Journal of Autoimmunity 23 (2004) 75e80

www.elsevier.com/locate/issn/08968411

Analysis of MHC genes in a Tunisian isolate with autoimmunethyroid diseases: implication of TNF �308 gene polymorphism

Noura Bougacha-Elleucha, Ahmed Rebaib, Mouna Mnif c, Hafedh Maknia,d,Mohamed Bellassouadc, Jomaa Jouidae, Mohamed Abidc, A. Hammadia,)

aLaboratoire de Genetique Moleculaire Humaine Faculte de Medecine, Avenue Majida Boulila, 3029 Sfax, TunisiabCentre de Biotechnologie Sfax, Tunisia

cService Endocrinologie, CHU Hedi Chaker, Sfax, TunisiadLaboratoire d’Histocompatibilite, CHU Hedi Chaker, Sfax, Tunisia

eDispensaire Bir Hfaı, Sidi Bouzid, Tunisia

Received 2 December 2003; revised 11 March 2004; accepted 26 March 2004

Abstract

Autoimmune thyroid diseases (AITDs), which include Hashimoto thyroiditis (HT), Graves’ disease (GD) and primary idiopathicmyxoedema (PIM), are recognized as multifactorial diseases. In this study, we have examined single and haplotypic genetic variation

across the major histocompatibility complex (MHC) in a Tunisian isolate with a high prevalence of AITDs (62 patients: 32 with GD,9 with HT and 21 with PIM). Genotyping was performed for HLA class I and II alleles as well as polymorphisms within tumornecrosis factor (TNF), lymphotoxin a (TLa) and heat shock protein (HSP70-02 and HSP70-hom) genes. Our results showedassociation of HLA-A2-B50-TNF 2 haplotype with AITDs ( p ¼ 0:045). Linkage analysis using Simwalk2 program has shown

significant result with TNF �308 gene polymorphism ( p ¼ 0:03). The FBAT has given evidence for genetic association with TNF�308 and HLA-DR gene polymorphisms. TNF 2 allele was associated with GD ( p ¼ 0:0011), whereas TNF 1, HLA-DR11 andDR12 ( p ¼ 0:0039, p ¼ 0:00089 and p ¼ 0:0056, respectively) were rather implicated in HT pathogenesis. Results found by

TDTeSTDT have confirmed the involvement of the TNF �308 gene polymorphism in AITD pathogenesis ( p! 10�9).� 2004 Elsevier Ltd. All rights reserved.

Keywords: MHC; Autoimmune thyroid diseases; TNF �308

1. Introduction

Autoimmune thyroid diseases (AITDs), which in-clude Hashimoto thyroiditis (HT), Graves’ disease (GD)and primary idiopathic myxoedema (PIM), are recog-nized as multifactorial diseases, arising from interactionsof environmental factors with multiple genes. In GD,the autoimmune process results in the production ofthyroid-stimulating antibodies and leads to hyperthy-roidism, whereas in HT, the end result is destruction ofthyroid cells and hypothyroidism. The search for

) Corresponding author. Tel.: +216-74-241-888; fax: +216-74-246-

946.

E-mail address: hammadi.ayadi@fmsf.rnu.tn (A. Hammadi).

0896-8411/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jaut.2004.03.011

susceptibility genes involves two major approaches,genome-wide screenings and association studies for can-didate genes. Genome-wide scans have identified manyregions linked to AITDs on different chromosomes[1e3]. Linkage or linkage disequilibrium of GD to themajor histocompatibility complex (MHC) has also beenreported in some populations [4e7]. However, evidencefor linkage was not strong enough to be replicated inother samples or populations [8e10]. This suggests thatdifferent MHC susceptibility loci exist between differentethnic groups and more than one gene was involved inthe genetic susceptibility to AITDs. Indeed, this regionharbors an important number of genes involved in manyautoimmune diseases (AID) [11]. The second approachhas also provided a great deal of information that manycandidate genes involved either in thyroid physiology

76 N. Bougacha-Elleuch et al. / Journal of Autoimmunity 23 (2004) 75e80

[12e16] or in immune response [6,17e19] are conciselyassociated with AITDs. Associations between AITDsand MHC have long been recognized in different popu-lations. GD is associated with the HLA haplotypeA*01-B*0801-DRB1*0301-DQA1*0501-DQB1*0201 [4e5,20e22], whereas autoimmune hypothyroidism has beenweakly associated with HLA-DRB1*03, *04 and *11/*12alleles [23e26]. However, the presence of important im-munoregulatory genes (TNF, HSP70, LMP, TAP) withinthe MHC raises the possibility that HLA associationsseen in AITDs are primarily due to genetic variation inthese immunoregulatory genes which harbor the majorsusceptibility locus. The entireMHCregionwhich has notbeen extensively investigated [23,27], would providemorepower in detecting susceptibility genes.

In order to analyze theMHC region in a rare Tunisianisolate (Akr), with a high prevalence of AITDs, we havegenotyped 134 Akr DNAs for HLA class I and II allelesand polymorphisms within TNF, LTa, HSP70-02 andHSP70-hom genes. Our results have given evidence forgenetic association with TNF �308 gene polymorphism.

2. Patients and methods

2.1. Subjects

Subjects were recruited from a geographic isolate insouth Tunisia named Akr with a high prevalence ofAITDs [28]. Regular clinical follow-up of this isolatesince 1992 has permitted an update of the pedigree. Thispedigree consists in 10 generations of 391 members in-cluding 62 patients subdivided into 32 patients affectedwith GD, 9 patients with HT and 21 patients with PIM(Table 1).

2.2. Clinical assessment

The diagnosis of GD was based on the presence ofbiochemical hyperthyroidism as indicated by a decreaseof TSH, an increase of T4 levels and positive TSHreceptor antibody, in association with diffuse goiter orthe presence of exophthalmos. The diagnosis of HT wasbased on the presence of thyroid hormone replacedprimary hypothyroidism, defined as a TSH level abovethe upper limits associated with positive titers of thyroidantibodies (anti-thyroid peroxidase, or anti-thyroglobu-lin) and a palpable goiter. PIM was diagnosed by thepresence of hypothyroidism requiring T3 or T4 replace-ment. Patients with PIM have an atrophic gland.

2.3. Genotyping methods

Genomic DNA was extracted from 10 ml of pe-ripheral blood lymphocyte (PBL) of Akr patients and

healthy Akr-related controls using standard methods[29]. Subjects were then genotyped for HLA class I andII, TNF �308 A/G, TNFe, TNFc, HSP70-02 andHSP70-hom gene polymorphisms. The location of thesegenes with respect to the HLA gene complex is shown inFig. 1. Genotyping of TNFe and TNFc was performedby PCR technique using specific primers [30]. Screeningfor TNF �308 A/G, HSP70-02 (C1267 A/G) andHSP70-hom (C2437 T/C) was performed by PCR-RFLP technique as previously described [31e32].

HLA class I and II DNA typing was performedby PCR-single standard polymorphism (PCR-SSP)strategy using commercial one lamda micro SSP�HLA class I A and class I B and generic HLA class IIDNA typing tray.

Table 1

Distribution of 62 AITDs patients from the Akr isolate in different

categories with regard to their disease status and clinical parameters

GD (n ¼ 32) HT (n ¼ 9) PIM (n ¼ 21)

Males 16 3 4

Females 16 6 17

Agea 40.48 (20e81) 45 (23e58) 57.15 (30e81)

Age of onseta 28.31 (13e70) 30 (7e50) 45.4 (40e65)

Associated diseases IDDM/

SGS/Addisson

IDDM/NGS Vitiligo

Goiter

Yes 21 5 1

No 11 4 20

Ophthalmopathy

Yes 22 5 0

No 10 4 21

Anti-Tg status

Positive 12 2 7

Negative 20 4 10

Unknown e 3 4

Anti-Tpo status

Positive 16 4 10

Negative 15 2 6

Unknown 1 3 5

a Mean age with the age range between parentheses.

DP EC G F

DMLMP2

TAP1LMP7

TAP2 C4C2 2 1 Hom LTβ TNF LTα

HSP70

Centromere Telomere

DQ DR B A

Fig. 1. Schematic representation showing the main genetic regions of

the human major histocompatibility complex. Polymorphisms within

genes shaded grey have been genotyped in this study.

77N. Bougacha-Elleuch et al. / Journal of Autoimmunity 23 (2004) 75e80

2.4. Disease models

GD and HT are two different diseases with overlapin the involved genes and some of the immunologicalmanifestations. In this study, we analyzed the dataunder three disease models: (i) all AITD patients wereconsidered as affected, (ii) only GD patients were con-sidered as affected and HT patients were considered asunaffected, and (iii) only HT patients were considered asaffected and GD patients were considered as unaffected.To evaluate the influence of different disease parameters,we have examined the possible implication of gene poly-morphisms tested owing to the following criteria: anti-TPO titers, TSH and FT4 levels, presence or not of clin-ical symptoms, as well as patient ages.

2.5. Statistical analyses

2.5.1. Linkage analysesExact nonparametric multipoint linkage analysis was

performed using Genehunter [33] version 1.3 (http://www.genome.wi.mit.edu/ftp/distribution/sofware/Gene-hunter/). For the purpose of computation feasibility, theAkr pedigree was split into 17 smaller pedigrees. We alsorun a multipoint nonparametric analysis on the pedigreeas a whole, to avoid missing positive linkage [34].This analysis was run using Simwalk2 version 2.82(http://watson.hgen.pitt.edu/): a program which allowslinkage analysis of complex pedigree with multipleinbreeding loops and of any size by using resamplingmethods [35].

2.5.2. Family based association testWe used the FBAT program [36] to test for associa-

tion in the presence of linkage or not [37]. FBATprovides a family of chi-square distributed score testsadjusting for population admixture and that have manyenhancements over the TDT approach as it can use datafrom any pedigree structure. We also used the TDTapproach [38,39] as implemented in Genehunter version1.3 to test for association and linkage. This programalso allows the calculation of multi-locus TDT to testfor transmission disequilibrium of haplotypes for two tofour markers. Exact p-values of the TDT were calcu-lated using 30,000 permutations.

3. Results

In order to study the MHC gene polymorphisms andwhether one or more MHC alleles or haplotype(s) areassociated or linked to GD, HT or AITD segregationin the Akr isolate, we have performed typing of thedescribed gene polymorphisms (HLA class I and II,

TNF �308 A/G, TNFe, TNFc, HSP70-02 and HSP70-hom) on 62 Akr affected and 72 unaffected individuals.

3.1. Linkage and association analyses

Results with Genehunter program did not reveal anysignificant linkage. However, a suggestive linkage wasfound with TNF �308 gene polymorphism (p ¼ 0:03)using Simwalk2 program. Analysis using haplotypicTDT implemented in Genehunter showed association ofHLA-A2-B50-TNF 2 (allele 2 of TNF �308) and AITD(p ¼ 0:045). We have also used the FBAT under dif-ferent modes of inheritance. Table 2 summarizes thesignificant results found for different polymorphismstested in the MHC region under different models. Theseresults show a particularly significant association be-tween TNF 2 allele and AITDs (p ¼ 0:004) underrecessive mode.

On the other hand, we have examined our data underthe different disease models previously described. Ac-cording to these analyses, the HLA-DR11 and HLA-DR12 alleles are strongly associated with HT (p ¼0:00089 and p ¼ 0:0056, respectively) under the recessivemode. The TNF �308 gene polymorphism remains sig-nificantly associated with the two forms of AITDs: GDand HT, but with different alleles reflecting allelic heter-ogeneity of AITDs. In fact, in the only GD model, theTNF�308participateswith theTNF2allele (p ¼ 0:0011),whereas in the only HT model, the association wasrather with the TNF 1 allele (p ¼ 0:0039). Our dataindicate that the most significant association results forTNF �308 were found under the recessive mode consis-tently across disease models (Table 3).

Table 2

Family based association test (FBAT) with significant statistical results

Markers AITDs GD HT

TNF �308

Allele TNF 2 TNF 2 TNF 1

Z 11.069* 3.244 2.883

p 0.0039 0.0011 0.0039

TNFe

Allele e e 2

Z e e 2.301

p O0.05 O0.05 0.0213

DR

Allele e e DR11 DR12 DR01

Z e e 3.323 2.77 11.385*

p O0.05 O0.05 0.0008 0.0056 0.01

DQ

Allele e e DQ03 DQ02

Z e e 2.195 �2.026

p O0.05 O0.05 0.028 0.0428

Z-values have a random distribution except those indicated by an *

which corresponds to a multiallelic test and follows a chi-square

distribution.

78 N. Bougacha-Elleuch et al. / Journal of Autoimmunity 23 (2004) 75e80

Table 3

Significant values found with TNF-308 under different models and recessive mode

Markers AITDs GD HT Susceptibility class Anti-TPo TSH FT4 Clinical symptoms

TNF �308

Allele TNF 2 TNF 2 TNF 1 TNF 2 TNF 2 TNF 1 TNF 2 TNF 2

Z 2.251 3.244 2.883 3.244 12.356* 2.519 2.896 8.552*

p 0.024 0.0011 0.0039 0.0011 0.002 0.0117 0.0037 0.0138

Z-values have a random distribution except those indicated by an * which corresponds to a multiallelic test and follows a chi-square distribution.

4. Discussion

Genetic predisposition in AITDs is most likely due tointeractions between several genes and is influenced byso-far unknown non-genetic factors. In order to mini-mize genetic heterogeneity, we have targeted our studyto a rare large multigenerational isolate with high pre-valence of AITDs from South of Tunisia. In this isolate,AITDs appear in association with five other AID(Table 1). This observation is well documented andconfirms again the existence of common susceptibilitygenes for AID as it was already reported [40].

The studied isolate has been used in a genome screenand a maximum lod score was found at marker D2S171in 2p21 [2]. Many candidate genes (IgVH, Cb TCR andCTLA-4) [41e42] were excluded for possible implicationin AITDs pathogenesis in this isolate. Moreover, wehave analyzed six microsatellite markers (D6S299,D6S285, HLA DQB1 CAR1/CAR2, D6S273, TNFaIR2/IR4 and D6S291) in 16 cM spanning the MHCregion and negative lod scores were found (unpublisheddata). Due to the complexity and the high polymor-phism of the MHC and its known implication in theAID susceptibility, we have undertaken a detailed anal-ysis spanning 1.51 cM of the MHC chromosomal region.

In our study, we have found evidence for associationbetween HT and three different DR alleles (HLA-DR11,DR12 and DR01) with varying degrees of contribution(Table 2). This can be explained by intrafamilial heter-ogeneity. Indeed, each allele was associated with manynuclear families belonging to Akr pedigree and not withothers. Family based association test has also shown anassociation between AITDs and TNF �308 gene poly-morphism (p ¼ 0:004).

Linkage analyses using Genehunter program havefailed to detect any linkage with MHC gene polymor-phisms in Akr isolate split into 17 smaller pedigrees.However, Simwalk2 program, which is able to analyzecomplex pedigree with multiple inbreeding loops of anysize, was more powerful and has revealed linkage ofAITDs with TNF �308 gene polymorphism (p ¼ 0:03).This finding is in good agreement with previous findingsthat positive linkage could be missed when notanalyzing the pedigree as a whole [34].

The TNFa gene polymorphism and expression havebeen found to be involved in many AID [43e48]. On the

other hand, few studies which have investigated thispolymorphism in GD pathogenesis have reportedpositive association with TNF 2 allele in Polish andCaucasian populations (p! 0:0001, OR ¼ 4:38; p ¼3! 10�5, OR ¼ 2:2, respectively) [49,23].

There are two allelic forms in the TNF �308 genepolymorphism, referred to as TNF 1 (G) and TNF 2 (A).In the studied isolate, the A allele was associated withGD, whereas the G allele was rather associated with HT.The TNF 2 allele (A) was reported to be associated withhigh constitutive and inducible levels of the TNFa chain[50]. The promoter region of TNFA, in reporter genesystems, showed that the TNF 2 allele creates a promotersequence which is a more potent transcriptional activatorthan the TNF 1 allele [51e52]. However, conflicting datawere found concerning the transcriptional capacity ofthis allele [53e54]. It has also been demonstrated that inGD, thyrocytes express ‘‘de novo’’ HLA-DR antigensfollowing stimulation by TNF, TSH and INFg [55].This ‘‘de novo’’ expression could be implicated in thedevelopment of the autoimmune process in AITDs.

This study has assessed the relative contribution ofvarious immunomodulatory genes within the MHC tosusceptibility to AITDs in an isolate with high preva-lence of AITDs. Even in the absence of a confirmeddirect functional effect for TNF �308 gene polymor-phism, our genetic data clearly identify the latter asa marker of susceptibility to AITDs: the TNF 2 allelewas associated with GD, whereas the TNF 1 and DR11alleles were rather associated with HT. The associationdata alone do not help to understand the pathologicalmechanism of these genes. Functional analysis of TNFand HLA genes should be investigated to better under-stand their role in AITDs pathogenesis. Moreover,sequencing of HLA alleles found in the associatedhaplotype (HLA-A2-B50-TNF 2) will be of great interestmainly if we will find particular alleles.

Acknowledgements

Thisworkwas supportedby theMinisterede laRecher-che Scientifique et de la Technologie (MRST) (Tunisia)and the International Centre for Engineering and Bio-technology (ICGEB) (Italy). We thank Mr AbdelmajidDammak for his helpful review of the manuscript.

79N. Bougacha-Elleuch et al. / Journal of Autoimmunity 23 (2004) 75e80

References

[1] Vaidya B, Kendall-Taylor P, Pearce SHS. Genetics of endocrine

disease. The genetics of autoimmune thyroid disease. J Clin

Endocrinol Metab 2002;87(12):5385e97.[2] Maalej A, Makni H, Ayadi F, Bellassouad M, Jouida J, Bougacha

N, et al. A full genome screening in a large Tunisian family

affected with thyroid autoimmune disorders. Genes Immun 2001;

2:71e5.[3] Tomer Y, Ban Y, Concepcion E, Barbesino G, Villanueva R,

Greenberg DA, et al. Common and unique susceptibility loci

in Graves and Hashimoto diseases: results of whole-genome

screening in a data set of 102 multiplex families. Am J Hum Genet

2003;73(4):736e47.

[4] Vaidya B, Imrie H, Perros P, Young ET, Kelly WF, Carr D, et al.

The cytotoxic T lymphocyte antigen-4 is a major Graves’ disease

locus. Hum Mol Genet 1999;8(Jul (7)):1195e9.[5] Heward JM, Allahabadia A, Daykin J, Carr-Smith J, Daly A,

Armitage M, et al. Linkage disequilibrium between the human

leukocyte antigen class II region of the major histocompatibility

complex and Graves’ disease: replication using a population case

control and family-based study. J Clin Endocrinol Metab 1998;83:

3394e7.

[6] Uno H, Sasazuki T, Tamai H, Matsumoto H. Two major genes,

linked to HLA and Gm, control susceptibility to Graves’ disease.

Nature 1981;292:768e70.

[7] Shields DC, Ratanachaiyavong S, McGregor AM, Collins A,

Morton NE. Combined segregation and linkage analysis of

Graves’ disease with a thyroid autoantibody diathesis. Am J

Hum Genet 1994;55:540e54.

[8] Tomer Y, Barbesino G, Keddache M, Greenberg DA, Davies TF.

Mapping of a major susceptibility locus for Graves’ disease

(GD-1) to chromosome 14q31. J Clin Endocrinol Metab 1997;82:

1645e8.

[9] Roman SH, Greenberg D, Rubinstein P, Wallenstein S, Davies

TF. Genetics of autoimmune thyroid disease: lack of evidence for

linkage to HLA within families. J Clin Endocrinol Metab 1992;74:

496e503.

[10] Ban Y, Davies TF, Greenberg DA, Concepcion ES, Tomer Y.

The influence of human leukocyte antigen (HLA) genes on

autoimmune thyroid disease (AITD): results of studies in

HLA-DR3 positive AITD families. Clin Endocrinol (Oxf) 2002;

57(1):81e8.

[11] Mungall AJ, Palmer SA, Sims SK, Edwards CA, Ashurst JL,

Wilming L, et al. The DNA sequence and analysis of human

chromosome 6. Nature 2003;425:805e11.[12] Cuddihy RM, Dutton CM, Bahn RS. A polymorphism in the

extra cellular domain of the thyrotropin receptor is highly

associated with autoimmune thyroid disease in females. Thyroid

1995;5:89e95.[13] Pirro MT, De Filippis V, Di Cerbo A, Scillitani A, Liuzzi A, Tassi

V. Thyroperoxydase microsatellite polymorphism in thyroid

diseases. Thyroid 1995;5:461e4.

[14] Tomer Y, Greenberg DA, Concepcion E, Ban Y, Davies TF.

Thyroglobulin is a thyroid specific gene for the familial autoim-

mune thyroid diseases. J Clin Endocrinol Metab 2002;87:404e7.

[15] Kotsa KD, Watson PF, Weetman AP. No association between

a thyrotropin receptor gene polymorphism and Graves’ disease in

the female population. Thyroid 1997;7:31e3.

[16] Hadj Kacem H, Rebai A, Kaffel N, Masmoudi S, Abid M, Ayadi

H. PDS is a new susceptibility gene to autoimmune thyroid

diseases: association and linkage study. J Clin Endocrinol Metab

2003;88(May (5)):2274e80.

[17] Balkemore AI, Watson PF, Weetman AP, Duff GW. Association

of Graves’ disease with an allele of the interleukin-1 receptor

antagonist gene. J Clin Endocrinol Metab 1995;80:111e5.

[18] Cuddihy RM, Bahn RS. Lack of an association between alleles of

intereleukin-1 alpha and interleukin-1 receptor antagonist genes

and Graves’ disease in a North American Caucasian population.

J Clin Endocrinol Metab 1996;81:4476e8.

[19] Demaine A, Welsh KI, Hawe BS, Farid NR. Polymorphism of the

T cell receptor beta-chain in Graves’ disease. J Clin Endocrinol

Metab 1987;65:643e6.

[20] Stenszky V, Kozma L, Balazs C, Rochlitz S, Bear JC, Farid NR.

The genetics of Graves’ disease: HLA and disease susceptibility.

J Clin Endocrinol Metab 1985;61:735e40.

[21] Payami H, Joe S, Farid NR, Stenszky V, Chan SH, Yeo PP, et al.

Relative predispositional effects (RPEs) of marker alleles with

disease: HLA-DR alleles and Graves’ disease. Am J Hum Genet

1989;45:541e6.

[22] Yanagawa T, Mangklabruks A, Chang YB, Okamoto Y, Fifalen

ME, Curran PG, et al. Human histocompatibility leukocyte

antigen-DQ-A1*0501 allele associated with genetic susceptibility

to Graves’ disease in a Caucasian population. J Clin Endocrinol

Metab 1993;76:1569e74.[23] Hunt PJ, Marshall SE, Weetman AP, Bunce M, Bell JI, Wass JA,

et al. Histocompatibility leukocyte antigens and closely linked

immunomodulatory genes in autoimmune thyroid disease. Clin

Endocrinol (Oxf) 2001;55(Oct (4)):491e9.[24] Moens H, Farid NR, Sampson L, Noel EP, Barnard JM.

Hashimoto’s thyroiditis is associated with HLA-DRw3. N Engl

J Med 1978;299:133e4.

[25] Farid NR, Sampson L, Moens H, Barnard JM. The association of

goitrous autoimmune thyroiditis with HLA-DR5. Tissue Antigens

1981;17:265e8.

[26] Thompson C, Farid NR. Post-partum thyroiditis and goitrous

(Hashimoto’s) thyroiditis are associated with HLA-DR4. Immu-

nol Lett 1985;11:301e3.

[27] Heward JM, Allahabadia A, Sheppard MC, Barnett AH,

Franklyn JA, Gough SC. Associaton of the large multifunc-

tional proteasome (LMP2) gene with Graves’ disease is a result

of linkage disequilibrium with the HLA haplotype

DRB1*0304-DQB1*02-DQA1*0501. Clin Endocrinol 1999;

51(1):115e8.[28] Makni H, Maalej A, Ayadi F, Abid M, Jouida J, Ayadi H. Suivi

clinique et biologique d’une famille a haute prevalence de maladies

auto-immunes thyroidiennes. La Tunisie Medicale 1996;74(10):

433e8.[29] Kawazaki E. Sample preparation from blood, cells and other

fluids. In: Innis M, Gelffand D, Snisky G, White T, editors. PCR

protocols. A guide to methods and application. San Diego:

Academic Press; 1990. p. 146e52.

[30] Udalova IA, Nedospasov SA, Webb GC, Chaplin DD, Tur-

etskaya RL. Highly informative typing of the human TNF locus

using six adjacent polymorphic markers. Genomics 1993;16(Apr

(1)):180e6.

[31] Wilson AG, di Giovine FS, Balkemore AIF, Duff GW. Single

base polymorphism in the human tumor necrosis factor alpha

(TNFa) gene detectable by NcoI restriction of PCR product.

Hum Mol Genet 1993;1:353e9.

[32] Milner CM, Campell RD. Polymorphic analysis of the three

MHC-linked HSP70 genes. Immunogenetics 1992;36:357e62.[33] Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric

and nonparametric linkage analysis: a unified multipoint ap-

proach. Am J Hum Genet 1996;58(Jun (6)):1347e63.

[34] Garner C, McInnes LA, Service SK, Spesny M, Fournier E, Leon

P, et al. Linkage analysis of a complex pedigree with severe

bipolar disorder, using a Markov chain Monte Carlo method. Am

J Hum Genet 2001;68(Apr (4)):1061e4.

[35] Sobel E, Lange K. Descent graphs in pedigree analysis:

applications to haplotyping, location scores, and marker-sharing

statistics. Am J Hum Genet 1996;58(Jun (6)):1323e37.

80 N. Bougacha-Elleuch et al. / Journal of Autoimmunity 23 (2004) 75e80

[36] Laird NM, Horvath S, Xu X. Implementing a unified approach

to family based tests of association. Genet Epidemiol 2000;

19(Suppl 1):36e42.

[37] Lake SL, Blacker D, Laird NM. Family based tests of association

in the presence of linkage. Am J Hum Genet 2000;67:1515e25.

[38] Spielman RS, McGinnis RE, Ewens WJ. Transmission test for

linkage disequilibrium: the insulin gene region and insulin-

dependent diabetes mellitus (IDDM). Am J Hum Genet 1993;

52:506e16.

[39] Curtis D, Sham PC. A note on the application of transmission

disequilibrium test when a parent is missing. Am J Hum Genet

1995;56:811e2.[40] Alkhateeb A, Steller GL, Old W, Talbert J, Uhlhorn C, Taylor M,

et al. Mapping of an autoimmunity susceptibility locus (AIS1) to

chromosome 1p31.3-p32.3. Hum Mol Genet 2002;11(6):661e7.[41] Fakhfakh F, Maalej A, Makni H, Abid M, Jouida J, Zouali M,

et al. Analysis of immunoglobulin VH and TCR Cb polymor-

phisms in a large family with thyroid autoimmune disorder. Exp

Clin Immunogenet 1999;16:185e91.[42] Maalej A, Bougacha N, Rebai A, BellassouadM, Ayadi-Makni F,

Abid M, et al. Lack of linkage and association between auto-

immune thyroid diseases and the CTLA-4 gene in a large Tunisian

family. Hum Immunol 2001;62:1245e50.[43] Yoon JW, Jun HS. Cellular and molecular pathogenic mecha-

nisms of insulin-dependent diabetes mellitus. Ann N Y Acad Sci

2001;928(Apr):200e11.

[44] Matsuno H, Yudoh K, Katayama R, Nakazawa F, Uzuki M,

Sawai T, et al. The role of TNF-alpha in the pathogenesis of

inflammation and joint destruction in rheumatoid arthritis (RA):

a study using a human RA/SCID mouse chimera. Rheumatology

(Oxf) 2002;41(Mar (3)):329e37.

[45] Romas E, Gillespie MT, Martin TJ. Involvement of receptor

activator of NFkappaB ligand and tumor necrosis factor-alpha in

bone destruction in rheumatoid arthritis. Bone 2002;30(Feb (2)):

340e6.

[46] Zuniga J, Vargas-Alrcion G, Hernandez-Pacheco G, Portal-

Celhay C, Yamamoto-Furusho JK, Granados J. Tumor necrosis

factor-alpha promoter polymorphisms in Mexican patients with

systemic lupus erythematosus (SLE). Genes Immun 2001;

2(Nov (7)):363e6.

[47] Mycko M, Kowalski W, Kwinkowski M, Buenafe AC, Szyman-

ska B, Tronczynska E, et al. Multiple sclerosis: the frequency of

allelic forms of tumor necrosis factor and lymphotoxin-alpha.

J Neuroimmunol 1998;84(Apr 15 (2)):198e206.

[48] Arias AI, Giles B, Eiermann TH, Sterry W, Pandey JP. Tumor

necrosis factor-alpha gene polymorphism in psoriasis. Exp Clin

Immunogenet 1997;14(2):118e22.

[49] Kula D, Jurecka-Tuleja B, Gubala E, Krawczyk A, Szpak S,

Jarzab M. Association of polymorphism of LTa and TNF genes

with Graves’ disease. Folia Histochem Cytobiol 2001;39(Suppl 2):

77e8.

[50] Wilson AG, Symons JA, Mc Dowell TL, di Giovani FS, Duff

GW. Effects of a tumor necrosis factor (TNFa) promotor base

transition on transcriptional activity. Br J Rheumatol 1994;33:

89e97.

[51] Kroeger KM, Carville KS, Abraham LJ. The �308 tumor

necrosis factor-alpha promoter polymorphism affects transcrip-

tion. Mol Immunol 1997;34:391e9.

[52] Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW.

Effects of apolymorphism in the human tumor necrosis factor

a promoter on transcriptional activation. Proc Natl Acad Sci

USA 1997;94:3195e9.

[53] Brinkman BM, Zuijdgeest D, Kaijzel EL, Breedved FC, Verweij

CL. Relevance of the tumor necrosis factor a (TNFa)-308

promoter polymorphism in TNFa gene regulation. J Inflamm

1996;46:32e41.

[54] Stuber F, Udalova IA, Book M, Drutskaya LN, Kuprash DV,

Turetskaya RL, et al. �308 tumor necrosis factor (TNF) poly-

morphism is not associated with survival in severe sepsis and is

unrelated to lipopolysaccharide inducibility of the human TNF

promoter. J Inflamm 1996;46(1):42e50.

[55] Kissonerghis AM, Grubeck-loebenstein B, Pirich K, Feldmann

M, Londei M. Tumor necrosis factor synergises with gamma

interferon on the induction of mRNA for DR alpha chain on

thyrocytes from Graves’ disesase and no toxic goitre. Autoimmu-

nity 1989;4(4):255e66.