A novel mutation of dipeptidyl aminopeptidase-like protein-6 in a family with suspicious idiopathic ventricular fibrillation

Abstract

Background

Sudden cardiac death (SCD) occurs in a broad spectrum of cardiac pathologies and is an important cause of mortality in the general population. Idiopathic ventricular fibrillation (IVF) is a rare but important factor resulting in SCD. It is diagnosed in a resuscitated cardiac arrest victim underlying unknown cause, with documented ventricular fibrillation. Previous studies have demonstrated that mutations in dipeptidyl aminopeptidase-like protein-6 (DPP6) and cardiac sodium channel Nav1.5 (SCN5A) are the most important genetic factors involve in IVF.

Aim

By using whole sequencing to identify the genetic lesion of a family with suspicious idiopathic ventricular fibrillation

Design

Prospective genetic study.

Methods

In this study, we employed whole-exome sequencing in combination with arrhythmia-related gene filtering to identify the genetic lesion for a family suffering from suspicious IVF, syncope and SCD. We then generated the plasmids of DPP6-pcDNA3.1+ (WT and c.1578G>C/p.Q526H). K_v4.3-pcDNA3.1+ was co-transfected together with/without DPP6-pcDNA3.1+ (WT and/or c.1578G>C/p.Q526H) into HEK293 cells to perform the patch clamp experiments.

Results

A novel missense mutation (c.1578G>C/p.Q526H) of DPP6 was identified and co-segregated with affected patients in this family. Patch clamp experiments suggested that this novel mutation might result in a gain of function and disturb the efflux of potassium ion.

Conclusion

Our study not only reported the second missense mutation of DPP6 in heart disease and expanded the spectrum of DPP6 mutations, but also contribute to the genetic diagnosis and counseling of families with suspicious IVF, syncope and SCD.

Introduction

Sudden cardiac death (SCD) is a serious heart disease and a leading cause of mortality worldwide.¹ At present, at least four most common categories of cardiac disorders, namely premature atherosclerosis, primary electrical disease, cardiomyopathies and thoracic aortic aneurysm or dissection, have been identified and all of them can lead to SCD.^2,3 Among these categories, the primary electrical disease accounts for the vast majority (up to 93%) of inherited SCD in patients with negative autopsy.^3,4 Mechanistically, the primary electrical disease is caused by molecular defects mostly in ion channels involved in cardiac action potential generation,^3,5 including Long QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation (IVF) and other cardiac conduction diseases.^6,7

IVF is defined as the spontaneous occurrence of ventricular fibrillation in the absence of known causes that may lead to SCD.⁸ The baseline electrocardiogram (ECG) of IVF is usually unnoticeable and ventricular fibrillation tends to be triggered by short-coupled ventricular extrasystoles originating from a single focus.⁹ According to previous studies, mutations in dipeptidyl aminopeptidase-like protein-6 (DPP6) and cardiac sodium channel Nav1.5 (SCN5A) are the most important genetic factors involve in IVF.^6,8 However, due to extensive genetic heterogeneity and complexity of a diverse genetic architecture, the mutation of causative genes in IVF patients are rarely reported.

In this study, we employed whole-exome sequencing in combination with arrhythmia-related gene-filtering to explore the possible causative gene for a family with SCD, syncope and suspicious IVF. A novel missense mutation (c.1578 G > C/p.Q526H) of DPP6 was identified and co-segregated with affected patients. Patch clamp experiments further confirmed that this novel mutation might result in a gain of function and disturb the efflux of potassium ion.

Materials and methods

Subjects

This study was approved by the review board of the Second Xiangya Hospital of the Central South University. The proband and her relatives who participated in the study gave written informed consent. After that, we investigated the medical histories of all 12 family members (Figure 1A). All the family members were examined by 12-lead ECG and B-mode ultrasound.

Figure 1.

The clinic and genetic data of the family. (A) Pedigree of the family affected with suspicious IVF, syncope and SCD. Squares indicate male members; circles, female members; closed symbols, the affected members; open symbols, unaffected members; arrow, proband. (B) The ECG records of the proband (II-5). (C) The ECG records of the proband’s brother (II-2). (D) Sanger sequencing results of the DPP6 mutation. Sequence chromatogram indicates a G to C transition of nucleotide 1578. (E) Alignment of multiple DPP6 protein sequences across species. The Q526 affected amino acid locates in the highly conserved amino acid region in different mammals (from Ensemble). The Q526 site was shown in red.

Whole-exome sequencing and co-segregation analysis

The genomic DNA was extracted by DNeasy blood and tissue kit (QIAGEN # 69506). The exome capture, high-throughput sequencing and common filtering were performed in the OE Biotech. Co., Ltd (Shanghai, China). All the exomes were captured by Agilent SureSelect Human All Exon V5 kits and sequenced by Illumina HiSeq 2000 platform. The strategies of data filtering referred to our previous study.¹⁰ All the mutations will be filtered by 1000 genomes database, EXAC database and ESP6500 database, as well as arrhythmia-related genes list (Supplementary Table S1). The co-segregation analysis was performed for all family members as we have described.¹⁰

Mutagenesis and cell transfection

The c.1578 G > C/p.Q526H mutation of DPP6 was introduced in the pcDNA3.1+ by PCR using QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) following manufacturer’s instructions. HEK293 cells were transiently transfected with DPP6-pcDNA3.1+ (WT or Mutation) and/or K_V4.3-pcDNA3.1+ by using Lipofectamine™ 2000 CD Transfection Reagent (Thermo Fisher Scientific), following the manufacturer’s instructions.

Electrophysiological recordings

Currents were recorded from transiently transfected HEK293 cells using the perforated amphotericin B-patch-clamp technique with an Axopatch 200B amplifier (Axon Instruments) as described.¹¹

Statistics

Data are presented as mean values ± SEM. Two-way ANOVA repetitive measurements test followed by Bonferroni test were used to assess statistical significance when appropriate. A value of P < 0.05 was considered significant.

Results

Clinic data

The proband (II-5), a 45-year-old farmer from Hunan province of Central-South China, was admitted in our hospital due to syncope during manual labor. ECG records showed sinus tachycardia, abnormal of PtfV1 and frequent ventricular PtfV1 (Figure 1B). The B-ultrasound showed no obvious malformations. We have done 24-h Holter monitoring and exercise testing for all the family members. However, none of the individuals displayed significant arrhythmias during 24-h Holter monitoring or exercise testing. All family members that were tested displayed a maximal heart rate response to exercise. No prominent U waves were observed at rest or during exercise testing.

Based on these evidences, the proband was finally diagnosed as suspicious IVF. Family history examination found that the proband’s father (I-1) died at 40 years old during sleep for the unknown reason. The brother (II-2) of the proband also had history of repeatable syncope, the ECG records also showed similar characters (Figure 1C). Also, the niece (III-2) of the proband also experienced a syncope 1 year ago. However, she did not accept the medical examination during the study. The patient (II-2) has received an implantable cardioverter-defibrillator (ICD). During follow-up, the patient did not report any syncopal episodes nor did the ICD record any episode of (non-)sustained ventricular tachycardia.

Genetic analysis identified a novel segregating mutation in DPP6

Whole-exome sequencing yielded 10.14 Gb data with 99.7% coverage of target region and 99.0% of target covered over 10 X. After alignment and single nucleotide variant calling, 54 417 variants were identified in the proband. We then performed the data filtering as we formerly described.¹⁰ Approximate 412 single nucleotide variants and indels were picked out. Next, we used the arrhythmia-related genes list to filter the rest variants, and a set of 13 variants in 12 genes were identified. Bioinformatics analysis by Mutationtaster, Polyphen-2 and SIFT were also carried out (Table 1). Based on the bioinformatics analysis results, in which only disease causing or damaging mutations from all three bioinformatics analysis tools are considered, TTN, XIRP1, ANK2 and DPP6 are candidates. Then based on the functions or potential diseases leaded by these genes and the symptom of the proband and other patients in this family, we chose DPP6 (c.1578 G > C/p.Q526H) as the potential pathogenic gene.

Table 1.

Variants identified by whole-exome sequencing in combination with arrhythmia-related gene-filtering in the family

CHR	POS	RB	AB	Gene Name	AA change	MutationTaster	Phentype-2	SIFT
1	236925804	A	G	ACTN2	NM_001103: exon21: c.A2570G: p.D857G	Disease causing (0.99)	Begin (0.316)	Tolerated (0.09)
2	73613083	TGCA	T	ALMS1	NM_015120: exon1: c.88_90del: p.30_30del	Polymorphism (0.99)	–	–
2	179408024	G	T	TTN	NM_003319: exon175: c.C69481A: p.P23161T	Disease causing (1.0)	Damaging (1.0)	Damaging (0)
2	179442857	C	G	TTN	NM_003319: exon150: c.G41190C: p.K13730N	Polymorphism (0.549)	Damaging (0.824)	Damaging (0)
3	39230821	T	C	XIRP1	NM_001198621: exon2: c.A116G: p.K39R	Disease causing (0.99)	Damaging (0.998)	Damaging (0.05)
3	123419535	G	A	MYLK	NM_053026: exon17: c.C2573T: p.A858V	Disease causing (1.0)	Damaging (0.994)	Tolerated (0.31)
4	114278984	G	A	ANK2	NM_001148: exon38: c.G9210A: p.M3070I	Disease causing (1.0)	Damaging (0.961)	Damaging (0.05)
6	76540265	A	G	MYO6	NM_001300899: exon5: c.391 + 3A>G	Disease causing (0.99)	–	–
7	154598734	G	C	DPP6	NM_130797: exon16: c.G1578C: p.Q526H	Disease causing (1.0)	Damaging (0.81)	Damaging (0.02)
9	101867537	T	TGGC	TGFBR1	NM_001306210: exon1: c.50_51insGGC: p.L17delinsLA	Polymorphism (0.99)	–	–
11	119170231	G	A	CBL	NM_005188: exon16: c.G2461A: p.E821K	Disease causing (1.0)	Damaging (0.986)	Tolerated (0.78)
12	22063801	C	T	ABCC9	NM_005691: exon7: c.G1123A: p.V375I	Disease causing (1.0)	Begin (0.129)	Tolerated (0.53)
12	33031969	G	C	PKP2	NM_001005242: exon3: c.224-3C>G	Disease causing (1.0)	–	–

CHR, Chromosome; POS, position; RB, reference sequence base; AB, alternative base identified.

Sanger sequencing indicated that only this novel missense mutation (c.1578 G > C/p.Q526H) of DPP6 co-segregated with the affected family members (Figure 1D). This novel mutation (c.1578 G > C/p.Q526H), corresponding to a glutamine-to-histidine substitution at position 526 of DPP6, was located in a highly conserved cysteine-rich domain of DPP6 (Figure 1E) and was not found in our 200 local control cohorts.¹⁰

Functional research revealed the novel mutation might lead to gain of function

The bioinformatics programs Mutationtaster, Polyphen-2 and SIFT predicted that this mutation is disease causing (Table 1), because DPP6 is an essential β-subunit of the human cardiac transient outward current encoded by Kv4.3. We then performed transfection of K_v4.3-pcDNA3.1+ alone or together with DPP6-pcDNA3.1+ (WT or Q526H) into HEK293 cells. The electrophysiological currents showed that the K_v4.3 + DPP6 (Q526H) mutation increase the current intensity more than K_v4.3 + DPP6 WT (* represents P < 0.05) (Figure 2A and B). Patch clamp experiments further confirmed that this novel mutation might result in a gain of function and disturb the efflux of potassium ion.¹²

Figure 2.

The electrophysiological recordings from B-patch-clamp experiments. (A) DPP6-Q526H+ K_v4.3 displays augmented I_to compared with DPP6-WT+ K_v4.3 and K_v4.3 alone, *represents P < 0.05. (B) The statistics results.

Discussion

DPP6 is an auxiliary subunit of the K_v4 family of voltage-gated K⁺ channels known to enhance channel surface expression and potently accelerate their kinetics.¹³ DPP6 consists of N-terminal, transmembrane domain, glycosylation domain, cysteine-rich domain and aminopeptidase domain.¹⁴ The novel mutation (c.1578 G > C/p.Q526H) is located in the cysteine-rich domain, which is responsible for ER-to-Membrane transport.¹⁴ Combined with patch clamp experiments, this novel mutation may lead to the conformation change of K_v4.3 and cause a gain of function in the heart. Regarding the previous study which has detected the first missense mutation (p.H332R) of DPP6 in heart disease, our study is consistent with this research that both mutations can result in a gain of function on K_v4.3 and lead to ventricular fibrillation.¹²

DPP6 can both regulate the activation of K_v4.2 and K_v4.3 in neurodevelopment and heart development.^15,16 The previous study has demonstrated that besides the heart disease, mutations in DPP6 can also lead to microcephaly and mental retardation.¹⁷ However, there was only one missense mutation has been identified in brain disease.¹⁷ At present, 10 DPP6 mutations have been identified in patients with different disease phenotypes.⁸ Our study not only identified a rare DPP6 missense mutation and expanded the spectrum of DPP6 mutations, but also further confirmed that mutations in DPP6 could lead to gain of function of K_v4.3 and result in IVF.

Genetic testing was not recommended in patients with IVF because of the low yield and high costs in the past.⁶ However, with the reduction of costs of genetic testing, it becomes more feasible. Now-a-days, large custom multigene panels have been created and are rapidly replacing targeted genetic screening based on phenotype.^18,19 According to recent studies in IVF, more and more novel gene mutations have been identified in patients with IVF and other overlapping phenotypes.²⁰ Moreover, an increasing number of mutations of uncertain clinical significance are detected, but the interpretation and clinical use of these mutations of uncertain clinical significance are still unclear.⁶

In fact, there were still some limitations in our studies. First, our patient was not challenged with sodium channel blockade, we cannot rule out Brugada syndrome by current clinic diagnosis.²¹ But in combination with EGC records and previous studies in DPP6,¹³ we believe the diagnosis of IVF was more appropriate. Second, although whole exome sequencing in combination with arrhythmia-related genes list has been applied to filter the candidate genes in many studies,²² there were some limitations of this filtering strategy. It cannot identify other candidate genes which was not exist in the arrhythmia-related genes list. This was the reason why we perform the functional research of DPP6 mutation (c.1578 G > C/p.Q526H), although we have found this mutation was co-segregated with the affected individuals.

In conclusion, using whole exome sequencing in combination with arrhythmia-related genes list filtering strategy, we identified the novel missense mutation (c.1578 G > C/p.Q526H) of DPP6 as the possible cause of suspicious IVF, syncope and SCD in a Chinese family. Patch clamp experiments also revealed that this mutation could lead to a gain of function and affect the efflux of potassium ion. Our study not only further supported the important role of DPP6 in IVF but also expanded the spectrum of DPP6 mutations and will contribute to the genetic diagnosis and counseling of families with suspicious IVF, syncope and SCD.

Acknowledgements

The authors thank all subjects for participating in this study.

Funding

This study was supported by the National Natural Science Foundation of China (81370394) and the Open-End Fund for Valuable and Precision Instruments of Central South University (CSUZC201737).

Supplementary material

Supplementary material is available at QJMED online.

Conflict of interest: None declared.

Author notes

D.-B. Ding and L.-L. Fan contributed equally to this work.