Skip to main content
NCBI home page
Molecular Syndromology logoLink to Molecular Syndromology
. 2025 Jun 10;17(2):112–122. doi: 10.1159/000546625

HIRA, NKX2-5, and GATA4 Alterations versus Cardiac Malformations Related to 22q11.2 Deletion Syndrome

Bruna Lixinski Diniz a, Desirée Deconte a, Ana Kalise Böttcher b, Rafaela Mergener a, Bruna Baierle Guaraná a, Natasha Malgarezi de Moraes a, Rafael Fabiano Machado Rosa c, Paulo Ricardo Gazzola Zen c,✉
PMCID: PMC13046320  PMID: 41940406

Abstract

Introduction

Congenital heart disease (CHD) comprises a wide spectrum of structural defects. However, the etiology of a large proportion of CHDs remains undefined. Among the genetic causes, 22q11.2 deletion syndrome is the condition which most stands out. This association is related to many cardiac embryonic development genes being in the chromosome 22 region, as well as being a region with a high probability of errors in gene recombination, influencing normal levels of gene expression and affecting a gene’s copy number.

Objective

This study aimed to compare molecular findings using multiplex ligation-dependent probe amplification assay in patients presenting CHD with a previous fluorescence in situ hybridization (FISH) diagnosis of 22q11.2DS versus patients without known genetic disorder.

Results

All patients had CHD and facial dysmorphia. Patients who had been previously diagnosed by FISH were found to have the exact same deletion size, low-copy-number repeat sequences and genes involved. GATA4 when deleted or duplicated in different exons (1 and 6) showed distinct congenital heart defect phenotypes. Patients who did not have their diagnosis defined by FISH showed different molecular results, ranging from normal findings to alterations in the GATA and NXK2 genes.

Conclusion

Molecular diversity in cardiac malformations is a reality and a great challenge since genotype-phenotype correlation is hindered. Therefore, new insights on that matter should be considered: 22q11.2 deletion syndrome should only be linked to the chromosome 22 region or is there a phenotype variability to be looked at that involves a broader genomic environment?

Keywords: HIRA gene, NKX2-5 gene, GATA4 gene, 22q11.2 deletion syndrome

Introduction

22q11.2 deletion syndrome (22q11.2DS), also known as DiGeorge syndrome (DGS) (OMIM #188400), is characterized by a microdeletion in the long arm of chromosome 22 and is widely known for its clinical and molecular heterogeneity [1]. Among the more than 180 clinical characteristics already described, congenital heart disease (CHD) stands out as one of the indicative pieces of evidence for beginning investigations into the syndrome [2].

CHDs constitute a developmental malformation of the heart, aorta, or other large blood vessels that make up the most common forms of major birth defects [3], affecting 1.3−1.7% of newborns per year in Brazil (Brazil Ministry of Health, 2021, http://tabnet.datasus.gov.br/cgi/deftohtm.exe?sinasc/cnv/nvuf.def). CHD has its genetic basis determined by the association of single or multiple genes, chromosomal changes, and multifactorial causes. Studies have demonstrated a high frequency of copy number variation (CNV) in the human genome, which are likely to contribute to genetic heterogeneity and phenotypic variability [4, 5]. Other genes outside the 22q region can also influence cardiac development. CNVs in GATA4 and NKX2–5 genes have been identified among mechanisms that may explain some CHDs, since all these are transcription factors (TFs) strongly involved in cardiogenesis [6].

Molecular biology and the arrival of several new technologies have made it possible to discover genes that can interact with each other or with external factors, generating a predisposition profile to the development of the disease [7, 8]. Overall, 60–80% of the individuals diagnosed with 22q11.2DS have cardiac malformation that can vary in severity [1, 2]. Tetralogy of Fallot and pulmonary atresia with ventricular septal defect (VSD) are regular findings in patients with 22q11.2DS; however, their prevalence is yet to be established [9]. Through the identification of CHD in these individuals, hypotheses were created that a mechanism that leads to an alteration in embryonic development could be involved [10].

Different cytogenetic methodologies have emerged and have been improving genetics research in the last decade. Fluorescence in situ hybridization (FISH) is the gold standard method for 22q11.2DS diagnosis. However, in the last 2 decades, multiplex ligation-dependent probe amplification (MLPA) and microarray analysis became effective tools alongside FISH [11, 12]. MLPA is a variation of the multiplex polymerase chain reaction method. It is used to identify CNVs, including deletions and duplications in CHDs predisposition genes [13].

The variety of molecular technologies allowed individuals to be diagnosed more frequently, providing new insights into the molecular mechanism behind 22q11.2DS. Furthermore, since the 22q11.2DS critical region has a complex molecular structure, combining more methodologies in 22q11.2DS molecular investigation may be a crucial ally in order to provide a more accurate diagnosis for patients as well as deepen our understanding of this syndrome’s challenging and intricate genetic roots [14].

Knowledge of normal cardiac development and the mechanisms of CHDs are essential to daily practice, as much for the daily evaluation of heart diseases as for genetic counseling [15]. This study assessed two groups of individuals presenting CHDs, suspected, or diagnosed with 22q11.2DS, who have previously used only one molecular approach. Our intent was to determine the deletion size of subjects previously diagnosed by FISH and investigate the genetic profile of individuals that were yet to be molecularly diagnosed.

Methods

The 20 subjects selected in this study are part of a large cohort previously described by Diniz et al. [16]. The clinical and cardiac characteristics can be found in the reference article. All patients had CHD and facial dysmorphia (skull, nose, eyes, and ears malformations). Participants were recruited from cardiac intensive care units of three health institutions in Southern Brazil.

Our research carried out screening with ten patients previously diagnosed with 22q11.2DS by FISH and randomized another ten patients who did not have a previous molecular diagnosis by the same methodology. CHDs were described based on echocardiography, cardiac catheterization, and surgical description following the classification suggested by Botto et al. [17].

Molecular cytogenetic testing by FISH was previously performed using the commercial Vysis LSI DiGeorge/TUPLE1 (Abbott Molecular) region dual-color probe, which identifies deletions of band 22q11.2. Blood samples in EDTA were taken for subsequent DNA extraction according to the PUREGENE protocol (https://web.emmes.com/study/hbb/public/EN-Gentra-PuregeneHandbook-2011.pdf).

For subjects with positive result for 22q11.2DS by FISH, MLPA assay was performed using the SALSA MLPA P250-B2 DiGeorge to determine deletion size throughout low-copy-number repeat sequences (LCRs) involved in the deletion. This kit detects not only deletions or duplications in the human 22q11.2 region, but also contains probes that can assess the copy number status at 4q, 8p, 9q, 10p, 17p, and 22q13 chromosome regions. All alterations detected are causative of DGS, DGS type II, or disorders with phenotypic characteristics of DGS. Probe sequences are shown in Table 1.

Table 1.

P250 probes arranged according to chromosomal location

Gene Partial sequence (24 nt adjacent to ligation site)
Cat eye syndrome (CES) region:
 IL17RA GCA​GAG​TTA​TCT-GTC​CTG​CAG​CTG
 SLC25A18 GCA​GTG​AGA​AGA-GTC​GAG​TGA​AGC
 BID CTA​CTG​GTG​TTT-GGC​TTC​CTC​CAA
 MICAL3 GAA​CTA​CCG​CCT-GTC​CCT​GAG​GCA
 USP18 CTC​AGT​CCC​GAC-GTG​GAA​CTC​AGC
End of CES region; Start DiGeorge (DGS) region; probes in region LCR22A – LCR22B:
 CLTCL1 TGT​TGC​CTT​GGT-GAC​CGA​GAC​CGC
 HIRA GGA​GCT​GCT​GAA-GGA​GCT​GCT​ACC
 CDC45 ATG​TTC​GTG​TCC-GAT​TTC​CGC​AAA
 CLDN5 TTC​GCC​AAC​ATT-GTC​GTC​CGC​GAG
 GP1BB CAC​AAC​CGA​GCT-GGT​GCT​GAC​CGG
 TBX1 CCG​GGT​GAA​GCT-TCG​CTG​GCT​GCC
 TBX1 TCC​CTT​CGC​GAA-AGG​CTT​CCG​GGA
 TXNRD2 GGA​GGG​TCA​GGA-GAG​GAG​CTG​CAG
 DGCR8 GGT​AAT​GGA​CGT-TGG​CTC​TGG​TGG
Probes in region LCR22B–LCR22C:
 ZNF74 CAG​GCA​GAT​TAT-TCC​TCG​ATG​CTG
 KLHL22 TCT​TCG​ATG​TTG-TGC​TGG​TGG​TGG
 MED15 (PCQAP) TGG​CAT​TTG​GAT-GAA​GAC​ACA​GGT
Probes in region LCR22C–LCR22D:
 SNAP29 AGG​AGC​AAG​ATG-ACA​TTC​TTG​ACC
 LZTR1 ATG​ATG​AAG​GAG-TTC​GAG​CGC​CTC
End of the commonly-deleted DiGeorge (DGS) region; probes in region LCR22D–LCR22E:
 HIC2 GTT​CCA​GCA​GAT-CTT​GGA​CTT​CAT
 PPIL2 GAA​GAG​CCC​TCA-ACC​AGT​GCC​ACT
 TOP3B GAG​ACA​TGA​TAA-AAT​CCA​GTC​CTT
Probes in region LCR22E–LCR22F:
 RSPH14 (RTDR1) GGT​GTG​TCA​TTT-TGA​CGT​CAT​CCC
 GNAZ TCA​CCA​TCT​GCT-TTC​CCG​AGT​ACA
 RSPH14 (RTDR1) CTC​CTT​GGA​GCT-TCC​CAT​TAA​CAT
 RAB36 AGC​TGG​ATG​CTT-GGA​CGC​GCC​GCT
Probes in region LCR22F–LCR22G:
 SMARCB1 CTT​CGG​GCA​GAA-GCC​CGT​GAA​GTT
 SMARCB1 CAT​CAG​CAC​ACG-GCT​CCC​ACG​GAG
Probe in region LCR22G–LCR22H:
 SNRPD3 CCG​GTG​AGG​TAT-ATC​GGG​GGA​AGC
4q35-qter
 SLC25A4 CAT​CAA​GAT​CTT-CAA​GTC​TGA​TGG
 KLKB1 ATG​CCC​AAT​ACT-GCC​AGA​TGA​GGT
8p23
 PPP1R3B ACC​GAG​CTC​CTA-GAC​AAC​ATT​GTG
 MSRA GCA​ACA​GAA​CAG-TCG​AAC​CTT​TCC
 GATA4 TGG​ATT​TTC​TCA-GAT​GCC​TTT​ACA
9q34.3
 EHMT1 AAA​TGC​TGC​AAA-GCA​CAC​TCA​GGA
 EHMT1 GGA​CCC​CGT​TGA-TGG​AAG​CAG​CCG
10p14
 GATA3 GAG​TGC​CTC​AAG-TAC​CAG​GTG​CCC
 GATA3 AAC​AGC​TCG​TTT-AAC​CCG​GCC​GCC
 TCEB1P3 TGT​AGA​CCA​CAT-GAT​GGA​GAT​TTG
 CELF2 (CUGBP2) GAC​ATT​CAC​TGT-GGA​AAT​TTG​GTG
 CELF2 (CUGBP2) TCC​CCC​GGT​CAT-GGT​CGG​AAA​AGG
 NEBL CTG​GGA​TCC​TTT-TCT​GTT​CAC​TCA
17p13.3
 RPH3AL AGG​CGG​AAT​GTG-ATG​GGG​AAC​GGC
 RPH3AL GTA​GTG​GAC​ACT-TGT​ACG​TGC​ACT
 GEMIN4 AAA​CAG​TGA​TAG-ACG​TCA​GCA​CAG
 YWHAE GCC​ACA​GGA​AAC-GAC​AGG​AAG​GAG
22q13.3
 ARSA GGA​GGA​TCA​GAT-CTC​CGC​TCG​AGA
 SHANK3 ACC​AAC​TGT​GAT-CAG​TGA​GCT​CAG
Open in a new tab

On the other hand, subjects with a negative result for 22q11.2DS by FISH, were tested by MLPA assay with the SALSA MLPA P311 – a kit that can detect deletions or duplications on genes related to CHD outside the 22q11 region. The assay follows the manufacturer’s recommendations (MRC-Holland, Amsterdam, The Netherlands). Probe sequences are shown in Table 2. Molecular analysis was performed by both ABI3130 sequencing and Coffalyser software (MRC-Holland). For each sample analyzed, commercial controls in triplicate were used.

Table 2.

P311 probes arranged according to chromosomal location

Region Partial sequence (24 nt adjacent to ligation site)
GATA4
 Upstream CAT​GCT​CAA​GAT-AGG​CAC​TGG​AGC
 Upstream GAG​GTT​CTT​CTT-TAA​AAT​CCA​TTC
 Exon 1 TTT​CTT​CCC​TTT-CTT​TGC​TCC​TTC
 Exon 3 CTC​AGT​AGA​TAT-GTT​TGA​CGA​CTT
 Exon 4 CTA​CAT​GAA​GCT-CCA​CGG​GGT​ACG
 Exon 5 AAG​AAC​CTG​AAT-AAA​TCT​AAG​ACA
 Exon 6 CAA​CTC​CAG​CAA-CGC​CAC​CAC​CAG
 Exon 7 CAC​AAG​GCT​ATG-CGT​CTC​CCG​TCA
 CTSB gene AAG​TGT​AGC​AAG-ATC​TGT​GAG​CCT
NKX2-5
 Exon 1 CCG​GCC​AAG​TGT-GCG​TCT​GCC​TTT
 Exon 1 AGG​TGA​GGA​GGA-AAC​ACA​GGC​CCC
 Exon 2 CGC​TCC​AGC​TCA-TAG​ACC​TGC​GCC
 Exon 2 CGG​GAT​TCC​GCA-GAG​CAA​CTC​GGG
TBX5
 Exon 1 GAC​GTT​GGA​AGA-AGA​CCT​GGC​CTA
 Intron 1 CTA​TTC​TGG​GTA-AGC​AGT​AAA​CCC
 Exon 2 GCC​TGA​CGC​AAA-AGA​CCT​GCC​CTG
 Exon 3 AAT​CAA​AGT​GTT-TCT​CCA​TGA​AAG
 Exon 5 TCC​TTC​CAG​AAA-CTC​AAG​CTC​ACC
 Exon 6 TAC​CAG​CCT​AGA-TTA​CAC​ATC​GTG
 Exon 8 GTG​AGG​CAA​AAA-GTG​GCC​TCC​AAC
 Exon 8 CCA​TTG​TAC​CAA-GAG​GAA​AGG​TGA
 Exon 9 AAG​AAG​ATT​CCT-TCT​ACC​GCT​CTA
 Exon 9 TTT​GCT​TTG​GTT-TTG​TCC​TGC​CTT
BMP4
 Exon 1 TGC​AGG​GAC​CTA-TGG​TGA​GCA​AGG
 Intron 1 CGC​AGG​CCG​AAA-GCT​GTT​CAC​CGT
 Exon 3 TGG​TAA​CCG​AAT-GCT​GAT​GGT​CGT
 Exon 4 AAC​ATC​TGG​AGA-ACA​TCC​CAG​GGA
CRELD1
 Exon 4 CAA​GTC​AGA​CTT-CGA​GTG​CCA​CCG
 Exon 11 GCA​TTC​CCC​ATC-TTA​ACT​GAT​TTA
22q11 region (DiGeorge)
 CDC45 ATG​TTC​GTG​TCC-GAT​TTC​CGC​AAA
 GP1BB CAC​AAC​CGA​GCT-GGT​GCT​GAC​CGG
 DGCR8 GAC​TCA​GCG​ACT-GCA​CCA​GTG​GCA
Open in a new tab

Results

We divided 20 individuals who presented CHD and facial dysmorphia into two groups: one with ten subjects who have already been diagnosed by FISH and a second group with ten subjects whose 22q11.2DS diagnosis have not been confirmed by the same methodology. We observed that all patients who had been previously diagnosed by FISH were found to have the exact same deletion size, LCRs and genes involved (MLPA P250 kit): a typical 3 Mb deletion comprising LCR22A–LCR22D (Fig. 1). On the other hand, patients who did not have their diagnosis defined by FISH showed different molecular results by MLPA (P311 kit), ranging from normal findings to alterations in the GATA4 and NXK2-5 genes.

Fig. 1.

Fig. 1.

Open in a new tab

Heart defects and molecular genetic results of both groups of patients analyzed. Red boxes indicate presence of cardiac malformation in each subject; shaded gray boxes indicate absence of cardiac malformation in each subject.

Both groups of subjects showed similar and distinct cardiological abnormalities. Eight heart defects were found only in the group previously diagnosed by FISH and nine heart defects were found only in the group that was yet to be diagnosed (Fig. 1). The three most common cardiac abnormalities observed in those diagnosed with 22q11.2DS were atrial septal defects (ASDs) (5/10), interventricular communication (4/10), and right aortic arch (4/10). GATA4 exon 1 deletion subject showed a unique cardiac phenotype that was not shared with any other group: double outlet left ventricle, non-restrictive interventricular communication, right ventricular hypoplasia, and tricuspid atresia. Also, GATA4 when deleted or duplicated in different exons (1 and 6, respectively) showed distinct congenital heart defect phenotypes between them.

In addition, subjects presenting no molecular alterations have been observed in the second group (6/10). CHDs in those patients were shared by both the first and second groups; however, we can highlight the following malformations as exclusive to patients without identification of molecular alterations: pulmonary valve stenosis, pentalogy of Fallot, mild tricuspid regurgitation.

Discussion

Researchers are aware of the complexity surrounding 22q11.2DS diagnosis. In developing countries, where access to molecular technologies is scarce, a patient’s phenotype is one of the most important indicators for the initial diagnosis of a genetic disease, and often the only stage of assessment. Brazil is no different. It was only in 2014 that the Sistema Único de Saúde (SUS) instituted financial incentives for applying some molecular methodologies to diagnose patients suspected of having a genetic condition (Ordinance No. 981, of May 21, 2014: https://bvsms.saude.gov.br/bvs/saudelegis/gm/2014/prt0981_21_05_2014.html). However, even today, health services face challenges in accessing these methodologies on a routine basis. Our research group has been studying cases of 22q11.2DS in partnership with health institutions in southern Brazil for more than a decade using different approaches. Here, all cases were selected based on CHD findings and facial dysmorphia as the first indication for 22q11.2DS molecular analysis.

22q11.2DS is characterized by haploinsufficiency, resulting from a hemizygous deletion in the region 11.2 on the long arm of chromosome 22, meaning that the gene alleles have no homologic counterparts [18]. The deletion types and sizes show a high degree of variability due to several LCRs flanking the deleted region. LCRs are specific DNA regions that are generally 10–300 kilobases (kb) in size and 95–97% similar to each other.

It is known that 4 LCRs (A, B, C, and D) define this region and due to their substantial sequence similarity, facilitate non-allelic meiotic homologous recombination, resulting in translocation, deletion, or unbalanced duplication [19]. The majority of patients show a 3 Mb heterozygous deletion comprising four repeats extending from LCR22A to LCR22D and involving approximately 40 genes [20]. The 1.5 Mb deletion between the LCR22A and LCR22B regions can be identified in ∼10% of affected individuals [19]. Between the 22q11.2 deletion group of our study, all patients presented the same deletion size comprising the following genes: CLTCL1, HIRA, CDC45-1, GP1BB, TBX1, TXNRD2, DGCR8, ZNF74, KLHL22, MED15, SNAP29, LZTR1. It means a typical deletion in LCR22A-LCR22D.

Histone cell cycle regulator (HIRA) (OMIM: 600237) gene is located between LCR22A and LCR22B and is the main gene targeted for FISH analysis when investigating this syndrome due to its significant impact in embryonic and cardiac development [21]. Based on this, we wondered why the subjects who had the HIRA deletion (identified by FISH) in our study were shown to have the same size of deletion, LCRs, and genes involved when analyzed by MLPA? So, every time we identify the HIRA deletion can we suggest that the patient has the typical deletion? New studies with this focus will be necessary to try to answer these questions including a significant number of affected patients. We tried to find studies with focus on “How HIRA deletion impacts the genes subsequent deletion in 22q region?” We found studies applying different investigation approaches such as whole-genome sequencing [22], MLPA assays [20], and reviews [23, 24]; however, none of them brought a discussion that could answer our questions about HIRA or could explain our “coinciding” results.

Other studies not related to 22q11.2DS demonstrated HIRA role in other biologic processes. HIRA encodes a histone chaperone H3.3 and plays an important role in the epigenetic regulation of gene expression, influencing transcription, genome integrity, cellular senescence, and genome reprogramming [25]. Dilg et al. [26] demonstrated by embryonic stem cell study that HIRA plays a major role in the cardiogenic mesoderm. Mutant HIRA embryos presented edema and cardiac malformations such as VSD, ASD, thin ventricular wall, and constricted pulmonary trunk (PT). These two studies help us confirm that HIRA has an important impact in embryonic development that could possibly explain the 22q11.2DS phenotype. However, we continue not understanding if HIRA deletion can indicate a typical deletion when using only FISH as a diagnostic tool.

It is known that individuals with the LCR22A–LCR22B deletion have the full spectrum of phenotypes that is also found in individuals carrying typical LCR22A–LCR22D deletions, suggesting that key phenotypes of 22q11DS are mostly due to a decrease in the gene’s dosage located within LCR22A–LCR22B region, i.e., where HIRA is located. Also, cardiovascular defects seem to be three times more frequent in LCR22A–LCR22B or LCR22A–LCR22D than distal deletions [24, 27]. Looking at this, is it possible that the subsequent genes deleted after the LCR22A-LCR22B region cannot interfere with the 22q11.2 deletion phenotype and are just the remaining result of the size of the complete deletion caused by the genomic rearrangement?

CNVs, which constitute either gross DNA deletions or duplications, were identified as being critical dosage-sensitive genome for cardiac development. Examples of this include the TBX1 deletion at 22q11.2, the GATA4 deletion at 8p23.1, and the NKX2-5 deletion at 5q35.1 [28, 29]. Our CHD results are quite diverse, as is commonly found in literature. The three most common cardiac abnormalities observed in those diagnosed with 22q11.2DS were ASDs (5/10), interventricular communication (4/10), and right aortic arch (4/10). According to literature, VSD, ASD, and TOF are the most common heart defects associated with chromosomal abnormalities [14, 30]. Although the number of patients presented in this study is small, our results corroborate the literature as the most CHD finding was ASD. At the same time, TOF was also present in the 22q11.2DS subjects (2/10) but also in GATA4 duplication and normal sample. Curiously, VSD was not reported in our cases, but it is important to note that VSD is one of the cardiac malformations that comprises TOF [14].

In addition, we were able to observe a distinct cardiological profile, especially for patients who were suspected of having 22q11.2DS but were diagnosed with a GATA4 deletion in exon 1. CHDs observed are shown in Figure 2. Also, patients who had GATA4 deletion in exon 6 shared the cardiac phenotype with those diagnosed with 22q11.2DS.

Fig. 2.

Fig. 2.

Open in a new tab

Congenital heart defect findings shared with 22q11.2 deletion, GATA4 deletion/duplication and NKX2-5 duplication. *LVSD was identified only in NKX2 duplication. PFO was identified just in NKX2-5 duplication and normal subjects.

The patients who did not have a diagnosis defined by neither FISH nor MLPA (6/10) showed congenital heart defects found only among this sample as well as CHDs that were shared with del 22, GATA4 exon 6 deletion, NKX2-5 duplication and GATA4 exon 1 duplication patients (Fig. 3). Again, GATA exon 1 deletion subject showed an unique cardiac phenotype.

Fig. 3.

Fig. 3.

Open in a new tab

Cardiac profile among subjects with no molecular diagnostic and subjects with genetic alterations. Converging and diverging heart defects phenotypes are illustrated according to the molecular alteration (red: 22q11.2 deletion; yellow:GATA4 exon 6 deletion; blue:NKX2-5 exon 1 and 2 duplication; green:GATA4 exon 1 duplication). CHD’s of subject’s without any molecular finding are pictured in gray.

CHD candidate genes share common features, but their properties lack statistical significance for definitive categorization [31]. Also, they can provide new knowledge about the essential regulatory molecules and pathways involved in cardiogenesis, complementing, and extending the wealth of information gained from studying heart development in experimental models. Studying spontaneous gene variants in sporadic CHD can reveal novel genes implicated in cardiogenesis while providing detailed information [32].

Outside of 22q11.2 region, other candidate genes such as GATA4 and NKX2-5 have also been proven to be responsible for heart development and diseases [33]. Mutations of cardiac TF genes, such as GATA4 and NKX2-5 were identified in genetic linkage studies of large affected families [34]. Studies have investigated the relationship between mutations at the GATA4 level and the appearance of a specific heart defect, mainly in syndromic cardiac septal defects. The main research directions are oriented toward the GATA4 gene and T-box TF [35]. GATA4 belongs to GATA family which consists of 6 structure-conserved TFs. Over 100 known mutation sites have been identified within its gene, which are related to the structural heart defect [36]. We have observed three subjects presenting duplication/deletion in GATA4 and at the same time, two of them shared cardiac findings (Fig. 2). Shaker et al. [37], identified nonsynonymous sequence variation in exon 1 of GATA4 in cases of septal defects: isolated VSD and combined VSD and ASD in Egyptian children. Wang et al. [38], observed two mutations in exon 1 and 6 of GATA4 in addition to other mutations in patients with VSD and minor anomalies. Other authors have described distincts variants in different GATA4 exons related to heart defects [39, 40]. GATA4 seems to be indeed an important cardiac regulator gene, no matter which exons alterations are involved and its investigation, and of 22q11.2 deletion syndrome in patients with CHDs constitute an important issue in prenatal diagnosis, contributing to family planning through appropriate genetic counseling and management of extracardiac symptoms [41].

GATA4 mediates target gene expression by forming complexes with other cardiac transcriptional factors, including NKX2-5, another regulator essential for proper cardiogenesis with its expression and functions overlapping with those of GATA4 during embryogenesis. Moreover, GATA4 and NKX2-5 can interact physically and regulate synergistically the expression of multiple important cardiac genes [42, 43]. NKX2-5 is a vertebrate member of the NK-2 class of homeobox genes and is expressed in early heart myocardiocyte progenitors and developing heart, as well as in the pharynx, spleen and stomach [44]. The main heart defects related to NKX2-5 are conotruncal abnormalities, such as tetralogy of Fallot [45, 46].

In this study, we found a patient presenting NKX2-5 duplication associated with interventricular communication, left ventricular systolic dysfunction and patent foramen ovale. However, all those defects were shared with 22q11.2 deletion and/or normal subjects. No studies linking NKX2-5 duplication and CHD were found in the literature. What we know is that the process of cardiac development is complex and that NKX2–5 does participate in the protein-protein interaction with other TFs. Approximately 50 different mutations in this gene have been identified to date, and only a few have been functionally characterized. The mutant NKX2-5 factor can regulate a few off-targets downstream to facilitate CHD development [47]. Perhaps in order to better understand the NKX2-5 influence in cardiogenesis we should look into the failure of cardiac TF networks resulting from genetic instability in cardiac cells [48]. As NKX2-5 mutations exhibit common inheritance patterns, screening could also identify family members who may also be at risk [47].

We’ve performed a two group analysis between individuals with the same clinical diagnosis but different molecular results. HIRA is a well-known gene on congenital heart defects phenotypes; however, further studies on how this gene interacts with others are still needed to elucidate why the same deletion size is often found on 22q11.2DS subjects. GATA4 also showed to be a key gene in CHD phenotypes. On the other hand, NKX2-5 duplications are yet to be clarified in the cardiogenesis context. The patients that remained undiagnosed could be further investigated for point mutations in the critical regions of the genes such as HIRA, GATA4, and NKX2 by next generation sequencing technologies. To sum up, molecular diversity in cardiac malformations is a reality and a great challenge since genotype-phenotype correlation is hindered. Therefore, new insights on that matter should be considered: 22q11.2 deletion syndrome should only be linked to the chromosome 22 region, or is there a phenotype variability to be looked at that involves a broader genomic environment?

Acknowledgment

The authors thank the families involved for their support for this study.

Statement of Ethics

This study involves human participants and was approved by REC Hospital da Criança Santo Antônio No. 2.315.917 and UFCSPA No. 3.577.284.

Participants gave informed consent to participate in the study before taking part.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work was funding by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, 17/2551-0001063-9), Programa de Extensão Universitária do Ministério da Educação e Cultura (PROEXT), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (302931/2019-8).

Author Contributions

B.L.D. designed experiments, performed methodology, and wrote the manuscript; D.D. helped contributed to data extraction and wrote the manuscript; A.K.B., R.M., and N.M. contributed to perform methodology and data extraction. B.B.G. performed the patients’ morphological assessment. R.F.M.R. and P.R.G.Z. provided feedback on the manuscript.

Funding Statement

This work was funding by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, 17/2551-0001063-9), Programa de Extensão Universitária do Ministério da Educação e Cultura (PROEXT), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (302931/2019-8).

Data Availability Statement

All data relevant to the study is included in the article.

References

  • 1. McDonald-McGinn DM, Sullivan KE, Marino B, Philip N, Swillen A, Vorstman JAS, et al. 22q11.2 deletion syndrome. Nat Rev Dis Primers. 2015;1:15071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Goldmuntz E. 22q11.2 deletion syndrome and congenital heart disease. Am J Med Genet C Semin Med Genet. 2020;184(1):64–72. [DOI] [PubMed] [Google Scholar]
  • 3. Tennant PW, Pearce MS, Bythell M, Rankin J. 20-year survival of children born with congenital anomalies: a population-based study. Lancet. 2010;375(9715):649–56. [DOI] [PubMed] [Google Scholar]
  • 4. Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, Andrews TD, et al. Global variation in copy number in the human genome. Nature. 2006;444(7118):444–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Liu Z, Wang J, Liu S, Deng Y, Liu H, Li N, et al. Copy number variation of GATA4 and NKX2-5 in Chinese fetuses with congenital heart disease. Pediatr Int. 2015;57(2):234–8. [DOI] [PubMed] [Google Scholar]
  • 6. Kinnunen S, Välimäki M, Tölli M, Wohlfahrt G, Darwich R, Komati H, et al. Nuclear receptor-like structure and interaction of congenital heart disease-associated factors GATA4 and NKX2-5. PLoS One. 2015;10(12):e0144145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Blue GM, Kirk EP, Sholler GF, Harvey RP, Winlaw DS. Congenital heart disease: current knowledge about causes and inheritance. Med J Aust. 2012;197(3):155–9. [DOI] [PubMed] [Google Scholar]
  • 8. Zaidi S, Brueckner M. Genetics and genomics of congenital heart disease. Circ Res. 2017;120(6):923–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Athanasiadis DI, Mylonas KS, Kasparian K, Ziogas IA, Vlachopoulou D, Sfyridis PG, et al. Surgical outcomes in syndromic tetralogy of Fallot: a systematic review and evidence quality assessment. Pediatr Cardiol. 2019;40(6):1105–12. [DOI] [PubMed] [Google Scholar]
  • 10. Takao A, Ando M, Cho K, Kinouchi A, Murakami Y. Etiology and morphogenesis of congenital heart disease. Futura pub Co; 1980; p. 253–69. [Google Scholar]
  • 11. Mutlu ET, Aykan HH, Karagöz T. Analysis of gene copy number variations in patients with congenital heart disease using multiplex ligation-dependent probe amplification. Anatol J Cardiol. 2018;20(1):9–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Chen CP, Huang JP, Chen YY, Chern SR, Wu PS, Su JW, et al. Chromosome 22q11.2 deletion syndrome: prenatal diagnosis, array comparative genomic hybridization characterization using uncultured amniocytes and literature review. Gene. 2013;527(1):405–9. [DOI] [PubMed] [Google Scholar]
  • 13. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002;30(12):e57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Diniz BL, Deconte D, Gadelha KA, Glaeser AB, Guaraná BB, de Moura AÁ, et al. Congenital heart defects and 22q11.2 deletion syndrome: a 20-year update and new insights to aid clinical diagnosis. J Pediatr Genet. 2023;12(2):113–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Bajolle F, Zaffran S, Bonnet D. Genetics and embryological mechanisms of congenital heart diseases. Arch Cardiovasc Dis. 2009;102:59–63. [DOI] [PubMed] [Google Scholar]
  • 16. Diniz BL, Santos AS, Glaeser AB, Guaraná BB, Lorea CF, Josahkian JA, et al. Congenital heart defects and dysmorphic facial features in patients suspicious of 22q11.2 deletion syndrome in southern Brazil. J Pediatr Genet. 2020;9(4):227–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Botto LD, May K, Fernhoff PM, Correa A, Coleman K, Rasmussen SA, et al. A population-based study of the 22q11.2 deletion: phenotype, incidence, and contribution to major birth defects in the population. Pediatrics. 2003;112(1 Pt 1):101–7. [DOI] [PubMed] [Google Scholar]
  • 18. Szczawińska-Popłonyk A, Schwartzmann E, Chmara Z, Głukowska A, Krysa T, Majchrzycki M, et al. Chromosome 22q11.2 deletion syndrome: a comprehensive review of molecular genetics in the context of multidisciplinary clinical approach. Int J Mol Sci. 2023;24(9):8317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Nogueira SI, Hacker AM, Bellucco FT, Christofolini DM, Kulikowski LD, Cernach MCSP, et al. Atypical 22q11.2 deletion in a patient with DGS/VCFS spectrum. Eur J Med Genet. 2008;51(3):226–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Gavril E-C, Popescu R, Nucă I, Ciobanu C-G, Butnariu LI, Rusu C, et al. Different types of deletions created by low-copy repeats sequences location in 22q11.2 deletion syndrome: genotype–phenotype correlation. Genes. 2022;13(11):2083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Gunjan A, Paik J, Verreault A. Regulation of histone synthesis and nucleosome assembly. Biochimie. 2005;87(7):625–35. [DOI] [PubMed] [Google Scholar]
  • 22. Zhao Y, Diacou A, Johnston HR, Musfee FI, McDonald-McGinn DM, McGinn D, et al. Complete sequence of the 22q11.2 allele in 1,053 subjects with 22q11.2 deletion syndrome reveals modifiers of conotruncal heart defects. Am J Hum Genet. 2020;106(1):26–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Morrow BE, McDonald-McGinn DM, Emanuel BS, Vermeesch JR, Scambler PJ. Molecular genetics of 22q11.2 deletion syndrome. Am J Med Genet A. 2018;176(10):2070–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Motahari Z, Moody SA, Maynard TM, LaMantia AS. In the line-up: deleted genes associated with DiGeorge/22q11. 2 deletion syndrome: are they all suspects? J Neurodev Disord. 2019;11(1):7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Jeanne M, Vuillaume ML, Ung DC, Vancollie VE, Wagner C, Collins SC, et al. Haploinsufficiency of the HIRA gene located in the 22q11 deletion syndrome region is associated with abnormal neurodevelopment and impaired dendritic outgrowth. Hum Genet. 2021;140(6):885–96. [DOI] [PubMed] [Google Scholar]
  • 26. Dilg D, Saleh RN, Phelps SE, Rose Y, Dupays L, Murphy C, et al. HIRA is required for heart development and directly regulates Tnni2 and Tnnt3. PLoS One. 2016;11(8):e0161096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Burnside RD. 22q11.21 deletion syndromes: a review of proximal, central, and distal deletions and their associated features. Cytogenet Genome Res. 2015;146(2):89–99. [DOI] [PubMed] [Google Scholar]
  • 28. McDonald-McGinn DM, Sullivan KE. Chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome). Medicine. 2011;90(1):1–18. [DOI] [PubMed] [Google Scholar]
  • 29. Glessner JT, Bick AG, Ito K, Homsy J, Rodriguez-Murillo L, Fromer M, et al. Increased frequency of de novo copy number variants in congenital heart disease by integrative analysis of single nucleotide polymorphism array and exome sequence data. Circ Res. 2014;115(10):884–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Bellucco FT, Belangero SI, Farah LM, Machado MV, Cruz AP, Lopes LM, et al. Investigating 22q11.2 deletion and other chromosomal aberrations in fetuses with heart defects detected by prenatal echocardiography. Pediatr Cardiol. 2010;31(8):1146–50. [DOI] [PubMed] [Google Scholar]
  • 31. Nappi F. In-depth genomic analysis: the new challenge in congenital heart disease. Int J Mol Sci. 2024;25(3):1734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Deciphering Developmental Disorders Study . Prevalence and architecture of de novo mutations in developmental disorders. Nature. 2017;542(7642):433–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Paige SL, Plonowska K, Xu A, Wu SM. Molecular regulation of cardiomyocyte differentiation. Circ Res. 2015;116(2):341–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Granados-Riveron JT, Pope M, Bu’lock FA, Thornborough C, Eason J, Setchfield K, et al. Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations. Congenit Heart Dis. 2012;7(2):151–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Moldovan E, Bănescu C, Cucerea M, Moldovan V, Gozar L, Puşcaşiu L. GATA4 rs61277615, rs73203482, and rs35813172 in newborns with transposition of the great arteries. Acta Med Okayama 2023;77:365–70. [DOI] [PubMed] [Google Scholar]
  • 36. Patient RK, McGhee JD. The GATA family (vertebrates and invertebrates). Curr Opin Genet Dev. 2002;12(4):416–22. [DOI] [PubMed] [Google Scholar]
  • 37. Shaker O, Omran S, Sharaf E, A Hegazy G, Mashaly M, E A Gaboon N. A novel mutation in exon 1 of GATA4 in Egyptian patients with congenital heart disease. Turk J Med Sci. 2017;47(1):217–21. [DOI] [PubMed] [Google Scholar]
  • 38. Wang J, Fang M, Liu XY, Xin YF, Liu ZM, Chen X, et al. A novel GATA4 mutation responsible for congenital ventricular septal defects. Int J Mol Med. 2011;28(4):557–64. [DOI] [PubMed] [Google Scholar]
  • 39. Tomita-Mitchell A, Maslen CL, Morris CD, Garg V, Goldmuntz E. GATA4 sequence variants in patients with congenital heart disease. J Med Genet. 2007;44(12):779–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Hussein I, El-Rubi M, Helmy N, Hussein H, El-Gerzawy A, Bassyouni R, et al. Genetic studies of congenital heart defects in Egyptian patients. Res J Med Med Sci. 2009;4:55–66. [Google Scholar]
  • 41. Floriani MA, Glaeser AB, Dorfman LE, Agnes G, Rosa RFM, Zen PRG. GATA 4 deletions associated with congenital heart diseases in south Brazil. J Pediatr Genet. 2021;10(2):92–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Li J, Liu WD, Yang ZL, Yuan F, Xu L, Li RG, et al. Prevalence and spectrum of GATA4 mutations associated with sporadic dilated cardiomyopathy. Gene. 2014;548(2):174–81. [DOI] [PubMed] [Google Scholar]
  • 43. McCulley DJ, Black BL. Transcription factor pathways and congenital heart disease. Curr Top Dev Biol. 2012;100:253–77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Lints TJ, Parsons LM, Hartley L, Lyons I, Harvey RP. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development. 1993;119(2):419–31. Erratum in: Development. 1993 Nov;119(3):969. [DOI] [PubMed] [Google Scholar]
  • 45. Goldmuntz E, Geiger E, Benson DW. NKX2.5 mutations in patients with tetralogy of Fallot. Circulation. 2001;104(21):2565–8. [DOI] [PubMed] [Google Scholar]
  • 46. Rauch R, Hofbeck M, Zweier C, Koch A, Zink S, Trautmann U, et al. Comprehensive genotype-phenotype analysis in 230 patients with tetralogy of Fallot. J Med Genet. 2010;47(5):321–31. [DOI] [PubMed] [Google Scholar]
  • 47. Chung IM, Rajakumar G. Genetics of congenital heart defects: the NKX2-5 gene, a key player. Genes. 2016;7(2):6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Benson DW, Silberbach GM, Kavanaugh-McHugh A, Cottrill C, Zhang Y, Riggs S, et al. Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. J Clin Investig. 1999;104(11):1567–73. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data relevant to the study is included in the article.

Articles from Molecular Syndromology are provided here courtesy of Karger Publishers

RESOURCES