Research priorities to prevent and treat diabetic foot ulcers-A digital James Lind Alliance Priority Setting Partnership.

AIM: To establish outcomes of a priority setting partnership between participants with diabetes mellitus and clinicians to identify the top 10 research priorities for preventing and treating diabetic foot ulcers (DFUs). METHODS: Due to the COVID-19 pandemic, the James Lind Alliance Priority Setting Partnership process was adapted into a digital format which involved a pilot survey to identify understandable uncertainties with high relevance for participants tested by calculating the content validity index; a main survey answered by 53 participants living with diabetes and 49 clinicians; and a final digital workshop to process and prioritise the final top 10 research priorities. RESULTS: The content validity index was satisfactory for 20 out of 25 uncertainties followed by minor changes and one additional uncertainty. After we processed the 26 uncertainties from the main survey and seven current guidelines, a list of 28 research uncertainties remained for review and discussion in the digital workshop. The final top 10 research priorities included the organisation of diabetes care; screening of diabetes, impaired blood circulation, neuropathy, and skin properties; vascular surgical treatment; importance of self-care; help from significant others; pressure relief; and prevention of infection. CONCLUSION: The top 10 research priorities for preventing and treating DFUs represent consensus areas from persons living with diabetes and clinicians to guide future research. These research priorities can justify and inform strategic allocation of research funding. The digitalisation of James Lind Alliance methodology was feasible.

Plasma circulating cell-free mitochondrial DNA in depressive disorders.

BACKGROUND: Plasma circulating cell-free mitochondrial DNA (ccf-mtDNA) is an immunogenic molecule and a novel biomarker of psychiatric disorders. Some previous studies reported increased levels of ccf-mtDNA in unmedicated depression and recent suicide attempters, while other studies found unchanged or decreased ccf-mtDNA levels in depression. Inconsistent findings across studies may be explained by small sample sizes and between-study variations in somatic and psychiatric co-morbidity or medication status. METHODS: We measured plasma ccf-mtDNA in a cohort of 281 patients with depressive disorders and 49 healthy controls. Ninety-three percent of all patients were treated with one or several psychotropic medications. Thirty-six percent had a personality disorder, 13% bipolar disorder. All analyses involving ccf-mtDNA were a priori adjusted for age and sex. RESULTS: Mean levels in ccf-mtDNA were significantly different between patients with a current depressive episode (n = 236), remitted depressive episode (n = 45) and healthy controls (n = 49) (f = 8.3, p<0.001). Post-hoc tests revealed that both patients with current (p<0.001) and remitted (p = 0.002) depression had lower ccf-mtDNA compared to controls. Within the depressed group there was a positive correlation between ccf-mtDNA and "inflammatory depression symptoms" (r = 0.15, p = 0.02). We also found that treatment with mood stabilizers lamotrigine, valproic acid or lithium was associated with lower ccf-mtDNA (f = 8.1, p = 0.005). DISCUSSION: Decreased plasma ccf-mtDNA in difficult-to-treat depression may be partly explained by concurrent psychotropic medications and co-morbidity. Our findings suggest that ccf-mtDNA may be differentially regulated in different subtypes of depression, and this hypothesis should be pursued in future studies.

Increased level of circulating cell-free mitochondrial DNA due to a single bout of strenuous physical exercise.

PURPOSE: Physical exercise is reported to affect the immune response in various ways. Thus, the levels of pro-inflammatory cytokines as well as the abundance of circulating leukocytes are changed. In this study, the occurence of circulating cell-free mitochondrial DNA (cfmtDNA) and nuclear DNA (nDNA) was investigated in connection with a single bout of strenuous physical exercise. METHODS: Healthy volunteers performed a controlled ergo-spirometry cycle test and venous blood samples were taken at different time-points to analyze the concentration of blood components before, during and after the test. The number of circulating leukocytes was measured, as well as secretion of the soluble urokinase activator receptor (suPAR). RESULTS: Cf-mtDNA significantly increased during exercise, compared to baseline values and after 30 and 90 min of rest. Circulating leukocytes increased during exercise, but returned to baseline levels afterwards. Surface expression of the urokinase plasminogen activating receptor (uPAR) on neutrophils decreased significantly during exercise. The concentration of suPAR tended to increase during exercise but only significantly after 90 min of rest. CONCLUSION: Increased concentration of cf-mtDNA indicates that cell damage takes place during high intensity training. Hypoxia and tissue damage are likely causes of cf-mtDNA from muscle cells. The levels of cf-mtDNA remain high during the initial rest, due to the decreasing numbers of leukocytes normally clearing the plasma from cf-mtDNA. The increased levels of suPAR further emphasize that strenuous physical exercise causes a reaction similar to inflammation. Further studies are needed to detect the source of increased cf-mtDNA and the corresponding increase of suPAR liberation.

HLA class I and genetic susceptibility to type 1 diabetes: results from the Type 1 Diabetes Genetics Consortium.

OBJECTIVE: We report here genotyping data and type 1 diabetes association analyses for HLA class I loci (A, B, and C) on 1,753 multiplex pedigrees from the Type 1 Diabetes Genetics Consortium (T1DGC), a large international collaborative study. RESEARCH DESIGN AND METHODS: Complete eight-locus HLA genotyping data were generated. Expected patient class I (HLA-A, -B, and -C) allele frequencies were calculated, based on linkage disequilibrium (LD) patterns with observed HLA class II DRB1-DQA1-DQB1 haplotype frequencies. Expected frequencies were compared to observed allele frequencies in patients. RESULTS: Significant type 1 diabetes associations were observed at all class I HLA loci. After accounting for LD with HLA class II, the most significantly type 1 diabetes-associated alleles were B*5701 (odds ratio 0.19; P = 4 × 10(-11)) and B*3906 (10.31; P = 4 × 10(-10)). Other significantly type 1 diabetes-associated alleles included A*2402, A*0201, B*1801, and C*0501 (predisposing) and A*1101, A*3201, A*6601, B*0702, B*4403, B*3502, C*1601, and C*0401 (protective). Some alleles, notably B*3906, appear to modulate the risk of all DRB1-DQA1-DQB1 haplotypes on which they reside, suggesting a class I effect that is independent of class II. Other class I type 1 diabetes associations appear to be specific to individual class II haplotypes. Some apparent associations (e.g., C*1601) could be attributed to strong LD to another class I susceptibility locus (B*4403). CONCLUSIONS: These data indicate that HLA class I alleles, in addition to and independently from HLA class II alleles, are associated with type 1 diabetes.

HLA genotyping in the international Type 1 Diabetes Genetics Consortium.

BACKGROUND: Although human leukocyte antigen (HLA) DQ and DR loci appear to confer the strongest genetic risk for type 1 diabetes, more detailed information is required for other loci within the HLA region to understand causality and stratify additional risk factors. The Type 1 Diabetes Genetics Consortium (T1DGC) study design included high-resolution genotyping of HLA-A, B, C, DRB1, DQ, and DP loci in all affected sibling pair and trio families, and cases and controls, recruited from four networks worldwide, for analysis with clinical phenotypes and immunological markers. PURPOSE: In this article, we present the operational strategy of training, classification, reporting, and quality control of HLA genotyping in four laboratories on three continents over nearly 5 years. METHODS: Methods to standardize HLA genotyping at eight loci included: central training and initial certification testing; the use of uniform reagents, protocols, instrumentation, and software versions; an automated data transfer; and the use of standardized nomenclature and allele databases. We implemented a rigorous and consistent quality control process, reinforced by repeated workshops, yearly meetings, and telephone conferences. RESULTS: A total of 15,246 samples have been HLA genotyped at eight loci to four-digit resolution; an additional 6797 samples have been HLA genotyped at two loci. The genotyping repeat rate decreased significantly over time, with an estimated unresolved Mendelian inconsistency rate of 0.21%. Annual quality control exercises tested 2192 genotypes (4384 alleles) and achieved 99.82% intra-laboratory and 99.68% inter-laboratory concordances. LIMITATIONS: The chosen genotyping platform was unable to distinguish many allele combinations, which would require further multiple stepwise testing to resolve. For these combinations, a standard allele assignment was agreed upon, allowing further analysis if required. CONCLUSIONS: High-resolution HLA genotyping can be performed in multiple laboratories using standard equipment, reagents, protocols, software, and communication to produce consistent and reproducible data with minimal systematic error. Many of the strategies used in this study are generally applicable to other large multi-center studies.

HLA DPA1, DPB1 alleles and haplotypes contribute to the risk associated with type 1 diabetes: analysis of the type 1 diabetes genetics consortium families.

OBJECTIVE: To determine the relative risk associated with DPA1 and DPB1 alleles and haplotypes in type 1 diabetes. RESEARCH DESIGN AND METHODS: The frequency of DPA1 and DPB1 alleles and haplotypes in type 1 diabetic patients was compared to the family based control frequency in 1,771 families directly and conditional on HLA (B)-DRB1-DQA1-DQB1 linkage disequilibrium. A relative predispositional analysis (RPA) was performed in the presence or absence of the primary HLA DR-DQ associations and the contribution of DP haplotype to individual DR-DQ haplotype risks examined. RESULTS: Eight DPA1 and thirty-eight DPB1 alleles forming seventy-four DPA1-DPB1 haplotypes were observed; nineteen DPB1 alleles were associated with multiple DPA1 alleles. Following both analyses, type 1 diabetes susceptibility was significantly associated with DPB1*0301 (DPA1*0103-DPB1*0301) and protection with DPB1*0402 (DPA1*0103-DPB1*0402) and DPA1*0103-DPB1*0101 but not DPA1*0201-DPB1*0101. In addition, DPB1*0202 (DPA1*0103-DPB1*0202) and DPB1*0201 (DPA1*0103-DPB1*0201) were significantly associated with susceptibility in the presence of the high risk and protective DR-DQ haplotypes. Three associations (DPB1*0301, *0402, and *0202) remained statistically significant when only the extended HLA-A1-B8-DR3 haplotype was considered, suggesting that DPB1 alone may delineate the risk associated with this otherwise conserved haplotype. CONCLUSIONS: HLA DP allelic and haplotypic diversity contributes significantly to the risk for type 1 diabetes; DPB1*0301 (DPA1*0103-DPB1*0301) is associated with susceptibility and DPB1*0402 (DPA1*0103-DPB1*0402) and DPA1*0103-DPB1*0101 with protection. Additional evidence is presented for the susceptibility association of DPB1*0202 (DPA1*0103-DPB1*0202) and for a contributory role of individual amino acids and DPA1 or a gene in linkage disequilibrium in DR3-DPB1*0101 positive haplotypes.

HLA DR-DQ haplotypes and genotypes and type 1 diabetes risk: analysis of the type 1 diabetes genetics consortium families.

OBJECTIVE: The Type 1 Diabetes Genetics Consortium has collected type 1 diabetic families worldwide for genetic analysis. The major genetic determinants of type 1 diabetes are alleles at the HLA-DRB1 and DQB1 loci, with both susceptible and protective DR-DQ haplotypes present in all human populations. The aim of this study is to estimate the risk conferred by specific DR-DQ haplotypes and genotypes. RESEARCH DESIGN AND METHODS: Six hundred and seven Caucasian families and 38 Asian families were typed at high resolution for the DRB1, DQA1, and DQB1 loci. The association analysis was performed by comparing the frequency of DR-DQ haplotypes among the chromosomes transmitted to an affected child with the frequency of chromosomes not transmitted to any affected child. RESULTS: A number of susceptible, neutral, and protective DR-DQ haplotypes have been identified, and a statistically significant hierarchy of type 1 diabetes risk has been established. The most susceptible haplotypes are the DRB1*0301-DQA1*0501-DQB1*0201 (odds ratio [OR] 3.64) and the DRB1*0405-DQA1*0301-DQB1*0302, DRB1*0401-DQA1*0301-DQB*0302, and DRB1*0402-DQA1*0301-DQB1*0302 haplotypes (ORs 11.37, 8.39, and 3.63), followed by the DRB1*0404-DQA1*0301-DQB1*0302 (OR 1.59) and the DRB1*0801-DQB1*0401-DQB1*0402 (OR 1.25) haplotypes. The most protective haplotypes are DRB1*1501-DQA1*0102-DQB1*0602 (OR 0.03), DRB1*1401-DQA1*0101-DQB1*0503 (OR 0.02), and DRB1*0701-DQA1*0201-DQB1*0303 (OR 0.02). CONCLUSIONS: Specific combinations of alleles at the DRB1, DQA1, and DQB1 loci determine the extent of haplotypic risk. The comparison of closely related DR-DQ haplotype pairs with different type 1 diabetes risks allowed identification of specific amino acid positions critical in determining disease susceptibility. These data also indicate that the risk associated with specific HLA haplotypes can be influenced by the genotype context and that the trans-complementing heterodimer encoded by DQA1*0501 and DQB1*0302 confers very high risk.