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Sequence Analysis of the Diabetes-Protective Human Leukocyte Antigen-DQB1¹0602 Allele in Unaffected, Islet Cell Antibody-Positive First Degree Relatives and in Rare Patients with Type 1 Diabetes^,1

Diabetes Research Institute (A.P., M.Z., C.R.) and the Department of Neurology (C.T.M.), University of Miami School of Medicine, Miami, Florida 33136; the First Department of Internal Medicine, Nagasaki University School of Medicine (E.K.), Nagasaki, Japan; the Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center (E.K., L.Y., S.B., G.S.E.), Denver, Colorado 80262; the Section of Endocrinology, Department of Internal Medicine, Yale School of Medicine (M.S.), New Haven, Connecticut 06510; the Division of Immunogenetics, Children’s Hospital of Pittsburgh, University of Pittsburgh (M.P., R.P.F., M.T.), Pittsburgh, Pennsylvania 15213; the Human Genetics Department, Roche Molecular Systems (M.A., J.A.N.), Alameda, California 94501; and the Children’s Hospital Oakland Research Institute (J.A.N., H.A.E.), Oakland, California 94609

Address all correspondence and requests for reprints to: Alberto Pugliese, M.D., Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Avenue, Miami, Florida 33136. E-mail: apuglies{at}mednet.med.miami.edu

	Abstract

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The human leukocyte antigen (HLA)-DQA1*0102/DQB1*0602/DRB1*1501 (DR2) haplotype confers strong protection from type 1 diabetes. Growing evidence suggests that such protection may be mostly encoded by the DQB1*0602 allele, and we reported that even first degree relatives with islet cell antibodies (ICA) have an extremely low diabetes risk if they carry DQB1*0602. Recently, novel variants of the DQB1*0602 and *0603 alleles were reported in four patients with type 1 diabetes originally typed as DQB1*0602 with conventional techniques. One inference from this observation is that DQB1*0602 may confer absolute protection and may never occur in type 1 diabetes. By this hypothesis, all patients typed as DQB1*0602 positive with conventional techniques should carry one of the above diabetes-permissive variants instead of the protective DQB1*0602. Such variants could also occur in ICA/DQB1*0602-positive relatives, with the implication that their diabetes risk could be significantly higher than previously estimated. We therefore sequenced the DQB1*0602 and DQA1*0102 alleles in all ICA/DQB1*0602-positive relatives (n = 8) previously described and in six rare patients with type 1 diabetes and DQB1*0602. We found that all relatives and patients carry the known DQB1*0602 and DQA1*0102 sequences, and none of them has the mtDNA A3243G mutation associated with late-onset diabetes in ICA-positive individuals. These findings suggest that diabetes-permissive DQB1*0602/3 variants may be very rare. Thus, although the protective effect associated with DQB1*0602 is extremely powerful, it is not absolute. Nonetheless, the development of diabetes in individuals with DQB1*0602 remains extremely unlikely, even in the presence of ICA, as confirmed by our further evaluation of ICA/DQB1*0602-positive relatives, none of whom has yet developed diabetes.

	Introduction

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TYPE 1 diabetes is an autoimmune disease resulting in pancreatic ß-cell destruction and absolute insulin deficiency (1, 2). The disease mostly develops in genetically susceptible individuals, and at least 50% of the genetic risk of developing diabetes is conferred by the IDDM1 susceptibility locus, mapped to specific alleles of the human leukocyte antigen (HLA)-DQ and DR loci (3, 4). These highly polymorphic loci encode the HLA-DQ and -DR class II antigens, heterodimers known to play a key role in antigen presentation and immune recognition (5). In Caucasians, the HLA-DQ molecules encoded by the DQA1¹0301/DQB1¹0302 and DQA1¹0501/DQB1¹0201 alleles on DR4 and DR3 haplotypes have the strongest association with the disease (6, 7, 8, 9), although DRB1 alleles significantly modulate DR4-associated susceptibility (9, 10, 11, 12, 13, 14, 15).

In contrast, HLA-DR2 haplotypes are rarely observed among patients with type 1 diabetes. Most of the HLA-DR2-associated type 1 diabetes in Caucasians is accounted for by three neutral haplotypes: DQA1¹0102/DQB1¹0502/DRB1¹1601, DQA1¹0103/DQB1¹0601/DRB1¹1501, and DQA1¹0103/DQB1¹0601/DRB1¹1502 (6, 16, 17, 18, 19, 20). The DQA1¹0102/DQB1¹0602/DRB1¹1501 haplotype, the most common DR2 haplotype among Caucasians, is the only DR2 haplotype conferring dominant and almost absolute protection from type 1 diabetes among Caucasian and other racial groups. Indeed, patients carrying this haplotype are extremely rare (4, 6, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). The characterization of both common and rare recombinant DR2 haplotypes observed in patients with type 1 diabetes also indicates that DQB1¹0602 is the only class II allele exclusively found on diabetes-protective DR2 haplotypes. This suggests that protection is mostly, although not exclusively, conferred by DQB1¹0602 (which together with DQA1¹0102 codes for a protective DQ heterodimer) (18, 31, 32). Moreover, our previous studies indicate that DQB1¹0602 confers strong diabetes protection even among islet cell antibodies (ICA)-positive first degree relatives of patients with type 1 diabetes (33). In fact, approximately 7% of ICA-positive relatives identified through autoantibody screening carry DQB1¹0602, and in our family study, none of such relatives has developed diabetes on follow-up. Because of the apparent dominance of the protective effect (19, 20) and the extremely low risk ascertained in prospective studies (33, 34), ICA/DQB1¹0602-positive first degree relatives are presently excluded from receiving treatment in the Diabetes Prevention Trial–Type 1, a major ongoing trial involving several centers in the United States (35).

Of interest, a few novel variants of the DQB1¹0602 and DQB1¹0603 alleles were recently reported by Hoover et al. (36) in four rare patients originally typed as DQB1¹0602-positive with conventional sequence-specific oligonucleotide (SSO) typing techniques (20). Such variants, not distinguished by the initial panel of SSO probes, appear to be permissive for the development of diabetes. This observation suggests the hypothesis that DQB1¹0602 may confer absolute protection and may never occur in patients with type 1 diabetes. If this hypothesis is, in fact, correct, all or most of those rare patients typed as DQB1¹0602 with conventional techniques could carry one of the above diabetes-permissive variants instead of the protective DQB1¹0602. It is also conceivable that such variants may occur in the previously described ICA/DQB1¹0602-positive relatives (33); if so, this could have significant prognostic implications, as their risk of developing diabetes may be higher than previously estimated.

In addition, a mitochondrial DNA mutation (mtDNA A3243G) has been recently associated with the development of late-onset diabetes in ICA-positive individuals and was also reported in patients with various forms of diabetes (37, 38, 39, 40, 41). The presence of such a mutation could be associated with the development of diabetes in ICA/DQB1¹0602-positive individuals.

We therefore investigated the occurrence of diabetes-permissive DQB1¹0602 variants and the mtDNA A3243G mutation in the eight ICA-positive relatives previously identified (33) and in six rare patients with type 1 diabetes, all typed as DQB1¹0602-positive with conventional techniques.

	Subjects and Methods

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Subjects

Unaffected ICA/DQB1¹0602-positive first degree relatives (n = 8) were identified through autoantibody screening of family members of patients with type 1 diabetes at the Joslin Diabetes Center and the Barbara Davis Center for Childhood Diabetes, as previously reported (33). All relatives were Caucasians. Their clinical characteristics and updated follow-up information are shown in Table 1. ICA positivity was defined as measurements of 20 Juvenile Diabetes Foundation units or more on at least two occasions, and the ICA titer shown in Table 1 is the highest level observed for each individual.

Genomic DNA samples were extracted from heparinized blood samples with phenol/chloroform. HLA typing for DQA1, DQB1, and DRB1 alleles was performed on all subjects by the PCR and hybridization with SSO probes as previously described (4, 33). For one patient, DRB1 alleles were determined by direct sequencing. The second exon of the DRB1 gene was amplified using forward primers for the DRB1¹15/16 or DRB1¹04 subtypes paired with a generic DRB1 reverse primer in separate PCR reactions. PCR products were cloned and sequenced with the same strategy described below for DQA1¹0102 and DQB1¹0602 alleles.

Sequencing exon 2 of the DQB1¹0602 and DQA1¹0102 alleles

The second exon of both the DQA1 and DQB1 genes was sequenced, because this exon encodes the extracytoplasmic polymorphic regions of the HLA-DQ molecule. The DQA1 gene was amplified from genomic DNA by PCR with primers 0102ex2-F 5'-CT GAC CAC GTT GCC TCT TGT-3' and 0102ex2-R 5'-ATT GGT AGC AGC GGT AGA GTT-3'. Primers were selected at the 5'- and 3'-termini of the second exon (intron sequences are not currently available) and amplify a 261-bp product (annealing, 58 C). The second exon of the DQB1 gene was amplified with primers 0602ex2-F 5'-TC CCC GCA GAG GAT TTC GTG T-3' and 0602ex2-R 5'-TCC TGC AGG GCG ACG ACG CTC ACC TCT CC-3' (annealing, 60 C). These primers amplify a 302-bp product including 8 and 18 bp of intronic sequences at the 5'- and 3'-ends, respectively, and allow distinction of the polymorphism reported at codon 9 by Hoover et al. and Williams et al. (36, 48). Primers MADQ4 5'-CCT GAC TGA CCG GCC GGT GAT-3' and UG71 5'-ACA TGT AAA ACG ACG GCCAGT TCT CCT CTG CAG GAT CCC GC-3' were also used and allowed discrimination of the codon 9 polymorphism as well (299-bp product; annealing, 55 C). The PCR products were cloned into the TA Cloning Vector (Invitrogen, San Diego, CA). DNA samples extracted from transformed colonies were screened by PCR using sequence-specific primers for the DQB1¹0602 and DQA1¹0102 alleles (PCR-SSP) as described by Olerup et al. (49). DNA samples from DQB1¹0602- and DQA1¹0102-positive colonies were then sequenced (both strands, using the SP6 and T7 primers). PCR products generated with the MADQ4/UG71 primer pair were sequenced directly using MADQ4 as primer and dye terminators FS (ABI). The other strand of each product PCR was also sequenced as a control using the dye primer FS kit (ABI, Foster City, CA) containing the -21M13 primer. Sequence analysis was performed on an ABI 373 automated sequencer.

Typing for the mtDNA A3243G gene mutation

Typing for the A3243G transfer ribonucleic acid^{Leu (UUR)} mutation was performed by PCR-restriction fragment length polymorphism as previously described (50). In brief, approximately 0.5 µg genomic DNA was subjected to PCR amplification (94 C for 60 s, 55 C or 60 s, 72 C for 45 s; 25 cycles) using primers for the mtDNA light strand positions 3116–3134 and heavy strand positions 3353–3333 (51). The DNA fragment produced by the PCR reaction was digested with the restriction enzyme HaeIII for 2 h at 37 C. The AG transition at position 3243 creates a new HaeIII site that is diagnostic for the mutation (50). The digestion products were electrophoresed on a 12% nondenaturing polyacrylamide gel and stained with ethidium bromide. This technique can detect mitochondrial mutations with low degree of heteroplasmy in peripheral blood (as low as 5%, below which any significant influence on the mitochondrial function in more relevant tissues is unlikely).

Autoantibody testing

The eight unaffected ICA/DQB1¹0602-positive relatives were tested for the presence of ICA with a standardized assay as previously described (33, 52). ICA positivity was defined as measurements of 20 Juvenile Diabetes Foundation units or more on at least two occasions. Serum samples from these relatives and the other subjects studied were also tested for the presence of autoantibodies against three major type 1 diabetes autoantigens, such as insulin, GAD65, and the tyrosine phosphatase-like ICA512 (or IA-2). Insulin autoantibodies (IAA) were determined with a fluid phase RIA using 600 µL serum, with duplicate determinations with and without unlabeled insulin for competition (53). The interassay coefficient of variation for the IAA assay is 10.3% at low positive values. The assay had a specificity of 91% and a sensitivity of 49% for new-onset patients less than age 30 yr in the 1995 Immunology of Diabetes Society (IDS) Combinatorial Workshop. Autoantibodies to GAD65 were measured in triplicate by RIA, using in vitro transcribed and translated GAD65 (clone provided by A. Lernmark) (54, 55). Autoantibody-bound GAD65 was precipitated with protein A-Sepharose. The interassay coefficient of variation of this assay is 6.5%. Assay specificity was 99%, and sensitivity was 83.7% for new-onset patients less than age 30 yr in the IDS Combinatorial Workshop. Autoantibodies to ICA512 were measured using the in vitro transcribed and translated labeled product of a clone termed ICA512bdc (amino acids 256–979 of ICA512/IA-2) (55, 56, 57, 58). Autoantibodies were measured in triplicate with protein A-Sepharose precipitation. This assay gave a specificity of 100% and a sensitivity of 74.4% for new-onset patients less than age 30 yr at the IDS Combinatorial Workshop. GAD65 and ICA512 autoantibody levels are expressed as an index calculated from the counts per min for the test sample and the positive and negative control samples. GAD65 and ICA512 autoantibodies were determined simultaneously with differential labeling (³⁵S for ICA512 and ³H for GAD65) in an automated 96-well ß-counter.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Sequencing exon 2 of the DQB1¹0602 and DQA1¹0102 alleles

We studied eight unaffected, ICA-positive first degree relatives (Table 1) and six rare patients with type 1 diabetes (Table 2), all previously typed as DQB1¹0602 positive with standard SSO typing techniques. We performed direct sequence analysis of the second exon of their DQB1 and DQA1 alleles to investigate whether any of the diabetes-permissive DQB1¹0602 variants recently described (36) occurred among these subjects. All subjects carried conventional DQB1¹0602 and DQA1¹0102 exon 2 sequences. Thus, all subjects carry DQA1 and DQB1 alleles coding for a diabetes-protective HLA-DQ heterodimer.

Typing for the mtDNA A3243G gene mutation

None of the subjects studied carried the mtDNA A3243G mutation reportedly associated with the development of late-onset diabetes in ICA-positive individuals.

Additional follow-up and autoantibody testing in ICA/DQB1¹0602-positive first degree relatives

All of the previously identified ICA/DQB1¹0602-positive relatives were tested to determine whether they express other autoantibodies besides ICA. The upper limits of normal are 42 nU/mL for IAA and indexes of 0.032 and 0.071 for GAD65 and ICA512 autoantibodies, respectively. Autoantibody levels shown in Tables 1 and 2 are the mean levels for each individual. As illustrated in Table 1, seven of eight relatives have GAD65 autoantibodies but only two of eight and one of eight have autoantibodies against insulin and ICA512, respectively. None of these relatives have developed type 1 diabetes to date with further extended follow-up (Table 1; mean follow-up ± SD, 8.55 ± 4.93 yr; a mean increase of 2.6 yr in follow-up length since our first report) (33).

	Discussion

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The HLA-DQB1¹0602 allele confers strong diabetes protection, even among ICA-positive first degree relatives of patients with type 1 diabetes (33). In our family study, approximately 7% of ICA-positive relatives were found to carry DQB1¹0602 using standard SSO typing techniques. However, such techniques cannot distinguish DQB1¹0602 from the putative diabetes-permissive DQB1¹0602/3 variants recently described by Hoover et al. (36). Moreover, a form of slowly progressive diabetes has been associated with late age of onset in Japanese ICA-positive patients carrying the mtDNA A3243G mutation (37, 38, 39). The presence of such DQB1¹0602 variants or of the above mtDNA mutation may have significant prognostic implications, as the risk of diabetes for ICA/DQB1¹0602-positive relatives would certainly be higher than previously estimated. We therefore investigated the occurrence of diabetes-permissive DQB1¹0602 variants and of the mtDNA A3243G mutation among all the previously identified, unaffected, ICA-positive first degree relatives, all identified as DQB1¹0602 positive (33). We found that all ICA-positive relatives studied have conventional sequences for both DQB1¹0602 and DQA1¹0102, thus coding for a protective HLA-DQ heterodimer. Moreover, none of them carries the mtDNA A3243G mutation reportedly associated with late-onset diabetes in ICA-positive subjects (37).

Among ICA/DQB1¹0602-positive relatives, 7 of 8 have GAD65 autoantibodies but only 2 of 8 and 1 of 8 have autoantibodies against insulin and ICA512, respectively (Table 1). Thus, ICA reactivity appears to be mainly directed against GAD65 in most ICA/DQB1¹0602-positive relatives (59, 60). In contrast, GAD65 autoantibodies were detected only in 2 of 13 ICA-positive patients from Japan with the mtDNA A3243G mutation, none of whom had DQB1¹0602 (37). As diabetes risk also correlates with the number of autoantigens targeted by antiislet immune responses (61), it is apparent that ICA/DQB1¹0602-positive relatives express a low risk phenotype with only limited loss of tolerance to islet cell autoantigens (60). Indeed, despite ICA positivity, none of these relatives has developed type 1 diabetes to date during further extended follow-up. Moreover, most relatives (5 of 8) are now over 40 yr old. Together, these observations confirm that ICA/DQB1¹0602-positive relatives are a distinct group of individuals with extremely low risk of diabetes, even at an older age. These findings validate our previous report that DQB1¹0602 significantly protects from type 1 diabetes even in the presence of ICA/GAD65 autoantibodies (33), an observation that has significant implications for the design of prevention trials.

We also investigated the occurrence of diabetes-permissive DQB1¹0602 variants in six rare patients with type 1 diabetes and DQB1¹0602 (as defined with conventional typing techniques). Although a number of patients with DQB1¹0602 may suffer from rare genetic forms of diabetes rather than type 1 diabetes [e.g. Wolfram’s syndrome, type 1 autoimmune polyendocrine syndrome, type 1B diabetes, and maturity onset diabetes of the young (MODY)], the clinical diagnosis of type 1 diabetes was confirmed in four of six patients by the presence of autoantibodies against ICA. The diagnosis was exclusively based on clinical criteria for the two patients identified through the Human Biological Data Interchange repository of families with type 1 diabetes (42). We did not observe the diabetes-permissive variants reported by Hoover et al. (36), and all six patients carry normal DQB1¹0602 and DQA1¹0102 sequences. Moreover, none of the patients has the A3243G mtDNA mutation. Thus, our findings do not support the hypothesis that the above variants may commonly occur and be specifically associated with diabetes development in conventionally typed DQB1¹0602-positive subjects.

Our results also imply that the protective effect associated with DQB1¹0602 is not absolute, as type 1 diabetes may develop in extremely rare cases in individuals carrying the DQB1¹0602 allele. Perhaps other polymorphisms regulating the phenotypic expression of DQB1¹0602, including polymorphisms in the DQA1/DQB1 promoter regions, or other genetic loci/environmental factors may modulate the protective effect associated with DQB1¹0602. For instance, patient 17865, an African-American with diabetes and SMS, has the DRB1¹1503 allele instead of the DRB1¹1501 allele. The DRB1¹1501 allele, usually found in linkage disequilibrium with DQB1¹0602 on protective DR2 haplotypes, may theoretically contribute to the protective effect and, conversely, the DQB1¹0602/DRB1¹1503 combination may be less protective. However, the analysis of recombinant haplotypes suggests that most of the protection derives from the DQB1¹0602 allele (18, 31, 32); moreover, one of the ICA-positive relatives (no. 1271) carries the unusual combination of DQB1¹0602 in cis with the diabetes-predisposing DRB1¹0301 allele (DR3) and has not developed diabetes despite having ICA/GAD65 autoantibodies for more than 12 yr. We also identified a patient with type 1 diabetes carrying DQB1¹0603 in cis with DQA1¹0102 and DRB1¹1501, once again suggesting that DQB1¹0602 is central to diabetes protection (data not shown).

In contrast to our findings, none of the five DQB1¹0602-positive patients with type 1 diabetes originally described by Baisch et al. (20) were found to carry DQB1¹0602 by Hoover et al. (36); one patient was initially mistyped and had DQB1¹0603, and four patients carry alleles with a sequence closely related to that of DQB1¹0602 (n = 1) and DQB1¹0603 (n = 3). Of note, DQB1¹0603 is usually found on haplotypes bearing the DQA1¹0103 and DRB1¹1301 (DR6) alleles, and such haplotypes are significantly less protective than the DQA1¹0102/DQB1¹0602/DRB1¹1501 (DR2) haplotype. It remains unclear whether the DQB1¹0603 variants described by Hoover (termed 0603a, 0603b, and 0603c) are linked to DRB1¹1501 (DR2) or DRB1¹1301 (DR6) alleles; however, in all instances those variants were found in cis with DQA1¹0103, and based on linkage disequilibrium, one would predict that none of those patients carries a DRB1¹1501 (DR2) haplotype. The allele variant directly related to DQB1¹0602, also reported by Williams et al. and now termed DQB1¹0611 (48), differs from DQB1¹0602 at codon 9, where a TA substitution determines a change in the amino acid sequence (PheTyr). Molecular modelling studies suggest that such sequence variation may alter the peptide-binding site of the HLA-DQ molecule (36), and it is also of interest that the same codon 9 polymorphism is shared with the most common susceptibility alleles (DQB1¹0302 and DQB1¹0201) and other diabetes-permissive alleles (DQB1¹0502, DQB1¹0604, and DQB1¹0603c) (36, 62). However, the same polymorphism is also found in alleles that are not associated with increased susceptibility (i.e. DQB1¹0603 and DQB1¹0301), and susceptible alleles such as DQB1¹0401 share codon 9 sequence with DQB1¹0602 instead. It should also be noted that none of the diabetes-permissive variants differs from DQB1¹0602 at positions 57 and 70, the combined variation of which reportedly modulates peptide binding and diabetes susceptibility (36, 62).

As regards the frequency of the diabetes-permissive variants, DQB1¹0611 is extremely rare, as it was detected in only 1 of 30 African-Americans and 0 of 62 non-Hispanic/Hispanic whites with DQB1¹0602 (48). No frequency data are available for the other variants (DQB1¹0603a, DQB1¹0603b, and DQB1¹0603c), but one would speculate that these alleles are probably very rare, as they would have been reported much earlier had they occurred at a significant frequency in the population. This is consistent with our findings confirming the presence of DQB1¹0602 in all of the relatives and patients studied. Nonetheless, given the significant prognostic implications associated with DQB1¹0602, the current typing protocols should be modified to allow the unequivocal identification of DQB1¹0602 when typing relatives of patients with type 1 diabetes. This can be easily achieved with sequence-specific primers that selectively amplify DQB1¹0602 (PCR-SSP) with a strategy based on the protocols of Olerup et al. (49) and Williams et al. (48), as illustrated in Table 3.

In conclusion, we show that all the ICA/DQB1¹0602-positive relatives previously described carry conventional DQB1¹0602 and DQA1¹0102 sequences coding for a protective heterodimer. Such relatives appear to have a low risk of type 1 diabetes, as also confirmed by their autoantibody profiles and extended follow-up data presented. Thus, our findings provide additional confirmation for the dramatic protective effect associated with DQB1¹0602, even among relatives with autoantibody positivity. However, our finding that DQB1¹0602 is present in rare patients with type 1 diabetes suggests that protection is not absolute, perhaps because of other unknown genetic or environmental factors that may modulate the protective mechanisms associated with DQB1¹0602. Finally, although putative diabetes-permissive DQB1¹0602 variants appear to be extremely uncommon, we suggest a simple typing strategy, based on the protocols of Olerup et al. (49) and Williams et al. (48), that can be easily applied to allow the unequivocal identification of the DQB1¹0602 allele when typing results are to be used for predicting type 1 diabetes.

	Acknowledgments

 
We thank Terry Smith, R.N., for her nursing skills, Lesley Kenyon for her technical assistance, and Mr. David Stenger for his assistance and support.

	Footnotes

 
¹ This work was supported by Grant MO1RR00069, General Clinical Research Centers Program, National Centers for Research Resources, NIH, by Grant DK32083, and by the Diabetes Research Institute Foundation.

Received August 7, 1998.

Revised February 3, 1999.

Accepted February 8, 1999.

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