JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 17:87-93 (1997)Mary Ann Liebert, Inc.

Analysis of an Interferon-y Gene (IFNG) Polymorphism inDanish and Finnish Insulin-Dependent Diabetes Mellitus

(IDDM) Patients and Control Subjects

FLEMMING POCIOT,1 RIITTA VEIJOLA,2 JESPER JOHANNESEN,1 PERNILLE M. HANSEN,1TOVE LORENZEN,1 ALLAN E. KARLSEN,1 HELENA REIJONEN,3 MIKAEL KNIP,2 J0RN NERUP,1

and THE DANISH STUDY GROUP OF DIABETES IN CHILDHOOD

ABSTRACT

A CA-repeat polymorphism within the first intron of the interferon (IFN)-y gene was analyzed. This poly-morphism was recently demonstrated to be associated with insulin-dependent diabetes mellitus (IDDM) inJapanese subjects. We typed 266 IDDM patients and 195 control subjects of Danish Caucasoid origin. No sig-nificant differences in alíele or genotype frequencies between patients and control were observed. In addition,we typed 168 IDDM and 110 control subjects of Finnish origin. A significant disease association of the studiedIFN-y allelic pattern was found (p = 0.029). Analysis of data according to HLA-DQB1 susceptibility status didnot reveal heterogeneity of risk at the IFN-y locus in either of the populations. Fifty-five Danish and 94 FinnishIDDM multiplex families with at least two affected siblings (660 individuals) were typed to test for transmis-sion disequilibrium (TDT). No evidence for overall transmission disequilibrium using either an alíele-wise (p =

0.42; combined data) or a genotype-wise analysis (p = 0.21; combined data) could be detected. Thus, the mod-est significance level observed in the Finnish case-control study and the failure to replicate it by the TDT pro-vide little support for the hypothesis that the IFN-y gene microsatellite is associated with IDDM.

IINTRODUCTION hesion molecules*18-19' on various cells, including pancreatic

beta cells. It may also modulate the processing of exogenousnsulin-dependent diabetes mellitus (IDDM) is caused by (MHC class II) antigens by human monocytes.(20) Transgenican immune-mediated, selective destruction of the pancreatic mice expressing IFN-y by pancreatic beta cells develop au-

beta cells triggered by unknown environmental factors in ge- toimmune diabetes/21>22) and postnatal anti-IFN-y treatmentnetically susceptible individuals. Recent studies have affirmed prevents pancreatic inflammation in this model.<23) Antibodiesthe polygenic nature and genetic heterogeneity of IDDM/1^1' to IFN-y also protect against diabetes development in NOD

Cytokines may mediate pancreatic beta cell damage during mice(24,25) and in BB rats/26' Finally, IFN-y is expressed inthe early stages of IDDM/5_7) The cytokine interferon (IFN)- late-stage insulitis of recent-onset IDDM patients.(27) The re-

y, alone or in combination with interleukin-1 (IL-1) or tumor ported actions of IFN-y are consistently diabetes promoting,necrosis factor (TNF)-cdß or both, was suggested to be cyto- and, therefore, the IFN-y gene (IFNG) is an obvious candidatetoxic to beta cells by our model of pathogenesis of IDDM/5,6) gene for conferring susceptibility to IDDM.This has been shown subsequently to be the case in rat, mouse, The IFNG is a single copy gene that contains three intronsand human pancreatic islets in vitro(8~13) at least in part via gen- and four exons encoding a polypeptide of 166 amino acids, 20eration of nitric oxide/8,14' IFN-y is a product of T lympho- of which constitute the signal peptide/28,29' and has been as-

cytes and natural killer (NK) cells, possesses unique im- signed to 12q24.12.(30) A dinucleotide repeat polymorphism ofmunomodulatory properties, and can affect also nonlymphoid this gene has been reported/31' This polymorphism was subse-tissues. In addition to the direct effect on beta cells, IFN-y may quently demonstrated to be associated with IDDM in a Japaneseplay a role in the pathogenesis of IDDM for the following rea- population*32' where the IDDM incidence is low.sons: IFN-y upregulates MHC class I,(15> class H/16'17' and ad- In the present study, we analyzed this polymorphism in an

'Steno Diabetes Center, DK-2820 Gentofte, Denmark.2University of Oulu, Department of Pediatrics, FIN-90220 Oulu, Finland.'University of Turku, Department of Virology, FIN-20520 Turku, Finland.

87

88 POCIOT ET AL.

IDDM intermediate-risk population of Danish origin and in an

IDDM high-risk Finnish population.

MATERIALS AND METHODS

SubjectsThe study population comprised the following subgroups: ( 1 )

266 IDDM patients of Danish Caucasoid origin. Of these, 55were identified as probands of IDDM multiplex families in a

nationwide population-based survey/33' All family members(257 individuals) from these families were typed in order toevaluate a possible distortion in transmission of disease-asso-ciated alíeles/34' The other IDDM patients were identifiedthrough the outpatient clinic at Steno Diabetes Center. They allfulfilled the WHO criteria for IDDM, and all were diagnosedbefore the age of 30 years. (2) The second group was 195 eth-nically matched, unrelated control individuals. None had a fam-ily history of IDDM or other endocrinopathies. (3) The thirdgroup was made up of 168 Finnish IDDM patients with youngage at diagnosis (mean age ± SD at diagnosis: 5.04 ± 3.28years). Of these, 94 were probands from IDDM multiplex fam-ilies identified at pédiatrie units in Finland. Parents and IDDM-affected siblings from these families (403 individuals) were

typed to evaluate transmission of disease-associated alíeles/34'The sporadic cases represent all the IDDM patients youngerthan 10 years at diagnosis (mean age ± SD at diagnosis: 6.3 ±3.2 years) who were admitted to the Department of Pediatrics,University of Oulu, Oulu, for initial treatment of IDDM since1989. (4) The fourth group was 110 Finnish healthy school-children from five municipalities in the Province of Oulu, whoserved as control subjects.

GenotypingDNA was prepared by standard procedures from leuko-

cytes/35' A CA repeat polymorphism of intron 1 of the IFN-ygene<31) was analyzed in the present study. The CA repeat was

amplified by PCR using the following primers: 5'-Biotin-AATTGA TTT TAT TCT TAC-3' and 5'-TTG CTG TAG GGTATT ATT-3'. PCR amplification was 94°C for 5 min, then 30cycles each consisting of 94°C for 60 sec, 42°C for 30 sec, and72°C for 40 sec, followed by 72°C for 10 min, performed in a

Perkin Elmer Cetus Thermal Cycler. The PCR mixture con-

tained 100 ng genomic DNA, 50 pM of each dNTP, 1 pM ofeach primer, and 1 U Taq polymerase/sample in 1 X PCR buffer(Promega Corp., Madison, WI) with 2 mM MgCb in a 20 mlreaction volume. Twenty microliters of stop buffer (98% for-mamide) was added to the PCR product, and the sample washeated for 2 min at 94°C and placed on ice. Eight microlitersof this mixture was analyzed on an 8% polyacrylamide gel at65 W. Afterward, the PCR products were transferred to an un-

charged nylon membrane (IMMOBILON-S, Millipore Corp.,Bedford, MA) and UV cross-linked (UV Stratalinker 2400,Stratagene, La Jolla, CA). The membrane was then blocked by10% SDS, 0.9% NaCl for 2-5 min, and then covered with 0.5%SDS, 0.9% NaCl containing 1 mg/ml streptavidin-HRP (Sigma,St. Louis, MO) for 4 min with gentle shaking. The membranewas washed twice in 0.5% SDS, 0.9% NaCl and once in wa-ter. The PCR products were detected using the ECL gene de-

tection reagents (Amersham, Buckinghamshire, UK) accordingto the manufacturer's directions. The membrane was exposedto x-ray film for 1-5 min.

SequencingFor preparation of single-stranded templates of PCR prod-

ucts, Dynabeads M-280 (Dynal A.S., Oslo, Norway) with chem-ically bound streptavidin were used according to the manufac-turer's instructions. Briefly, 20 pi of washed beads suspensionwas used per sequence reaction. The PCR product was incu-bated with the beads for 15 min at room temperature, with fre-quent resuspension. Then, the beads were washed with TE-buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0),resuspended in 10 pi 0.1 M NaOH, and incubated for 5 min atroom temperature. The beads were pelleted in an MPC-E mag-net (Dynal AS), washed, and resuspended in 7 pi dH^O.Sequencing was performed using the Sequenase kit, version 2.0(U.S. Biochemical Corp., Cleveland, OH) and 35S-dATP(Amersham).

HLA-DQB1 typingDQB1 typing of the Danish material was performed using

standard procedures, as previously reported/4' The DQB 1 typ-ing of the Finnish material was based on solution hybridizationwith lanthanide-labeled oligonucleotide probes detected withtime-resolved fluorometry, as described in detail elsewhere/36'Sequence-specific oligonucleotide probes for identification ofthe DQB 1*0302, 0201, 0301, and 0602/3 alíeles were used inthe analysis.

Statistical analysisCategorical comparisons were made by the ^-test or Fisher's

exact test, whichever was appropriate. Five percent (two-tailed)was chosen as level of significance. Odds ratios (OR) were cal-culated by Woolf s formula/37' An extended transmission dis-equilibrium test (TDT) for multiallele marker loci was used toassess transmission from heterozygous parents to offspring bythe software package EDTD version 1.1Z38'

RESULTS

A total of five different sized alíeles were identified in thepresent study (Fig. 1 ). Each of these alíeles varied with one CArepeat (11-15), as verified by sequence analysis (Fig. 2). In ad-dition, we identified a single nucleotide substitution, just up-stream of the CA repeat sequence, in position 1348 of the pub-lished sequence/28' This T —» A transversion, which introducedan extra CA repeat, did not show disease restriction or absolutelinkage to any of the CA-repeat alíeles and was not further in-vestigated.

No differences were found between familial and nonfamil-ial IDDM cases in either of the populations, and these sub-groups were analyzed together. The global allelic distributionin the Danish population is shown in Table 1. No significantdifferences in allelic or genotype frequencies (not shown) were

observed (p = 0.73 and p = 0.53, respectively). As thestrongest association with IDDM was reported in Japanese sub-

IFN-y GENE POLYMORPHISM IN IDDM 89

FIG. 1. PCR products of the CA repeat within the first intronof the IFN-y gene. Five alíeles were detected and designatedA11-A15 according to the number of repeats. Genotypes rep-resenting all five alíeles are shown.

jects with clinical onset £ 10 years of age, the Danish popula-tion was stratified accordingly (Table 1). Still, no significantdifferences in allelic and genotype frequencies were observed(p = 0.7 and p = 0.5, respectively).

The allelic distribution in the Finnish population is shown inTable 2. A significant difference in the global allelic distribu-tion was observed between patients and controls (p = 0.029),and a trend for difference in genotype distribution (not shown)was found (p = 0.077). The difference in allelic distribution

A12/A12 A13/13 A13/13TGCA TGCA TGCA

^Hh WKbb <MMPK «• 1 - p —•* Si

-

—

[CA]n

was due to an increased frequency of the A12 allel and a de-creased frequency of the A14 and A15 alíeles in patients.Consequently, different frequencies of various genotypes were

observed in the two groups: 61.9% A12 positive patients vs.

49.1% controls (p = 0.035) (OR 1.7, 95% CI 1.05-2.7) and25.0% A14-positive and/or A15-positive patients vs. 41.8%controls (p = 0.003) (OR 0.46, 95% CI 0.31-0.67). The geno-type frequencies in both Danish and Finnish control and IDDMpopulations did not deviate from the expected values by Hardy-Weinberg equilibrium.

To investigate if there was any difference at IFNG accordingto HLA-DQB associated susceptibility in the Finnish population,a direct comparison was made of the DQB 1*0201/DQB 1*0302-positive, the DQB 1 *0302-positive/DQB 1*0201 -negative, theDQBl*0201-positive/DQBl*0302-negative, and the DQB1*-0201-negative/DQBl*0302-negative diabetic subjects. No het-erogeneity of risk at IFNG according to the HLA-DQB 1 statuscould be demonstrated (p = 0.36, x2 = 13.06, 12df) (Table 3),although the DQB1*0201/X group had a higher frequency of theA12 alíele compared with the other groups (p = 0.02, uncor-

rected). Individuals positive for DQB 1*0602/3 (n = 14) were

studied separately and did not differ from IDDM patients withother studied HLA-DQB 1 specificities (data not shown). In theDanish population, HLA-DQB 1 data were available from 156IDDM subjects. Stratifying for DQB1 status did not reveal anysignificant differences in this material (data not shown).

To further investigate the IFNG polymorphism in relation toIDDM, we typed 55 Danish and 94 Finnish IDDM multiplexfamilies with at least two IDDM-affected offspring and per-formed an extended TDT for a marker with multiple alíeles/38'One hundred eighteen and 218 transmissions from Ijeterozy-gous patients were observed in the Danish and the Finnish fam-ily material, respectively. First, a genotype-wise analysis was

performed. In this analysis, every heterozygous parental geno-type was considered separately, and it was determined if eachalíele of the genotype was transmitted to affected offspring on

50% of occasions. Second, an analysis was performed to testfor preferential transmission of certain alíeles across genotypes.Initially, the Danish and Finnish family data sets were analyzedseparately. No differences between the two data sets were ob-served, and the data were pooled (Table 4). No evidence foroverall transmission disequilibrium using either the allele-wise(p = 0.42) or the genotype-wise (p = 0.21) analysis was found.Also, no evidence for a distorted transmission of individual al-íeles was found (Table 5).

When comparing the two populations, differences were

found between control subjects (p < 0.00001) as well as be-tween IDDM patients (age at onset :£ 10 years) (p = 0.03).

DISCUSSION

FIG. 2. Sequence of the CA repeat region from three indi-viduals. Two individuals are A13/A13 homozygous and one isA12/A12 homozygous. The CA repeats are indicated at the left.A new polymorphism, aA^T transition, was detected by se-

quencing this region. The position of this single base substitu-tion is indicated with an arrow. The first individual is A/A, thenext is A/T, and the individual to the right is T/T.

In the present study, we found evidence for an associationof a CA-repeat polymorphism within the first intron of the IFN-y gene and IDDM in a Finnish but not in a Danish population.We were able to identify only 5 alíeles of this polymorphismdespite typing a very large number of unrelated individuals (n =

888), control and IDDM subjects, from two Scandinavian pop-ulations. This was in contrast to the original report describingthe polymorphism/31' where 6 alíeles were reported with the

90 POCIOT ET AL.

Table 1. Allelic Distribution and Frequencies of CA-Repeat Polymorphismof IFN-y Gene in Danish Control and IDDM Subjects"

Alíele All A12 A13 A14 A15

Control subjects (n = 390) 0 183 166 27 140 0.469 0.426 0.069 0.036

IDDM patients (n = 532) 2 253 222 30 250.004 0.476 0.417 0.056 0.047

IDDM patients diagnosed 0 149 127 18 16<10 years (n = 310) 0 0.481 0.410 0.058 0.052

an is number of chromosomes, x2—

2-78 (4 df); p = 0.6 for comparing control subjects and IDDM patients.X2 = 1.48 (3 df); p = 0.7 for comparing control subjects and IDDM patients with age at onset before 10 years.

apparent lack of the A14 alíele (our nomenclature) but the ad- We were not able to demonstrate any association of IFNGditional identification of alíeles corresponding to Al 6 and A17. polymorphism with IDDM in the Danish population. TheThat study was based on analysis of 54 chromosomes of unre- strongest association with IDDM in Japanese individuals was

lated individuals of African, Asian, and European origin/31' In reported for patients with young age at diagnosis, that is, ^10the Japanese population, the presence of 8 alíeles was re- years/32'Therefore, the Danish data were analyzed with respectported/32' However, the exact length of the PCR-identified al- to age at diagnosis of IDDM. Still, no evidence for an IDDMleles or the number of CA repeats of the different alíeles were association of IFNG polymorphism was found. In contrast, datanot reported, and comparison of data, including evaluation of from the Finnish population demonstrated a significant IDDMthe IDDM association, from the different studies is, therefore, association based on allelic frequency and a trend based on

difficult. Nevertheless, the CA repeat polymorphism of the first genotypic frequency. The frequency of the A12 alíele was in-intron of the IFNG locus seems to vary considerably between creased in patients, and A12-positive genotypes were signifi-ethnically different populations. In the present study, a highly cantly more frequent in patients, whereas the frequency of thesignificant difference in allelic distribution was observed be- AI*4 alíele was increased in controls, and A14-positive geno-tween the Danish and Finnish control subjects. types were significantly more frequent in controls.

Table 2. Allelic Distribution and Frequencies of IFN-y GenePolymorphism in Finnish Control and IDDM Subjects"

Alíele All A12 A13 A14 A15

Control subjects (n = 220) 5 67 99 27 220.023 0.305 0.450 0.123 0.100

IDDM patients (n = 336) 9 139 143 25 200.027 0.414 0.426 0.074 0.060

an is number of chromosomes, x2 = 10.75 (4 df); p = 0.029 for comparing control subjects and IDDM pa-tients.

Table 3. Comparison of Allelic Frequencies of IFNG in Finnish IDDMSubjects According to HLA-DQB 1 Status (Heterogeneity of Risk)

Alíele AU A12 A13 A14 A15

DQB 1*0201-DQB l*0302(n = 53)a 4 46 43 6 70.038 0.434 0.406 0.057 0.066

DQB1*0302/Xb(n = 81) 4 59 77 11 110.025 0.364 0.475 0.068 0.068

DQB1*0201/XC (n = 22) 0 26 12 4 20.591 0.273 0.091 0.045

Xd/Xd (n = 12) 1 10 10 3 00.042 0.417 0.417 0.125

an is number of individuals.bNon-DQB 1*0201.cNon-DQB 1*0302.dNon-DQB 1*0201, non-DQB 1 *0302.X2 = 13.06 (12 df); p = 0.36.

IFN-y GENE POLYMORPHISM IN IDDM 91

Table 4. Observed Heterozygous Parental Genotypesand Frequencies of Transmitted Alleles in Combined

Danish and Finnish Family Material

Parental genotype*

A11/A12A11/A13A11/A15A12/A13A12/A14A12/A15A13/A14A13/A15A14/A15

411

2184

25392939

3

First alíele ofgenotype transmitted0

(n)

232

1039

231517

2

"X2 for genotype TDT = 12.03 (9 df); p = 0.21.V TDT = 3.88 (4 df); p = 0.42.

in the Finnish case-control study and the failure to replicate itby TDT provide little support for the hypothesis that the IFNGmicrosatellite is associated with IDDM.

However, to firmly establish or exclude the possible role ofIFNG polymorphism in IDDM susceptibility, other ethnic pop-ulations need to be studied. Furthermore, it is necessary to con-

firm whether it is the same alíele demonstrating disease asso-

ciation in the Finnish and the Japanese populations, and, finally,the possible functional significance of this polymorphismshould be studied.

ACKNOWLEDGMENTS

The skillful technical assistance of Bodil Bosmann is greatlyappreciated. The Danish material was in part collected by theDanish Study Group of Diabetes in Childhood (DSGD). F.P. isthe recipient of a research grant from the Danish MedicalResearch Council (J.no. 12-1857-1).

We found no heterogeneity of risk at IFNG according toHLA-DQB 1 status, although we cannot ignore the fact that theA12 alíele is more frequent in the DQB1*0201/X group.Previous reports have observed that non-MHC susceptibilitygenes exert their maximum effect in diabetic subjects with thelowest MHC class Il-associated susceptibility/39^*2' This in-cludes studies of populations of Finnish origin/41'42' The pre-sent case-control data may suggest an independent (of MHC),although minor, contribution of IFNG to IDDM susceptibilityin the Finnish population. However, analysis of 55 Danish and94 Finnish families with multiple children affected with IDDMrevealed no evidence for linkage by the TDT of the studiedpolymorphism. Based on this analysis, the observed differencein A12 alíele (or other alleles) frequency is likely to be of lim-ited relevance with regard to conferring susceptibility to IDDM.There is substantial reason to believe that techniques for test-

ing for association, such as the TDT, that use family-based con-

trols are much more sensitive and less prone to error than are

case-control studies/43^15' The differences observed in theFinnish case-control study may, therefore, derive from (an un-

known) population stratification. If the data of Awata et al/32'claiming a highly significant association between IFNG andJapanese IDDM patients diagnosed before 10 years of age, are

reconsidered once the p values are corrected (both for allelesanalyzed and number of tests performed), the statistical signif-icance would be substantially reduced to almost marginal sig-nificance. In conclusion, the modest significance level observed

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Table 5. Transmission for Individual Alleles inCombined Danish and Finnish Data Sets"

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PassedNot passedX2p values

7100.5290.47

137115

1.9210.17

121142

1.6770.20

3225

0.8600.35

3944

0.3010.58

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92 POCIOT ET AL.

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Address reprint requests to:Dr. Flemming Pociot

Steno Diabetes CenterDK-2820 Gentofte

Denmark

Received 23 July 1996/Accepted 4 October 1996