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The HHEX gene is not related to congenital heart disease in 296 Chinese patients 
 
The HHEX gene is not related to congenital heart disease in 296 Chinese patients
  Xiao-Peng Deng, Li-Xi Zhao, Bin-Bin Wang, Jing Wang, Long-Fei Cheng, Zhi Cheng, Pei-Su Suo, Hui Li, Xu Ma
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The HHEX gene is not related to congenital heart disease in 296 Chinese patients

Xiao-Peng Deng, Li-Xi Zhao, Bin-Bin Wang, Jing Wang, Long-Fei Cheng, Zhi Cheng, Pei-Su Suo, Hui Li, Xu Ma

Shenyang and Beijing, China

Author Affiliations: Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang 110004, China (Deng XP, Li H); Graduate School of Peking Union Medical College, Beijing 100730, China (Zhao LX, Wang BB, Wang J, Cheng LF, Cheng Z, Suo PS, Ma X); National Research Institute for Family Planning, Beijing 100081, China (Zhao LX, Wang BB, Wang J, Cheng LF, Cheng Z, Suo PS, Ma X); World Health Organization Collaborating Centre for Research in Human Reproduction, Beijing 100081, China (Ma X)

Corresponding Author: Hui Li, Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, No. 36 Sanhao Street, Heping District, Shenyang 110004, China (Tel: +86 024 83955179; Fax: +86 024 83955092; Email: FCKLIHUI@126.com)

doi: 10.1007/s12519-013-0430-4

Background: The hematopoietically expressed homeobox (HHEX) gene is an important determinant of mammalian heart development. This study aimed to identify the potential mutations of the gene in Chinese patients with congenital heart disease (CHD).

Methods: We collected 296 CHD patients and 200 controls, and classified the cardiac deformities. Then we conducted sequence analyses of the HHEX gene in those patients.

Results: In all the CHD patients, we did not find any causative mutations in the coding region of the HHEX gene.

Conclusion: To our knowledge, this is the first study to examine the HHEX gene in non-symptomatic CHD cases, and this has expanded our knowledge about its etiology.

Key words: congenital heart disease; genetics; HHEX; transcription factor

World J Pediatr 2013;9(3):278-280


Introduction

Cngenital heart disease (CHD) is related to abnormal cardiac development and is a major cause of morbidity and mortality in human newborns, affecting 1%-2% of live births.[1] Transcription factors are known to play significant roles in the complex biological processes governing cardiac morphogenesis. Homeobox genes play critical roles in regulating the tissue-specific gene expression that is essential for tissue differentiation, as well as in determining temporal and spatial patterns of development. Mutations in the mouse hematopoietically expressed homeobox (Hhex) gene results in abnormal cardiac development and defective vasculogenesis.[2]

The human hematopoietically expressed homeobox (HHEX) gene spans about 5.7 kb on chromosome 10q24, comprises four exons, and encodes a 270-amino-acid protein.[3,4] Studies[2,5,6] on the functions of the HHEX gene in avian, murine, Xenopus, zebrafish and human cells have shown that the essential roles of the HHEX gene are similar in all vertebrate species. In mice, Hhex is expressed in the developing blood island, contributing to the murine cardiovascular system. Hhex is also expressed in the endothelium of the developing vasculature, in the developing heart and, in the ventral foregut endoderm. The HHEX gene is a major cardiac determinant, responsible for mammalian heart development. It also functions as a transcriptional repressor that correlates with heart-inducing activities.[2,7]

We hypothesized that the HHEX gene possibly contributed to the development of CHD in humans. We attempted to identify potential pathogenic HHEX mutations in 296 Chinese children with CHD, thereby providing insights into its etiology.

Methods

Study population

In this study there were 296 children with non-symptomatic CHD and 200 controls without cardiac phenotype. All of the children were recruited from Lanzhou University. Informed consent was obtained from patients' parents or guardians. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committee of the National Research Institute for Family Planning. Clinical assessment of the patients included anthropometric measurement, physical examination for dysmorphism and malformation, and radiological evaluation. The patients also underwent chest X-ray examination, electrocardiogram, and ultrasonic echocardiogram.

DNA analysis

Genomic DNA was extracted from peripheral blood leukocytes using a QIAamp blood kit (Qiagen, Hilden, Germany). The HHEX is located on 10q24 and is encoded by four exons. For mutational analysis, the four exons and nearby introns were amplified using polymerase chain reaction and three pairs of HHEX-specific primers (Table 1). Amplicons were sequenced using appropriate primers and a BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequencing was carried out on an automated sequencer (ABI 3730XL; Applied Biosystems).

Results

Clinical and molecular findings from patients with cardiac defects are shown in Table 2. Ventricular septal defects, atrial septal defects, patent ductus arteriosus, tetralogy of Fallot, pulmonary atresia or stenosis, double outlet right ventricle, pulmonary hypertension, and other complex cardiac malformations are all indicated. Sequence analysis of the HHEX did not show any non-synonymous variance in the coding regions.

Discussion

Homeobox genes are an evolutionarily conserved class of transcription factors that are key regulators during developmental processes, such as regional specification, patterning and differentiation. Moreover, homeobox genes play crucial roles in specifying cell identity and cell positioning during embryonic development. Mutations in these genes can cause dramatic developmental defects.[8,9]

Animal experiments have shown the significance of the HHEX in vertebrate cardiac development. Loss of the HHEX expression in the endoderm blocks heart development in Xenopus embryos. Similarly, knocking out Hhex in the mouse results in structural heart defects such as ventricular septal defect, valve hypoplasia, and thin myocardia.[2] However, the cardiac phenotypes of CHD in our study included ventricular septal defects, but there was lack of evidence of other defects detected in mice with Hhex mutations. The absence of HHEX mutations could be caused by selection bias owing to different cardiac defects in our study. Antagonists of canonical Wnt/β-catenin signaling have a vital role in the mesoderm during the initiation of cardiogenesis in mouse and Xenopus embryos.[7,10,11] Transcriptional repression of the HHEX correlated with heart-inducing activities in tissues adjacent to the heart forming region in Xenopus, chick and mouse embryos, suggesting an evolutionarily conserved cardiogenic function for the HHEX.[7,12-14] As the HHEX is highly conserved between humans and mice, we speculate that CHD patients might contain mutations in the HHEX.

In this study, we screened potential causative mutations in the HHEX in Chinese children with CHD. However, we did not find any pathogenic mutations. Previously, Balasubramanian[15] analyzed the sequence of the HHEX in three unrelated patients with pancreatic agenesis and congenital heart defects by direct sequencing, also found no mutation.

The negative results of direct sequencing in this study did not suggest abnormal functions of the HHEX protein in CHD may be disregarded. Trans-acting factors and cis-acting elements, which are critical for gene transcription and protein synthesis, could affect the expression of the HHEX. Abnormal cardiac morphogenesis may occur when spatial and temporal transcription of the HHEX is altered, or when the mRNA is not correctly spliced. Over-expression of the HHEX in Xenopus laevis causes disruption to developing vascular structures and an increase in the number of vascular endothelial cells.[16]

A 21-year study from the Czech Republic reported 1604 cases of prenatal diagnosis for CHD. The study showed that only 479 (29.9%) of the 1604 prenatally diagnosed fetuses were alive at the end of the study.[17] In the present study, we did not find any diagnostic mutations in the the HHEX coding regions. Possibly, there are two reasons for this finding. First, any mutation in the coding sequence of the HHEX results in early and severe cardiac defects, similar to those observed in Hhex knockout mice. Second, it may be simply due to the relationship between the HHEX and the risk of CHD in Chinese patients.

In conclusion, our understanding of the functions of the HHEX remains limited, and its exact role in heart development is still unknown. Therefore, further study is required to determine the important role of the HHEX during heart development, which may give an insight into the etiology of heart defects.

Funding: This work was supported by the National Basic Research Program of China (2010CB529500), the Doctorate Tutor Program Foundation of Ministry of Education (20092104110011) and the scientific and technology program of Liaoning Province (2011225017).

Ethical approval: This study was approved by the Ethics Committee of the National Research Institute for Family Planning.

Competing interest: There were no competing interests.

Contributors: Deng XP and Zhao LX contributed equally to this paper: performing the experiment and drafted the manuscript. Zhao LX participated in study design and coordination and helped to draft the manuscript. Wang BB did manuscript revision. Wang J carried out the molecular genetic studies. Cheng LF, Cheng Z, and Suo PS participated in sample collection.

References

1   Hoffman JI, Kaplan S. The incidence of congenital heart disease. J Am Coll Cardiol 2002;39:1890-1900.

2   Hallaq H, Pinter E, Enciso J, McGrath J, Zeiss C, Brueckner M, et al. A null mutation of Hhex results in abnormal cardiac development, defective vasculogenesis and elevated Vegfa levels. Development 2004;131:5197-5209.

3   Morgutti M, Demori E, Pecile V, Amoroso A, Rustighi A, Manfioletti G. Genomic organization and chromosome mapping of the human homeobox gene HHEX. Cytogenet Cell Genet 2001;94:30-32.

4   Crompton MR, Bartlett TJ, MacGregor AD, Manfioletti G, Buratti E, Giancotti V, et al. Identification of a novel vertebrate homeobox gene expressed in haematopoietic cells. Nucleic Acids Res 1992;20:5661-5667.

5   Ghosh B, Ganea GR, Denson LA, Iannucci R, Jacobs HC, Bogue CW. Immunocytochemical characterization of murine Hex, a homeobox-containing protein. Pediatr Res 2000;48:634-638.

6   Thomas PQ, Brown A, Beddington RS. Hex: a homeobox gene revealing peri-implantation asymmetry in the mouse embryo and an early transient marker of endothelial cell precursors. Development 1998;125:85-94.

7   Foley AC, Mercola M. Heart induction by Wnt antagonists depends on the homeodomain transcription factor Hex. Genes Dev 2005;19:387-396.

8   Harvey RP. NK-2 homeobox genes and heart development. Dev Biol 1996;178:203-216.

9   Newman CS, Krieg PA. Tinman-related genes expressed during heart development in Xenopus. Dev Genet 1998;22:230-238.

10 Marvin MJ, Di Rocco G, Gardiner A, Bush SM, Lassar AB. Inhibition of Wnt activity induces heart formation from posterior mesoderm. Genes Dev 2001;15:316-327.

11 Lickert H, Kutsch S, Kanzler B, Tamai Y, Taketo MM, Kemler R. Formation of multiple hearts in mice following deletion of beta-catenin in the embryonic endoderm. Dev Cell 2002;3:171-181.

12 Arai A, Yamamoto K, Toyama J. Murine cardiac progenitor cells require visceral embryonic endoderm and primitive streak for terminal differentiation. Dev Dyn 1997;210:344-353.

13 Schneider VA, Mercola M. Spatially distinct head and heart inducers within the Xenopus organizer region. Curr Biol 1999;9:800-809.

14 Yatskievych TA, Pascoe S, Antin PB. Expression of the homebox gene Hex during early stages of chick embryo development. Mech Dev 1999;80:107-109.

15 Balasubramanian M, Shield JP, Acerini CL, Walker J, Ellard S, Marchand M, et al. Pancreatic hypoplasia presenting with neonatal diabetes mellitus in association with congenital heart defect and developmental delay. Am J Med Genet A 2010;152A:340-346.

16 Newman CS, Chia F, Krieg PA. The XHex homeobox gene is expressed during development of the vascular endothelium: overexpression leads to an increase in vascular endothelial cell number. Mech Dev 1997;66:83-93.

17 Marek J, Tomek V, Skovránek J, Povysilová V, Samánek M. Prenatal ultrasound screening of congenital heart disease in an unselected national population: a 21-year experience. Heart 2011;97:124-130.

Received May 29, 2012Accepted after revision August 13, 2012

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