Whole‐exome sequencing identifies a novel mutation of GPD1L (R189X) associated with familial conduction disease and sudden death

Abstract Cardiac conduction disease (CCD) is a serious disorder and the leading cause of mortality worldwide. It is characterized by arrhythmia, syncope or even sudden cardiac death caused by the dysfunction of cardiac voltage‐gated channel. Previous study has demonstrated that mutations in genes encoding voltage‐gated channel and related proteins were the crucial genetic lesion of CCD. In this study, we employed whole‐exome sequencing to explore the potential causative genes in a Chinese family with ventricular tachycardia and syncope. A novel nonsense mutation (c.565C>T/p.R189X) of glycerol‐3‐phosphate dehydrogenase‐like (GPD1L) was identified and co‐segregated with the affected family members. GPD1L is a crucial interacting protein of SCN5A, a gene encoded sodium channel α‐subunit Nav1.5 and mainly associated with Brugada syndrome (BrS). The novel mutation (c.565C>T/p.R189X) may result in a premature stop codon at position 189 in exon 4 of the GPD1L gene and lead to functional haploinsufficiency of GPD1L due to mRNA carrying this mutation will be degraded by nonsense‐mediated mRNA decay, which has been confirmed by Western blot in HEK293 cells transfected HIS‐GPD1L plasmid. The levels of GPD1L decreasing may disturb the function of Nav1.5 and induce arrhythmia and syncope in the end. In conclusion, our study not only further supported the important role of GPD1L in CCD, but also expanded the spectrum of GPD1L mutations and will contribute to the genetic diagnosis and counselling of families with CCD.


Introduction
Cardiac conduction diseases (CCDs) are an inherited cardiac disease that may present as a primary electrical disease or be associated with structural heart disease [1]. Diseases like long-QT syndrome (LQTS), short-QT syndrome (SQTS), BrS are relatively common among CCD with the clinical manifestations of arrhythmia, syncope or even sudden cardiac death (SCD) [2]. Mutations in genes that encoding the cardiac voltage-gated channels or their modify proteins are the main pathogeny of CCD. Among that, the cardiac sodium current is primarily conducted through the sodium channel protein Na v 1.5 that encoded by SCN5A [3,4]. Meanwhile, GPD1L protein can also influence sodium current by modify the sodium channel. GPD1L was first described in 2002 [5] and then identified as a new causative gene of BrS in 2007 [6]. Later, GPD1L mutations were also found in patients with sudden infant death (SID) [7]. To date, only several mutations have been found in GPD1L that associated with CCD, mainly BrS.
In this study, we investigated a family with arrhythmia, dilated cardiomyopathy (DCM) and syncope. An obvious autosomal-dominant inheritance has been observed in this family. We have employed whole-exome sequencing (WES) in combination with arrhythmiarelated gene-filtering to explore the possible causative gene for this family, a novel nonsense heterozygous mutation (c.565C>T/ p.Arg189*) of GPD1L was identified may underlie the inherited arrhythmia. Western blot in HEK293 cells transfected HIS-GPD1L plasmid further confirmed that the levels of GPD1L (c.565C>T/p.Arg189*) expression was degraded compared with the controls (Wt).

Subjects
This study was approved by the review board of the Second Xiangya Hospital of the Central South University. The proband and his relatives who participated in the study had been given written informed consent. After that, we investigated medical histories of all nine family members. Blood was collected from the affected proband and his family members. Subjects were examined by 12-lead echocardiogram (ECG) and B-mode ultrasound.

Method
DNA extraction, exome sequencing and filtering Genomic DNA was extracted by DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). The main part of WES was performed in the Novogene Bioinformatics Institute (Beijing, China). The exomes were captured by means of Agilent SureSelect Human All Exon V6 kits, and the platform of high-throughput sequencing was performed in Illumina HiSeq X-10. Filtering strategies conformed to our previous study [10]. And the genes list is provided in Table S1.

Mutation validation and co-segregation analysis
Sanger sequencing was performed to confirm potential causative variants in the family. Segregation analysis was performed in all family members. Primer pairs used to amplify fragments encompassing individual variants were designed by Primer 5, and the sequences of PCR primers will be provided upon request.

GPD1L expression vectors
The wild-type GPD1L cDNA with C-terminal flag and His-tag in the pEnter was designed by ourself. The R189X-GPD1L nonsense mutation was engineered into the vector above using the TaKaRa MutanBEST Kit (Takara Bio, Otsu, Shiga, Japan).

Western blot experiments
Protein samples were resolved on 4-12% Bis-Tris NuPAGE gels, followed by standard Western blotting with anti-His-tag (Cell Signaling Technology, Danvers, MA, USA) and anti-b-actin (Cell Signaling Technology). Chemiluminescent signals were scanned, and integrated density values were calculated with a chemiluminescent imaging system (Alpha Innotech, San Leandro, CA, USA).

Clinical feature
We described a Chinese family with multiple complex phenotypes including ventricular tachycardia and DCM (Fig. 1A, Table 1). The proband, a 20-year-old man, had an unprovoked palpitation from 2002 and diagnosed as left ventricular tachycardia in 2003, then discharged after radiofrequency ablation. During follow-up, he still felt palpitation and attacked by syncope once in 2014. The ECG showed ventricular tachycardia (Fig. 1B). So he re-hospitalized in 2014 for further treatment. The ultrasonic cardiogram revealed preserved biventricular enlargement (left atrial, LA 29 mm, left ventricle, LV 58 mm, right atrial, RA 32 mm, right ventricle, RV 36 mm) and LV systolic dysfunction (EF 48%). His mother and uncle were also suffered from syncope for several times. Additional, his mother and grandfather suddenly died during sleep for unknown reason.
Genetic analysis identified a novel segregating mutation in GPD1L WES yielded 10.21 Gb data with 99.7% coverage of target region and 99.0% of target covered over 109. After alignment and single nucleotide variant calling, 54,727 variants were identified in the proband. Approximate 374 single nucleotide variants and indels were picked out after filtering. Next, we used the arrhythmia-related genes list to filter the rest variants, and a set of 10 variants in nine genes were identified. Bioinformatics analysis by MutationTaster, PolyPhen-2 and SIFT also carried out ( Table 2).
Sanger sequencing indicated that only a novel nonsense heterozygous mutation (c.565C>T/p.Arg189*) of GPD1L co-segregated with the affected family members (Fig. 1C). This novel mutation, resulting in a premature stop codon at position 189 in exon 5 of the GPD1L gene, was also not found in our 200 local control cohorts, dbSNP and Exome Variant Server database (http://evs.gs.washington.edu/EVS/).

Functional characterization of mutant R189X GPD1L
The nonsense mutation of p.Arg189* was introduced into GPD1L cDNA clone as described in Methods. Mutant and wild-type plasmids were transfected into the HEK293 cells, and Western Blot was used to examine the expression of mutant and wild-type His-GPD1L by anti-His. R189X mutation showed no expression of His-GPD1L in contrast to WT protein, whereas b-tubulin level was unchanged (Fig. 2A).

Discussion
In this study, we employed WES to explore the genetic lesion of a family with ventricular tachycardia, DCM, syncope and sudden death. A novel heterozygous nonsense mutation (c.565C>T/ p.Arg189*) of GPD1L was identified and co-segregated with the affected family members. This mutation was absent from the 1000 genomes, dbSNP144 and 200 local normal controls. Functional analysis revealed that this variant is deleterious. Western blot in HEK293 cells transfected HIS-GPD1L plasmid further confirmed that the levels of GPD1L (R189X) expression was degraded compared with the controls (Wt). Based on the clinic phenotypes and genetic study, we can further make a definite diagnosis of BrS (a coved-type ECG, syncope and family history of sudden death before 45 years old) to the proband and the affected family members.
In fact, just 16% of BrS patients can be explained by SCN5A mutation [11], with the other genes accounting for <5% of patients [12]. As for GPD1L mutation, Makiyama et al. [13] have study 80 unrelated Japanese patients and screened for GPD1L mutations, no non-synonymous mutations were found, and only one synonymous mutation and one intronic variant were identified, as well as a single nucleotide polymorphism detected in four patients. That seems the contribution of GPD1L mutations to BrS is probably rare in Japan. Winkel et al. [14] enrolled 66 non-referred SCD patients born in Denmark and screened for genetic variants in the eight major genes including GPD1L. One rare variant of R220H (unknown significance) was found in an infant, who also carry another rare variant of SCN5A. In China, Liu et al. [15] were also found no non-synonymous mutations in 123 medico-legal autopsy-negative sudden unexplained nocturnal death syndrome cases from Southern China. That seems the contribution of GPD1L to BrS is very rare and our study further provide a novel rare mutation of GPD1L.
To date, three mutations have been identified in GPD1L associated with BrS, including A280V, I124V and R189X (present study). Meanwhile three mutations, E83K, I124V and R273C were found associated with SID (Fig. 2B). Interestingly, the mutation I124V was reported twice to associate with SID and suspected BrS [16], respectively. Different from A280V, R189X nonsense mutation may trigger nonsensemediated mRNA decay (NMD) to prevent the production of truncated proteins [17,18].
In conclusion, we report a novel rare GPD1L mutation (p.Arg189*) in a Chinese family with ventricular tachycardia, DCM, syncope and sudden death. Functional research indicated that this nonsense may lead to functional haploinsufficiency of GPD1L due to mRNA carrying this mutation degraded by nonsense-mediated mRNA decay, which is confirmed by Western blot in HEK293 cells transfected HIS-GPD1L plasmid. Our study not only further confirms the clinical diagnosis of BrS and provides a rare case, but also expands the spectrum of GPD1L mutations and may provide insight into genetic diagnosis and counselling of families with CCD and BrS.