subjects included in this study were identified through two different diabetes registry studies conducted 13 years apart : the colorado type 1 diabetes registry and the colorado site of the search for diabetes in youth study . the colorado type 1 diabetes registry was a statewide registry developed to ascertain all new cases of type 1 diabetes in 0- to 17-year - olds in colorado from 1 january 1978 to 31 december 1988 ( 10,11 ) . search is a multicenter , population - based observational registry that ascertained new cases of physician - diagnosed diabetes in youth 019 years of age from 1 january 2002 forward ( 12 ) . a detailed description of this population ( 2 ) , the colorado type 1 diabetes registry ( 10,11 ) , and search have been published ( 12 ) . completeness of case ascertainment was assessed by the capture - recapture method ( 13,14 ) and estimated to be 9697% over time ( 2 ) . youth aged 517 years identified with incident type 1 diabetes during the period of 19781988 and 20022004 were eligible to participate in this analysis . in 19781988 , all hispanic youth ( n = 120 ) and a similar size random sample of nhw ( n = 122 ) youth identified by the colorado type 1 diabetes registry were eligible for genetic typing , and 41 hispanic ( 34% ) and 59 nhw youth ( 48% ) had hla measured ( 15 ) . in 20022004 , all youth over the age of 4 years of hispanic ( n = 87 ) or nhw ( n = 512 ) origin were eligible for genetic typing . a total of 39 hispanic ( 45% ) and 225 nhw ( 44% ) youth participated . age at diagnosis , sex , and family history of diabetes were assessed to determine whether the youth with hla measured were representative of the overall registered population in each period and racial / ethnic group . family history of diabetes was obtained from medical record abstraction ( 71% in 19781988 and 85% in 20022004 ) or from self - administered questionnaires completed within 5 years from the date of diagnosis ( 17% in 19781988 and 3% in 20022004 ) . children who had a sibling , parent , or grandparent with any type of diabetes were defined as having a positive family history . to assess whether the sample of youth with available hla typing was representative of the entire patient population of youth with type 1 diabetes diagnosed in these two time periods , demographic and clinical characteristics of study participants in 20022004 , the only significant difference between those with and without hla typing was a higher frequency of a positive family history of diabetes in nhw participants with typing available . however , no significant relationship was noted between having the highest - risk hla genotype ( drb1 * 03-dqb1 * 02/drb1 * 04-dqb1 * 03 ) and having a positive family history of diabetes in either time period ( p = 0.96 ) . these data suggest that our samples of nhw and hispanic youth with measured hla are likely representative of the larger eligible population with type 1 diabetes in both periods , or that they differ in characteristics not strongly related with the frequency of high - risk hla genotypes . therefore , we believe that any observed trends or lack thereof in the frequency and distribution of hla genotypes over time in our population are not likely to be due to selection bias . all participants provided informed consent and both the colorado type 1 diabetes registry and search were approved by relevant institutional review boards . drb1 and dqb1 typing was performed using onelambda pcr assays that were standard for the time period in which they were used ( 19781988 and 20022004 ) ( 16 ) . the main difference between assays was the number of dqb1 alleles identified that had sequence - specific oligonucleotides ( ssos ) available for typing . there were seven dqb1 ssos identified for typing in 19781988 , and there were 100 dqb1 ssos identified for typing in 20022004 . due to the difference in the number of dqb1 alleles identified and the availability of ssos for typing these alleles over the two periods , only the gene locus was used in the analysis to compare genotypes ( e.g. , dqb1 * 02 vs. dqb1 * 0201 ) . specific oligonucleotide probes used in 20022004 allowed for a larger number of samples to be typed at one time compared with the prior assay ( 17,18 ) . previous validation studies based on onelambda luminex sso typing yielded similar results when compared with the earlier sso assay ( 19 ) . genotypes assessed in both time periods were categorized as high risk ( drb1 * 03-dqb1 * 02/drb1 * 04-dqb1 * 03 ) ; moderate to low risk ( drb1 * 04-dqb1 * 03/drb1 * 04-dqb1 * 03 , drb1 * 04-dqb1 * 03/x , where x is not drb1 * 03-dqb1 * 02 , and drb1 * 04-dqb1 * 03/unknown ) ; low risk ( drb1 * 03-dqb1 * 02/drb1 * 03-dqb1 * 02 , drb1 * 03-dqb1 * 02/x , where x is not drb1 * 04-dqb1 * 03 , and drb1 * 03-dqb1 * 02/unknown ) ; and neutral risk ( x / x or x / unknown ) . descriptive univariate analysis was used to determine the frequency of the genotypes for each period . statistics were used to compare frequencies of hla haplotypes by race and period and genotypes by high - risk ( drb1 * 03-dqb1 * 02/drb1 * 04-dqb1 * 03 ) versus all other genotypes . multivariate logistic regression was used to adjust for potential confounders ( onset age , sex , family history of diabetes , and race / ethnicity ) and assess temporal trends . high - risk was compared with all other genotypes using all other genotypes as the referent group . drb1 and dqb1 typing was performed using onelambda pcr assays that were standard for the time period in which they were used ( 19781988 and 20022004 ) ( 16 ) . the main difference between assays was the number of dqb1 alleles identified that had sequence - specific oligonucleotides ( ssos ) available for typing . there were seven dqb1 ssos identified for typing in 19781988 , and there were 100 dqb1 ssos identified for typing in 20022004 . due to the difference in the number of dqb1 alleles identified and the availability of ssos for typing these alleles over the two periods , only the gene locus was used in the analysis to compare genotypes ( e.g. , dqb1 * 02 vs. dqb1 * 0201 ) . additionally , the current pcr sequence specific oligonucleotide probes used in 20022004 allowed for a larger number of samples to be typed at one time compared with the prior assay ( 17,18 ) . previous validation studies based on onelambda luminex sso typing yielded similar results when compared with the earlier sso assay ( 19 ) . genotypes assessed in both time periods were categorized as high risk ( drb1 * 03-dqb1 * 02/drb1 * 04-dqb1 * 03 ) ; moderate to low risk ( drb1 * 04-dqb1 * 03/drb1 * 04-dqb1 * 03 , drb1 * 04-dqb1 * 03/x , where x is not drb1 * 03-dqb1 * 02 , and drb1 * 04-dqb1 * 03/unknown ) ; low risk ( drb1 * 03-dqb1 * 02/drb1 * 03-dqb1 * 02 , drb1 * 03-dqb1 * 02/x , where x is not drb1 * 04-dqb1 * 03 , and drb1 * 03-dqb1 * 02/unknown ) ; and neutral risk ( x / x or x / unknown ) . descriptive univariate analysis was used to determine the frequency of the genotypes for each period . statistics were used to compare frequencies of hla haplotypes by race and period and genotypes by high - risk ( drb1 * 03-dqb1 * 02/drb1 * 04-dqb1 * 03 ) versus all other genotypes . multivariate logistic regression was used to adjust for potential confounders ( onset age , sex , family history of diabetes , and race / ethnicity ) and assess temporal trends . high - risk was compared with all other genotypes using all other genotypes as the referent group . a total of 364 youth ( 100 in 19781988 , and 264 in 20022004 ) with new - onset type 1 diabetes at age 517 years had hla genes measured as described above . table 2 shows the frequency of hla class ii drb1-dqb1 genotypes by time period and ethnicity . more children ( both nhw and hispanic ) carried the high - risk genotype ( drb1 * 03-dqb1 * 02/dr04-dqb1 * 03 ) in 19781988 than in 20022004 ( p = 0.05 ) . conversely , more children in 20022004 carried the moderate to low risk drb1 * 04-dqb1 * 03 ( homo- or heterozygous ) genotypes than in 19781988 ( p = 0.04 ) . there was no significant difference by time period or ethnicity in frequency of the neutral risk , drb1 * 03-dqb1 * 02 ( homo- or heterozygous ) , or x / x genotypes . using multiple logistic regression analysis ( table 2 ) , we assessed the association between having the high - risk genotype ( versus all other genotypes ) and time period , controlling for age , sex , race / ethnicity , and family history of diabetes . there was a 40% lower odds for carrying the high - risk genotype in 20022004 versus 19781988 ( odds ratio 0.60 [ 95% ci 0.360.99 ] , p = 0.05 ) . a decreasing trend over the two time periods was noted for both nhws ( 13% ) and hispanics ( 6% ) . conversely , there was a 70% greater chance of carrying the moderate- to low - risk genotypes in 20022004 versus 19781988 ( 1.7 [ 1.12.8 ] , p = 0.03 ) . no significant interactions between ethnicity and time period on the odds of having the highest risk genotype were noted . when stratified according to age - group ( table 3 ) , the most dramatic decreasing trend in the prevalence of the high - risk genotype occurred in those aged 59 years ( 51% [ 19781988 ] vs. 32% [ 20022004 ] , p = 0.05 ) with a coincident increasing trend in the proportion with the drb1 * 04-dqb1 * 03 ( homo- or heterozygous ) genotype in the same age - group ( 19% [ 19781988 ] to 40% [ 20022004 ] , p = 0.03 ) . we found that the distribution of hla genotype changed in youth with type 1 diabetes diagnosed 27 years apart . having type 1 diabetes at age 517 years in 20022004 was associated with an 11% difference or 0.6-fold lower odds for carrying the high - risk hla genotype ( drb1 * 03-dqb1 * 02/drb1 * 04-dqb1 * 03 ) compared with having type 1 diabetes in 19781988 . the largest proportional difference ( 19% ) over time in the high - risk genotype was found in 5- to 9-year - olds , suggesting that environmental pressures may have a greater impact on type 1 diabetes risk at earlier ages . this is the first documentation of such a trend in a representative biracial sample of youth with type 1 diabetes in the u.s . our study supports previous findings from finland ( 9 ) and the u.k ( 8) , both including caucasian adults with childhood - onset type 1 diabetes diagnosed up to 80 years apart . study showed that the frequency of the high - risk genotype was 12% lower in adults diagnosed up to 15 years of age in 19852002 compared with those diagnosed between 19221946 and 21% lower in those diagnosed under 5 years of age ( 8) . the finnish study showed that the frequency of the high - risk genotype was 7.1% lower and that of protective genotypes was 7.2% higher in adults with childhood - onset type 1 diabetes diagnosed after 1990 compared with those diagnosed before 1965 ( 9 ) . two older studies conducted in finland in the 1980s reported a decrease in hla - dr3 by 25% for those diagnosed with type 1 diabetes in the 1980s compared with those diagnosed in the 1960s ( 6,7 ) . neither of these studies provided information on the size or representativeness of the referent population . more importantly , two studies ( 8,9 ) measured hla in adults who were diagnosed with type 1 diabetes as children in the first half of the 20th century , when early mortality associated with type 1 diabetes was high . therefore , the frequency and distribution of hla genotypes in this sample may be significantly influenced by factors associated with survival of type 1 diabetes . an underestimate of the proportional distribution of high - risk hla genotypes in earlier time periods ( due to a potential association of high - risk hla genes with increased mortality ) may have resulted in underestimating the true temporal trends in the proportion of type 1 diabetes cases with high - risk hla genotypes in these earlier studies . in contrast , our study genotyped youth with type 1 diabetes relatively close to their disease onset and thus is not affected by survivor bias . in addition , we were able to provide evidence that the sample included in the analysis was representative of the colorado population of youth with type 1 diabetes in both time periods . importantly , although the same method was used for typing , due to the specific time periods in which they were performed , two slightly different assays were used , namely , onelambda labtype luminex sso in 20022004 and onelambda sso in 19781988 . we were unable to validate the results obtained with the earlier sso assay due to the lack of stored dna samples and inability to recontact participants . however , previous studies ( 19,21 ) suggest comparable agreement in serologic specificity with an 85% allele agreement . due to this and the difference in the number of allele ssos available for typing over time , only the gene locus was used in this analysis . by not using expanded allele information , we potentially introduced some misclassification , likely increasing the proportion with the high - risk genotype . however , misclassification would be similar for both time periods and therefore unlikely to influence our analysis of trends . because for 20022004 we did have allele information on 100 dqb1 ssos , we estimated the magnitude of the potential misclassification , and only four ( 1.5% ) cases were misclassified as having the highest - risk genotype in 20022004 using the expanded allele information only . in addition , there were 12 youth from 19781988 that did not have complete genotypes . this categorization could have led to some misclassification . specifically , if the unknown haplotype was drb1 * 03-dqb1 * 02 or drb1 * 04-dqb1 * 03 , youth would have been classified as high risk based on the typed haplotype or as heterozygous for the above haplotypes . for example , in the extreme situation where all youth with the unknown haplotype in the first time period would have been in fact at  high - risk ,  the proportion of youth with the highest risk genotype in 19781988 would have been 51% ( instead of 39% ) . this would have made our trend estimates even more substantial ( i.e. , a 23% decrease in the proportion with high - risk genotype ) . there are several hypotheses proposed to explain the increase in type 1 diabetes incidence in youth and the earlier age at onset . overload  hypotheses suggest that an increase in body mass in genetically susceptible individuals accelerates -cell decline or stresses ( overloads ) the -cells , leading to an early age at type 1 diabetes diagnosis ( 22,23 ) . the  hygiene hypothesis  postulates that the decreasing early life exposure to infectious agents in westernized societies has led to an impairment in the maturation of the immune system , thus permitting an increased occurrence of immune - mediated disorders , such as asthma and type 1 diabetes ( 24 ) . given that there is an increasing incidence of type 1 diabetes in colorado youth ( 2 ) , our findings support the hypothesis that environmental pressures may be increasing the disease penetrance despite a shift in the distribution of hla genotypes from high to moderate - low risk . although this and previous studies ( 69 ) evaluating the distribution and frequency of hla susceptibility genes over time have provided evidence in support of this hypothesis , the nature of environmental factors responsible for the increasing incidence of type 1 diabetes remains unknown .
objective  type 1 diabetes is associated with a wide spectrum of susceptibility and protective genotypes within the hla class ii system . 
 it has been reported that adults diagnosed with youth - onset type 1 diabetes more recently have been found to have fewer classical high - risk hla class ii genotypes than those diagnosed several decades ago . 
 we hypothesized that such temporal trends in the distribution of hla - dr , dq genotypes would be evident , and perhaps even stronger , among 5- to 17-year - old hispanic and non - hispanic white ( nhw ) youth diagnosed with type 1 diabetes in colorado between 1978 and 2004.research design and methods  hla - dr , dq was typed using pcr and sequence - specific oligonucleotide hybridization in 100 youth diagnosed during the period of 19781988 and 264 diagnosed during 20022004 . 
 logistic regression was used to adjust for confounders and assess temporal trends.resultsthe frequency of the highest - risk genotype ( drb1 * 03-dqb1 * 02/drb1 * 04-dqb1 * 03 ) was higher ( 39% ) in children diagnosed during the period 19781988 than in those diagnosed during 20022004 ( 28% ) . 
 a similar pattern was observed in nhws and hispanics.conclusionswe found that high - risk hla genotypes are becoming less frequent over time in youth with type 1 diabetes of nhw and hispanic origin . 
 this temporal trend may suggest that increasing environmental exposure is now able to trigger type 1 diabetes in subjects who are less genetically susceptible .