Transient Neonatal Diabetes, ZFP57,and Hypomethylation ofMultipleImprinted LociA detailed follow-up

SUSANNE E. BOONEN, MD, PHD1,2

DEBORAH J.G. MACKAY, PHD3,4

JOHANNE M.D. HAHNEMANN, MD, PHD1

LOUISE DOCHERTY, PHD3,4

KAREN GRØNSKOV, PHD1

ANNA LEHMANN, PHD4

LISE G. LARSEN, MD5

ANDREAS P. HAEMERS , MD6

YVES KOCKAERTS, MD7

LUTGARDE DOOMS, MD8

D~uNG CH�I V~u, MD9

C.T. BICH NGOC, MD9

PHUONG BICH NGUYEN, MD10

OLGA KORDONOURI, MD11

FRIDA SUNDBERG, MD12

PINAR DAYANIKLI, MD, FAAP13

VIJITH PUTHI, MD14

CARLO ACERINI, MD15

AHMED F. MASSOUD, MD, MBBS, FRCP,

FRCPCH16

ZEYNEP T€UMER, MD, PHD1

I. KAREN TEMPLE, MD4,17

OBJECTIVEdTransient neonatal diabetes mellitus 1 (TNDM1) is the most common cause ofdiabetes presenting at birth. Approximately 5% of the cases are due to recessive ZFP57mutations,causing hypomethylation at the TNDM locus and other imprinted loci (HIL). This has conse-quences for patient care because it has impact on the phenotype and recurrence risk for families.We have determined the genotype, phenotype, and epigenotype of the first 10 families to alerthealth professionals to this newly described genetic subgroup of diabetes.

RESEARCH DESIGN AND METHODSdThe 10 families (14 homozygous/compoundheterozygous individuals) with ZFP57 mutations were ascertained through TNDM1 diagnostictesting. ZFP57 was sequenced in probands and their relatives, and the methylation levels atmultiple maternally and paternally imprinted loci were determined. Medical and family historieswere obtained, and clinical examination was performed.

RESULTSdThe key clinical features in probands were transient neonatal diabetes, intrauterinegrowth retardation, macroglossia, heart defects, and developmental delay. However, the findingof two homozygous relatives without diabetes and normal intelligence showed that the pheno-type could be very variable. The epigenotype always included total loss of methylation at theTNDM1 locus and reproducible combinations of differential hypomethylation at other mater-nally imprinted loci, including tissue mosaicism.

CONCLUSIONSdThere is yet no clear genotype–epigenotype–phenotype correlation toexplain the variable clinical presentation, and this results in difficulties predicting the prognosisof affected individuals. However, many cases have a more severe phenotype than seen in othercauses of TNDM1. Further cases and global epigenetic testing are needed to clarify this.

Diabetes Care 36:505–512, 2013

Imprinted genes are characterized by aparent of origin expression patterncontrolled by epigenetic modifica-

tions, including DNA methylation withindifferentially methylated regions (DMRs).The normal methylation pattern of DMRsis established in the germlines and main-tained during embryogenesis. Imprintedgenes are crucial in normal growth anddevelopment. Currently, 73 human im-printed genes are known (http://igc.otago.ac.nz). Aberrant methylation ofthese DMRs results in imprinting disor-ders such as transient neonatal diabetesmellitus 1 (TNDM1; MIM 601410),Prader-Willi syndrome (MIM 176270),Angelman syndrome (MIM 105830),Beckwith-Wiedemann syndrome (BWS;MIM 130650), and Silver-Russell syn-drome (SRS; MIM 180860). However,the complex mechanisms underpinningestablishment and maintenance of pri-mary DMRs are not fully understood.

Until very recently, our knowledgewas limited to imprinting disorders inwhich a single locus was exposed to ab-normal methylation. It is now clear thatsome patients have methylation aberra-tions affecting multiple imprinted loci,which we termed hypomethylation ofmultiple imprinted loci (HIL) (1–3).This phenomenon is heterogeneous,and there are likely genetic and environ-mental causes.

We described the first heritable globalimprinting disorder compatible with life

c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c

From the 1Center for Applied Human MolecularGenetics, The Kennedy Center, Glostrup, Den-mark; 2The Wilhelm Johannsen Centre forFunctional Genome Research, University of Co-penhagen, Copenhagen, Denmark; the 3WessexRegional Genetics Laboratory, Salisbury HealthCare Trust, Salisbury, U.K.; 4HumanGenetics andGenomic Medicine, Faculty of Medicine, Univer-sity of Southampton, Southampton, U.K.; the5Department of Clinical Pathology, NæstvedHospital, Næstved, Denmark; the 6Department ofInternal Medicine-Endocrinology, Maria HospitalNoord Limburg, Overpelt, Belgium; the 7Depart-ment of Endocrinology and Diabetes, CampusAndré Dumont, Genk, Belgium; the 8Departmentof Paediatrics, University of Liege, Liege, Belgium;

the 9Department of Medical Genetics, Metabolismand Endocrinology, National Hospital of Pediat-rics, Hanoi, Vietnam; the 10Department of Medi-cine, Austin Health/Northern Health, Universityof Melbourne, Melbourne, Victoria, Australia;the 11Diabetes Centre for Children and Adoles-cents, Children’s Hospital at the Bult, Hannover,Germany; the 12Department of Paediatrics, TheQueen Silvia Children’s Hospital, Gothenburg,Sweden; the 13American Hospital, Nisantasi,Istanbul, Turkey; the 14Department of Paedi-atrics, Peterborough and Stamford HospitalsFoundation Trust, Cambridgeshire, U.K.; the15Department of Paediatrics, Addenbrooke’sHospital, Cambridge, U.K.; 16Children’s Services,Northwick Park Hospital, London, U.K.; and the

17Wessex Clinical Genetics Service, UniversityHospital Southampton NHS Trust, Southampton,U.K.

Corresponding author: SusanneE. Boonen, susaboon@rm.dk.

Received 31May 2012 and accepted 13August 2012.DOI: 10.2337/dc12-0700This article contains Supplementary Data online at

http://care.diabetesjournals.org/lookup/suppl/doi:10.2337/dc12-0700/-/DC1.

© 2013 by the American Diabetes Association.Readers may use this article as long as the work isproperly cited, the use is educational and not forprofit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ fordetails.

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C l i n i c a l C a r e / E d u c a t i o n / N u t r i t i o n / P s y c h o s o c i a l R e s e a r c hO R I G I N A L A R T I C L E

in patients presenting with TNDM1 dueto HIL. The patients have total loss ofmethylation (LOM) at the maternallymethylated TNDM1 DMR (6q24) (4). Inaddition, they have a mosaic pattern ofhypomethylation affecting other mater-nally methylated loci, primarily PEG3/ZIM2 (19q13.4) and GRB10 (7p12.2).Homozygous or compound heterozygousDNA sequence mutations in the ZFP57were identified in more than half of thesepatients (5). The probands with ZFP57mutations have a more complex TNDM1phenotype compared with patients withTNDM due to other causes. The findingof heritable mosaic epigenetic aberrationsassociated with ZFP57mutations indicatesthat the gene may be involved in mainte-nance of DNAmethylation at imprinted re-gions during the early multicellular stagesof human development. In mouse, zfp57has a clear role inmethylationmaintenanceat imprinted loci in both the germline andpostfertilization but is also implicated inmethylation establishment in the develop-ing oocyte (6).

ZFP57 is localized at chromosome6p22.1 spanning an 8.6-kb genomic re-gion. The gene is organized in six exonsencoding the ZFP57 protein consisting of516 amino acids (Fig. 1). The ZFP57 pro-tein is a Kr€uppel-associated box domain(KRAB) zinc finger protein with a KRAB Aand KRAB B domain encoded by exons 4

and 5, respectively. The seven zinc fingersof the C2H2 type are encoded by exon 6.

In mice, expression of zfp57 has beendemonstrated in embryonic stem cells(expression levels declining with cell dif-ferentiation), testes, and ovary and in ratin the nervous system (7–9). Recent stud-ies indicate that zfp57, via interactionwith its cofactor KRAB-associated protein1, recruits DNA methyltransferases and isrequired for the maintenance of methyla-tion of DNA imprints, as well as of histo-nes in embryonic stem cells (10,11).

HIL also occurs in some patients withBWS associated with hypomethylation ofKCNQ1OT1 DMR (11p15.5) (12–15) andSRS due to hypomethylation ofH19 DMR(11p15.5) (14,16). In some of these pa-tients, the hypomethylation can involvepaternally or maternally methylatedDMRs, in contrast to ZFP57-relatedTNDM1HIL patients, in whom the hypo-methylation occurs only at maternally im-printed loci. However, no underlyinggenetic defect for BWS or SRS HIL casesis so far known, except for a single casewith a homozygous sequence mutation inthe NLRP2 gene in the mother of two sib-lings with BWS and hypomethylation atKCNQ1OT1 DMR, in which one of thepatients also had hypomethylation atPEG1/MEST DMR (7q32) (17). We andothers (18,19) have sequenced ZFP57 in27 BWS patients with hypomethylation of

KCNQ1OT1 DMR and 30 SRS patientswith hypomethylation at H19 DMR, re-spectively, and could not find disease-associated alterations, but these studiesdid not necessarily include cases withHIL.

In this study, we present the detailedgenotype, phenotype, and epigenotype ofthe first 10 families with ZFP57-relatedTNDM1 HIL and discuss these findingsin the context of the current knowledgeof the molecular mechanisms involved.

RESEARCH DESIGN ANDMETHODSdThe study was conduc-ted in accordance with the Helsinki Dec-laration. Informed consent was obtainedfrom all participants (parental consentobtained for children).

Clinical investigationsClinical features of families 1–7 were pre-viously described in brief (5), and family 1was described extensively (3). Updatedandmore detailed medical and family his-tories were obtained for families 1–3, 6,and 7; in families 1, 2, 3, 6, and 7, addi-tional members were examined. Families8–10 have not been reported previously.

Laboratory investigationsZFP57 sequencing and methylation stud-ies were carried out on genomic DNA ex-tracted from peripheral blood leukocytes.

Figure 1dA: Structure of the ZFP57 gene. The gray boxes represent the six exons of ZFP57. The thin black lines between the exons represent theZFP57 introns. B: Known sequence alterations in ZFP57 are shown as predicted protein alterations. The amino acid numbers are indicated above,and each alteration is indicated below. ZF, zinc finger.

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Abnormal methylation results were con-firmed with pyrosequencing of bisulfite-induced polymorphisms. In one patient(II-3, family 1), methylation studieswere carried out on DNA from multipletissues (cultured cells from the chorionicvillus sampling at gestational age 11 + 2weeks, postpartumplacental tissue, umbil-ical cord and peripheral blood leukocytes,and skin biopsies from surgically removedsixth fingers). For the families 1–7, labora-tory results for the probands and parentswere published previously (5).

ZFP57 analysesZFP57 sequence alterations were investi-gated by direct sequencing of two PCRamplification products, which includedexon 1 (49 bp) and exons 2–6 (4.8 kb),respectively. Exons 2–6 were amplifiedtogether in order to overcome amplifica-tion problems due to exon 6, which con-tained the repetitive zinc finger sequence.The primerswere: 59-CAGAGGAGTGGG-GACAACAT-39 and 59- CTAGCGC-TACTTGGGACCAG-39 for exon 1 and59-CCCAGGCTGGTGTTGTTACT-39and 59-ATGCTCACTGCCTCCTTTGT-39for exons 2–6. PCRwas carried out using aslightly optimized protocol described

previously (5). Shortly, DNA was ampli-fied using Phusion hot start high fidelitypolymerase (New England Biolabs) andthe GC buffer provided with the additionof DMSO to a final concentration of 3%.The PCR conditions were: initial denatur-ation at 988C (3 min), 29 cycles of 988C(10 s), 508C (10 s), and 728C (1 min 45 s),and final extension at 728C (5 min).

Methylation-specific PCR andpyrosequencingMethylation status was targeted to spe-cific imprinted loci: TNDM1, GRB10,PEG1/MEST, KCNQ1OT1, H19, DLK1(14q32), SNRPN (15q12), PEG3/ZIM2,and NESPAS/GNAS-AS1 (20q13.2) DMRsusing methylation-specific PCR and pyro-sequencing when necessary, as previouslydescribed (5,20).

Ethical approvalFamily 1 was recruited as part of a DanishImprinting and Methylation study, ethi-cal approval number H-D-2008–079 andDanish Data Protection Agency number2008-41–2565. Families 2–10 were re-cruited as part of the U.K. clinical localresearch network study, Imprinting

Disorders Finding Out Why, ethics ap-proval number 07/H0502/85.

RESULTS

Molecular geneticsIn 14 individuals from 10 TNDM1 HILfamilies, ZFP57 sequence mutations wereidentified on both alleles. The mutations innine individuals from families 1–7 werepreviously published (5). In this study,we included additional family members(families 1–7) and three new families (fam-ilies 8–10) (Fig. 2). Two families have twohomozygous affected individuals (families1 and 6), and in family 2, two relatives withno TNDM1 history are homozygotes. Sixfamilies (families 1–6) are consanguineous.In eight families (families 1–6, 8, and 10),the affected individuals are homozygous,and in families 7 and 9, the probands arecompound-heterozygous. The 10 differentsequence changes include 5 missense mu-tations (families 4, 5/7, 6, 9, and 10), 4frame-shift mutations (families 2, 3, 7,and 8/9), and a nonsense mutation (family1). Two mutations were observed twice:p.R228H in families 5 and 7 andp.L133HfsX49 in families 8 and 9 (Sup-plementary Table 1) (21). The missense

Figure 2dPedigrees of the 10 ZFP57-related HIL syndrome families.

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mutations in families 4, 5/7, 6, and 10 af-fect conserved residues, and the first three in-volve histidine, which is a metal ion-bindingamino acid. The missense mutation infamily 9 affects a nonconserved aminoacid. Amino acid changes in zinc fingerdomains or KRAB domains are predictedto affect DNA binding of the ZFP57 pro-tein and its possible function, respectively.In nine families (families 1 and 3–10), theZFP57 mutations are in exon 6 and infamily 2 in exon 5 (Fig. 1).

Methylation studiesThe individuals with homozygous andcompound heterozygous ZFP57 muta-tions havemosaic patterns of hypomethy-lation regarding both the number andcombination of affected imprinted lociand the extent of hypomethylation ineach locus (Supplementary Table 1).The epigenetic signature of ZFP57 homo-zygous and compound heterozygous in-dividuals includes total LOM of TNDM1DMR, partial hypomethylation of PEG3DMR, and partial or complete hypome-thylation of GRB10 DMR. Additionally,some individuals have partial hypome-thylation of the other one to three loci,PEG1, KCNQ1OT1, and NESPAS/GNAS-AS1 DMRs. There is no obvious corre-lation between methylation levels ofaffected loci and the type of ZFP57 muta-tion. Intrafamilial divergences exist re-garding the number of affected loci(families 1 and 6) and the extent of hypo-methylation at the individual locus (fam-ily 2) (Supplementary Table 1). Thisremains unexplained. We found no aber-rant methylation at the maternally meth-ylated SNRPN DMR or at the paternallymethylated H19 or DLK1 DMRs.

Clinical featuresThe key clinical features of the 12 ZFP57homozygous and compound heterozy-gous TNDM1 HIL individuals are neona-tal diabetes mellitus (NDM), intrauterinegrowth retardation (IUGR), macroglossia,and developmental delay (Table 1). De-tailed clinical features of each of theseplus the two asymptomatic ZFP57 homo-zygous relatives with HIL are shown inSupplementary Table 2. The 12 affectedindividuals were ascertained throughTNDM, which was indistinguishablefrom other causes. Three out of four pa-tients over the age of 4 years had diabetesrelapse (Table 1). Ten out of the 12 af-fected individuals had IUGR with a birthweight less than or equal to the secondpercentile, and in the two remaining

affected individuals, birth weight wasless than the 25th percentile (Table 1).In six out of seven children, postnatalgrowth showed rapid catchup. Addition-ally, the affected individuals showed var-ious combinations of developmentaldelay and/or congenital anomalies. Re-current features in the affected individu-als were: macroglossia, umbilical hernia,congenital heart disease, ear lobe abnor-malities, hemihypertrophy of extremities,central nervous system structural abnor-malities and developmental delay, or pec-tus carinatum (Supplementary Table 2).

Clinical variabilityZFP57-related HIL shows inter- and intra-familial clinical variability. One of themost intriguing variations is the nonoc-currence of diabetes (Supplementary Ta-ble 2) in two homozygous individuals, I-2and II-1 in family 2, who are the father andsister of the homozygous proband II-3. Allthree have total LOM of TNDM1 DMR(Supplementary Table 1); however, bothI-2 and II-1 are healthy with no signs ofdiabetes and are developmentally normal.I-2 is 39 years of age and has normal glu-cose tolerance testing. Likewise, there isno history of diabetes in II-1, though shedid have a low birth weight and postnatalgrowth catchup. There is previous evi-dence from families with inherited dupli-cations of 6q24 that relatives at genetichigh risk do not always present withTNDM1 (4,22,23), and there are reportsof cases with paternal uniparental disomyof chromosome 6 (UPD6) in older pa-tients, who have no history of TNDM1(24). Presumably, our two healthyZFP57-related HIL individuals are at riskfor developing noninsulin-dependent dia-betes later in life as individuals with 6q24duplications and no history of NDM de-veloped diabetes later in life (4,22,23).

The clinical phenotype was particu-larly severe in two affected individuals (II-2 in family 1 and II-1 in family 3) who hadsevere developmental delay and struc-tural brain abnormalities involving anabsent/hypoplastic corpus callosum. Infamily 1, the homozygous sister, II-1,has a much milder phenotype than II-2who was severely ill and died at 11months (Supplementary Table 2). Bothfamilies 1 and 3 are consanguineous,thus possibly other deleterious recessivegenes may contribute to the phenotypes.

Clinical features of heterozygotesTwenty-five individuals with heterozy-gous ZFP57 mutations were identified in

the 10 ZFP57-related TNDM1 HIL fami-lies. We have some clinical information on15. None of these had TNDM1, and to thebest of our knowledge, 12 of the 15 had anormal phenotype and development.Three heterozygous individuals (I-2 andII-3 in family 1 and II-1 in family 7) hadsymptoms needing further investigation:I-2 in family 1 developed gestational dia-betes mellitus in her third pregnancy. Weare only aware of two other heterozygotestested for diabetes: II-3 in family 1 hadblood glucose measured neonatally, andI-2 in family 10 had oral glucose tolerancetests due to diabetes in several relatives. Allmeasurements were normal. II-3 in family1 had bilateral postaxial polydactyly likeher homozygous sister, II-1, did. Other-wise, II-3 has a normal phenotype andnormal development by the age of 5months. Her methylation status was nor-mal (including skin fibroblasts from thebilateral sixth digits; data not shown),and her placenta was extensively exam-ined with normal results (clinical andhistological examination and immu-nohistochemical staining for p57kip2 pro-tein). No other family members hadpolydactyly, and it seems likely to be un-related. In the nonconsanguineous family7, II-1 shared a neurologic phenotypewithprogressive contractures with his com-pound heterozygous brother, II-3. How-ever, II-1 also had severe developmentaldelay and facial dysmorphic features pos-sibly related to prematurity and not seen inII-3. The reason for the neurologic find-ings remains unexplained despite exten-sive investigations. The phenotype of II-3is described in Supplementary Table 2.With the limited knowledge available, weconclude that the few symptoms observedin the heterozygotes are likely due to othercauses.

CONCLUSIONSdIn general, ZFP57-related TNDM1 HIL individuals seem tohave a more complex phenotype com-pared with patients with isolated LOMof TNDM1 DMR (Table 1), and there aremany possible explanations.

However, for HIL patients ascer-tained through other imprinting syn-dromes, there are conflicting reports ofthe phenotypic consequences. In somepatients ascertained with BWS (12,14,15)or SRS (14), no additional clinical featuresrelated to HIL compared with single-locus hypomethylation were noted. Forexample, there was no clinical history ofTNDM1 in the BWS (13) or SRS (14)HIL patients who had total or partial

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LOM of TNDM1DMR. However, the clin-ical presentation of BWS was modified insome BWS HIL patients (13), and two pa-tients with SRS HIL due to LOM of H19DMR had additional features includinglanguage delay (16). Furthermore, among3 of 77 SRS patients with hypomethyla-tion at both H19 DMR and KCNQ1OT1DMR, 1 had an umbilical hernia that isnot typical for SRS, but a common featurefor BWS (25). Azzi et al. (14,26) suggestedthat in cases with comparable levels of hy-pomethylation at multiple loci, one locusmay have an (epi)dominant effect over theother(s).

The clinical variability observed inthis cohort may be explained by the lociinvolved in HIL. Apart from TNDM1DMR, the other loci most consistentlyinvolved were GRB10 and PEG3. GRB10is a negative regulator of IGF signalingand is imprinted in fetal skeletal muscleand other postnatal tissues. It has long

been a candidate for SRS associated withUPD7 (27). Although the affected indi-viduals in the ZFP57-related TNDM1HIL cohort consistently had a low birthweight, all of them had weight catchupand normal last height measurements.As this pattern is also observed in mostcases with TNDM1 due to isolated geneticaberrations at 6q24, this cannot be attrib-uted to the changes at GRB10 DMR. Ourknowledge is even less about the clinicalimpact of hypomethylation of PEG3DMRand PEG1 DMR. The paternal knockoutof peg1 causes IUGR and a behavioral de-fect in mice (28). In contrast to this, muchis known about hypomethylation ofKCNQ1OT1 DMR and its associationwith BWS (29). Partial hypomethylationat this locus was only identified in three ofthe affected individuals [II-1 and II-2 infamily 1 (3) and II-1 in family 6]. All threehadmacroglossia, the first two also had anumbilical defect, and one of these

additionally had bilateral ear lobe creases,all of which are typical BWS features.However, macroglossia, umbilical defect,and ear creases were also seen in five in-dividuals with normal methylation ofKCNQ1OT1 DMR. Calcium metabolismwas investigated in the two affected indi-viduals with partial hypomethylation atNESPAS DMR (II-1 in family 1 and II-3in family 7), which is part of the compleximprinted GNAS cluster and is associatedwith pseudohypoparathyroidism type IB(MIM 603233) (30). However, no consis-tent abnormality of calcium metabolismwas demonstrated. It still remains a pos-sibility that methylation abnormalities atother loci not tested are responsible forthe clinical differences between patients.

Another possible mechanism for theextreme clinical variability is divergentextent of hypomethylation at the in-dividual locus and the tissues involved(26). We found, however, no obvious

Table 1dComparison of phenotypic features in ZFP57-related TNDM1 HIL individuals (ZFP57mut/mut) and individuals with isolated6q24 TNDM1 of unknown cause and UDP6 or duplication 6q24

Features

ZFP57mut/mut individuals TNDM1: isolated

Affected individuals Nonaffected individuals LOM 6q24*

n/p % n/p % n/p %

Multiple hypomethylation 12/12 100 2/2 100 0/12 0TNDM1 12/12† 100† 0/2 0 12/12 100IUGR (below 2nd percentile) 10/12 83 1/2‡ 50 7/11 64IUGR (below 25th percentile) 12/12 100 1/2‡ 50 9/11 82Macroglossia 8/11 73 nk d 5/10 50Developmental delay 5/11 45 0/2 0 0/8 0Congenital heart disease 4/11 36 0/2 0 0/10 0Umbilical hernia 3/11 27 0/2 0 0/11 0One or more other minor congenitalabnormalityx 8/11 73 0/2 0 3/11 27

Epilepsy 2/10|| 20|| 0/2 0 0/11 0Visual impairment 2/10 20 1/2{ 50 0/8 0Hearing impairment 1/10 10 0/2 0 0/8 0Brain imaging abnormality 2 structural, brain

abnormalities, 1 normalCT, 11 not imaged

d None imaged d None imaged d

Age at presentation of diabetes,average (days) 20.2 (n = 12) d n/a d 9.7 (n = 10) d

Age at presentation of diabetes,median (days) 14.5 (n = 12) d n/a d 4 (n = 10) d

Age of recovery, average (months) 5.4 (n = 10) d n/a d 4 (n = 6) dAge of recovery, median (months) 4.3 (n = 10) d n/a d 2 (n = 6) dRecurrence of diabetes, patients age .4years (persons) 3/4 75 n/a d 3/5 60

Age of recurrence of diabetes (average years) 8 (2 y 8 m–11 y) (n = 3) d n/a d 13.7 (11–16 y) (n = 3) d

CT, computed tomography; m, months; n/a, not applicable; nk, not known; n/p, number of observations for number of individuals; y, years; ZFP57mut/mut, HILindividuals with homo- or compound-heterozygous mutations of ZFP57 (n = 14 individuals). *Figures from Wessex Regional Genetics Laboratory, SalisburyHospital, Salisbury, U.K., with the cutoff date 22 December 2010. †Include two children with NDM and no recovery yet. ‡Birth weight only known for one of theZFP57mut/mut nonaffected individuals. xEar lobe abnormality, asymmetry, pectus carinatum, polydactyly, and tracheomalacy. ||Two other affected individuals hada single seizure neonatally or in childhood. {One ZFP57mut/mut nonaffected individual had episodic diplopia in his late thirties.

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relationship between themethylation pat-tern and the phenotype. All of the homo-zygous individuals in family 2 withdivergent penetrance of diabetes (I-2, II-1,and the proband II-3) had total LOM ofTNDM1 DMR and partial LOM of PEG3DMR. At GRB10 DMR, however, the pro-band (II-3) had total LOM, whereas thetwo healthy individuals (I-2 and II-1)only had partial hypomethylation. How-ever, this potential association was notconfirmed in other individuals (the pro-bands in families 3–5, III-2 in family 6,and probands in families 8–10) with thesame epigenetic signature and TNDM1(Supplementary Tables 1 and 2).

In family 1, methylation analyses inII-1 and II-2 of blood leukocytes showedpartial LOM of KCNQ1OT1 DMR but wasnormal at this locus in skin fibroblasts (3).However, this remains a possible reasonfor variability but is very difficult to test inhumans.

There is evidence that in mice, zfp57is a maternal-zygotic effect gene (6), and

this may be impacting in humans. Thematernal effect is described as the phe-nomenon in which a phenotype in theprogeny is caused by a genetic alterationin the maternal genome rather than analteration of its own (31,32). Whenzfp57 expression in mice was lost only inthe zygote (zfp572 /2 offspring fromzfp57+/2 mothers), partial hypomethyla-tion in some embryonic maternally meth-ylated and some paternally methylatedDMRs and peri- and neonatal lethalitywas observed in themajority of homozygousoffspring. In contrast, in the zfp572/2

offspring from zfp572/2 mothers, therewas highly penetrant midgestation lethal-ity, and the developing embryo had com-plete LOM at the above-mentionedDMRs.Family 6 may be an example for maternal-zygotic effect, in which a homozygouschild (III-2) was born to a homozygousmother (II-1) who was herself born atterm with TNDM1 (Supplementary Table2). Besides TNDM1, III-2 only had minormalformations (a ventricular septal defect

that closed spontaneously and a minoresophageal hernia). She had no evidenceof a more severe phenotype, and neitherwas her degree of LOM higher when com-pared with other affected individuals(Supplementary Table 1). As zfp572/2 off-spring of zfp572/2 female mice die inutero (6), the survival of III-2 may be ex-plained by the nature of the ZFP57 mis-sense mutation. Some residual activity ofZFP57 may be retained with a missensemutation, whereas in mice, both zfp57alleles are knocked out. It is also possiblethat ZFP57-related HIL individuals com-prise the milder end of a phenotypic spec-trum, in which the full-blown formis prenatally lethal (4). Interestingly, fam-ily 2 is the only family with nonoccurrenceof diabetes in individuals with homozy-gous ZFP57 mutations. A possible expla-nation is related to the ZFP57 mutationitself; family 2 is the only family in whichthe ZFP57mutation in this particular fam-ily affects the KRAB domain, whichshould be essential for the association of

Figure 3dZFP57-related HIL is a small subgroup of NDM.ABCC8, gene coding for the Kir6.2 subunit of the ATP-sensitive potassium channel of theb-cells of the pancreas;KCNJ11, gene coding for the SUR1 subunit of the ATP-sensitive potassium channel of theb-cells of the pancreas; n, number offirst members identified in each pedigree at Wessex Regional Genetics Laboratory, Salisbury Hospital, Salisbury, U.K., with the cutoff date 22December 2010; 6q24, chromosome 6q24; Type 1, TNDM1; Type 2, TNDM2 (MIM 610374); Type 3, TNDM3 (MIM 610582); ZFP57mut/mut, HILindividuals with homo- or compound-heterozygous mutations of ZFP57. Hypomethylation DMRHIL, n = 33 (four of these had insufficient sample toinvestigate more loci than TNDM locus).

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KRAB-associated protein 1 with the DNAmethyltransferases (11). Further func-tional investigations would be needed toverify this.

NDM can be divided into permanentNDM and TNDM (Fig. 3). More than 50%of NDM cases are transient (33). TNDMis a rare entity with an incidence of;1:200,000 (34–36). Of these transientdiabetic cases, 6q24 abnormalities ac-count for 60–70% (33,37). Individualswith ZFP57-related TNDM1 HIL accountfor only a small fraction of all individualswith TNDM. As a diagnostic pathway anddifferential diagnosis, 6q24 testing is thefirst step for neonates (,3 weeks) pre-senting with diabetes and the secondstep (following sequencing the potassiumchannel genes KCNJ11 and ABCC8) inchildren .3 weeks (38). Three knowngenetic mechanisms of 6q24 TNDM1 ex-ist: UPD6, paternal duplications of 6q24,and hypomethylation of TNDM1 DMR(4). In the latter group, the hypomethyla-tion is isolated to TNDM1 DMR in 41%(4) (12 of 29 pedigrees, Fig. 3) or partof a more complex hypomethyla-tion, TNDM1 HIL, in 59% (4) (17 of 29pedigrees). Homozygous or compound-heterozygous ZFP57mutations are foundin 59% (10 of 17) of these TNDM1 HILpedigrees. We recommend that all pa-tients with TNDM1 due to LOM atTNDM1 DMR are tested for HIL and formutations in ZFP57. The results may haveconsequences for reproductive risk, and,in families with ZFP57 mutations, prena-tal genetic testing is possible. However,genetic counseling is difficult becausethe clinical phenotype is variable.

In conclusion, homozygous andcompound-heterozygous mutations inZFP57 cause a human imprinting syn-drome as yet only ascertained throughpresentation with TNDM1. It causes anincreased risk of low birth weight, macro-glossia, and developmental delay; how-ever, the phenotype is highly variable,ranging from death at 11 months to nor-mal health in the fourth decade. We foundno evidence of abnormal clinical pheno-types caused by ZFP57 heterozygosity.The findings in humans are less severethan those in the knockout mouse model,as reflected by the expanded phenotypicdescription of the first 10 families identi-fied with this disorder, but there remainmany challenges before our knowledge issecure enough to provide definite clinicaladvice for families. Patients with unusualimprinting syndromes are an importantresource for novel biological insights into

epigenetic mechanisms and the regulationof human gene expression.

AcknowledgmentsdTheWilhelm JohannsenCentre for Functional Genome Research wasestablished by the Danish National ResearchFoundation. This work was supported by agrant from the Danish Agency for Science,Technology and Innovation and the Universityof Copenhagen. Furthermore, financial sup-port was provided by the “Direktør JacobMadsen og Hustru Olga Madsens Fond” and“Kong Christian den Tiendes Fond” and a grantfunded by Diabetes UK.No potential conflicts of interest relevant to

this article were reported.S.E.B. wrote the manuscript and researched

data. D.J.G.M., L.Doc., and I.K.T. researcheddata, contributed to discussion, and edited themanuscript. J.M.D.H., K.G., and Z.T. con-tributed to discussion and edited the manu-script. A.L., L.G.L., A.P.H., Y.K., L.Doo., D.C.V.,C.T.B.N., P.B.N.,O.K., F.S., P.D., V.P., C.A., and A.F.M.researched data and edited the manuscript.S.E.B. and I.K.T. are the guarantors of this workand, as such, had full access to all the data in thestudy and take responsibility for the integrity ofthe data and the accuracy of the data analysis.The authors thank the patients and families

for their participation in the study; ProfessorJudith Goodship, Medical Genetics, NewcastleUniversity, U.K.; Consultant Dr. Helen Firth,Department of Medical Genetics, Cambridge,U.K.; Professor Sian Ellard, Royal Devon &Exeter Hospital, Devon, U.K.; Dr. Gunilla Drake,Department of Neurology, The Queen SilviaChildren’s Hospital, Gothenburg, Sweden; andConsultant Birthe S. Olsen, Department of Pae-diatrics, Glostrup Hospital, Glostrup, Denmark,who referred the patients to the research study.

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