GATA4 screening in Iranian patients of various ethnicities affected with congenital heart disease: Co‐occurrence of a novel de novo translocation (5;7) and a likely pathogenic heterozygous GATA4 mutation in a family with autosomal dominant congenital heart disease

Abstract Background Congenital heart disease (CHD) is the most common birth defect and a major health problem around the world. However, its exact etiology has remained unclear. Among various genetic contributing factors, GATA4 transcription factor plays a significant role in the CHD pathogenesis. In this study, GATA4 coding sequence was screened in Iranian patients of various ethnicities. Methods Sixty six individuals with familial CHD referred to our center were recruited in this study. After receiving written informed consent from each individual or their parents, chromosomal analyses and GATA4 variant screening were performed. Pathogenicity of the suspected variants was evaluated using available online software tools: CADD, Mutation Taster, SIFT, and PolyPhen‐2. Results A total of twelve GATA4 variants were detected including five intronic, 2 exonic and 3 polymorphisms as well as 2 missense mutations, the c.1220C>A and c.1309G>A. Unlike the c.1220C>A, the likely pathogenic heterozygous c.1309G>A has not been previously associated with any phenotype. Here, we not only report, for the first time, a c.1309G>A‐related CHD, but also report a novel de novo balanced translocation, 46,XY,t(5;7)(qter13;qter11), in the same patient which may have influenced the disease severity. Conclusion From screening GATA4 sequence in 66 Iranian patients of various ethnicities, we conclude that cytogenetic analysis and PCR‐direct sequencing of different candidate genes may not be the best approach for genetic diagnosis in CHD. Applying novel approaches such as next‐generation sequencing (NGS) may provide a better understating of genetic contributing factors in CHD patients for whom conventional methods could not reveal any genetic causative factor.


| INTRODUC TI ON
Congenital heart disease (CHD) is the most common birth defect with various incidence of 4-50 per 1000 live births, worldwide, 1 consisting different types of cardiac malformations from severe forms such as tetralogy of Fallot (TOF) and transposition of the great arteries (TGA) to mild forms such as bicuspid aortic valve (BAV). 2 Although 8% of CHD cases are estimated to result from chromosomal abnormalities and single gene disorders 3 , the exact etiology of CHD is mostly unknown (about 75% of cases). 4 Previous studies indicated some transcription factors which play significant role in heart development including GATA binding protein 4 (GATA4), NOTCH, myosin heavy chain 6 (MYH6), NK2 homeobox 5 (NKX2-5), Zic family member 3 (ZIC3), T-box 5 (TBX5), and T-box 20 (TBX20). [5][6][7] Among these transcription factors, GATA4, located on 8p23.1, is expressed in endocardial, myocardial, and mesenchymal cells for suitable cardiac septation. 8 As a critical zinc finger transcription factor, it is important in different types of CHD causality, that is, TOF, ventricular septal defect (VSD), atrioventricular septal defect (AVSD), atrial septal defect (ASD), patent ductus arteriosus (PDA), and pulmonary valve stenosis (PS). 5,6,9,10 Functional experiments in different animal models such as mice, fly, and fish demonstrated that any change in GATA4 sequence can affect cardiac development. 11,12 Full length of GATA4 cDNA is 3371 bp, and it contains 6 exons. The GATA4 protein has 442 amino acids and binds to the GATA motif of target genes involved in cardiogenesis. 13 To date, based on Human Gene Mutation Database (HGMD) (www.hgmd.cf.ac.uk), 114 mutations in GATA4 gene have been reported in the CHD cases.
Given there are few studies [14][15][16] which surveyed CHD etiology in Iranian population, we evaluated the entire coding sequence of GATA4 gene in sixty-six familial CHD patients. This study is the first report of a clinical significance of a pathogenic GATA4 mutation in an Iranian patient with BAV. Also, this study is the first report of a novel balanced translocation 5,7 that may or may not related with heart defect phenotype in the same patient.

| Study subjects and samples
Sixty-six patients affected from any types of CHD, as confirmed by echocardiography, who had been referred to the Rajaei Heart Center (Tehran, Iran) between November of 2015 and the end of 2017 as well as some of their family members (from fifty-five pedigrees) were enrolled in this study. A comprehensive genetic counseling was performed for all families. We previously investigated the genetic cause of CHD in these patients using sequence analysis of the NKX2-5 17 , ZIC3, NODAL, FOXH1, GJA1 genes, and MLPA (multiplex ligation-dependent probe amplification) for common reported deletions and array CGH (array comparative genomic hybridization) for genomic imbalances; no change was detected. In this study, karyotype analysis, whole-exome sequencing (WES) in the case (CHD-7) that we found structural changes in his chromosomes, and GATA4 variant were checked; briefly, peripheral blood from all the patients was collected in heparin and EDTA tubes, both. Heparinized blood was used for karyotyping, based on the standard protocols as described previously 18

| Primers, PCR, and direct Sanger sequencing
Five millilitre peripheral blood from each subject was collected in EDTA-containing tubes, and genomic DNA was extracted according to our in-house method based on the standard salting-out technique.
To amplify the entire GATA4 coding sequence as well as exon-intron boundaries, six primer pairs were designed (Table S1)

| Whole-exome sequencing
DNA sample of candidate case (CHD-7) was subjected to WES at Macrogen (Seoul, South Korea). 10 ng of DNA was applied for exome enrichment by SureSelect XT Library Prep Kit. WES was performed on an Illumina HiSeq 4000 according to the manufacturer's protocol (Illumina) and generating paired-end reads, that is, read quality of >20 and depth of >5 were applied for further analyses.
It should be noted that the overall coverage of the whole exome (read depth 1X) was 99.8% but this was only 68.4% coverage with a read depth of 50X, more than 90% reads have average Phred scores above 20, the mean of per sequence quality scores was more than 36, per sequence GC content was ~59%, and the sequence length distribution was about 150 bp.

| Cytogenetic analysis
Cytogenetic analysis of the patients for genome and chromosomal mutations appeared normal in all, but one, the CHD-7, for whom G-banding revealed a novel balanced translocation between chromosomes 5 and 7 (46,XY,t(5;7)(qter13;qter11)) in 20 metaphase spreads that were studied. Segregation analysis showed the absence of that translocation in both parents. To investigate the presence of any other pathogenic genomic mutation linked to CHD, WES was performed on the CHD-7 patient. Subsequently, a heterozygous c.1309G>A (p.Gly437Arg) variation in GATA4 coding sequence was identified. This case is further described below. A 14-year-old boy to whom we are referring as CHD-7 throughout the study was admitted to our hospital suffering from shortness of breath. Clinical examination and echocardiography unraveled the presence of BAV, as shown in Figure 1A. Through genetic counseling, the history of CHD was confirmed in the pedigree. Proband's mother (III-8 in Figure 1B), a 40-year-old lady, had been diagnosed with ASD at the age of 28. Her 62-year-old mother (II-4 in Figure 1B) was also suffering from the shortness of breath, started when she was 50 years old. However, she did not agree to participate in this study.
None of the other family members who were studied including II-3, III-7, and IV-3 individuals ( Figure 1B Table 1, and CHD types in our studied population are indicated in Table S2.

| D ISCUSS I ON
Sixty six CHD patients of various ethnicities within the Iranian population were screened in this study. Since our center is one the major country's referral hospitals for cardiogenetic diseases, the studied patients were of diverse ethnicities including Fars, Azeri, Kurd, Lur, Gilaki, Mazandarani, Arab, Turkmen, and Baloch. The presence of the disease in the patient was confirmed by expert cardiologists.
G-banding screening of all the patients resulted in identification of a de novo balanced chromosomal translocation, t(5;7) (qter13;qter11), in a 14-year-old boy with BAV. None of his parents including the mother who is suffering from ASD carry the translocation. Using WES data for CHD-7, we tried to identify chromosomal breakpoint locations but we failed. 30 In addition, no CHD-related CNV was identified throughout the chromosomes 5 and 7, as far as our WES data could have provide. Through WES of the mentioned patient (CHD-7), a heterozygous c.1309G>A (p.Gly437Arg) variation in GATA4 (one of the important CHD-associated genes) was identified to which no defined phenotype has been correlated.
The p.Gly437Arg mutation in our patient is predicted likely pathogenic based on most amino acid change predictors such as CADD (score: 32). It should be noted that another mutation at the same base, c.1309G>T (p.Gly437Trp), is described in ExAc with predicted pathogenicity. The presence of the variant was confirmed in the patient's mother and grandmother who seem to be affected from a different types of CHD. Such a phenotypic difference could raise the hypothesis that the patient's phenotype may have been modified by the presence of the chromosomal translocation. Though we stopped further investigation due to the unwillingness of the family to further participate in the study, this chromosomal abnormality should be noted in genetic counseling as individuals with balanced chromosomal translocations might have fertility difficulties such as infertility or giving birth to babies with congenital anomalies, despite normal phenotypes. 31 To the best of our knowledge, 16 c.1138G>A in their studied patient/control groups that has no effect in the CHD pathology. c.1129A>G was also reported previously in normal controls as well as CHD patients in some documents. 29,39,40 Variable expressivity has been reported in GATA4-causing CHDs. 41 In a study by Tomita-Mitchell et al, 9  F I G U R E 1 Clinical and GATA4 genetic analysis of the CHD-7 patient. A, Echocardiogram of the patient having bicuspid aortic valve (BAV). B, The CHD-7 patients in which the proband (IV-2) and his affected mother (III-8) are indicated with filled symbols. The proband's grandmother (II-4) was suspected for CHD but did not agree with further examination. C, The G-banded karyotype of the CHD-7 patient. Arrow is pointing to the position of the identified t(5;7)(qter13;qter11) balanced translocation. D, Partial chromatograms show the results of GATA4 exon 7 sequencing in CHD-7 patient (IV-2), proband's mother (III-8 In conclusion, here we report for the first time a de novo balanced translocation may or may not related to the CHD phenotype. We also present c.1309G>A as the BAV causing likely pathogenic mutation in GATA4 gene. As mentioned before, no clinical evidence for that mutation has been presented so far. Correlating GATA4 mutations to phenotypes in congenital BAV, ASD, and VSD provides a ground for early diagnosis of these defects in families who have affected individuals.

AUTH O R S ' CO NTR I B UTI O N S
SK, TSH, and NM wrote the article. SK carried out the experiments. NM, AB, MM, and HA contributed to patient's diagnosis.
HRZ and MCA performed computational analysis of the data. SK, TSH, and NM contributed to project management, genetic analyses, interpretation of data, revision of the initial manuscript, and final approval.

E TH I C A L A PPROVA L
Informed consent has been obtained by the authors.
F I G U R E 2 GATA4 genetic and clinical analysis of the CHD-33 pedigree. A, The family pedigree in which proband at the age of 6 months old (III-1) displayed ventricular septal defect (VSD); also, his father (II-5) was affected with VSD. B, Presents echocardiogram of the patient (III-1) with VSD.