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 hypercholesterolemia 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 < 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>A), a previously undescribed single-nucleotide deletion in the SIDT1 gene [c.2426del (p.Leu809CysfsTer2)], new missense variant c.10313C>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.

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 ß-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>A substitution (IVS5 -1G>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. Key words: human; maturity onset diabetes of the young; MODY2; glucokinase gene; next-generation sequencing; genetic analysis; bioinformatics.

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.

Polymorphism of the GLIS3 gene in a Caucasian population and among individuals with carbohydrate metabolism disorders in Russia.

OBJECTIVE: Earlier, GLIS3 gene polymorphisms have been shown to be associated with the development of maturity onset diabetes of the young (MODY). We screened GLIS3 gene sequences among patients with MODY to identify probably pathogenic variants by whole-exome sequencing. We estimated frequency of rare single-nucleotide variants in the coding region of GLIS3 in a Caucasian population and among individuals with carbohydrate metabolism disorders in Russia. RESULTS: We identified 15 single-nucleotide variants in GLIS3. Three rare variants (minor allele frequency < 1%) rs806052, rs143051164, and rs149840771 were genotyped in 126 cases of MODY, in 188 patients with type 2 diabetes mellitus (DM2), and 564 randomly selected Caucasian individuals in Russia. A heterozygous rs806052 variant was identified in one patient with DM2; c.1270T frequency was 0.003. Prevalence of rs143051164 c.844G was 0.003 in the control population and 0.004 and 0.003 in MODY and DM2 samples, respectively. Prevalence of rs149840771 c.2096A was 0.003 and 0.004 in the control population and among MODY patients, respectively. In DM2 patients, rs149840771 c.2096A was not identified. We did not detect any associations of rs806052, rs143051164, and rs149840771 with carbohydrate metabolism disorders among patients with MODY and DM2 in Russia.

A Case of Maturity Onset Diabetes of the Young (MODY3) in a Family with a Novel HNF1A Gene Mutation in Five Generations.

Diabetes mellitus with autosomal dominant inheritance, i.e., maturity-onset diabetes of the young (MODY), is a genetic form of diabetes mellitus. The MODY phenotype is associated with gene mutations leading to pancreatic ß-cell dysfunction. Here, we present the clinical case of a 50-year-old proband with familial diabetes mellitus in five generations (proband, her mother, grandmother, great-grandfather, and son). This disease is most likely associated with the novel Ser6Arg mutation in the HNF1A gene, which was identified in four family members. The mutation was not detected in MODY patients (126 subjects), in patients with type 2 diabetes mellitus (188 subjects), and in a general population sample (564 subjects).

[Blood Levels of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in Men from Different Population Groups and Its Relation to Unfavorable Long-Term Prognosis].

AIM: of the study was to investigate blood levels of proprotein convertase subtilisin/kexin type 9 (PCSK9) in men from different population subgroups, their associations with cardiovascular risk factors and with unfavorable 7-years long-term prognosis. MATERIAL AND METHODS: The study included three subgroups of men from a population sample of residents of Novosibirsk, 44-73 years old, not receiving lipid-lowering drugs: subgroup of population proper (183 men), subgroup with hypercholesterolemia (46 men), and subgroup with hypocholesterolemia (18 men). Blood level of PCSK9 was determined by ELISA using the test-systems "Human Proprotein Convertase 9/PCSK9 Immunoassay". Study endpoints (myocardial infarction, cardiovascular death) were registered during 7 years after baseline examination of subgroups using the data of the Registers of myocardial infarction and cardiovascular mortality. RESULTS: Distribution of PCSK9 protein in subgroups with hyper- and hypocholesterolemia was normal. In the subgroup of population proper it was abnormal with leftward shift. PCSK9 protein concentration in the subgroup with hypercholesterolemia was 1.2 times higher than in the population subgroup. PCSK9 protein level correlated significantly with blood levels of total cholesterol (CH), low density lipoprotein (LDL) CH, and glucose. Only 15% of PCSK9 variability was due to the influence of other factors (R Square=0.155, p<0.001). Factors with significant influence on blood level of PCSK9 protein were levels of high density lipoprotein CH (=0.238, p=0.023), triglycerides (=0.253, p=0.049) and LDL CH (=0.751, p=0.009). Multivariate regression analysis revealed significant independent association of PCSK9 protein levels with cardiovascular death during period of registration (7-years) (p=0.048, OR=1.01). This result indicates that in men increase of blood level of PCSK9 protein by 1ng/ml independently of other parameters increases relative risk of cardiovascular death during following 7 years by 1%.

Association of Level of Proprotein Convertase Subtilisin/Kexin Type 9 with Intima-Media Thickness in Patients with Familial Hypercholesterolemia.

We studied association of PCSK9 protein with the carotid artery intima-media thickness in patients with familial hypercholesterolemia (N=53; age 49.9±6.9 years) treated with statins. Blood level of PCSK9 protein was measured by ELISA; ultrasonography of the carotid arteries with measurement of the thickness of the intima-media complex of the common carotid arteries in the distal segment for 10 mm from the bifurcation on the far wall of the vessel was performed in on-line mode. The mean values were calculated for both sides, the maximum mean value was included in the analysis. It was shown that PCSK9 levels positively correlate with carotid artery intima-media thickness in patients with familial hypercholesterolemia.

[Proprotein Convertase Subtilisin/Kexin-Type 9 (PCSK9) New Opportunities of Lipid-Lowering Therapy].

In the literature review covered issues opening protein-proprotein convertase, subtilisin/kexin-type9 (PCSK9), its modern terminology, the results of its biochemical, molecular and genetic studies, metabolic regulation, functions and clinical findings in the blood content ofPCSK9 in lipid disorders and clinical pharmacological studies of monoclonal antibodies to this protein for the correction of lipid metabolism of major interest for cardiology and lipidology.

[Molecular genetics of maturity-onset diabetes of the young]. / Molekuliarnaia genetika MODY.

To verify the type of diabetes mellitus (DM) remains an extremely important problem in endocrinology, as along with types 1 and 2 DM there are rarer hereditary types of DM, including maturity-onset diabetes of the young (MODY). The latter is a genetic type of DM, which is characterized by an autosomal dominant inheritance. Eleven types of MODY (MODY 1 to MODY13) are identified; each is associated with mutations in the certain gene: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, KCNJ11 and ABCC8. A molecular genetic testing for suspected MODY is conducted to verify the diagnosis and to define a subtype of MODY, patient management tactics, to predict the outcome of the disease and its complications in relation to the found subtype of MODY. It is also important to seek mutation causing MODY in terms of the early detection of MODY in the first-degree relatives of a proband, appropriate therapy of the disease, and prevention of its complications.

CETP gene polymorphism in the caucasian population of West Siberia and in groups contrast by total serum cholesterol levels.

TaqIB polymorphism of the gene encoding cholesterol ester transporting protein (CETP) was analyzed in the Caucasian population of West Siberia and in groups contrast by total serum cholesterol content. The patients were selected for the study from the main sample of HAPIEE project (9600 examined subjects aged 45-69 years, 50% men). Analysis was carried out in 293 patients with high levels of total cholesterol (>300 mg/dl), 293 patients with normal and low levels of total cholesterol (<200 mg/dl), and 265 patients represented the population sample (mean level of total cholesterol 235.8±43.9 mg/dl). The frequencies of B1B1, B1B2, and B2B2 genotypes in the population were 27.5, 54.8, and 17.7%, respectively. The incidence of allele B2 was 45.1, 45.2, and 50.2% in the population and in groups with normal and high total cholesterol levels, respectively (p>0.05). Associations of CETP gene TaqIB (rs708272) polymorphism with HDL cholesterol levels was detected in groups with high and low total cholesterol levels (p=0.014 and p=0.008). CETP gene TaqIB polymorphism B2B2 genotype was associated with high level of HDL cholesterol and a more favorable lipid profile.

[Key laboratory diagnostic biomarkers of coronary atherosclerosis].

Laboratory lipid and lipoprotein biomarkers (total cholesterol - CH, triglycerides - TG, low-density and high-density lipoprotein cholesterol- LDL-CH, HDL-CH, apolipoproteins B and A1 - apoB, apoA1), carbohydrate biomarkers (plasma glucose, basal insulin), high sensitive C-reactive protein (hsCRP) and oxidative biomarkers (basal level of lipid peroxidation [LPO] products in LDL, LDL resistance to oxidation in vitro, oxidative modification of apoLDL and level of LDL lipophilic antioxidants) were studied in 388 men aged 42-70 years: 96 citizens of Western Siberia with angiographically documented coronary atherosclerosis and coronary heart disease (CHD); 292 men of population sample of citizens of Novosibirsk, including 44 men with CHD confirmed by standardized criteria and methods. Significant associations were found of coronary atherosclerosis and CHD with laboratory diagnostic biomarkers like blood levels of HDL-CH, TG, apoB, apoA1, basal insulin, hsCRP and basal level of LPO products in LDL and LDL resistance to oxidation.

Apolipoprotein E gene polymorphism in men with coronary atherosclerosis in Siberia.

Polymorphism of coding fragment of APOE gene was analyzed in two groups of men. The main group consisted of 77 residents of the West Siberian region aged 45-65 years with coronary atherosclerosis (documented by coronary angiography) without acute coronary syndrome with stable effort angina, functional class II-IV. The reference group consisted of 350 residents of Novosibirsk, aged 45-69 years. Statistically significant associations between genotypes of APOE gene coding part polymorphism and some key lipid risk factors (blood total and LDL cholesterol, atherogenic index, etc.) for coronary atherosclerosis were found in male residents of the West Siberian region. Elevated total mean level of cholesterol was detected in male residents of Novosibirsk with the APOE genotypes containing ε4 allele.

Association of cold receptor TRPM8 gene polymorphism with blood lipid indices and anthropometric parameters in Shorians.

We analyzed single nucleotide polymorphisms of the cold receptor TRPM8 gene as genetic markers of blood serum lipid indices in Shorians. Associations were found between rs11562975 (L250L) TRPM8 gene mononucleotide polymorphism with total cholesterol and LDL cholesterol and between rs28901637 (P249P) and HDL cholesterol. No associations of P249P and L250L with triglyceride level were found. L250L polymorphism was associated with anthropometric parameters characterizing lipid metabolism (hip and waist circumferences). The TRPM8 gene is likely to be involved in the regulation of lipid metabolism.

[Genetic aspects of digestive diseases. Part 1].

The paper presents the data available in the literature on mutations in known genes in pancreatitis, such as cationic trypsinogen (PRSS1), pancreatic secretory trypsin inhibitor (PSTI/SPINK1), cystic fibrosis (CFTR), and apolipoprotein E (APOE) genes, as well as the new candidate gene--chymotrypsinogen (CTRC). It also gives the results of the authors studies estimating the spread of the mutations in the PRSS1 (2.5%), PSTI/SPINK1 (3.3%), and CFTR (0.8%) genes, as well as APOE polymorphism in patients with pancreatitis. It is shown that the E4 allele of the APOE gene was more frequently identified in patients with acute pancreatitis than in those with chronic pancreatitis (0.143 +/- 0.05 and 0.026 +/- 0.02, respectively; p < 0.05). An overview is given of 7 major classes of candidate genes implicated in the pathogenesis of cholesterol cholelithiasis (CL): hepatic enzymes regulating blood lipid composition; receptors of lipoproteins, hepatic and intestinal membrane and intracellular transport proteins; factors regulating the transcription of lipids and bile salts, cholecystokinin and its receptors, and mucin. In the authors' epidemiological study, the spread of APOE alleles and genotypes did not differ in women with and without CL; low molecular-weight apolipoprotein(a) isoforms (B, S2) were significantly found in patients with CL than in those without CL; the spread of the CG genotype in the TRPM8 gene was significantly lower in women with cholesterol CL than that in the Novosibirsk population. These polymorphisms have been proved to be associated with bile cholesterol concentrations in women with cholesterol CL. The opposite effect of the APOE4 allele on gallbladder stone formation processes is demonstrated, by using the APOE polymorphism as an example, which shows it necessary to examine each specific population to elicit a possible association between the polymorphism of different genes and gastrointestinal tract diseases.

[Sudden cardiac death and polymorphism of genes-candidates of cardiovascular diseases].

Aim of the study was to elucidate genetic markers associated with elevated risk of sudden cardiac death (SCD). We collected autopsy material during 1999 - 2001 in a process of forensic medical pathologo-anatomical examination of corpses of 182 men who had died suddenly in Octyabrsky district of Novosibirsk in the age of 25 - 64 years (mean age 53,6 +/- 7,9 years). We studied polymorphisms of the following genes: angiotensin converting enzyme - , glycoprotein IIb/IIIa - GPIIb/IIIa, alpha2b adrenoreceptor - ADRA2B, beta(1)-adrenoreceptor - ADRB1. Control comprised samples of population of men aged 25 - 35 and 55 - 64 years from the same district of Novosibirsk examined within framework of international WHO project MONICA. Comparison of frequencies of genotypes of polymorphism A1/A2 of GPIIb/IIIa gene in combined sample of population and group with SCD revealed in SCD group lowering of portion of A2/A2 homozygotes (5.0 and 1.2%, respectively, =0.029) and elevation of portion of A1/A2 heterozygotes (18.7 and 28.3%, respectively, =0.027). Odds ratio for a heterozygote to enter SCD group was 1.71 (95% confidence interval 1.0 to 2.77). Comparison of frequencies of genotypes and alleles of polymorphism A145G of ADRB1 gene in combined sample of population and group with SCD did not reveal any difference. Comparison of frequencies of polymorphism I/D of ACE gene in combined sample of population and group with SCD revealed significant lowering of frequencies of genotype I/I in SCD group (22.0 and 13.8%, respectively, p=0.033). There were no significant differences between SCD group and control in frequencies of studied polymorphism of alpha2b-adrenoreceptor gene.

[APOE gene polimorphism and lithogenicity of the bile in the women with cholelithiasis].

AIM: to study the frequency of alleles and genotypes of APOE gene in women with gallstone disease (GSD), and also to research the association of the APOE gene polymorphism and bile lithogenicity indices. MATERIALS AND THE METHODS: were investigated 104 women after cholecystectomy about verified cholesterol gallstones, and 176 women from the female Novosibirsk population, they were control group for the comparison of the frequency of APOE alleles and genotypes. Gallbladder bile (bile cholesterol, common bile acids) was investigated in women with GSD. Blood serum lipids and APOEgene polymorphism were studied in all women. RESULTS: APOE gene polymorphism in the women with GSD did not differ from those registered in control group from women population. At the presence of APOE4 allele at the women with GSD the significant increase of gallbladder biliary cholesterol level was marked. In the women with GSD with various APOE genotypes average levels of blood serum lipids, common bile acids and bile cholesterol indices were similar.

[The spectrum of mutations in the low-density lipoprotein receptor gene in the Russian population].

The spectrum of mutations in the low-density lipoprotein (LDL) receptor gene was studied in a sample of hypercholesterolemia patients of Caucasoid origin from the population of Russia. The examined patients were 45 to 49-years-old and had the highest level of total serum cholesterol in this age group. Seven previously non-described mutations have been revealed in exon 9 (R410G; M412V) and in exon 12 (Y/Y576; N/N591; L605V; L605R; A612G). Twelve previously described mutations have been identified in exons 2 (C/C27), 5 (C261F; E240X), 6 (E288K), 8 (A391T), 9 (E418G; L432R; D433E), 11 (G/G549; E558K; L/L568), and 12 (G592E). Only one of these mutations was previously described in Russia in a clinical sample of patients with familial hypercholesterolemia. The spectrum of LDL receptor gene mutations in the population sample of patients with hypercholesterolemia significantly differs from the mutation spectrum in patients with familial hypercholesterolemia (clinical samples). Sequencing of the LDL receptor gene is a highly efficient method for identifying the markers of hypercholesterolemia predisposition in a population.

[Polymorphism of the gene of angiotensin-converting enzyme and apolipoprotein E gene in long-livers of Novosibirsk city]. / Polimorfizm gena angiotensin-prevrashchaiushchego fermenta i gena apolipoproteina E u dolgozhitelei goroda Novosibirska.

The levels of polymorphism of genes of angiotensin converting enzyme (ACE) and apolipoprotein E (Apo E) were studied in elderly and long-living people in Novosibirsk. The results of the study in the investigated group (97 subjects) were compared with polymorphism of these genes in Novosibirsk population group aged 25-64 who were investigated in MONICA Project survey and had DNA data base formed. Frequency of D/D genotype among senile and long-living men was 5.9%. It is 5 times lower than in men 55-64 years of age (p = 0.04). Similar decrease of this gene frequency was also found in women of the same age. In men older than 83 years of age 4 times lowering of 3/4 genotype of Apo E gene and 2 times increasing of frequency of 2/3 genotype were revealed when comprising frequency of these genotypes in people of middle age. In subjects of senile age and long-livers of both sexes genotype 4/4 was not revealed. Lipid levels were more favorable in women with genotype 2/3 of Apo E gene (comparatively lower mean level of total cholesterol and higher level of HDL cholesterol) if compared with genotypes 3/3 and 3/4.