Role of SCN5A coding and non-coding sequences in Brugada syndrome onset: What's behind the scenes?

Abstract

Background: Brugada syndrome (BrS) is a rare inherited cardiac arrhythmia associated with a high risk of sudden cardiac death (SCD) due to ventricular fibrillation (VF). BrS is characterized by coved-type ST-segment elevation in the right precordial leads (V1-V3). Mutations in SCN5A gene coding for the α-subunit of the NaV1.5 cardiac sodium channel are identified in 15-30% of BrS cases. Genetic testing of BrS patients generally involves sequencing of the protein-coding portions and flanking intronic regions of SCN5A. This excludes the 5'UTR and 3'UTR from the routine genetic testing.

Methods: We here screened the coding sequence, the flanking intronic regions as well as the 5' and 3'UTR regions of SCN5A gene and further five candidate genes (GPD1L, SCN1B, KCNE3, SCN4B, and MOG1) in a Tunisian family diagnosed with BrS.

Results: A new SCN5A-Q1000K mutation was identified along with two common polymorphisms (H558R and D1819). Multiple genetic variants were identified on the SCN5A 3'UTR, one of which is predicted to create additional microRNA binding site for miR-1270. Additionally, we identified the hsa-miR-219a-rs107822. No relevant coding sequence variant was identified in the remaining studied candidate genes.

Conclusions: The absence of genotype-phenotype concordance within all the identified genetic variants in this family gives extra evidences about the complexity of the disease and suggests that the occurrence and prognosis of BrS is most likely controlled by a combination of multiple genetic factors, rather than a single variant. Most SCN5A variants were localized in non-coding regions hypothesizing an impact on the miRNA-target complementarities.

Keywords: 3′UTR; Brugada syndrome; Genotype-phenotype correlation; SCN5A; microRNAs.

Fig. 1

Family kindred and mutations/polymorphisms distribution. (A): Family kindred. Q1000K, H558R and D1819D distribution is displayed, illustrating a lack of genotype-phenotype correlation. Black cells refer to symptomatic BrS patients presenting with spontaneous BrS type 1 ECG, Gray cells refer to asymptomatic patients presenting with induced BrS type 1 ECG and white cells refer to patient presenting no BrS symptoms nor type 1 BrS ECG. (B,C): 12 leads ECG recorded from II-1 and III-2 patients respectively. Note the ST segment elevation in the precordial leads V1, V2 and V3 in II-1 patient ECG. Both II-1 and III-2 patients are carriers of the Q1000K mutation. However, N-III-2 patient presents no symptoms for BrS nor Type 1 ECG which could be due to its young age and/or the incomplete penetrance of the disease. (D) Clinical profiles of subjects belonging to Family N. Note that there is a heterogeneous clinical profile within the symptomatic patients. Abbreviations used:M: male; S: Spontaneous; *: Not applicable; N: Negative; P: Positive; C: Confirmed; I: Induced.

Fig. 2

SCN5A nucleotide changes among the family members. (A): A new missense variant c.2998C > A was revealed in Exon 17 leading to the p.Q1000K substitution. Q1000 is conserved among species which may predict a crucial functional impact of Q1000K mutation. (B) Localization of the three identified amino acid changes among the whole Nav1.5 protein structure. Note that Q1000K is localized in the conserved binding site of MOG1 which predicts a functional consequence. (C): Summary of the SCN5A mutations and polymorphisms distribution among the family members.

Fig. 3

SCN5A 3′UTR nucleotide changes and impact on micrRNAs binding sites. (A): Sequencing results of exon 28 reveals two already reported successive polymorphisms T83948A and C83949T. Together, these polymorphisms create a forth miR- 1270 binding site within SCN5A 30UTR as detailed. Note that the surrounding region to T83948A and C83949T polymorphisms is highly conserved predicting a functional importance. (B) Luciferase assays showed that miR-1270 gain of function decreases the luciferase activity in cultured human fibroblasts. (C) Similar effects to the Luciferase essays were observed in a cellular context; Over-expression of miR- 1270 in HL-1 cardiomyocytes demonstrated a significant decrease of Scn5a mRNA quantity (∗p < 0.05, ∗∗p < 0.01) 6 hours post-transfection. qRT-PCR of two control genes Nkx2.5 and Mef2c in HL1 cells overexpressing miR-1270 displayed no difference of expression in transfected cells compared to control condition. Black bars refer to control condition (non-transfected cells) and patterned bars to the experimental condition (transfected cells). P (D) Sequencing of miR-219a precursor and flanking regions identified the heterozygous alleles of rs107822 SNP which is a substitution C > T localized 36 bp upstream the miR-219a precursor sequence. (E) Distribution of rs107822 nucleotide change among the family members.