin-2 gene and shared by all affected family members. It was not listed on any of available public CNVs databases and was confirmed by quantitative real-time PCR. This is the first SNP array-based genome-wide study leading to the identification of a CNV segregating with the disease phenotype in an ARVC family. This result underscores the importance of performing additional analysis for possible genomic deletions/duplications in ARVC patients without point mutations in known disease genes.     

Long QT syndrome with a high mortality rate caused by a novel G572R missense mutation in KCNH2

In a four-generation family with long QT syndrome, syncopes and torsades de pointes ventricular tachycardia (TdP) were elicited by abrupt awakening in the early morning hours. The syndrome was associated with a novel KCNH2 missense mutation, G572R, causing the substitution of a glycine residue at position 572, at the end of the S5 transmembrane segment of the HERG K(+)-channel, with an arginine residue. This segment is involved in the channel pore and the mutation may cause a reduction in the rapidly activating delayed rectifier K+ current (Ikr), or changed gating properties of the ion channel, leading to prolonged cardiac repolarization. The electrocardiograms of affected persons showed prolonged QT interval and notched T waves. Despite treatment with atenolol, 200 mg twice daily, the proband still experienced TdP episodes. Three untreated relatives of the proband died suddenly, and unexpectedly, at 18, 32, and 57 years of age. The G572R mutation is thus associated with a high mortality rate, and the clinical presentation illustrates that some mutations may not be controllable by just beta-blockade.     

良性家族性新生儿惊厥 KCNQ2 基因新突变

目的 对一个中国良性家族性新生儿惊厥(benign familial neonatal convulsions，BFNC)家系进行基因诊断，并探讨其分子发病机理。方法 对该家系进行详细的临床检查及疾病基因的连锁分析。应用聚合酶链反应(polymerase chain reaction，PCR)-DNA直接测序，并用PCR-单链构象多态(single strand conformation polymorphism，SSCP)对先证者、家系内16人及家系外72名无血缘关系的正常入进行KCNQ2基因突变分析。结果 连锁分析提示该家系与KCNQ2基因连锁，并排除与KCNQ3基因连锁。PCR—DNA直接测序在先证者发现．KCNQ2基因突变193ldelG，PCR—SSCP发现家系内其他患者均出现与先证者相同的异常SSCP条带，而72名正常人未出现此异常条带。结论 KCNQ2基因突变是中国人BFNC的发病原因之一，193ldelG是国内外未曾报道过的新突变，连锁分析结合基因突变分析可对BFNC患者进行基因诊断。     

A novel point mutation (Pro84 ← Ser) of the low density lipoprotein receptor gene in a family with moderate hypercholesterolemia

To obtain insight into the possibility that genetic variation of the structure of the low density lipoprotein (LDL) receptor protein could result in subtle changes of serum cholesterol levels, we used single-strand conformation polymorphism (SSCP) to screen all 18 exons of the LDL receptor gene in a panel of subjects with moderate hypercholesterolemia. One novel mutation, replacing C to T at nucleotide 313 and predicted to cause a substitution of serine for proline at codon 84, was identified in a single proband. A convenient PCR assay based on the use of primer-introduced restriction fragment length polymorphism was set up for the detection of this mutation. However, the pathophysiologic significance of the Pro84 → Ser replacement remains to be clarified, as serum LDL cholesterol levels were not significantly higher in mutation carriers vs. non-carriers in the affected family, and no other proband was identified, on screening of DNA samples from 350 Finns. The Pro84 → Ser mutation represents the second single-amino acid change of the LDL receptor protein so far reported which is not associated with the clinical phenotype of familial hypercholesterolemia.     

Two related Dutch families with a clinically variable presentation of cardioskeletal myopathy caused by a novel S13F mutation in the desmin gene

Desmin-related myopathy is characterised by skeletal muscle weakness often combined with cardiac involvement. Mutations in the desmin gene have been described as a cause of desmin-related myopathy (OMIM 601419). We report here on two distantly related Dutch families with autosomal dominant inheritance of desmin-related myopathy affecting 15 family members. A highly heterogeneous clinical picture is apparent, varying from isolated dilated cardiomyopathy to a more generalised skeletal myopathy and mild respiratory problems. Morphological analysis of muscle biopsies revealed intracytoplasmic desmin aggregates (desmin and p62 staining). In both families we identified an identical novel pathogenic heterozygous missense mutation, S13F, in the 'head' domain of the desmin gene which cosegregates with the disease phenotype. This is the 5th reported missense mutation located at the 'head' domain of the desmin gene and the first reported Dutch family with desmin-related myopathy. This article illustrates the importance of analysing the desmin gene in patients with (familial) cardiac conduction disease, dilated cardiomyopathy and/or a progressive skeletal myopathy resembling limb-girdle muscular dystrophy.     

X-连锁迟发性脊椎骨骺发育不良家系SEDL基因突变研究

目的：确定中国汉族中一个X－连锁迟发性脊椎骨骺发育不良（spondyloepiphyseal dyskplasia tarda,SEDL)大家系SEDL基因突变类型，探讨SEDL发病的分子基础。方法：用聚合酶链反应扩增产物双向直接测序方法检测了患者构成SEDL基因可读框的第3－6外显子及相邻侧翼区的DNA序列，将测序结果与GenBank公布的SEDL基因正常序列对比找出突变。然后在家系其他成员中证实该突变。结果：在2例患者（Ⅳ15、Ⅴ3）SEDL基因第2内含子剪接受体处发现了IVS2－2A→C突变，4个外显子的核苷酸序列未见改变。该突变在4例女性携带者得到证实，她们的基因型表现为野生型与突变型杂合现象。家系中2名未受累男性和15名无关健康个体未检测到这一突变。在该家系还检测出4个无症状的携带者。结论：首次发现SEDL基因IVS2－2A→C突变。该突变引起SEDL基因第2内含子3'端剪接受体改变，使之不能与外显子3正常剪接，可能是SEDL发病的分子基础。检测该突变可进行基因诊断，有重要的临床价值。     

Screening the human bradykinin B2 receptor gene in patients with cardiovascular diseases: Identification of a functional mutation in the promoter and a new coding variant (T21M)

To elucidate if genetic variants in the bradykinin B₂ receptor (B₂) gene occur that could affect receptor expression and function, we screened for mutations in the promoter and in the coding region of the human B₂ gene. In our initial study we analyzed 92 consecutive, unrelated subjects (including 25 patients with hypertrophic cardiomyopathy, 18 patients with dilated cardiomyopathy (DCM), 25 patients with hypertension, 18 patients with coronary heart disease, and 6 patients with valvular heart disease) using nonradioactive polymerase chain reaction–single-strand conformation polymorphism analysis as mutation screening method. We detected eight as yet unknown polymorphic sites in the promoter region of the B₂ gene (−845 C/T, −704C/T, −649 insG, −640 T/C, −536 C/T, −412 C/G, −143 C/T and −78 C/T) with allele frequencies between 0.5 and 13%. One of them (−412 C/G) destroys a Sp1 binding site and abolishes protein binding to this Sp1 site in human umbilical vein endothelial cells and human vascular smooth muscle cells. In the protein-coding region one new coding variant (T21M) with the potential to create a truncated receptor isoform was detected. We determined the frequency of the promoter variant at position −412 (C → G) and the newly identified coding variant (T21M) in extended samples of 69 patients with HCM, 163 patients with DCM, 109 patients with hypertension, and 173 healthy anonymous blood donors. The promoter variant (−412C/G) was found in one blood donor and the T21M mutation was not found in the control population. Therefore, it appears that these mutations are rare events and the determination of clinical significance will be a demanding task in the future. Am. J. Med. Genet. 80:521–525, 1998. © 1998 Wiley-Liss, Inc.