A novel mutation of Hyaluronan synthase 2 gene in Chinese children with ventricular septal defect

Abstract

As a major product of extracellular matrix (ECM), Hyaluronic acid (HA) is involved in early cardiac development and mainly synthesized by Hyaluronan synthase 2 (HAS2) during embryogenesis. Targeted deletion of HAS2 gene in mice led to obvious cardiac and vascular defects. To clarify the potential association of the mutation in HAS2 with the development of congenital heart disease (CHD), in this study, we sequenced the coding region of HAS2 and identified a novel non-synonymous variant c.A1496T (p.Glu499Val) in one of 100 non-syndromic Ventricular Septal Defect (VSD) patients. The variant was not observed in 250 controls. In addition, to determine the contribution of HAS2 variant in VSD, we compared HA content in supernatant using HA quantitative analysis and found that the mutation obviously affected the HA synthetic activity of HAS2. To our knowledge, this is the first time that the mutation in HAS2 was found in Chinese VSD patients, which suggested that HAS2 may be involved in the etiology of non-syndromic VSD and have the vital function in the development of heart septum.

Figure 1. Mutation analysis of the HAS2 gene.

The sequence chromatogram (reverse strand) shows a heterozygous T>A transition which resulted in the substitution of glutamic acid by valine at codon 499. The black arrows show the wild-type (the controls) and mutant point (patient).

Figure 2. The alignment of the HAS2 sequence with the corresponding segments in different species was shown.

It indicates that glutamic acid at position 499 is highly conserved. The black arrow indicates high conservation.

Figure 3. Effect of E499V mutation on subcellular localization of HAS2.

293T cells were transfected with empty vector pEGFP-N1, pEGFP-HAS2 wild-type, and the E499V mutant separately. After 30 h, transfected cells were then analyzed with an inverted fluorescence microscope to study the protein localization.

Figure 4. Effect of E499V mutation on HA synthesis. 293T cells were transfected with empty vector pcDNA3.1 (+), pcDNA3.1-HAS2 wild-type and the E499V mutant separately.

HA content in media was determined as described in the methods section. Results are representative of three separate experiments. The significance of differences was calculated using the independent-samples t test. *p<0.05, **p<0.01 versus empty vector pcDNA3.1 (+); #p<0.05, ##p<0.01 versus wild-type.