subjects in the present study are part of a longitudinal study of the etiology of type 2 diabetes among the gila river indian community in arizona , where most of the residents are pima indians or of the closely related tohono o'odham tribe . diabetes status was determined by an oral glucose tolerance test according to the criteria of the world health organization ( 10 ) . the initial genetic study was conducted in a population - based sample of full - heritage pima indians ( n = 3,501 ) , where 1,561 subjects had type 2 diabetes ( 37% male , age at the last exam 49  14 years , and bmi 39  8 kg / m ) and 1,940 subjects were nondiabetic ( 46% male , age at the last exam 31  15 years , and bmi 36  8 kg / m ) . independent replication was assessed in 3,723 subjects from the same longitudinal study who were of mixed native american heritage ( reported heritage , on average , was one - half pima and three - quarters native american ) . the replication sample had 750 subjects with type 2 diabetes ( 41% male , age at the last exam 42  14 years , and bmi 38  9 kg / m ) and 2,973 nondiabetic subjects ( 47% male , age at the last exam 24  11 years , and bmi 34  8 additional replication was assessed in a case - control sample from the old order amish ( 139 diabetic subjects and 347 subjects with normal glucose tolerance ) as previously described ( 11 ) . among the full - heritage pima indians , 415 subjects ( 58% male , age 27  6 years , and bmi 34  8 kg / m ) had undergone detailed metabolic testing for risk factors that predict type 2 diabetes . these individuals were determined to be nondiabetic , and acute insulin response was only analyzed in subjects who had normal glucose tolerance . glucose tolerance was determined by a 75-g oral glucose tolerance test ( ogtt ) with measurements of fasting and 30 , 60 , 120 , and 180-min plasma glucose and insulin concentrations ( 12 ) . the acute insulin response to intravenous glucose was measured on a separate day from the ogtt . blood samples were collected prior to a 25-g glucose intravenous bolus infusion and at 3 , 4 , 5 , 6 , 8 , and 10 min after infusion . the acute insulin response was calculated as the mean increment in plasma insulin concentrations from 35 min ( 13 ) . insulin sensitivity was assessed using the hyperinsulinemic - euglycemic clamp technique as previously described ( 12,13 ) . body composition was estimated by underwater weighing until january 1996 and by dual - energy x - ray absorptiometry ( dpx-1 ; lunar radiation ) thereafter ( 14 ) . dna from 24 pima indians ( 12 nondiabetic and aged > 45 years ; 12 diabetic with onset age < 25 years ) was sequenced using a big dye terminator ( applied biosystems ) on an automated dna capillary sequencer ( model 3730 ; applied biosystems ) . genotyping was done using the snplex genotyping system 48-plex ( applied biosystems ) on an automated dna capillary sequencer ( model 3730 ; applied biosystems ) for the native american samples and taqman genotyping assays ( applied biosystems ) for the old order amish samples . in the pima study , statistical analyses were performed using the software of the sas institute ( cary , nc ) . the general association of genotypes with type 2 diabetes was assessed by logistic regression analysis and was adjusted for covariates ( age , sex , and birth year ) . the model was fit with a generalized estimating equation technique to account for correlation among siblings . genotype was analyzed as a numeric variable representing the number ( 0 , 1 , 2 ) of copies of a given allele . the association of quantitative traits with genotypes was analyzed by linear regression using the generalized estimating equation procedure to account for correlation among siblings . p values were adjusted for potential confounding covariates . in the replication study , which included individuals of mixed ancestry , these estimates were derived by the method of hanis et al . ( 15 ) from 39 informative markers with large differences in allele frequency between populations ( 16 ) . linkage disequilibrium ( ld ) and haplotype blocks were estimated by haploview ( version 3.32 ) . for the amish study , the odds ratio ( or ) is derived from a logistic regression model , while the p value is based on the normal - liability threshold model implemented in solar to account for relationships among individuals . among the full - heritage pima indians , 415 subjects ( 58% male , age 27  6 years , and bmi 34  8 kg / m ) had undergone detailed metabolic testing for risk factors that predict type 2 diabetes . these individuals were determined to be nondiabetic , and acute insulin response was only analyzed in subjects who had normal glucose tolerance . glucose tolerance was determined by a 75-g oral glucose tolerance test ( ogtt ) with measurements of fasting and 30 , 60 , 120 , and 180-min plasma glucose and insulin concentrations ( 12 ) . the acute insulin response to intravenous glucose blood samples were collected prior to a 25-g glucose intravenous bolus infusion and at 3 , 4 , 5 , 6 , 8 , and 10 min after infusion . the acute insulin response was calculated as the mean increment in plasma insulin concentrations from 35 min ( 13 ) . insulin sensitivity was assessed using the hyperinsulinemic - euglycemic clamp technique as previously described ( 12,13 ) . body composition was estimated by underwater weighing until january 1996 and by dual - energy x - ray absorptiometry ( dpx-1 ; lunar radiation ) thereafter ( 14 ) . dna from 24 pima indians ( 12 nondiabetic and aged > 45 years ; 12 diabetic with onset age < 25 years ) was sequenced using a big dye terminator ( applied biosystems ) on an automated dna capillary sequencer ( model 3730 ; applied biosystems ) . genotyping was done using the snplex genotyping system 48-plex ( applied biosystems ) on an automated dna capillary sequencer ( model 3730 ; applied biosystems ) for the native american samples and taqman genotyping assays ( applied biosystems ) for the old order amish samples . in the pima study , statistical analyses were performed using the software of the sas institute ( cary , nc ) . the general association of genotypes with type 2 diabetes was assessed by logistic regression analysis and was adjusted for covariates ( age , sex , and birth year ) . the model was fit with a generalized estimating equation technique to account for correlation among siblings . genotype was analyzed as a numeric variable representing the number ( 0 , 1 , 2 ) of copies of a given allele . the association of quantitative traits with genotypes was analyzed by linear regression using the generalized estimating equation procedure to account for correlation among siblings . p values were adjusted for potential confounding covariates . in the replication study , which included individuals of mixed ancestry , ( 15 ) from 39 informative markers with large differences in allele frequency between populations ( 16 ) . linkage disequilibrium ( ld ) and haplotype blocks were estimated by haploview ( version 3.32 ) . for the amish study , the odds ratio ( or ) is derived from a logistic regression model , while the p value is based on the normal - liability threshold model implemented in solar to account for relationships among individuals . sequencing of the mbl2 gene ( all four exons , three introns , and 2 kb of the upstream region ) in 24 pima indians identified 37 variants . rs5030737 ( arg52cys ) , rs1800450 ( gly54asp ) , and rs1800451 ( gly57glu)commonly referred to as a / d , a / b , and a / c , respectively ( 12 ) . three known promoter snps , rs11003125 , rs7096206 , and rs7095891 , previously classified as 550 g > c ( h / l ) ,  221 g > c ( y / x ) , and + 4 c > t ( p / q ) , respectively , were also identified ( 3 ) . from these 37 snps in the mbl2 gene , 12 tag snps were selected based on the tagger algorithm ( haploview 3.32 , using r  0.8 to indicate redundancy [ supplementary fig . 1 , available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1593/dc1 ] ) . to additionally analyze variation flanking mbl2 ( 25 kb flanking each side of the sequenced region , chromosome 10:5417014654228466 ) , 20 database snps that serve as tag snps in the chinese hapmap ( r  0.8 ; minor allele frequency 0.1 ) were genotyped in pima indians , and it was determined that seven snps could serve as tag snps ( defined above ) in pima samples ( supplementary fig . all 19 tag snps ( 12 in mbl2 and 7 flanking ) were genotyped in the sample of 3,501 full - heritage pima indians for association analysis with type 2 diabetes ( table 1 ) . the missense snp rs1800450 ( gly54asp , designated a / b ) and the promoter snp rs11003125 ( designated h / l ) were in high ld ( d = 0.99 ; r = 0.71 ) and were associated with type 2 diabetes ( p = 0.002 and 0.0007 , respectively , adjusted for age , sex , birth year , and family membership ; table 1 ) . these two tag snps captured several additional snps within intron 2 and the region near mbl2 . associations of mbl2 tag snps with type 2 diabetes in pima indians nineteen tag snps ( r 0.8 ) were selected from 57 snps that span mbl2 ( 8.3 kb ) and approximately 25 kb flanking each side of the gene . allele frequency ( af ) is presented as frequency of the risk allele . or is expressed as per copy of the risk allele ( and thus , by definition , is > 1 ) . sequences flanking the two novel snps are as follows : novel : promoter , tttcatggatgggtgtgtgc[g / a]tgcatgcacgtgtctgtgtg ; novel : 3utr , catgactgcacagtaatttc[g / a]tctgtttataaacattgtat . to determine whether the association with type 2 diabetes in the full - heritage pima indians could be replicated in other native americans , rs1800450 and rs11003125 were further genotyped in a nonoverlapping sample of 3,723 subjects who were predominately of mixed native american heritage . the promoter snp rs11003125 reproducibly associated with type 2 diabetes ( p = 0.04 , adjusted for age , sex , birth year , and heritage ) ( table 1 ) . combining the initial and replication samples ( n = 7,224 ) provided the strongest evidence for association with type 2 diabetes for rs11003125 ( p = 9.2  10 ) ( table 1 ) . the combined sample also showed a significant association for rs1800450 ( p = 0.001 ) ( table 1 ) , but this significant association was largely driven by the initial full - heritage pima sample , with only a nonsignificant association in the same direction ( p = 0.18 ) identified in the largely mixed - heritage replication group . to determine whether variants in mbl2 had a significant effect on diabetes in non  native american populations , rs1800450 and rs11003125 were genotyped in an amish sample of 139 diabetic subjects and 347 subjects with normal glucose tolerance . consistent with the native american samples , rs11003125 was associated with type 2 diabetes in the amish ( or 1.51 ; p = 0.004 , adjusted for age , sex , and family structure ) ( table 2 ) , but the frequency of risk allele g was lower in the amish than in pima indians ( 0.45 vs. 0.77 , respectively ) . the snp rs1800450 ( gly54asp ) was also associated with type 2 diabetes in the amish ( or 2.38 , adjusted p = 0.003 ) ( table 2 ) , but the frequency of risk allele glycine was comparable in the amish and pima indians ( 0.87 vs. 0.83 ) . in contrast , these associations do not appear to replicate in the large caucasian diabetes genetics replication and meta - analysis ( diagram ) ( 19 ) . neither of these snps were directly genotyped in genome - wide association ( gwa ) studies from the diabetes genetics initiative ( dgi ) and the wellcome trust case control consortium ( wtccc ) , which are two of the three large studies from which the meta - analysis was derived ; however , a proxy , rs1838065 , which we determined to have an r = 1 with rs11003125 ( based on our genotyping of 90 caucasians ) , was not associated with type 2 diabetes in diagram ( p = 0.35 , table 2 ) . combining the negative diagram data together with the positive amish and native american data rendered the overall combined association nonsignificant ( p = 0.17 ) ( table 2 ) . association of promoter rs1103125 and rs1800450 ( gly54asp ) with type 2 diabetes in pima indian , amish , and diagram subjects a combined test was conducted by the inverse variance method . ors are per copy of the risk allele identified in the full - heritage pima sample . phet is the p value for the null hypothesis that the ors are the same for the combined groups . * diagram results based on rs1838065 , which has an r of 1.0 with rs1103125 . data across a larger genomic region encompassing mbl2 could also be obtained from prior gwa studies in pima indians ( 20 ) , dgi ( 21 ) , wtccc ( 22 ) , and diagram ( 19 ) . for example , a 400 kb region encompassing the 8.3 kb mbl2 ( chromosome 10 : 5400702754401287 ) yielded 101 snps that were previously genotyped in a gwa study of pima indians . only one gwa study snp ( rs1838065 ) was located within mbl2 ( intron 2 ) . the pairwise ld pattern of these 101 snps and their association with early - onset type 2 diabetes in pima subjects who were analyzed in this prior gwa ( 300 early - onset diabetes case and 334 control subjects ) are shown in supplementary fig . 2a and b. gwa study snp rs1838065 , which is in near - perfect ld with the promoter rs11003125 ( r = 0.99 ) , was associated with early - onset type 2 diabetes ( defined as diabetes onset 25 years of age ) in the gwa study ( adjusted p = 0.0006 ) , as were several nearby gwa study snps ( adjusted p = 0.00070.005 ) ( supplementary fig . 2 ) , which were in high ld among themselves ( r = 0.660.99 ) but in low ld with rs1838065 ( r = 0.300.37 ) . in contrast with rs1838065 , these additional gwa study snps ( tagged by rs920727 ) had only a borderline association with type 2 diabetes ( defined as diabetes at any age ) in the current larger study of 3,501 full - heritage pima subjects ( rs920727 , p = 0.05 ) ( table 1 ) . gwa study data across this region in the dgi and wtccc studies and diagram are shown in supplementary fig . overall , there were no consistent associations with type 2 diabetes across the region in any of these caucasian studies . when gwa study results of pimas were compared with other populations in the mbl2 region , rs1838065 was not significant in diagram . snps tagged by rs17587392 in caucasians had a borderline significance with type 2 diabetes in diagram ( p = 0.05 ) ( supplementary fig . 3 ) but was nearly monomorphic ( minor allele frequency < 0.001 ) and therefore uninformative in full - heritage pima indians , while snp rs11003132 , which had a borderline association with type 2 diabetes in wtccc ( p = 0.05 ) ( supplementary fig . 3 ) , was not associated with type 2 diabetes in pima indians ( table 1 ) . the observation that specific mbl2 snps had replicated associations with type 2 diabetes in native americans and a small group of amish subjects , who are of european descent but were not associated with diabetes in the large diagram , is unexpected . , both the pima indians and the old order amish represent far more homogeneous populations compared with the study population of diagram . it is also possible that susceptibility genes for common diseases may have larger effects in these populations compared with others as a result of segregation of high - penetrance alleles that are rare or nonexistent in the general population , gene - gene or gene - environment interactions , or the absence of other susceptibility genes whose effects could mask other genes . to aid in validating the positive associations with type 2 diabetes , we further investigated whether these variants in mbl2 were associated with metabolic risk factors that predict type 2 diabetes among 415 nondiabetic , full - heritage pima indians . for both rs11003125 and rs1800450 , the allele associated with higher risk for diabetes ( g and glycine , respectively ) was associated with a higher 2-h plasma glucose concentration ( adjusted p = 0.0005 and 0.04 , respectively ) and higher 2-h plasma insulin concentration ( adjusted p = 0.0008 and 0.003 , respectively ) during an oral glucose tolerance test ( table 3 ) . the risk allele glycine for rs1800450 was additionally associated with a lower acute insulin response to an intravenous glucose bolus infusion ( adjusted p = 0.004 ) among subjects who had normal glucose tolerance ( n = 281 ) ( table 3 ) . metabolic characteristics of full - heritage nondiabetic subjects grouped by genotypes of promoter rs11003125 or rs1800450 ( gly54asp ) variants data are means  se unless otherwise indicated . plasma insulin concentrations ( fasting , 2 h , and 30 min ) , rates of glucose disappearance during the low - dose insulin stimulation , and the acute insulin response were log transformed before analyses to approximate a normal distribution . the p value for percent body fat was adjusted for age , sex , and family membership . the p value for the acute insulin response was adjusted for age , sex , percent body fat , and rate of glucose disappearance during the low - dose insulin stimulation . all remaining p values ( except for age ) were adjusted for age , sex , percent body fat , and family membership . previous functional studies have shown that serum mbl levels are greatly influenced by variants within the mbl2 gene . three promoter variants ( rs11003125 [ h / l ] , rs7096206 [ y / x ] , and rs7095891 [ p / q ] ) and three missense variants ( rs5030737 [ a / d ] , rs1800450 [ a / b ] , and rs1800451 [ a / c ] ) were previously associated with mbl deficiency ( 3,23 ) . several of these variants are in high ld , so a limited number of haplotypes are present in humans . hypa ( g - g - c - gly ) haplotype carriers have the highest serum concentration of mbl , typically from 1,400 to 2,500 g / l , whereas lypb ( c - g - c - asp ) haplotype carriers have the lowest serum concentration : 20400 g / l ( 3,20 ) . since rs7096206 ( y / x ) and rs7095891 ( p / q ) are very rare in pima indians ( minor allele frequency < 0.03 ) , these high versus low serum level haplotypes can essentially be determined from a two - snp haplotype of the promoter rs11003125 ( h haplotype analysis for rs11003125 and rs1800450 was performed in the combined sample of 7,224 predominately pima indians . the haplotype g - glycine ( ha ) , carrying both the g nucleotide ( designated h allele ) of the promoter rs11003125 and glycine ( designated a allele ) of rs1800450 , was associated with increased risk for type 2 diabetes in pima indians ( haplotype frequency of g - glycine 0.78 in diabetic vs. 0.71 in nondiabetic ; or 1.25 [ 95% ci 1.121.33 ] , p = 0.0001 , adjusted for age , sex , and birth year [ heritage estimate in the replication group ] ) . because this haplotype g - glycine is highly concordant with the g ( h ) allele of rs11003125 ( r = 0.99 ) , it is difficult to statistically distinguish the single genotypic versus haplotypic effects . when both snps are included in a single model , there is a significant association with rs11003125 ( or 1.29 [ 95% ci 1.081.54 ] ; p = 0.005 ) conditional on the effect at rs1800450 , but there is little association with rs1800450 conditional on the effect at rs1103125 ( 0.94 [ 0.771.16 ] ; p = 0.61 ) . it thus appears that the promoter rs11003125 is the stronger predictor of diabetes , with little additional information from rs1800450 . the prevalence of type 2 diabetes was plotted in the full - heritage pima indian ( n = 3,081 ) ( fig . 1b ) groups according to the genotypes of rs11003125 and rs1800450 . ordering the genotypic groups according to their association with serum mbl levels showed that subjects homozygous for both g and glycine alleles had a higher prevalence of diabetes than did subjects homozygous for the c allele and either homozygous or heterozygous for the aspartic acid alleles ( ptrend = 8.4  10 in the combined analysis of 6,585 predominately pima subjects , adjusted for age , sex , and birth year , and in the replication group , heritage ) . in caucasians , eskimos , africans , south americans , and native americans , the g - glycine ( ha ) haplotype is associated with higher mbl serum level ( 3,2426 ) . prevalence of type 2 diabetes in the full - heritage pima indian group ( a ) ( n = 3,081 ) and the replication mixed heritage group ( b ) ( n = 3,504 ) according to genotypes of promoter rs11003125 and rs1800450 ( gly54asp ) . * either homozygous or heterozygous for the asp alleles . the present study demonstrates that an allele for mbl2 , which arises predominately from the promoter snp rs11003125 ( g allele ) , predicts a higher serum level of mbl2 and is associated with increased risk for type 2 diabetes , increased 2-h plasma glucose and 2-h plasma insulin , and decreased insulin secretion in some populations . consistent with our observations , high mbl2 levels have recently been reported to be associated with high a1c levels in the strong heart study , a longitudinal study of cardiovascular disease among native americans ( 26 ) . snp rs11003125 is in high ld with other snps that map within intron 2 and a flanking region of mbl2 ; therefore , the contribution of other functional snps can not be ruled out . nevertheless , both high ld and diabetes association were restricted to the region in and near mbl2 , suggesting that evidence for association with type 2 diabetes is more likely derived from mbl2 rather than other genes in the region . although the physiologic mechanisms underlying the association of mbl2 levels with diabetes are unknown , it has previously been shown that mbl2 plays a dual role in modifying inflammatory responses ( 27 ) . deficiency of mbl has been linked to increased risk of developing type 1 diabetes ( 6 ) , insulin resistance , and obesity ( 9 ) as a result of a chronic infectious state or low - grade inflammation . mbl2 could also affect metabolic pathways through stimulating fatty acid oxidation in skeletal muscle ( 28 ) or reducing release of tumor necrosis factor- , interleukin-1 , and interleukin-6 ( 29 ) . in contrast , increased mbl levels could lead to an overly activated complement system , thereby inducing inflammatory damage or interweaving a complex autoimmune process ( 30 ) . consistent with the latter effect , high mbl levels have been associated with increased risk for insulin resistance in pregnancy ( 31 ) and late - onset of rheumatoid arthritis ( 5 ) . however , our study indicates that mbl2 variants are more likely to influence type 2 diabetes via an effect on insulin secretion rather than on insulin action , suggesting that inflammatory damage in pancreatic -cell function may be involved . additional studies are needed to investigate the impact of this gene on specific type 2 diabetes related  pathways and disease susceptibility in non  native american groups .
objectivembl2 encodes the mannose - binding lectin , which is a key player in the innate immune system and has recently been found to play a role in insulin resistance and development of type 1 diabetes and gestational diabetes mellitus . to assess the role of mbl2 in diabetes susceptibility , 
 this gene was analyzed in the pima indian population , which has a high prevalence of type 2 diabetes.research design and methodsnineteen tag single nucleotide polymorphisms ( snps ) were genotyped in a population - based sample of 3,501 full - heritage pima indians , and selected snps were further genotyped in independent samples of native american ( n = 3,723 ) and old order amish ( n = 486 ) subjects.resultstwo variants , a promoter snp ( rs11003125 ) at 550 bp with a risk allele frequency of 0.77 and a gly54asp ( rs1800450 ) with a risk allele frequency of 0.83 , were associated with type 2 diabetes in the full - heritage pima indians ( odds ratio 1.30 per copy of the g allele for rs1103125 , p = 0.0007 , and 1.30 per copy of the glycine allele for rs1800450 , p = 0.002 , adjusted for age , sex , birth year , and family membership ) . 
 these associations replicated in an independent native american sample ( 1.19 , p = 0.04 , for rs11003125 ) and a caucasian sample , the old order amish ( 1.51 , p = 0.004 , for rs1103125 and 2.38 , p = 0.003 , for rs1800450 ) . among pima indians with normal glucose tolerance , the diabetes risk allele glycine of gly54asp was associated with a decreased acute insulin response to an intravenous glucose bolus infusion ( p = 0.004 , adjusted for age , sex , percent body fat , glucose disposal under physiological insulin stimulation , and family membership).conclusionsour data suggest that the functional variants in mbl2 contribute to type 2 diabetes susceptibility in both native americans and the old order amish .