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1.  Risk of Pediatric Celiac Disease According to HLA Haplotype and Country 
The presence of HLA haplotype DR3–DQ2 or DR4–DQ8 is associated with an increased risk of celiac disease. In addition, nearly all children with celiac disease have serum antibodies against tissue transglutaminase (tTG).
We studied 6403 children with HLA haplotype DR3–DQ2 or DR4–DQ8 prospectively from birth in the United States, Finland, Germany, and Sweden. The primary end point was the development of celiac disease autoimmunity, which was defined as the presence of tTG antibodies on two consecutive tests at least 3 months apart. The secondary end point was the development of celiac disease, which was defined for the purpose of this study as either a diagnosis on biopsy or persistently high levels of tTG antibodies.
The median follow-up was 60 months (interquartile range, 46 to 77). Celiac disease autoimmunity developed in 786 children (12%). Of the 350 children who underwent biopsy, 291 had confirmed celiac disease; an additional 21 children who did not undergo biopsy had persistently high levels of tTG antibodies. The risks of celiac disease autoimmunity and celiac disease by the age of 5 years were 11% and 3%, respectively, among children with a single DR3–DQ2 haplotype, and 26% and 11%, respectively, among those with two copies (DR3–DQ2 homozygosity). In the adjusted model, the hazard ratios for celiac disease autoimmunity were 2.09 (95% confidence interval [CI], 1.70 to 2.56) among heterozygotes and 5.70 (95% CI, 4.66 to 6.97) among homozygotes, as compared with children who had the lowest-risk genotypes (DR4–DQ8 heterozygotes or homozygotes). Residence in Sweden was also independently associated with an increased risk of celiac disease autoimmunity (hazard ratio, 1.90; 95% CI, 1.61 to 2.25).
Children with the HLA haplotype DR3–DQ2, especially homozygotes, were found to be at high risk for celiac disease autoimmunity and celiac disease early in childhood. The higher risk in Sweden than in other countries highlights the importance of studying environmental factors associated with celiac disease. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others.)
PMCID: PMC4163840  PMID: 24988556
2.  GAD65 Autoantibodies Detected by Electrochemiluminescence Assay Identify High Risk for Type 1 Diabetes 
Diabetes  2013;62(12):4174-4178.
The identification of diabetes-relevant islet autoantibodies is essential for predicting and preventing type 1 diabetes (T1D). The aim of the current study was to evaluate a newly developed electrochemiluminescence (ECL)-GAD antibody (GADA) assay and compare its sensitivity and disease relevance with standard radioassay. The assay was validated with serum samples from 227 newly diagnosed diabetic children; 68 prediabetic children who were prospectively followed to T1D; 130 nondiabetic children with confirmed islet autoantibodies to insulin, GAD65, IA-2, and/or ZnT8 longitudinally followed for 12 ± 3.7 years; and 181 age-matched, healthy, antibody-negative children. The ECL-GADA assay had a sensitivity similar to that of the standard GADA radioassay in children newly diagnosed with T1D, prediabetic children, and high-risk children with multiple positive islet autoantibodies. On the other hand, only 9 of 39 nondiabetic children with only a single islet autoantibody (GADA only) by radioassay were positive for ECL-GADA. GADA not detectable by ECL assay is shown to be of low affinity and likely not predictive of future diabetes. In conclusion, the new ECL assay identifies disease-relevant GADA by radioassay. It may help to improve the prediction and correct diagnosis of T1D among subjects positive only for GADA and no other islet autoantibodies.
PMCID: PMC3837058  PMID: 23974918
3.  Early Diabetic Nephropathy 
Diabetes Care  2013;36(11):3678-3683.
Diabetic nephropathy (DN) is a major cause of mortality in type 1 diabetes. Reduced insulin sensitivity is a well-documented component of type 1 diabetes. We hypothesized that baseline insulin sensitivity would predict development of DN over 6 years.
We assessed the relationship between insulin sensitivity at baseline and development of early phenotypes of DN—microalbuminuria (albumin-creatinine ratio [ACR] ≥30 mg/g) and rapid renal function decline (glomerular filtration rate [GFR] loss >3 mL/min/1.73 m2 per year)—with three Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations over 6 years. Subjects with diabetes (n = 449) and without diabetes (n = 565) in the Coronary Artery Calcification in Type 1 Diabetes study had an estimated insulin sensitivity index (ISI) at baseline and 6-year follow-up.
The ISI was lower in subjects with diabetes than in those without diabetes (P < 0.0001). A higher ISI at baseline predicted a lower odds of developing an ACR ≥30 mg/g (odds ratio 0.65 [95% CI 0.49–0.85], P = 0.003) univariately and after adjusting for HbA1c (0.69 [0.51–0.93], P = 0.01). A higher ISI at baseline conferred protection from a rapid decline of GFR as assessed by CKD-EPI cystatin C (0.77 [0.64–0.92], P = 0.004) and remained significant after adjusting for HbA1c and age (0.80 [0.67–0.97], P = 0.02). We found no relation between ISI and rapid GFR decline estimated by CKD-EPI creatinine (P = 0.38) or CKD-EPI combined cystatin C and creatinine (P = 0.50).
Over 6 years, a higher ISI independently predicts a lower odds of developing microalbuminuria and rapid GFR decline as estimated with cystatin C, suggesting a relationship between insulin sensitivity and early phenotypes of DN.
PMCID: PMC3816872  PMID: 24026551
4.  Insulin Sensitivity and Insulin Clearance are Heritable and Have Strong Genetic Correlation in Mexican Americans 
Obesity (Silver Spring, Md.)  2014;22(4):1157-1164.
We describe the GUARDIAN (Genetics UndeRlying DIAbetes in HispaNics) consortium, along with heritability estimates and genetic and environmental correlations of insulin sensitivity and metabolic clearance rate of insulin (MCRI).
Design and Methods
GUARDIAN is comprised of seven cohorts, consisting of 4336 Mexican-American individuals in 1346 pedigrees. Insulin sensitivity (SI), MCRI, and acute insulin response (AIRg) were measured by frequently sampled intravenous glucose tolerance test in four cohorts. Insulin sensitivity (M, M/I) and MCRI were measured by hyperinsulinemic-euglycemic clamp in three cohorts. Heritability and genetic and environmental correlations were estimated within the family cohorts (totaling 3925 individuals) using variance components.
Across studies, age and gender-adjusted heritability of insulin sensitivity (SI, M, M/I) ranged from 0.23–0.48 and of MCRI from 0.35–0.73. The ranges for the genetic correlations were 0.91 to 0.93 between SI and MCRI; and −0.57 to −0.59 for AIRg and MCRI (all P<0.0001). The ranges for the environmental correlations were 0.54 to 0.74 for SI and MCRI (all P<0.0001); and −0.16 to −0.36 for AIRg and MCRI (P <0.0001−0.06).
These data support a strong familial basis for insulin sensitivity and MCRI in Mexican Americans. The strong genetic correlations between MCRI and SI suggest common genetic determinants.
PMCID: PMC3968231  PMID: 24124113
insulin sensitivity; insulin clearance; heritability; genetic correlation; environmental correlation
5.  Methods, Quality Control and Specimen Management in an International Multi-Center Investigation of Type 1 Diabetes: TEDDY 
The Environmental Determinants of Diabetes in the Young (TEDDY) is a multi-center, international prospective study (n = 8,677) designed to identify environmental triggers of type 1 diabetes (T1D) in genetically at-risk children from age 3 months until 15 years. The study is conducted through six primary clinical centers located in four countries. As of May 2012, over three million biological samples and 250 million total data points have been collected which will be analyzed to assess autoimmunity status, presence of inflammatory biomarkers, genetic factors, exposure to infectious agents, dietary biomarkers, and other potentially important environmental exposures in relation to autoimmunity and progression to T1D. The vast array and quantity of longitudinal samples collected in the TEDDY study present a series of challenges in terms of quality control procedures and data validity. To address this, pilot studies have been conducted to standardize and enhance both biospecimen collection and sample obtainment in terms of autoantibody collection, stool sample preservation, RNA, biomarker stability, metabolic biomarkers, and T-cell viability. This paper details the procedures utilized to standardize both data harmonization and management when handling a large quantity of longitudinal samples obtained from multiple locations. In addition, we provide a description of the available specimens that serve as an invaluable repository for the elucidation of determinants in T1D focusing on autoantibody concordance and harmonization, transglutaminase autoantibody (tGA), inflammatory biomarkers (T-cells), genetic proficiency testing, RNA lab internal quality control testing, infectious agents (monitoring cross contamination, virus preservation, and nasal swab collection validity), and HbA1c testing.
PMCID: PMC3992860  PMID: 23674484
quality control; data integrity; stool sample preservation; RNA; biomarker stability; metabolic biomarkers; T-cell viability
6.  Impaired Renal Function Further Increases Odds of 6-Year Coronary Artery Calcification Progression in Adults With Type 1 Diabetes 
Diabetes Care  2013;36(9):2607-2614.
To determine whether baseline estimated glomerular filtration rate (eGFR) and albumin-to-creatinine ratio (ACR) independently predict coronary artery calcification (CAC) progression, and to determine how eGFR changes over 6 years in adults with type 1 diabetes compared with nondiabetic adults.
The Coronary Artery Calcification in Type 1 Diabetes study participants (n = 1,066) with complete data for eGFR assessment at baseline and 6 years were included. Three Chronic Kidney Disease Epidemiology Collaboration equations (serum creatinine, cystatin C, and both) were used to estimate eGFR. The association of baseline ACR and eGFR with CAC progression was analyzed using multiple logistic regression.
Increasing categorical baseline ACR (<10, 10–30, and >30 µg/mg) predicted CAC progression in participants with type 1 diabetes (odds ratio [OR], 2.15; 95% CI, 1.50–3.09; 7.19 [3.90–13.26]; and 18.09 [8.48–38.62]), respectively, compared with nondiabetic subjects. Baseline eGFR <60 mL/min/1.73 m2 also predicted CAC progression (OR, 5–7, compared with nondiabetic participants). ORs for CAC progression were higher in women than in men when using the cystatin C–based Chronic Kidney Disease Epidemiology Collaboration equations. Participants with type 1 diabetes had greater eGFR decreases over 6 years than nondiabetic participants using cystatin C–based equations.
Although increasing ACR or decreasing eGFR predicts CAC progression, coronary atherosclerosis progresses faster in people with type 1 diabetes even in the absence of diabetic kidney disease. These findings emphasize the interaction between kidney disease and cardiovascular disease in type 1 diabetes and highlight the public health importance of lowering cardiorenal risk in people with type 1 diabetes.
PMCID: PMC3747879  PMID: 23835686
7.  Prospective Association Between Inflammatory Markers and Progression of Coronary Artery Calcification in Adults With and Without Type 1 Diabetes 
Diabetes Care  2013;36(7):1967-1973.
The role of inflammation in the increased risk of cardiovascular disease in type 1 diabetes is unclear. We examined the association of inflammation and progression of coronary artery calcification (CAC)—a marker of subclinical atherosclerosis—in adults with and without type 1 diabetes.
A nested case-control study was performed within the prospective cohort of the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. Participants underwent two CAC measurements ∼2.5 years apart. Case subjects (n = 204) were those with significant progression of CAC. Control subjects (n = 258) were frequency-matched to case subjects on diabetes status, sex, age, and baseline CAC status. Inflammatory marker assessments were performed on stored blood samples from baseline. A principal components analysis (PCA) was performed and a composite score derived from that analysis. The composite score was constructed by assigning a value of 1 for each PCA component where at least one of the markers exceeded the 75th percentile (range 0–4). Conditional logistic regression was used for the matching strategy.
The first two components of the PCA were modestly (odds ratio 1.38 [95% CI 1.08–1.77] and 1.27 [1.02–1.59], respectively) associated with CAC progression after adjustment for other risk factors. The composite score was more strongly associated with CAC progression for those with elevated markers in three or four of the principal components compared with those with none.
Measures of inflammation were associated with progression of CAC in a population of adults with and without type 1 diabetes.
PMCID: PMC3687315  PMID: 23340891
8.  The Next Big Idea 
Diabetes Technology & Therapeutics  2013;15(Suppl 2):S2-29-S2-36.
George S. Eisenbarth will remain in our memories as a brilliant scientist and great collaborator. His quest to discover the cause and prevention of type 1 (autoimmune) diabetes started from building predictive models based on immunogenetic markers. Despite his tremendous contributions to our understanding of the natural history of pre-type 1 diabetes and potential mechanisms, George left us with several big questions to answer before his quest is completed.
PMCID: PMC3676661  PMID: 23786296
9.  Infant Exposures and Development of Type 1 Diabetes Mellitus 
JAMA pediatrics  2013;167(9):808-815.
The incidence of type 1 diabetes mellitus (T1DM) is increasing worldwide, with the most rapid increase among children younger than 5 years of age.
To examine the associations between perinatal and infant exposures, especially early infant diet, and the development of T1DM.
The Diabetes Autoimmunity Study in the Young (DAISY) is a longitudinal, observational study.
Newborn screening for human leukocyte antigen (HLA) was done at St. Joseph’s Hospital in Denver, Colorado. First-degree relatives of individuals with T1DM were recruited from the Denver metropolitan area.
A total of 1835 children at increased genetic risk for T1DM followed up from birth with complete prospective assessment of infant diet. Fifty-three children developed T1DM.
Early (<4 months of age) and late (≥6 months of age) first exposure to solid foods compared with first exposures at 4 to 5 months of age (referent).
Risk for T1DM diagnosed by a physician.
Both early and late first exposure to any solid food predicted development of T1DM (hazard ratio [HR], 1.91; 95% CI, 1.04–3.51, and HR, 3.02; 95% CI, 1.26–7.24, respectively), adjusting for the HLA-DR genotype, first-degree relative with T1DM, maternal education, and delivery type. Specifically, early exposure to fruit and late exposure to rice/oat predicted T1DM (HR, 2.23; 95% CI, 1.14–4.39, and HR, 2.88; 95% CI, 1.36–6.11, respectively), while breastfeeding at the time of introduction to wheat/barley conferred protection (HR, 0.47; 95% CI, 0.26–0.86). Complicated vaginal delivery was also a predictor of T1DM (HR, 1.93; 95% CI, 1.03–3.61).
These results suggest the safest age to introduce solid foods in children at increased genetic risk for T1DM is between 4 and 5 months of age. Breastfeeding while introducing new foods may reduce T1DM risk.
PMCID: PMC4038357  PMID: 23836309
10.  The effect of insurance status and parental education on glycemic control and cardiovascular disease risk profile in youth with Type 1 Diabetes 
Adult studies have shown a correlation between low socioeconomic status and Type 1 Diabetes complications, but studies have not been done in children to examine the effect of socioeconomic status on risk for future complications. This study investigates the relationship between insurance status and parental education and both glycemic control and cardiovascular disease (CVD) risk factors in youth with type 1 diabetes.
A cross-sectional study of 295 youth with established type 1 diabetes who underwent examination with fasting blood draw and reported insurance status and parental education.
Youth with type 1 diabetes and public insurance had higher hemoglobin A1c (HbA1c), body mass index, hs-CRP, and blood pressure (p < 0.05) than those with private insurance. Insulin regimen varied between insurance groups, and differences in HbA1c and CVD risk factors, except for diastolic blood pressure (DBP), were no longer evident after controlling for insulin regimen. Parental education was not associated with HbA1c or CVD risk factors.
Youth with type 1 diabetes and public insurance have worse glycemic control and elevated CVD risk factors compared to those with private insurance, but this was no longer seen when insulin regimen was controlled for. Further research is needed to look at differences between those with public insurance and private insurance that contribute to differences in type 1 diabetes outcomes, and to identify modifiable risk factors in pediatric patients in order to focus earlier interventions to decrease and prevent future diabetes complications.
PMCID: PMC4064822  PMID: 24955334
Type 1 diabetes; Socioeconomic status; Parental education; Hemoglobin A1c; Cardiovascular disease
11.  Components of metabolic syndrome and 5-year change in insulin clearance - The Insulin Resistance Atherosclerosis Study (IRAS) 
Diabetes, obesity & metabolism  2013;15(5):10.1111/dom.12049.
Cross-sectional evidence indicates that abdominal adiposity, hypertension, dyslipidemia and glycemia are associated with reduced metabolic clearance of insulin (MCRI). Little is known about the progression of MCRI and whether components of metabolic syndrome are associated with the change in MCRI. In this study, we examined the association between components of metabolic syndrome and the 5-year change of MCRI.
Methods and Materials
At baseline and 5-year follow-up, we measured fasting plasma triglycerides (TG), high density lipoprotein (HDL)-cholesterol, blood pressure (BP), waist circumference (WC) and fasting blood glucose (FBG) in 784 non-diabetic participants in the Insulin Resistance Atherosclerosis Study. MCRI, insulin sensitivity (SI) and acute insulin response (AIR) were determined from frequently sampled intravenous glucose tolerance tests.
We observed a 29% decline of MCRI at follow-up. TG, systolic BP and WC at baseline were inversely associated with a decline of MCRI regression models adjusted for age, sex, ethnicity, smoking, alcohol consumption, energy expenditure, family history of diabetes, BMI, SI and AIR (β= −0.057 [95% CI −0.11, −0.0084] for TG, β= −0.0019 [95% CI −0.0035, −0.00023] for systolic BP, β= −0.0084 [95% CI −0.013, −0.0039] for WC; all p<0.05). Higher HDL-cholesterol at baseline was associated with an increase in MCRI (multivariable-adjusted β= 0.0029 [95% CI 0.0010, 0.0048], p=0.002). FBG at baseline was not associated with MCRI at follow-up (multivariable-adjusted β= 0.0014 [95% CI −0.0026, 0.0029]).
MCRI declined progressively over 5 years in a non-diabetic cohort. Components of metabolic syndrome at baseline were associated with a significant change in MCRI.
PMCID: PMC3810428  PMID: 23216702
12.  Fasting Blood Glucose-A Missing Variable for GFR-Estimation in Type 1 Diabetes? 
PLoS ONE  2014;9(4):e96264.
Estimation of glomerular filtration rate (eGFR) is one of the current clinical methods for identifying risk for diabetic nephropathy in subjects with type 1 diabetes (T1D). Hyperglycemia is known to influence GFR in T1D and variability in blood glucose at the time of eGFR measurement could introduce bias in eGFR. We hypothesized that simultaneously measured blood glucose would influence eGFR in adults with T1D.
Longitudinal multivariable mixed-models were employed to investigate the relationships between blood glucose and eGFR by CKD-EPI eGFRCYSTATIN C over 6-years in the Coronary Artery Calcification in Type 1 diabetes (CACTI) study. All subjects with T1D and complete data including blood glucose and cystatin C for at least one of the three visits (n = 616, 554, and 521, respectively) were included in the longitudinal analyses.
In mixed-models adjusting for sex, HbA1c, ACEi/ARB, protein and sodium intake positive associations were observed between simultaneous blood glucose and eGFRCYSTATIN C (β±SE:0.14±0.04 per 10 mg/dL of blood glucose, p<0.0001), and hyperfiltration as a dichotomous outcome (OR: 1.04, 95% CI: 1.01–1.07 per 10 mg/dL of blood glucose, p = 0.02).
In our longitudinal data in subjects with T1D, simultaneous blood glucose has an independent positive effect on eGFRCYSTATIN C. The associations between blood glucose and eGFRCYSTATIN C may bias the accurate detection of early diabetic nephropathy, especially in people with longitudinal variability in blood glucose.
PMCID: PMC4004575  PMID: 24781861
13.  Improving coeliac disease risk prediction by testing non-HLA variants additional to HLA variants 
Gut  2013;63(3):415-422.
The majority of coeliac disease (CD) patients are not being properly diagnosed and therefore remain untreated, leading to a greater risk of developing CD-associated complications. The major genetic risk heterodimer, HLA-DQ2 and DQ8, is already used clinically to help exclude disease. However, approximately 40% of the population carry these alleles and the majority never develop CD.
We explored whether CD risk prediction can be improved by adding non-HLA-susceptible variants to common HLA testing.
We developed an average weighted genetic risk score with 10, 26 and 57 single nucleotide polymorphisms (SNP) in 2675 cases and 2815 controls and assessed the improvement in risk prediction provided by the non-HLA SNP. Moreover, we assessed the transferability of the genetic risk model with 26 non-HLA variants to a nested case–control population (n=1709) and a prospective cohort (n=1245) and then tested how well this model predicted CD outcome for 985 independent individuals.
Adding 57 non-HLA variants to HLA testing showed a statistically significant improvement compared to scores from models based on HLA only, HLA plus 10 SNP and HLA plus 26 SNP. With 57 non-HLA variants, the area under the receiver operator characteristic curve reached 0.854 compared to 0.823 for HLA only, and 11.1% of individuals were reclassified to a more accurate risk group. We show that the risk model with HLA plus 26 SNP is useful in independent populations.
Predicting risk with 57 additional non-HLA variants improved the identification of potential CD patients. This demonstrates a possible role for combined HLA and non-HLA genetic testing in diagnostic work for CD.
PMCID: PMC3933173  PMID: 23704318
Coeliac Disease; Genetic Testing; Hla; Molecular Genetics; Celiac Disease
15.  Relationship of Insulin Sensitivity, Insulin Secretion, and Adiposity With Insulin Clearance in a Multiethnic Population 
Diabetes Care  2012;36(1):101-103.
We aimed to examine insulin clearance, a compensatory mechanism to changes in insulin sensitivity, across sex, race/ethnicity populations, and varying states of glucose tolerance.
We measured insulin sensitivity index (SI), acute insulin response (AIR), and metabolic clearance rate of insulin (MCRI) by the frequently sampled intravenous glucose tolerance test in 1,295 participants in the Insulin Resistance Atherosclerosis Study.
MCRI was positively related to SI and negatively to AIR and adiposity across sex, race/ethnicity populations, and varying states of glucose tolerance, adiposity, and family history of diabetes. Differences in MCRI by race/ethnicity (lower in African Americans and Hispanics compared with non-Hispanic whites) and glucose tolerance were largely explained by differences in adiposity, SI, and AIR.
Insulin sensitivity, insulin secretion, and adiposity are correlates of insulin clearance and appear to explain differences in insulin clearance by race/ethnicity and glucose tolerance status.
PMCID: PMC3526225  PMID: 22933441
16.  Evidence of Stage- and Age-Related Heterogeneity of Non-HLA SNPs and Risk of Islet Autoimmunity and Type 1 Diabetes: The Diabetes Autoimmunity Study in the Young 
Previously, we examined 20 non-HLA SNPs for association with islet autoimmunity (IA) and/or progression to type 1 diabetes (T1D). Our objective was to investigate fourteen additional non-HLA T1D candidate SNPs for stage- and age-related heterogeneity in the etiology of T1D. Of 1634 non-Hispanic white DAISY children genotyped, 132 developed IA (positive for GAD, insulin, or IA-2 autoantibodies at two or more consecutive visits); 50 IA positive children progressed to T1D. Cox regression was used to analyze risk of IA and progression to T1D in IA positive children. Restricted cubic splines were used to model SNPs when there was evidence that risk was not constant with age. C1QTNF6 (rs229541) predicted increased IA risk (HR: 1.57, CI: 1.20–2.05) but not progression to T1D (HR: 1.13, CI: 0.75–1.71). SNP (rs10517086) appears to exhibit an age-related effect on risk of IA, with increased risk before age 2 years (age 2 HR: 1.67, CI: 1.08–2.56) but not older ages (age 4 HR: 0.84, CI: 0.43–1.62). C1QTNF6 (rs229541), SNP (rs10517086), and UBASH3A (rs3788013) were associated with development of T1D. This prospective investigation of non-HLA T1D candidate loci shows that some SNPs may exhibit stage- and age-related heterogeneity in the etiology of T1D.
PMCID: PMC3866813  PMID: 24367383
17.  Early Childhood Infections and the Risk of Islet Autoimmunity 
Diabetes Care  2012;35(12):2553-2558.
Type 1 diabetes is a common chronic childhood disease, and the incidence is increasing globally. Childhood infections are considered a potential environmental trigger of type 1 diabetes. Alternatively, improved hygiene and reduced childhood infections could explain the increase in type 1 diabetes in developed countries. The association of reported illnesses during infancy and later development of islet autoimmunity (IA) were examined in the Diabetes Autoimmunity Study in the Young.
Complete illness interviews through 9 months of age were collected for 1,729 children—1,174 without a family history of type 1 diabetes and 555 with a first-degree relative with type 1 diabetes. Persistent IA was defined as positive antibodies to insulin, glutamic acid decarboxylase, or tyrosine phosphatase on at least two consecutive study visits.
There were 109 children with persistent IA among the 1,729 children with illness records. A greater number of gastrointestinal illnesses were associated with an increased risk of IA, but only among children who were exposed to gluten-containing grains (wheat or barley) either <4 months of age (hazard ratio 1.37 [95% CI 1.22–1.55]; P < 0.0001) or ≥7 months of age (1.12 [1.05–1.19]; P = 0.0005) compared with 4–6 months of age (P for interaction = 0.02). There were no associations of upper respiratory symptoms, respiratory illnesses, or fevers with IA.
Specific pathogens such as enteroviruses or rotavirus may increase the risk of IA in the presence of existing inflammation induced by diet.
PMCID: PMC3507568  PMID: 23043167
18.  Evidence of Gene-Gene Interaction and Age-at-Diagnosis Effects in Type 1 Diabetes 
Diabetes  2012;61(11):3012-3017.
The common genetic loci that independently influence the risk of type 1 diabetes have largely been determined. Their interactions with age-at-diagnosis of type 1 diabetes, sex, or the major susceptibility locus, HLA class II, remain mostly unexplored. A large collection of more than 14,866 type 1 diabetes samples (6,750 British diabetic individuals and 8,116 affected family samples of European descent) were genotyped at 38 confirmed type 1 diabetes-associated non-HLA regions and used to test for interaction of association with age-at-diagnosis, sex, and HLA class II genotypes using regression models. The alleles that confer susceptibility to type 1 diabetes at interleukin-2 (IL-2), IL2/4q27 (rs2069763) and renalase, FAD-dependent amine oxidase (RNLS)/10q23.31 (rs10509540), were associated with a lower age-at-diagnosis (P = 4.6 × 10−6 and 2.5 × 10−5, respectively). For both loci, individuals carrying the susceptible homozygous genotype were, on average, 7.2 months younger at diagnosis than those carrying the protective homozygous genotypes. In addition to protein tyrosine phosphatase nonreceptor type 22 (PTPN22), evidence of statistical interaction between HLA class II genotypes and rs3087243 at cytotoxic T-lymphocyte antigen 4 (CTLA4)/2q33.2 was obtained (P = 7.90 × 10−5). No evidence of differential risk by sex was obtained at any loci (P ≥ 0.01). Statistical interaction effects can be detected in type 1 diabetes although they provide a relatively small contribution to our understanding of the familial clustering of the disease.
PMCID: PMC3478521  PMID: 22891215
19.  Dysregulated Toll-Like Receptor–Induced Interleukin-1β and Interleukin-6 Responses in Subjects at Risk for the Development of Type 1 Diabetes 
Diabetes  2012;61(10):2525-2533.
We tested the hypothesis that altered Toll-like receptor (TLR) signaling may be involved in early stages of type 1 diabetes (T1D). To do so, we analyzed TLR-induced interleukin (IL)-1β and IL-6 responses in freshly isolated peripheral blood mononuclear cells (PBMNCs) from seropositive compared with seronegative subjects. Similar frequencies of myeloid dendritic cells (mDCs), plasmacytoid DCs (pDCs), and monocytes were observed in seropositive and seronegative subjects. Subjects with autoantibodies had increased proportions of monocytes expressing IL-1β ex vivo. Activating PBMNCs with TLR3, TLR4, or TLR7/8 agonists in vitro led to increased percentages of IL-1β–expressing monocytes and mDCs from seropositive versus seronegative subjects. TLR ligation also resulted in a diminished IL-6 response in seropositive individuals as lower frequencies of IL-6–expressing monocytes and mDCs were induced. The dysregulated TLR-induced IL-1β and IL-6 pathways were more readily detectable in children aged <11 years and from 11 to <21 years, respectively, and did not involve altered HbA1c or the presence of one or more autoantibodies. Finally, subjects with autoantibodies had lower amounts of serum chemokine (C-X-C motif) ligand 10 compared with autoantibody-negative subjects. Our data may imply that alterations in innate immune pathways are detectable in genetically susceptible individuals and could be linked with the early course of T1D.
PMCID: PMC3447890  PMID: 22751696
20.  Performance of HbA1c as an Early Diagnostic Indicator of Type 1 Diabetes in Children and Youth 
Diabetes Care  2012;35(9):1821-1825.
The aim of this study was to evaluate HbA1c as an alternative criterion for impaired glucose tolerance (IGT) or type 1 diabetes (T1D) in high-risk subjects <21 years of age.
Subjects <21 years of age who participated in the prospective DPT-1, TEDDY, TRIGR, and Type 1 Diabetes TrialNet Natural History (TrialNet) studies and had an HbA1c within 90 days of an OGTT with a 2-h plasma glucose (2-hPG) measure were included. An OGTT of 140–199 mg/dL defined IGT, and an OGTT with 2-hPG ≥200 mg/dL or fasting plasma glucose ≥126 mg/dL defined diabetes. HbA1c ≥5.7% defined IGT, and HbA1c ≥ 6.5% defined diabetes. Receiver-operating characteristic curve analysis was used to assess diagnostic accuracy of HbA1c compared with OGTT.
There were 587 subjects from DPT-1, 884 from TrialNet, 91 from TEDDY, and 420 from TRIGR. As an indicator for IGT, HbA1c sensitivity was very low across the studies (8–42%), and specificity was variable (64–95%). With HbA1c ≥6.5% threshold used for T1D diagnosis, the sensitivity was very low and specificity was high (sensitivity and specificity: DPT-1 24 and 98%, TrialNet 28 and 99%, TEDDY 34 and 98%, and TRIGR 33 and 99%, respectively). The positive predictive value of HbA1c ≥6.5% for the development of T1D was variable (50–94%) across the four studies.
HbA1c ≥6.5% is a specific but not sensitive early indicator for T1D in high-risk subjects <21 years of age diagnosed by OGTT or asymptomatic hyperglycemia. Redefining the HbA1c threshold is recommended if used as an alternative criterion in diagnosing T1D.
PMCID: PMC3425003  PMID: 22699293
21.  An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype 
Haptoglobin (Hp) is an abundant serum protein which binds extracorpuscular hemoglobin (Hb). Two alleles exist in humans for the Hp gene, denoted 1 and 2. Diabetic individuals with the Hp 2-2 genotype are at increased risk of developing vascular complications including heart attack, stroke, and kidney disease. Recent evidence shows that treatment with vitamin E can reduce the risk of diabetic vascular complications by as much as 50% in Hp 2-2 individuals. We sought to develop a rapid and accurate test for Hp phenotype (which is 100% concordant with the three major Hp genotypes) to facilitate widespread diagnostic testing as well as prospective clinical trials.
A monoclonal antibody raised against human Hp was shown to distinguish between the three Hp phenotypes in an enzyme linked immunosorbent assay (ELISA). Hp phenotypes obtained in over 8000 patient samples using this ELISA method were compared with those obtained by polyacrylamide gel electrophoresis or the TaqMan PCR method.
Our analysis showed that the sensitivity and specificity of the ELISA test for Hp 2-2 phenotype is 99.0% and 98.1%, respectively. The positive predictive value and the negative predictive value for Hp 2-2 phenotype is 97.5% and 99.3%, respectively. Similar results were obtained for Hp 2-1 and Hp 1-1 phenotypes. In addition, the ELISA was determined to be more sensitive and specific than the TaqMan method.
The Hp ELISA represents a user-friendly, rapid and highly accurate diagnostic tool for determining Hp phenotypes. This test will greatly facilitate the typing of thousands of samples in ongoing clinical studies.
PMCID: PMC3717392  PMID: 23492570
diabetes; ELISA; haptoglobin phenotype; pharmacogenomics; vitamin E
22.  Serum proteomics reveals systemic dysregulation of innate immunity in type 1 diabetes 
Proteomics analysis identifies human serum proteins involved with innate immune responses, complement activation, and blood coagulation that are diagnostic for type 1 diabetes.
Using global liquid chromatography-mass spectrometry (LC-MS)–based proteomics analyses, we identified 24 serum proteins that were significantly variant between those with type 1 diabetes (T1D) and healthy controls. Functionally, these proteins represent innate immune responses, the activation cascade of complement, inflammatory responses, and blood coagulation. Targeted verification analyses were performed on 52 surrogate peptides representing these proteins, with serum samples from an antibody standardization program cohort of 100 healthy control and 50 type 1 diabetic subjects. 16 peptides were verified as having very good discriminating power, with areas under the receiver operating characteristic curve ≥0.8. Further validation with blinded serum samples from an independent cohort (10 healthy control and 10 type 1 diabetics) demonstrated that peptides from platelet basic protein and C1 inhibitor achieved both 100% sensitivity and 100% specificity for classification of samples. The disease specificity of these proteins was assessed using sera from 50 age-matched type 2 diabetic individuals, and a subset of proteins, C1 inhibitor in particular, were exceptionally good discriminators between these two forms of diabetes. The panel of biomarkers distinguishing those with T1D from healthy controls and those with type 2 diabetes suggests that dysregulated innate immune responses may be associated with the development of this disorder.
PMCID: PMC3549705  PMID: 23277452
23.  Replication and Further Characterization of a Type 1 Diabetes-Associated Locus at the Telomeric End of the Major Histocompatibility Complex 
Journal of diabetes  2011;3(3):238-247.
We recently reported an association with type 1 diabetes of a telomeric MHC SNP rs1233478. As further families have been analyzed in the Type 1 Diabetes Genetics Consortium (T1DGC), we sought to test replication of the association and with more data analyze haplotypic associations.
Research Design and Methods
We have since analyzed an additional 2,717 case and 1,315 control chromosomes from the T1DGC, with HLA-typing and data for 2,837 SNPs across the MHC region.
We confirmed the association of rs1233478 [new data only: p=2.2E-5, OR=1.4]. We also found two additional SNPs nearby which were significantly associated with type 1 diabetes (new data only rs3131020: p=8.3E-9, OR=0.65; rs1592410 p=2.2E-8, OR=1.5). For studies of type 1 diabetes in the MHC region it is critical to account for linkage disequilibrium with the HLA genes. Logistic regression analysis of this new data indicated that the effects of rs3131020 and rs1592410 on type 1 diabetes risk are independent of HLA alleles (rs3131020: p=2.3E-3, OR=0.73; rs1592410: p=2.1E-3, OR=1.4). Haplotypes of 12 SNPs (including the three highly significant SNPs) stratify diabetes risk (high risk, protective, and neutral), with high risk haplotypes limited to approximately 20,000 base pairs in length. The 20,000 base pair region is telomeric of the UBD gene and contains LOC729653, a hypothetical gene.
We believe that polymorphisms of the telomeric MHC locus LOC729653 may confer risk for type 1 diabetes.
PMCID: PMC3610173  PMID: 21631897
genetic association studies; major histocompatibility complex; type 1 diabetes
24.  Effectiveness of an Informational Video Method to Improve Enrollment and Retention of a Pediatric Cohort 
Contemporary Clinical Trials  2011;33(2):273-278.
The Environmental Determinants of Diabetes in the Young (TEDDY), a multinational epidemiological study, is designed to identify environmental exposures triggering autoimmunity and type 1 diabetes (T1D) in children at increased genetic risk. The objective of this analysis was to evaluate the use of an informational video in the enrollment and retention of eligible participants at the Colorado TEDDY clinical center.
Study Design and Setting
Eligible participants were divided into two groups based on the inclusion of the video in the enrollment materials: the No-Video Group (n=449) did not receive the video and were contacted between 7/1/07 and 6/30/08. The Video Group (n=494) received the video and were contacted between 7/1/08 and 6/30/09. Multiple logistic regression compared the enrollment rates (percent of eligible subjects deciding to enroll) of those who received the video compared to those who did not. Kaplan-Meier survival analysis and a multivariate Cox proportional hazards model compared the differences in study retention, as defined by active participation fifteen months after the baseline visit at three months of age.
Both groups were demographically similar. The enrollment rate was significantly higher for the Video Group (56.9%) compared to the No-Video Group (49.9%). Differences remained significant with adjustment for other known factors. A difference in retention between the two groups was not observed.
Methods and materials increasing understanding and more accurately informing participants of what is involved in participation may increase enrollment in a prospective observational study.
PMCID: PMC3268864  PMID: 22101229
pediatric observational study; enrollment; informed consent; video methods; type 1 diabetes mellitus
25.  Effects of Non-HLA Gene Polymorphisms on Development of Islet Autoimmunity and Type 1 Diabetes in a Population With High-Risk HLA-DR,DQ Genotypes 
Diabetes  2012;61(3):753-758.
We assessed the effects of non-HLA gene polymorphisms on the risk of islet autoimmunity (IA) and progression to type 1 diabetes in the Diabetes Autoimmunity Study in the Young. A total of 1,743 non-Hispanic, white children were included: 861 first-degree relatives and 882 general population children identified as having high-risk HLA-DR/DQ genotypes for type 1 diabetes. Of those, 109 developed IA and 61 progressed to diabetes. Study participants were genotyped for 20 non-HLA polymorphisms, previously confirmed as type 1 diabetes susceptibility loci. PTPN22 and UBASH3A predicted both IA and diabetes in regression models controlling for family history of type 1 diabetes and presence of HLA-DR3/4-DQB1*0302 genotype. In addition, PTPN2 predicted IA whereas INS predicted type 1 diabetes. The final multivariate regression models for both IA and type 1 diabetes included PTPN22, UBASH3A, and INS, in addition to family history of type 1 diabetes and HLA-DR3/4. In general population children, the most frequent combinations including these five significant predictors conferred hazard ratio of up to 13 for IA and >40 for type 1 diabetes. Non-HLA susceptibility alleles may help estimate risk for development of type 1 diabetes in the general population. These findings require replication in different populations.
PMCID: PMC3282811  PMID: 22315323

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