Estimated frequency and plasma resistin in each haplotype defined by <i>RETN</i> SNP-420, and SNP-358 genotypes.

<p>Frequency and plasma resistin for each haplotype defined by <i>RETN</i> SNP-420 and SNP-358 genotypes were estimated by SNPstats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009718#pone.0009718-Sole1" target="_blank">[29]</a>. Estimated differences of plasma resistin compared to a reference haplotype, C-G, are shown in each haplotype.</p

Nuclear proteins specifically recognized a difference in one base at SNP-358 but not at SNP-638.

<p>EMSA was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009718#s4" target="_blank"><i>Methods</i></a>. A labeled probe representing <i>RETN</i> promoter sequences around SNP-358 contained G (major allele) or A (minor allele) at SNP-358 (<b>³²</b>P-358G or <b>³²</b>P-358A, respectively), and that around SNP-638 contained G (major allele) or A (minor allele) at SNP-638 (<b>³²</b>P-638G or <b>³²</b>P-638A, respectively). Each probe was incubated with a nuclear extract of THP-1 cells in the absence (−) or presence of a 200-fold molar excess of unlabeled competitor double-stranded oligonucleotides indicated (−358G, −358A, −638G, or −638A). The arrow points to the band that specifically bound to the probe with G at SNP-358.</p

Characteristics of the population studied (<i>n</i> = 2,019).

<p>Values are means±SD, or <i>n</i> (%). Number of subjects who measured immunoreactive insulin was 1,958. HOMA-IR (homeostasis model assessment insulin resistance index) was calculated as glucose (mg/dl) x insulin (µU/ml)/405. CVD indicates cardiovascular disease, including stroke, myocardial infarction, and angina pectoris.</p

Multiple regression analysis involving plasma resistin (ng/ml) as a dependent variable and each SNP as an independent variable (adjusted for age, gender, and BMI).

<p>Multiple regression analyses involving plasma resistin (ng/ml) as a dependent variable and each SNP genotype, age (years), gender (male = 0, female = 1), and BMI as independent variables were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009718#s4" target="_blank">Methods</a>. For each SNP genotype, S/S was compared to N/N, and N/S was compared to N/N, respectively where the major and minor alleles based on the allele frequency were defined by normal (N), and susceptibility (S) alleles, respectively.</p

Plasma resistin in subjects with each combination of <i>RETN</i> SNP-420, and SNP-358 genotypes.

<p>Plasma resistin (mean±SD) (ng/ml) is shown for each combination of SNP-420, and SNP-358 genotypes. The number of subjects is shown in parenthesis. Plasma resistin was higher in subjects with SNP-420G/G and SNP-358A/A genotypes than those with any other genotypes (ANOVA, <i>P</i><0.001; Scheffe, <i>P</i><0.001 compared to each of the other genotypes). N/A, not applicable because no subjects had this combination.</p

Association between each SNP around <i>RETN</i> and plasma resistin in the general Japanese population.

<p>ANOVA is used for statistical analysis (<i>n</i> = 2,019). HWE, Hardy-Weinberg Equilibrium. *, these <i>P</i> values remained significant after Bonferroni's correction (raw <i>P</i> value ×8).</p

The <i>RETN</i> promoter with the G-A haplotype defined by SNP-420 and SNP-358 showed the highest activity in THP-1 cells.

<p>The <i>RETN</i> promoter reporter including −450/206 of <i>RETN</i> with each haplotype defined by SNP-420 and SNP-358 was transiently transfected into THP-1 human monocytes. Luciferase activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009718#s4" target="_blank">Methods</a>. Relative luciferase activities are shown as the mean fold induction ± SE relative to the activity of the reference promoter reporter with the C-G haplotype. The data represent eight independent experiments with triplicate wells for each condition. ANOVA, <i>P</i> = 0.002. *, Scheffe, <i>P</i> = 0.008 compared to C-G. The base numbers were shown where the translation initiation site was defined as +1.</p

SNP-358, and SNP-638 were nearly in complete LD, and were tightly correlated with SNP-420 in the general Japanese population.

<p>The 8 SNPs around <i>RETN</i> were genotyped in 2,019 subjects in the general Japanese population, and LD between each pair of these SNPs were analyzed by Haploview <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009718#pone.0009718-Barrett1" target="_blank">[28]</a>. One LD block consisting of SNP-638, SNP -420, SNP-358, and SNP+157 was defined by the confidence interval analysis. Each square represents a pairwise value of <i>D</i>', with the standard gradation (black indicates LOD> = 2 and <i>D</i>' = 1; none indicates, LOD<2 and <i>D</i>' = 1; white indicates LOD<2 and D' <1, gray indicates LOD> = 2 and <i>D</i>'<1). The <i>r²</i> between SNP-638 and SNP-358 was 0.98. The <i>r²</i> between SNP-420 and SNP-358 or SNP-638 was 0.50, and 0.51, respectively.</p

Clinical and Genetic Characteristics of Non-Insulin-Requiring Glutamic Acid Decarboxylase (GAD) Autoantibody-Positive Diabetes: A Nationwide Survey in Japan

<div><p>Aims</p><p>Glutamic acid decarboxylase autoantibodies (GADAb) differentiate slowly progressive insulin-dependent (type 1) diabetes mellitus (SPIDDM) from phenotypic type 2 diabetes, but many GADAb-positive patients with diabetes do not progress to insulin-requiring diabetes. To characterize GADAb-positive patients with adult-onset diabetes who do not require insulin therapy for >5 years (NIR-SPIDDM), we conducted a nationwide cross-sectional survey in Japan.</p><p>Methods</p><p>We collected 82 GADAb-positive patients who did not require insulin therapy for >5 years (NIR-SPIDDM) and compared them with 63 patients with insulin-requiring SPIDDM (IR-SPIDDM). Clinical and biochemical characteristics, HLA-DRB1-DQB1 haplotypes, and predictive markers for progression to insulin therapy were investigated.</p><p>Results</p><p>Compared with the IR-SPIDDM group, the NIR-SPIDDM patients showed later diabetes onset, higher body mass index, longer duration before diagnosis, and less frequent hyperglycemic symptoms at onset. In addition, C-peptide, LDL-cholesterol, and TG were significantly higher in the NIR-SPIDDM compared to IR-SPIDDM patients. The NIR-SPIDDM group had lower frequency of susceptible HLA-DRB1*04:05-DQB1*04:01 and a higher frequency of resistant HLA-DRB1*15:01-DQB1*06:02 haplotype compared to IR-SPIDDM. A multivariable analysis showed that age at diabetes onset (OR = 0.82), duration before diagnosis of GADAb-positive diabetes (OR = 0.82), higher GADAb level (≥10.0 U/ml) (OR = 20.41), and fasting C-peptide at diagnosis (OR = 0.07) were independent predictive markers for progression to insulin-requiring diabetes. An ROC curve analysis showed that the optimal cut-off points for discriminating two groups was the GADAb level of 13.6 U/ml, age of diabetes onset of 47 years, duration before diagnosis of 5 years, and fasting C-peptide of 0.65 ng/ml.</p><p>Conclusions</p><p>Clinical, biochemical and genetic characteristics of patients with NIR-SPIDDM are different from those of IR-SPIDDM patients. Age of diabetes onset, duration before GADAb-positivity, GADAb level, and fasting C-peptide at diagnosis must be carefully considered in planning prevention trials for SPIDDM.</p></div

Frequency of the HLA DRB1-DQB1 haplotype in NIR-SPIDDM, IR-SPIDDM, and healthy controls.

<p>Frequency of the HLA DRB1-DQB1 haplotype in NIR-SPIDDM, IR-SPIDDM, and healthy controls.</p