Identification and Functional Characterization of a Novel SOX4 Mutation Predisposing to Coffin-Siris Syndromic Congenital Heart Disease

Abstract

Background/Objectives: Congenital heart disease (CHD) occurs in ~1% of all live neonates globally, rendering it the most prevalent developmental anomaly affecting humans; this condition confers substantial infant morbidity and mortality worldwide. Although there is ample evidence to suggest a paramount genetic basis for CHD, the genetic etiologies underpinning the majority of CHD remain elusive. In the present study, SOX4 was selected as a significant candidate gene for human CHD, mainly because SOX4 is abundantly expressed in both human and murine hearts during embryogenesis, and the knockout of Sox4 in mice causes embryonic demise predominantly attributable to cardiovascular developmental defects. Methods: Sequencing analysis of SOX4 was fulfilled in 248 probands affected with various types of CHD and the available relatives of the identified variation carrier as well as 262 unrelated healthy individuals. Functional analysis of the mutant SOX4 protein was conducted by utilizing a dual-reporter gene system. Results: a novel heterozygous SOX4 variation, NM_003107.3:c.331G>T;p.(Glu111*), was discovered in a male proband with Coffin-Siris syndromic CHD. Genetic investigation of the proband's available relatives revealed that the truncating variation co-segregated with the phenotype in the whole family. The nonsense variation was absent from 262 healthy controls. Functional analysis demonstrated that the Glu111*-mutant SOX4 lost transactivation on NKX2.5 and GATA4, two well-established genes that are causative factors for CHD. Moreover, the Glu111* mutation nullified the synergistic transactivation between SOX4 and TBX20, another CHD-causing gene. Conclusions: These findings support SOX4 as a causative gene accountable for familial Coffin-Siris syndromic CHD in humans. These findings may aid in developing personalized preventive and therapeutic strategies for patients with Coffin-Siris syndromic CHD.

Keywords: SOX4; biological assay; congenital heart disease; human molecular genetics; patent ductus arteriosus; transcription regulation.

Figure 1

Pedigree of the family with congenital heart disease. The family was arbitrarily designated as Family CHD-001. Roman–Arabic numerals were used to identify family members. CHD: congenital heart disease; +: a carrier of the identified heterozygous SOX4 mutation (c.331G>T, equal to p.Glu111*); and −: a non-carrier.

Figure 2

Representative SOX4 sequence chromatograms from the family members and schemas of structural domains of SOX4. (A) Sequence chromatograms displaying the heterozygous SOX4 mutation (c.331G>T) identified in a family member with patent ductus arteriosus (mutant) along with the wild-type control (G/G) in a healthy individual (wild-type). (B) Schemas delineating the critical structural domains of wild-type SOX4 and its mutant with the Glu111* mutation noted. COOH, carboxyl-terminus; TAD, transactivation domain; HMG, high-mobility group; and NH2, amino-terminus. Here a blue color line indicates cytosine (C), a red color line indicates thymine (T), a green color line indicates adenine (A), and a black color line indicates guanine (G).

Figure 3

No transactivation of NKX2.5 by Glu111*-mutant SOX4. In cultured HeLa cells transfected with the appropriate expression plasmids, dual-reporter gene gauges of the NKX2.5 promoter activity revealed that the Glu111*-mutant SOX4 lost transactivation of NKX2.5. For each gene expression construct, three independent cellular transfection experiments were executed.

Figure 4

No synergistic transactivation between Glu111*-mutant SOX4 and TBX20. In cultured 293 cells transfected with the appropriate expression plasmids, dual-luciferase examinations of the GATA4 promoter activity unveiled that the Glu111*-mutant SOX4 lost the ability to transactivate GATA4 alone or in synergy with TBX20. For each gene expression plasmid, three independent cellular transfection experiments were fulfilled.

Figure 5

Schematic diagram of SOX4 protein showing pathogenic variants responsible for Coffin–Siris syndrome. The SOX4 variants reported to cause Coffin–Siris syndrome without cardiovascular developmental deformities are displayed above the protein, while the SOX4 variants reported to cause Coffin–Siris syndrome with cardiovascular developmental malformations are shown below in red. COOH, carboxyl-terminus; TAD, transactivation domain; HMG, high-mobility group; and NH2, amino-terminus. Here * means a stop codon.