Evidence of Gene-Gene Interaction and Age-at-Diagnosis Effects in Type 1 Diabetes

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.Knowledge of the genetic architecture of type 1 diabetes has increased recently owing to large-scale genome-wide association (GWA) studies (1–3). Estimates of the contributions of the HLA region and numerous non-HLA loci across the genome now account for a sizeable proportion of familial clustering of the disorder (4–6). However, there remains substantial familial clustering that is not explained by the known loci (likely to be in excess of 40%) (4–6). Interactions between risk loci beyond that of a multiplicative model on the odds ratio (OR) scale (or additive on the log odds scale (7)) could account for some of the “missing heritability.” In addition, the existence of differential effects according to age-at-diagnosis and sex remains relatively unexplored.The HLA region on chromosome 6p21 is the major source of familial clustering in type 1 diabetes (4). HLA-DRB1 and HLA-DQB1 are associated with ORs in excess of 10 for susceptible genotypes (or less than 0.1 for protective genotypes) (8). The risk genotype HLA-DRB1*03/HLA-DRB1*04-HLA-DQB1*0302 (referred to as DR3/DR4-DQ302) with greatest effect has been shown to have the highest frequency in the individuals with youngest onset (9). An age-at-diagnosis interaction has also been reported for HLA-DRB1*04 (10) and the HLA class I alleles HLA-A*24 and HLA-B*39 (11,12).In contrast, reports of age-at-diagnosis interaction effects at non-HLA loci are contradictory, with positive reports largely confined to studies involving small sample sets (3,13–15). Similarly, reports of gene–gene interaction of type 1 diabetes–associated regions are also mainly conflicting (16–19), we presume due to inadequate sample sizes, with most positive reports likely to be false because the false-discovery rate would be high in these underpowered studies. The only convincing gene–gene interaction reported, is between a major non-HLA locus, protein tyrosine phosphatase nonreceptor type 22 (PTPN22) and DR3/DR4-DQ302 genotypes (20–23).The incidence of childhood type 1 diabetes is similar in males and females, unlike other autoimmune diseases such as Graves disease, celiac disease, or multiple sclerosis. Despite similar frequencies of childhood type 1 diabetes by sex, there have been reports of genetic risk factors differing between males and females (22,24).Given that most studies of gene–gene interaction, age-at-diagnosis effects, and sex effects on type 1 diabetes risk have not been addressed in sufficiently well-powered studies, the Type 1 Diabetes Genetics Consortium (T1DGC) has collected more than 16,000 type 1 diabetes–affected samples and tested them for interaction effects with sex and age-at-diagnosis at 38 non-HLA type 1 diabetes–associated regions (Supplementary Table 2). Gene–gene interaction was also tested between HLA class II and the 38 non-HLA loci. With this very large sample set, the study had at least 80% power to detect effects as small as an interaction OR = 1.12 for sex and 1.19 for interactions with age-at-diagnosis or HLA. These calculations assume a multiplicative (log additive) effects model, an OR = 1.15 for association with type 1 diabetes for the test locus and a minor allele frequency of 0.2 and α = 0.0004. In contrast, with 5,000 samples, which is twice as large as any other study testing for interaction effects in type 1 diabetes published to date, the study would only be powered at 80% to detect interaction effects larger than an OR = 1.3 with sex (with the same assumptions as above). For age-at-diagnosis interaction, an OR ≥ 1.37 could be detected; for HLA interaction, an OR ≥ 1.38 could be detected (Supplementary Figs. 1–6).     

Prospective, Randomized, Multi-Center Trial of Antibody Induction Therapy in Simultaneous Pancreas-Kidney Transplantation

A randomized, multicenter, prospective study was conducted at 18 pancreas transplant centers in the United States to determine the role of induction therapy in simultaneous pancreas-kidney (SPK) transplantation. One hundred and 74 recipients were enrolled: 87 recipients each in the induction and noninduction treatment arms. Maintenance immunosuppression consisted of tacrolimus, mycophenolate mofetil, and corticosteroids. There were no statistically significant differences between treatment groups for patient, kidney, and pancreas graft survival at 1-year. The 1-year cumulative incidence of any treated biopsy-confirmed or presumptive rejection episodes (kidney or pancreas) in the induction and noninduction treatment arms was 24.6% and 31.2% (p = 0.28), respectively. The 1-year cumulative incidence of biopsy-confirmed, treated, acute kidney allograft rejection in the induction and noninduction treatment arms was 13.1% and 23.0% (p = 0.08), respectively. Biopsy-confirmed kidney allograft rejection occurred later post-transplant and appeared to be less severe among recipients that received induction therapy. The highest rate of Cytomegalovirus (CMV) viremia/syndrome was observed in the subgroup of recipients who received T-cell depleting antibody induction and received organs from CMV serologically positive donors. Decisions regarding the routine use of induction therapy in SPK transplantation must take into consideration its differential effects on risk of rejection and infection.     

Fronto-limbic white matter microstructural changes in psychiatrically healthy adults with childhood trauma

Childhood trauma (CT) may influence brain white matter microstructure; however, few studies have examined the differential impact of distinct CT types on white matter microstructure in psychiatrically healthy adults living in a developing country. In adults without significant medical or psychiatric disorders, we investigated the association(s) between CT, including abuse and neglect, and fractional anisotropy (FA) of limbic tracts previously shown to be associated with CT. Participants underwent diffusion tensor imaging and completed the Childhood Trauma Questionnaire. Multivariate analysis of variance models were used to test the effects of total overall CT, as well as CT subtypes, on FA in six fronto-limbic tracts, adjusting for age, sex, and educational level. The final sample included 69 adults (age 47 ± 17 years; 70% female). Overall, CT had a significant main effect on FA for tracts of interest (p < .001). Greater CT severity was associated with lower FA for the bilateral and left stria terminalis (uncorrected) as well as the bilateral, left, and right anterior limb of the internal capsule (ALIC; corrected). Exposure to total non-violent/deprivational trauma specifically was associated with lower FA of the bilateral, left, and right ALIC, suggesting that distinct types of CT are associated with differential white matter changes in apparently healthy adults. The ALIC predominantly carries fibers connecting the thalamus with prefrontal cortical regions. Microstructural alterations in the ALIC may be associated with functional brain changes, which may be adaptive or increase the risk of accelerated age-related cognitive decline, maladaptive behaviors, and subsyndromal psychiatric symptoms.     

Risk Factors for Postoperative Hemorrhage after Vitrectomy for Diabetic Retinopathy

Objective: To identify the potential risk factors for postvitrectomy diabetic vitreous hemorrhage (PDVH). Methods: A matched case-control nested into a retrospective follow-up study was done to review the surgical results in 68 consecutive eyes undergoing primary pars plana vitrectomy for vitreous hemorrhage. The eyes were divided into two groups based on the presence of PDVH (19 cases and 49 controls) and were matched on surgeon and the date of surgery. Twenty-three factors related to the preoperative examination and eight factors related to the operative procedure were analyzed. Statistical analysis was based on conditional logistic regression models with PDVH as the dependent variable. The mean follow-up interval was six months. Results: The factors associated with the incidence of PDVH were iris neovascularization (OR = 9.8, P = 0.03), lower extremity amputations (OR = 8.3, P = 0.02) and the use of antihypertensive agents within three months before vitrectomy (OR = 0.2, P = 0.04). Phakic and aphakic eyes of diabetic patients with lower extremity amputations would have a 70% probability of developing PDVH. This probability would have dropped to 30–40% had they been taking antihypertensive treatment. Conclusions: Iris neovascularization and lower extremity amputations increase the risk of PDVH. Antihypertensive treatment before vitrectomy decreases this risk.     

Method of heart transplantation for treatment of hypoplastic left heart syndrome

Technical details of investigational orthotopic cardiac transplantation for management of hypoplastic left heart syndrome in a neonate are presented. Extracorporeal perfusion technique and need for extensive aortic arch reconstruction are emphasized. Although this experience was with a subhuman primate (baboon) donor, source of donor graft makes little difference with regards to the unique technical aspects of cardiac transplantation in a ductus-dependent newborn infant with a diminutive aortic arch.     

Phenotypic expression of histocompatibility antigens in human primary tumours and metastases

HLA class I and II expression was studied on 244 (177 primary and 67 metastatic) solid human tumours of different origin. Alkaline immunophosphatase (APAAP) and immunoperoxidase were used on cryostatic sections to stain MHC antigens. Monomorphic MoAbs were used against class I heavy chain, 2-microglobulin, DR, DQ and DP molecules.Class I expression was homogeneous on colon, melanoma and epidermoidal primitive tumours. Loss of HLA class I antigens was more frequent on basal cell carcinomas and sarcomas and was related to tumour differentiation on larynx carcinoma. Class I expression was heterogeneous on breast, larynx and stomach primitive neoplasias. Class I negative tumours were more frequent on metastatic than on primitive melanomas. Divergence of class I between primary tumours and autologous metastases was observed on melanomas, larynx and colorectal carcinomas.Class II expression was heterogeneous on all tumours and in a large number of cases was associated with high intensity of leukocytic infiltrate. HLA-DR expression was higher than HLA-DP and HLA-DQ (DR>DP>DQ) and was related to tumour progression. Four human tumour cell lines were modulated with recombinant interferon- for HLA class I and II antigens. Different HLA profiles were obtained: increased class I and II expression, increased class II or a low response.Finally, class I genes from 22 tumours were compared with autologous normal cells by Southern blot analysis: 12 tumours were class I positive and 10 negative. No clear differences in RFLP were observed that could be associated with class I rearrangement. The results are discussed in relation to the role that histocompatibility antigens may play in tumour progression and invasiveness.     

Abnormal calcaemic response to PTH in the uraemic rat without secondary hyperparathyroidism

BACKGROUND: Skeletal resistance to the calcaemic action of parathyroid hormone(PTH) is an important pathogenic factor in the development ofsecondary hyperparathyroidism. Since parathyroidectomy normalizesthe calcaemic response to PTH in uraemic animals, the increasein PTH levels has been advanced as a cause of skeletal resistanceto the calcaemic action of PTH. This study was designed to evaluatein uraemic rats the effect of normal PTH levels on the calcaemicresponse to PTH. METHODS: To maintain normal PTH levels, rats were parathyroidectomized(PTX) and rat 1–34 PTH was infused at a rate of 0.022µg/100 g per hour via a subcutaneously implanted miniosmoticpump; this rate of infusion was considered to be the normalPTH replacement dose since it normalized serum calcium and phosphorusin PTX rats with normal renal function. Two separate studieswere performed. In the first study, rats were maintained ona moderate-phosphorus (0.6%) diet and rats were divided intofour groups: (I) normal; (II) uraemic; (III) PTX with normalPTH replacement; and (IV) uraemic with PTX and normal PTH replacement.In a second study, the groups were the same except that a high-phosphorus(1.2%) diet was given to increase the magnitude of hyperparathyroidismin rats with intact parathyroid glands; an additional group(V) identical to group IV except that rats received daily calcitriolwas included. After 14 days, rats received a 48-h infusion ofhigh-dose rat 1–34 PTH (0.11 µg/100 g per hour)to evaluate the calcaemic response to PTH. RESULTS: The calcaemic response to PTH was similar in normal rats andPTX rats with PTH replacement on both a moderate and high-phosphorusdiet. In uraemic rats, the calcaemic response to PTH was decreasedand the maintenance of normal PTH levels by PTH replacementdid not correct the decreased calcaemic response to PTH; moreover,calcitriol supplementation did not improve the calcaemic responseto PTH. Finally, hypocalcaemia was observed in uraemic ratswith PTH replacement and was more profound than in rats on ahigh-phosphorus diet. CONCLUSIONS: This study demonstrates that the maintenance of a normal PTHlevel in uraemic rats did not correct the impaired calcaemicresponse to PTH, suggesting that factors intrinsic to uraemia,independent of phosphorus, calcitriol, and PTH participate inthe decreased calcaemic response to PTH in uraemia.     

Biodegradable micro- and nanoparticles as long-term delivery vehicles for gentamicin

Micro- and nanoparticles of poly(lactide-co-glycolide) (PLGA) loading gentamicin were prepared by a solvent evaporation method with the aim of obtaining appropriate vectors for systemic administration. Microspheres presented mean diameters below 3 microm and nanoparticles showed homogeneous sizes with a diameter of 320 nm. Drug loading was more efficient in the case of microencapsulation. The more hydrophilic copolymers with carboxyl-end groups yielded higher microparticle loadings, reaching encapsulation efficiencies up to 9.2 microg mg(-1) of polymer (502H, 503H or 75:25H). Nanoparticles made of 502H PLGA also achieved an acceptable level of encapsulation (6.2 microg mg(-1)). Particles prepared by using the solvent evaporation method showed no aggregation after hydration, in contrast to the microparticles prepared by spray-drying which showed fast and high auto-aggregation. In vitro release profiles revealed that 503H microspheres showed the highest burst during the first hour, while the most sustained release was for microparticles of 502H copolymer (40% of gentamicin remained in the formulation after 28 days). In summary, microspheres made of 502H, 503H and 75:25H and nanoparticles of 502H showed the best potential properties for systemic use in the treatment of intra-cellular gentamicin-susceptible pathogens.