Chromatin remodeller CHD7 is required for GABAergic neuron development by promoting PAQR3 expression

Mutations in the chromatin remodeller‐coding gene CHD7 cause CHARGE syndrome (CS). CS features include moderate to severe neurological and behavioural problems, clinically characterized by intellectual disability, attention‐deficit/hyperactivity disorder and autism spectrum disorder. To investigate the poorly characterized neurobiological role of CHD7, we here generate a zebrafish chd7 ^−/− model. chd7 ^−/− mutants have less GABAergic neurons and exhibit a hyperactivity behavioural phenotype. The GABAergic neuron defect is at least in part due to downregulation of the CHD7 direct target gene paqr3b, and subsequent upregulation of MAPK/ERK signalling, which is also dysregulated in CHD7 mutant human cells. Through a phenotype‐based screen in chd7 ^−/− zebrafish and Caenorhabditis elegans, we show that the small molecule ephedrine restores normal levels of MAPK/ERK signalling and improves both GABAergic defects and behavioural anomalies. We conclude that chd7 promotes paqr3b expression and that this is required for normal GABAergic network development. This work provides insight into the neuropathogenesis associated with CHD7 deficiency and identifies a promising compound for further preclinical studies.     

CHD7, the gene mutated in CHARGE syndrome,regulates genes involved in neural crest cell guidance

Heterozygous loss of function mutations in CHD7 (chromodomain helicase DNA-binding protein 7) lead to CHARGE syndrome, a complex developmental disorder affecting craniofacial structures, cranial nerves and several organ systems. Recently, it was demonstrated that CHD7 is essential for the formation of multipotent migratory neural crest cells, which migrate from the neural tube to many regions of the embryo, where they differentiate into various tissues including craniofacial and heart structures. So far, only few CHD7 target genes involved in neural crest cell development have been identified and the role of CHD7 in neural crest cell guidance and the regulation of mesenchymal-epithelial transition are unknown. Therefore, we undertook a genome-wide microarray expression analysis on wild-type and CHD7 deficient (Chd7 ^Whi/+ and Chd7 ^Whi/Whi ) mouse embryos at day 9.5, a time point of neural crest cell migration. We identified 98 differentially expressed genes between wild-type and Chd7 ^Whi/Whi embryos. Interestingly, many misregulated genes are involved in neural crest cell and axon guidance such as semaphorins and ephrin receptors. By performing knockdown experiments for Chd7 in Xenopus laevis embryos, we found abnormalities in the expression pattern of Sema3a, a protein involved in the pathogenesis of Kallmann syndrome, in vivo. In addition, we detected non-synonymous SEMA3A variations in 3 out of 45 CHD7-negative CHARGE patients. In summary, we discovered for the first time that Chd7 regulates genes involved in neural crest cell guidance, demonstrating a new aspect in the pathogenesis of CHARGE syndrome. Furthermore, we showed for Sema3a a conserved regulatory mechanism across different species, highlighting its significance during development. Although we postulated that the non-synonymous SEMA3A variants which we found in CHD7-negative CHARGE patients alone are not sufficient to produce the phenotype, we suggest an important modifier role for SEMA3A in the pathogenesis of this multiple malformation syndrome.     

Association of Aminoacyl-tRNA Synthetases Gene Polymorphisms with the Risk of Congenital Heart Disease in the Chinese Han Population

Aminoacyl-tRNA synthetases (ARSs) are in charge of cellular protein synthesis and have additional domains that function in a versatile manner beyond translation. Eight core ARSs (EPRS, MRS, QRS, RRS, IRS, LRS, KRS, DRS) combined with three nonenzymatic components form a complex known as multisynthetase complex (MSC).We hypothesize that the single-nucleotide polymorphisms (SNPs) of the eight core ARS coding genes might influence the susceptibility of sporadic congenital heart disease (CHD). Thus, we conducted a case-control study of 984 CHD cases and 2953 non-CHD controls in the Chinese Han population to evaluate the associations of 16 potentially functional SNPs within the eight ARS coding genes with the risk of CHD. We observed significant associations with the risk of CHD for rs1061248 [G/A; odds ratio (OR) = 0.90, 95% confidence interval (CI) = 0.81–0.99; P = 3.81×10⁻²], rs2230301 [A/C; OR = 0.73, 95%CI = 0.60–0.90, P = 3.81×10⁻²], rs1061160 [G/A; OR = 1.18, 95%CI = 1.06–1.31; P = 3.53×10⁻³] and rs5030754 [G/A; OR = 1.39, 95%CI = 1.11–1.75; P = 4.47×10⁻³] of EPRS gene. After multiple comparisons, rs1061248 conferred no predisposition to CHD. Additionally, a combined analysis showed a significant dosage-response effect of CHD risk among individuals carrying the different number of risk alleles (P _trend = 5.00×10⁻⁴). Compared with individuals with “0–2” risk allele, those carrying “3”, “4” or “5 or more” risk alleles had a 0.97-, 1.25- or 1.38-fold increased risk of CHD, respectively. These findings indicate that genetic variants of the EPRS gene may influence the individual susceptibility to CHD in the Chinese Han population.     

CHD7 Deficiency in “Looper”, a New Mouse Model of CHARGE Syndrome,Results in Ossicle Malformation,Otosclerosis and Hearing Impairment

CHARGE syndrome is a rare human disorder caused by mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). Characteristics of CHARGE are varied and include developmental ear and hearing anomalies. Here we report a novel mouse model of CHD7 dysfunction, termed Looper. The Looper strain harbours a nonsense mutation (c.5690C>A, p.S1897X) within the Chd7 gene. Looper mice exhibit many of the clinical features of the human syndrome, consistent with previously reported CHARGE models, including growth retardation, facial asymmetry, vestibular defects, eye anomalies, hyperactivity, ossicle malformation, hearing loss and vestibular dysfunction. Looper mice display an otosclerosis-like fusion of the stapes footplate to the cochlear oval window and blepharoconjunctivitis but not coloboma. Looper mice are hyperactive and have vestibular dysfunction but do not display motor impairment.     

Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.     

Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis

We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. BglII restriction fragments were size-selected to 500–600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. We constructed RRBS libraries from murine ES cells and from ES cells lacking DNA methyltransferases Dnmt3a and 3b and with knocked-down (kd) levels of Dnmt1 (Dnmt[1^kd,3a^−/−,3b^−/−]). Sequencing of 960 RRBS clones from Dnmt[1^kd,3a^−/−,3b^−/−] cells generated 343 kb of non-redundant bisulfite sequence covering 66212 cytosines in the genome. All but 38 cytosines had been converted to uracil indicating a conversion rate of >99.9%. Of the remaining cytosines 35 were found in CpG and 3 in CpT dinucleotides. Non-CpG methylation was >250-fold reduced compared with wild-type ES cells, consistent with a role for Dnmt3a and/or Dnmt3b in CpA and CpT methylation. Closer inspection revealed neither a consensus sequence around the methylated sites nor evidence for clustering of residual methylation in the genome. Our findings indicate random loss rather than specific maintenance of methylation in Dnmt[1^kd,3a^−/−,3b^−/−] cells. Near-complete bisulfite conversion and largely unbiased representation of RRBS libraries suggest that random shotgun bisulfite sequencing can be scaled to a genome-wide approach.     

In Situ Patrolling of Regulatory T Cells Is Essential for Protecting Autoimmune Exocrinopathy

Background

Migration of T cells, including regulatory T (Treg) cells, into the secondary lymph organs is critically controlled by chemokines and adhesion molecules. However, the mechanisms by which Treg cells regulate organ-specific autoimmunity via these molecules remain unclear. Although we previously reported autoimmune exocrinopathy resembling Sjögren''s syndrome (SS) in the lacrimal and salivary glands from C-C chemokine receptor 7 (CCR7)-deficient mice, it is still unclear whether CCR7 signaling might specifically affect the dynamics and functions of Treg cells in vivo. We therefore investigated the cellular mechanism for suppressive function of Treg cells via CCR7 in autoimmunity using mouse models and human samples.

Methods and Findings

Patrolling Treg cells were detected in the exocrine organs such as lacrimal and salivary glands from normal mice that tend to be targets for autoimmunity while the Treg cells were almost undetectable in the exocrine glands of CCR7 ^−/− mice. In addition, we found the significantly increased retention of CD4⁺CD25⁺Foxp3⁺ Treg cells in the lymph nodes of CCR7 ^−/− mice with aging. Although Treg cell egress requires sphingosine 1-phosphate (S1P), chemotactic function to S1P of CCR7−/− Treg cells was impaired compared with that of WT Treg cells. Moreover, the in vivo suppression activity was remarkably diminished in CCR7 ^−/− Treg cells in the model where Treg cells were co-transferred with CCR7 ^−/− CD25^-CD4⁺ T cells into Rag2 ^−/− mice. Finally, confocal analysis showed that CCR7⁺Treg cells were detectable in normal salivary glands while the number of CCR7⁺Treg cells was extremely decreased in the tissues from patients with Sjögren''s syndrome.

Conclusions

These results indicate that CCR7 essentially governs the patrolling functions of Treg cells by controlling the traffic to the exocrine organs for protecting autoimmunity. Characterization of this cellular mechanism could have clinical implications by supporting development of new diagnosis or treatments for the organ-specific autoimmune diseases such as Sjögren''s syndrome and clarifying how the local immune system regulates autoimmunity.     

Tip30 controls differentiation of murine mammary luminal progenitor to estrogen receptor-positive luminal cell through regulating FoxA1 expression

Estrogen receptor-alpha positive (ER⁺) breast cancers comprise the majority of human breast cancers, but molecular mechanisms underlying this subtype of breast cancers remain poorly understood. Here, we show that ER⁺ mammary luminal tumors arising in Tip30^−/−MMTV-Neu mice exhibited increased enrichment of luminal progenitor gene signature. Deletion of the Tip30 gene increased proportion of mammary stem and progenitor cell populations, and raised susceptibility to ER⁺ mammary luminal tumors in female Balb/c mice. Moreover, Tip30^−/− luminal progenitors displayed increases in propensity to differentiate to mature ER⁺ luminal cells and FoxA1 expression. Knockdown of FoxA1 expression in Tip30^−/− progenitors by shRNA specific for FoxA1 reduced their differentiation toward ER⁺ mature luminal cells. Taken together, our results suggest that TIP30 is a key regulator for maintaining ER⁺ and ER⁻luminal pools in the mammary luminal lineage, and loss of it promotes expansion of ER⁺ luminal progenitors and mature cells and ER⁺ mammary tumorigenesis.     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Receptor-independent Role of Urokinase-Type Plasminogen Activator in Pericellular Plasmin and Matrix Metalloproteinase Proteolysis during Vascular Wound Healing in Mice

It has been proposed that the urokinase receptor (u-PAR) is essential for the various biological roles of urokinase-type plasminogen activator (u-PA) in vivo, and that smooth muscle cells require u-PA for migration during arterial neointima formation. The present study was undertaken to evaluate the role of u-PAR during this process in mice with targeted disruption of the u-PAR gene (u-PAR^−/−). Surprisingly, u-PAR deficiency did not affect arterial neointima formation, neointimal cell accumulation, or migration of smooth muscle cells. Indeed, topographic analysis of arterial wound healing after electric injury revealed that u-PAR^−/− smooth muscle cells, originating from the uninjured borders, migrated over a similar distance and at a similar rate into the necrotic center of the wound as wild-type (u-PAR^+/+) smooth muscle cells. In addition, u-PAR deficiency did not impair migration of wounded cultured smooth muscle cells in vitro. There were no genotypic differences in reendothelialization of the vascular wound. The minimal role of u-PAR in smooth muscle cell migration was not because of absent expression, since wild-type smooth muscle cells expressed u-PAR mRNA and functional receptor in vitro and in vivo. Pericellular plasmin proteolysis, evaluated by degradation of ¹²⁵I-labeled fibrin and activation of zymogen matrix metalloproteinases, was similar for u-PAR^−/− and u-PAR^+/+ cells. Immunoelectron microscopy of injured arteries in vivo revealed that u-PA was bound on the cell surface of u-PAR^+/+ cells, whereas it was present in the pericellular space around u-PAR^−/− cells. Taken together, these results suggest that binding of u-PA to u-PAR is not required to provide sufficient pericellular u-PA–mediated plasmin proteolysis to allow cellular migration into a vascular wound.     

A Transgenic Myogenic Cell Line Lacking Ryanodine Receptor Protein for Homologous Expression Studies: Reconstitution of Ry1R Protein and Function

Abstract. CCS embryonic stem (ES) cells possessing two mutant alleles (ry₁r−/ry₁r−) for the skeletal muscle ryanodine receptor (RyR) have been produced and injected subcutaneously into severely compromised immunodeficient mice to produce teratocarcinomas in which Ry₁R expression is absent. Several primary fibroblast cell lines were isolated and subcloned from one of these tumors that contain the knockout mutation in both alleles and exhibit a doubling time of 18–24 h, are not contact growth inhibited, do not exhibit drastic morphological change upon serum reduction, and possess the normal complement of chromosomes. Four of these fibroblast clones were infected with a retrovirus containing the cDNA encoding myoD and a puromycin selection marker. Several (1–2 μg/ml) puromycin-resistant subclones from each initial cell line were expanded and examined for their ability to express myoD and to form multinucleated myotubes that express desmin and myosin upon removal of mitogens. One of these clones (1B5 cells) was selected on this basis for further study. These cells, upon withdrawal of mitogens for 5–7 d, were shown by Western blot analysis to express key triadic proteins, including skeletal triadin, calsequestrin, FK506-binding protein, 12 kD, sarco(endo)plasmic reticulum calcium–ATPase1, and dihydropyridine receptors. Neither RyR isoform protein, Ry₁R (skeletal), Ry₂R (cardiac), nor Ry₃R (brain), were detected in differentiated 1B5 cells. Measurements of intracellular Ca²⁺ by ratio fluorescence imaging of fura-2–loaded cells revealed that differentiated 1B5 cells exhibited no responses to K⁺ (40 mM) depolarization, ryanodine (50–500 μM), or caffeine (20–100 mM). Transient transfection of the 1B5 cells with the full-length rabbit Ry₁R cDNA restored the expected responses to K⁺ depolarization, caffeine, and ryanodine. Depolarization-induced Ca²⁺ release was independent of extracellular Ca²⁺, consistent with skeletal-type excitation–contraction coupling. Wild-type Ry₁R expressed in 1B5 cells were reconstituted into bilayer lipid membranes and found to be indistinguishable from channels reconstituted from rabbit sarcoplasmic reticulum with respect to unitary conductance, open dwell times, and responses to ryanodine and ruthenium red. The 1B5 cell line provides a powerful and easily managed homologous expression system in which to study how Ry₁R structure relates to function.     

A Polycomb Group Protein Is Retained at Specific Sites on Chromatin in Mitosis

Epigenetic regulation of gene expression, including by Polycomb Group (PcG) proteins, may depend on heritable chromatin states, but how these states can be propagated through mitosis is unclear. Using immunofluorescence and biochemical fractionation, we find PcG proteins associated with mitotic chromosomes in Drosophila S2 cells. Genome-wide sequencing of chromatin immunoprecipitations (ChIP–SEQ) from mitotic cells indicates that Posterior Sex Combs (PSC) is not present at well-characterized PcG targets including Hox genes in mitosis, but does remain at a subset of interphase sites. Many of these persistent sites overlap with chromatin domain borders described by Sexton et al. (2012), which are genomic regions characterized by low levels of long range contacts. Persistent PSC binding sites flank both Hox gene clusters. We hypothesize that disruption of long-range chromatin contacts in mitosis contributes to PcG protein release from most sites, while persistent binding at sites with minimal long-range contacts may nucleate re-establishment of PcG binding and chromosome organization after mitosis.     

DNA polymerases ν and θ are required for efficient immunoglobulin V gene diversification in chicken

The chicken DT40 B lymphocyte line diversifies its immunoglobulin (Ig) V genes through translesion DNA synthesis–dependent point mutations (Ig hypermutation) and homologous recombination (HR)–dependent Ig gene conversion. The error-prone biochemical characteristic of the A family DNA polymerases Polν and Polθ led us to explore the role of these polymerases in Ig gene diversification in DT40 cells. Disruption of both polymerases causes a significant decrease in Ig gene conversion events, although POLN^−/−/POLQ^−/− cells exhibit no prominent defect in HR-mediated DNA repair, as indicated by no increase in sensitivity to camptothecin. Polη has also been previously implicated in Ig gene conversion. We show that a POLH^−/−/POLN^−/−/POLQ^−/− triple mutant displays no Ig gene conversion and reduced Ig hypermutation. Together, these data define a role for Polν and Polθ in recombination and suggest that the DNA synthesis associated with Ig gene conversion is accounted for by three specialized DNA polymerases.     

Genetic analysis of the CHD7 gene in Korean patients with CHARGE syndrome

CHARGE syndrome is an autosomal dominant congenital disorder known to be caused by the haploinsufficiency of the CHD7 gene. Heterozygous mutations in the CHD7 gene have been identified in approximately 60–70% of patients clinically diagnosed with CHARGE syndrome. Although there have been many reports on the mutational spectrum of the CHD7 gene in patients with CHARGE syndrome worldwide, little is known about this syndrome in the Korean population. In this study, three Korean patients with CHARGE syndrome including one patient with Patau syndrome were evaluated for genetic analysis of the CHD7 gene using direct sequencing of all 38 exons and the flanking intronic regions. One nonsense and two novel missense mutations were identified in the CHD7 gene. Clinical symptoms caused by the missense mutations were much milder compared to the nonsense mutation, confirming the previously determined genotype–phenotype correlation in CHARGE syndrome. Our study demonstrates the importance of mutational screening of CHD7 in patients who have been diagnosed with other syndromes but display clinical features of CHARGE syndrome.     

Genetic Variation in the 3'-Untranslated Region of NBN Gene Is Associated with Gastric Cancer Risk in a Chinese Population

NBN plays a crucial role in carcinogenesis as a core component for both homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA double-strand breaks (DSBs) repair pathways. Genetic variants in the NBN gene have been associated with multiple cancers risk, suggesting pleiotropic effect on cancer. We hypothesized that genetic variants in the NBN gene may modify the risk of gastric cancer. To test this hypothesis, we evaluated the association between four potentially functional single nucleotide polymorphisms in NBN and gastric cancer risk in a case–control study of 1,140 gastric cancer cases and 1,547 controls in a Chinese population. We found that the A allele of rs10464867 (G>A) was significantly associated with a decreased risk of gastric cancer (odds ratio [OR] = 0.81, 95% confidence interval [95% CI] = 0.71–0.94; P = 4.71×10⁻³). Furthermore, the association between A allele of rs10464867 and decreased risk of gastric cancer was more significantly in elder individuals (per-allele OR = 0.72[0.59–0.88], P = 1.07×10⁻³), and male individuals (per-allele OR = 0.73[0.62–0.87], P = 3.68×10⁻⁴). We further conducted a haplotype analysis and identified that the NBN A_rs10464867G_rs14448G_rs1063053 haplotype conferred stronger protective effect on gastric cancer (OR = 0.76[0.65–0.89], P = 6.39×10⁻⁴). In summary, these findings indicate that genetic variants at NBN gene may contribute to gastric cancer susceptibility and may further advance our understanding of NBN gene in cancer development.     

Role of Chd7 in zebrafish: a model for CHARGE syndrome

CHARGE syndrome is caused by mutations in the CHD7 gene. Several organ systems including the retina, cranial nerves, inner ear and heart are affected in CHARGE syndrome. However, the mechanistic link between mutations in CHD7 and many of the organ systems dysfunction remains elusive. Here, we show that Chd7 is required for the organization of the neural retina in zebrafish. We observe an abnormal expression or a complete absence of molecular markers for the retinal ganglion cells and photoreceptors, indicating that Chd7 regulates the differentiation of retinal cells and plays an essential role in retinal cell development. In addition, zebrafish with reduced Chd7 display an abnormal organization and clustering of cranial motor neurons. We also note a pronounced reduction in the facial branchiomotor neurons and the vagal motor neurons display aberrant positioning. Further, these fish exhibit a severe loss of the facial nerves. Knock-down of Chd7 results in a curvature of the long body axis and these fish develop irregular shaped vertebrae and have a reduction in bone mineralization. Chd7 knockdown also results in a loss of proper segment polarity illustrated by flawed efnb2a and ttna expression, which is associated with later vascular segmentation defects. These critical roles for Chd7 in retinal and vertebral development were previously unrecognized and our results provide new insights into the role of Chd7 during development and in CHARGE syndrome pathogenesis.     

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a +/? Mouse Model

Background

Loss-of-function mutations in SCN5A, the gene encoding Na_v1.5 Na⁺ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a ^+/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/Principal Findings

Based on ECG, 10-week-old Scn5a ^+/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na⁺ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a ^+/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a ^+/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a ^+/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a ^+/− mice had similar Na_v1.5 mRNA but higher Na_v1.5 protein expression, and moderately larger I_Na current than severely affected Scn5a ^+/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a ^+/− mice than in mildly affected ones.

Conclusions

Scn5a ^+/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a ^+/− mice, phenotype severity correlates with wild-type Na_v1.5 protein expression.