Novel pathogenic gata6 variant associated with congenital heart disease, diabetes mellitus and necrotizing enterocolitis

ABSTRACT BACKGROUND Pathogenic _GATA6_ variants have been associated with congenital heart disease (CHD) and a spectrum of extracardiac abnormalities, including pancreatic agenesis,

congenital diaphragmatic hernia, and developmental delay. However, the comprehensive genotype-phenotype correlation of pathogenic _GATA6_ variation in humans remains to be fully understood.

METHODS Exome sequencing was performed in a family where four members had CHD. In vitro functional analysis of the _GATA6_ variant was performed using immunofluorescence, western blot, and

dual-luciferase reporter assay. RESULTS A novel, heterozygous missense variant in _GATA6_ (c.1403 G > A; p.Cys468Tyr) segregated with affected members in a family with CHD, including

three with persistent truncus arteriosus. In addition, one member had childhood onset diabetes mellitus (DM), and another had necrotizing enterocolitis (NEC) with intestinal perforation. The

p.Cys468Tyr variant was located in the c-terminal zinc finger domain encoded by exon 4. The mutant protein demonstrated an abnormal nuclear localization pattern with protein aggregation and

decreased transcriptional activity. CONCLUSIONS We report a novel, familial _GATA6_ likely pathogenic variant associated with CHD, DM, and NEC with intestinal perforation. These findings

expand the phenotypic spectrum of pathologic _GATA6_ variation to include intestinal abnormalities. IMPACT * Exome sequencing identified a novel heterozygous _GATA6_ variant (p.Cys468Tyr)

that segregated in a family with CHD including persistent truncus arteriosus, atrial septal defects and bicuspid aortic valve. Additionally, affected members displayed extracardiac findings

including childhood-onset diabetes mellitus, and uniquely, necrotizing enterocolitis with intestinal perforation in the first four days of life. * In vitro functional assays demonstrated

that _GATA6_ p.Cys468Tyr variant leads to cellular localization defects and decreased transactivation activity. * This work supports the importance of _GATA6_ as a causative gene for CHD and

expands the phenotypic spectrum of pathogenic _GATA6_ variation, highlighting neonatal intestinal perforation as a novel extracardiac phenotype. You have full access to this article via

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22 May 2024 INTRODUCTION The GATA family of transcription factors encode for zinc finger DNA binding proteins that play critical regulatory roles in organogenesis.1 Among this family,

GATA4, GATA5, and GATA6 have been implicated in cardiovascular morphogenesis and expressed in overlapping but distinct spatiotemporal patterns during development.2,3 Pathogenic variation in

_GATA4_, _GATA5_ and _GATA6_ have been reported in a spectrum of human congenital heart disease (CHD).4,5 Pathogenic variants in _GATA4_ were first reported in individuals with cardiac

septal defects including atrial septal defects (ASD), ventricular septal defects (VSD), and atrioventricular septal defects (AVSD).6 The CHD phenotypes associated with pathologic _GATA4_

variants have expanded to include pulmonary stenosis (PS), tetralogy of Fallot (TOF) and bicuspid aortic valve (BAV).7,8,9,10,11 The primary cardiac phenotype associated with _GATA5_

pathogenic variants is BAV although additional phenotypes include VSD, ASD, TOF and double outlet right ventricle (DORV).3,12,13,14,15,16 The expression pattern of GATA6 during embryogenesis

partially overlaps with GATA4 and GATA5 in the heart and other tissues, including the digestive system and the extraembryonic endoderm.17,18,19 In particular, GATA6 plays a role in the

developing second heart field (SHF) and in the recruitment of cardiac neural crest cells, which together form the cardiac outflow tract.20 _GATA6_ variants in humans were first discovered in

individuals with persistent truncus arteriosus (PTA).21 Subsequently, _GATA6_ pathogenic variants have been identified in other malformations of cardiac outflow tract defect, including TOF,

pulmonary atresia with ventricular septal defect (PA/VSD), DORV and transposition of the great arteries (TGA).22,23,24,25,26,27 Similar to GATA4 and GATA5, _GATA6_ variants have been found

in individuals with a variety of CHD phenotypes including ASD, VSD, PS, BAV and patent ductus arteriosus (PDA).28,29,30 Unique from _GATA4_ and _GATA5_, _GATA6_ pathogenic variants are found

to be associated with extracardiac abnormalities in humans. Consistent with this, GATA6 plays a critical role for regulating gene expression in the development of endoderm from which the

pancreas and gut are derived.18,19 Pancreatic agenesis was initially identified as an extracardiac phenotype in individuals with CHD who had _GATA6_ variants.31 Pancreatic abnormalities are

the most common extracardiac features associated with _GATA6_ pathogenic variation, which range from severe hypoplasia or agenesis of the pancreas with neonatal or childhood-onset diabetes

mellitus (DM) to a mild phenotype such as adult-onset DM.32,33,34,35 Additional extracardiac malformations have also been reported, including hepatobiliary and gastrointestinal

abnormalities.31,32,36,37 Gastrointestinal abnormalities associated with _GATA6_ variants include congenital diaphragmatic hernia, but evidence regarding the association between _GATA6_

pathogenic variation and congenital anomalies affecting gut such as small intestine, colon and rectum is less common.37 Accordingly, the broad phenotypic spectrum and genotype-phenotype

correlations for genetic variation in _GATA6_ for cardiac and extracardiac diseases remains to be defined. Here, we identified a novel, heterozygous missense variant in _GATA6_ that

segregated with disease in a family with a spectrum of CHD and extracardiac phenotypes using exome sequencing. The father had PTA while three children also had CHD, including PTA, ASD, BAV

and interrupted aortic arch. In addition, the father had childhood-onset DM and one child had necrotizing enterocolitis (NEC) with intestinal perforation. Using in vitro assays, we

demonstrate that the GATA6 mutant protein displays functional deficits in nuclear localization and reduced transcriptional activity. This work highlights the phenotypic spectrum for

_GATA6_-associated disease from CHD and DM to even gastrointestinal abnormalities. METHODS ETHICS STATEMENT Family members were recruited under an approved Nationwide Children’s Hospital

Institutional Review Board protocol (#IRB09-00339). Written informed consent was obtained from study subjects 18 years of age and older and from parents for study subjects under 18 years of

age, with assent obtained from subjects 9-17 years of age. DNA ISOLATION Blood samples or saliva samples (Oragene) were obtained and DNA was isolated using Gentra Puregene Kit (Qiagen,

Hilden, Germany) as previously described.38 EXOME LIBRARY CONSTRUCTION AND EXOME SEQUENCING Exome libraries were constructed using Agilent Clinical Research Exome Kit v1 (Agilent

Technologies, CA). Paired-end 150 base pair reads were generated for exome-enriched libraries sequenced on Illumina NovaSeq to a targeted depth of 100x coverage. EXOME SEQUENCING DATA

PIPELINE We analyzed and annotated exome sequencing data using methods as previously described.38 VARIANT ANALYSIS Variant filtering on frequency, effect, and predicted impact, and

segregation with disease was performed as described in Manivannan et al. 2020.39 _GATA6_ genomic region flanking the identified variant was amplified using primers GATA6.gen.For and

GATA6.gen.Rev (Supplementary Table S1) to confirm the presence of the variant using Sanger sequencing method. The novel _GATA6_ variant identified was assessed for pathogenicity using the

American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG/AMP) guidelines.40 The predicted three-dimensional structure of the mutant protein was created

using Missense3D.41 The wildtype and mutant protein structures were visualized and attribution of the residue position to the protein function was analyzed using Jmol

(https://jmol.sourceforge.net/). Gibbs free energy calculations were performed using DynaMut2.42 PLASMID CONSTRUCTION Full length human _GATA6_ cDNA (Refseq ID NM_005257) was obtained from

Harvard Plasmid Database. _GATA6_ coding region with the 3’UTR sequence was amplified using PCR with primers FP.GATA6.BamHI and RP.GATA6.EcoRI (Supplementary Table S1), and cloned into

_BamHI/EcoRI_ site in the pIRES-mCherry plasmid (a gift from Ellen Rothenberg, Addgene plasmid # 80139). Then, _GATA6_-coding region, 3ʹUTR and IRES mCherry were excised using _BamHI/EcoRI_

and introduced in frame with the EGFP tag in a modified pEGFP-C1 plasmid (Clontech) to generate the pEGFP-GATA6-IRES-mCherry plasmid. When transfected, this construct leads to the production

of an EGFP tagged GATA6 protein and independently translated mCherry from the same transcript. The EGFP tag distinguishes the overexpressed cDNA from _GATA6_, while the independently

produced mCherry acts as a readout of mRNA transcription and also serves as a transfection control allowing direct comparison of cellular levels of overexpressed GATA6 protein variant. To

generate the variant, site-directed mutagenesis was used with pEGFP-GATA6-IRES-mCherry as the template and using the primers FP.GATA6.C468Y and RP.GATA6.C468R (Supplementary Table S1), and

the Agilent Quickchange II kit.43 All vectors constructed were verified by sequencing. CELL CULTURE HEK293 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L

Glucose, 4 mM L-Glutamine, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate (ATCC 30–2002), supplemented with 10% fetal bovine serum, 100 I.U./mL penicillin and 100 (μg/mL) streptomycin

at 37 °C incubator with 5% CO2. Cells were transfected with 2 μg of the plasmid with Lipofectamine 3000 reagent (Thermo Fisher Scientific, #L3000015) with Opti-MEM medium (Thermo Fisher

Scientific, #31985-070) according to manufacturer’s recommendations. Transfection media was removed five hours post-transfection and replaced by normal growth media. Cells were collected for

western blotting or immunofluorescence analysis 48 h after transfection. WESTERN BLOTTING Cell lysates were prepared from cultured HEK293 cells using RIPA Lysis and Extraction Buffer

(ThermoFisher Scientific, #89900) supplemented with Halt Protease Inhibitor Cocktail (ThermoFisher Scientific, #87785). BCA Protein Assay Kit (ThermoFisher Scientific, #23227) was used to

estimate protein concentration. Cell lysates were mixed with 6X Laemmli SDS-Sample Buffer (Boston BioProducts, #BP-111R) containing β-mercaptoethanol and boiled for 5 min. Protein samples

were separated in 4 to 20% Mini-PROTEAN TGX Precast Gels (Bio-Rad, #4561094), transferred into a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, #1620177) and blocked with 5% nonfat milk

in Tris-Buffered Saline containing 0.1% Tween 20 (TBST). Membranes were probed with primary antibodies against GATA6 (1:500, R&D Systems, #AF1700), GAPDH (1:1000, Novus Biologicals,

#NB300-221). After probing with primary antibodies, membranes were further probed with horseradish peroxidase–conjugated anti-rabbit and anti-mouse secondary antibodies (Vector Laboratories,

#PI-1000 and PI-2000). Western blots were developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, #32106). Restore Western Blot Stripping Buffer (Thermo Fisher

Scientific, #21059) was used for re-probing with different primary antibodies following the manufacturer’s protocol. Protein levels were quantified by densitometric analysis using ImageJ

software and normalized to GAPDH. LUCIFERASE TRANSACTIVATION ASSAYS HEK293 cells were transiently transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific, #L3000015) with 150 

ng of atrial natriuretic factor (ANF) luciferase reporter and with 300 ng of GATA6 wildtype or GATA6 p.Cys468Tyr, using previously described methods.15,23 Luciferase activity was measured 48

 h after transient transfection according to the manufacturer’s instructions (Promega, Madison, WI). The luciferase signal was normalized to the _Renilla_ signal to control for variation in

transfection efficiency. Three independent experiments were performed in triplicate. IMMUNOFLUORESCENCE Cultured HEK293 cells were fixed with 2.5% PFA 48 h after transient transfection and

permeabilized with Phosphate-Buffered Saline Triton (PBST, PBS containing 0.1% TritonX100). After permeabilization, non-specific immunoreactions were blocked using 1% BSA in PBST for 1 h at

room temperature and incubated overnight with primary antibodies: Chicken anti-GFP (1:1000, Abcam, #ab13970) and Rabbit anti-mCherry (1:1000, Abcam, #ab167453). After primary antibody

incubation, cells were washed with PBST and incubated with anti-chicken and anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 and 568 (ThermoFisher Scientific, #A11039 and

#A11011) for 1 h in the dark. Nuclei were stained with DAPI (1.5 μg/ml; Sigma-Aldrich, #D9542). Keyence BZ-X800 Fluorescence microscope was used to capture images. Expression of EGFP

tagged-GATA6 wildtype or GATA6 p.Cys468Tyr protein and protein aggregation was quantified using ImageJ by counting the number of particles by counting the nuclei (DAPI) in the same image, as

previously described.44 STATISTICAL ANALYSIS All experiments were performed at least in triplicate. Statistical analysis was performed in the GraphPad Prism 9 software package using

Two-tailed Student’s _t_ test. _P_ values of ≤0.05 were considered as statistically significant. RESULTS FAMILY WITH MULTIPLE MEMBERS AFFECTED WITH CONGENITAL HEART DISEASE We identified a

family where four members displayed CHD along with additional non-cardiac diseases (Fig. 1a). The father had PTA and childhood-onset DM. Among the four children, three had CHD. Two children

had PTA while another ASD, BAV and mild right ventricle (RV) hypoplasia (Fig. 1b). The proband (III-4) was prenatally diagnosed with PTA (Collett and Edwards type II)45 due to the family

history of CHD in the father. She was born at 37 weeks of gestation to a 28 year old mother. The birthweight was 2580 g with Apgar scores of 7 and 8. On the first day of life, she was

transferred to the cardiothoracic intensive care unit (CTICU) for the management of PTA. In addition to PTA, ASD, mild hypoplasia of tricuspid valve and RV were noted by echocardiogram and

later demonstrated by cardiac MRI. She was made nil per os (NPO) on admission but allowed limited self-advance feedings with breastmilk. She was hemodynamically stable breathing in room air,

with adequate near-infrared spectroscopy and diastolic blood pressures without cardiovascular or respiratory compromise. However, at 44 h after birth, a routine morning X-ray showed

pneumatosis, dilated bowel loops and portal venous gas. Clinically, she also developed abdominal distension and was diagnosed with NEC. She was made NPO and piperacillin/tazobactam was

started. At four days of age, an exploratory laparotomy revealed necrotic bowel of the distal ileum and distal right colon along with perforation of the proximal transverse colon. She

underwent bowel resection, and end ileostomy was performed in the right lower quadrant with the mesentery of the small bowel facing medially. Surgical pathology report revealed marked

mucosal necrosis and acute inflammation with regions of full-thickness bowel necrosis in the ileum, right colon and proximal transverse colon. After recovering from NEC, she underwent

bilateral pulmonary artery (PA) banding on 40 days of age. She then underwent ileostomy takedown and anastomosis for restoration of continuity, and gastrostomy tube placement at 65 days of

age. Subsequently, she developed complete colonic obstruction requiring multiple surgeries, including an emergent exploratory laparotomy (99 days old) and recreation of ileostomy (105 days

old). She was discharged from the hospital at 181 days of age. She underwent complete repair of PTA with VSD closure, utilizing a 12 mm Hancock valved conduit as the right ventricular to

pulmonary artery conduit, patch augmentation of both pulmonary arteries and ASD closure at the age of nine months without complication and was discharged in six days. Due to NEC and its

complications, she developed short bowel syndrome and was able to achieve enteral autonomy by the age of 15 months. Subsequently, she underwent ostomy takedown and ileocolonic reanastomosis

creation to increase bowel length at 19 months of age. She recovered from the operation and the clinical course was uneventful for one year. Of note, the tricuspid valve remains mildly

hypoplastic while the mild RV hypoplasia resolved by echocardiogram at four years of age. She had normal karyotype of 46, XX, normal results of 22q11 FISH and SNP microarray. The father

(II-1), a 35 year old man, was diagnosed with PTA (Collett and Edwards type II) and at 3 months of age underwent complete repair of PTA with closure of ASD. Furthermore, he was diagnosed

with DM at the age of 14 (by report) and is insulin-dependent. The sibling (III-2) was diagnosed with PTA with interrupted aortic arch (type B) and bicuspid truncal valve with mild stenosis

at birth due to cyanosis. In addition, there was hypoplasia of the tricuspid valve, RV, and proximal branch pulmonary arteries along with a large ASD. She died at two weeks of age from

circulatory failure at another institution. She had normal karyotype of 46, XX, normal results of 22q11 FISH and SNP microarray. The other sibling (III-3) was diagnosed with large ASD, PDA

and mild hypoplasia of tricuspid valve, RV, and pulmonary valve at birth due to heart murmur and family history of CHD. In addition,  left pulmonary artery (LPA) hypoplasia with associated

proximal LPA stenosis was noted. At six months of age, she underwent implantation of LPA stent and PDA occlusion with Amplatzer Vascular Plug II. She was then diagnosed with supraventricular

tachycardia. Subsequently, electrophysiology study showed atrioventricular nodal reentrant tachycardia (AVNRT) and she underwent repeated LPA stent dilation. Given her arrhythmia, she did

not undergo ASD closure. Her recent echocardiogram demonstrated moderate RV dilation and bidirectional shunting through a moderate-sized ASD with her baseline oxygen saturations of 93% at

the age of eight years. Notably, BAV with mild aortic valve insufficiency and no aortic valve stenosis as well as mild to moderate dilatation of the aortic root and ascending aorta were also

detected. She is being followed to determine the plan for ASD closure. Her chromosome analysis showed a normal female karyotype of 46, XX and FISH result showed no deletion of 22q11.

IDENTIFICATION OF NOVEL MISSENSE VARIANT IN GATA6 BY EXOME SEQUENCING Due to the multiple family members affected with CHD, exome sequencing was performed on the proband-parent trio (III-4,

II-1, II-2) as well as the affected sibling (III-3), the unaffected sibling (III-1) and unaffected paternal grandparents (I-1 and I-2). Sequencing data were analyzed using our previously

published pipeline, Churchill, for calling variants.46 Variants were prioritized using minor allele frequency (MAF) (≤0.001) across population in gnomAD, sequencing quality filtering,

predicted functional impact, and segregation of candidate variants (Supplementary Fig. S1).47,48,49,50,51,52,53,54 This approach resulted in the identification of a single heterozygous

missense variant in _GATA6_ (NM_005257.5: c.1403 G > A; p.Cys468Tyr) that segregated in the family, i.e. present in affected family members and absent from unaffected members. Therefore,

we focused on the _GATA6_ variant since it was the only candidate disease-causing variant in the family. We confirmed presence of the heterozygous _GATA6_ variant in the proband (III-4),

affected father (II-1), and affected sibling (III-3) and its absence in the unaffected mother (II-2) and unaffected sibling (III-1) by Sanger sequencing (Fig. 1c). Further, testing of

paternal grandparents (I-1 and I-2) demonstrated that this was a de novo variant in the father II-1 (Fig.1c). This _GATA6_ variant was not found in control populations in gnomAD database.

The variant in _GATA6_ was rated likely pathogenic according to ACMG/AMP guidelines (Table 1). The _GATA6_ variant occurs within the C-terminal zinc finger (ZF) domain (Fig. 1d).

Cross-species alignment of GATA6 protein demonstrates highly conserved cysteine at position 468 (Fig. 1e). Three-dimensional protein structure of C-terminal ZF in GATA6 showed that the

structure introduced by ZF being bound to these four cysteines is imperative to proper functioning in GATA6 wildtype protein. Substitution of the cysteine at codon 468 with a tyrosine

changes the ZF structure from four closed coordination sphere to three open coordinate system (Fig. 1f). This structural modeling suggests that _GATA6_ p.Cys468Tyr variant likely severely

impacts the stability of the zinc finger. IN VITRO _ANALYSIS OF THE GATA6_ P.CYS468TYR VARIANT To assess the structural and functional impact of _GATA6_ variant, we generated an EGFP-tagged

human _GATA6_ cDNA construct that also expresses mCherry, allowing for simultaneous evaluation of mRNA stability and protein stability (Fig. 2a). Site-directed mutagenesis was performed on

this construct to introduce the _GATA6_ p.Cys468Tyr variant. Using this construct, we tested the stability of the _GATA6_ p.Cys468Tyr variant. Expression constructs of _GATA6_ wildtype and

_GATA6_ p.Cys468Tyr variant were transiently transfected into HEK293 cells. Immunofluorescence analysis of EGFP-tagged _GATA6_ constructs that overexpress mCherry from the same transcript

after transfection in HEK293 cells demonstrated that the GATA6 wildtype localized to the nucleus, whereas the GATA6 p.Cys468Tyr variant exhibited an abnormal localization pattern in the

nucleus (Fig. 2b). There was no significant difference in the production of mCherry between the wildtype and variant constructs, suggesting that the transfection and transcription of the

GATA6 variant construct was comparable to the wildtype GATA6 construct (Fig. 2b). To identify and quantify protein aggregates, the number of particles divided by the number of nuclei was

counted. The number of particles/nucleus was significantly increased in GATA6 p.Cys468Tyr variant as compared to GATA6 wildtype, suggesting aggregation of GATA6 p.Cys468Tyr protein (Fig. 

2c). Western blot analyses showed reduced protein expression in GATA6 p.Cys468Tyr variant protein compared to GATA6 wildtype protein (Fig. 2d). In transient transfection transactivation

assays using the GATA6-dependent cardiac enhancer, ANF, upstream of a luciferase reporter, the GATA6 p.Cys468Tyr mutant protein demonstrated significantly decreased transcriptional activity

when compared with wildtype GATA6 (Fig. 2e). Together, these in vitro functional analyses indicated that _GATA6_ p.Cys468Tyr variant leads to functional deficits of the GATA6 protein and

likely impacts the regulation of downstream target genes during heart development. DISCUSSION Here, we investigated the genetic etiology of a familial case of CHD, predominantly PTA. Using

an exome sequencing approach, we identified a novel, heterozygous missense _GATA6_ variant (p.Cys468Tyr) which segregated with affected family members. In vitro functional analyses

demonstrated that _GATA6_ p.Cys468Tyr variant leads to cellular localization defects and decreased transactivation activity when compared with wildtype _GATA6_. While the spectrum of disease

phenotypes found in family members harboring the likely pathogenic variant included CHD and childhood onset DM, as reported by others,28 it also includes NEC and intestinal perforation

within the first week of life. These findings support the importance of _GATA6_ as a causative gene for CHD and highlights a novel extracardiac phenotype, early NEC, that can be associated

with _GATA6_ variants. This study will contribute to the growing literature about the association of pathogenic GATA6 variation with intestinal abnormalities as well as cardiac and

pancreatic malformations (Fig. 3). _GATA6_ variants have been identified in human CHD.55 The prevalence of _GATA6_ variants ranged from 0.5-2.0% in non-familial CHD in initial reports with a

higher frequency in cardiac outflow tract defects such as PTA and TOF.21,22,23,24,25,26,27,56 Recent exome sequencing studies in a large CHD cohort with more than 4000 CHD patients

conducted by the Pediatric Cardiac Genome Consortium (PCGC) discovered 9 heterozygous de novo variants in _GATA6_ including loss of function (LoF) and damaging missense variants.57,58 CHD

associated with _GATA6_ pathogenic variants is mainly cardiac outflow tract malformations, such as PTA, TOF and DORV, along with septal defects in some patients (Supplementary Table S2). In

addition, approximately 40% of these CHD patients with _GATA6_ variants also had extracardiac phenotypes, including pancreatic agenesis, congenital diaphragmatic hernia, and

neurodevelopmental disorders. Approximately 25% of previously reported _GATA6_ missense variants associated with CHD were located in the C-terminal DNA-binding zinc finger domain encoded by

exon 4 (amino acids 435-477). Interestingly, pancreatic malformations and congenital diaphragmatic hernia were observed in 9 out of 11 CHD patients who had _GATA6_ missense mutations within

exon 4 that encoding the C-terminal DNA-binding zinc finger domain (amino acids 435-477), whereas these extracardiac abnormalities were not found in any of the 32 patients with _GATA6_

missense variants in the other exons.59 Consistent with these findings, the novel _GATA6_ missense variant (p.Cys468Tyr) identified in our study was located in the C-terminal zinc finger

domain encoded by exon 4, leading to both cardiac and extracardiac phenotypes. The cysteine at codon 468 in the C-terminal zinc finger domain is highly conserved among the GATA family

members and plays an important role in the formation of the core zinc module for the GATA binding site recognition and protein interactions.60,61 The p.Cys468Tyr variant presumably disrupts

the core zinc module, leading to defects in the DNA binding as well as interactions with transcription factors, which thus causes dysregulation of GATA6 downstream target genes implicated in

multiple cell lineages mainly associated with cardiovascular and digestive organs. This works highlights early NEC as a new gastrointestinal phenotype associated with _GATA6_ variants. To

the best of our knowledge, two cases with CHD and intestinal abnormalities associated with _GATA6_ exon 4 missense mutations (p.Thr452Ala and p.Asn466Asp) and one case with _GATA6_ LoF

mutation were previously reported (Fig. 3), although NEC was not observed in either case.31,62 Remarkably, the proband developed NEC with intestinal perforation on the second day of life

without evidence of circulatory failure. NEC has been described in PTA with pulmonary overcirculation, which typically becomes clinically apparent over the first few days to weeks of life as

the pulmonary vascular resistance decreases.63 The pathophysiology of NEC is multifactorial, with premature birth being a primary determining factor as it is almost exclusively found in

preterm infants and its incidence is inversely correlated to gestational age at birth.64,65 Other risk factors associated with the development of NEC include bacterial infection, maternal

cocaine use, hypoxia and CHD primarily single ventricle physiology and left ventricular outflow tract obstructive lesions and episodes of systemic perfusion failure or shock.66,67,68 The

proband in our study had PTA, but did not have any of these risk factors including prematurity or episodes of systemic circulatory failure. Our institutional feeding protocol considers

respiratory status, respiratory support, near-infrared spectroscopy and diastolic blood pressures, which are surrogate markers for end organ or gut perfusion. Accordingly, we postulate that

NEC with intestinal perforation in the first four days of life in this patient is part of the _GATA6_ extracardiac phenotypic spectrum, although we cannot rule out the possibility that the

more severe CHD phenotype might influence the onset of NEC when compared to other reported cases with _GATA6_ exon 4 missense variants that did not have NEC (Fig. 3). The molecular

mechanisms how the _GATA6_ mutations cause PTA have been well investigated using genetically engineered mouse models.20 GATA6 is critical for the development of SHF and in the recruitment of

cardiac neural crest cells, contributing to the formation of the cardiac outflow tract by regulating semaphorin 3 C (_SEMA3C_) and plexin A2 (_PLXNA2_) during development.19,20,21 Further,

a molecular network involving _GATA6_, _FOXC1/2_, _TBX1_, _SEMA3C_, and _FGF8_ play important roles in the interaction between SHF and cardiac neural crest cells, and the failure of this

interaction results in PTA.69,70 Our study showed an abnormal localization pattern and decreased transcriptional activity of a novel GATA6 p.Cys468Tyr mutant protein that results in PTA

likely due to the disruption of the above molecular network during cardiovascular development. We also demonstrated protein aggregates of a novel GATA6 p.Cys468Tyr mutant protein. Protein

aggregates have been studied as causes underlying contractile dysfunction and human cardiomyopathy associated with genetic mutations, including _RBM20_, desmins, αB-crystallin, and COP9

signalosome subunit 8 (CSN8).71,72,73,74 In addition, a TBX1 mutant protein identified in a patient with TOF showed nuclear and cytoplasmic aggregates in transfected cells.75 However, the

mechanisms by which protein aggregates of genetic variants contribute to CHD in cardiac development remain unclear. Further studies are warranted to investigate the role of protein

aggregates underlying CHD phenotypes associated with _GATA6_ variants. The molecular mechanisms by which _GATA6_ variants cause NEC has not been explored. GATA6 is known to play a role in

intestinal development as conditional deletion in the developing intestine results in loss of the normal architecture of the intestinal villus with altered cell populations.76,77,78 While

intestinal abnormalities associated with _GATA6_ pathologic variants have been reported, including intestinal malrotation as well as protein losing enteropathy,28,79 the genetics of NEC

remains largely unknown.80 Large sequencing-based pilot studies have identified _NOD2_ (nucleotide-binding oligomerization domain containing 2), inducing autophagy pathway, and _SIGIRR_

(single immunoglobulin interleukin-1 related receptor), a modulator of Toll-like receptor-interleukin 1 receptor signaling, as potential loci for NEC.81,82 Since excessive inflammation is a

feature of NEC, several pro-inflammatory and cytokine genes have also been investigated, showing variable results.83 Additionally, the expression of tight junction proteins-coding genes such

as zonula occludens-1 (ZO-1) is shown to be significantly downregulated in intestinal specimens from NEC patients and animal models.84,85 Recently, investigators demonstrated that

conditional deletion of _Gata6_ in the gut epithelium significantly impacted intestinal barrier integrity and decreased expression of ZO-1, leading to enhanced intestinal permeability and

susceptibility to gut inflammation. Further, GATA6 directly modulates ZO-1 expression by binding ZO-1 promoter in human colon epithelial cells.86 Notably, decreased GATA6 expression was also

seen in the intestinal epithelium of patients with inflammatory bowel diseases.86 These observations indicate a role for Gata6 in the function of the intestinal epithelial barrier and

immune-inflammatory responses, and loss of GATA6 may contribute to the development of NEC by altering the expression of tight junction proteins-coding gene. Given the genetic heterogeneity

of NEC, candidate pathway-targeted or unbiased genomic sequencing approaches are needed to identify novel candidate genes for NEC. Our findings provide novel insights into understanding the

genetic basis underlying NEC associated with CHD and expand the phenotypic spectrum associated with _GATA6_ variants to intestinal abnormalities such as NEC and intestinal perforation. This

study has several limitations that should be noted. First, the in vitro functional assays of the _GATA6_ p.Cys468Tyr variant did not investigate the mechanistic role of the _GATA6_ variant

by evaluation of specific GATA6 target genes or interacting proteins that are specific for cardiac development and CHD. Although pathogenic variation in _GATA6_ is a well-accepted genetic

etiology of CHD and the altered cysteine is located in a highly conserved zinc finger, these additional experimental studies would strengthen our findings. Second, the functional studies

were in vitro assays, therefore, the findings may not translate to the developing human heart, intestine and pancreas. Additional in vivo animal studies are required to confirm the role of

_GATA6_ and this novel _GATA6_ variant on cardiac morphogenesis, as well as intestinal and pancreas development. Lastly, immunofluorescence analysis of both GATA6 wildtype and p.Cys468Tyr

demonstrated an abnormal nuclear localization pattern, however, further analyses are warranted to determine the nature of the protein aggregates and examination of protein expression in

cellular compartments by Western blot. In conclusion, we describe a novel heterozygous _GATA6_ variant (p.Cys468Tyr) resulting in a spectrum of CHD and extracardiac abnormalities. The family

members presented PTA, ASD, BAV, and uniquely, NEC with intestinal perforation. Our findings expand the genotypic and phenotypic spectrum for likely pathogenic variation in _GATA6_,

highlighting gut abnormalities. The combination of early neonatal intestinal perforation and CHD should lead clinical providers to consider the possibility of _GATA6_ pathogenic variation,

which could have clinical implications in the intensive care unit as well as prediction of associated phenotypes and genetic counseling. DATA AVAILABILITY Data presented in current

publication have been deposited in and are available in dbGaP database under dbGaP accession phs002010.v1.p1. Some restrictions apply for dbGaP, as data is available to researchers who meet

access criteria. Data access is through the dbGaP website and researchers apply for data access. Supplied accession number should be used to search the dataset on the website:

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references ACKNOWLEDGEMENTS We thank the family members for study participation, Gloria Zender in Cell Line Core and Samantha Fichtner and Jade Hayden in Heart Center Clinical Research Core.

FUNDING This work was supported in part by funding from National Institutes of Health (R01 HL109758) to V.G., K.L.M, and P.W. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Center for

Cardiovascular Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA Jun Yasuhara, Sathiya N. Manivannan, Uddalak Majumdar, Mona Aljuhani, Katherine

Myers, Kim L. McBride & Vidu Garg * The Heart Center, Nationwide Children’s Hospital, Columbus, OH, USA Jun Yasuhara, Sathiya N. Manivannan, Uddalak Majumdar, Mona Aljuhani, Katherine

Myers, Corey Stiver, Mark Galantowicz, Kim L. McBride & Vidu Garg * Institute for Genomic Medicine, Nationwide Children’s Hospital, Columbus, OH, USA David M. Gordon, Patrick J. Lawrence

 & Peter White * Department of Pediatrics, The Ohio State University, Columbus, OH, USA Corey Stiver, Amee M. Bigelow, Kim L. McBride, Peter White & Vidu Garg * Division of Pediatric

Cardiology, Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan Hiroyuki Yamagishi * Division of Genetic and Genomic Medicine, Nationwide Children’s Hospital,

Columbus, OH, USA Kim L. McBride * Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA Vidu Garg Authors * Jun Yasuhara View author publications You can also

search for this author inPubMed Google Scholar * Sathiya N. Manivannan View author publications You can also search for this author inPubMed Google Scholar * Uddalak Majumdar View author

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Lawrence View author publications You can also search for this author inPubMed Google Scholar * Mona Aljuhani View author publications You can also search for this author inPubMed Google

Scholar * Katherine Myers View author publications You can also search for this author inPubMed Google Scholar * Corey Stiver View author publications You can also search for this author

inPubMed Google Scholar * Amee M. Bigelow View author publications You can also search for this author inPubMed Google Scholar * Mark Galantowicz View author publications You can also search

for this author inPubMed Google Scholar * Hiroyuki Yamagishi View author publications You can also search for this author inPubMed Google Scholar * Kim L. McBride View author publications

You can also search for this author inPubMed Google Scholar * Peter White View author publications You can also search for this author inPubMed Google Scholar * Vidu Garg View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS J.Y. and V.G. conceived and designed the study. K.M, C.S., K.L.M, and V.G. enrolled patients. J.Y.,

M.A., K.M., and C.S. collected clinical data. J.Y., S.N.M., and U.M. performed and acquired experimental data. J.Y., S.N.M., D.M.G., P.J.L, and P.W. performed bioinformatics analyses. J.Y.,

S.N.M., U.M., D.M.G., P.J.L, K.L.M., P.W., and V.G. analyzed and interpreted the data. J.Y. and V.G. drafted the manuscript. C.S., A.M.B., M.G., H.Y., K.L.M., P.W., and V.G. critically

reviewed the manuscript. All authors read and approved the final version of the manuscript for publication. CORRESPONDING AUTHOR Correspondence to Vidu Garg. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing interests. INFORMED CONSENT Statement of written consent was obtained as described in the Ethics statement. ADDITIONAL INFORMATION PUBLISHER’S NOTE

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ARTICLE CITE THIS ARTICLE Yasuhara, J., Manivannan, S.N., Majumdar, U. _et al._ Novel pathogenic _GATA6_ variant associated with congenital heart disease, diabetes mellitus and necrotizing

enterocolitis. _Pediatr Res_ 95, 146–155 (2024). https://doi.org/10.1038/s41390-023-02811-y Download citation * Received: 27 February 2023 * Revised: 11 August 2023 * Accepted: 21 August

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