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1.  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
2.  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
3.  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
4.  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
5.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  Novel Urinary Protein Biomarkers Predicting the Development of Microalbuminuria and Renal Function Decline in Type 1 Diabetes 
Diabetes Care  2012;35(3):549-555.
To define a panel of novel protein biomarkers of renal disease.
Adults with type 1 diabetes in the Coronary Artery Calcification in Type 1 Diabetes study who were initially free of renal complications (n = 465) were followed for development of micro- or macroalbuminuria (MA) and early renal function decline (ERFD, annual decline in estimated glomerular filtration rate of ≥3.3%). The label-free proteomic discovery phase was conducted in 13 patients who progressed to MA by the 6-year visit and 11 control subjects, and four proteins (Tamm-Horsfall glycoprotein, α-1 acid glycoprotein, clusterin, and progranulin) identified in the discovery phase were measured by enzyme-linked immunosorbent assay in 74 subjects: group A, normal renal function (n = 35); group B, ERFD without MA (n = 15); group C, MA without ERFD (n = 16); and group D, both ERFD and MA (n = 8).
In the label-free analysis, a model of progression to MA was built using 252 peptides, yielding an area under the curve (AUC) of 84.7 ± 5.3%. In the validation study, ordinal logistic regression was used to predict development of ERFD, MA, or both. A panel including Tamm-Horsfall glycoprotein (odds ratio 2.9, 95% CI 1.3–6.2, P = 0.008), progranulin (1.9, 0.8–4.5, P = 0.16), clusterin (0.6, 0.3–1.1, P = 0.09), and α-1 acid glycoprotein (1.6, 0.7–3.7, P = 0.27) improved the AUC from 0.841 to 0.889.
A panel of four novel protein biomarkers predicted early renal damage in type 1 diabetes. These findings require further validation in other populations for prediction of renal complications and treatment monitoring.
PMCID: PMC3322681  PMID: 22238279
19.  The Association between IgG4 Antibodies to Dietary Factors, Islet Autoimmunity and Type 1 Diabetes: The Diabetes Autoimmunity Study in the Young 
PLoS ONE  2013;8(2):e57936.
Infant dietary exposures have been linked to type 1 diabetes (T1D) development. IgG4 antibody responses to food antigens are associated with food intolerances but have not been explored prospectively in the period preceding T1D.
Using a case-cohort design, IgG4 antibodies to ß-lactoglobulin, gluten, and ovalbumin were measured in plasma collected annually from 260 DAISY participants. Of those, 77 developed islet autoimmunity (IA), defined as positive for either insulin, GAD65 or IA-2 autoantibodies on two consecutive visits, and 22 developed T1D.
In mixed model analysis adjusting for HLA-DR status, T1D family history, age and ethnicity, higher ß-lactoglobulin IgG4 concentrations were associated with shorter breastfeeding duration (beta = −0.03, 95% Confidence Interval: −0.05, −0.006) and earlier first cow’s milk exposure (beta = −0.04, 95% Confidence Interval: −0.08, 0.00). Higher gluten IgG4 was associated with older age at gluten introduction (beta = 0.06, 95% Confidence Interval: 0.00, 0.13). In proportional hazards analysis adjusting for HLA-DR status, T1D family history and ethnicity, IgG4 against individual or multiple dietary antigens throughout childhood were not associated with IA. In addition, mean antigen-specific IgG4 concentrations in infancy (age <2 years) were not associated with risk of IA nor progression to T1D. Higher ovalbumin IgG4 at first IA positive visit was marginally associated with progression to T1D (Hazard Ratio: 1.39, 95% Confidence Interval: 1.00, 1.92).
We found no association between the IgG4 response to β-lactoglobulin, gluten, and the development of either IA or T1D. The association between higher ovalbumin and progression to T1D in children with IA should be explored in other populations.
PMCID: PMC3585253  PMID: 23469110
20.  Distinguishing Persistent Insulin Autoantibodies With Differential Risk 
Diabetes  2011;61(1):179-186.
A subset of children develops persistent insulin autoantibodies (IAA; almost always as the only islet autoantibody) without evidence of progression to diabetes. The aim of the current study was the development and characterization of the performance of a nonradioactive fluid phase IAA assay in relation to standard IAA radioassay. We developed a nonradioactive IAA assay where bivalent IAA cross-link two insulin moieties in a fluid phase. The serum samples positive for anti-islet autoantibodies from 150 newly diagnosed patients with diabetes (Barbara Davis Center plus Diabetes Autoantibody Standardization Program [DASP] workshop) and 70 prediabetic subjects who were followed to diabetes were studied. In addition, sequential samples from 64 nondiabetic subjects who were persistently IAA+ were analyzed. With 99th percentile of specificity, the new assay with the technology from Meso Scale Discovery Company (MSD-IAA) detects as positive 61% (61 of 100) of new-onset patients and 80% (56 of 70) of prediabetic patients compared with our current fluid phase micro-IAA radioassay (mIAA; 44 and 74%, respectively). In addition, MSD-IAA demonstrated better sensitivity than our mIAA from blinded DASP workshop (68 vs. 56% with the same 99% specificity). Of 64 IAA+ nondiabetic subjects, 25% (8 of 32) who had only IAA and thus the low risk for progression to diabetes were positive with MSD-IAA assay. In contrast, 100% (32 of 32) high-risk children (IAA plus other islet autoantibodies) were positive with MSD-IAA. The IAA detectable by radioassay, but not MSD-IAA, were usually of lower affinity compared with the IAA of the high-risk children. These data suggest that a subset of IAA with current radioassay (not MSD-IAA) represents biologic false positives in terms of autoimmunity leading to diabetes. We hypothesize that factors related to the mechanism of loss of tolerance leading to diabetes determine high affinity and MSD-IAA reactivity.
PMCID: PMC3237666  PMID: 22124462
21.  The Environmental Determinants of Diabetes in the Young (TEDDY): Genetic Criteria and International Diabetes Risk Screening of 421,000 infants 
Pediatric Diabetes  2011;12(8):733-743.
The TEDDY study seeks to identify environmental factors influencing the development of type 1 diabetes (T1D) using intensive follow-up of children at elevated genetic risk. The study requires a cost-effective yet accurate screening strategy to identify the high-risk cohort.
The TEDDY cohort was identified through newborn screening using HLA class II genes based on criteria established with pre-TEDDY data. HLA typing was completed at six international centers using different genotyping methods that can achieve >98% accuracy.
TEDDY developed separate inclusion criteria for the general population (GP) and first degree relatives (FDR) of T1D patients. The FDR eligibility includes nine haplogenotypes (DR3/4, DR4/4, DR4/8, DR3/3, DR4/4b, DR4/1, DR4/13, DR4/9 and DR3/9) for broad HLA diversity, while the GP eligibility includes only the first four haplogenotypes with DRB1*0403 as an exclusion allele. TEDDY has screened 414,714 GP infants, of which 19,906 (4.8%) were eligible, while 1,415 of the 6,333 screened FDR infants (22.2%) were eligible. High resolution confirmation testing of the eligible subjects indicated that the low-cost and low-resolution genotyping techniques employed at the screening centers yielded an accuracy of 99%. There were considerable variations in eligibility rates among the centers for GP (3.5% – 7.4%) and FDR (19% – 32%) subjects. The eligibility rates among US ethnic groups were 0.9%, 1.3%, 5.0% and 6.9% for Asians, Black, Caucasians and Hispanics, respectively.
Different low-cost and low-resolution genotyping methods are useful for the efficient and accurate identification of a high-risk cohort for follow-up based on the TEDDY HLA inclusion criteria ( NCT00279318).
PMCID: PMC3315186  PMID: 21564455
genetic screening; prediction; autoimmunity; type 1 diabetes; population-based
22.  Erythrocyte membrane omega-3 fatty acid levels and omega-3 fatty acid intake are not associated with conversion to type 1 diabetes in children with islet autoimmunity: The Diabetes Autoimmunity Study in the Young (DAISY) 
Pediatric diabetes  2011;12(8):669-675.
We investigated whether omega-3 fatty acid intake and erythrocyte membrane omega-3 fatty acid levels are associated with conversion to type 1 diabetes in children with islet autoimmunity (IA).
The Diabetes Autoimmunity Study in the Young is following children at increased genetic risk for type 1 diabetes for the development of persistent IA, as defined as being positive for glutamic acid decarboxylase 65, i, or insulin autoantibodies on two consecutive visits, and then for the development of type 1 diabetes, as diagnosed by a physician. One hundred and sixty-seven children with persistent IA were followed for a mean of 4.8 yr, and 45 of these developed type 1 diabetes at a mean age of 8.7 yr. Erythrocyte membrane fatty acids (as a percent of total lipid) and dietary fatty acid intake (estimated via food frequency questionnaire) were analyzed as time-varying covariates in proportional hazards survival analysis, with follow-up time starting at detection of the first autoantibody.
Neither dietary intake of omega-3 fatty acids nor omega-6 fatty acids were associated with conversion to type 1 diabetes, adjusting for human leukocyte antigen (HLA)-DR, family history of type 1 diabetes, age at first IA positivity, maternal age, maternal education, and maternal ethnicity. Adjusting for HLA-DR, family history of type 1 diabetes and age at first IA positivity, omega-3 and omega-6 fatty acid levels of erythrocyte membranes were not associated with conversion to type 1 diabetes.
In this observational study, omega-3 fatty acid intake and status are not associated with conversion to type 1 diabetes in children with IA.
PMCID: PMC3475955  PMID: 21435137
dietary intake; IA; omega-3 fatty acids; type 1 diabetes mellitus
23.  Obesity and Coronary Artery Calcium in Diabetes: The Coronary Artery Calcification in Type 1 Diabetes (CACTI) Study 
Diabetes Technology & Therapeutics  2011;13(10):991-996.
The aim was to examine whether excess weight is associated with coronary artery calcium (CAC), independent of metabolic parameters in adults with type 1 diabetes (T1D).
Subjects between 19 and 56 years of age with T1D (n=621) from the Coronary Artery Calcification in Type 1 Diabetes study were classified as abnormal on four metabolic parameters: blood pressure ≥130/85 mm Hg or on antihypertensive treatment; high-density lipoprotein-cholesterol of <40 mg/dL for men or <50 mg/dL for women; triglycerides of ≥150 mg/dL; or C-reactive protein of ≥3 μg/mL. Study participants with two or more abnormal parameters were classified as metabolically abnormal. Weight categories by body mass index were normal (<25 kg/m2), overweight (25 to <30 kg/m2), and obese (≥30 kg/m2). CAC was measured at two visits 6.0±0.5 years apart. Progression of CAC was defined as an increase in square root transformed CAC volume of ≥2.5 mm3 or development of clinical coronary artery disease.
Among subjects with T1D, 48% of normal, 61% of overweight, and 73% of obese participants were classified as metabolically abnormal (P<0.0001). Overweight and obesity were independently associated with presence of CAC, independent of presence of metabolically abnormal. Obesity but not overweight was associated with CAC progression, independent of the other cardiovascular risk factors.
Although obesity is known to increase cardiovascular disease risk through inducing metabolic abnormalities such as dyslipidemia, hypertension, and inflammation, it is also a strong predictor of subclinical atherosclerosis progression in adults with T1D independent of these factors.
PMCID: PMC3182677  PMID: 21770813
24.  Extrapancreatic Autoantibody Profiles in Type I Diabetes 
PLoS ONE  2012;7(9):e45216.
Type I diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-producing β-cells in the pancreas. Although several islet cell autoantigens are known, the breadth and spectrum of autoantibody targets has not been fully explored. Here the luciferase immunoprecipitation systems (LIPS) antibody profiling technology was used to study islet and other organ-specific autoantibody responses in parallel. Examination of an initial cohort of 93 controls and 50 T1D subjects revealed that 16% of the diabetic subjects showed anti-gastric ATPase autoantibodies which did not correlate with autoantibodies against GAD65, IA2, or IA2-β. A more detailed study of a second cohort with 18 potential autoantibody targets revealed marked heterogeneity in autoantibody responses against islet cell autoantigens including two polymorphic variants of ZnT8. A subset of T1D subjects exhibited autoantibodies against several organ-specific targets including gastric ATPase (11%), thyroid peroxidase (14%), and anti-IgA autoantibodies against tissue transglutaminase (12%). Although a few T1D subjects showed autoantibodies against a lung-associated protein KCNRG (6%) and S100-β (8%), no statistically significant autoantibodies were detected against several cytokines. Analysis of the overall autoantibody profiles using a heatmap revealed two major subgroups of approximately similar numbers, consisting of T1D subjects with and without organ-specific autoantibodies. Within the organ-specific subgroup, there was minimal overlap among anti-gastric ATPase, anti-thyroid peroxidase, and anti-transglutaminase seropositivity, and these autoantibodies did not correlate with islet cell autoantibodies. Examination of a third cohort, comprising prospectively collected longitudinal samples from high-risk individuals, revealed that anti-gastric ATPase autoantibodies were present in several individuals prior to detection of islet autoantibodies and before clinical onset of T1D. Taken together, these results suggest that autoantibody portraits derived from islet and organ-specific targets will likely be useful for enhancing the clinical management of T1D.
PMCID: PMC3448600  PMID: 23028856
25.  Cross-Sectional and Longitudinal Changes of Glucose Effectiveness in Relation to Glucose Tolerance 
Diabetes Care  2011;34(9):1959-1964.
Glucose effectiveness (SG), the capacity of glucose to enhance its own disposition, is an independent predictor of future diabetes. However, there are data on cross-sectional and longitudinal changes of SG and its components, basal insulin effect on SG (BIE) and SG at zero insulin (GEZI), but the natural course of SG has not been described in a large population.
SG was measured at baseline in 1,265 participants (aged 40–69 years) and at the 5-year examination in 827 participants in the Insulin Resistance Atherosclerosis Study (IRAS) using the frequently sampled intravenous glucose tolerance test. None of these participants were treated with glucose-lowering agents.
In cross-sectional analyses, SG, BIE, and GEZI deteriorated with worsening of glucose tolerance (P < 0.001 for all three associations). In longitudinal analyses among subjects with normal glucose tolerance (NGT) at baseline, SG, BIE, and GEZI declined in those who progressed to impaired glucose tolerance (IGT) or diabetes (P < 0.001 for all three measures). More modest longitudinal changes were demonstrated in individuals with IGT. The transition back to NGT (as opposed to no change) compared with the transition to diabetes was statistically significant for SG (P = 0.049) and BIE (P = 0.042) and was not a statistically significant trend for GEZI (P = 0.332). In individuals with diabetes, only BIE had a significant decline (P = 0.003).
SG, BIE, and GEZI decline in subjects whose glycemic status worsens. SG and GEZI deteriorate more in the initial stages of the disease process.
PMCID: PMC3161274  PMID: 21788626

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