pubim1

The project aims to study the genetic factors associated with diabetes of the young, which include insulin dependent diabetes mellitus (IDDM) and ketosis resistant diabetes of the young (KRDY). The ultimate aim of the project is to decipher ways that can be used for the diagnosis of the prediabetics in the high risk groups and devise approaches to inhibit autoimmunity. The basic problem with the IDDM patients is that by the time they report to the clinic, the insulin producing beta cells of the pancreases have been damaged to a considerable extent. So, the only treatment left is to give insulin which takes care of the daily insulin requirement but does not stop autoimmunity. Hence, there is a need to identify these patients earlier in life for better monitoring and some kind of immune intervention to inhibit autoimmunity. Altered peptides ligands have been designed to be used in vitro to inhibit autoimmune T-cell responses.

Since the disease has been shown to have genetic predisposition, to be able to identify prediabetics, the project aims to study (i) the HLA polymorphism in IDDM and KRDY patients from North India and ethnically matched controls, (ii) the polymorphism of 5’-INS gene (IDDM2) or Insulin linked polymorphic region (ILPR) in IDDM and KRDY patients and controls, (iii) the autoantibody profile to insulin and islet cells and C-peptide levels in IDDM and KRDY patients and controls, (iv) the association of HLA with the type of autoantibodies found in the patients and (v) to design and use peptides in vitro to inhibit autoimmune T-cell responses.

After doing the DR generic typing, all the patients and control samples were processed for group specific amplifications and hybridizations for DRB1*02, DRB1*04 and DR52 associated DRB1*03, DRB1*08, *11, *12, *13 and *14 since each one of these groups is a cluster of alleles. 88 samples from IDDM patients and 94 normal healthy controls have been typed for SNPs in TNF-a, IFN-g, IL-10 -1082, -819 and –590, TGFb codon 10 and 25 and IL-6 since these cytokine genes only showed significant associations done in the preliminary study on 58 IDDM patients and 34 normal healthy controls reported last year.

More blood samples from diabetes patients have been studied for in vitro inhibition of autoimmune T-cell responses by peptides. Initially we were doing Elispots only for IFN-g, IL-4 and IL-10. However, we have also started doing Elispots for TNF-a and IL-12 to check if there is any reduction in the cells producing these cytokine after priming with synthetic peptides. Data on some of the peptides look promising, since they induce a reduction in IFN-g spots and increase in IL-4 spots (in Elispot assays), suggesting a shift from Th1 to Th2 phenotype, which may have therapeutic implications. However, complete data with other cytokines will be analysed later to avoid biases.

The genetic data has been analysed for DRB1 alleles and insulin linked polymorphic region (INS-VNTR) present in IDDM patients and healthy controls.

Fisher’s exact test was used to determine the significance of differences between the patient and control groups. P values were corrected wherever required by multiplying them with the number of alleles tested for the locus. Relative risks (RR) were calculated using Woolf’s method, with Haldane’s modification wherever the numbers were 5 or less.

Table-1 shows the frequencies of HLA-DRB1 alleles showing significant differences in 100 IDDM patients and 94 healthy controls. DRB1*0301, was significantly increased in the patients as compared to controls (p < 10-8). Two alleles of DR4, DRB1*0401 and DRB1*0405 were significantly increased in the patients and DRB1*0403 and 0404 were increased significantly in the controls. DRB1*0701 was also significantly increased in the controls (p < 7x10-6) even after correction.

Table-1: HLA DRB1 alleles significantly different in IDDM patients and controls

HLA DRB1	DIABETES N=100 No.	%	CONTROLS N=94 No.	%	p value	RR
DRB1*0301	76	76	21	22.34	10^-8*	11
DRB1*0401	9	9	1	1.06	0.01^**	6.44
DRB1*0403	2	2	9	9.6	0.02^**	0.254
DRB1*0404	1	1	6	6.38	0.05**	0.207
DRB1*0405	7	7	1	1.06	0.03**	5
DRB1*0409	1	1	0	0	0.5	2.84
DRB1*0701	7	7	30	31.9	7x10-6*	0.16
DRB1*1501	6	6	11	11.7	0.125	0.481
DRB1*1502	14	14	11	11.7	0.233	1.22
DRB1*1503	1	1	0	0	0.5	2.84
DRB1*1506	0	0	4	4.25	0.05**	0.012

* Corrected p (pc) value is significant
** Corrected p (pc) value is not significant

Table-2 shows the homozygosity and heterozygosity of DRB1*0301 and DRB1*04 alleles significantly associated with IDDM. Thirty two percent of the patients studied were homozygous for DRB1*0301 as compared to 2% controls and this difference was highly significant. The number of homozygous DRB1*0301 was also more than that expected under Hardy-Weinberg equilibrium (32 as compared to an expected number of 29.16). Heterozygous DRB1*0301/ *0405 and DRB1*0301/ *0401 were significantly increased in the patients as compared to controls who lacked this heterozygous combination.

Table-2: Homozygosity or heterozygosity of DRB1*0301 and DRB1*04 alleles in IDDM patients and
controls

Sl. No.	DRB1 Genotype	DIABETES N=100 No.	%	CONTROLS N=94 No.	%	p value	RR
1.	0301, 0301	32	32	2	2.12	10^-8*	17.55
2.	0301, 0405	6	6	0	0	0.01**	13
3.	0301, 0401	6	6	0	0	0.01**	13
4.	0301, 0409	1	1	0	0	0.52	2.84
5.	S.N. 1+2+3	44	44	2	2.12	< 10^-8*	29.14
6.	S.N. 2+3	12	12	0	0	2x10^-4*	26.29
7.	0301, X1	31	31	19	20.21	0.05**	1.77
8.	0403, X	2	2	8	8.5	0.04**	0.219
9.	0401, X	3	3	1	1.06	0.33	2.23
10.	0404, X	1	1	5	5.32	0.09	0.25
11.	0405, X	1	1	1	1.06	0.74	0.94
12.	0403,0404	0	0	1	1.06	0.48	0.31
13.	0301, 0701	5	5	4	4.25	0.53	1.16
14.	0701, X	2	2	26	27.65	10^-7*	0.066
15.	Non DR3,42	17	17	58	61.7	10^-8*	0.127

1. X means any allele for DRB1 other than DRB1*0301 or DRB1*04, also includes DRB1*0701. 2. Non DR3, 4 samples include DRB1*0701. * Corrected p (pc) value is significant. ** Corrected p (pc) value is not significant.

Table-3 shows the phenotypes and genotypes of Insulin VNTR in 110 IDDM patients and 95 healthy controls. While the frequency of class-I VNTR was increased significantly in the patients, class-III VNTR was increased significantly in the controls. However, when the genotypes were studied, class I homozygosity was considerably increased in the patients as compared to controls (p < 2X10-8), giving a relative risk of 7.8. Class I, III heterozygosity was significantly increased in the controls (p < 10-5). Although Class III homozygosity was observed only in 10.52% of the controls, it was significantly increased in the controls as compared to the patients.

Table-3: Insulin linked variable number of Tandem Repeats in IDDM patients and controls

INS-VNTR Alleles	DIABETES N=110 No.	%	CONTROLS N=95 No.	%	p value	RR
Class I	108	98.2	85	89.47	0.008*	6.35
Class III	64	58.2	87	91.57	2 x 10^-8*	0.13
Genotypes
Class I, I	46	41.8	8	8.42	2x10^-8*	7.8
Class I, III	62	56.4	77	81.05	10-5*	0.301
Class III, III	2	1.8	10	10.52	0.008*	0.157

Ninety five of IDDM patients and 72 of the healthy controls were typed for both the HLA-DRB1 as well as INS-VNTR loci. Table-4 shows simultaneous presence of HLA DRB1*0301 in homozygous or heterozygous state along with homozygosity or heterozygosity of Class I/III INS-VNTR. Homozygous Class-I INS-VNTR along with homozygous or heterozygous DRB1*0301 were significantly increased in the IDDM patients (p < 10-8) with a Relative Risk of 70.81 (Serial No. 7 in Table-4). In fact, none of the controls had homozygous Class-I INS-VNTR along with DRB1*0301 in homozygous or heterozygous state. Since DRB1*0301 homozygosity is significantly increased in the patients, homozygous DRB1*0301 and heterozygous DRB1*0301 only with DRB1*0401 and DRB1*0405 along with heterozygous INS-VNTR may also be considered as predisposing since it gives a relative risk of 10.55. And if we add all these predisposing combinations i.e. serial no.12 in Table-4, 50.53% of the patients as compared to only 1.4% of the controls had these combinations giving a relative risk of 48.67. Thus the two independently assorting alleles at two different chromosomes increase the risk of developing IDDM.

Thus, the results in this study show that: (1) homozygous or heterozygous DRB1*0301 along with homozygous Class-I INS-VNTR and (2) homozygous DRB*0301 and heterozygous DRB1*0301 only with DRB1*0401 or DRB1*0405 with heterozygous Class-I/III INS-VNTR may be used to predict a prediabetic before the onset of the disease in North Indian high risk group. However, typing a larger cohort may be required to confirm such a major increase in risk.

Table-4: Simultaneous presence of DRB1*0301 and DRB1*04 alleles and INS-VNTR alleles in IDDM patients and controls

Sl. No.	DRB1 Genotype	INS-VNTR Genotype	DIABETES N=95 No.	%	CONTROLS N=72 No.	%	p	Relative Risk
1.	0301, 0301	I, I	14	14.73	0	0	0.0002*	25.8
2.	0301, X1	I, I	13	13.68	0	0	0.0004*	23.72
3.	0301, 0401	I, I	3	3.15	0	0	0.181	5.49
4.	0301, 0405	I, I	1	1.05	0	0	0.572	2.3
5.	S.N. 1+2	27	28.42	0	0	0	3x10^-8*	58.21
6.	S.N. 1+2+3	30	31.58	0	0	0	<10^-8*	67.51
7.	S.N. 1+2+3+4	31	32.63	0	0	0	<10^-8*	70.81
8.	0301, 0301	I, III	10	10.52	1	1.4	0.01**	5.85
9.	0301, 0405	I, III	4	4.21	0	0	0.1	7.13
10.	0301, 0401	I, III	3	3.15	0	0	0.181	5.5
11.	8+9+10	17	17.89	1	1.4	0	3x10^-4*	10.55
12.	S.N. 7+11	48	50.53	1	1.4	0	<10^-8*	48.67
13.	0301, X	I, III	22	23.15	16	22.22	0.504	1.12
14.	0301, X	III, III	0	0	3	4.2	0.07	0.1
15.	0301, 0409	I, III	1	1.05	0	0	0.57	2.3
16.	0405, X	I, I	1	1.05	1	1.4	0.678	0.76
17.	0401, X	I, I	1	1.05	0	0	0.57	2.3
18.	0403, 0404	I, I	0	0	1	1.4	0.43	0.25
19.	0401, X	I, III	2	2.1	0	0	0.32	3.87
20.	0403, X	I, III	0	0	4	5.6	0.03**	0.08
21.	0403, X	III, III	1	1.05	0	0	0.57	2.3
22.	0403, 0403	I, III	0	0	1	1.4	0.43	0.25
23.	0404, X	III, III	0	0	1	1.4	0.43	0.25
24.	0403, X	I, I	0	0	1	1.4	0.43	0.25
25.	0404, X	I, I	0	0	1	1.4	0.43	0.25
26.	0301, 0301	III, III	1	1.05	1	1.4	0.68	.76
27.	0701, X	I, I	1	1.05	1	1.4	0.677	0.76
28.	0701, X	I, III	0	0	15	20.83	10-7*	0.019
29	Non DR3,42	I, I	9	9.47	3	4.2	0.155	2.18
30.	Non DR3,41	I, III	8	8.42	35	48.6	10-8*	0.097
31.	Non DR3,4	III, III	0	0	3	4.2	0.07	0.1

1 and 2 same as in Table-2.
* Corrected p (pc) value is significant
** Corrected p (pc) value is not significant

1. Goswami R, Brown M, Kochupillai N, Gupta N, Rani R, Kifor O and Chattopadhay N (2003) Prevalence of calcium sensing receptor autoantibodies in patients with sporadic idiopathic hypoparathyroidism. Eur J Endocrinol 150:9-18.

2. Rani R, Sood A and Goswami R (2004) Molecular basis of predisposition to develop type 1 diabetes mellitus in North Indians. Tissue Antigens (in press).

3. Louie LG, Silver EW, Direskeneli GS, Schmelzer K, Kearney FC, Crowley J, Spiroski MZh, Dzhekova P, Peste-Tsilimidou C, Spyropoulou-Vlachou MS, Stavropoulos-Giokas CG, Yilmaz V, Lazidou P, Polyminidis Z, Slavcev A, Costeas PA, Koumouli A, Goldberg AC, Ioschpe R, Uboldi de Capei M, Dametto E, Tang J, Kaslow RA, Mehra NK, Kaur G, Martinetti M, Chae GT, Balladares S, Camacho A, Gorodezky C, Lin M, Hansen J, Schultz KA, Amirzargar AA, Rani R, Piancatelli D, Adorno D, Pratsidou-Gertsi P, Tsakalidis D, Petrek M, Tseng LH, Nelson MP, Thomson G and Mytilineos J (2004) Worldwide variation in cytokine genes. Tissue Antigens (in press).

1. Rani R, Tiwari D and Goswami R (2004) Cytokine gene polymorphism in IDDM patients form North India: Report from the 13IHWG Cytokine Disease Component. In: HLA 2002, Immunobiology of the human MHC (Eds: John A Hansen & Bo Dupont), IHWG Press (in press).