Glutathione- S-transferase micro and theta gene polymorphisms as new risk factors of atopic bronchial asthma.

The genetic polymorphism of glutathione- S-transferase M1 (GSTM1) and glutathione- S-transferase T1 (GSTT1) genes and the cytochrome P4501A1 gene responsible for xenobiotic conjugating enzymes of the phase II and phase I detoxification system were studied by PCR-RFLP in the blood spots of 109 patients with atopic bronchial asthma and 90 healthy individuals. GSTM1 gene deletion (GSTM1(0/0)) was detected in 47.8% of individuals in the control group and in 76.1% of asthmatic patients. Individuals without the GSTM1 gene were at approximately 3.5--fold higher risk of developing asthma. The proportion of GSTT1(0/0) genotypes was significantly higher in the group of asthmatics (67.0%) than in controls (23.3%). The proportion of individuals with a deficiency in both GSTM1 and GSTT1 gene activity was more than four times higher in asthmatic patients than in the control group (54.1% and 12.2%, respectively). The frequency of the Ile-Val polymorphism of the CYP1A1 gene was similar in controls and asthmatic patients. This study shows the association of atopic bronchial asthma with GSTM1(0/0), GSTT1(0/0) genotypes.

Characterization of a novel 21-kb deletion, CFTRdele2,3(21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central and East Europe.

We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, viz., a deletion that is frequently observed in Central and Eastern Europe. The mutation, termed CFTRdele2,3(21 kb), deletes 21,080 bp spanning introns 1-3 of the CFTR gene. Transcript analyses have revealed that this deletion results in the loss of exons 2 and 3 in epithelial CFTR mRNA, thereby producing a premature termination signal within exon 4. In order to develop a simple polymerase chain reaction assay for this allele, we defined the end-points of the deletion at the DNA sequence level. We next screened for this mutation in a representative set of European and European-derived populations. Some 197 CF patients, including seven homozygotes, bearing this mutation have been identified during the course of our study. Clinical evaluation of CFTRdele2,3(21 kb) homozygotes and a comparison of compound heterozygotes for deltaF508/CFTRdele2,3(21 kb) with pairwise-matched deltaF508 homozygotes indicate that this deletion represents a severe mutation associated with pancreatic insufficiency and early age at diagnosis. Current data show that the mutation is particularly common in Czech (6.4% of all CF chromosomes), Russian (5.2%), Belorussian (3.3%), Austrian (2.6%), German (1.5%), Polish (1.5%), Slovenian (1.5%), Ukrainian (1.2%), and Slovak patients (1.1%). It has also been found in Lithuania, Latvia, Macedonia and Greece and has sporadically been observed in Canada, USA, France, Spain, Turkey, and UK, but not in CF patients from Bulgaria, Croatia, Romania or Serbia. Haplotype analysis has identified the same extragenic CF-haplotype XV-2c/KM. 19 "A" and the same infrequent intragenic microsatellite haplotype 16-33-13 (IVS8CA-IVS 17bTA-IVS 17bCA) in all examined CFTRdele2,3(21 kb) chromosomes, suggesting a common origin for this deletion. We conclude that the 21-kb deletion is a frequent and severe CF mutation in populations of Eastern- and Western-Slavic descent.

Local and distant transfection of mdx muscle fibers with dystrophin and LacZ genes delivered in vivo by synthetic microspheres.

Patterns of dystrophin and beta-galactosidase expression were examined in mdx mice after i.m. injections of synthetic microspheres (MF-2) loaded with full-length (pHSADy) or mini-dystrophin gene (pSG5dys) cDNA plasmid constructs or with LacZ marker gene (pCMV-LacZ). A single injection of 25 microg pHSADy into quadriceps femoris muscle resulted in 6.8% of dystrophin positive myofibers (DPM) in a given muscle; 8.4% of DPM in glutaeus muscle and 4.3% of DPM in quadriceps femoris muscle of contralateral limb on day 21 after exposure compared with only 0.6% DPM in intact (non-injected) mdx mice. A high proportion of DPM (17.6% and 10.8%, respectively) was registered in both injected and contralateral muscles after mini- gene cDNA administration. MF-2/dystrophin cDNA particles were detected by FISH analysis in about 60-70% of myofiber nuclei in muscles of injected and contralateral limbs 7 days after application. The presence of human dystrophin cDNA and its products in all skeletal muscles and in different internal organs was proven by PCR and RT-PCR analysis. Patches of beta-galactosidase expression were abundant in injected muscle, and frequent in the contralateral and other skeletal muscles as well as in diaphragm, heart and lungs. High levels of dystrophin cDNA expression, and an efficient distant transfection effect with preferential intranuclei inclusion of MF-2 vehicle, are very encouraging for the development of a new constructive strategy in gene therapy trials of DMD.

Possible involvement of arylamine N-acetyltransferase 2, glutathione S-transferases M1 and T1 genes in the development of endometriosis.

Wide inter-individual variation of expression of compound metabolic enzymes is determined by polymorphism and may predispose the development of diseases provoked by environmental factors. The combined analysis of phase II detoxification system genes: arylamine N-acetyltransferase 2 (NAT2), and glutathione S-transferases (GST) M1 and T1 was carried out in patients with minimal/mild (group I; n = 36) and moderate/severe endometriosis (group II; n = 29) and controls (n = 72) of French origin, using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The results show a significant difference between patients and controls with regard to NAT2 gene polymorphism (P < 0.05). This is mainly due to the high percentage of slow acetylator genotypes (SA) in patients compared with controls (60.0 versus 38.9%; P < 0.02) with a distinct preponderance in subjects with minimal/mild endometriosis (69.4%, P < 0.005) where there is a significantly elevated frequency of slow allele S1 (NAT2*5) (P = 0.05). Significantly increased proportions of GSTM1-deficient genotypes were found in both groups of patients, in comparison with the controls (75.0 and 79.3% versus 45.8%; P < 0. 0001). A preponderance of GSTT1-negative subjects among patients was also detected, but did not appear significant. We suggest the involvement of both NAT2 and GSTM1 detoxification system genes in the pathogenesis of endometriosis and the possible impact of NAT2 gene polymorphism in the development of different forms of this disease.

Bacterial beta-galactosidase and human dystrophin genes are expressed in mouse skeletal muscle fibers after ballistic transfection.

Ballistic transfection, based on cell and tissue bombardment by the tungsten and gold microparticles covered with the gene DNA, was used for the delivery of a bacterial beta-galactosidase and a full-length cDNA copy of the human dystrophin genes into mouse skeletal muscles. CMV-lacZ, SV40-lacZ, LTR-lacZneo and full-length cDNA dystrophin (pDMD-1, approximately 16 kb) in eukaryotic expression vector pJ OMEGA driven by mouse leukaemia virus promotor (pMLVDy) were used throughout the studies. Musculus glutaeus superficialis of C57BL/6J and quadriceps femoris of mdx male mice were opened surgically under anesthesia and bombarded by means of the gene-gun technique originally developed by us. Different mixtures of gold and tungsten particles at ratios of 4:1, 1:1, 1:4 were applied. X-gal assay revealed marked beta-gal activity, both in total muscles and whole muscle fibers on histological sections, up to three months after transfection. The most intensive staining was observed after SV40-lacZ delivery. No staining was detected with LTR-lacZneo DNA as well as in untreated muscles. The higher tungsten particle concentration in the bombardment mixture correlated with more intense X-gal staining. At the gold/tungsten ratio of 1:4 the microparticles penetrated the musculus glutaeus superficialis and transfected the underlying musculus glutaeus medius as well. Immuno-cytochemical assay for human dystrophin revealed dystrophin positive myofibers (DPM) in the bombarded area up to two months after transfection. The proportion of DMP varied from 2.5% on day 17 up two 5% on day 60 after bombardment compared to only 0.5% in the control mdx mice. These results suggest the applicability of particle bombardment for gene delivery into muscle fibers.

Peculiarities of the GSTM1 0/0 genotype in French heavy smokers with various types of chronic bronchitis.

A homozygous gene deletion of the glutathione S-transferase M1 (GSTM1) locus of genomic DNA from blood spots was studied by the polymerase chain reaction in a group of French heavy smokers (n = 361), which included patients with severe chronic bronchitis (SCB; n = 87), moderate chronic bronchitis (MCB: n = 102) and hard smokers (HS) with no permanent clinical symptoms of chronic bronchitis (n = 172). The GSTM1 0/0 genotype was found in 71.3% and 65.7% of cases in SCB and MCB, respectively, compared with only 47.1% in the control HS group (P = 0.0002). This latter figure (47.1%) is consistent with the average GSTM1 deletion frequency in French Caucasians. Moreover, the results showed a significant difference in the distribution of the GSTM1 0/0 genotype for both the SCB and MCB groups against the control HS group, according to gender (SCB: P = 0.001; MCB: P = 0.005), age (SCB: P = 0.0001; MCB: P = 0.005) and smoking history (SCB: P = 0.0001; MCB: P = 0.005). Thus, individuals homozygous for the GSTM1 gene deletion, especially in the under-41 age group (SCB: P = 0.001; MSB: P = 0.04) with an average smoking history of 16-30 pack-years (SCB: P = 0.002; MSB: P = 0.01) are more prone to chronic lung diseases, such as SCB and MCB, than are GSTM1 +/+ or 0/+ subjects. Population screening of young people for the identification of GSTM1 0/0 subjects, with special emphasis on smoking habits, might be useful (1) for the early detection of individuals at high risk of lung complications caused by environmental toxins and pollutants and (2) in clinical practice, in order to prevent the development of chronic bronchitis, which is a common disease.

Proportion of the GSTM1 0/0 genotype in some Slavic populations and its correlation with cystic fibrosis and some multifactorial diseases.

A homozygous gene deletion at the glutathione S-transferase M1 (GSTM1) locus of genomic DNA from blood spots was studied by PCR in the group of Slavic populations from the north-western and central-eastern regions of European Russia and in patients with lung cancer (LC), other tumors (OT), endometriosis (E), alcoholic cirrhosis (AC), cystic fibrosis (CF) and chronic bronchitis (CB). The frequencies of the GSTM1 0/0 genotype were 38.8% and 67.5% for both population groups, respectively. The proportion of the GSTM1 gene deletion genotype was estimated as significantly increased in LC (81%), OT (65%), E (81%), AC (77.3%), and in CB (73.6%) patients with symptoms of CB confirmed by X-ray but not in CB patients without X-ray evidence of disease (40.9%). A definite preponderance of GSTM1-0 homozygotes (51.1%) has been registered in CF patients of the pancreatic sufficient group with clear-cut pulmonological manifestations but not in those of the pancreatic insufficient group with predominantly intestinal or mixed clinical symptoms (41.2% and 37.5%, respectively). Earlier clinical manifestations and death before the age of 5 years are typical for GSTM1-deleted CF patients. These data support the notion that GSTM1 deletion should be considered as a convenient genetic marker for the early detection of groups at higher risk of many diseases caused by environmental and genetic factors, where manifestation depends on the lack of detoxification. High levels of GSTM1 0/0 genotypes in E patients favor the substantial contribution of certain environmental toxins in the pathogenesis of this widespread disease.

Cytogenetic and molecular findings in patients with Turner's syndrome stigmata.

Cytogenetic and DNA analysis in 12 people with stigmata of Turner's syndrome was carried out. Cytogenetic analysis of these patients showed two subjects with 46,X, i(Xq) karyotypes, one with 45,X/46,X, i(Xq), one with 46,X,t(X;Y), and eight with 45,X/46,X,mar. Molecular analysis of DNA samples was performed in nine out of 12 patients with marker chromosomes. PCR analysis with oligoprimers specific for SRY, DYZ1, or DYZ3 loci identified Y chromosome material in five patients in the latter group. The X chromosome origin of the marker chromosome was proved by FISH technique with biotin labelled pericentromeric X chromosome specific probe in four other patients. These results show that patients with a number of Turner's syndrome stigmata usually do not have a typical XO karyotype but have some structural chromosomal aberrations involving the X or Y chromosomes. Combined application of cytogenetic, molecular cytogenetic (FISH), and PCR techniques significantly improves the precision of marker chromosome identification and thus might be of practical importance for the proper management and treatment of affected patients. Peculiarities of pathological manifestations of different karyotypes bearing structural abnormalities of the X or Y chromosomes in patients with Turner's syndrome stigmata, as well as feasible genetic mechanisms of sex determination and differentiation abnormalities in these subjects, are briefly discussed.

X;Y translocation in a girl with short stature and some features of Turner's syndrome: cytogenetic and molecular studies.

A 13 year old girl referred for chromosome analysis because of disproportionate short stature (short neck, curved legs, pectus excavatum) with an initial clinical diagnosis of Turner's syndrome was found to have the karyotype 46,X, + der(X) in 100% of her blood lymphocytes. By means of conventional differential staining (QFH/AcD, FPG, and RBA banding) supplemented with distamycin A treatment, the karyotype of the proband was interpreted as 46,X,t(X;Y) (p22.3;q11). The rearranged marker X chromosome was found to be active in 91% of lymphocytes studied. PCR analysis with Y chromosome specific oligoprimers showed the presence of some Y chromosome long arm DNA in both lymphocyte and gonadal tissue biopsy cells. At laparoscopy the patient was found to have small gonads with a rudimentary uterus and fallopian tubes. Histological examination of gonadal tissue showed primary follicles with dystrophic changes of the germ cells and numerous follicular cysts (polycystic ovaries). The proband's phenotype and its correlation with the genetic imbalance of the rearranged X chromosomes, as well as with non-random t(X;Y) chromosome inactivation, are briefly discussed.

Allele frequencies and molecular diagnosis in haemophilia A and B patients from Russia and from some Asian republics of the former U.S.S.R.

RFLP analysis of some intra- and extra-genic polymorphic sites of Factor VIII (FVIII) and Factor IX (FIX) genes with relevant DNA probes or by polymerase chain reaction (PCR) was carried out in Slavic populations from the European part of Russia and also in the native ethnic groups of Uzbekistan and Kazahstan. The allele frequencies for the HindIII (intron 19) and XbaI (intron 22) polymorphic sites (PSs) in the FVIII gene were very similar in the two populations studied, but different for the intron 13 (CA)n repeat. Significant variations in the TaqI (intron d) and DdeI (intron a) polymorphisms of the FIX gene were evident between the Russian and Asian populations. Two unusual alleles (4.35 and 4.2 kb) for the extragenic PS St14/TaqI were registered in Slavs and one new allele (380 bp) for the DdeI polymorphic site of FIX was discovered in both Asian populations. Altogether, 210 haemophilia A (HA) and 24 haemophilia B (HB) families were subjected to molecular studies. So far, 160 HA and 12 HB families have been found to be informative for DNA analysis. Carrier status was ascertained in 42 HA and 6 HB female relatives, and rejected in 52 and 10 of them, respectively. The origin of some HA and HB mutations was traced with relevant polymorphic markers in several at-risk families. Prenatal diagnosis was accomplished in 28 HA and three HB families, resulting in the identification of 20 affected male fetuses.

Potential of chaotic chemical systems in nanotrace analysis based on the Belousov-Zhabotinskii reaction (BrO(-)(3)-malonic acid-ferroin). Determination of manganese(II).

A new method for the determination of trace amounts of manganese(II) in aqueous solutions by its affect on some types of the chaotic regimes in the Belousov-Zhabotinskii reaction has been proposed. The method is based on the high sensitivity of some types of chaotic regimes of the BZ reaction to small perturbations of initial conditions (e.g., low concentrations of some metal ions). The experimentally obtained detection limit for manganese is 3 pg/ml.

Two new mutations detected by single-strand conformation polymorphism analysis in cystic fibrosis from Russia.

Single-strand conformation polymorphism (SSCP) analysis followed by direct sequencing of exons containing ATP-binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was performed on 80 Russian DNA samples. Two new alterations--S1196X (exon 19) and W1282R (exon 20)--and two novel polymorphisms--1525-61 (intron 9) and 1716+12 T-C (intron 10)--were identified. Mutation S1196X changes a TCA codon to TGA and destroys an EcoRI site. Alteration W1282R results from a T-to-C change at position 3976. It was found in one Russian patient and creates an AciI site; however, it is unclear whether this is a disease-causing mutation or a polymorphism. Polymorphism 1525-61 results from an A-to-G change. Alteration 1716+12 T-C was found in a Moldovian patient and creates a new MaeII site. It is not known whether this alteration affects the splicing of the mRNA. The previously described A4002G polymorphism was encountered in approximately 9% of Russian CF chromosomes. In addition, we have found the previously described 3732delA mutation in 7 CF chromosomes, making it the second (after delta F508) most frequent mutation in the Russian population.

Five years' experience of prenatal diagnosis of cystic fibrosis in the former U.S.S.R.

From a total of 490 cystic fibrosis (CF) high-risk families under supervision (mostly Russian Slavs from the European part of the country), DNA data including both direct screening for some CF gene (CFTR) mutations (delF508, G551D and 1677delTA) and allelic polymorphism studies with tightly CF linked DNA markers were collected from 261 families. All full families (129) and 86 CF families with a decreased index child were found to be either fully (42 per cent) or partially (40 per cent) informative for DNA analysis. Prenatal diagnosis (PD) was carried out in 161 CF families. Microvillar enzyme (MVE) assay was applied to all 140 PD at the second trimester either as a single test (88) or in conjunction with DNA analysis (52). The frequency of false-negative results of the MVE assay was 1.3 per cent and that of false-positive results, as judged by the albumin meconium test, was 5.0 per cent. Ambiguous results of MVE analysis were found in 30 cases, 12 of which were verified by DNA analysis. Molecular diagnosis of CF at the first trimester was carried out in 21 cases and four pregnancies were terminated. Altogether, 39 pregnancies with a predicted high risk of CF fetuses were terminated. The low average frequency of delF508 in CF chromosomes of Russian Slavs (50 per cent), its remarkable inter-population variation, and the significant proportion of at-risk families without an affected child determine the necessity of combined molecular and biochemical (MVE assay) approaches for efficient prenatal diagnosis of CF in the former U.S.S.R.

Frequency of the F508 deletion in cystic fibrosis patients from the European part of the USSR.

The frequency of the F508 deletion (delta F508) has been analyzed in 189 cystic fibrosis (CF) patients from the European part of the USSR, viz. 127 northern Slavonians (Leningrad region), 30 southern Slavonians (the Ukraine), 10 central Slavonians (Moscow region), 14 Moldavians (Kishenev region) and 8 Lithuanians (Vilnius region). The distribution of CF+ chromosomes with and without delta F508 varied significantly in the different ethnic groups studied and correlated with the clinical manifestation of CF. The overall frequency of delta F508 in Slavonian patients is equal to 62.5%, approximately 90% of them being heterozygous or homozygous for this mutation. The frequency of the deletion among 99 Slavonian patients with severe disease manifestation (pancreatic insufficiency, PI) is equal to 67.5%, only 12 patients having pancreatic sufficiency (PS, 17.5%). The highest value of delta F508 (77.4%) is registered in PI/CF patients of the southern Slavonian group; it is much less frequent (about 57%) in relevant groups of Slavonians from the northern and central parts of the country. Unusually low frequencies (24% and 26%) of delta F508 are detected in a few samples of Lithuanian and Moldavian CF patients, respectively. All delta F508+CF-chromosomes of Slavonian origin are associated with haplotypes 2.2.2. defined by the restriction fragment length polymorphism sites KM19/PstI, CS.7/Hin6I and MP6d-9/MspI, although a high proportion (about 25%) of unknown mutations is associated with the same haplotype. Haplotype B (allele 1XV2c/TaqI; allele 2 KM19/PstI) accounts for 91% of delta F508+CF chromosomes. Our data are consistent with the hypothesis of a single origin and subsequent diffusion of this major CF mutation; however, its interpopulational dissemination in Eastern Europe does not follow the suggested south-east to north-west gradient in Western Europe. The significance of these data for prenatal diagnosis and carrier screening of CF mutations is briefly discussed.

Detection of three rare frameshift mutations in the cystic fibrosis gene in an African-American (CF444delA), an Italian (CF2522insC), and a Soviet (CF3821delT).

We have identified three new frameshift mutations in the CFTR gene in patients with cystic fibrosis (CF). The first one involves the deletion of an adenine nucleotide in exon 4 in an African-American patient (CF444delA), the second involves the insertion of a cytosine nucleotide in exon 13 in an Italian patient (CF2522insC), and the third results from the deletion of a thymidine nucleotide in exon 19 in a Soviet patient (CF3821delT). Each mutation is predicted to result in premature termination of the CFTR protein.