Whole-exome sequencing identifies novel LEPR mutations in individuals with severe early onset obesity.

OBJECTIVE: Obesity is a major public health problem that increases the risk for a broad spectrum of co-morbid conditions. Despite evidence for a strong genetic contribution to susceptibility to obesity, previous efforts to discover the relevant genes using positional cloning have failed to account for most of the apparent genetic risk variance. DESIGN AND METHODS: Deploying a strategy combining analysis of exome sequencing data in extremely obese members of four consanguineous families with segregation analysis, we screened for causal genetic variants. Filter-based analysis and homozygosity mapping were used to identify and prioritize putative functional variants. RESULTS: Two novel frameshift mutations in the leptin receptor in two of the families were identified. CONCLUSIONS: These results provide proof-of-principle that whole-exome sequencing of families segregating for extreme obesity can identify causal pathogenic mutations. The methods described here can be extended to additional families segregating for extreme obesity and should enable the identification of mutations in novel genes that predispose to obesity.

Basal insulin requirements on continuous subcutaneous insulin infusion during the first 12 months after diagnosis of type 1 diabetes mellitus.

INTRODUCTION: While the endogenous first-phase insulin response has disappeared by the time of diagnosis of type 1 diabetes mellitus (T1DM), anecdotal evidence suggests that these patients can continue to have a second-phase insulin response during the first 12 months after diagnosis. We hypothesized that patients who are started on continuous subcutaneous insulin infusion (CSII) at the time of diagnosis of T1DM would have a lower basal insulin requirement than the 40-60% usually expected. METHODS: We analyzed 38 patients with T1DM, age 9.9 +/- 6.4 years, 71% male, who were started on CSII within the first month of diagnosis. RESULTS: Average basal insulin requirements were 47-49% of total daily dose during the first 12 months after diagnosis and decreased from 0.30 U/kg/day at diagnosis to 0.20 U/kg/day by 12 months. Baseline percentage of basal insulin was significantly correlated with hemoglobin A1c at baseline and at six months. The percentage of basal insulin requirement at 12 months after diagnosis was significantly correlated with baseline body mass index (BMI) and current BMI. No other correlations between percentage of basal insulin requirements and any other factors were seen. CONCLUSION: Our data suggest that, even though some endogenous insulin production remains during the first year after diagnosis of T1DM, the distribution of basal versus total daily insulin requirements remains the same as in the general population of people with diabetes. There may be benefits to starting patients on a higher basal rate at time of diagnosis for overall glycemic control during the first six months. Further research is needed to optimize starting insulin doses to maximize their potential in preserving beta-cell function.

Human CD1D gene expression is regulated by LEF-1 through distal promoter regulatory elements.

CD1d-expressing cells present lipid Ag to CD1d-restricted NKT cells, which play an important role in immune regulation and tumor rejection. Lymphoid enhancer-binding factor-1 (LEF-1) is one of the regulators of the Wnt signaling pathway, which is a powerful regulator in cellular growth, differentiation, and transformation. There is little evidence connecting Wnt signaling to CD1d expression. In this study, we have identified LEF-1 as a regulator of the expression of the gene encoding the human CD1d molecule (CD1D). We found that LEF-1 binds specifically to the CD1D promoter. Overexpression of LEF-1 in K562 or Jurkat cells suppresses CD1D promoter activity and downregulates endogenous CD1D transcripts, whereas knockdown of LEF-1 using LEF-1-specific small interfering RNA increases CD1D transcripts in K562 and Jurkat cells but there are different levels of surface CD1d on these two cell types. Chromatin immunoprecipitation showed that the endogenous LEF-1 is situated at the CD1D promoter and interacts with histone deacetylase-1 to facilitate the transcriptional repressor activity. Knockdown of LEF-1 using small interfering RNA potentiates an acetylation state of histone H3/H4, supporting the notion that LEF-1 acts as a transcriptional repressor for the CD1D gene. Our finding links LEF-1 to CD1D and suggests a role of Wnt signaling in the regulation of the human CD1D gene.

Chronic mucocutaneous candidiasis in APECED or thymoma patients correlates with autoimmunity to Th17-associated cytokines.

Chronic mucocutaneous candidiasis (CMC) is frequently associated with T cell immunodeficiencies. Specifically, the proinflammatory IL-17A-producing Th17 subset is implicated in protection against fungi at epithelial surfaces. In autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED, or autoimmune polyendocrine syndrome 1), CMC is often the first sign, but the underlying immunodeficiency is a long-standing puzzle. In contrast, the subsequent endocrine features are clearly autoimmune, resulting from defects in thymic self-tolerance induction caused by mutations in the autoimmune regulator (AIRE). We report severely reduced IL-17F and IL-22 responses to both Candida albicans antigens and polyclonal stimulation in APECED patients with CMC. Surprisingly, these reductions are strongly associated with neutralizing autoantibodies to IL-17F and IL-22, whereas responses were normal and autoantibodies infrequent in APECED patients without CMC. Our multicenter survey revealed neutralizing autoantibodies against IL-17A (41%), IL-17F (75%), and/ or IL-22 (91%) in >150 APECED patients, especially those with CMC. We independently found autoantibodies against these Th17-produced cytokines in rare thymoma patients with CMC. The autoantibodies preceded the CMC in all informative cases. We conclude that IL-22 and IL-17F are key natural defenders against CMC and that the immunodeficiency underlying CMC in both patient groups has an autoimmune basis.

Interferon autoantibodies associated with AIRE deficiency decrease the expression of IFN-stimulated genes.

Neutralizing autoantibodies to type I, but not type II, interferons (IFNs) are found at high titers in almost every patient with autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), a disease caused by AIRE gene mutations that lead to defects in thymic T-cell selection. Combining genome-wide expression array with real time RT-PCR assays, we here demonstrate that antibodies against IFN-alpha cause highly significant down-regulation of interferon-stimulated gene expression in cells from APECED patients' blood by blocking their highly dilute endogenous IFNs. This down-regulation was lost progressively as these APECED cells matured in cultures without neutralizing autoantibodies. Most interestingly, a rare APECED patient with autoantibodies to IFN-omega but not IFN-alpha showed a marked increase in expression of the same interferon-stimulated genes. We also report unexpected increases in serum CXCL10 levels in APECED. Our results argue that the breakdown of tolerance to IFNs in AIRE deficiency is associated with impaired responses to them in thymus, and highlight APECED as another autoimmune disease with associated dysregulation of IFN activity.

Resting energy expenditure in insulin resistance falls with decompensation of insulin secretion in obese children.

BACKGROUND: Low resting energy expenditure (REE) and respiratory quotient (RQ) have been shown in adults to predispose to obesity and diabetes mellitus. AIM: To correlate REE and RQ in 73 obese children and young adults (body mass index [BMI] 37 +/- 10 kg/m2) with measures of insulin secretion and resistance (IR) indices, percent carbohydrate and fat oxidation, and prolactin and leptin levels. DESIGN: During a 3-day admission, REE and RQ were determined by indirect calorimetry. Blood chemistries and oral glucose tolerance test (OGTT) were obtained, and intravenous glucose tolerance test (IVGTT) modified by tolbutamide was conducted after an overnight fast, permitting calculation of acute insulin response (AIR), insulin resistance (SiIVGTT), and disposition index (DI). RESULTS: Patients fell into two groups according to their SiIVGTT: those with normal insulin sensitivity (NIS) and those with insulin resistance (IR). IR patients were subdivided on the basis of DI (cut-off value 0.13 min(-1)) into compensated (CIR) or decompensated (DIR) groups. CIR patients had higher RQ, REE corrected by BMI, AIR, and carbohydrate oxidation and lower fat oxidation than NIS and DIR patients. REE correlated positively with BMI, leptin, and AIR, and negatively with SiIVGTT. CONCLUSIONS: Findings in the CIR and DIR groups support the correlation of REE with metabolic changes consistent with an increased risk of diabetes mellitus.

Insulin receptor autoimmunity and insulin resistance.

The frequency of insulin receptor autoantibodies (IR-ab) was determined among adolescents and young adults with documented insulin resistance syndrome (IRS) with and without concomitant autoimmunity. The study population was comprised of 61 patients with obesity, acanthosis nigricans and insulin resistance (simple IRS); 12 with IRS and other autoimmune problems (lupus erythematosus, rheumatoid arthritis, dermatomyositis) (type B insulin resistance); six with autoimmune polyglandular syndrome type 2; and 40 healthy controls. Using our newly developed radiobinding assay system, we found no control positive while 25% of the patients with type B IRS (3/12) were positive, as expected. However, we found that 9.8% of the patients with simple IRS (6/61) were also reproducibly positive. All the latter patients with positive IR-ab were female with ovarian hyperandrogenism. The phenotype of those affected was otherwise unremarkably different from those without IR-ab. Our findings suggest that autoimmunity to insulin receptors may be causal in IRS especially for females with ovarian hyperandrogenism, and that IR-ab may be found in IRS besides those previously defined by the type B phenotype. Determining the level of IR-ab in childhood onset IRS may provide mechanistic insights into the genesis of insulin resistance and lead to novel treatment approaches.

Characterization of insulin resistance syndrome in children and young adults. When to screen for prediabetes?

CONTEXT: Insulin resistance syndrome (IRS) is associated with the development of type 2 diabetes mellitus (DM2). However, it is unclear which individuals with insulin resistance will develop DM2. AIM: To study the prevalence of IRS in childhood and to identify the group with the highest risk of further progression to DM2. METHODS: In a cross-sectional study, 86 obese individuals underwent an intravenous glucose tolerance test (IVGTT). Insulin resistance index (Si(IVGTT)), acute insulin response (AIR) and disposition index (DI) were calculated from IVGTT. RESULTS: For analysis the participants were divided into insulin-sensitive (IS) (n = 25, 13.3 +/- 5.9 yr) and insulin-resistant (IR) groups on the basis of having an Si(IVGTT) greater or lesser than 4.5 x 10(-4) mU/ml/min, respectively. The IR group was then subdivided according to DI, with the standard cut-off value of 0.13 min(-1), into compensated IR (CIR) (n = 37, 13.0 +/- 3.5 yr) and decompensated IR (DIR) (n = 24, 21.9 +/- 12.6 yr) groups. The frequency of IRS was 43% in children, 78% in adolescents and 83.6% in adults. Decompensated insulin response first appeared during adolescence. The frequency of decompensation increased from 22% in adolescence to 67% in adulthood. The DIR group had increased triglycerides (TG) and urinary free cortisol levels. CONCLUSIONS: The frequency and severity of IR increases with age. Decompensation first presents in adolescence with low AIR and elevated TG. Decompensated adolescents are the group at highest risk for further progression to DM2.

Effect of metformin and rosiglitazone in a prepubertal boy with Alström syndrome.

UNLABELLED: Alström syndrome (AS) is an autosomal recessive disorder characterized by progressive pigmentary retinopathy, sensorineural hearing loss, fatty liver infiltration, obesity, insulin resistance and early-onset type 2 diabetes mellitus (DM2). Early onset of insulin resistance and DM2 are key components of this syndrome. AIM: To study the effect of early initiation of the insulin sensitizer metformin combined with rosiglitazone in a patient with AS with impaired glucose tolerance. PATIENT: An 8 year-old boy with AS presented with acanthosis nigricans and insulin resistance at the age of 6 years. He had progressive excessive weight gain from 9 months of age. By the age of 1 year he developed photosensitivity, blindness and nystagmus. At the age of 5.5 years, his body mass index (BMI) was above the 95th percentile. He developed impaired glucose tolerance at 6 years of age and treatment with metformin was initiated. After 8 months of treatment with metformin he developed DM2. The dose of metformin was increased, and rosiglitazone added. METHODS: A 2-hour oral glucose tolerance test (OGTT) and a rapid intravenous glucose tolerance test (IVGTT) were performed before treatment was initiated, after treatment with metformin and at the end of 1 year of combination therapy with metformin and rosiglitazone to calculate quantitative insulin sensitivity check index (QUICKI) and acute insulin response (AIR). For mutation analysis, all exons and splice site sequences of the ALMS1 gene were amplified and sequenced. RESULTS: Metformin treatment alone at the stage of impaired glucose tolerance did not prevent progression to DM2. However, metformin at a higher dose and in combination with rosiglitazone resulted in improvement of pancreatic beta-cell function, shown by markedly improved first-phase insulin response to glucose measured by AIR. The patient was found to have two heterozygous nonsense mutations in ALMS1, 8008 C-->T Ter, R2670X, and 11449 C-->T Ter, Q3817X. These alterations cause premature stops and result in a truncated ALMS1 protein. CONCLUSION: We suggest that early initiation of combined therapy comprising a high dose of metformin plus rosiglitazone may be valuable in managing insulin resistance and DM2 in children with AS.

Childhood obesity and insulin resistance.

Insulin resistance (IR) in childhood has importance to the understanding and prevention of the growing epidemic of insulin resistance syndrome (IRS) in adults with attendant obesity, type 2 diabetes (T2DM), atherosclerotic diseases, hypertension, gout, non-alcoholic, steato-hepatitis (NASH), gall bladder disease, nephropathy, polycystic ovarian disease (PCOS), infertility and premature senility. The severity of IR and its' complications in children unfortunately and usually progresses in their pubertal transition to adulthood; affected young children are more likely than adults to have underlying causal monogenic disorders; the sequence of natural history and events give insights into disease pathogeneses, and optimal life style choices that last are best made during the early formative years. Some features of IR in children discussed herein are: a strong tendency to low birth weight for gestational age, adverse effects of adrenarche and therapeutic steroid therapy, predisposition to premature pubarche, acanthosis nigricans, tall stature despite pituitary GH suppression, allergic diathesis, hyperandrogenism and PCOS, dyslipidemia and fatty liver disease, and diagnosis by clinical and biochemical markers of IR including insulin regulated hepatic hormonal binding proteins such as IGFBP-1. The national preoccupation with the "metabolic syndrome" T2DM and obesity, should be appropriately directed to an improved understanding of IR in children and their management, if the looming health crisis in affected adults is to be seriously addressed. The nation is facing its' first generation of children who will be less healthy and die younger than the previous generation (Marks (2005) Presentation to the American Association of Diabetes Educators 32nd Annual Meeting and Exhibition, August 10-13, Washington, DC).

Insulin-like growth factor binding protein-1 to screen for insulin resistance in children.

BACKGROUND: Diabetes and atherosclerosis are burgeoning health problems complicating obesity-associated insulin resistance (IR). Early detection of IR in children is a key to preventative strategies. Since peripheral insulin levels insensitively reflect hepatic insulin fluxes, we studied the insulin-regulated hepatic insulin-like growth factor binding proteins (IGFBPs)-1 and -3 as possible screening markers of childhood IR. METHODS: The tolbutamide-modified frequently sampled intravenous glucose tolerance test (FSIVGTT) and the oral glucose tolerance test (OGTT) were performed in 118 subjects < 21 years old with obesity. The relationships between insulin sensitivity index by minimal modeling (SiIVGTT), other Sis derived from fasting and OGTT insulin and glucose values, and the candidate serum markers were sought. RESULTS: Significant correlation was found between IGFBP-1 and SiIVGTT, similar to the correlations of insulin sensitivity indices with SiIVGTT. In children < or = 10 years old, correlation of IGFBP-1 with SiIVGTT was the strongest. All (100%) subjects with IR defined by SiIVGTT < 4.5 +/- 0.5 x 10(-4) min(-1) /(microIU/mL) had inappropriately low IGFBP-1 levels. IGFBP-3 was not correlated with SiIVGTT. CONCLUSIONS: IGFBP-1 levels decrease with obesity and IR. We propose that in young subjects, especially children under the age of 10 years, IGFBP-1 is a convenient and sensitive marker of IR, whereas elevated fasting insulin is less sensitive but more specific.

Insulin pump therapy from the time of diagnosis of type 1 diabetes.

BACKGROUND: This study was designed to test the feasibility and efficacy of continuous subcutaneous insulin infusion (CSII) being instituted within 1 month of diagnosis of type 1 diabetes mellitus (T1DM). METHODS: Twenty-eight consecutive patients with newly diagnosed T1DM with a mean age of 12.1 +/- 6.2 years were placed on CSII, as early as within 1 day of their diagnosis. All accepted CSII when offered it, and none elected to discontinue CSII after follow-up periods of up to 3 years. RESULTS: Hemoglobin A1c levels declined from an initial mean of 10.5 +/- 2.4% to between 6.5% and 7.4% over the next 18 months, at a mean insulin requirement of 0.33 units/kg/day at 3 months, which gradually rose to 0.58 units/kg/day by 18 months. Endogenous insulin secretion, measured by C-peptide values, remained stable during the first 12 months after diagnosis. There was no significant weight gain for the duration of the study (20.7 kg/m(2) vs. a peak of 22.1 kg/m(2) at 12 months, P = 0.54). CONCLUSIONS: The study provided a positive experience with CSII as the initial insulin replacement therapy in newly diagnosed patients with T1DM with excellent clinical outcomes and apparent prolongations of the honeymoon period. It remains to be proven by random patient assignment whether endogenous insulin secretion is better preserved with CSII as an initial ongoing treatment modality and whether long-term complications are reduced by this approach.

Different outcomes of neonatal thyroid function after Graves' disease in pregnancy: patient reports and literature review.

Graves' disease in pregnancy is a rare condition that directly affects neonatal thyroid function. We describe three newborns born to mothers with Graves' disease and discuss differences in outcomes and management. The first infant presented with a goiter at birth but was euthyroid and did not require therapy. The second infant presented with thyroid storm and the third infant present with neonatal hyperthyroidism, and both required treatment with antithyroid drugs. There was documented elevation of maternal and infant thyroid stimulating hormone immunoglobulin (TSI) levels in all three infants. Management of an infant born to a mother with Graves' disease should include monitoring of both maternal and neonatal thyroid function, and maternal TSI levels during pregnancy. Treatment may be needed if the newborn is symptomatic. With clearance of maternal antibodies and antithyroid drugs, manifestations of abnormal thyroid function in the neonate gradually regress, including eventual resolution of a goiter, if initially present.

Insulin resistance syndrome in children.

The insulin resistance syndrome (syndrome X, metabolic syndrome) has become the major health problem of our times. Associated obesity, dyslipidemia, atherosclerosis, hypertension, and type 2 diabetes conspire to shorten life spans, while hyperandrogenism with polycystic ovarian syndrome affect the quality of life and fertility of increasing numbers of women. Whereas a growing number of single genetic diseases affecting satiety or energy metabolism have been found to produce the clinical phenotype, strong familial occurrences, especially in racially prone groups such as those from the Indian subcontinent, or individuals of African, Hispanic, and American Indian descents, together with emerging genetic findings, are revealing the polygenetic nature of the syndrome. However, the strong lifestyle factors of excessive carbohydrate and fat consumption and lack of exercise are important keys to the phenotypic expression of the syndrome. The natural history includes small for gestational age birth weight, excessive weight gains during childhood, premature pubarche, an allergic diathesis, acanthosis nigricans, striae compounded by gynecomastia, hypertriglyceridemia, hepatic steatosis, premature atherosclerosis, hypertension, polycystic ovarian syndrome, and focal glomerulonephritis appearing increasingly through adolescence into adulthood. Type 2 diabetes, which develops because of an inherent and/or an acquired failure of an insulin compensatory response, is increasingly seen from early puberty onward, as is atheromatous disease leading to coronary heart disease and stroke. A predisposition to certain cancers and Alzheimer's disease is also now recognized. The looming tragedy from growing numbers of individuals affected by obesity/insulin resistance syndrome requires urgent public health approaches directed at their early identification and intervention during childhood. Such measures include educating the public on the topic, limiting the consumption of sucrose-containing drinks and foods with high carbohydrate and fat contents, and promoting exercise programs in our nation's homes and schools.

Preservation of C-peptide secretion in subjects at high risk of developing type 1 diabetes mellitus--a new surrogate measure of non-progression?

Individuals at high risk of developing type 1 diabetes mellitus can be identified using immunologic, genetic, and metabolic parameters. In the Diabetes Prevention Trial-1 (DPT-1), annual intravenous infusions of low doses of regular insulin, together with daily subcutaneous injection of a single low dose of Ultralente insulin at nighttime, failed to prevent or delay the onset of type 1 diabetes in high-risk non-diabetic relatives. In our study, we attempted to achieve beta-cell rest by administering higher doses of neutral protamine Hagedorn (NPH) insulin twice daily to high-risk non-diabetic subjects in an effort to prevent or delay the onset of the disease. The maximum tolerable dose was given with the dose reduced for any hypoglycemia (mean dose 0.33 +/- 0.15; range 0.09-0.66 units/kg/d). We treated 26 subjects who were confirmed to have islet cell antibodies (ICAs) and a low first-phase insulin response (FPIR) to intravenous glucose. Fourteen had normal glucose tolerance and 12 impaired glucose tolerance (IGT). The median duration of follow-up was 5.5 yr. Diabetes occurred in 10 of 12 subjects with IGT and five of 14 subjects with normal glucose tolerance. The cumulative incidence of diabetes was the same as with that seen in a matched, observation group (subjects followed prospectively as part of the University of Florida natural history studies) (age, sex, ICA, insulin autoantibodies, duration of ICA prior to enrollment, FPIR, and glucose intolerance; p = 0.39), as was the rate of progression (p = 0.79). There was a higher rate of progression to diabetes in the group with abnormal glucose tolerance at baseline than in those with normal baseline glucose tolerance (p = 0.003). Interestingly, in non-progressors, as opposed to progressors, there was no fall in C-peptide (peak and area under the curve) production regardless of the type of tolerance testing (mixed meal, oral or intravenous) over time (p < 0.001). In this study, in the dose and regimen of NPH insulin used, insulin did not delay or prevent the development of type 1 diabetes. However, preservation of C-peptide production in the prediabetic period appears to indicate non-progression to clinical disease and may serve as a new surrogate for determining response to preventative efforts.

The relationship between immune-mediated Type 1 diabetes mellitus and ethnicity.

OBJECTIVE: To determine the proportion of adults with newly diagnosed Type 1 diabetes that had immune-mediated disease (IMD). SUBJECTS AND METHODS: Two hundred fifteen patients with hyperglycemia and symptoms of insulin deficiency were screened for antibodies [islet cell antibodies (ICA), insulin autoantibodies (IAA), and glutamatic acid decarboxylase antibodies (GADA(65))] and HLA DR/DQ markers of IMD. RESULTS: The mean age was 34 years and the mean BMI was 30.0 kg/m(2); 21.8% were non-Hispanic white (NHW), 27% were Latin American (LA), 47.9% were African American (AA), and 3.3% of other (OT) ethnic origins. Sixty individuals (28%) had one or more autoantibodies [Ab(+)]: 62% of NHW, 19% of AA, and 19% of LA. BMI was significantly lower (P<.01) in Ab(+). At least one HLA-DQ allele for susceptibility for IMD was identified in 52% of Ab(+). CONCLUSIONS: These results suggest that islet cell autoantibodies and HLA markers of susceptibility for IMD are frequent in individuals of varying ethnic backgrounds, older age at onset, and higher body weight. Since early insulin therapy could delay what may otherwise be rapid progression to complete insulinopenia in patients with IMD, screening of adult diabetic patients for IMD at diagnosis may be of benefit to long-term management.

Immune-mediated disease and secondary failure to oral therapy in type 2 diabetes mellitus.

PURPOSE: To determine the proportion of adults with type 2 diabetes, who developed secondary failure to oral medications that had immune-mediated diabetes (IMD). SUBJECTS AND METHODS: One hundred and eight subjects who failed to oral therapy were screened for autoantibodies (ICA, IAA, IA-2A, and GADA(65)) and HLA DR/DQ markers of IMD. RESULTS: Mean age was 49 years and mean body mass index (BMI) was 31.5 kg/m(2); 21.3% were non-Hispanic White (NHW), 32.3% Latin American (LA), and 43.2% African American (AA). Fourteen percent had one or more autoantibodies [Ab(+)]: five NHW, three LA, and seven AA. Ab(+) patients were younger (P=.03) and had lower body weight (P=.05) than Ab(-) patients. HLA markers of susceptibility for IMD were identified in 64% Ab(+) and in 43% Ab(-) (chi(2), P=.46). CONCLUSIONS: These results suggest that secondary failure to oral therapy can be due to continuing IMD in obese, type 2 diabetic individuals of varying ethnic backgrounds than those who have been traditionally associated with autoimmune type 1 diabetes. Screening for markers for IMD at diagnosis could be a useful way to predict those likely to develop secondary failure to oral therapy and may assist in the selection of medical therapy.

Oral insulin therapy to prevent progression of immune-mediated (type 1) diabetes.

Repeated ingestion of insulin has been suggested as an immune tolerization therapy to prevent immune-mediated (type 1) diabetes. We performed a placebo-controlled, two-dose, oral insulin tolerance trial in newly diagnosed (< 2 years) diabetic patients who had required insulin replacement for less than 4 weeks and were found to have cytoplasmic islet cell autoantibodies (ICAs). No oral hypoglycemic agents were permitted during the trial. Endogenous insulin reserves were estimated at six-month intervals by plasma C-peptide responses to a mixed meal. Positive ICAs were found in 262 (31%) of the 846 patients screened. Of the 197 who agreed to participate, 187 could be followed for 6 to 36 months. Endogenous insulin retention was dependent upon initial stimulated C-peptide response, age at diabetes onset, and numbers of specific islet cell autoantibodies found. Oral insulin improved plasma C-peptide responses in patients diagnosed at ages greater than 20 years, best seen at the low (1 mg/day) over the high (10 mg/day) insulin dose (P = .003 and P = .01, respectively). In patients diagnosed before age 20 years, the 1 mg dose was ineffective, whereas the 10 mg dose actually accelerated C-peptide loss (P = .003). There were no adverse effects. If confirmed, these findings suggest that diabetic patients over age 20 years with ICA evidence of late-onset immune-mediated diabetes should be considered for oral insulin at 1 mg/day to better retain endogenous insulin secretion.

Screening strategies for the identification of multiple antibody-positive relatives of individuals with type 1 diabetes.

The objective of this study was to determine the extent to which different screening strategies could identify a population of nondiabetic relatives of a proband with type 1 diabetes who had two or more immunologic markers from the group consisting of islet cell antibodies (ICA), micro insulin autoantibodies (MIAA), GAD65 autoantibodies (GAA), and ICA512 autoantibodies (ICA512AA). Relatives of subjects with type 1 diabetes were screened for ICA as part of the Diabetes Prevention Trial-Type 1. A total of 71,148 samples were also tested for GAA and ICA512AA. IAA results were available on 17,207 of these samples using a protein A/protein G MIAA assay as well. The study population was defined to be those in which all four antibodies were tested. There were 1010 (5.9%) relatives with a single autoantibody on initial screening and 394 (2.3%) with two or more autoantibodies. GAA was more sensitive than ICA [GAA, 91% (357 of 394); ICA, 82% (324 of 394)] in the detection of multiple antibody-positive individuals. The addition of ICA512AA to GAA as a screening test increased sensitivity to 97% (381 of 394), whereas adding ICA512AA to ICA as a screening test increased sensitivity to 93% (367 of 394). GAA and ICA identified somewhat nonoverlapping subgroups of multiple antibody-positive subjects. Thus, the substitution of GAA or ICA for the other failed to detect 8-17% of multiple antibody subjects. Higher ICA titers were associated with increased percentages of multiple antibody-positive subjects; 86% of subjects having Juvenile Diabetes Foundation titers of at least 160 were positive for two or more antibodies. A screening strategy combining GAA and ICA512AA resulted in a higher sensitivity than using any marker individually, although statistically it was not significantly higher than using GAA alone. Screening for any three antibodies guaranteed that all multiple antibody-positive subjects were detected. Screening for two antibodies at one time and testing for the remaining antibodies among those who are positive for one resulted in a sensitivity of 99% for GAA and ICA, 97% for GAA and MIAA or GAA and ICA512AA, 93% for ICA512AA and ICA, 92% for MIAA and ICA, and 73% for ICA512AA and MIAA. From a laboratory perspective, screenings for GAA, ICA512AA, and MIAA are semiautomated tests with high throughput that, if used as initial screen, would identify at first testing 67% of the 2.3% of multiple antibody-positive relatives (100% if antibody-positive subjects are subsequently tested for ICA) as well as 4.7% of relatives with a single biochemical autoantibody, some of whom may convert to multiple autoantibody positivity on follow-up. Testing for ICA among relatives with one biochemical antibody would identify the remaining 33% of multiple antibody-positive relatives. Further follow-up and analysis of actual progression to diabetes will be essential to define actual diabetes risk in this large cohort.