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.

The value of bacterial metagenomic analysis in post-surgical examination of gallstones.

Gallstone disease is one of the most common causes of hospitalization for gastrointestinal diseases in the world. Recent studies have examined the presence of bacteria in the formation of stones. Our main goal was to determine the overall composition of gallstone microflora. Gallstones were obtained from 24 patients during laparoscopic cholecystectomy from which DNA were extracted. Composition of bacterial flora was evaluated on 16 s rDNA sequencing technique. In the vast majority of samples, bacteria were present, and four groups could be differentiated regarding the flora. Overall composition shows that 87% of the stones were cholesterol/mixed type of gallstone. Additionally, potentially harmful microorganisms (Streptococcus, Clostridium and Kocuria) that could cause post-surgery complications were identified in several patients. The obtained results indicate that this technique may be useful in analyzing the type of stones and in pinpointing the presence of pathogenic bacteria.

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.

Genetic diversity of urinary bladder cancer and the risk of recurrence based on mutation analysis.

The aim of the study was to assess the genetic diversity of bladder cancer and determine the suitability of a proposed molecular marker panel to monitor the course of bladder cancer patients. The study involved 185 patients with diagnosed bladder cancer. The genetic diversity of the bladder cancer was evaluated by the prevalence of mutations in the TP53, HRAS, FGFR3 and WWOX genes. Mutations were detected in 62.2% of the tumor samples. The most frequently mutated genes were FGFR3 (49.7%) and TP53 (16.2%). No mutation was observed in the WWOX gene. FGFR3 mutations, contrary to TP53, correlated with lower tumor stage and grade, and the presence of multiple tumors. The risk of death was significantly higher in patients with TP53 mutant tumors (HR=3.12; 95%CI: 1.14-7.27; p=0.006) but lower in patients with FGFR3 mutations (HR=0.36; 95%CI: 0.15-0.87; p=0.002). None of the investigated genes was an independent predictor of disease-specific survival, recurrence-free survival or progression-free survival. The results confirm the existence of two alternative pathways of bladder cancer. However the presence of a high percentage of wild type variants in the higher stages of the disease suggest the existence of another pathway of molecular changes leading to the development of bladder cancer. Molecular analysis may have prognostic value and may facilitate the assignment of patients to appropriate forms of treatment - especially in the case of patients with a T1 tumor, where different mutational patterns were observed in each grade.

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.

Lymph node metastases in papillary thyroid cancer detected by quantitative real-time polymerase chain reaction for thyroglobulin and cytokeratine-19.

Papillary thyroid cancer (PTC) metastases in the lymph nodes (LNs) were detected by real-time polymerase chain reaction (PCR) for TG and cytokeratin 19 (CK19), and the obtained results were compared with histopathology. 107 LNs from 34 PTC patients were divided into four blocks by a special cutting device - 2 for histopathology, while the other 2 were tested by quantitative real-time PCR. Metastases were detected in 20 nodes from 10 (29.4%) patients. TG and CK19 expression levels differed vastly between nodes with and without metastatic cells. ddCt of TG in the genetic material extracted from N0 nodes was 9.97 ±4.20, while in nodes with metastases ddCt was 0.91±4.20 (p < 0.0001). Cytokeratin 19 showed similar results with expression level (ddCt) in N0 nodes of 10.96 ±2.58 vs. 7.73 ±3.63 in nodes with metastases (p < 0.0001). Evaluation of the utility of both parameters showed efficient differentiation of node involvement in the case of TG, with area under the ROC curve (AUC) equal to 0.91 (95% CI: 0.85-0.96). Cytokeratin 19 also allowed for a degree of differentiation but its diagnostic efficacy was lower (AUC 0.76, 95% CI: 0.64-0.88). The combined TG and CK19 quantitative real-time PCR could be used to select a previously missed group of patients with nodal involvement undetectable by standard histopathology.

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.

HbA1c-based diabetes diagnosis among patients with glucokinase mutation (GCK-MODY) is affected by a genetic variant of glucose-6-phosphatase (G6PC2).

AIMS: Genetic variation at the rs560887 locus of the glucose-6-phosphatase, catalytic 2 gene (G6PC2) is known to affect regulation of fasting glycaemia. We determined the rs560887 genotype of patients with monogenic diabetes and glucokinase gene mutations (GCK-MODY) and correlated the genotypes with HbA(1c) levels. METHODS: Patients from families with GCK-MODY were recruited from two large cohorts from Poland (n = 128) and the Czech Republic (n = 154). Genotypes at the rs560887 polymorphic site in G6PC2 were examined using real-time quantitative polymerase chain reaction. The effect of rs560887 genotype on age at diagnosis of GCK-MODY and initial HbA(1c) levels were evaluated separately within both cohorts. Following that, a meta-analysis of rs560887 genotype-HbA(1c) associations of both Polish and Czech cohorts was performed to confirm homogeneity of findings and validate cohort-specific results. RESULTS: GG homozygosity at rs560887 was associated with marginally elevated HbA(1c) levels (P = 0.07 in both cohorts). The effects observed in both groups were very homogeneous (Q = 0.18; P = 0.68). Meta-analysis showed that GG homozygosity at rs560887 was associated with mean HbA(1c) levels higher by 2.4 mmol/mol (0.24%), 95% CI 0.5-4.4 mmol/mol (0.05-0.44%) than in individuals with other genotypes. Additionally, meta-analysis of both cohorts showed that GG homozygous individuals had higher odds of reaching the 48 mmol/mol (6.5%) diagnostic threshold of diabetes; (odds ratio 1.90; 95% CI 1.07-3.36; P = 0.03). No such effects were observed for age at diagnosis of diabetes. CONCLUSIONS: Variation at the rs560887 locus of G6PC2 is associated with worse glycated haemoglobin levels in individuals with GCK mutations; GG homozygotes are more likely to meet diagnostic criteria for diabetes based on HbA(1c) level.

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.

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.

EXAFS studies on the reaction of gold (III) chloride complex ions with sodium hydroxide and glucose.

EXAFS and QEXAFS experiments were carried out at Hasylab laboratory in DESY center (X1 beamline, Hamburg, Germany) to monitor the course of the hydrolysis reactions of [AuCl(4)](-) complex ions as well as their reduction using glucose. As a result, changes in the spectra of [AuCl(4)](-) ions and disappearance of absorption Au-L(3) edge were registered. From the results of the experiments we have carried out, the changes in bond lengths between Au(3+) central ion and Cl(-) ligands as well as the reduction of Au(3+) to metallic form (colloidal gold was formed in the system) are evident. Good quality spectra obtained before and after the reactions gave a chance to determine the bond length characteristic of Au-Cl, Au-OH and Au-Au pairs. Additionally, the obtained results were compared with the simulated spectra of different gold (III) complex ions, possibly present in the solution. Finally, the mechanism of these reactions was suggested. Unfortunately, it was not possible to detect the changes in the structure of gold (III) complex ions within the time of reaction, because of too high rates of both processes (hydrolysis and reduction) as compared with the detection time.

Molecular background and clinical characteristics of HNF1A MODY in a Polish population.

PURPOSE: Knowing the molecular background of monogenic diabetes in affected individuals influences the clinical practice. Mutations in the HNF1A gene are the most frequent cause of MODY. The aim of the present study was to identify the genetic and clinical characteristics of HNF1A MODY in a Polish population, and the prevalence of diabetic complications and renal malformations. METHODS: We identified 47 families with the early-onset, autosomal-dominant form of diabetes that met the criteria of MODY. Mutation screening involved direct sequencing of the HNF1A gene. Patients' characteristics included clinical data, anthropometric measurements and biochemical parameters. The search for renal malformations involved ultrasound examination of all HNF1A mutation carriers. RESULTS: We identified 13 HNF1A MODY families and examined 56 mutation carriers, including 46 diabetic patients. The average HbA(1c) level among the diabetics was 7.5%. We identified diabetic retinopathy in 47.7% of the MODY patients, while diabetic nephropathy was present in 25%. In five HNF1A mutation carriers from three families, renal developmental malformations were identified, including one functioning kidney in two (3.6%) of them. CONCLUSION: This first systematic search for HNF1A mutations in a Polish population revealed that they are a frequent cause of MODY. In this population, HNF1A mutation carriers were characterized by a high prevalence of diabetic complications. In addition, renal developmental abnormalities were found in some mutation carriers.

Prevalence of Retinopathy in Adult Patients with GCK-MODY and HNF1A-MODY.

We aimed to assess the prevalence of diabetic retinopathy (DR) in adult patients with GCK-MODY and HNF1A-MODY in Poland and to identify biochemical and clinical risk factors associated with its occurrence.We examined 74 GCK mutation carriers, 51 with diabetes and 23 with prediabetes, respectively, and 63 patients with HNF1A-MODY. Retinal photographs, 12 for each patient, were done by a fundus camera. Signs of DR were graded according to the DR disease severity scale. Statistical tests were performed to assess differences between the groups and logistic regression was done for the association with DR.The mean age at examination was 34.5±14.8 and 39.9±15.2 in the GCK-MODY and HNF1A-MODY groups, respectively. Mild nonproliferative DR (NPDR) was found in one patient with the GCK mutation and likely concomitant type 1 diabetes, whereas DR was diagnosed in 15 HNF1A-MODY patients: 9 with proliferative, 3 with moderate NPDR and 2 with mild NPDR. In univariate logistic regression analysis in the HNF1A-MODY group, significant results were found for diabetes duration, fasting glycemia, HbA1c, arterial hypertension, age at the examination, and eGFR. The strongest independent predictors of DR in HNF1A-MODY were markers of glucose control: HbA1c (OR: 2.05, CL%95: 1.2-3.83, p=0.01) and glucose (p=0.006, OR: 1.40, CL%95: 1.12-1.83) analyzed in 2 separated models. Additionally, arterial hypertension independently predicted DR (OR: 9.06, CL%95: 1.19-98.99, p=0.04) in the model with HbA1c as glycaemic control marker.In conclusion, DR of any degree was not present in our GCK-MODY group, while in spite of young age almost every fourth subject with HNF1A-MODY showed signs of this complication.

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.

Comparison of serological and molecular (PCR-SSP) techniques of HLA-DR typing in clinical laboratory routine.

Advances in molecular biology techniques allowed for introduction of PCR-based methods for HLA typing. In routine HLA typing for organ transplantation serological method is still being used as a standard, although molecular techniques are applied more and more often. The aim of our study was to compare HLA-DR typing using traditional serological method and PCR-SSP methodology in routine clinical laboratory. HLA-DR typing was performed using standard microcytotoxicity assay and PCR-SSP method in 28 patients referred to our Transplantation Immunology Unit for HLA typing. Comparison of results obtained by both methods revealed no discrepancies in 5 patients, in 12 patients the PCR-SSP typing showed additional DR antigens or splits of antigens. In 11 patients serological typing turned out to be impossible because of technical problems. Molecular PCR typing allowed for precise antigen determination in all the patients. Comparing both methods we found PCR-SSP HLA typing method very useful in routine HLA-DR determination, especially valuable in patients, in whom some problems in serological testing are expected.