Genetic advances in sporadic tetralogy of Fallot: discovery of novel HAND1 promoter variants

One of the most common forms of congenital heart disease is Tetralogy of Fallot (TOF). The global birth prevalence of TOF is 3.3–3.9 per 100,000 live births.¹ TOF is accompanied by a tetrad of anomalies, which include a ventricular septal defect (VSD), pulmonary stenosis, an aorta that overrides the ventricular septum, and right ventricular hypertrophy.² The diagnosis of TOF can be made prenatally, usually discovered on an anatomy ultrasound. TOF may also be discovered postnatally in a neonate who presents with cyanosis of unknown etiology. Postnatal echocardiography is the gold standard to confirm the anatomy and diagnosis of TOF along with other possible associated anomalies. The exact embryologic pathogenesis of how TOF occurs is unknown, however, it is hypothesized that defective partitioning of the conotruncus during septation accompanied by incomplete rotation results in two unequally sized vessels resulting in a conal septum that deviates far anteriorly according to Wise-Faberowski, et al.³. Several variants of TOF exist along with additional anomalies, including anomalous pulmonary venous drainage, atrial septal defect, and an interrupted inferior vena cava, to name a few.³ Additionally, there are genetic conditions associated with TOF including DiGeorge, CHARGE, VACTERL, and Trisomy 21.³ Efforts have been made to identify cellular mutations contributing to TOF, including Jagged1 (Jag1) mutations⁴ and mutations in the ZFPM2/FOG2 gene.⁵ Despite these findings, there continues to be a gap in knowledge regarding factors contributing to the cellular pathogenesis of TOF.

Since the contributing factors to TOF development are unclear, there is a need to investigate abnormalities occurring at the cellular level. HAND1 (eHAND, HXT, Thing 1) and HAND2 (dHAND, HED, Thing 2) are bHLH transcription factors that are involved in determining the formation of both the right and left ventricles.⁶ In this journal issue, Qi, et al.⁷, build on their prior discoveries of variants in the gene promoter regions such as ISL1, MYH6, and CITED2 in TOF patients, and hypothesize that variants in the HAND1 gene promoter could play a role in the formation of TOF. Their study evaluates variants of the HAND1 gene promoter region in the pathogenesis of TOF, which has not been explored in this population. Herein, we comment on the strengths and limitations of the Qi et al. translational study.⁷

The authors employ a robust study design, obtaining ethics board approval and utilizing a large sample size. They also ensure the controls have no echocardiographic evidence of heart disease and carefully account for potential confounders such as age and sex. Qi, et al.⁷ clearly state their hypothesis that “variants in HAND1 gene promoter may also be involved in the formation of TOF” and test it by investigating the variants in the HAND1 gene promoter region in TOF patients by comparing patients to healthy controls. The authors provide an in-depth explanation of DNA sample extraction and sequence analysis along with plasmid construction, cell culture, and cell transfection. The authors utilize HEK-293 cells, probably owing to their reliable fast growth and propensity for transfection. Additionally, Sanger sequencing is used, which is the gold standard for accurately detecting single nucleotide variants and small insertions/deletions.⁸ The authors are very meticulous in articulating the details of the study, including cell mediums and cell plating, which enhances the study’s reproducibility. Specific internal controls are utilized and described for each experiment, including Renilla luciferase that provides an internal control value to which expression of the experimental firefly luciferase reporter gene may be normalized.⁹ Each experiment described was independently repeated by the authors three times. To demonstrate the reproducibility of the results in HL-1 cells (atrial myocyte cell lines), they also replicated the experiment in HEK-293 cells (human embryonic kidney cells), which is an additional strength of the study. The JASPAR database is utilized to predict whether variants in the HAND1 promoter region would disrupt or create transcription factor binding sites. The authors also further analyze the three identified TOF variants using electrophoretic mobility shift analysis (EMSA) analysis, showing the effect of the mutant genes on the binding of paralogous transcription factors. The results of this additional analysis suggest the three variants significantly alter the binding ability of transcription factors that may be important in cardiac embryogenesis, providing tangible information that can assist with future studies. Qi, et al.⁷ discovered all three variants identified in the promoter region of the HAND1 gene decreased the transcriptional activity of HAND1 gene in the in vitro assay, causing altered cellular function.

This study’s strengths lie in its robust methodology listing intricate details of each experiment performed, making a compelling case for the validity of the findings and the reproducibility of the experiments. It effectively summarizes findings on all identified variants, along with discovering three new variants found exclusively in TOF patients. However, some limitations exist. The study evaluated the transcriptional activity of the HAND1 gene in vitro; however, the study lacks an in vivo model to validate these findings. The authors discuss this limitation briefly and that further confirmation of discovered variants is needed in animal models. They also discuss the need for evaluating the interaction of the identified variants in the promoter region of the HAND1 gene with downstream genes, which would need to be further verified to strengthen the study findings. It is unclear if the authors had any criteria for the inclusion and exclusion of study participants. While the authors note the absence of a familial history of genetic disorders among participants, it remains unclear whether the cases and controls were free of non-cardiac anomalies. This detail could be significant, as five other variants in the promoter region of the HAND1 gene were identified in both cases and controls, suggesting that HAND1 gene perturbations may play a role in non-cardiac anomalies. Furthermore, the recruitment of subjects was limited to Chinese participants, reducing the generalizability of their findings. Lastly, the authors discuss the findings of three new variants; however, they do not give the prevalence of these findings amongst the studied TOF patients. Nevertheless, given the gap in knowledge regarding factors contributing to the cellular pathogenesis of TOF, the authors deserve recognition on this study for discovering three variants in the promoter region of HAND1 genes that could be analyzed further in future studies. However, as emphasized, in vivo studies are highly recommended to help further validate the authors’ findings.