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Endocrine Genetics (P.V., H.O.-B., M.P., R.G., M.R., C.P.) and Molecular Endocrinology (C.G.G.) Laboratories, McGill University-Montreal Children’s Hospital Research Institute; and Department of Pediatrics, Division of Endocrinology (P.V., H.O.-B., M.P., R.G., M.R., C.G.G., C.P.), McGill University, Montreal, Quebec, Canada H3H 1P3
Address all correspondence and requests for reprints to: Dr. Constantin Polychronakos, Endocrine Genetics Laboratory, McGill University, Montreal Children’s Hospital Research Institute, 2300 Tupper Street, Room E-316, Montreal, Quebec, Canada, H3H 1P3. E-mail cpolyc{at}po-box.mcgill.ca
Abstract
Type 1 diabetes results from autoimmune destruction of the insulin-producing pancreatic ß cells. The insulin gene (INS) is also expressed in human thymus, an ectopic expression site likely involved in immune tolerance. The IDDM2 diabetes susceptibility locus maps to a minisatellite composed of a variable number of tandem repeats situated 0.5 kb upstream of INS. Chromosomes carrying the protective long INS variable number of tandem repeats alleles (class III) produce higher levels of thymic INS mRNA than those with the predisposing, short class I alleles. However, complete silencing of thymic INS transcripts from the class III chromosome was found in a small proportion of heterozygous human thymus samples. We hypothesized that the specific class III alleles found on these chromosomes silence rather than enhance thymic insulin expression. To test the prediction that these alleles are predisposing, we developed a DNA fingerprinting method for detecting two putative "silencing" alleles found in two thymus samples (S1, S2). In a set of 287 diabetic children and their parents we found 13 alleles matching the fingerprint of the S1 or S2 alleles. Of 18 possible transmissions, 12 of the S1–S2 alleles were transmitted to the diabetic offspring, a frequency of 0.67, significantly higher than the 0.38 seen in the remaining 142 class III alleles; P = 0.025. This confirms our prediction and represents an additional level of correlation between thymic insulin and diabetes susceptibility, which supports a thymic enhancer effect of the INS variable number of tandem repeats as the mechanism of IDDM2 and refines the contribution of IDDM2 genotyping to diabetes risk assessment.
INSULIN HAS AN important place among the antigens involved in the autoimmune process that results in type 1 diabetes. Of the known autoantigens, insulin has the highest expression specificity for pancreatic ß cells, and it is the only antigen whose gene has been mapped to a genetic susceptibility locus. Anti-insulin antibodies are most often the first to appear in prediabetic individuals (1), and, in rodent models, autoimmune diabetes can be prevented by peripheral tolerance induction to insulin (2, 3).
Central T-cell tolerance is determined in the thymus, where cells with autoreactive T-cell receptor rearrangements are deleted. The discovery that the thymus expresses small amounts of insulin (and other proteins with tissue-restricted expression) has generated interest in the role of central mechanisms in immune tolerance to these autoantigens (reviewed in Ref. 4). Direct evidence for this comes from studies in which thymus grafts can transfer tolerance to allo- (5) or xeno- (6) antigens transgenically expressed under the insulin promoter to nontransgenic syngeneic recipients. Indirect evidence for the importance of this mechanism in the pathogenesis of type 1 diabetes comes from studies of the IDDM2 locus of type 1 diabetes susceptibility.
IDDM2 maps 0.5 kb upstream of the insulin gene
(INS), to a repeat polymorphism that consists of a variable
number of tandem repeats (VNTR) of the consensus sequence
ACAGGGGTGTGGGG (7, 8). The number of repeats ranges from
30 to more than 150 in Caucasian chromosomes, and this, combined with
slight sequence variations in the repeat unit (Fig. 3
), results in a
complex, hypervariable allele system. About 80% of Caucasoid alleles
are in the range of 30–44 repeats (class I), and virtually all of the
rest are longer than 110 repeats (class III). Intermediate lengths
(class II) are rare.
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The cause of this monoallelic silencing is, at present, not known but it may be due to genomic imprinting, the property of certain genes to be expressed only from the copy inherited from a parent of a given sex. Human INS and mouse Ins2 are located in syntenic domains that contain several genes that are imprinted in both species. Ins2 expresses both copies in pancreas, but has exclusive paternal expression in mouse yolk sac (23). We hypothesized that INS may be silenced in thymus by parental imprinting, but only on chromosomes carrying specific alleles within class III (allele-restricted imprinting). Alternatively, some class III alleles could act as thymus-specific silencing elements regardless of parental origin. Either version of this hypothesis predicts that those specific alleles predispose to diabetes through decreased thymic insulin expression, in contrast to class III as a whole, which is protective.
Direct testing of the hypothesis that the silencing is due to the VNTR allele in cis is a major aim of our laboratory, but is not simple. We are accumulating more thymus samples but the alleles are infrequent, and it will require a large number to reach statistical proof. In vitro testing with reporter constructs is hampered by the fact that the effect is thymus specific and insulin-expressing cells in this organ are rare, incompletely characterized (4), and no cell lines exist. While the effort for direct proof is underway, we decided to address the hypothesis through one testable major prediction it makes: if these specific alleles silence insulin in the thymus, they must be predisposing to diabetes.
To pursue this, we first precisely defined the class III alleles associated with INS silencing in two of our thymus samples by size and restriction fingerprinting. We then examined the transmission frequency of class III alleles indistinguishable from these "silencing" alleles (which we call S1 and S2) from heterozygous parents to diabetic offspring. We predicted that S-type alleles would be transmitted to diabetic offspring more often than the rest of class III.
Research Design and Methods
Sample sources and preparation
DNA was extracted from blood samples collected from 167 patients who either attended the Montreal Children’s Hospital diabetes clinic, or participated in the Minneapolis branch of a multicenter study of the natural history of diabetic nephropathy in type 1 diabetes. Samples were obtained with signed, informed consent and approval by the Institutional Review Board of the respective institutions. In addition, we examined 120 DNA samples from 60 diabetic sibling pairs from the Human Biological Data Interchange (HBDI). DNA form both parents was available in all cases. All patients developed insulin-dependent diabetes under the age of 19, except for two HBDI patients who were 29 and 34 at onset but were included because the other sibling in the pair had a young onset. All patients were of mixed European background.
Human fetal thymus tissues were obtained at the time of pregnancy termination with written consent from the mother, approved by the Institutional Review Board of the Maisonneuve-Rosemont Hospital. The tissue was flash-frozen and pulverized under liquid nitrogen to extract DNA and RNA. INS mRNA from class I vs. III chromosomes was quantitatively differentiated by RT-PCR using a transcribed PstI polymorphism in tight linkage disequilibrium with the VNTR, as we described previously (16, 18).
PCR protocol for all classes of INS VNTR alleles
The PCR reaction for amplification of all classes of INS VNTR alleles contained approximately 100–200 ng of DNA, 0.2 mM of each dNTP, 1 µCi P32-dCTP, 1 mM MgCl2, ammonium PCR reaction buffer, 0.4 U ID-Zyme thermostable polymerase, and 100 ng sense and antisense primer. ID-Zyme contains a proprietary mixture of high-fidelity polymerases. We used the primers described by Bennett et al. (9), as well as an additional pair of internal primers that could amplify both genomic and cloned DNA: Sense, 5'-GGCATCTTGGCATCCGGGACTG-3'; Antisense, 5'-GCAGGGCGGGGCTCTTTGCGCTG-3'.
The PCR was carried out for 25 cycles of: 94 C/30 sec denaturing, 62
C/30 sec annealing, and 70 C/3 min 30 sec with a 4-sec extension per
cycle extension step. Products, internally labeled with
32P-dCTP, were visualized by 8% PAGE and
autoradiography. To distinguish small differences in size, PCR product
from the S1 and S2 alleles were loaded in every two to four lanes,
interspersed throughout the unknown samples (Fig. 2). All class III
alleles analyzed were either larger than both S1 and S2, smaller than
S1 and S2 or equal in size to either S1 or S2. Thus, the S1 allele is
only slightly larger than the S2 allele, and there are no allele sizes
between them that can be resolved by our method. Furthermore, each
class III allele was loaded into two separate wells of the
polyacrylamide gel for comparison to S1 and S2 separately. Those that
were equal in size to S1 were always larger than the S2 allele, and
those that were equal in size to S2 were always smaller than S1 allele.
This indicates that the same alleles behave in a consistent manner
between separate loadings in PAGE and that our method can consistently
identify small differences in migration distance.
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S1 and several non-S alleles were directionally cloned by double digestion of the PCR product with NcoI and PstI. Cloned and genomic DNA template gave identical results and were used interchangeably for PCR amplification. The identity and integrity of the cloned S1 allele was repeatedly demonstrated by showing electrophoretic co-migration of PCR amplification products (n > 15). Similarly, both cloned and genomic DNA demonstrated the same restriction fragment length polymorphism band pattern after MspI digestion (MspI recognizes an uncommon variant of the repeat unit; Refs. 8 and 12), suggesting the same arrangement of internal repeat sequences.
The highly repetitive nature of the VNTR precludes the use of restriction subcloning or internal primers for sequencing. Therefore, the S1 allele and a class III allele associated with enhanced thymic insulin expression (E1), were sequenced by generating a series of overlapping unidirectional deletions from a NcoI-PstI fragment subcloned into the pGEM-T vector using the Exo-Size deletion kit, (New England Biolabs, Inc., Beverly, MA). Exonuclease III digestion of the construct linearized by double digestion with SphI and NcoI, both on the 5' end of the insert, shortens the insert but not the vector, which is protected by the 3' overhang left by SphI.
DNA fingerprinting of class III alleles
Alleles of the same size could be further distinguished using two highly polymorphic MspI fragments (700–800 bp and 400–500 bp) that, in class I/III individuals, are always located above the largest class I allele digestion fragment. This was determined by comparing the MspI digestion fragments in a number of class I/III individuals obtained from PCR product where only the class I allele is amplified (using the previously described PCR conditions) (9, 18) and product from our PCR protocol which amplifies both class I and III alleles.
Results
PCR amplification of all INS VNTR classes
Our protocol was successful in amplifying all INS-VNTR classes
(Fig. 1), an important technical advance
in the study of the IDDM2 susceptibility locus. Due to the
high GC-rich content and repetitive nature of this sequence, resulting
in a highly stable intramolecularly folded structure
(24, 25, 26), there have been, to date, no published reports
of successful PCR amplification of the long class III alleles.
Codominant segregation of class III alleles within families confirmed
the high fidelity of the method and the stability of these alleles
within families, as illustrated in Fig. 4.
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First, in DNA from the two thymus samples that showed monoallelic expression, we sequenced the nonrepetitive part of the INS promoter/enhancer region from just downstream of the VNTR to the first INS exon. No unknown sequence variant was found that could account for the silencing of one allele in this region. However, the possibility remains that S1 or S2 may be merely markers for a polymorphic silencing element outside the promoter region we sequenced. We then proceeded to define the VNTR in these two chromosomes where INS was apparently silenced.
Class III alleles ranged in size from 1.8–2.5 kb. The S1 and S2 silencing alleles were in the middle of the range, at around 150 repeats (2.1 kb), and probably differing from each other by a single repeat. S1 was distinguishably longer than S2, without observable alleles between the two.
Class III alleles identical in size but different in terms of
composition in variants of the repeat unit were further distinguished
by restriction fingerprinting (Fig. 2
).
The PCR product was digested with MspI, whose recognition
site (5'-CCGG-3') is present only in certain of the less common
variants. Alleles of identical size often had fingerprints that could
be distinguished using the two bands that could always be seen above
the class I allele bands. Most MspI digests of class III
alleles had two major bands clustered around 700–800 and 400–500 bp,
plus a number of smaller or less common bands. The exception were class
III alleles that were part of the unusual haplotype termed "very
protective haplotype" by Bennett et al. (9),
which were richer in MspI sites and gave no band over 350
bp, confirming their derivation from a different ancestral chromosome.
These are the same alleles recently termed class III.b by Stead and
Jeffreys (27), whose rich content in
MspI-containing repeats explains the absence of visible long
bands in the restriction digest.
Transmission analysis of "silencing" alleles in type 1 diabetes
Using both size and MspI fingerprint, we
determined that 7 of the 908 parental chromosomes in families
with at least one type 1 diabetes-affected child carried alleles
indistinguishable from S1 and 6 from S2. Among 31 thymus
samples showing enhanced expression from the class III chromosome (Ref.
18 and an additional 21 informative fetal thymus samples
tested subsequently), 30 were distinct from S1–S2 by size alone,
whereas only 1 allele associated with enhancing (termed E1) had
identical size and fingerprint to S1. The two alleles were sequenced
and found to differ in 4 of 150 repeats (Fig. 3), none of which affected
the MspI restriction pattern. In any event, alleles
identical to S1 or S2 by both size and fingerprint but which are not
associated with INS silencing are too infrequent (1 of 33)
to interfere with a statistical testing of the hypothesis that S1 and
S2 predispose to diabetes.
We proceeded to test our hypothesis by genotyping the diabetic children of all parents who had a class III allele. There were no S/S homozygous genotypes, therefore, all parents who had a "silencing" (S) allele were heterozygous for one S and one "nonsilencing" INS VNTR allele (either class I or nonS class III). Alleles present in the child were counted as transmitted, and those absent as nontransmitted. In families with more than one diabetic child, each opportunity for transmission was counted separately in this way.
The results of the transmission analysis are summarized in
Table 1. Non-S class III alleles
were transmitted at a frequency significantly less than 0.5, as
expected from the known dominant protective effect of class III as a
whole. As our hypothesis had predicted, S alleles behaved as
predisposing rather than protective: they were much more frequently
transmitted than all other class III alleles (P = 0.025
by Fisher’s exact test). Because in most cases the transmitting
parent’s other allele was a class I, S-type alleles seem to behave
as more predisposing than even class I alleles, as our hypothesis would
predict based on thymic insulin expression levels. Due to the small
sample size, we were not able to evaluate over-transmission of S-type
alleles in terms of parental origin, and the question of imprinting
vs. parent-of-origin independent silencing remains open.
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Our previous findings have led to the hypothesis that the expression of insulin (or antigenic epitopes thereof) in the thymus is important in insulin-specific T-cell tolerance, and that the effect of the IDDM2 locus is due to allele dependence of the levels of such expression: higher expression would better induce immune tolerance to INS-encoded antigens, resulting in a decreased probability of an autoimmune response against the pancreatic ß cells. The dose-responsiveness of thymic T-cell precursor fate is supported by studies in vitro that have demonstrated positive selection of thymocytes at low concentrations of a selecting peptide in fetal thymus organ cultures, while increasing concentrations of the selecting peptide (20, 21) or peptide-major histocompatibility complexes (22) resulted in decreased positive selection and induction of negative selection.
A direct corollary of this hypothesis is that the rare individuals who silence INS on the class III-bearing chromosome in the thymus would be predisposed to diabetes. If this silencing depends on the cis-effect of specific alleles within class III, either through the direct transcriptional effect of S1 or S2, or because these alleles are markers for a sequence variant outside the regions sequenced, those alleles will be predisposing. Alternatively, silencing could simply require the presence of any class III allele, the difference between silencing and enhancing being determined by genetic influences in trans or environmental factors. Our data clearly support the model of a specific genetic effect in cis by confirming its one key prediction. This finding justifies the major investment in effort needed to directly prove the effect of these alleles in thymic insulin-producing cells.
Our characterization of two of these INS silencing-associated class III alleles by PCR amplification and restriction fingerprinting was an important technical advance that allows distinguishing a large number of different alleles within class III. Recently, the diversity of INS-VNTR alleles was demonstrated with minisatellite variant repeat-PCR by Stead and Jeffreys (27). This method defines the position of the six most common repeat variants within a given allele and has demonstrated the presence of 89 distinct patterns in 171 class-III bearing chromosomes. In light of these data, it is unlikely that all alleles identified by our restriction fingerprinting as either S1 or S2 represent the exact same DNA sequence. It seems more likely that these patterns are markers for some common structural feature that confers the functional property of INS silencing in the thymus. To explain the observed phenomenon, this structure-function relationship must be subtle, as illustrated by a comparison of the two alleles we sequenced, E1 and S1. In the thymus samples from which they were obtained, these alleles are associated with, respectively, enhanced or silenced INS expression, yet they only differ in 4 of the 150 repeat units.
A meaningful discussion of how these subtle differences could affect function would require understanding of the mechanism by which individual INS VNTR alleles could exercise transcriptional effects on INS. Such understanding is currently sketchy. The INS VNTR has a complex secondary structure (24, 25, 26) that may interact with transcriptional complexes and whose precise nature may be influenced by relatively subtle changes in size and the exact repeat composition.
Whatever the mechanism of polymorphic INS silencing is, it may involve parental imprinting. Ins2 is imprinted in mouse yolk sack (23) but in both mouse and human pancreas, INS has demonstrated biallelic expression (9, 13, 16, 23). Polymorphic parental imprinting of INS as a cause of monoallelic silencing in the thymus could not be evaluated as parental DNA was not available in any of the five monoallelic samples. If imprinting is the mechanism involved in silencing of thymic INS expression, then it likely requires the presence of specific class III alleles in cis (allele-restricted imprinting). Therefore, silencing-type alleles may only be predisposing when transmitted from a parent of a specific sex. This is a focus of ongoing work in our laboratory.
Functional heterogeneity among alleles within class III has three important implications: 1) the additional concordance between diabetes susceptibility and thymic INS expression lends further support to the latter as the biological mechanism involved in IDDM2; 2) it supports the INS VNTR as the disease locus in IDDM2, a concept not yet proven beyond all doubt (28); and 3) it enhances the contribution of IDDM2 to the predictive power of DNA testing for diabetes risk.
Acknowledgments
Many thanks to Tracey Hollingdrake, Danielle Drouin, Tammi Dalzel, M. Mauer, and Jacqueline Dufresne for patient recruitment and sample collection. We also thank George Eisenbarth and Sunanda Babu for making the HBDI samples available to us.
Footnotes
This work was supported by the Juvenile Diabetes Foundation International and the Quebec Diabetes Association (QDA). P.V. was supported by a Doctoral Research Award from the Medical Research Council of Canada and by the QDA.
Abbreviations: E1, Enhancing-type allele 1; HBDI, Human Biological Data Interchange; INS, human insulin gene; Ins2, mouse insulin gene; S1/S2, silencing-type allele 1 or 2; VNTR, variable number of tandem repeats.
Received November 17, 2000.
Accepted April 17, 2001.
References
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  B. Faideau, J.-P. Briand, C. Lotton, I. Tardivel, P. Halbout, J. Jami, J. F. Elliott, P. Krief, S. Muller, C. Boitard, et al. Expression of Preproinsulin-2 Gene Shapes the Immune Response to Preproinsulin in Normal Mice J. Immunol., January 1, 2004; 172(1): 25 - 33. [Abstract] [Full Text] [PDF]
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