The next-generation sequencing (NGS) has become a routine method for diagnostics of inherited disorders. However, assessment of the discovered variants may be challenging, especially when they are not predicted to change the protein sequence. Here we performed a functional analysis of 20 novel or rare intronic and synonymous glucokinase (GCK) gene variants identified by targeted NGS in 1,130 patients with maturity-onset diabetes of the young. Human Splicing Finder, ver 3.1 and a precomputed index of splicing variants (SPIDEX) were used for in silico prediction. In vitro effects of GCK gene variants on splicing were tested using a minigene expression approach. In vitro effect on splicing was shown for 9 of 20 variants, including two synonymous substitutions. In silico and in vitro results matched in about 50% of cases. The results demonstrate that novel or rare apparently benign GCK gene variants should be regarded as potential splicing mutations.

BACKGROUND: The basis of early ultrasound (US) diagnosis of diabetic fetopathy (DF) in pregnant with gestational diabetes mellitus (GDM) is the forehanded detection of macrosomia, especially its asymmetric forms. In pregnant with GDM on a diet therapy, the detection of macrosomia may be an indication for starting the insulin therapy. In pregnant with hyperglycemia due to mutation in the glucokinase gene (GCK), US fetal growth dynamics helps to assume the fetal genotype, as well as to stratify the risks of insulin therapy. AIM: To determine the prognostic significance of asymmetric form of macrosomia and the value of the coefficients of proportionality for the diagnosis of DF in pregnant with GDM, including hyperglycemia due to mutation in the GCK gene. MATERIALS AND METHODS: US fetometry was performed in 95 pregnant with GDM (including 22 pregnant with hyperglycemia caused by mutation in the GCK gene) (main group) and 427 healthy pregnant women (control group). Estimated fetal weight, standard fetometric indicators and coefficients of proportionality were evaluated. Retrospective analysis of US predictors of macrosomia was carried out after evaluating the weight of the newborn and clarifying the signs of DF. RESULTS: In the group with GDM, 51 (53.7%) pregnant had children with phenotypic symptoms of DF, including macrosomia – 66,7% (34 children). We found statistically significant differences in fetal weight between the control group and the main group who gave birth to children with DF starting from 32 weeks. The coefficients of proportionality (femur length/abdominal circumference and the head circumference/abdominal circumference), characterizing the formation of the asymmetric macrosomia were significantly from 34 weeks (Р<0,05). CONCLUSION: The most effective predictive fetometric indicators for the diagnosis of fetal macrosomia are the dimensions of fetal abdomen and fetal weight > 90 percentile for gestational age. A specific sign of DF in pregnant with GDM is the asymmetric macrosomia. In pregnant with a mutation in the GCK gene, the tendency to macrosomia was revealed only in the absence of a mutation in the fetus, but insulin therapy in the presence of a similar mutation in fetus did not lead to a significant decrease in its percentile ranges.

AIMS:Diabetes in pregnancy may be associated with monogenic defects of beta-cell function, frequency of which depends on ethnicity, clinical criteria for selection of patients as well as methods used for genetic analysis. The aim was to evaluate the contribution and molecular spectrum of mutations among genes associated with monogenic diabetes in non-obese Russian patients with diabetes in pregnancy using the next-generation sequencing (NGS).METHODS:188 non-obese pregnant women with diabetes during pregnancy were included in the study; among them 57 subjects (30.3%) met the American Diabetes Association (ADA) criteria of preexisting pregestational diabetes (pre-GDM), whereas 131 women (69.7%) fulfilled criteria of gestational diabetes mellitus (GDM). A custom NGS panel targeting 28 diabetes causative genes was used for sequencing. The sequence variants were rated according to the American College of Medical Genetics and Genomics (ACMG) guidelines.RESULTS:In total, 23 pathogenic, 18 likely pathogenic and 16 variants of uncertain significance were identified in 59/188 patients (31.4%). The majority of variants (38/59) were found in GCK gene. No significant differences in the number of variants among the two study groups (pre-GDM and GDM) were observed.CONCLUSIONS:The study suggests that frequency of monogenic variants of diabetes might be underestimated, which warrants a broader use of genetic testing, especially in pregnancy.

Objective — the research was aimed at studying clinical and molecular genetic characteristics of the most common subtypes of MODY (1—3) detected by NGS. Material and methods. The study included 312 patients (162 boys and 150 girls) aged 3 months to 25 years with suspected MODY. Inclusion criteria were as follows: carbohydrate metabolism disorders of varying severity, negative titer of ICA, IA2, and GAD autoantibodies, preserved secretion of endogenous insulin. NGS technique was used for molecular genetic studies. Custom DNA Panel was used for the multiplex PCR and sequencing using the Ion Ampliseq technique. Custom Diabetes Panel included 28 genes (13 MODY candidates genes and other diabetes-associated genes). Non-synonymous mutations that were not previously described were rated as «probably pathogenic» if they had minor allele frequency of <0.1% and «pathogenic» when assessed against the ANNOVAR database. Results. Mutations in MODY candidate genes were detected in 178 (57.1%) probands. Of these, 99 mutations in GCK 99 gene were found in 129 (41.4%) probands and 77 relatives, 20 mutations in HNF1A gene were found in 19 (6.1%) probands and 14 relatives, 8 mutations in HNF4A gene — in 9 (2.9%) probands and 3 relatives. All detected mutations were heterozygous. MODY1 subtype was not previously described in the Russian Federation. Conclusions. The Russian population is dominated by MODY2 subtype. Only MODY2 is characterized by typical clinical presentation. NGS is a highly effective method in the diagnosis of MODY.

Background . Gestational diabetes (GDM) due to GCK gene mutations is the most frequent form of monogenic diabetes mellitus (DM) presenting during pregnancy. It has been suggested that the use of insulin in pregnancies with fetuses carrying GCK mutations may lead to intrauterine growth retardation. In the present study we evaluated the effect of insulin therapy during pregnancy on birth weight and length in the offsprings of mothers with GDM due to GCK mutations. Aims . The aim was to study birth weight and length in offsprings of mothers with gestational diabetes mellitus due to mutations in GCK, depending on the therapy during pregnancy. Materials and methods . The study included 38 patients with GDM caused by GCK gene mutations (18.7%) and the 45 offsprings. To define molecular basis of GDM in pregnant women we used a targeted NGS. ‘Diabetes panel’ genes were sequenced using a custom Ion Ampliseq gene panel and PGM semiconductor sequencer (Ion Torrent). To found the same mutations in their offsprings was used Sanger sequencing. All children were divided into 3 groups depending of their genotype and therapy received by the mothers during pregnancy. Results . We found statistically significant differences in birth length (p=0.04) and weight (p=0,031) depending on the genotype of the child and therapy in the mother. The risk of macrosomia was shown in non-mutation-carrying offsprings only. The birth weight in children with GCK gene mutations whose mothers received insulin during pregnancy was significantly lower. However, the birth weight remained in the normal range. Conclusions . Since prenatal diagnostics in the mothers with GCK gene mutations is not always justified, we recommend insulin therapy in order to prevent fetal macrosomia, which, however, should be less aggressive than in GDM due to other causes.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a rare X-linked disorder of water balance, which was first described in two unrelated male infants with severe symptomatic hyponatremia. NSIAD is caused by activating mutations of the arginine vasopressin receptor 2 ( AVPR2 ) gene, resulting in impaired reabsorption of free water, which leads to increased osmolarity of urine, plasma hypo-osmolarity, and persistent hyponatremia. We report, for the first time in the domestic literature, a case of isolated euvolemic hyposmolality hyponatremia in a child with oligodipsia. Because hypocorticism and renal pathology were excluded, and there was no antidiuretic hormone hypersecretion, NSIAD was suggested. A molecular genetic study revealed a new mutation L312S, not described earlier, in the seventh transmembrane domain of the AVPR2 gene. Pathogenicity of the mutation was proved by a functional study. We provide the clinical and laboratory characteristics of SIAD and the main principles of treatment. This disease should be considered in the differential diagnosis of hyponatremia syndrome.