A dual-reporter, diagnostic vector for prostate cancer detection and tumor imaging.

Detection of prostate-specific antigen (PSA) as a screening strategy for prostate cancer is limited by the inability of the PSA test to differentiate between malignant cancer and benign hyperplasia. Here, we report the use of a cancer-specific promoter, inhibition of differentiation-1 (Id1), to drive a dual-reporter system (Ad5/3-Id1-SEAP-Id1-mCherry) designed for detection of prostate cancer using a blood-based reporter-secreted embryonic alkaline phosphatase (SEAP) and tumor visualization using a fluorescent reporter protein, mCherry. In human prostate tumors, Id1 levels are correlated with increased Gleason grade and disease progression. To evaluate the performance of the dual-reporter system, a prostate cell panel with varying aggressive phenotypes was tested. Following infection with the Ad5/3-Id1-SEAP-Id1-mCherry vector, expression of the SEAP and mCherry reporters was shown to increase with increasing levels of cellular Id1. No correlation was observed between Id1 and PSA. To evaluate in vivo performance, flank tumors were grown in athymic male mice using three prostate cancer cell lines. Following intra-tumoral injection of the vector, tumors formed by cells with high Id1 had the greatest reporter expression. Interestingly, tumors with the lowest levels of Id1 and reporter expression produced the greatest amounts of PSA. These data support the use of Ad5/3-Id1-SEAP-Id1-mCherry as a predictor of prostate cancer malignancy and as a strategy for tumor localization.

Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus.

The helix-loop-helix (HLH) protein NEUROD1 (also known as BETA2) functions as a regulatory switch for endocrine pancreatic development. In mice homozygous for a targeted disruption of Neurod, pancreatic islet morphogenesis is abnormal and overt diabetes develops due in part to inadequate expression of the insulin gene (Ins2). NEUROD1, following its heterodimerization with the ubiquitous HLH protein E47, regulates insulin gene (INS) expression by binding to a critical E-box motif on the INS promoter. Here we describe two mutations in NEUROD1, which are associated with the development of type 2 diabetes in the heterozygous state. The first, a missense mutation at Arg 111 in the DNA-binding domain, abolishes E-box binding activity of NEUROD1. The second mutation gives rise to a truncated polypeptide lacking the carboxy-terminal trans-activation domain, a region that associates with the co-activators CBP and p300 (refs 3,4). The clinical profile of patients with the truncated NEUROD1 polypeptide is more severe than that of patients with the Arg 111 mutation. Our findings suggest that deficient binding of NEUROD1 or binding of a transcriptionally inactive NEUROD1 polypeptide to target promoters in pancreatic islets leads to the development of type 2 diabetes in humans.

A Bayesian population analysis of the development of type 2 diabetes in the offspring of diabetic parents.

Disease progression of type 2 diabetes (T2D) has received considerable attention, but little is known about the disease development of T2D. The purposes of this study were to identify disease development variables (DDV) for development of T2D and to compare corresponding models for disease development. All subjects included in this study were the offspring of diabetic parents and were followed up to 25 years. Repeated fasting blood samples were collected during the follow-up. Longitudinal data of four DDVs, namely fasting blood glucose (FBG), fasting serum insulin (FSI), homeostatic model assessment of insulin resistance (HOMA-IR) and body mass index (BMI) were recorded and compared. According to the diabetes status at the end of the follow-up, the data analysis involved a progressor group of 25 subjects, and a non-progressor group of 127 subjects. The temporal changes in the four DDVs over the time course of the disease development were evaluated by a single-slope and a dual-slope population-based Bayesian model. A dual-slope model based on FBG was found to be the best disease development model. For non-progressors, the FBG baseline stayed at 69.2 [66.5, 72.1] mg/dl (Bayes estimate [95% Bayesian credible set]) and increased with age by a rate of 0.227 mg/dl [0.149, 0.3] per year. For the progressors, the FBG increase with age the same rate as non-progressors and started to have an additional increase of 2.27 [0.505, 4.52] mg/dl per year, starting 8.73 [-10.8, -6.93] years before the diagnosis of T2D. No significant longitudinal increasing or decreasing temporal pattern was found for FSI, HOMA-IR and BMI by the population-based Bayesian approach. The proposed model, which enables a quantitative, time-based evaluation of the development of T2D in this higher risk population, may be used to quantify the effect of interventions/prevention strategies such as drug treatment and lifestyle changes.

T1-Enhanced MRI-visible nanoclusters for imaging-guided drug delivery.

Iron oxide nanoparticles with extremely low dimensions have recently been explored as positive (T1) contrast agent for magnetic resonance imaging (MRI). However, their small sizes lead to fast renal clearance and limit their use in elongated in vivo tracking or therapy monitoring. In this paper, we present a state of art approach to forming nanoclusters by crosslinking ultrasmall iron oxide nanoparticles with bovine serum albumin. This novel design not only maintains the T1 performance of the ultrasmall nanoparticles, but also significantly increases their blood circulation times from 15 minutes to over two hours. Our breast tumor model study also exhibited enhanced contrast at tumor sites for more than 24 hours. The ability of maintaining the T1 performance of the ultrasmall nanoparticles is significant, because previous studies have shown complete T1 loss or signal decrease upon polymer encapsulation. This design also shows great potential in encapsulating model drug molecules, which will greatly benefit the field of imaging-guided drug delivery.

Effect of diet on excretion of estrogens in pre- and postmenopausal women.

Fecal, urinary, and plasma estrogens and plasma androgens were studied in healthy pre- and postmenopausal vegetarian and omnivorous women. Dietary histories of the subjects revealed that omnivores consumed a higher percentage of total protein and fat from animal sources. The total 72-hr fecal excretion as measured by dry weight was higher for vegetarians. Preliminary results indicate that vegetarian women excrete 2 to 3 times more estrogens in feces than do omnivores and that omnivores have about 50% higher mean plasma level of unconjugated estrone and estradiol than vegetarians. Estriol-3-glucuronide, a compound that is formed upon reabsorption of free estriol from the intestine, is found in lower concentrations in the urine of vegetarians. These data suggest that in vegetarians a greater amount of the biliary estrogens escape reabsorption and are excreted with the feces. The differences in estrogen metabolism may explain the lower incidence of breast cancer in vegetarian women.

Biodistribution Study of Intravenously Injected Cetuximab-IRDye700DX in Cynomolgus Macaques.

PURPOSE: The use of receptor-targeted antibodies conjugated to photosensitizers is actively being explored to enhance treatment efficacy. To facilitate clinical testing, we evaluated cetuximab conjugated to IRDye700DX (IR700) in cynomolgus macaques. PROCEDURES: Total IR700 and intact cetuximab-IR700 were measured in 51 tissues at 2 and 14 days after intravenous injection of 40 and 80 mg/kg cetuximab-IR700, respectively, and compared with an unlabeled cetuximab-dosed control group (two each per sex per time point per group). RESULTS: The IR700 retrieved from all tissues at 2 and 14 days after dosing was estimated at 34.9 ± 1.8 and 2.53 ± 0.67% of the total dose, respectively. The tissues with the highest levels of intact cetuximab-IR700 at 2 days after dosing were the blood, lung, and skin. Formalin-fixed paraffin-embedded tissue sections at 2 days after dosing showed the highest IR700 signals in the axillary lymph node, mammary gland, and gall bladder. CONCLUSIONS: Both IR700 and intact cetuximab-IR700 biodistributions were consistent with known epidermal growth factor receptor (EGFR) expression, and changes between 2 and 14 days were consistent with rapid metabolism and excretion of the cetuximab-IR700.

Genetic predisposition to diabetic nephropathy. Evidence for a role of the angiotensin I--converting enzyme gene.

In search of genetic determinants of susceptibility to diabetic nephropathy, we examined the association between DNA sequence differences at the locus of angiotensin I-converting enzyme (ACE) and renal complications in 151 insulin-dependent diabetes mellitus (IDDM) patients with a diabetes duration of 16-21 years. This nested case-control study included 77 normoalbuminuric control subjects (albumin excretion rate < 30 micrograms/min) and 74 cases with evidence of nephropathy ranging from microalbuminuria to overt proteinuria. DNA from each of these patients was genotyped at the ACE locus by a three-allele restriction fragment-melting polymorphism (RFMP) (Dde I), which we described recently, and a two-allele insertion/deletion recognized as an Xba I restriction fragment-length polymorphism, which has been shown by other investigators to be associated with serum levels of ACE and with risk of myocardial infarction. The least common allele of the Dde I RFMP was significantly more frequent among cases with nephropathy than among normoalbuminuric control subjects (12.8 vs. 4.5%, P < 0.05). The deletion in the ACE gene was also more frequent in case than in control subjects (56.1 vs. 47.4%), but the difference was not statistically significant (P < 0.25) with this sample size. To determine the independence of these associations, the two polymorphisms were analyzed jointly to identify Xba I/Dde I haplotypes. As might be expected, carriers of the Xba I/Dde I '+ =' haplotype had a fourfold risk of developing diabetic nephropathy (odds ratio [OR] 4.0, 95% confidence interval [CI] 1.5-11.0). However, this did not explain all of the excess Xba I '+' allele among cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Determinants of IDDM and perinatal mortality in children of diabetic mothers.

Offspring of women with insulin-dependent diabetes mellitus (IDDM) have a lower risk of developing IDDM than offspring of men with IDDM (1). To determine whether the risk of diabetes in offspring of diabetic mothers has changed after dramatic improvements in perinatal survival of these infants, we undertook a follow-up study of 1602 pregnancies of 739 women with IDDM who were patients at the Joslin Diabetes Center. Improvements in perinatal survival were abrupt rather than gradual. During the two decades before 1961, perinatal mortality was stable around 23%. After a sudden drop in 1961, it stabilized around 14% until 1975, when it was brought down to 4%, where it has remained. Of the 1391 offspring who survived the neonatal period, IDDM has developed in 21, a cumulative risk of 2.1 +/- 0.5% (SE) by age 20 yr. This is one-third the risk previously reported for offspring of fathers with IDDM and is independent of the calendar time of the births (1). The risk of diabetes in offspring of diabetic mothers is increased in young mothers and is otherwise independent of risk factors for perinatal mortality in this series. We conclude that there is no evidence that selective loss of diabetes-susceptible fetuses in perinatal deaths is a mechanism for the lower incidence of IDDM in the offspring of mothers with IDDM than in those of fathers with IDDM. The principal alternative mechanism is that exposure in utero to an affected mother can protect a fetus from developing IDDM later in life. Induction of immunologic tolerance to the autoantigens of the beta-cells is a plausible mechanism for this protective effect.

Risk of IDDM in children of diabetic mothers decreases with increasing maternal age at pregnancy.

Offspring of women with insulin-dependent diabetes mellitus (IDDM) have a significantly lower risk of IDDM than the offspring of men with IDDM. Furthermore, a negative association of the risk of IDDM in the offspring with maternal age at delivery has been reported. This study tested the association with maternal age in an independent set of families (n = 103) in which the mother had at least one pregnancy before and after the onset of IDDM. In the 304 offspring, the mean +/- SE risk of IDDM by age 20 was 6.0 +/- 2.4% for those born at maternal ages less than 25 yr, whereas, the risk was significantly lower (0.7 +/- 0.7%) for those born at older maternal ages (P = 0.03). These 304 offspring were combined with a sample of 1391 offspring previously reported for a multivariate analysis of other factors related to pregnancy. In the combined analysis, the risk of IDDM in offspring born at maternal ages greater than 25 yr was one-fifth that for offspring born to younger mothers. The risk of IDDM in the offspring was not significantly related to birth order, mother's age at first pregnancy, or the interval between pregnancies for subsequent ones. The risk for the children born before the mother's onset of diabetes was higher than that for those exposed in utero to her diabetes, but the difference did not reach statistical significance. In conclusion, although genetic factors are important determinants of susceptibility to IDDM, exposure to maternal diabetes protects offspring from IDDM during the first 2 decades of life.(ABSTRACT TRUNCATED AT 250 WORDS)

Risk factors for progression of background retinopathy in long-standing IDDM.

Risk factors for the development of severe forms of diabetic retinopathy were examined prospectively in a group of 153 patients with long-standing insulin-dependent diabetes mellitus. During a 4-yr follow-up study, 34 individuals progressed to preproliferative and proliferative retinopathy. The risk of progression to severe forms of diabetic retinopathy was determined by the degree of background diabetic retinopathy and several systemic factors. It increased steeply with hemoglobin A1c, declined proportionally with increasing age, and was dramatically different in patients with diastolic blood pressure below versus above 70 mmHg. Although the mechanisms of action of these systemic factors are unclear, the findings emphasize the multifactorial nature of the pathogenesis of severe forms of eye lesions.

APOE polymorphisms and the development of diabetic nephropathy in type 1 diabetes: results of case-control and family-based studies.

The goal of this study was to examine the association between known polymorphisms in the apolipoprotein E gene (APOE) and diabetic nephropathy (DN) in type 1 diabetes. We used both a case-control comparison and a family-based study design known as the transmission/disequilibrium test (TDT). For the case-control comparison, we collected DNA from 223 subjects with clinically diagnosed DN and 196 control subjects with normoalbuminuria and long-duration type 1 diabetes (> or = 15 years). For the family-based study, we obtained DNA from both parents of 154 DN subjects and 81 control subjects. The frequency of the epsilon2 allele of exon 4 of APOE was significantly higher in DN subjects than in control subjects. The risk of DN was 3.1 times higher (95% CI 1.6-5.9) in carriers of this allele than in noncarriers. In the family study, heterozygous parents for the E2 allele preferentially transmitted epsilon2 to DN offspring (64 vs. 36%, P < 0.03). Four additional polymorphisms (i.e., -491 A/T, -219 G/T, IE1 G/C, and APOCI insertion/deletion [I/D]) that flank the APOE locus were not associated with DN in either the case-control comparison or in the family-based study. In conclusion, the results of the case-control as well as the family-based study provide evidence that the epsilon2 allele of APOE increases the risk of DN in type 1 diabetes. The molecular mechanisms underlying this risk are unclear at present.

Factors modifying the risk of IDDM in offspring of an IDDM parent.

Offspring of mothers with insulin-dependent diabetes mellitus (IDDM) have a much lower risk of IDDM than do offspring of diabetic fathers, and this risk is particularly low for offspring born to diabetic mothers over the age of 25 years. To determine whether increasing maternal age also protects the offspring of IDDM fathers from IDDM, we surveyed 367 IDDM fathers (IDDM onset before age 35) who first came to the Joslin Clinic (Boston, MA) between 1945 and 1969. Of the 840 offspring of these men, IDDM developed in 28 before the age of 20, giving a cumulative risk of 5.1 +/- 1.0% (means +/- SE). Because this is similar to the result of our earlier study of IDDM fathers, the two groups were combined to give 1,084 offspring, 39 having IDDM (cumulative risk of IDDM 5.4 +/- 0.9% by age 20), for comparison with our cohort of 1,391 offspring of 739 IDDM mothers. In that cohort, IDDM developed in 20 offspring before the age of 20 years, giving a cumulative risk of 2.1 +/- 0.5%. The risk of diabetes in offspring was higher if the parent's IDDM was diagnosed before age 11 than if it was diagnosed later: 9.3 compared with 4.0% (P = 0.006) for the offspring of IDDM fathers and 2.7 compared with 1.8% for the offspring of IDDM mothers (P = 0.06). In the families in which the father's IDDM was diagnosed after age 11, a protective effect of maternal age > or = 25, similar to that in families of IDDM mothers, seems to be present.(ABSTRACT TRUNCATED AT 250 WORDS)

Familial clustering of insulin sensitivity.

This study's objective was to determine whether there is familial clustering of insulin sensitivity (SI) or insulin-independent glucose uptake (SG), which would be evidence that they are genetically determined traits. Outpatients had a 3-h intravenous glucose tolerance test. Nondiabetic individuals (n = 183), ranging in age from 16 to 60 yr, were from 105 families that had 2 parents with non-insulin-dependent diabetes mellitus. Of these families, 62 contributed 1 offspring, 21 contributed 2, 13 contributed 3, 6 contributed 4, and 2 and 1 contributed 5 and 6, respectively. The minimal model of glucose disposal and the glucose and insulin values from the intravenous glucose tolerance tests were used to estimate SI and SG. The intraclass correlation coefficient was used to compare the within-family variability of SI and SG with the respective between-family distributions. The intraclass correlation coefficients were 0.26 (P = 0.008) for SI and 0.081 (P = 0.45) for SG. SI and SG were uncorrelated (r = -0.059, P = 0.42). The intraclass correlation of SI could not be explained by familial clustering of fasting insulin or ideal body weight. Finally, the 10 families with the lowest values of SI had a significantly higher within-sibship variability of SI than the other 33 families (P less than 0.001, F test). SI but not SG showed familial clustering, which is consistent with a polygenic determinant of SI. In addition, a large within-family variability of SI in some families is compatible with a major gene effect with a dominant mode of inheritance.(ABSTRACT TRUNCATED AT 250 WORDS)

Risk of early-onset proliferative retinopathy in IDDM is closely related to cardiovascular autonomic neuropathy.

Determinants of proliferative diabetic retinopathy (PDR) that occur during the 2nd decade of insulin-dependent diabetes mellitus (IDDM) (early-onset PDR) were investigated in a nested case-control study. From an inception cohort of patients with juvenile-onset IDDM that now has 15-21 yr diabetes duration, the patients with PDR (cases, n = 74) were selected for study along with a random sample of the patients in the cohort without PDR (control subjects, n = 88). The risk of PDR was associated with poor glycemic control during the first 12 yr of diabetes. Relative to patients in the first quartile of the index of hyperglycemia, those in higher quartiles and nonattenders had a four- to fivefold risk of developing PDR. A striking relationship with cardiovascular autonomic neuropathy (CAN) was found. Relative to patients without CAN, patients with significant and mild CAN had odds ratios of 77.5 and 34.6, respectively. Patients with albumin excretion rates greater than 30 micrograms/min had moderately increased risk of PDR (ranging from 4-fold for microalbuminuria to 7-fold for proteinuria). In contrast, patients with impaired renal function had an extremely high risk of PDR. All 20 of these patients were cases, therefore the odds ratio was infinite. All three factors (poor glycemic control, CAN, and various stages of nephropathy) were associated with PDR in multiple logistic regression analysis. However, in models including glycemic control, the association between microalbuminuria or proteinuria and PDR was weakened. In conclusion, our findings are consistent with a hypothesis that the level of glycemia is a primary determinant of early-onset PDR.(ABSTRACT TRUNCATED AT 250 WORDS)

Segregation analysis of urinary albumin excretion in families with type 2 diabetes.

Elevated urinary albumin excretion (UAE) is a predictor of the development of nephropathy and cardiovascular mortality. To study whether genetic factors may determine UAE, we examined familial aggregation of UAE in 96 large multigenerational pedigrees ascertained for type 2 diabetes. A total of 1,269 subjects had UAE measured as the urinary albumin-to-creatinine ratio (ACR). This included 630 subjects with type 2 diabetes and 639 subjects without diabetes. A significant correlation (Spearman's correlation 0.34, P < 0.001) was found between the median ACR values determined separately in nondiabetic and diabetic members of the same family. To determine whether this familial aggregation of ACR could be explained by the transmission of 1 or more major genes and thus be suitable for gene mapping studies, segregation analyses were performed. In these analyses, ACR was modeled as a continuous trait with the inclusion of age, sex, and duration of diabetes as covariates. Likelihood ratio tests were performed to test competing hypotheses, and Akaike's information criterion was used to determine the most parsimonious models. The Mendelian model with multifactorial inheritance was supported more strongly than Mendelian inheritance alone. These analyses suggested that the best model for ACR levels was multifactorial with evidence for a common major gene. When the analyses were repeated for diabetic subjects only, the evidence for Mendelian inheritance was improved, although a single major locus with additional multifactorial effects was more strongly supported. The results from the current study suggest that levels of UAE are determined by a mixture of genes with large and small effects as well as other measured covariates, such as diabetes.

Exclusion of insulin receptor substrate 2 (IRS-2) as a major locus for early-onset autosomal dominant type 2 diabetes.

We investigated whether variability at the insulin receptor substrate (IRS)-2 locus plays a role in the etiology of early-onset autosomal dominant type 2 diabetes. By means of radiation hybrid mapping, we placed the human IRS-2 gene on 13q at 8.6 cRays from SHGC-37358. Linkage between diabetes and two polymorphic markers located in this region (D13S285 and D13S1295) was then evaluated in 29 families with early-onset autosomal dominant type 2 diabetes. Included were 220 individuals with diabetes, impaired glucose tolerance, or gestational diabetes (mean age at diabetes diagnosis 36 +/- 17 years) and 146 nondiabetic subjects. Overall, strongly negative logarithm of odds (LOD) scores for linkage with diabetes were obtained by multipoint parametric analysis (LOD score -45.4 at D13S285 and -40.9 at D13S1295). No significant evidence of linkage was obtained under the hypothesis of heterogeneity or by nonparametric methods. Fourteen pedigrees for which linkage could not be excluded (LOD score > -2.0) were screened for mutations in the IRS-2 coding region by dideoxy fingerprinting. However, no mutations segregating with diabetes could be detected in these families. These data indicate that IRS-2 is not a major gene for early-onset autosomal dominant type 2 diabetes, although a role of mutations in the promoter region cannot be excluded at this time.

Major susceptibility locus for nephropathy in type 1 diabetes on chromosome 3q: results of novel discordant sib-pair analysis.

Diabetic nephropathy (DN) clusters in families with type 1 diabetes and the degree of clustering suggests that a major gene having a common disease allele may be responsible. To investigate the chromosomal regions containing genes for the renin-angiotensin system, we performed a linkage study using pairs of siblings with type 1 diabetes who were discordant for DN. Theoretical considerations supported by simulation studies indicated that such discordant pairs, rather than the usual concordant pairs, would be more effective in detecting a major susceptibility gene for DN. We applied this novel strategy to test for linkage between DN and chromosomal regions containing genes for the ACE, angiotensinogen (AGT), and angiotensin II type 1 receptor (AT1). Two polymorphic markers were genotyped in the vicinity of each of the three loci in 66 discordant sib pairs and were analyzed with multipoint methods. The regions containing ACE and AGT loci were not linked with DN, while the region containing the AT1 locus showed linkage with DN. As a result of these positive findings, eight additional polymorphic markers spanning a 63-cM region around AT1 locus were genotyped. Linkage was demonstrated between DN and a 20-cM region that includes AT1 (P = 7.7 x 10(-5)), an obvious candidate gene for DN. To investigate whether AT1 could account for the observed linkage, we sequenced all exons, splicing junctions, and the promoter region and examined the identified polymorphisms/mutations for association with DN using the transmission disequilibrium test. Four new polymorphisms in the gene were found, but neither these nor previously described polymorphisms were associated with DN. Thus, while our study does not implicate AT1 itself in the etiology of DN, it provides very strong evidence that a 20-cM region around AT1 contains a major locus for susceptibility to DN.

Shortened telomere length in white blood cells of patients with IDDM.

IDDM is a polygenic and autoimmune disorder in which subsets of white blood cells (WBCs) are engaged in the destruction of beta-cells of the pancreas. The mechanisms that account for the abnormal behavior of these cells in IDDM are not fully understood. By measuring the mean length of telomeres of WBCs from patients with IDDM, we tested the concept that telomeres might play a role in IDDM. We examined the lengths of the terminal restriction fragments (TRFs) of DNA of WBCs from 234 white men comprising 54 patients with IDDM, 74 patients with NIDDM, and 106 control subjects. When adjusted for age, the TRF length from WBCs of patients with IDDM was significantly shorter than that of nondiabetic control subjects (mean +/- SE: 8.6 +/- 0.1 vs. 9.2 +/- 0.1, P = 0.002). No significant difference was observed between the TRF length from WBCs of patients with NIDDM versus nondiabetic subjects. Neither the duration nor the complications of IDDM (i.e., nephropathy and hypertension) had an effect on the TRF length of WBCs from patients with IDDM. The shortened TRF length of WBCs of patients with IDDM likely reflects a marked reduction in the TRF length of subsets of WBCs that play a role in the pathogenesis of IDDM.

Type 2 diabetes locus on 12q15. Further mapping and mutation screening of two candidate genes.

We recently reported evidence of a novel type 2 diabetes locus placed on chromosome 12q15 between markers D12S375 and D12S1684 (Diabetes 48:2246-2251, 1999). Four multigenerational families having logarithm of odds (LOD) scores >1.0 in the original analysis were genotyped for 11 additional markers in this interval to refine this mapping; this allowed us to narrow the linked region to the interval between markers D12S1693 and D12S326. In a multipoint parametric analysis using the VITESSE software, the LOD score for linkage at this location reached 3.1 in one of these families. This interval contains the gene for protein tyrosine phosphatase receptor type R (PTPRR)--a protein that may be involved in both insulin secretion and action. After determining PTPRR exon-intron structure, we identified several polymorphisms in this gene but no mutation segregating with diabetes. The search for mutations was also negative for carboxypeptidase M (CPM)--another candidate gene mapped to this region. In summary, our data provide further evidence for the existence of a type 2 diabetes locus on chromosome 12q15. This locus, however, does not appear to correspond to the PTPRR or CPM, although a contribution of mutations in regulatory regions cannot be excluded at this time.

A nonlinear effect of hyperglycemia and current cigarette smoking are major determinants of the onset of microalbuminuria in type 1 diabetes.

Cigarette smoking and poor glycemic control are risk factors for diabetic nephropathy in type 1 diabetes. However, the specifics of the relation of these risk factors to the onset of this complication have not been elucidated. To investigate these issues, we followed for 4 years 943 Joslin Clinic patients aged 15-44 years with type 1 diabetes who had normoalbuminuria during the 2-year baseline period. Microalbuminuria developed in 109 of the 943 individuals, giving an incidence rate of 3.3/100 person-years. The risk of onset of microalbuminuria was predicted somewhat more precisely by the measurements during the 1st and 2nd years preceding onset than by all the measurements during the longer (4-year) interval, suggesting attenuation of the impact of past hyperglycemia over time. Point estimates of the incidence rate (per 100 person-years) according to quartiles of HbA(1c) during the 1st and 2nd years preceding the outcome were 1.3 in the 1st, 1.5 in the 2nd, 3.1 in the 3rd, and 6.9 in the 4th (P = 1.3 x 10(-9)). Point estimates of the incidence rate (per 100 person-years) according to smoking status were 7.9 for current smokers, 1.8 for past smokers, and 2.2 for those who had never smoked (P = 2.0 x 10(-7)). In a multiple logistic model, the independent effects of HbA(1c) level and cigarette smoking remained highly significant, but their magnitudes were reduced. Using the same covariates in a generalized additive model, we examined the shape of the relationship between HbA(1c) and onset of microalbuminuria and found significant nonlinearity in the logarithm of odds scale (P = 0.04). The slope was steeper with HbA(1c) >8% than <8%. Furthermore, the change in the slope was magnified among current smokers. In conclusion, patients with type 1 diabetes who smoke and have an HbA(1c) >8% have the highest risk of onset of microalbuminuria.