Corrected p value (pc) not significant, @ Corrected p value (pc) significant,

Controls No. (%)

*CC (L)* 11 (4.7) 1 (0.78) 0.03# 4.3 (1. 21-

*Low secretor* 8 (3.4) 1 (0.8) 0.11 3.17 (0.87-

**genotypes and haplotypes** 

*, IL-10, IL-6* and *TGF-*

genes interact in integrated networks. Since only *TNF-*

is encoded on chromosome 6p21.3, *IFN-*

T1D No. (%)

*IL-6* is encoded on 7p21 and *TGF-*

*TNF-, IFN-*

cytokine genes.

Genotype / haplotype

*AA (L)* <sup>41</sup>

*TA+TT (H)* <sup>56</sup>

*GG+GC (H)* <sup>86</sup>

*Low secretor* <sup>45</sup>

*High Secretor* <sup>52</sup>

*High Secretor* <sup>89</sup>

ACC,ACC; ACC,ATA = Low secretors.

Table 2. Simultaneous presence of *TNF-*

genotypes and haplotypes (Kumar et al., 2007).

CG,CC, CC,CC = Low secretors.

*IL-10* 

*TGF-β1* 

genotypes.

mRNA levels of insulin than *Class-I* in thymii of fetuses, suggesting poor expression of thymic INS expression resulting in poor thymic education for insulin in people with homozygous *Class-I,I* and *class-I,III VNTRs* resulting in break of tolerance in predisposed individuals. However, higher expression of insulin in the thymii of individuals with homozygous *class-III, III* may be able to facilitate immune tolerance induction, as a mechanism for dominant protective effect of *Class-III* alleles (Pugliese et al., 1997, Vafiadis et al., 1997).

Our results are contrary to that of Veijola et al. (Veijola et al., 1995) on Finnish children who showed that both 5' and 3' INS loci showed an association with T1D in *non-DR3/non-DR4* patients (Veijola et al., 1995). However, in our studies only 9.47% of the non-*DR3/DR4*  patients were homozygous for *Class-I INS-VNTR* as compared to 4.2% controls and this difference was not significant statistically. Julier et al. (Julier et al., 1991), on the other hand, had reported that the risk contributed by the INS region was increased in *DR4-*positive patients. Again, in our study only 6.32% of the patients as compared to 1.39% of controls had *INS-VNTR class-I* homozygosity with *DRB1\*04:01* and *DRB1\*04:05* alleles and this difference was not significant statistically.

#### **4. Cytokine genes**

Cytokines are the coordinators of the immune system that interact in integrated networks and functions of one cytokine may be modulated or substituted by another (Bidwell et al., 1999). A cascade of cytokines are involved in pro-inflammatory auto-immune responses in T1D. Single nucleotide polymorphisms (SNPs) in different pro-inflammatory and antiinflammatory cytokine genes at certain defined regions have been shown to be associated with differential amount of their production (Asderakis et al., 2001, Awad et al., 1998, Bittar et al., 2006, Burzotta et al., 2001, Fishman et al., 1998, Louis et al., 1998, Pociot et al., 1993). Pro-inflammatory cytokines and their integrated influences are known to regulate complex immune responses during autoimmune destruction of tissues (Rabinovitch, 1994). Hence it is necessary that they are studied and analysed in context of each other and not in isolation from each other. We had reported for the first time the integration and interaction of *TNF-* gene with other cytokine genes and *HLA-DRB1* and *B* loci alleles (Kumar et al., 2007).

We studied the cytokine gene polymorphism using XIIIth International Histocompatibility Workshop's (IHWC, Heidelberg kit) and One lambda's cytokine typing kits (Canoga Park, CA, USA) based on Polymerase Chain reaction (PCR) with sequence specific primers (PCR-SSP). PCR-SSPs were done for *IFN- (A+874T)* (14), *TNF- (G-308A)* (15), *IL-6 (G-174C)* (9), *IL-10 (A-1082G, T-819C, C-592A*) (16), and *TGF1 (Tcdn10C, Gcdn25C*) (11). *T→C* substitution in nucleotide 29, codon 10 of the first exon of TGF1, changes the amino acid Leu → Pro. Similarly *G→ C* substitution in nucleotide 74, codon 25 of first exon of *TGF1*, changes the amino acid Arg → Pro. However, since we are studying the SNPs in the two codons, we will refer to the SNPs in codons 10 and 25 hereafter. and not the resultant amino acids to avoid any confusion and to maintain consistency with the other SNPs.

Our results showed that the high producing genotype of *TNF--308GA* and *AA* were significantly increased and low producing genotype *GG* was significantly reduced in T1D patients as compared to controls (p < 7 x10-6). None of the other cytokine genes showed any significant difference between the patients and controls.

mRNA levels of insulin than *Class-I* in thymii of fetuses, suggesting poor expression of thymic INS expression resulting in poor thymic education for insulin in people with homozygous *Class-I,I* and *class-I,III VNTRs* resulting in break of tolerance in predisposed individuals. However, higher expression of insulin in the thymii of individuals with homozygous *class-III, III* may be able to facilitate immune tolerance induction, as a mechanism for dominant protective effect of *Class-III* alleles (Pugliese et al., 1997, Vafiadis et

Our results are contrary to that of Veijola et al. (Veijola et al., 1995) on Finnish children who showed that both 5' and 3' INS loci showed an association with T1D in *non-DR3/non-DR4* patients (Veijola et al., 1995). However, in our studies only 9.47% of the non-*DR3/DR4*  patients were homozygous for *Class-I INS-VNTR* as compared to 4.2% controls and this difference was not significant statistically. Julier et al. (Julier et al., 1991), on the other hand, had reported that the risk contributed by the INS region was increased in *DR4-*positive patients. Again, in our study only 6.32% of the patients as compared to 1.39% of controls had *INS-VNTR class-I* homozygosity with *DRB1\*04:01* and *DRB1\*04:05* alleles and this

Cytokines are the coordinators of the immune system that interact in integrated networks and functions of one cytokine may be modulated or substituted by another (Bidwell et al., 1999). A cascade of cytokines are involved in pro-inflammatory auto-immune responses in T1D. Single nucleotide polymorphisms (SNPs) in different pro-inflammatory and antiinflammatory cytokine genes at certain defined regions have been shown to be associated with differential amount of their production (Asderakis et al., 2001, Awad et al., 1998, Bittar et al., 2006, Burzotta et al., 2001, Fishman et al., 1998, Louis et al., 1998, Pociot et al., 1993). Pro-inflammatory cytokines and their integrated influences are known to regulate complex immune responses during autoimmune destruction of tissues (Rabinovitch, 1994). Hence it is necessary that they are studied and analysed in context of each other and not in isolation from each other. We had reported for the first time the integration and

We studied the cytokine gene polymorphism using XIIIth International Histocompatibility Workshop's (IHWC, Heidelberg kit) and One lambda's cytokine typing kits (Canoga Park, CA, USA) based on Polymerase Chain reaction (PCR) with sequence specific primers (PCR-

 *(A+874T)* (14), *TNF-*

29, codon 10 of the first exon of TGF1, changes the amino acid Leu → Pro. Similarly *G→ C*

→ Pro. However, since we are studying the SNPs in the two codons, we will refer to the SNPs in codons 10 and 25 hereafter. and not the resultant amino acids to avoid any

significantly increased and low producing genotype *GG* was significantly reduced in T1D patients as compared to controls (p < 7 x10-6). None of the other cytokine genes showed any

substitution in nucleotide 74, codon 25 of first exon of *TGF*

confusion and to maintain consistency with the other SNPs.

significant difference between the patients and controls.

Our results showed that the high producing genotype of *TNF-*

gene with other cytokine genes and *HLA-DRB1* and *B* loci alleles

*1 (Tcdn10C, Gcdn25C*) (11). *T→C* substitution in nucleotide

 *(G-308A)* (15), *IL-6 (G-174C)* (9), *IL-10* 

*1*, changes the amino acid Arg

*-308GA* and *AA* were

al., 1997).

difference was not significant statistically.

SSP). PCR-SSPs were done for *IFN-*

*(A-1082G, T-819C, C-592A*) (16), and *TGF*

**4. Cytokine genes** 

interaction of *TNF-*

(Kumar et al., 2007).

#### **4.1 Simultaneous presence of TNF- genotypes with IFN-, IL-6, IL-10 and TGF-1 genotypes and haplotypes**

*TNF-, IFN-, IL-10, IL-6* and *TGF-1* genes are localized on different chromosomes. *TNF-* is encoded on chromosome 6p21.3, *IFN-* is encoded on 12q14, *IL-10* is encoded on 1q31-q32, *IL-6* is encoded on 7p21 and *TGF-1* is encoded on 19q13.2. However, the products of these genes interact in integrated networks. Since only *TNF-* showed a significant association with T1D, we studied whether simultaneous presence of *TNF-* genotypes with different genotypes of the other cytokines in an individual could suggest an interaction between these cytokine genes.


N=Total number of samples studied, \$Number of control samples studied for IL-6 were 127, one sample could not be typed due to PCR failure. TNF-α GA/AA have been combined as high secretor genotypes.
