[Familial hypercholesterolemia in St. Petersburg: diversity of mutations argues against a strong founder effect].

Examination of low-density lipoprotein (LDL) receptor, its promoter, and major exon-intron boundaries from a sample of patients with familial hypercholesterolemia (FH) from 74 probands of St. Petersburg revealed 34 mutations and 8 widely spread polymorphisms at this locus. Only four mutations were considered silent, while the other 30 are likely associated with familial hypercholesterolemia (FH). Mutations in the LDL receptor gene, inducing the disease, were identified in 41 (55%) out of 74 families with FH. Mutation R3500Q in apolipoprotein B (APOB) gene was not detected in all probands. Therefore in the families lacking mutations hypercholesterolemia was induced by mutations in the introns of the LDL receptor gene or by other genetic factors. Nineteen mutations causing disease progression were described in St. Petersburg for the first time, while 18 of them are specific for Russia. Among Ashkenazi Jews, major mutation G197del was detected in 30% (7 out of 22) of patients with FH. In the Slavic population of St. Petersburg, no major mutations were detected. Only five mutations were identified in two families, while 24 were found in isolated families. These data are indicative of the lack of a strong founder effect for FH in the St. Petersburg population.

[Novel BRCA1 gene mutations in breast cancer patients from St. Petersburg].

Screening of patients with familial breast cancer from St. Petersburg for BRCA1 gene mutations resulted in identification of three mutations (414del3, 276delA, and A622V) and two polymorphisms (P871L and S1436S). Mutations 4146del3 and 276delA are novel, never previously described elsewhere. Deletion 2761delA produces a reading frame shift, premature protein synthesis termination and can cause predisposition for breast cancer. Deletion 414de13 does not cause a frame shift, but can result both in the disappearance of amino acid residue (D1343del) in the BRCA1 protein and in alteration of folding of the protein, entailing loss of its functional activity. Two variants of nucleotide sequence observed in the number of patients were classified as DNA polymorphisms (P871L and S1436S) rather than mutations as they were not tightly associated with the increased risk of breast cancer.

[Mutations and polymorphisms in the genes for myocilin and optineur in as the risk factors of primary open-angle glaucoma].

A collection of DNA samples obtained from primary open-angle glaucoma (POAG) patients from St. Petersburg was analyzed for single-strand conformation polymorphism (SSCP) to reveal sequence variants in exon 3 of the myocilin gene (MYOC/TIGR) and in exons 4 and 5 of the optineurin gene (OPTN), where most of the mutations revealed worldwide are located. The Q368X mutation (c. 1102 C --> T) in exon 3 of MYOC/TIGR was detected in 1.2% (2/170) of the POAG patients from St. Petersburg, i.e., with the frequency close to that observed in other world populations. Three known polymorphisms in exon 3 of MYOC/TIGR, Y347Y (c. 1041 T --> C) (12.4%), T325T (c. 975 G --> A) (0.6%), and K398R (c. 1193 A --> G) (0.6%) were also detected. No statistically significant differences in frequencies of these polymorphisms were revealed between the POAG patient and control groups. The L41L polymorphism (c. 433 G --> A) in exon 4 of OPTN was detected in 2.9% of probands and in 1% of controls. The frequency of heterozygotes for the M98K polymorphism (c. 603 T --> A) in the OPTN exon 5 was statistically significantly higher (P = 0.036; Fisher's exact test) among the POAG patients (6.5%) than among the controls (1%). In the sample examined the E50K mutation, typical of the patients with pseudonormal intraocular pressure glaucoma, was not found.

[Prevalence of widespread BRCA1 gene mutations in patients with familial breast cancer from St. Petersburg].

Ten variants different from the canonical nucleotide sequence (GenBank, U14680) has been identified when studying the mutation spectrum in gene BRCA1. Six of them (5382insC, 2963del10, 3819de15, 3875del4, 2274insA, and R1203X) cause premature termination of protein synthesis, thus predisposing to breast cancer. A missense mutation E1250K is presumed to be a factor of predisposition to cancer. We classified three variants of nucleotide sequence found in some patients as DNA polymorphisms S694S, L771L, and E1038G. The 5382insC and 3819de15 mutations have been detected in four and two families, respectively. Five of the mutations detected have not been found in Russia before. However, all mutations except for 2963del10 have been found in other populations of the world, which indicates their long evolutionary history. Two mutations found in patients from St. Petersburg (5382insC and 3875de14) have also been found in oncological patients from other regions of the Russian Federation.

[Instability of repetitive units of foreign centromeric satellite DNA in transgenic mice and transfected cells]. / Analiz nestabil'nosti povtoriaiushchikhsia edinits chuzherodnoi tsentromernoi satellitnoi DNK u transgennykh myshei i v transfektnykh kletkakh.

Cytologically detectable instability of centromeric satellite DNA may cause hereditary disorders in human. To study the mechanisms of such instability, two transgenic mouse lines and 11 clones of transfected F9 mouse embryonic teratocarcinoma cells were obtained with the 3.8-kb repetitive unit (Sat) of Bos taurus satellite DNA IV. Intergeneration and somatic instability of exogenous satellite DNA (satDNA) was observed in transgenic mice and transfected cells as a change in nucleotide sequence of an internal Sat region approximately 1000 bp in size. Since Sat was in the hemizygous state in both cases by the experimental protocol, the instability was attributed to intra-allelic processes. Intergeneration instability probably took place in the premeiotic period of gametogenesis or in early embryo development and led to prenatal death of transgenic embryos after at least one generation. No direct or inverse correlation was observed between methylation and instability of Sat. The results testify that submicroscopic changes in highly repetitive noncoding DNA sequences may already affect the genome function in higher eukaryotes.

[Protein of Escherichia coli interacting specifically with human low density lipoproteins]. / Belok kishechnoi palochki, spetsificheski vzaimodeistvuiushchii s lipoproteinami nizkoi plotnosti cheloveka.

Escherichia coli 48 kDa protein interacting specifically with human low-density lipoproteins is described. The dissociation constant of this highly specific interaction was found to be equal to 4 mkg LDL per 1 ml or 7.3 x 10 M, which is comparable with the dissociation constant of the complex formed by LDL and human LDL receptor. A protocol for purifying the E. Coli binding protein was developed and antibodies against this purified protein were raised. The absence of sequences with homology to the ligand-binding repeats of the human LDL receptor in E. Coli proteome was shown by computer analysis of E. Coli genome. A conclusion was made that binding of the human LDL with specific E. Coli protein is thus mediated by other sequences and by another mechanism different from that, which occurs in human cells during the interaction of lipoproteins with their specific receptor. The establishment of specific interaction between E. Coli protein and human LDL can turn out to be useful in the future for purifying lipoproteins of a specific class and for administering plasmapheresis in patients with severe hyperlipoproteinemia.

[Identification of novel missense mutation G571E, novel silent mutation H229H, nonsense mutation C74X, and four single nucleotide polymorphisms in the low-density lipoprotein receptor in patients with familial hypercholesterolemia from St. Petersburg]. / Identifikatsiia novoi missens-mutatsii G571E, novoi molchashchei mutatsii H229H, nonsens-mutatsii C74X i chetyrekh odnonukleotidnykh polimorfizmov v gene retsptora lipoproteinov nizkoi plotnosti u patsientov s semeinoi giperkholesterinemiei Sankt-Peterburga.

Novel missense mutation G571E (c.1775 G > A), novel silent mutation H229H (c.750 C > T), and nonsense mutation C74X (c.285 C > A), earlier described in Japan but unknown in Russia, were identified in the low-density lipoprotein (LDL) receptor gene in St. Petersburg familial hypercholesterolemia in patients. The analyzed group of patients was shown to be polymorphic in many positions of the LDL receptor gene, namely: c.1171 G/A, c.1773 T/C, c.2177 C/T, and c.2231 G/A.

[Four new mutations and polymorphic variants of the low density lipoprotein receptor in patients with familial hypercholesterolemia in Saint Petersburg]. / Chetyre novye mutatsii i polimorfnye varianty gena retseptora lipoproteinov nizkoi plotnosti u patsientov s semeinoi giperkholesterinemiei Sankt-Peterburga.

In a collection of DNA samples from 100 unrelated patients with clinical features of familial hypercholesterolemia (FH), a search for mutations of exons 4 and 10 of the low-density lipoprotein (LDL) receptor gene was performed using heteroduplex and single-strand conformational polymorphism (SSCP) analyses followed by sequencing of amplified DNA fragments. Four new mutations of the LDL receptor gene were identified: C146R (c.499 T > C), A130P (c.451 G > C), G128G (c.477 T > C), and C188Y (c.626 G > A). Mutation A130P was assigned to the same chromosome with allele variant 447C. Two polymorphic sites in exon 10 of the LDL receptor gene (1413G/A and 1545C/T) were found in the Russian population for the first time. Based on the data obtained, familial hypercholesterolemia was confirmed in seven patients.

[Search for frequently encountered mutations in genes predisposing to breast cancer]. / Poisk chasto vstrechaiushchikhsia mutatsii v genakh predraspolozhennosti k raku molochnoi zhelezy.

DNA of oncological patients, including Ashkenazi Jews and Slavs, living in St. Petersburg was collected, and the resultant collection was screened for three common mutations of genes BRCA1 and BRCA2 by means of heteroduplex analysis. The mutation 5382insC in exon 20 of the BRCA1 gene was found in four unrelated patients, including three Slavs and one Ashkenazi Jew, with a positive family history of breast cancer. The mutations 185delAG and 6174delT in the BRCA1 and BRCA2 genes, respectively, which are typical of Ashkenazi Jewish patients with breast cancer, were not found in the patients of either ethnicity living in St. Petersburg, although the 6174delT mutation was found in the control group of Ashkenazi Jews. A new 12-nucleotide duplication g.71741ins12nt found in intron 20 of the BRCA1 gene was described. The high frequency of the 5382insC mutation in the BRCA1 gene in patients with familial breast cancer in both St. Petersburg and Moscow indicates that Russian families with the history of breast cancer should be primarily tested for this mutation.

[The detection of transgenic animals using a polymerase chain reaction in situ]. / Obnaruzhenie transgennykh zhivotnykh pri pomoshchi polimeraznoi tsepnoi reaktsii in situ.

The technique for detecting both foreign and host specific DNA sequences inside nuclei and chromosomes of single cells of transgenic mice with the help of polymerase chain reaction (PCR) in situ is described. The mouse preimplantation and postimplantation embryonic and adult cells were studied. The methodology is described in detail with particular attention to the optimization of composition of reaction mixture, kind of fixation and preliminary denaturation of target DNA. The reaction takes only several hours and needs no sophisticated equipment.

Persistence of human mitochondrial DNA throughout the development to the blastocyst of mouse zygotes microinjected with human mitochondria.

The conditions for transfer of human mitochondria into fertilised mouse ova were elaborated. Species-specific primers were designed to discriminate human mitochondrial DNA (mtDNA) and the endogenous mtDNA in the preimplantation embryos. Human mitochondria isolated from the HepG2 cell line were microinjected into murine zygotes, and the latter cultured for 96 h to the blastocyst stage. The polymerase chain reaction allowed the detection of human mtDNA at every stage of embryo cleavage. In some cases a clear disparity in distribution of human mtDNA among blastomeres was evident.

Two novel low-density lipoprotein receptor gene mutations (E397X and 347delGCC) in St. Petersburg familial hypercholesterolemia.

Familial hypercholesterolemia (FH), a monogenic disease known to be caused by low-density lipoprotein receptor (LDLR) gene mutations, results in the development of premature atherosclerosis and coronary artery disease in affected individuals. The spectrum of LDLR gene mutations in Russia is poorly known. Using polymerase chain reaction (PCR)-single-strand conformational polymorphism (SSCP) analysis, followed by DNA sequencing, we have screened selected exons of the LDLR gene in 80 unrelated St. Petersburg FH patients for the presence of mutations. Two new LDLR gene mutations, 347delGCC and E397X, were characterized among individuals with familial hypercholesterolemia in St. Petersburg. The carriers of both mutations possessed highly elevated blood serum cholesterol. Cosegregation of E397X mutation and LDLR gene RFLP haplotypes with hyperlipidemia was demonstrated by family study. Both mutations seem to be specific to Slavic patients.

Expression of cDNA fragment encoding ligand-binding domain of human low density lipoprotein receptor in Escherichia coli cells.

To study structure-function relationships in low density lipoprotein receptor (LDLR), a key protein in human cholesterol metabolism, it is reasonable to operate with separate protein domains. To obtain highly purified functionally active LDLR ligand-binding domain, we have cloned the corresponding LDLR cDNA fragment in two expression plasmid vectors of Escherichia coli. We have developed methods to purify fusion and practically individual recombinant proteins and characterized the obtained products biochemically. Antibodies raised against fused with beta-galactosidase and individual recombinant protein have been shown to be efficient in identification of LDLR protein in crude lysates of human fibroblasts (cell line HT-1080).

[Obtaining and properties of recombinant protein G]. / Poluchenie i svoistva rekombinantnogo belka G.

A gene for G protein from Streptococcus strain G148 was cloned in Escherichia coli, which gave rise to several plasmids. One plasmid containing a 1.5 kb insert coding for entire G protein with 63 kD. This protein had both an IgG binding capacity and albumin-binding activity. The second plasmid containing a 0.7 kD insert coded for protein with MM of 38 kD and had only an IgG-binding activity. The third coding for protein with 25 kD has only albumin-binding activity. After subcloning the 1.5-kb insert into the other vector pSP65 and analysing the nucleotide sequence of this insert both in pSP65 vector, the authors came to the conclusion that the proteins obtained are fusion protein of G protein and beta-galactosidase. All the proteins were prepared by affinity chromatography on IgG sepharose or on HSA sepharose. The interaction between G protein and polyclonal and monoclonal IgG of the reactions between G protein and human IgG have determined.

[The cloning and expression of the phospholipase D gene from Corynebacterium pseudotuberculosis]. / Klonirovanie i ékspressiia gena fosfolipazy D iz Corynebacterium pseudotuberculosis.

In a number of consecutive gene engineering operations a DNA fragment having a size of about 2.8 kb was cloned in Escherichia coli by means of Blue-script II SK+ used as vector. The insert contained pld gene coding the synthesis of phospholipase D, one of the key factors of C. pseudotuberculosis virulence. The stable and active expression of this gene in E.coli was achieved. High phospholipase A activity was accumulated in the periplasmatic space. The molecular weight of the synthesized protein was 31 kD. The product obtained by gene engineering methods was found to possess the biological activity of the natural product: it induced the hemolysis of sheep red blood cells in the presence of equi factor of Rhinococcus equi and inhibited the hemolytic activity of Staphylococcus aureus beta-hemolysin (phospholipase C). The pld gene cloned in these experiments differed from that of another C.pseudotuberculosis strain. Further research is underway with a view of searching for the limits of pls gene.

[The cloning and expression of the gene for Staphylococcus aureus beta-hemolysin]. / Klonirovanie i ékspressia gena beta-gemolizina Staphylococcus aureus.

The gene coding the synthesis of beta-hemolysin (phospholipase C) has been cloned from S.aureus strain 126/89. A highly active Escherichia coli producer has been obtained, its capacity of synthesizing phospholipase C exceeding that of the natural strain 37-fold. Phospholipase C (beta-hemolysin) obtained from the natural and gene-engineering producers have been found to be identical in their enzymatic and molecular characteristics. The nucleotide sequence of the full plc gene which codes peptide consisting of 333 acid radicals has been established. In S.aureus strains of different origin this gene has been found to have a conservative character.

[Creation and analysis of a bank of chromosomal genes of Streptococcus group A]. / Sozdanie i analiz banka khromosomnykh genov Streptokokka gruppy A.

The representative genomic library of chromosomal genes has been constructed for streptococcus group A serotype M48 strain 1/64 on the vector lambda L 47.1. Screening of the obtained genomic library by hybridization and immunological techniques revealed about 50 clones producing the streptococcal antigens (extracellular nonidentified products and non-type specific structural streptococcal proteins). Among the recombinant clones three were found to harbour the genetic determinants for M-protein. One the clones contains a determinant coding for epitopes crossreacting with antisera to M-proteins of other serotypes and a protective epitope. The presence of the latter was tested in an indirect bactericidal test.

[Identification of the gene of type A erythrogenic toxin in strains of group A Streptococcus]. / Identifikatsiia gena éritrogennogo toksina tipa A v shtammakh streptokokka gruppy A.

The article specifies the mechanisms of toxigenicity in clinical streptococci strains. The production of type A erythrogenic toxin is found to be related to the toxigenicity gene expression localized in moderate bacteriophage genome. The possibility of using DNA probes to assess the degree of toxigenicity is discussed together with the relationship between the toxigenicity and the gene dose.

[Current data on the cloning of virulence factors of pathogenic Streptococci]. / Sovremennye dannye o klonirovanii faktorov virulentnosti patogennykh streptokokkov.

The data on the cloning of the main pathogenic determinants of group A streptococci including M-protein, erythrogenic toxin, streptokinase, streptolysin O are analyzed. The scientific importance and the possible ways to use the data obtained after cloning are discussed. The hypothesis on the operon system of a number of streptococcal virulence factors regulation is discussed. Construction of vector systems for streptococcal genes cloning is summarized.