Charles Shaw-Smith,1 Elisa De Franco,1 Hana Lango Allen,1 Marta Batlle,2,3 Sarah E. Flanagan,1

Maciej Borowiec,4 Craig E. Taplin,5 Janiëlle van Alfen-van der Velden,6 Jaime Cruz-Rojo,7

Guiomar Perez de Nanclares,8 Zosia Miedzybrodzka,9 Grazyna Deja,10 Iwona Wlodarska,11

Wojciech Mlynarski,4 Jorge Ferrer,2,3,12 Andrew T. Hattersley,1 and Sian Ellard1

GATA4 Mutations Are a Causeof Neonatal and Childhood-Onset DiabetesDiabetes 2014;63:2888–2894 | DOI: 10.2337/db14-0061

The GATA family zinc finger transcription factors GATA4and GATA6 are known to play important roles in the de-velopment of the pancreas. In mice, both Gata4 and Gata6are required for pancreatic development. In humans,GATA6 haploinsufficiency can cause pancreatic agenesisand heart defects. Congenital heart defects also are com-mon in patients with GATA4 mutations and deletions, butthe role of GATA4 in the developing human pancreas isunproven. We report five patients with deletions (n = 4) ormutations of theGATA4 genewhohave diabetes and a vari-able exocrine phenotype. In four cases, diabetes presentedin the neonatal period (age at diagnosis 1–7 days). A denovo GATA4 missense mutation (p.N273K) was identifiedin a patient with complete absence of the pancreas con-firmed at postmortem. This mutation affects a highly con-served residue located in the second zinc finger domain ofthe GATA4 protein. In vitro studies showed reduced DNAbinding and transactivational activity of the mutant protein.We show that GATA4 mutations/deletions are a cause ofneonatal or childhood-onset diabetes with or without exo-crine insufficiency. These results confirm a role for GATA4in normal development of the human pancreas.

The GATA family of transcription factors, including GATA4and GATA6, plays important roles in the development of

several endodermally and mesodermally derived organs.Heterozygous mutations in GATA6 have previously beenassociated with congenital heart malformations (1), and,more recently, they were found to be the major cause ofpancreatic agenesis in humans (2). Further studies haveshown that patients with GATA6mutations have a variablespectrum of diabetes manifestations, ranging from com-plete pancreatic agenesis to adult-onset diabetes withoutexocrine insufficiency (3).

GATA4 is a zinc finger transcription factor closely relatedto GATA6. Mutations in GATA4 have been described infamilial and sporadic cases of heart malformation (4,5), al-though nonpenetrance is common (6). More than 120 casesof chromosome rearrangements resulting in interstitial orterminal deletions of chromosome 8p including GATA4 havebeen reported. The phenotypes associated with these dele-tions include congenital heart malformations, dysmorphicfeatures, and mental retardation (reviewed in Wat et al. [6]).

Mouse studies have suggested a role for Gata4 in pan-creatic development. Gata42/2 mice die prior to pancreasspecification due to defective extraembryonic tissues for-mation (7,8), but investigation of Gata42/2 mice rescuedfrom embryonic lethality shows agenesis of the ventral pan-creas (9). Gata4+/2 mice are phenotypically normal (7,8).

1Institute of Biomedical and Clinical Science, University of Exeter Medical School,Exeter, U.K.2Genomic Programming of Beta-Cells Laboratory, Institut d’Investigacions BiomèdiquesAugust Pi i Sunyer, Barcelona, Spain3CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain4Department of Paediatrics, Oncology, Haematology and Diabetology, MedicalUniversity of Lodz, Lodz, Poland5Department of Pediatrics, Seattle Children’s Hospital, University of Washington,Seattle, WA6Department of Pediatrics, Radboud University Nijmegen Medical Centre, Nijmegen,the Netherlands7Unidad de Endocrinología Pediátrica Hospital, Universitario Doce de Octubre,Madrid, Spain8Molecular (Epi)Genetics Laboratory, Hospital Universitario Araba-Txagorritxu,BioAraba, Vitoria-Gasteiz, Spain

9Division of Applied Medicine, University of Aberdeen, Aberdeen, U.K.10Department of Paediatrics, Paediatric Endocrinology and Diabetes, SilesianMedical University, Katowice, Poland11Center for Human Genetics, KU Leuven, Leuven, Belgium12Department of Medicine, Imperial College London, London, U.K.

Corresponding author: Sian Ellard, sian.ellard@nhs.net.

Received 14 January 2014 and accepted 27 March 2014.

C.S.-S. and E.D.F. contributed equally to this work.

© 2014 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

2888 Diabetes Volume 63, August 2014

GENETIC

S/G

ENOMES/P

ROTEOMIC

S/M

ETABOLOMIC

S

In human embryos, GATA4 is detected in the area of thedeveloping pancreas at 29–31 dpc (CS12), at the same timeof the early transcription factor PDX1, suggesting a role ofGATA4 in human pancreas development (10). There is onereport of a GATA4missense mutation (p.R319W) in a childwith pancreatic agenesis and an atrial septal defect (11).The proband’s father and sister were also heterozygous forthe mutation, which was shown to impair protein functionin vitro. Both had a congenital heart defect but no diabetes,so a causal link between GATA4 and pancreatic agenesiscould not be proven and evidence to confirm a key role forGATA4 in development of the human pancreas is lacking.

We investigated the role of GATA4 in human pancreaticdevelopment by studying three patients with diabetes andchromosome 8p deletions spanning the gene and searchingfor deletions or intragenic GATA4mutations in 186 patientswith neonatal diabetes (NDM) of unknown etiology.

RESEARCH DESIGN AND METHODS

Patient CohortPatients were defined from two sources: 1) One hundredeighty six probands diagnosed with NDM before 6 monthsin whom no genetic diagnosis had been established wereselected from a cohort of 867 patients with NDM whowere referred from 73 countries (the relevant clinical in-formation was provided by the referring clinician); and 2)a cohort of patients with diabetes and known chromosomalabnormalities (n = 13).

GATA4 Mutation AnalysisWe used a custom targeted next-generation sequencing(NGS) panel test (12) to screen the coding sequence andexon-intron boundaries of GATA4 in the NDM cohort.The assay is able to detect partial and whole-gene dele-tions in addition to base substitutions and indels. In twocases, there was insufficient DNA for targeted NGS andmutation testing was carried out by Sanger sequencing.We also sequenced the GATA4 pancreatic and endodermalenhancers described by Rojas and colleagues (13,14).Primer sequences are available on request. Sequencingreactions were run on an ABI 3730 capillary machine(Applied Biosystems) and analyzed using Mutation Surveyorv3.98 (SoftGenetics) (reference sequence NM_002052.3).Parent-proband relationships were confirmed using thePowerPlex kit (Promega).

Deletions of GATA4Deletions of GATA4 were either cytogenetically visiblerearrangements including chromosome 8p in patientsfrom the known chromosomal abnormalities cohort ordetected by reduced copy number on the targeted NGSassay in the NDM cohort. The extent of small GATA4deletions was determined using the Affymetrix Genome-Wide Human SNP Array 6.0 (Santa Clara, CA), containingnearly 1 million copy number probes. Genotyping andwithin-batch copy number calling were performed byAROS Applied Biotechnology (Aarhus, Denmark). We vi-sualized regions of reduced copy number state within

Affymetrix Chromosome Analysis Suite and identifiedprobes marking deletion breakpoints.

Functional StudiesMammalian expression vector pIRES(+)-GATA4 was usedto perform DpnI mediated site2directed mutagenesis togenerate GATA4 N273K (c.819C.A) mutation. Two in-dependent clones were created, sequenced, and assayedin transient transfection assays in quadruplicates on threeindependent experiments, as described in Boj et al. (15).Lipofectamine 2000 was used to transfect HeLa cells with0.15 mg of pGL3-WNT2 promoter luciferase construct (16)and 2-ng pRL Renilla luciferase reporter vector, in conjunc-tion with different amounts of empty vector (pIRES-hrGFP), pIRES-GATA4, or pIRES-GATA4-N273K, to reach0.5-mg total DNA. Firefly and Renilla luciferase activitywere assayed using the Dual-Luciferase Reporter Assay Sys-tem (Promega). Binding of nuclear lysates from cells express-ing GATA4 and GATA4-N273K protein to 32P-labeledoligonucleotides, including binding sites for GATA4, wasperformed as described (15). Sequences of oligonucleotidesused in this assay include a predicted GATA binding site inthe pancreatic (P2) HNF4A proximal promoter. Specificityof retardation complex was assessed by preincubating nu-clear extracts with 100-fold excess wild-type or mutantunlabeled oligonucleotides, or GATA4 antiserum (GATA4C-20; Santa Cruz Biotechnology, sc-1237). Immunoblotanalysis was used to verify that the expression efficienciesfor vectors encoding wild-type and N273K GATA4 weresimilar.

RESULTS

Molecular GeneticsWe report three patients with diabetes and a knownchromosomal abnormality resulting in GATA4 haploinsuf-ficiency. Cases 1 and 2 have terminal 8p deletions of 12.5or 17 Mb, and Case 3 has an unbalanced translocationresulting in a large (;15 Mb) terminal 8p deletion togetherwith a duplication of the tip of chromosome 9 (Fig. 1). Thepossibility of an intragenic or regulatory GATA4 mutationon the nondeleted 8p allele or a GATA6 mutation wasexcluded by sequence analysis.

We then tested 186 patients with NDM for intragenicmutations or deletions of GATA4 and found two mutations.A whole-gene deletion in Case 4 was detected by targetedNGS assay and shown to be a 1 Mb interstitial deletion(chr8:10796092–11934660) that includes 8 additional genes(Fig. 1) by copy number analysis of genome-wide SNPs.A novel heterozygous missense mutation (c.819C.A,p.N273K) that affects a highly conserved amino acid lo-cated in the second zinc finger of the GATA4 protein wasfound in Case 5 (Fig. 2). The mutation is predicted as like-ly to be pathogenic by Alamut (Interactive Biosoftware,Rouen, France). Mutations in GATA6, PDX1, and PTF1Ahad previously been excluded in both cases.

All five patients were born to unaffected parents andtesting in four cases showed that the GATA4 deletion or

diabetes.diabetesjournals.org Shaw-Smith and Associates 2889

missense mutation had arisen de novo (Table 1). Four ofthe five patients were diagnosed with diabetes during thefirst week of life, giving an overall frequency of GATA4mutations in our NDM cohort of 4/867 (0.5%).

Functional Analysis of the Novel Missense MutationTo test whether the p.N273K mutation truly affectsGATA4 function, we examined the ability of wild-typeand mutant proteins to bind and activate target sites.Electromobility shift assays showed that wild-type GATA4

exhibits high affinity binding to a GATA binding site inthe pancreatic HNF4A promoter and that the p.N273Kmutation impairs this interaction (Fig. 3A). Furthermore,transfected plasmids expressing GATA4 with the p.N273Kmutation showed a 4.3-fold decrease in the ability to ac-tivate expression of a promoter that responds to GATA4(16) (Fig. 3B). These results indicate that the p.N273Kmutation affects GATA4 transactivational activity by dis-rupting the ability of this protein to bind target recogni-tion sequences.

Figure 1—Schematic diagram indicating the extent of the deletion on chromosome 8p in Cases 124 and gene content (Hg19) of theminimal deleted interval.

Figure 2—Schematic diagram showing the genomic and protein position of the GATA4 missense mutation.

2890 GATA4 Mutations and Neonatal Diabetes Diabetes Volume 63, August 2014

Tab

le1—Clinicald

etailsofthe

five

casesdescrib

ed

Case

Geneticdefect

Denovo

Status

Age

atdiagnosis

Birth

weight/

gestationCurrentage

Pancreas

Card

iacmalform

ationsNeurocognitiveab

normalities

Other

abnorm

alitiesClinical

Biochem

istry(fecalelastase)

Imaging

18p

22pter

del

[12.5Mb]

Yes

Diab

etic13

years2,440

g/40

weeks

17years

Nosym

ptom

sof

exocrineinsufficiency

284.1mg/g

Pancreatic

hypop

lasiaof

bod

yand

tail(M

RCP)

AVcanal

defect,

pulm

onarystenosis,mitraland

tricuspid

abnorm

alities

Glob

aldevelop

mental

delay,

epilep

sy

28p

23.3–

22del

[17Mb]

Yes

PNDM

7days

1,780g/

gestationalage

notavailab

le

1year

Nosym

ptom

sof

exocrineinsufficiency

N/A

Pancreaticim

agingnorm

al(USSand

CT)

ASD,VSD,

pulm

onarystenosis

Psychom

otorretard

ation,norm

albrain

MRI

38p

23.3–

22del

[15Mb]

Yes

TNDM

1day

1,810g/

38weeks

10years

Nosym

ptom

sof

exocrineinsufficiency

132.2mg/g

Not

possib

leto

visualizepancreas(USS)

SmallA

SD,

smallV

SD,

pulm

onarystenosis

Mental

retardation,

MRIb

rain:sub

epend

ymal

calcification

48p

23.1del

[1Mb]

Not

maternallyinheritedbut

nopaternalsam

ple

available

TNDM

2days

1,680g/

36weeks

7years

Onpancreatic

enzyme

supplem

entation

17mg/g

Pancreatic

hypop

lasia(M

RI)

ASD,

pulm

onarystenosis

Mild

dysp

hasia,visual

percep

tiondeficit,

andslight

difficulty

inhearing

discrim

ination

Mild

hyposp

adias,

mild

liverenlargem

ent,and

riseof

liverenzym

es

5GATA

4c.819C

.A;

p.N273K

Yes

Pancreaticagenesis

1day

1,240g/

34weeks

Deceased

N/A

N/A

Com

plete

absence

ofpancreas(PM)

None

Abnorm

alwhite

matter

develop

ment

Died

ofmultiorganfailure(day

4)

ASD,atrial

septal

defect;

AV,atrioventricular;

CT,

computed

tomography;

MRCP,magnetic

resonancecholangiop

ancreatography;

MRI,magnetic

resonanceim

aging;PM,postm

ortem;

PNDM,perm

anentNDM;TN

DM,transient

NDM;USS,ultrasound

scan;VSD,ventricular

septald

efect.

diabetes.diabetesjournals.org Shaw-Smith and Associates 2891

Clinical Characteristics

Pancreatic PhenotypeThe clinical characteristics are shown in Table 1. The en-docrine phenotype was variable. Two patients had NDMdiagnosed in the first week of life, which remitted tem-porarily but relapsed after 6 months and 7 years. Twopatients had permanent diabetes diagnosed at 1 weekor 13 years. Case 1 was diagnosed at 13 years and pre-sented with an HbA1c of 114.2 mmol/mol (12.6%) follow-ing a 6-month history of polyuria and polydipsia, but wasnot ketotic. No pancreatic autoantibodies were detectedand C-peptide production was preserved 3 years’ post-diagnosis (stimulated C-peptide 3.8 ng/mL). Case 5 wasborn prematurely (34 weeks’ gestation), diagnosed withdiabetes on the first day of life, and died of multiple organfailure at four days of age. The four patients with NDMhad low birth weight (,3rd centile) consistent with in-sulin deficiency in utero. All five were treated with insulin.

The pancreatic exocrine phenotype was also variable.There was complete absence of the pancreas on post-mortem examination of Case 5. Case 4 had exocrinepancreatic insufficiency (fecal elastase = 17 mg/g) and re-ceived exocrine supplementation. To assess subclinicalexocrine insufficiency in the three patients not receivingexocrine supplementation, we measured fecal elastase andperformed pancreatic imaging. There was reduced pancreatic

size in two patients and low fecal elastase in one (132 mg/g;normal range .200 mg/g).

Extrapancreatic PhenotypeCongenital heart malformations were present in the fourcases with GATA4 deletions and ranged from septaldefects associated with pulmonary stenosis to atrioven-tricular canal defect, but they were not seen in Case 5 atpostmortem. Developmental delay and neurocognitivedefects were reported in all five patients.

DISCUSSION

Our study reports five patients with GATA4 mutationsand a variable phenotype of transient or permanent di-abetes diagnosed in neonates or during childhood. Theexocrine pancreatic phenotype ranged from completeagenesis to hypoplasia with subclinical exocrine insuffi-ciency or normal exocrine function. Additional featuresincluded neurocognitive defects and congenital heart mal-formations. The variable phenotype was not correlatedwith the size of the deletion, consistent with the variablepenetrance of GATA4 mutations/deletions reported in theliterature. Both patients with pancreatic agenesis (thisstudy and D’Amato et al. [11]) had missense mutationsrather than deletions, but the absence of diabetes in twoheterozygous relatives suggests that a dominant-negativeeffect is unlikely (11). Our results indicate that GATA4

Figure 3—The p.N273K mutation disrupts GATA4 function. A: Electrophoretic mobility shift assay showing that HeLa cells transfected withwild-type (WT) GATA4, but not the GATA4 p.N273K mutation, exhibit strong binding to a predicted GATA recognition sequence in thepancreatic (P2) HNF4A promoter. Binding disappears upon exposure to3100 excess WT competitor oligonucleotides and supershifts withantiserum for GATA4, but is not affected by oligonucleotides with a mutation in the GATA consensus sequence (Mut) or by IgG. B: A WNT2promoter reporter construct that contains a high-affinity GATA4 binding site shows transactivation by cotransfection with WT GATA4. Thisis markedly decreased when cotransfecting the p.N273K plasmid. Control transfections were performed with an empty expression vector.*P < 0.05 (Student t test). C: Immunoblot analysis shows comparable expression efficiency for vectors encoding WT and N273K GATA4.

2892 GATA4 Mutations and Neonatal Diabetes Diabetes Volume 63, August 2014

mutations/deletions are a rare cause of NDM, accountingfor 0.5% of our international series (4/867 cases). Theobservation that postzygotic GATA4 mutations in embry-onic heart tissue can cause congenital heart defects (16)implies a similar mechanism for NDM, but is impossibleto prove without access to pancreatic tissue.

The novel GATA4 missense p.N273K mutation ishighly likely to be pathogenic because it affects a highlyconserved amino acid within one of the zinc fingers andhas arisen de novo, and cell-based studies of the mutantprotein demonstrated impaired DNA binding and reducedtransactivational activity. A different mutation affectingthe same residue (p.N273S) was previously reported inthe heart tissue of a deceased patient with severe cardiacabnormalities (17), consistent with a pathogenic role ofthis mutation albeit with interindividual differences inthe phenotypic expressivity.

The GATA4 deletions in our cohort ranged from 1 to17 Mb with an overlapping interval that includes 8 addi-tional genes. No mutations in these genes have previouslybeen reported to cause NDM, although previous reportsdescribe an association between diabetes and this chro-mosome 8p region, with a possible critical region at 8p23(18). Several candidate genes have been proposed, includ-ing PPP1R3B (19) and BLK (20), but not GATA4. BLKsequencing was included within the targeted NGS assaybut no mutations were found. Follow-up studies of indi-viduals with 8p deletions and intragenic GATA4 muta-tions are required to ascertain the incidence of diabetesoutside the neonatal period.

Conditional simultaneous knockout of Gata4 andGata6 in mouse pancreas causes pancreatic agenesis,whereas only mild early developmental defects are ob-served in mice in which only Gata6 or Gata4 are ablated,suggesting redundant roles for Gata4 and Gata6 in mousepancreatic development (21,22). Recent evidence also sug-gests that mouse pancreas development is sensitive toGata4 and Gata6 dosage, as mice retaining only oneGata4 or Gata6 allele exhibit pancreatic developmentaldefects that are not observed with double homozygousmutant mice. These observations in mice should be con-trasted with our results showing that in humans haploin-sufficiency of either GATA6 (2,3) or GATA4 can causeimpaired pancreatic development resulting in a wide spec-trum of diabetes manifestations. Similar discrepancies inhuman/mouse phenotypes have been observed in otherendodermal transcription factors involved in pancreas de-velopment such as Hnf1a, Hnf1b, and Hnf4a. Existingknowledge suggests that although the transcription fac-tors that regulate pancreas development in humans andmice are very often the same, haploinsufficiency appearsto cause much more severe manifestations during humanas opposed to mouse pancreas development.

Intragenic GATA4 mutations or deletions causingGATA4 haploinsufficiency have been described in morethan 140 patients with congenital heart malformations.In contrast, diabetes only has been reported in six patients

with GATA4mutations or deletions (our study and D’Amatoet al. [11]), suggesting a lower penetrance of the pancre-atic phenotype compared with the cardiac phenotype.The genetic and/or environmental factors underlyingthis phenotypic variability are not understood. Animalstudies have shown that knockout of one copy of Gata4in mouse strains with different genetic backgroundresults in a marked difference in the cardiac phenotypeobserved, ranging from absence of pathogenic phenotypeto complex congenital heart defects (23). This evidencesuggests that additional genetic factors contribute to thepenetrance of the cardiac phenotype. A similar mechanismin humans would explain the difference in the pancreaticand extrapancreatic phenotypes observed in patients withGATA4 mutations or deletions. In our study, we were notable to identify any variant in the coding or regulatoryregions of GATA4 or GATA6 that could account for thepresence of the pancreatic phenotype in our patients. Fur-ther studies will be needed to assess the possible contribu-tion of other genes to the variable phenotype associated tomutations in this transcription factor.

In summary, our results show that GATA4 mutationsare a rare cause of NDM and pancreatic agenesis. This isan important diagnosis to make as it confers an increasedrisk for future offspring being affected with NDM and/orcongenital heart defects. Testing for intragenic mutationsor deletions of GATA4 should therefore be considered inpatients with NDM or childhood-onset diabetes and con-genital heart defects in whom no GATA6 mutation hasbeen identified. This study confirms a role for GATA4 inthe development of the human pancreas.

Acknowledgments. The authors thank the families who participated inthis study.Funding. The research leading to these results received funding from theEuropean Community’s 7th Framework Programme (FP7/2007-2013) undergrant agreement number FP7-PEOPLE-ITN-2008 (Marie Curie Initial TrainingNetwork, Biology of Liver and Pancreatic Development and Disease) and grantagreement number 223211 (Collaborative European Effort to Develop DiabetesDiagnostics), Ministerio de Economía y Competitividad (SAF2011-27086), Diabe-tes UK (ref. 11/0004193), and the Wellcome Trust. S.E. and A.T.H. are employedas core members of staff within the National Institute for Health Research–fundedExeter Clinical Research Facility. S.E., J.F., and A.T.H. are Wellcome Trust SeniorInvestigators and A.T.H. is a National Institute for Health Research Senior In-vestigator. M.B. and W.M. are supported by National Science Center, Poland(NCN) grant 2011/01/M/NZ5/02815 and by Innovative Economy OperationalProgramme–Activity 1.2 (the TEAM Programme coordinated by the Foundationfor Polish Science).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. C.S.-S. researched the clinical data, contributedto discussion, and wrote the manuscript. E.D.F. researched the molecular ge-netics data, contributed to discussion, and wrote the manuscript. H.L.A., S.E.F.,M.Bo., G.P.d.N., and I.W. researched the molecular genetics data and reviewedthe manuscript. M.Ba. and J.F. performed the functional studies, contributed todiscussion, and reviewed the manuscript. C.E.T., J.v.A.-v.d.V., J.C.-R., Z.M., andG.D. researched the clinical data and reviewed the manuscript. W.M. researchedthe clinical and molecular data, contributed to discussion, and reviewed the

diabetes.diabetesjournals.org Shaw-Smith and Associates 2893

manuscript. A.T.H. directed the research, contributed to discussion, and reviewedthe manuscript. S.E. directed the research, contributed to discussion, and wrotethe manuscript. S.E. is the guarantor of this work and, as such, had full access toall the data in the study and takes responsibility for the integrity of the data andthe accuracy of the data analysis.

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2894 GATA4 Mutations and Neonatal Diabetes Diabetes Volume 63, August 2014