Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

Cis-acting mutation affecting GJA5 transcription is underlying the Melanotic within-feather pigmentation pattern in chickens.

Melanotic (Ml) is a mutation in chickens that extends black (eumelanin) pigmentation in normally brown or red (pheomelanin) areas, thus affecting multiple within-feather patterns [J. W. Moore, J. R. Smyth Jr, J. Hered. 62, 215-219 (1971)]. In the present study, linkage mapping using a back-cross between Dark Cornish (Ml/Ml) and Partridge Plymouth Rock (ml+/ml+ ) chickens assigned Ml to an 820-kb region on chromosome 1. Identity-by-descent mapping, via whole-genome sequencing and diagnostic tests using a diverse set of chickens, refined the localization to the genomic region harboring GJA5 encoding gap-junction protein 5 (alias connexin 40) previously associated with pigmentation patterns in zebrafish. An insertion/deletion polymorphism located in the vicinity of the GJA5 promoter region was identified as the candidate causal mutation. Four different GJA5 transcripts were found to be expressed in feather follicles and at least two showed differential expression between genotypes. The results showed that Melanotic constitutes a cis-acting regulatory mutation affecting GJA5 expression. A recent study established the melanocortin-1 receptor (MC1R) locus and the interaction between the MC1R receptor and its antagonist agouti-signaling protein as the primary mechanism underlying variation in within-feather pigmentation patterns in chickens. The present study advances understanding the mechanisms underlying variation in plumage color in birds because it demonstrates that the activity of connexin 40/GJA5 can modulate the periodic pigmentation patterns within individual feathers.

Conjugated activation of myocardial-specific transcription of Gja5 by a pair of Nkx2-5-Shox2 co-responsive elements.

The sinoatrial node (SAN) is the primary pacemaker in the heart. During cardiogenesis, Shox2 and Nkx2-5 are co-expressed in the junction domain of the SAN and regulate pacemaker cell fate through a Shox2-Nkx2-5 antagonism. Cx40 is a marker of working myocardium and an Nkx2-5 transcriptional output antagonized by Shox2, but the underlying regulatory mechanisms remain elusive. Here we characterized a bona fide myocardial-specific Gja5 (coding gene of Cx40) distal enhancer consisting of a pair of Nkx2-5 and Shox2 co-bound elements in the regulatory region of Gja5. Transgenic reporter assays revealed that neither element alone, but the conjugation of both elements together, drives myocardial-specific transcription. Genetic analyses confirmed that the activation of this enhancer depends on Nkx2-5 but is inhibited by Shox2 in vivo, and its presence is essential for Gja5 expression in the myocardium but not the endothelial cells of the heart. Furthermore, chromatin conformation analysis showed an Nkx2-5-dependent loop formation between these two elements and the Gja5 promoter in vivo, indicating that Nkx2-5 bridges the conjugated activation of this enhancer by pairing the two elements to the Gja5 promoter.

Association of Polymorphism in SCN5A, GJA5, and KCNN3 Gene with Sudden Cardiac Death.

We studied association of single-nucleotide SCN5A (rs1805124), GJA5 (rs35594137), and KCNN3 (rs13376333) polymorphisms and sudden cardiac death. Humans died suddenly from cardiac causes (N=379) and unrelated sex- and age-matched control subjects were genotyped. No significant intergroup differences were found in the frequency of rs1805124 and rs13376333 genotypes and alleles. In women under 50 years, enhanced risk of sudden cardiac death was associated with rs35594137 GG genotype (OR=3.6; 95%CI=1.2-10.4; p=0.022), while in older women it was associated with rs35594137 AA genotype (OR=3.0; 95%CI=2.3-3.9; p=0.041). In women under 50 years, GA rs35594137 genotype was associated with a protective effect against sudden cardiac death (OR=0.3; 95%CI=0.1-0.8; p=0.036). Thus, GJA5 gene rs35594137 polymorphism is significantly associated with sudden cardiac death in the examined group.

Two polymorphisms in the Cx40 promoter are associated with hypertension and left ventricular hypertrophy preferentially in men.

BACKGROUND: Lack of connexin40, a gap junction protein expressed in endothelial and renin-producing cells, results in hypertension and cardiac hypertrophy in mice due to unleashed renin production caused by disruption of the pressure-induced feedback inhibition. We analysed human GJA5 consisting of two exons (exon1A or 1B and exon2) in a selected cohort identified by a single nucleotide polymorphism (SNP) in the GJA5 intron for polymorphisms and putative association with hypertension and left ventricular hypertrophy (LVH). METHODS: Individuals carrying a SNP in the intron of GJA5 (rs791295) were selected from the MONICA/KORA cohort (n = 1677) and searched for GJA5 polymorphisms. We accessed DNA of 178 probands, of which 26 suffered from LVH, 112 were hypertensive and 29 normotensive (unknown: 11). RESULTS: Sequencing of the GJA5 coding region did not reveal alterations suggesting the expression of functional connexin40 in all probands. Sequencing of the upstream region of transcript 1A including exon1A revealed two previously described linked SNPs (rs35594137 -44G>A; rs11552588 + 71A>G) at an increased frequency. Moreover, the rare genotype was significantly associated with hypertension and LVH with a preponderance in men. Functional analysis in a reporter gene assay verified promoter activity, however, it was unchanged by the identified SNPs after expressing respective reporter constructs in HeLa and human endothelial cells. CONCLUSION: We suggest to consider the -44G>A SNP upstream of the connexin40 transcript 1A indeed as a risk factor for hypertension in men. However, the underlying mechanisms remain unclear but animal data suggest that renin-producing cells may be involved and contribute to hypertension.

A novel GJA5 variant associated with increased risk of essential hypertension.

OBJECTIVES: Gap junction protein alpha 5 (GJA5), also termed connexin 40 (Cx40), exerts a pivotal role in the mediation of vascular wall tone and two closely-linked polymorphisms in the GJA5 promoter (-44G>A and +71A>G) have been associated with enhanced susceptibility to essential hypertension (EH) in men. The present investigation aimed to ascertain whether a novel common polymorphism within the upstream regulatory region of GJA5 (transcript 1B), -26A>G (rs10465885), confers an increased risk of EH. METHODS: For this investigation, 380 unrelated patients with EH and 396 unrelated normotensive individuals employed as control persons were enrolled from the Chinese Han-ethnicity population, and their GJA5 genotypes and plasma renin concentrations were determined by Sanger sequencing and an automated chemiluminescent immunoassay, respectively. The functional effect of the GJA5 variant was explored in cultured murine cardiomyocytes by dual-light reporter gene analysis. RESULTS: The GJA5 variant conferred a significantly increased risk for EH (OR: 2.156; 95% CL: 1.661-2.797, P < 0.0001), and significantly increased plasma renin levels were measured in patients with EH in comparison with control individuals (46.3±7.2 vs 37.4±6.9, P < 0.0001). A promoter-luciferase analysis revealed significantly diminished activity of the promoter harboring the minor allele for this variation in comparison with its wild-type counterpart (165.67±16.85 vs 61.53±8.67, P = 0.0007). CONCLUSIONS: These findings indicate that the novel variant upstream of the GJA5 gene (-26A>G) confers a significantly increased vulnerability of EH in humans, suggesting potential clinical implications for precisive prophylaxis and treatment of EH.

Phenotypic Variability Associated with a Large Recurrent 1q21.1 Microduplication in a Three-Generation Family.

Recurrent copy number variants of the q21.1 region of chromosome 1 have been associated with variable clinical features, including developmental delay, mild to moderate intellectual disability, psychiatric and behavioral problems, congenital heart malformations, and craniofacial abnormalities. A subset of individuals is clinically unaffected. We describe a unique 3-generation family with a large recurrent 1q21.1 microduplication (BP2-BP4). Our observations underline the incomplete penetrance and phenotypic variability of this rearrangement. We also confirm the association with congenital heart malformations, chronic depression, and anxiety. Furthermore, we report a broader range of dysmorphic features. The extreme phenotypic heterogeneity observed in this family suggests that additional factors modify the clinical phenotype.