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Auioimmunify. 1992, Vol. 14. pp. 97-100 Reprints available directly from the publisher Photocopying permitted by license only

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POLYMORPHISM STUDY OF TCRa AND y GENES IN INSULIN DEPENDENT DIABETES MELLITUS (IDDM) MULTIPLEX FAMILIES P. AVOUSTIN', L. BRIANT2, C. DE PREVAL' and A. CAMBON-THOMSEN2 'INSERM Uniik 100, CHU Purpan F-31052 Toulouse cedex. 'CRPG-CNRS, UPR 8291, CHU Purpan F-31300 Toulouse

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(Received May 13,1992; in final form Augusi 18,1992) T-cell receptor (TCR)a and ygenes polymorphisms were analysed by Restriction Fragment Length Polymorphism (RFLP) in 10 Insulin Dependent Diabetes Mellitus (IDDM) multiplex families. TCRa and y alleles distribution does not significantly differ between affected and non affected children. Furthermore there was no excess of C a or Vyallele sharing in affected sib pairs. Therefore the T-cell receptor a and ychain alleles studied do not seem to affect IDDM susceptibility per se.

KEY WORDS: Insulin dependent diabetes mellitus IDDM, T-cell Receptor TCR, multiplex families, RFLP.

INTRODUCTION Numerous previous studies have demonstrated that Major Histocompatibility Complex (MHC) class I1 alleles influence IDDM susceptibility. The most striking association in Caucasoid populations was observed with the HLA-DQw8 allele (DQB 1*0302) which is found in over 70% of diabetic patients'. Since, heterodimers involving DQA and DQB alleles have been described'. However, despite the strong association with HLA alleles, several studies indicate that genes outside the HLA region contribute to IDDM susceptibility3. The strong HLA class I1 association with IDDM and the physical interaction between HLA antigens and T-cell receptor (TCR) in the process of antigen presentation suggest a role for specific TCR molecules in this autoimmune disease. Therefore good candidates of susceptibility factors out of the HLA region are T-cell receptor (TCR) genes. Several TCR polymorphism studies have already been performed in IDDM families and populations and the discrepant results are still a matter of discussion4. T-cell receptors are involved in recognition of foreign antigen in conjunction with the major histocompatibility complex determinants. The most common form of the TCR is a heterodimer of one a and one p chain expressed on the surface of mature T-cells. Moreover, a second TCR yl6 is only expressed at the surface of a subset of T lymphocytes which does not possess the classical T-cell receptor. The function of TCR lymphocytes ~6 still remains elusive. The presence of T cells at the site of lesions in other autoimmune diseases points to the possibility of involvement of such yl6 T cells in the immunopathological processs. The description of TCR Vy polymorphisms6 provides a tool for testing such a hypothesis; this has

never been done in IDDM multiplex families so far. The genes that encode the a and y TCR are located respectively on chromosome 14 and 7. They are composed of variable (V), joining (J) and constant (C) gene segments. In order to test the implication of germ-line TCR gene region in the genetic susceptibility to the disease, we analysed T C R a and ?genetic markers among 10 multiplex families. We have not been able to evidence any correlation between polymorphisms of TCR genes and IDDM susceptibility. MATERIAL AND METHODS Families

The ten IDDM multiplex families (60 members and 25 IDDM patients) included at least 2 affected children and 2 had 3 affected children (Table 1). In 3 families one parent was affected. These families except family number 818 were already studied for HLA-DR, -DQw RFLP in the 5th Genetic Analysis Workshop (GAW5)'. The families were selected mainly in the area of Toulouse (Southern France) except families number 821 and 822 originating from Denmark. The control population studied for TCR Vygenes polymorphisms is composed of 53 healthy unrelated individuals (blood donors and laboratory personal in the area of Toulouse, France). RFLP Studies I)

TCR C a polymorphisms were studied after digestion of DNA samples with TaqI restriction endon-

P. AVOUSTIN ET AL.

98

uclease. The restriction fragments were separated by electrophoresis in 0.7% agarose gels and transferred onto nylon membranes (Genescreen plus, NEN). Filters were prehybridized 12 hr and then hybridized with PGASB probe labelled with 32P dCTP (Amersham). The polymorphisms detected are located in the 5' non coding region of the C a gene and generate 2 restriction fragments of 7.2 and 2.1 kb.

2) TCR Vypolymorphisms were also defined after digestion with the TaqI restriction endonuclease and hybridization with the pV3S probe6. The V y I probe is a 1.2 kb Sac1 fragment containing TRGV3 from clone lambda SH4.This probe cross-hybridizes to the 9 V y genes belonging to subgroup 19. This polymorphism defines 6 restriction fragments, 4 of them result from restriction site polymorphisms (alleles TRGVI*B 1, TRGVI"B2, TRGVI*B5, TRGVI*B6), the fifth corre-

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Table 1 Vyand Cagenotypes in 10 multiplex IDDM familes

F

M

I

AI/A2 B 1/83

AIIAI BIB1

A I/A2 81/83

803 C a alleles Vyalleles

2

3

AIIA2 BIB1

AI/A2 BIB1

*

4

5

A1/A2 BIB1

AI/A2 BIB1

A I/A2 BIB3

AIIAI BIB2

A1/A2 BIB1

*

Ca alleles

AI/A2 B 1/82

A I/A I BIB1

AI/A2

*

*

*

AIIAI BIB1

AI/AI BIB1

AUAI BID1

AI/AI BID1

AIIAI 81/81

*

*

*

AI/A2 BIB1

A I/A2 BIB1

AIIA? BIB1

A2/A2

A2iA2 BIB1

*

*

AI/AI BIB1

AI/AI BIB3

A I/A I B 1/83

AI/AI B 1/83

AI/AI

*

*

A I/A2 BIB1

AI/A2 BIB1

A2/A2 BIB1

A2/A2 BIB1

A2/A2 BIB1

AlIA2 BIB1

A I/A 1 BID1

A 1/A2 BIB1

AlIA2 81/81

A 1/A2 BIB1

A I/A2 BIB1

AI/A2 B 1/82

AI/A2 BIB1

A2/A2 B 1/82

A I/A2 BIB1

805 C a alleles V y allleles 806

C a alleles Vyalleles 809

C a alleles V yalleles 811

C a alleles V y alleles 814

Ca alleles V y alleles

C a alleles V y alleles

*

Ca alleles

AI/AI BIB1

AI/A2 BIB2

Al/A2 BIB1

AI/AI 83/83

A1/A2 F:/8 I

A I /A2 83/83

C a alleles Vyalleles

AI/AI BIB1

AIIAI BIB1

AIIAI BIB1

A I/A I BIB1

NT

*

*

*

822

AIIAI B 1/82

*

82 I

Vyalleles

BI/BI

*

818

AIIAI BIB1

*

* BIB1

7

*

804

Vyalleles

6

* AI/A2 BIB2

AI/AI BIB1

AI/A2 B3/8 I

A I/A I NT

*

Fdathcr. M=mother. number I to 7 children lrom the eldest. indicutes diabetic individuals. 81: TRGVI*BI.82: TRGVI.82 and 83: TRGVI*BI. A I: 2. I kb Ca allele and A?: 7.2 kb Ca sllele. NT: not tested.

*

A l/A I

BIB1

*

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TCRa and y GENES IN IDDM

99

sponds to a deletion of the Vy4 and Vy5 (allele TRGVI*B3). The sixth correspond to an insertion of 6 kb with an additional Vy gene between Vy3 and v y4.

sib pairs and in pairs comprising the index case and the first unaffected sib.

In TRGVI*B3 haplotypes the Vy4 (6.0 kb) and V y 5 (4.2 kb) TaqI bands were absent in the hybridization pattern. The TRGVI*B4, TRGVI*B5 and TRGVI*B6 haplotypes were only observed in the Tunisian, Lebanese and Black African populations" (Figure 1).

RESULTS AND DISCUSSION

The allelic frequencies for the Ca polymorphisms among affected and non affected sibs in the 10 multiplex families are given in Table 2. A non significant excess of the 2.1 kb C a allele ( ~ ~ 0 . was 2 ) observed among affected children. Although more 2.1/2.1 kb homozygous genotypes were observed among diabetic Statistical analysis children than among unaffected sibs, this difference The TCR C a and Vy alleles frequencies were esti- was not significant. In addition TCR allele sharing between IDDM mated by direct counting among the proband (the affected sib pairs and between each index case and the eldest diabetic) and the first healthy sib within each first unaffected sib was analysed. Only 8 families family. At least 10 diabetic and 8 unaffected children among 10 were fully informative: one family with 3 were compared. The distribution of these alleles 7 families with 2 affected sibs. All affected sibs and among each group was tested by X2 test. Allele sharing was studied in informative families in affected sibs shared at least one allele: among affected sibpairs 5 shared 1 allele and 5 shared 2; among affectedhnaffected sibpairs 6 shared 2 alleles and 2 shared only 1. No evidence of correlation was found between IDDM susceptibility and TCR gene polymorphism. At the functional level, the C a region is not directly involved in the TCR recognition properties. The RFLP detectable differences could reflect variations within the V a region genes in linkage disequilibrium with C a RFLP. However as two other IDDM multiplex family TCR analysis"*'2 concluded to non linkage between TCR C a and V a polymorphisms and IDDM susceptibility, our data support the same conclusion. The TCR Vy gene polymorphisms were studied among the 10 multicase diabetic families and among 53 unrelated healthy individuals. This analysis of TCR y genes polymorphisms among diabetic and non diabetic children did not show any difference (Table 3). The TRGVI*B 1 allele was the most frequent allele among diabetic (80%) and healthy sibs (75%). When these data are compared with allelic frequencies observed among 53 unrelated healthy individuals no significant difference is found. A random Vyallele sharing between the first IDDM

Figure 1 V y TCR gene polymorphism profile in 5 unrelated individuals. The four polymorphic bands are indicated by m o w s . Lane I , 3 and 5 correspond to TRGVI*B I homozygous individuals. TRGVI*BI is characterized by 4.4 and 6.2 kb fragments. Lane 2 corresponds to TRGVI*B3 homozygous individual. TRGVI*B3 is characterized by the absence of 4.4 and 6.2 kb corresponding to the deletion of Vy4 and Vy5. Lane 4 correspond to TRGVI* B IflRGVI*B? heterozygous individual. TRGVI*B2 is characterized by the presence of the 22.2 kb fragment.

Table ' 2 Allelic frequencies defined by TaqI/Ca TCR gene polymorphism among first affected children, first non affected children and control population'-' TCR Ca alleles

Diabetic

Non diabetic

(n=20)

(n=16)

Control population (n=32)

13 (65%) 7 (35%)

7 (43.5%) 9 (56.5%)

21 (66%) I I (34%)

2.1 kb 7.1 kb n=numkr of haplotypes.

P. AVOUSTIN ET AL.

100

Table 3 Allelic frequencies defined by the TaqIIVy TCR gene polymorphism among First affected children and first non affected children V y alleles

(n=20)

Non diabetic (ti= 16)

Control population (n= 106)

16 (80%) 0 (-1 4 (20%)

12 (75%) 2 (12.5%) 2 ( I 2.5%)

83 (78.3%) 10 (9.5%) 13 (12.2%)

Diabetic

TRGVI*BI TRGVI*B2 TRGVI*B3

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n-number of haplotypes

affected sibs and the other affected or unaffected sibs was also observed. Our data do not document any significant correlation of the C a and V y polymorphisms studied with IDDM. These results are in agreement with the other TCR family studies performed1’*”.This first trial of TCR Vypolymorphisms in IDDM does not encourage to continue such studies. We conclude that, if allelic variation in the TCRa and y genes plays a role in IDDM susceptibility, it must be a very modest one, much smaller than allelic polymorphism in the HLA region. Mature TCR repertoire is not encoded entirely in germ-line TCR genes but results from somatic diversification mechanisms including addition or deletion of nucleotides. Sheehy el d.’* suggested that the importance of TCR alleles in IDDM susceptibility results from different strategies by which HLA and TCR molecules achieve antigen binding diversity. HLA polymorphisms result from allelic diversity whereas TCR are generated by combinatorial and junctional diversity. However studies of germ-line TCR genes polymorphisms probably cannot provide the best way of approaching the role of T-cells in the autoimmune process which should be studied at the functional level.

References I . Nepom GT. HLA and type I diabetes. lmmunol Today 1990; 11: 314-15 2. Khalil I, d’Auriol L, Gobet M. Morin L. Lepage V. Deschamps

I, Park MS. Degos L, Galibert F, Hors J. A combination of HLA-DQ Beta Asp57 negative and HLA-DQ alpha Arg52 positive confers susceptibility to insulin dependent diabetes mellitus. JClin Invest 1990; 85: 1315-1321 3. Green A. Epidemiological and public health aspects of insulintreated diabetes in Denmark. Diaberes Care 1985; 1 suppl 8: 77-8 I 4. Hillbert J, Olerup 0. Germ line polymorphism of TCR genes and disease susceptibility-fact or hypothesis. Immunol foday 1992; 13: 47-49 5 . Kjelsen-Kragh J. Ouayle A. Kalvenes C, Forre 0, Sorkaar D, Vinje 0, Thoen J, Natvig JB. T gammddelta cells in juvenile rheumatoid arthritis and rheumatoid arthritis. Scand J Immunol 1990; 32: 65 1-655 6. Lefranc MP, Forster A, Rabbits TH. Rearrangement of two distinct T-cell chain variable-region genes in human DNA. Nature 1986; 319: 420-422 7. Cox NJ, Gogolin KJ, Horvath VJ, Barker DJ, Wright E, Tran T. Skolnick MH. Boehm BO. Fehsel K, Bertrams J, Hodge TW, Acton RT, McGill J, Elbein SC, Permutt MA, de Pr&al C, Avoustin P, Cambon-Thomsen A, Robinson DM, Holbeck SL, Nepom GT, Schneider PM. Rittner C, Toyoda H, Rotter JI, Spielman R. Restriction fragment length polymorphisms of the HLA-DR, HLA-DQ and insulin gene regions in IDDM: the GAW 5 data. Genet Epidemiol 1989; 6: 2 1-26 8. Rabbits TH, Lefranc MP, Stinson MA, Sims JE. Schroeder T, Steinmetz M,Spurr NL. Solomon E, Goodfellow PN. The chromosomal location of T-cell receptor genes and a T-cell rearranging gene: possible correlation with specific translocations in human T-cell leukemia. EMBO J 1985; 4: 1461-1465 9. Forster A, Huck S, Ghanem N, Lefranc MP, Rabbits TH. New subgroups in the human T-cell rearranging V y gene locus. EMBOJ 1987; 6: 1945-1950 10. Ghanem N, Buresi C. Moisan JP, Bensmana M, Chuchana P. Huck S . Lefranc G, Lefranc MP. Deletion, insertion and restriction site polymorphism of the T-cell receptor gamma variable locus in French, Lebanese, Tunisian and Black African populations. Immunogenetics 1989; 30: 350-360 1 I . Concannon P. Wright JA, Wright LG. Sylvester DR, Spielman RS. T-cell receptor genes and insulin-dependent diabetes mellitus (IDDM): No evidence for linkage from affected sib pair. Am J Hum Genet 1990; 47: 45-52 12. Sheehy MJ, Meske LM, Emler CA, Rowe JR, Neme de Gimenez MH, lngle CA. Chan A. Trucco M. Mak TW.Allelic T-cell complexes have little or no influence on susceptibility to type I diabetes. Hum lmmunol 1989; 26: 261-27 1 13. Zerbib A, Molvig J, Thomsen H. Coppin M. Cambon-Thomsen A. Sommer E, Nerup J, de Prival C. Restriction fragment length polymorphisms of HLA and non HLA genes in DR3/4 heterozygous Danish insulin-dependent diabetic patients and healthy individuals. Disease Markers 1989; 7 : 2 7 4 1

Polymorphism study of TCR alpha and gamma genes in insulin dependent diabetes mellitus (IDDM) multiplex families.

T-cell receptor (TCR) alpha and gamma genes polymorphisms were analysed by Restriction Fragment Length Polymorphism (RFLP) in 10 Insulin Dependent Dia...
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