Identification of TBX2 and TBX3 variants in patients with conotruncal heart defects by target sequencing

Abstract

BackgroundConotruncal heart defects (CTDs) are heterogeneous congenital heart malformations that result from outflow tract dysplasia; however, the genetic determinants underlying CTDs remain unclear. Increasing evidence demonstrates that dysfunctional TBX2 and TBX3 result in outflow tract malformations, implying that both of them are involved in CTD pathogenesis. We screened for TBX2 and TBX3 variants in a large cohort of CTD patients (n = 588) and population-matched healthy controls (n = 300) by target sequencing and genetically analyzed the expression and function of these variants.ResultsThe probably damaging variants p.R608W, p.T249I, and p.R616Q of TBX2 and p.A192T, p.M65L, and p.A562V of TBX3 were identified in CTD patients, but none in controls. All altered amino acids were highly conserved evolutionarily. Moreover, our data suggested that mRNA and protein expressions of TBX2 and TBX3 variants were altered compared with those of the wild-type. We screened PEA3 and MEF2C as novel downstream genes of TBX2 and TBX3, respectively. Functional analysis revealed that TBX2R608W and TBX2R616Q variant proteins further activated HAS2 promoter but failed to activate PEA3 promoter and that TBX3A192T and TBX3A562V variant proteins showed a reduced transcriptional activity over MEF2C promoter.ConclusionsOur results indicate that the R608W and R616Q variants of TBX2 as well as the A192T and A562V variants of TBX3 contribute to CTD etiology; this was the first association of variants of TBX2 and TBX3 to CTDs based on a large population.Electronic supplementary materialThe online version of this article (10.1186/s40246-018-0176-0) contains supplementary material, which is available to authorized users.

Whole genome sequencing delineates regulatory and novel genic variants in childhood cardiomyopathy

“…For additional confirmation of a difference in the mRNA expression level of the gene harboring the variant compared to the wild type sequences, we determined the relative mRNA expression using qRT-PCR 112 . Total RNA was extracted from patient LV myocardium using mirVana™ PARIS™ RNA and native protein purification Kit (Invitrogen, Carlsbad, California, USA) following the manufacturer’s protocol.…”

A loss‐of‐function mutation p.T256M in NDRG4 is implicated in the pathogenesis of pulmonary atresia with ventricular septal defect (PA/VSD) and tetralogy of Fallot (TOF)

“…and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, human) was used as an internal control [31,36]. Primer sequences of NDRG4, GAPDH, P27, cyclin D1, cyclin E, caspase 3 and caspase-9 were listed in Supplementary Table S1.…”

“…The proteins were subjected to 10% SDS-PAGE and were then transferred onto nitrocellulose membranes (Millipore, USA) and immunostained with rabbit anti-NRDG4 antibody (1:500, Novus, USA), rabbit anti-GAPDH antibody (1:5000, Proteintech, USA) rabbit anti-P27 antibody (1:1000, CST, USA) at 4 °C overnight. The membranes were incubated with HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) secondary antibody (1:10000, Proteintech, USA) [31,36].…”

“…Cell nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI) (Servicebio, China). A Leica SP8 microscope was used for image analysis [31,36].…”

“…Gene sequencing and variant analysis were performed as described previously [21,22]. Briefly, we used the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) to extract the genomic DNA of participants in accordance with the manufacturer's instructions.…”