Computational methods that allow predicting the effects of nonsynonymous substitutions are an integral part of exome studies. Here, we validated and improved their specificity by performing a comprehensive bioinformatics analysis combined with experimental and clinical data on a model of glucokinase (GCK): 8835 putative variations, including 515 disease-associated variations from 1596 families with diagnoses of monogenic diabetes (GCK-MODY) or persistent hyperinsulinemic hypoglycemia of infancy (PHHI), and 126 variations with available or newly reported (19 variations) data on enzyme kinetics. We also proved that high frequency of disease-associated variations found in patients is closely related to their evolutionary conservation. The default set prediction methods predicted correctly the effects of only a part of the GCK-MODY-associated variations and completely failed to predict the normoglycemic or PHHI-associated variations. Therefore, we calculated evidence-based thresholds that improved significantly the specificity of predictions (≤75%). The combined prediction analysis even allowed to distinguish activating from inactivating variations and identified a group of putatively highly pathogenic variations (EVmutation score <−7.5 and SNAP2 score >70), which were surprisingly underrepresented among MODY patients and thus under negative selection during molecular evolution. We suggested and validated the first robust evidence-based thresholds, which allow improved, highly specific predictions of disease-associated GCK variations.

Background: The protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene encodes lymphoid tyrosine phosphatase (LYP), which is expressed primarily in lymphoid tissues. The functional but geographically highly variable PTPN22 single-nucleotide polymorphisms (SNPs), particularly c.1858C>T, contribute to the onset and progression of autoimmunity-associated diseases and facilitate the expression of disease-associated autoantibodies. In Central Europe, 17-25% of patients with monogenic diabetes (maturity-onset diabetes of the young, MODY) transiently express islet cell autoantibodies. Methods: We addressed the links between the functional and geographically variable PTPN22 SNPs with MODY manifestation and the expression of islet cell autoantibodies in 276 MODY patients who originated from four regions (the Czech Republic, Israel, Japan and Brazil). Results: The frequency of PTPN22 polymorphisms in the MODY patients was similar to those in geographically matched healthy populations, with the exception of c.788G>A, the minor allele frequency of which was significantly elevated in the Czech hepatocyte nuclear factor 1-α (HNF1A) MODY patients [odds ratio (OR) 4.8, 95% confidence interval (CI) 2.2-10.7] and the Brazilian MODY patients (OR 8.4, 95% CI 1.8-39.1). A barely significant increase in the c.788G>A minor allele was also detected in the islet cell autoantibody-positive Czech MODY patients. However, c.788A behaves as a loss-of-function mutant in T cells, and thus protects against autoimmunity. Conclusions: MODY patients (including islet cell autoantibody-positive cases) do not display any increase in autoimmunity-associated PTPN22 alleles. The absence of autoimmunity-associated PTPN22 alleles was also demonstrated in latent autoimmune diabetes in adults, which suggests that the slow kinetics of the onset of autoantibodies is subject to a regulation that is different from that experienced in type 1 diabetes and other autoimmune disorders.

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Background: A portion of adults with humoral immune changes have clinical diabetes that is initially not insulin-requiring (latent autoimmune diabetes of the adult, LADA). One of the genes strongly associated with autoimmune diabetes is PTPN22. We hypothesized that the manifestation and clinical features of LADA are linked to functional variants of PTPN22. Methods: We genotyped allelic frequencies of 1 protective and 3 risk-associated PTPN22 variants in 156 Czech LADA patients, 194 type 2 diabetes mellitus patients with LADA-like progression to insulinotherapy and 324 type 1 diabetes mellitus patients, and subsequently examined the associations of PTPN22 variants with the expression of autoantibodies and other clinical features of LADA. Results: We challenged the paradigm that stated that the PTPN22 c.1858T allele serves as a risk allele for LADA, although we confirmed its risk status in the geographically matched T1DM cohort. In contrast, the frequencies of other PTPN22 alleles (c.–1123C, c.788A and c.1970-852C) differed significantly from the healthy controls. We confirmed gender-related differences in the frequency of some PTPN22 polymorphisms (but not c.1858C>T) in LADA. The particular PTPN22 alleles and genotypes were associated with specific clinical features of the examined patients (autoantibodies, HbA_1c and age at diagnosis of diabetes). Conclusions: The variability in PTPN22 haplotypes suggests that the genetic signature of LADA is independent and should not be considered a hybrid form of T1DM and T2DM. Further studies should elucidate the associations with clinical characteristics of the LADA patients and focus on the newly emerging types of diabetes with the disease onset in early to mid-adulthood.

Collyriclum faba (Plagiochiida: Collyriclidae) adults occur in pairs within subcutaneous cysts. Here, we tested the extensive C. faba infrapopulation for five DNA loci known to display variability among Central European C. faba individuals. The infrapopulation tested shared 100% similarity in four of the five mitochondrial and nuclear DNA loci tested. Contrariwise, the internal transcribed spacer 1 (ITS1) loci in all but one individual differed from each other. We found only 0.0-1.5 base substitutions per 1,000 sites within the cysts, while we found 0.7-9.0 substitutions between the cysts of the single host and 3.0-9.0 substitutions when comparing C. faba individuals isolated from different host individuals. We observed the most of the ITS1 variability within 48 bp repetitive sequences featured by the chi-like sequence 5'-GCTTGTCTGCC-3' at their beginning. Similarly to the extensive C. faba infrapopulation examined, we determined the presence of highly variable number of repetitive sequences within the ITS1 locus of C. faba isolated from multiple host species and from various geographic locations. While similar variability was observed earlier in mutually unrelated specimens of several Schistosomatidae and Microphallidae species, here, we for the first time document it among multiple individuals of a single infracommunity possessing single mitochondrial haplotype. Lower ITS1 evolutionary divergence rates observed between individuals within the cysts when compared to those between the cysts suggest that the recombination occurs at multiple stages of the life cycle. We propose DNA recombination involving chi-like sequences to serve as a general feature shared by multiple families of digenetic trematodes to increase genetic diversity of their polyembryonic populations infecting their definitive hosts.