SOX7 loss-of-function variation as a cause of familial congenital heart disease

Abstract

Introduction: As the most frequent type of birth defect in humans, congenital heart disease (CHD) leads to a large amount of morbidity and mortality as well as a tremendous socioeconomic burden. Accumulating studies have convincingly substantiated the pivotal roles of genetic defects in the occurrence of familial CHD, and deleterious variations in a great number of genes have been reported to cause various types of CHD. However, owing to pronounced genetic heterogeneity, the hereditary components underpinning CHD remain obscure in most cases. This investigation aimed to identify novel genetic determinants underlying CHD.

Methods and results: A four-generation pedigree with high incidence of autosomal-dominant CHD was enrolled from the Chinese Han race population. Using whole-exome sequencing and Sanger sequencing assays of the family members available, a novel SOX7 variation in heterozygous status, NM_031439.4: c.310C>T; p.(Gln104*), was discovered to be in co-segregation with the CHD phenotype in the whole family. The truncating variant was absent in 500 unrelated healthy subjects utilized as control individuals. Functional measurements by dual-luciferase reporter analysis revealed that Gln104*-mutant SOX7 failed to transactivate its two important target genes, GATA4 and BMP2, which are both responsible for CHD. In addition, the nonsense variation invalidated the cooperative transactivation between SOX7 and NKX2.5, which is another recognized CHD-causative gene.

Conclusion: The present study demonstrates for the first time that genetically defective SOX7 predisposes to CHD, which sheds light on the novel molecular mechanism underpinning CHD, and implies significance for precise prevention and personalized treatment in a subset of CHD patients.

Keywords: Congenital heart disease; SOX7; dual-luciferase analysis; medical genetics; transcriptional regulation.

Figure 1

A SOX7 variation leading to familial congenital heart disease. A. Pedigree displaying autosomal dominant inheritance of patent ductus arteriosus in Family 1. Genotype of each family member is marked with + or -, of which + indicates a member carrying the heterozygous SOX7 mutation, while-indicates a member without the given SOX7 mutation. B. Sequence chromatograms of the family members. The heterozygous nucleotide change of c.310C>T in the SOX7 gene was proved by Sanger sequencing in the members affected with CHD including the proband (IV-1, mutant), predicted to yield a truncated SOX7 protein (Gln104*) as compared to the unaffected members including the proband’s father (III-1, wild type). Each codon of SOX7 is underlined with its encoded amino acid shown above. A black arrow points to the position of the altered nucleotide or the corresponding wild-type nucleotide in the SOX7 gene. C. Schematic representations illustrating the structural domains of human SOX7 proteins with deletion of 285 amino acids at the carboxyl-terminus of mutant SOX7 protein. TAD: transcriptional activation domain; HMG: high mobility group.

Figure 2

Nullified transactivation function of Gln104*-mutant SOX7. In cultured COS-7 cells transfected with eukaryotic expression plasmids, biologic analysis of transcriptional activation of the BMP2 promoter-driven firefly luciferase reporter by wild-type SOX7 (SOX7) or Gln104*-mutant SOX7 (Gln104*), singly or together, revealed that SOX7 normally transactivated the promoter of the target gene BMP2, whereas Gln104* failed to do so. Here unpaired Student’s t test was used. **Denotes P < 0.01, and *denotes P < 0.001, when compared wto an equal amount of SOX7.

Figure 3

Synergistic transactivation between SOX7 and NKX2.5 abrogated by the variation. In transfected HeLa cells, measurement of activation of the GATA4 promoter-driven firefly luciferase reporter by wild-type SOX7 (SOX7) or Gln104*-mutant SOX7 (Gln104*), alone or together with NKX2.5, unveiled that the synergistic transcriptional activation between NKX2.5 and SOX7 was disrupted by the Gln104* mutation. Here unpaired Student’s t test was used. **Indicates P < 0.001, and *indicates P < 0.005, in comparison to their wild-type counterparts.