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Mechanisms of genetic susceptibility to type I diabetes: beyond HLA

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Abstract

An individual’s predisposition to Type I diabetes (T1D) is largely determined by complex interactions between several genetic loci and other, nonheritable factors. In T1D, the HLA locus has been known for decades to contribute 50% of the inherited risk. Outside the HLA are many proposed candidate loci with smaller effects, but only two confirmed candidate genes, the INS-VNTR and the CTLA-4 genes, which together do not contribute more than 15% of the risk. Because of the high frequency of the disease-associated DNA variants of these genes, understanding the biological mechanisms of such DNA variation in the context of T1D can have tremendous impact on the development of preventive therapeutics. However, establishing a causal relationship between common DNA variations and disease-predisposing functional effects is not trivial and remains difficult, as the effects are expected to be subtle. The variable-number tandem-repeat (VNTR) region upstream of the insulin gene is known to mediate expression in the thymus and pancreas, whereas various polymorphisms in the 5′ and 3′ regulatory regions of CTLA-4 are thought to alter gene expression and a coding A49G polymorphism exerts effects on post-translational processing. This review details the latest efforts in elucidating the functional mechanisms that explain the genetic association of the INS-VNTR and CTLA-4 genes with T1D.

Introduction

Type I diabetes (T1D) is characterized by the autoimmune destruction of β-cells in the pancreas, leading to complete insulin deficiency. There is a large body of evidence suggesting that an individual’s predisposition to developing the chronic disorder is determined largely by one’s genetic makeup. The Human Leukocyte Antigen (HLA) region is the major locus, conferring up to 40–50% of the susceptibility and has been known for over two decades. Of the large number of loci found to be linked by genome-wide scans, only two have been confirmed by the transmission disequilibrium test (TDT) and narrowed down to a single gene. These are the insulin-linked variable number of tandem repeats (INS-VNTR) region and the Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) gene (corresponding, respectively, to the IDDM2 locus and to what had been previously reported as IDDM7, IDDM12, and IDDM13). Together, these two genes confer an inheritable disease risk no larger than 15%. Establishing a causal relationship between common DNA polymorphisms and disease-predisposing functional effect remains a challenge. This review will focus on what is currently known about how DNA variation at the two T1D-associated genes outside the HLA affect human biology to modulate T1D risk.

Section snippets

The INS-VNTR and type I diabetes

Evidence for the involvement of the region near the Insulin gene on Chr11p15 with T1D has long been present [1]. The association of this region with T1D can be attributed with a large degree of probability to the variable number of tandem repeats (VNTR) located in the 5′ upstream region of the insulin gene [2], [3]. The 4.1 kb region encompassing the insulin gene and its flanking regions also contains several other polymorphisms in linkage disequilibrium (LD); all of which are located outside

INS-VNTR and function

The INS-VNTR was postulated early on to exert its effect through transcriptional regulation (in cis) since it does not encode for protein and is directly upstream of the insulin gene promoter (Fig. 1). Due to the location of the paternally expressed [12] Insulin-like Growth factor 2 (IGF2) gene just downstream of the insulin gene, a possible regulatory effect of the VNTR on IGF2 was an alternative possibility.

Initially, insulin gene expression as well as IGF2 gene expression were studied in

Insulin VNTR and thymic insulin expression

A more attractive mechanism for the VNTR effect was identified when ours and other groups looked at insulin expression in the thymus and correlated expression with INS-VNTR. The thymus plays a critical role in determining central tolerance by deleting autoreactive T-cell receptor rearrangements. Of the many putative autoantigens involved in the diabetes disease process, insulin has the highest specificity for the β-cells of the pancreas. In the NOD mouse the many T-cell clones isolated from

Silencing alleles

Although in most thymi examined by our group [24] and by Pugliese et al. [23], insulin expression was expressed by both alleles (in class I/III heterozygotes), 5 thymi out of 22 heterozygous individuals expressed only one allele [23], [24]. All silenced alleles were expressed in cis to a class III VNTR allele. By using a PCR-based restriction fingerprinting analysis that allowed us to distinguish between individual alleles within class III, we were able to demonstrate that those rare alleles

CTLA-4

The 2q31–35 interval in humans, syntenic with the NOD mouse type I diabetes locus Idd5 on chromosome 1, contains at least three type I Diabetes loci (IDDM7, IDDM12, and IDDM13) [33], [34], [35]. Within this 23cM interval, the 2q33 region (previously designated IDDM12,) is linked and associated to autoimmune disease. This region encompasses several potential candidate genes namely, CD28, CTLA-4, and ICOS (Fig. 2) all of which are important players in immune regulation and function and could be

CTLA-4s role in immunity and autoimmunity

CTLA-4s function as a negative regulator of T-cell function makes its association with type I diabetes highly plausible. There are two known isoforms of CTLA-4 in humans: the full-length CTLA-4 is a transmembrane glycoprotein expressed transiently on the surface of activated CD4 and CD8 T-cell populations, although it has also been detected on B-cells and is the main isoform found in adult thymocytes. The soluble CTLA-4 isoform is generated by alternative splicing of the transmembrane domain

The signal peptide polymorphism, an A49G SNP (threonine to alanine substitution) mediates differential expression of cell-surface CTLA-4

The G49 allele of the signal peptide has been consistently overtransmitted from heterozygous parents to affected offspring by the transmission disequilibrium test (TDT) in multiple ethnic groups [34], [52].

Functional evidence in support of this association was initially reported in two studies which showed increased proliferation of T-cells in individuals homozygous for the predisposing G49 allele at the signal peptide coding SNP [68], [69]. In addition, they also showed that this allele

The CTLA-4 promoter

In a case-control study by Ligers et al., CTLA-4 cell-surface levels were quantified by flow cytometry in multiple sclerosis patients vs. matched-controls. When the data were analyzed by promoter and signal peptide (C-318T;A49G) haplotype, significantly higher surface expression in both patients and controls for the T/C;A/A haplotype was found and not for any other haplotype [71]. They also compared lymphocyte expression levels from 44 heterozygous individuals at the promoter (T/C) vs. 186

The CTLA-4 3′UTR

Another obvious candidate for functional effect is the (AT)n repeat at the 3′UTR, a region known to be involved in RNA stability. Regulation of mRNA decay is an important mechanism by which the level of gene expression is controlled. AU-rich elements (AREs) commonly associated with mRNA degradation process, particularly the AUUUA pentamer and the nonamer UUAUUUA(U/A)(U/A), are not present in the CTLA-4 3′UTR. Since these are not always required for a functional ARE, as is the case of the zif278

The alternatively spliced soluble CTLA-4

There has been much excitement recently over a study in which a newly identified SNP, A6230G, in the 3′ flanking region of CTLA-4 was found to be highly associated with Graves’ disease [55]. In type I diabetes the predominance of this SNP was weaker. In three individuals heterozygous for the A49G SNP the authors report higher soluble CTLA-4 mRNA from the disease protective haplotype (A49;A6230) than from the predisposing haplotype (G40;G6230) in unstimulated CD4 T-cells [55]. The observation

Conclusion

This short overview of the functional evaluation of two type I diabetes susceptibility loci known outside the HLA locus (out of a yet unknown number) illustrates the complexity of genetic predisposition to type I diabetes and its functional underpinnings. Much work remains to be done for the discovery the remaining loci and the elucidation of the mechanism by which they alter biology to predispose to disease. It is hoped that such knowledge will permit the rational development of preventive

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      Citation Excerpt :

      The development of immune-based diabetes (henceforth referred to here as T1A) involves a degree of susceptibility. This susceptibility is mostly inherited, predominantly in the HLA genotypes DR and DQ, and to a lesser extent in a host of other genetic loci [29]. Recent genetic mapping studies and gene-phenotype studies shed light on the possible origins of T1A by indicating potential disease-relevant biological pathways [30].

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