Identification of rare variants in candidate genes associated with monogenic diabetes in polish mody-x patients.

Purpose: Monogenic diabetes (MD) is caused by a mutation in a single gene and accounts for approximately 2.5-6% of all diabetes cases. Maturity-onset diabetes of the young (MODY) is the most common form of MD. To date, 14 different genes have been identified and associated with the presence of MODY phenotype. However, the number of potential candidate genes with relevance to beta cell function and glucose metabolism is increasing as more research is published. The aim of the study was to identify potentially causative variants in selected candidate genes in patients with a clinical diagnosis of MD. Methods: Targeted Next-Generation Sequencing (tNGS) on Illumina NextSeq 550 platform involving Agilent SureSelectQXT Target Enrichment protocol for 994 patients with suspected MD was performed. In the next step, the sequencing data of 617 patients with no pathogenic variants in main MD-related genes were reanalysed for the presence of causative variants in six candidate genes (MTOR, TBC1D4, CACNA1E, MNX1, SLC19A2, KCNH6). The presence of the selected variants was confirmed by Sanger sequencing. Results: Seven heterozygous possibly damaging variants were identified in four candidate genes (MTOR, TBC1D4, CACNA1E, MNX1). Five changes were assessed as novel variants, not previously described in available databases. None of the described variants were present among patients previously diagnosed with MODY diabetes due to causative, pathogenic variants in known MODY-related genes. Conclusions: The results obtained seem to confirm the effectiveness of the NGS method in identifying potentially causative variants in novel candidate genes associated with MODY diabetes.

Novel potentially pathogenic variants detected in genes causing intellectual disability and epilepsy in Polish families.

BACKGROUND: Intellectual disability (ID) affects 1-3% of the world population. The number of genes whose dysfunctions cause intellectual disability is increasing. In addition, new gene associations are constantly being discovered, as well as specific phenotypic features for already identified genetic alterations are being described. The aim of our study was to search for pathogenic variants in genes responsible for moderate to severe intellectual disability and epilepsy, using a panel of targeted next-generation sequencing (tNGS) for diagnosis. METHODS: The group of 73 patients (ID, n=32; epilepsy, n=21; ID and epilepsy, n=18) was enrolled in the nucleus DNA (nuDNA) study using a tNGS panel (Agilent Technologies, USA). In addition, high coverage mitochondrial DNA (mtDNA) was extracted from the tNGS data for 54 patients. RESULTS: Fifty-two rare nuDNA variants, as well as 10 rare and 1 novel mtDNA variants, were found in patients in the study group. The 10 most damaging nuDNA variants were subjected to a detailed clinical analysis. Finally, 7 nuDNA and 1 mtDNA were found to be the cause of the disease. CONCLUSIONS: This shows that still a very large proportion of patients remain undiagnosed and may require further testing. The reason for the negative results of our analysis may be a non-genetic cause of the observed phenotypes or failure to detect the causative variant in the genome. In addition, the study clearly shows that analysis of the mtDNA genome is clinically relevant, as approximately 1% of patients with ID may have pathogenic variant in mitochondrial DNA.

Evaluation of the Oral Bacterial Genome and Metabolites in Patients with Wolfram Syndrome.

In Wolfram syndrome (WFS), due to the loss of wolframin function, there is increased ER stress and, as a result, progressive neurodegenerative disorders, accompanied by insulin-dependent diabetes. The aim of the study was to evaluate the oral microbiome and metabolome in WFS patients compared with patients with type 1 diabetes mellitus (T1DM) and controls. The buccal and gingival samples were collected from 12 WFS patients, 29 HbA1c-matched T1DM patients (p = 0.23), and 17 healthy individuals matched by age (p = 0.09) and gender (p = 0.91). The abundance of oral microbiota components was obtained by Illumina sequencing the 16S rRNA gene, and metabolite levels were measured by gas chromatography-mass spectrometry. Streptococcus (22.2%), Veillonella (12.1%), and Haemophilus (10.8%) were the most common bacteria in the WFS patients, while comparisons between groups showed significantly higher abundance of Olsenella, Dialister, Staphylococcus, Campylobacter, and Actinomyces in the WFS group (p < 0.001). An ROC curve (AUC = 0.861) was constructed for the three metabolites that best discriminated WFS from T1DM and controls (acetic acid, benzoic acid, and lactic acid). Selected oral microorganisms and metabolites that distinguish WFS patients from T1DM patients and healthy individuals may suggest their possible role in modulating neurodegeneration and serve as potential biomarkers and indicators of future therapeutic strategies.

Next- generation sequencing is an effective method for diagnosing patients with different forms of monogenic diabetes.

AIM: Monogenic diabetes (MD) represents 5-7% of antibody-negative diabetes cases and is a heterogeneous group of disorders. METHODS: We used targeted next-generation sequencing (NGS) on Illumina NextSeq 550 platform involving the SureSelect assay to perform genetic and clinical characteristics of a study group of 684 individuals, including 542 patients referred from 12 Polish Diabetes Centers with suspected MD diagnosed between December 2016 and December 2019 and their 142 family members (FM). RESULTS: In 198 probands (36.5%) and 66 FM (46.5%) heterozygous causative variants were confirmed in 11 different MD-related genes, including 31 novel mutations, with the highest number in the GCK gene (206/264), 22/264 in the HNF1A gene and 8/264 in the KCNJ11 gene. Of the 183 probands with MODY1-5 diabetes, 48.6% of them were diagnosed at the pre-diabetes stage and most of them (68.7%) were on diet only at the time of genetic diagnosis, while 31.3% were additionally treated with oral hypoglycaemic drugs and/or insulin. CONCLUSIONS: In summary, the results obtained confirm the efficacy of targeted NGS method in the molecular diagnosis of patients with suspected MD and broaden the spectrum of new causal variants, while updating our knowledge of the clinical features of patients defined as having MD.

Genetic evaluation of patients with Alström syndrome in the Polish population.

Alström syndrome (AS) is a rare syndromic form of obesity and type 2 diabetes (T2D) in children coexisting with retinal dystrophy and disorders of many organs caused by the mutations in ALMS1 gene. Aim of this study was to identify the causative mutations in ALMS1 in a group of 12 patients of Polish origin with clinical symptoms of AS, and their 21 first-degree relatives. Using DNA sequencing, nine different mutations including three novel were identified. These mutations were not present in 212 Polish individuals with no symptoms of AS, subjected to whole-exome sequencing and collected in a national registry. Looking for genotype-phenotype relationships, we confirmed a severe phenotype in a boy with homozygous mutation in exon 16, and a relationship between a presence of T2D and mutations in exon 19. Evaluation of the type of mutation and its clinical effects gives hope for earlier diagnosis of AS in future patients and more advanced therapeutic approaches for patients with already diagnosed AS.

Glycemic variability in patients with Wolfram syndrome is lower than in type 1 diabetes.

AIMS: Wolfram syndrome (WFS) is diagnosed as coexistence of diabetes mellitus and optic atrophy, where pancreatic beta cell destruction is associated with neurodegeneration. Typically, WFS necessitates insulin treatment similar to type 1 diabetes (T1D), but the mechanism of beta cell mass reduction leading to hyperglycemia is different. METHODS: The aim of the study was to assess glycemic variability using the continuous glucose monitoring (CGM) system in seven pediatric patients with genetically confirmed WFS and compare the results with data obtained from 21 propensity score-matched patients with T1D. The "GlyCulator" application was used for the calculation of glycemic variability indices. RESULTS: CGM recordings showed similarities in glycemic variability among WFS patients, but differing from those of the T1D group. Coefficient of variation (%CV), CONGA4h, and GONGA6h were significantly (p < 0.05) lower in WFS patients (28.08 ± 7.37, 54.96 ± 11.92, and 55.99 ± 10.58) than in T1D patients (37.87 ± 14.24, 74.12 ± 28.74, p = 0.02, and 80.26 ± 35.05, respectively). In WFS patients, the percentage of values above 126 mg/dL was 69.79 (52.08-77.43), whereas in patients with T1D, the percentage was significantly lower-47.22 (35.07-62.85, p = 0.018). Curiously, a tendency toward a lower percentage of measurements below 70 mg/dL was noted in the WFS group [0 (0-7.29)] in comparison with the T1D group [6.25 (0-18.06), p = 0.122]. WFS patients had a significantly higher C-peptide level (0.31 ± 0.2 ng/mL) than T1D patients (0.04 ± 0.04 ng/mL; p = 0.006). CONCLUSIONS: Patients with WFS show smaller glycemic variability than individuals with T1D, and this may be associated with persistent residual insulin secretion.

Delayed recognition of Wolfram syndrome frequently misdiagnosed as type 1 diabetes with early chronic complications.

AIMS: Improvements in diagnostic methods and greater genetic awareness have brought remarkable progress in the recognition of monogenic forms of diabetes, including Wolfram syndrome (WFS). WFS is diagnosed based on clinical criteria of coexistence of diabetes mellitus and optic atrophy, and confirmed by molecular analysis; however, the condition is still sometimes misdiagnosed. To begin to understand the reasons for misdiagnosis, we conducted a retrospective analysis of WFS patients who were originally misdiagnosed. MATERIALS AND METHODS: The medical histories of 13 pediatric patients with clinical misdiagnosis of type 1 diabetes and early chronic complications made in the years 1995-2010 and who were subsequently correctly diagnosed with WFS based on genetic testing in 2008-2011 were analyzed. RESULTS: The average age of the patients at diabetes onset was 5 (4.4-6.3) years, and the mean HbA1c level at diagnosis was 9.1±2.3%. Initially, all of these patients were treated as having type 1 diabetes with progressive visual impairment despite good metabolic control (mean HbA1c 7.5±1.3%). Diagnosis of optic atrophy was made at an average age of 9 (5.9-11.5) years, which corresponds to 4 years after diabetes recognition (p=0.002). At the time of genetic analysis, the average age of the patients was 16 (12-18.7) years, which corresponds to 7 years after recognition of coexistence of diabetes mellitus and optic atrophy (p=0.007). CONCLUSIONS: Delays of at least 7 years occurred before recognition of WFS among a cohort of pediatric patients with diabetes. All patients with WFS were primarily misdiagnosed as having type 1 diabetes.

Prevalence of monogenic diabetes amongst Polish children after a nationwide genetic screening campaign.

AIMS/HYPOTHESIS: The aim of this study was to study dynamic changes in the prevalence of different types of diabetes in paediatric populations in Poland, with a specific focus on monogenic diabetes (MD). METHODS: Using epidemiologic data (PolPeDiab Collaboration) and nationwide genetic test results (TEAM Programme), we compared the prevalence of type 1, type 2 and cystic fibrosis-related diabetes (CFRD) and MD. Genetically confirmed MD included MODY, neonatal diabetes and Wolfram and Alström syndromes. The study covered all children aged 0-18 years treated for diabetes between 2005 and 2011 in three regions, inhabited by 23.7% (1,989,988) of Polish children, with a low prevalence of childhood obesity (<5%). RESULTS: The prevalence of type 1 diabetes showed a continuous increase, from 96 to 138/100,000 children. The prevalence of type 2 diabetes and CFRD also increased, from 0.3 to 1.01/100,000 children and from 0.1 to 0.95/100,000 children, respectively. The prevalence of MD was stable at between 4.2 and 4.6/100,000 children, accounting for 3.1-4.2% of children with diabetes, with glucokinase (GCK)-MODY being the most frequent type, amounting to 83% of patients with MD. The percentage of positive test results decreased with the number of referrals, suggesting that children with the highest probability of MD were referred initially, followed by those with a less clear-cut phenotype. The prevalence of neonatal diabetes equalled 1 in 300,000 children. CONCLUSIONS/INTERPRETATION: The prevalence of MD in a paediatric population with a low prevalence of obesity remains stable and is nearly fivefold higher than that of type 2 diabetes and CFRD, justifying a need for increased access to genetic diagnostic procedures in diabetic children.

Doubling the referral rate of monogenic diabetes through a nationwide information campaign--update on glucokinase gene mutations in a Polish cohort.

In order to improve recruitment efficiency of patients with monogenic diabetes in Poland, in September 2010 a nationwide advertising campaign was launched to inform multiple target groups interested or participating in pediatric diabetologic care. Promotional actions aimed at informing physicians, patients, parents and educators were carried out through nationwide newspapers, medical and patient-developed websites and educational conference presentations. Recruitment efficiency was compared between September 2010 (publication of the first report on project's results) and the following 12 months. The number of families and patients referred to genetic screening was increased by 92% and 96% respectively nearly reaching the numbers recruited throughout the initial 4 years of the project. Participation of non-academic centers was also significantly increased from 2.3% to 7.5% (p = 0.0005). DNA sequencing and Multiplex Ligation-dependant Probe Amplification of the glucokinase gene resulted in finding 50 different mutations. Among those mutations, 19 were novel variants, which included: 17 missense mutations (predicted to be pathogenic according to bioinformatic analysis), 1 nonsense mutation and 1 mutation affecting a consensus intronic splice site. Advertising actions directed at increasing recruitment efficiency are a powerful and possibly neglected tool in screening for rare genetic disorders with a clinically defined phenotype.

Novel glucokinase mutations in patients with monogenic diabetes - clinical outline of GCK-MD and potential for founder effect in Slavic population.

Glucokinase (GCK) gene mutations are the causative factor of GCK-MD (monogenic diabetes) characterized by a mild clinical phenotype and potential for insulin withdrawal. This study presents the results of a nationwide genetic screening for GCK-MD performed in Poland. A group of 194 patients with clinical suspicion of GCK-MD and 17 patients with neonatal diabetes were subjected to GCK sequencing. Patients negative for GCK mutations were subjected to multiplex ligation-dependent probe amplification (MLPA) to detect deletions or insertions. A total of 44 GCK heterozygous mutations were found in 68 probands (35%). Among those, 20 mutations were novel ones: A282fs, D198V, E158X, G246V, G249R, I348N, L165V, L315Q, M115I, N254S, P284fs, Q338P, R377L, R43C, R46S, S212fs, S212P, T255N, V406A and Y214D. No abnormalities were detected in MLPA analysis. Homozygous D278E mutation was found in one patient with neonatal diabetes. The most frequently observed combinations of symptoms typical for GCK-MD were mild diabetes and/or fasting hyperglycaemia (98.3%), positive C-peptide at diagnosis (76%) and dominant mode of inheritance (59%). This study outlines numerous novel mutations of the GCK gene present in white Caucasians of Slavic origin. Thorough clinical assessment of known factors associated with GCK-MD may facilitate patient selection.

Wolfram syndrome in the Polish population: novel mutations and genotype-phenotype correlation.

OBJECTIVE: Wolfram syndrome is a rare form of diabetes mellitus associated with optic atrophy and disorders of different organs (e.g. diabetes insipidus, hearing loss, ataxia, anaemia and many others). This syndrome is caused by recessive mutations in the wolframin gene (WFS1) localized on chromosome 4p16·1. The aim of this study was to identify the causative mutations in WFS1 in a group of Polish patients with suspected Wolfram syndrome. PATIENTS AND MEASUREMENTS: Nine patients with clinical symptoms consistent with Wolfram syndrome (at least diabetes mellitus and optic atrophy) and 22 first-degree relatives were examined. The molecular analysis was carried out by direct sequencing of the exons, the exon-intron junctions, and the 5' and 3' untranslated regions of WFS1. RESULTS: Nine different mutations in WFS1 (five of them novel) were identified in the nine patients. Six patients were homozygous for the following mutations: V412fs, S443R, W539X, V659fs. They developed diabetes at a mean age of 5·2 years. Three patients were compound-heterozygous for the following mutations: S167fs, Q392X, Y513fs, W648X, V779G. They developed diabetes at a mean age of 6·5 years. CONCLUSIONS: Mean age of diagnosis of diabetes among the Polish patients was typical for Wolfram syndrome; however, compound-heterozygous patients were slightly older at diabetes onset.

Immunogenicity of different brands of human insulin and rapid-acting insulin analogs in insulin-naïve children with type 1 diabetes.

AIMS: To determine (i) whether insulin preparations produced by three companies induce the same immune responses in insulin-naïve children with type 1 diabetes (T1DM); (ii) if switching from human insulin to rapid-acting insulin analogs influences this immune response; and (iii) if different insulin brands produce different clinical results during the first 2 yr after T1DM diagnosis. METHODS: Insulin antibodies (IA) were measured for 140 patients aged 1.4-17.6 yr. Regular human insulin, neutral protamine Hagedorn (NPH) human insulin, and rapid-acting insulin analogs (lispro or aspart) taken by the patients were produced by one of three companies: Bioton, Poland (A), Eli Lilly, USA (B) and NovoNordisk, Denmark (C). RESULTS: Positive IA levels were found in 112 patients (80.0%) at baseline and in 137 (97.9%) at 6 and at 24 months after T1DM diagnosis. There was no difference in IA levels among patients taking insulin preparations produced by different companies at 6 months (mean ± SD, A 27.8 ± 15.7%; B 25.3 ± 15.4%; C 24.5 ± 14.2; p = 0.54) or at 24 months (A 25.6 ± 17.8%; B29.6 ± 17.0%; C 26.2 ± 17.0%; p = 0.52); HbA(1c) and daily insulin dose did not differ significantly either. After 24 months, IA levels were similar for those who had used human insulin (mean ± SD, 25.7 ± 17.2%) and for those that had added rapid-acting analogs (28.1 ± 17.3%, p = 0.41). CONCLUSIONS: Three brands of insulin preparations did not differ with respect to immunogenicity. Rapid-acting analogs did not increase IA levels in patients previously treated with human insulin only. Patients using insulin preparations of different brands did not differ with respect to daily insulin dose or HbA(1c) .

[Clinical remission in type 1 diabetes mellitus]. / Remisja kliniczna w przebiegu cukrzycy typu 1.

Type 1 diabetes mellitus is caused by autoimmune destruction of beta cells of the Langerhans islets. At the early stage of the disease in some patients clinical remission is observed. Until now no unequivocal criteria of complete or partial remission in diabetes have been established. In order to better define this phenomenon, individual, metabolic and genetic factors as well as immunological markers, which can influence the rate and length of remission, should be considered in greater detail. The state of remission is, however, transitory and all attempts to extend it result in immunosuppressive therapy. The latest investigations also point to the influence of the insulin treatment on the prevalence and duration of the clinical remission in type 1 diabetes mellitus. These studies seem to be fully justified, taking into consideration the mechanisms of interference of exogenous insulin in the immunological process.