we studied 1,297 patients with type 1 diabetes from the nationwide finnish diabetic nephropathy study ( finndiane ) .
patients were required to have an onset of diabetes before 35 years of age and initiation of permanent insulin treatment within 1 year of diagnosis .
patients were classified into four groups based on the urinary albumin excretion rate ( aer ) or the presence of end - stage renal disease ( esrd ) ( table 1 ) .
patients with normal aer were required to have a duration of diabetes over 15 years .
serum concentration of mbl was determined in 1,064 patients ( available in the supplemental methods at http://diabetes.diabetesjournals.org/cgi/content/full/db08-1495/dc1 ) .
for nondiabetic control subjects , 701 finnish blood donors ( 49% men ; mean age 46.1 years ) from all over the country were applied and compared with an age- and sex - matched subgroup of patients with type 1 diabetes ( n = 701 ; 49% men ; mean age 43.9 years ) .
the power of our study sample was calculated using the genetic power calculator ( http://pngu.mgh.harvard.edu/purcell/gpc/ ) ( 21 ) .
clinical characteristics of the patients grouped by their nephropathy status data are means  sd , median ( interquartile range ) , or percent unless otherwise indicated .
patients were classified into three groups based on the aer in at least two of three consecutive overnight or 24-h urine collections . a fourth patient group consisted of patients on dialysis ( n = 34 ) and patients who had received a kidney transplant ( n = 144 ) and were thus classified as having esrd .
the 24-h aer and serum creatinine values were derived from the last central laboratory measurements .
therefore , single values may exceed or fall behind the thresholds for the classification due to effects of treatment .
estimated glomerular filtration rate ( egfr ) was calculated using the cockcroft - gault formula adjusted for body surface area ( ref .
< 0.0001 versus microalbuminuria .  p < 0.01 versus microalbuminuria . p < 0.0001 versus macroalbuminuria .
dbp , diastolic blood pressure ; dnp , diabetic nephropathy ; sbp , systolic blood pressure .
we selected 17 snps using the hapmap database release 21a ( ceu ) ( http://www.hapmap.org/ ) and the haploview tagger program ( http://www.broad.mit.edu/mpg/haploview/index.php ) ( 22 ) from a 23-kb region covering the 6.3-kb mbl2 gene and the 5 untranslated region and 3 untranslated region .
snps were chosen to capture all known hapmap snps having a minor allele frequency ( maf ) > 0.01 in the ceu population with pairwise r  0.8 . additionally , three functional snps ( allele c , allele d , and variant
comparisons between groups were performed using the mann - whitney u test because mbl concentrations were not normally distributed .
these analyses were done using the spss v. 15.0 software ( spss , chicago , il ) .
allele frequencies and genotype distributions for single snps were compared between the control subjects and the matched subset of patients with type 1 diabetes as well as between patients with macroalbuminuria and patients with type 1 diabetes but normal aer .
we also compared patients having esrd with patients with normal aer and compared a combined group of patients with macroalbuminuria or esrd with patients with normal aer .
an allelic association  test and three genotypic tests ( general , dominant , and recessive models ) were performed for each snp .
additionally , analyses with adjustment for potential confounders ( age , sex , a1c , diabetes duration , and history of smoking ) were performed .
the plink analysis program , version 1.00 , was applied in all analyses ( http://pngu.mgh.harvard.edu/purcell/plink/ ) ( 23 ) . for haplotype association analyses ,
we selected 17 snps using the hapmap database release 21a ( ceu ) ( http://www.hapmap.org/ ) and the haploview tagger program ( http://www.broad.mit.edu/mpg/haploview/index.php ) ( 22 ) from a 23-kb region covering the 6.3-kb mbl2 gene and the 5 untranslated region and 3 untranslated region .
snps were chosen to capture all known hapmap snps having a minor allele frequency ( maf ) > 0.01 in the ceu population with pairwise r  0.8 . additionally , three functional snps ( allele c , allele d , and variant
comparisons between groups were performed using the mann - whitney u test because mbl concentrations were not normally distributed .
these analyses were done using the spss v. 15.0 software ( spss , chicago , il ) .
allele frequencies and genotype distributions for single snps were compared between the control subjects and the matched subset of patients with type 1 diabetes as well as between patients with macroalbuminuria and patients with type 1 diabetes but normal aer .
we also compared patients having esrd with patients with normal aer and compared a combined group of patients with macroalbuminuria or esrd with patients with normal aer .
an allelic association  test and three genotypic tests ( general , dominant , and recessive models ) were performed for each snp .
additionally , analyses with adjustment for potential confounders ( age , sex , a1c , diabetes duration , and history of smoking ) were performed .
the plink analysis program , version 1.00 , was applied in all analyses ( http://pngu.mgh.harvard.edu/purcell/plink/ ) ( 23 ) . for haplotype association analyses ,
the 17 tag snps genotyped captured the 68 hapmap snps having an maf>0.01 with a mean pairwise r of 0.97 .
all snps were in hardy - weinberg equilibrium ( p > 0.01 ) both in the healthy control subjects ( supplementary table 1 ) and in type 1 diabetic patients ( data not shown ) .
the frequencies of the exon 1 variants were 0.13 ( b ) , 0.06 ( c ) , and 0.006 ( d ) in the healthy subjects , which is in line with the previously published frequencies in the finnish population ( 17 ) .
of the patients with diabetes , 30.6% were heterozygous carriers of one of these variants ( a / o genotype ) and 5.8% were homozygotes or compound heterozygotes ( o / o genotype ) .
the median serum concentration of mbl was higher in patients with macroalbuminuria ( 1,881 g / l [ interquartile range 6083,124 ] ) than in patients with normal aer ( 1,548 g / l [ 5142,635 ] ) ; p = 0.019 ( fig .
1 ) . supplementary table 2 shows the distribution of the mbl2 diplotypes and the corresponding median mbl concentrations .
as expected , the carriers of the exon 1 variants and the individuals homozygous for the x promoter polymorphism had clearly reduced mbl concentrations , with a median of 397 g / l . when the patients were stratified both by nephropathy status and mbl2 diplotype category , it was evident that patients with macroalbuminuria had significantly higher mbl concentrations than patients with normal aer for two of the six ( ya / ya : p = 0.0002 ; ya / yo : p = 0.003 ) diplotype categories ( supplementary table 2 and fig .
box plot diagram showing the distribution of serum mbl concentrations , for patients with type 1 diabetes , stratified by nephropathy status .
black horizontal lines , median values ;  , interquartile ranges ;  , outliers .
1,881 g / l [ interquartile range 6083,124 ] ) than in patients with normal aer ( 1,548 g / l [ 5142,635 ] ) , p = 0.019 .
the median concentration was 1,645 g / l ( 5313,220 ) in patients with microalbuminuria and 1,359 g / l ( 3432798 ) in patients with esrd , p = 0.17 and p = 0.45 , respectively , compared with patients with normal aer .
distribution of serum mbl concentrations in patients with normal aer and patients with macroalbuminuria , stratified by the mbl2 diplotype .
there were no significant differences in allele frequencies or genotype distributions between the patients with type 1 diabetes and nondiabetic control subjects ( supplementary table 3 ) .
although nominal evidence of allelic association ( p = 0.010.04 ) was seen for four snps ( rs930507 , rs2384045 , rs11003132 , and rs11003137 ) , the p values did not remain significant after correction for the number of tests performed .
men and women were also tested separately , but no significant p values were achieved ( data not shown ) .
association analyses showed no significant differences in allele frequencies or genotype distributions between patients with macroalbuminuria and patients with type 1 diabetes but normal aer ( supplementary table 4 ) . when patients with esrd were compared with patients with normal aer ,
the maf of this snp was 0.19 in patients with normal aer and 0.29 in patients with esrd ( p = 0.00009 ) .
the genotype distribution of this snp was also significantly different between these groups ( p = 0.0002 ) , with homozygous individuals being more common among esrd patients ( 10 vs. 3% ) .
the same snp had a p value of 0.006 in the genotypic test when a combined group of patients with macroalbuminuria or esrd was compared with patients with normal aer .
the analyses were also performed with adjustment for potential confounders ( supplementary table 4 ) .
all snps , except rs10824793 , were associated with mbl concentration ( supplementary table 5 ) .
the snps showing the strongest associations were rs7899547 ( p = 3.0  10 ) , rs1031101 ( tagging the b allele ; p = 8.0  10 ) , rs2384045 ( p = 2.2  10 ) , and rs920727 ( p = 7.6  10 ) .
the promoter variants h / l ( rs11003125 ) and p / q ( tagged by rs920724 ) had p values of 8.8  10 and 6.1  10 , respectively , whereas the alleles c and d had p values of 1.5  10 and 9.0  10 , respectively .
the analyses were also performed with adjustment for potential confounders ( supplementary table 5 ) .
the haplotypes constructed using the haploview program were studied for association with type 1 diabetes and diabetic nephropathy .
the distribution of the mbl2 diplotypes , diplotype categories , and the high- and low - mbl genotypes in each patient group is presented in table 2 .
there were no differences in the frequency of the high - mbl genotypes between the patients with normal aer ( 0.60 ) and macroalbuminuria ( 0.61 ; p = 0.39 ) .
similarly , when the distribution of the six diplotype categories was analyzed , no differences were seen ( p = 0.63 ) .
distribution of the mbl2 genotypes within the different patient groups data are n or n ( percent ) .
the mbl2 haplotypes were determined based on the co - occurrence of the three promoter variants ( h / l , rs11003125 ; x / y , rs7096206 ; and p / q , rs7095891 ) and the three exon 1 variants ( b , rs1800450 ; c , rs1800451 ; and d , rs5030737 ) .
diplotypes were grouped into six categories ( ya / ya , ya / xa , xa / xa , ya / yo , xa / yo , and yo / yo ) based on the x / y polymorphism and the presence of any of the exon 1 variants ( b , c , and d ) , collectively designated with o , and further into low- and high - mbl genotypes . there were no differences in the frequency of the high - mbl genotypes between the patients with normal aer and macroalbuminuria . however , the frequency of the high - mbl genotypes within the group of esrd patients ( 0.54 ) was lower than in patients with normal aer ( 0.60 ) , although this difference was not significant ( p = 0.10 ) .
the 17 tag snps genotyped captured the 68 hapmap snps having an maf>0.01 with a mean pairwise r of 0.97 .
all snps were in hardy - weinberg equilibrium ( p > 0.01 ) both in the healthy control subjects ( supplementary table 1 ) and in type 1 diabetic patients ( data not shown ) .
the frequencies of the exon 1 variants were 0.13 ( b ) , 0.06 ( c ) , and 0.006 ( d ) in the healthy subjects , which is in line with the previously published frequencies in the finnish population ( 17 ) .
of the patients with diabetes , 30.6% were heterozygous carriers of one of these variants ( a / o genotype ) and 5.8% were homozygotes or compound heterozygotes ( o / o genotype ) .
the median serum concentration of mbl was higher in patients with macroalbuminuria ( 1,881 g / l [ interquartile range 6083,124 ] ) than in patients with normal aer ( 1,548 g / l [ 5142,635 ] ) ; p = 0.019 ( fig .
1 ) . supplementary table 2 shows the distribution of the mbl2 diplotypes and the corresponding median mbl concentrations .
as expected , the carriers of the exon 1 variants and the individuals homozygous for the x promoter polymorphism had clearly reduced mbl concentrations , with a median of 397 g / l . when the patients were stratified both by nephropathy status and mbl2 diplotype category , it was evident that patients with macroalbuminuria had significantly higher mbl concentrations than patients with normal aer for two of the six ( ya / ya : p = 0.0002 ; ya / yo : p = 0.003 ) diplotype categories ( supplementary table 2 and fig .
box plot diagram showing the distribution of serum mbl concentrations , for patients with type 1 diabetes , stratified by nephropathy status .
black horizontal lines , median values ;  , interquartile ranges ;  , outliers . the median serum concentration of mbl was higher in patients with macroalbuminuria ( median
1,881 g / l [ interquartile range 6083,124 ] ) than in patients with normal aer ( 1,548 g / l [ 5142,635 ] ) , p = 0.019 .
the median concentration was 1,645 g / l ( 5313,220 ) in patients with microalbuminuria and 1,359 g / l ( 3432798 ) in patients with esrd , p = 0.17 and p = 0.45 , respectively , compared with patients with normal aer .
distribution of serum mbl concentrations in patients with normal aer and patients with macroalbuminuria , stratified by the mbl2 diplotype .
there were no significant differences in allele frequencies or genotype distributions between the patients with type 1 diabetes and nondiabetic control subjects ( supplementary table 3 ) .
although nominal evidence of allelic association ( p = 0.010.04 ) was seen for four snps ( rs930507 , rs2384045 , rs11003132 , and rs11003137 ) , the p values did not remain significant after correction for the number of tests performed .
men and women were also tested separately , but no significant p values were achieved ( data not shown ) .
association analyses showed no significant differences in allele frequencies or genotype distributions between patients with macroalbuminuria and patients with type 1 diabetes but normal aer ( supplementary table 4 ) . when patients with esrd were compared with patients with normal aer , significant evidence of association
the maf of this snp was 0.19 in patients with normal aer and 0.29 in patients with esrd ( p = 0.00009 ) .
the genotype distribution of this snp was also significantly different between these groups ( p = 0.0002 ) , with homozygous individuals being more common among esrd patients ( 10 vs. 3% ) .
the same snp had a p value of 0.006 in the genotypic test when a combined group of patients with macroalbuminuria or esrd was compared with patients with normal aer .
the analyses were also performed with adjustment for potential confounders ( supplementary table 4 ) .
all snps , except rs10824793 , were associated with mbl concentration ( supplementary table 5 ) .
the snps showing the strongest associations were rs7899547 ( p = 3.0  10 ) , rs1031101 ( tagging the b allele ; p = 8.0  10 ) , rs2384045 ( p = 2.2  10 ) , and rs920727 ( p = 7.6  10 ) . the promoter variants
h / l ( rs11003125 ) and p / q ( tagged by rs920724 ) had p values of 8.8  10 and 6.1  10 , respectively , whereas the alleles c and d had p values of 1.5  10 and 9.0  10 , respectively .
the analyses were also performed with adjustment for potential confounders ( supplementary table 5 ) .
the haplotypes constructed using the haploview program were studied for association with type 1 diabetes and diabetic nephropathy .
the distribution of the mbl2 diplotypes , diplotype categories , and the high- and low - mbl genotypes in each patient group is presented in table 2 .
there were no differences in the frequency of the high - mbl genotypes between the patients with normal aer ( 0.60 ) and macroalbuminuria ( 0.61 ; p = 0.39 ) .
similarly , when the distribution of the six diplotype categories was analyzed , no differences were seen ( p = 0.63 ) .
distribution of the mbl2 genotypes within the different patient groups data are n or n ( percent ) .
the mbl2 haplotypes were determined based on the co - occurrence of the three promoter variants ( h / l , rs11003125 ; x / y , rs7096206 ; and p / q , rs7095891 ) and the three exon 1 variants ( b , rs1800450 ; c , rs1800451 ; and d , rs5030737 ) . diplotypes were grouped into six categories ( ya / ya , ya / xa , xa / xa , ya / yo , xa / yo , and yo / yo ) based on the x / y polymorphism and the presence of any of the exon 1 variants ( b , c , and d ) , collectively designated with o , and further into low- and high - mbl genotypes .
there were no differences in the frequency of the high - mbl genotypes between the patients with normal aer and macroalbuminuria .
however , the frequency of the high - mbl genotypes within the group of esrd patients ( 0.54 ) was lower than in patients with normal aer ( 0.60 ) , although this difference was not significant ( p = 0.10 ) .
our study showed no evidence of association between the mbl2 snps and type 1 diabetes , although weak association signals ( uncorrected p = 0.010.04 ) were seen for four snps .
importantly , using tag snps , we have captured information on the whole gene and its surroundings , whereas the other studies addressed only the functional exon 1 variants . in a danish study ( 6 ) , genotypes producing high mbl concentrations were more common in patients having diabetic nephropathy than in patients with normal aer .
we found no evidence of such an association , although our study sample was about double the size ( 6 ) .
furthermore , we thoroughly studied association between both single mbl2 snps and haplotypes and nephropathy , but all the results were negative . in accordance with previous studies ( 6,7 )
, we showed that the median serum mbl concentration was significantly higher in patients with macroalbuminuria compared with the patients with normal aer .
this result persisted even when the patients were stratified by the mbl diplotype categories , although the difference was significant in only two of them .
thus , it seems that the high serum mbl concentration in patients with nephropathy is likely to reflect some still unknown pathogenic event such as chronic low - grade inflammation .
further studies are needed to resolve whether elevated mbl concentrations are a marker associated with some other contributing factor or a consequence of nephropathy or diabetic microvascular complications in general .
the minor allele of the snp rs920727 was more common in esrd patients than in the other patients 0.29 vs. 0.190.20 ) .
furthermore , the frequency of the high - mbl genotypes was somewhat lower in the esrd patients than in the other groups ( 0.54 vs. 0.600.61 ) , potentially explaining the relatively low mbl concentration in the esrd patients .
however , we do not consider these differences as an indication of a causal association with esrd phenotype .
most likely they are due to chance or even signs of selective mortality . supporting this hypothesis
, evidence exists that mortality of patients with type 2 diabetes is higher among individuals with an mbl concentration 1,000 g / l ( 24 ) .
moreover , most of the esrd patients in our study have received a kidney transplant , and graft rejection is less common for the group of patients with low mbl concentrations ( 25 ) .
many of the snps showing strong association with mbl concentration ( including the esrd - associated rs920727 ) did not belong to the group of snps forming the common mbl2 haplotypes .
some of these variants may have an independent effect on the mbl concentration or tag other still unknown functional variants .
however , this somewhat surprising association pattern can mainly be explained by ld between the associated and haplotype - forming snps ( supplementary table 6 ) .
our study sample has a reasonably high power to detect associations with relatively common snps ( population frequency > 20% ) with modest effects . the previously described higher median mbl concentration in patients with macroalbuminuria ( 6,7 ) was evident also in our sample . we have thoroughly covered the common variation within the mbl2 gene in carefully characterized finnish type 1 diabetic patients .
we conclude that although most of the mbl2 snps studied were associated with the mbl concentration , neither any single snp nor any of their haplotype combinations confer risk of type 1 diabetes or diabetic nephropathy .
 objectivemannose - binding lectin ( mbl ) is an essential component of the acute - phase immune response and may thus play a role in the pathogenesis of type 1 diabetes and diabetic nephropathy . 
 the serum concentration of mbl is mainly genetically determined , and elevated concentrations have been associated with both type 1 diabetes and diabetic nephropathy . 
 previous genetic studies have not been conclusive due to the small number of patients and polymorphisms studied . 
 we investigated whether mbl2 polymorphisms are associated with type 1 diabetes or diabetic nephropathy and whether patients with nephropathy have elevated mbl concentrations as indicated previously . 
 furthermore , we studied the association between mbl2 polymorphisms and mbl concentration.research design and methodswe genotyped 20 mbl2 single nucleotide polymorphisms ( snps ) in a large , well - characterized finnish case - control sample consisting of 1,297 patients with type 1 diabetes with or without nephropathy and 701 nondiabetic individuals . 
 the serum concentration of mbl was available for 1,064 patients.resultswe found that 19 snps were associated with the mbl concentration ( p = 3  10817  104 ) . 
 mbl concentrations were higher in patients with macroalbuminuria compared with patients without nephropathy even when the patients were stratified by the mbl2 genotypic background in accordance with previous studies . 
 however , no evidence of association between any of the snps or their haplotype combinations and type 1 diabetes or diabetic nephropathy was observed.conclusionsalthough most of the mbl2 snps studied were associated with the mbl concentration , no common variations ( neither single snps nor their haplotype combinations ) confer risk of type 1 diabetes or diabetic nephropathy . 
RESEARCH DESIGN AND METHODS
SNP selection and genotyping.
Statistical analyses of MBL concentrations.
Association analyses.
RESULTS
SNP genotyping.
MBL concentrations.
Association analyses between single SNP and type 1 diabetes.
Association analyses between single SNPs and nephropathy.
Association analyses between single SNPs and MBL concentrations.
Haplotype and diplotype analyses.
DISCUSSION
Supplementary Material