Results of targeted sequencing of the <i>PRL, PRLR, PRLHR</i> genes in young women with non-tumor hyperprolactinemia

Aim. To study the spectrum of variants in the PRL, PRLR, PRLHR genes in women of reproductive age with non-tumor hyperprolactinemia. Material and methods. In women with non-tumor hyperprolactinemia (n = 15), targeted high-throughput sequencing of the PRL, PRLR, and PRLHR genes was performed. The target panel of genes included coding regions and adjacent splicing sites. Results. When analyzing the PRL, PRLR, PRLHR genes, a number of rare and common variants were identified. The common variant rs1205955 was found in the PRL gene (MAF А = 0.279). For the PRLR gene, a rare variant rs185353023 was identified in the 3’UTR (MAF А/С = 0.003) and 12 common variants. For the PRLHR gene, 10 common variants have been identified. The maximum number of variants was localized in the 3’UTR region and introns. Conclusions. For the first time in Russia, targeted high-throughput sequencing of the PRL, PRLR, PRLHR genes was performed, the results of which did not reveal obvious pathological variants in the studied genes in women with high prolactin content of non-tumor origin. The discovered polymorphism in these genes makes it possible to further study its association with impaired function of the prolactin link of hormonal regulation

GCK-MODY diabetes course in persons over 18 years of age: prospective observation

Most young patients with hyperglycemia have type 1 diabetes and type 2 diabetes but up to 10% of all cases of the disease occur in MODY (Maturity Onset Diabetes of the Young). Published abstracts show features of the debut, laboratory and genetic characteristics of MODY in the Russian population. However there is a small amount of data on the clinical course of this nosology in the Russian Federation.Aim: To investigate the characteristics of the 3-year course of GCK-MODY diagnosed after 18 years.Materials and methods: 85 probands and 46 relatives of the first and second degrees of kinship with a clinical diagnosis of GCK-MODY were examined: biochemical and hormonal blood tests, ultrasound, molecular genetic studies. Patients were invited for a follow-up visit 3 years after verification of the pathogenic mutations associated with GCK-MODY. Examination, biochemical and hormonalanalyzes , ultrasound were done in second visit.Results: The diagnosis GCK-MODY was verified by a molecular genetic study in 25 probands (29.4%). In 33 of 46 (71.7%) relatives of patients with GCK-MODY were diagnosed identical mutations. In 31 patients with GCK-MODY diagnosed after 18 years, a dynamic observation was performed for three years. Most patients over 18 years of age did not have clinical manifestations of carbohydrate metabolism disorders when diagnosing GCK-MODY and follow up visit. Skin rashes and allergic reactions prevailed among concomitant pathologies. Patients with GCK-MODY had preserved β-cell secretion, HbA1c targets were achieved. Low fasting hyperglycemia prevailed which persisted even after treatment correction. Among the characteristics of carbohydrate metabolism, biochemical, lipid and hormonal parameters during GCK-MODY verification and after three years of observation no significant differences were obtained, which indicates a stable course of the disease. Half of the patients achieved normoglycemia by rational nutrition, two people with GCK-MODY within three years after determining the diagnosis were transferred from insulin therapy to oral glucose-lowering drugs. Among oral glucose-lowering drugs prior to GCK-MODY verification most patients used metformin, 3 years later — dipeptidyl peptidase-4 inhibitors.Conclusion. The results of a three-year follow-up of a group of patients with GCK-MODY demonstrate a non-progressive course of this type of diabetes with stable indicators of carbohydrate metabolism and low fasting hyperglycemia that persists after 3 years of observation. With the verification of GCK-MODY and the achievement of the target values of glycated hemoglobin and postprandial glycaemia by rational nutrition, even if a low level of fasting hyperglycemia is determined, the prescription of oral glucose-lowering drugs is not indicated in most cases

Searching for new genes associated with the familial hypercholesterolemia phenotype using whole-genome sequencing and machine learning

One of the most common congenital metabolic disorders is familial hypercholesterolemia. Familial hyper-cholesterolemia is a condition caused by a type of genetic defect leading to a decreased rate of removal of low-density lipoproteins from the bloodstream and a pronounced increase in the blood level of total cholesterol. This disease leads to the early development of cardiovascular diseases of atherosclerotic etiology. Familial hypercholesterolemia is a monogenic disease that is predominantly autosomal dominant. Rare pathogenic variants in the LDLR gene are present in 75–85 % of cases with an identified molecular genetic cause of the disease, and variants in other genes (APOB, PCSK9, LDLRAP1, ABCG5, ABCG8, and others) occur at a frequency of &lt; 5 % in this group of patients. A negative result of genetic screening for pathogenic variants in genes of the low-density lipoprotein receptor and its ligands does not rule out a diagnosis of familial hypercholesterolemia. In 20–40 % of cases, molecular genetic testing fails to detect changes in the above genes. The aim of this work was to search for new genes associated with the familial hypercholesterolemia phenotype by modern high-tech methods of sequencing and machine learning. On the basis of a group of patients with familial hypercholesterolemia (enrolled according to the Dutch Lipid Clinic Network Criteria and including cases confirmed by molecular genetic analysis), decision trees were constructed, which made it possible to identify cases in the study population that require additional molecular genetic analysis. Five probands were identified as having the severest familial hypercholesterolemia without pathogenic variants in the studied genes and were analyzed by whole-genome sequencing on the HiSeq 1500 platform (Illumina). The whole-genome sequencing revealed rare variants in three out of five analyzed patients: a heterozygous variant (rs760657350) located in a splicing acceptor site in the PLD1 gene (c.2430-1G&gt;A), a previously undescribed single-nucleotide deletion in the SIDT1 gene [c.2426del (p.Leu809CysfsTer2)], new missense variant c.10313C&gt;G (p.Pro3438Arg) in the LRP1B gene, and single-nucleotide deletion variant rs753876598 [c.165del (p.Ser56AlafsTer11)] in the CETP gene. All these variants were found for the first time in patients with a clinical diagnosis of familial hypercholesterolemia. Variants were identified that may influence the formation of the familial hypercholesterolemia phenotype

Association between leukocyte telomere length and specific antibody levels after vaccination against tick-borne encephalitis

The primary objective of personalized vaccination is to induce an efficient immune defense while avoiding excessive immunization. Hence, it necessitates the development of methods for predicting the magnitude of the immune response prior to vaccination. Telomere length can be considered as a promising prognostic parameter for assessing the immune response to vaccination. The aim of the work was to analyze the possible association between leukocyte telomere length and specific antibody levels after vaccination against tick-borne encephalitis. The study included 55 men and 40 women who had not previously been vaccinated against tick-borne encephalitis and had no contacts with ticks. Vaccination was carried out with the EnceVir vaccine. One month after vaccination, the level of specific IgG antibodies against tick-borne encephalitis virus was analyzed using the VektoVKE-IgG-strip test system and leukocyte telomere length was measured using real-time quantitative PCR. According to the intensity of vaccine-elicited immune responses, patients were divided into three groups: unresponsive (IgG level 0–100 IU/ml), slightly responsive (IgG level 101–200 IU/ml) and highly responsive (IgG level above 200 IU/ml). The telomere length, at least at trend level (p &lt; 0.1), correlated with the response to vaccination as well as age, educational level and the presence of emotional stress. Using a general linear model, an association between telomere length and immune response to vaccination against tick-borne encephalitis at trend level (p &lt; 0.1) was found only in women. Using a pairwise comparison, it was found that telomere length was significantly higher in highly responsive women than in unresponsive women. Hence, an association between leukocyte telomere length and specific antibody levels after vaccination against tick-borne encephalitis was identified in women. Therefore, peripheral blood leukocyte telomere length can be considered as a promising marker for predicting lymphocyte proliferative responses and the magnitude of vaccine-elicited cellular immune responses

Screening of West Siberian patients with primary congenital glaucoma for CYP1B1 gene mutations

Primary congenital glaucoma (PСG) is a visual organ pathology that leads to progressive blindness and poor vision in children. Its main cause is an anomaly of the anterior chamber angle. Most cases of PСG are sporadic, but familial cases with an autosomal recessive (predominantly) and autosomal dominant (rare) type of inheritance have been described. Congenital glaucoma is a rare condition (1 case per 10,000–20,000 newborns), but its prevalence is substantially higher (up to 1 case per 250 newborns) in countries where consanguineous marriages are common. Mutations in the CYP1B1 gene, which encodes cytochrome P450 1B1, are the most common cause of autosomal recessive primary congenital glaucoma. This enzyme is known to be involved in retinoic acid metabolism and is necessary for normal eye development. The aim of this work was to assess the polymorphism of the CYP1B1 gene among West Siberian patients with primary congenital glaucoma. Direct automatic Sanger sequencing of exons and adjacent splicing sites of the CYP1B1 gene was carried out in 28 people with the PCG phenotype from a West Siberian region. As a result, in the sample of the white population we examined, pathogenic variants previously described in other ethnic groups were revealed: E387K (rs55989760), R444* (rs377049098), R444Q (rs72549376), and P437L (rs56175199), as well as novel single-nucleotide deletion p.F114Lfs*38 in the CYP1B1 gene. The latter can cause a frame shift, changed amino acid composition, and a formation of truncated in the protein. None of the detected mutations were found in the control sample of ophthalmologically examined individuals without PCG (100 people). Variants R444* (rs377049098) and R444Q (rs72549376) were not found in the general population sample either (576 randomly selected West Siberia residents). All the detected mutations caused the development of the autosomal recessive form of primary congenital glaucoma. The most severe clinical phenotype was observed in carriers of mutations in codon 444 of the gene. Consequently, in children with signs of increased intraocular pressure, molecular genetic analysis of the CYP1B1 gene is advisable for early diagnosis and timely initiation of PCG therapy

A rare splice site mutation in the gene encoding glucokinase/hexokinase 4 in a patient with MODY type 2

The article presents a variant of maturity onset diabetes of the young type 2, caused by a rare mutation in the GCK gene. Maturity onset diabetes of the young (MODY) is a hereditary form of diabetes with an autosomal dominant type of inheritance, an onset at a young age, and a primary defect in pancreatic β-cell function. This type of diabetes is different from classical types of diabetes mellitus (DM1 and DM2) in its clinical course, treatment strategies, and prognosis. Clinical manifestations of MODY are heterogeneous and may vary even among members of the same family, i. e., carriers of identical mutations. This phenotypic variation is due to the interaction of mutations with different genetic backgrounds and the influence of environmental factors (e. g., lifestyle). Using next-generation sequencing technology, the c.580–1G&gt;A substitution (IVS5 –1G&gt;A, rs1554335421) located in an acceptor splice site of intron 5 of the GCK gene was found in a proband. The identified variant cosegregated with a pathological phenotype in the examined family members. The GCK gene encodes glucokinase (hexokinase 4), which catalyzes the first step in a large number of glucose metabolic pathways such as glycolysis. Mutations in this gene are the cause of MODY2. The illness is characterized by an insignificant increase in the fasting glucose level, is a well-controlled disease without medication, and has a low prevalence of micro- and macrovascular complications of diabetes. The presented case of MODY2 reveals the clinical significance of a mutation in the splice site of the GCK gene. When nonclassical diabetes mellitus is being diagnosed in young people and pregnant women, genetic testing is needed to verify the diagnosis and to select the optimal treatment method

Полиморфизм гена APOA5 у пациентов с первичной гиперлипидемией

Aim. To study APOA5 genetic polymorphism in Caucasoid patients with familial hypercholesterolemia.Methods. Sample of patient with familial hypercholesterolemia (43 unrelated Caucasoid persons) was formed using Dutch Lipid Clinic Network Criteria. Targeted sequencing of genome DNA was performed by NimbleGen SeqCap EZ Choice kit on pyrosequencer Roche Junior GS (Roche, Switzerland).Results. In patients with familial hypercholesterolemia, 8 substitutions were identified in the APOA5 gene: rs2075291, rs3135506, rs2072560, rs2266788, rs3135507, rs34089864, rs619054, and rs651821, that are known to be associated with dyslipidemia. One novel substitution Ala169Asp was found. It is responsible for changing the charge of a domain for lipid droplets binding in the APOA5 protein. There were no differences in the frequencies of the ApoA5*2 intragenic haplotype, which has been recently reported to be associated with an increased triglyceride levels in patients with familial hypercholesterolemia and the population.Conclusion. Genetic variants ofAPOA5, common in patients with familial hypercholesterolemia, may be involved in the formation of the pathological phenotype of dyslipidemia. However, a more accurate assessment oftheir contribution is required to differentiate patients with familial hypercholesterolemia according to their triglycerides level.Цель. Анализ генетического полиморфизма гена APOA5 среди пациентов европеоидного происхождения с семейной гиперхолестеринемией.Материалы и методы. Выборка из 43 неродственных пациентов европеоидного происхождения с семейной гиперхолестеринемией (СГХС) сформирована с использованием диагностического критерия DutchLipidClinicNetwork. Таргетное секвени-рование геномной ДНК выполнено с помощью набора NimbleGenSeqCapEZChoicekit на пиросеквенаторе RocheJuniorGS (Roche, Швейцария).Результаты. У пациентов с СГХС выявлены восемь замен в гене APOA5 - rs2075291, rs3135506, rs2072560, rs2266788, rs3135507, rs34089864, rs619054, rs651821, -для которых ранее показана ассоциация с дислипидемией, а также одна ранее не описанная замена Ala169Asp, которая может менять заряд сайта связывания белка с липидными каплями в гепатоцитах. Не выявлено различий в частоте внутригенного гаплотипа ApoA5*2, для которого ранее показана ассоциация с повышенным уровнем триглицеридов, между пациентами c СГХС и популяцией.Заключение. Генетические варианты APOA5, распространенные у пациентов с СГХС, могут участвовать в формировании патологического фенотипа дислипидемии, однако для более точной оценки их вклада целесообразно проводить дифференциацию пациентов с СГХС по уровню триглицеридов

NEW ASPECTS OF THE USE OF PROTEIN TISSUE-SPECIFIC MARKERS IN THE ESTIMATION OF SEVERITY OF COMMUNITY-ACQUIRED PNEUMONIA

The purpose of the study was to assess the associations of proteins of pulmonary surfactants A (SP-A) and D (SP-D) with the severity of community-acquired pneumonia. Material and methods. A survey of 188 patients diagnosed with community-acquired pneumonia (CAP). The average age of the examined patients was 54.3 ± 16.5 years (M ± SD). In 102 patients (54.3 %) severe CAP was verified. All patients underwent clinical, functional, diagnostic and laboratory studies. Results and discussion. It was determined that SP-A and SP-D content was significantly higher in patients with severe CAP compared to patients with a mild course of this disease. The direct association of severe CAP with an elevated SP-AP level, directly associated with an increase in the concentration of C-reactive protein in the blood and erythrocyte sedimentation rate, have been confirmed in early studies. Using partial correlation analysis, it was established that the severe course of CAP is directly related to SP-A (r = 0.221; p = 0.003) and SP-D content (r = 0.262; p &lt; 0.001) regardless of age, smoking, and body weight. Thus, direct associations of SP-A and SP-D with a severe course of CAP reflect the high pathogenetic significance of these protective factors in infectious lung damage

Molecular-genetic mechanisms of the interaction between processes of cell response to mechanical stress and neuronal apoptosis in primary open-angle glaucoma

Glaucoma is a chronic and progressive disease, which affects more than 60 million people worldwide. Primary open-angle glaucoma (POAG) is one of the most common forms of glaucoma. For example, about 2.71 million people in the USA had primary open-angle glaucoma in 2011. Currently POAG is a major cause of irreversible vision loss. In patients with treated open-angle glaucoma the risk of blindness reached to be about 27 %. It is known that the death of optic nerve cells can be triggered by mechanical stress caused by increased intraocular pressure, which induces neuronal apoptosis and is observed in patients with POAG. Currently, there is a large number of scientific publications describing proteins and genes involved in the pathogenesis of POAG, including neuronal apoptosis and the cell response to mechanical stress. However, the molecular- genetic mechanisms underlying the pathophysiology of POAG are still poorly understood. Reconstruction of associative networks describing the functional interactions between these genes/proteins, including biochemical reactions, regulatory interactions, transport, etc., requires the use of methods of automated knowledge extraction from texts of scientific publications. The aim of the work was the analysis of associative networks, describing the molecular-genetic interactions between proteins and genes involved in cell response to mechanical stress (CRMS), neuronal apoptosis and pathogenesis of POAG using ANDSystem, our previous development for automated text analysis. It was shown that genes associated with POAG are statistically significantly more often represented among the genes involved in the interactions between CRMS and neuronal apoptosis than it was expected by random reasons, which can be an explanation for the effect of POAG leading to the retinal ganglion cell death

Результаты секвенирования нового поколения у мужчин с пограничным удлинением интервала QT (пилотное исследование)

Highlights. Probably causal mutations of QT interval prolongation in genes associated with LQTS were found in men of the Siberian population.Aim. To detect and study mutations in individuals with borderline prolongation of the QT interval in Siberian males.Methods. The study was conducted on the material of the international project HAPIEE in the period from 2003 to 2005 and screening of young people aged 25–44, performed in Novosibirsk. The total sample of men was 1353 people aged 25 to 69 years. From each age subgroup (25–29, 30–34, ..., 65–69 years old) 2–3 samples with the highest QT values were selected . The study group consisted of 30 men who subsequently underwent sequencing of a panel of genes. The search for mutations was carried out in genes associated with long QT syndrome (LQTS): KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, KCNJ2, CACNA1, SCN4B, KCNJ5, ANK2, CAV3, SNTA1, AKAP9, CALM1 and CALM2. All identified single nucleotide variants were verified by direct Sanger sequencing.Results. Three rare variants in the LQTS genes have been identified: p.P197L of the KCNQ1 gene, p.R176W, and p.D1003GfsX116 of the KCNH2 gene.Conclusion. In Caucasian men from the Novosibirsk population with borderline prolongation of the QT interval, probably causal substitutions in the LQTS genes – KCNH2 and KCNQ1, contributing to the prolongation of the QT interval, were found. To clarify the spectrum and frequency of occurrence of various mutations in genes, life-threatening arrhythmias in the population, additional studies are needed on extended samples.Основные положения. У мужчин сибирской популяции обнаружены, вероятно, причинные мутации удлинения интервала QT в генах, ассоциированных с LQTS.Цель. Обнаружить и изучить мутации у мужчин сибирской популяции с пограничным удлинением интервала QT.Материалы и методы. Исследование проведено на материале международного проекта HAPIEE в период с 2003 по 2005 г. и скрининга молодых людей 25–44 лет, выполненного в Новосибирске. Общая выборка мужчин составила 1 353 человека в возрасте от 25 до 69 лет. Из каждой возрастной подгруппы (25–29, 30–34, …, 65–69 лет) выбрано по 2–3 образца с наибольшими значениями QTc. Исследуемая группа состояла из 30 мужчин, которым в дальнейшем выполнено секвенирование панели генов. Поиск мутаций проведен в генах, ассоциированных с синдромом удлиненного интервала QT (LQTS): KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, KCNJ2, CACNA1, SCN4B, KCNJ5, ANK2, CAV3, SNTA1, AKAP9, CALM1 и CALM2. Все выявленные однонуклеотидные варианты проверены методом прямого секвенирования по Сэнгеру.Результаты. Идентифицированы три редких варианта в генах LQTS: p.P197L гена KCNQ1, p.R176W и p.D1003GfsX116 гена KCNH2.Заключение. У мужчин европеоидной популяции, жителей Новосибирска, с пограничным удлинением интервала QT обнаружены вероятные причинные замены в генах LQTS – KCNH2 и KCNQ1, способствующие пролонгации интервала QT. Для уточнения спектра и частоты встречаемости различных мутаций в генах, жизнеугрожающих аритмий в популяции необходимы дополнительные исследования на расширенных выборках