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Objective: to study the anamnestic, clinical and laboratory features of the acute period of ischemic stroke (IS) and to determine the risk factors for its development in young patients.Patients and methods. Clinical and statistical processing of data of 256 patients aged 18 to 44 years included, who had IS, confirmed by computed and/or magnetic resonance imaging of the brain in the acute period, was carried out. Furthermore, in 154 patients and in 117 healthy participants, who made up the control group, eight polymorphisms of the thrombophilic spectrum genes were determined – FGB: -455G>A, F2: 20210G>A, F5: 1691G>A, F7: 10976G>A, F13: 103 G>T, ITGA2: 807C>T, ITGB3: 1565 T>C, PAI-1: -675 5G>4G.Results and discussion. 154 (60.15%) patients demonstrated good recovery (achievement of a level of ≤2 points on the Rankin scale by the patient). None of the patients died during their hospitalization. In the evaluated group of patients, we identified allelic variants of the thrombophilic spectrum genes and gene-gene combinations, the carriage of which increased the likelihood of IS development at the young age by 1.74 and 2.19 times, respectively. Taking into consideration additional examination methods, the pathogenetic variant of IS according to the TOAST classification was verified in 226 (88%) patients.Conclusion. In IS at a young age a detailed assessment of risk factors is required, including an analysis of carrier variants and combinations of procoagulant and prothrombotic spectrum gene polymorphisms.

The aim of this study was to identify the most frequent yeasts in the oral cavity of adult individuals without immune disorders and to associate the presence of these oral yeasts with different characteristics of each individual. Oral rinse samples were obtained from 96 healthy adults and cultured in Sabouraud dextrose agar media and CHROMagar. Yeasts were identified by sequencing the D1/D2 region of the 28S rRNA gene. Probable association among the socio-demographic characteristics, body mass index, family and personal medical history, oral hygiene, tobacco and/or alcohol consumption habits and presence of oral fungi was analyzed. Contingency tables and logistic regression were employed to evaluate possible relationships between the presence of oral fungi and mixed colonization with these variables. 57.3% of the healthy individuals had oral yeasts and 21.8% had mixed colonization. The most prevalent yeasts were Candida albicans (52%), C. parapsilosis (17.9%), and C. dubliniensis (7.57%). Yeasts with most frequently mixed colonization were C. albicans and C. parapsilosis. No relationships were found among the variables analyzed. However, the presence of mixed colonization was related to the presence of dental prostheses (P less than 0.006), dental apparatuses (P=0.016) and O'Leary index (P=0.012). This is the first study that characterized oral yeasts in Colombian healthy individuals, determined the most prevalent oral yeasts C. albicans, C. parapsilosis and C. dublinensis and an association of mixed colonization with the use of dental prostheses and aparatology and poor hygiene. © 2019 by the Journal of Biomedical Research.

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present ‘AnnoTALE’, a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities.

Background: Familial amyloidotic polyneuropathy (FAP) TTR Val30Met is a lethal autosomal dominant sensorimotor and autonomic neuropathy due to a substitution of methionine for valine at position 30 of the transthyretin (TTR) gene. Amyloid, composed of mutated TTR, is deposited in the peripheral nervous system, myocardium and kidneys. Considerable variability in the age of onset and penetrance of the disease occurs in different countries. Penetrance in Sweden, Cyprus and Portugal is 2%, 28% and 80% respectively. Environmental and genetic factors are believed to contribute to this variability. So far, no single modifier gene has been unequivocally associated with age of onset or penetrance. Methods: Candidate modifier genes were chosen from among those coding for chaperone proteins co-localized with TTR deposits in peripheral nerves. Seventy one TTRVal30Met carriers, 51 affected and 20 asymptomatic, belonging to 22 unrelated Greek-Cypriot families, and 59 normal controls were recruited for this study. Sequencing of the coding regions of TTR, serum amyloid P (APCS) and complement C1Q (A, B and C) genes was performed and APOE genotypes were determined. We searched for correlations between various polymorphisms of chaperone proteins and age of disease onset. Results: Four new and 4 previously described single nucleotide substitutions were identified. One polymorphic site in C1QA (rs172378) and one in C1QC (rs9434) as well as the ε2 allele correlated with age of onset (p < 0.05). Conclusions: Our study has identified polymorphisms which may influence the FAP-TTR Val30Met phenotype. Identifying modifier genes and their protein products may contribute to therapeutic advances. © 2009 Elsevier B.V. All rights reserved. 284 1-2 158 162 Cited By :16

Background: Premature ovarian failure (POF) is characterized by hypergonadotropic amenorrhoea before the age of 40. Inhibin α-subunit (INHα) gene is proposed as a candidate gene due to its role in negative feedback control of FSH. Methods: Polymorphism -16 C>T of INHα gene was studied in 61 POF patients and 82 controls above 40 years old (C > 40). Substitution 769G>A was studied in 59 POF patients, 76 C > 40 and 73 controls below 40 years old (C < 40). Results: No significant difference in risk of POF development for -16T allele was found when comparing idiopathic POF (I-POF) with C > 40 (Odds ratio = 1.46; 95% confidence interval = 0.63-3.19). Implication of -16C>T polymorphism in serum inhibin levels was analysed in 46 controls, and no significant differences (P > 0.05) were found between CC and CT + TT genotype groups when comparing either mid-follicular phase Pro-αC and inhibin B values or mid-luteal phase Pro-αC and inhibin A values. Heterozygosity for substitution 769G>A was found in 1 of 59 POF woman, 2 of 76 C > 40 and 6 of 73 C < 40. Presence of this su bstitution in a relevant number of control subjects is herein described for the first time. Conclusion: Our results indicate that -16C>T and 769G>A variants in INHα gene may not be associated to POF disease. © 2006 Oxford University Press. Fil:Sundblad, V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Charreau, E.H. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

The chelonians are, in general, important for the economy of the traditional populations of the Amazon region, especially as a source of animal protein. Furthermore, sub-products, such as eggs and fat, are utilized in the manufacture of cosmetics, and the plastron and carapace are used in the manufacture of adornments. The freshwater turtle species Podocnemis sextuberculata, locally known as "iaçá" or "pitiú", is widely distributed in the Amazon Basin in Brazil and also in Colombia and Peru. This species is on the International Union for Conservation of Nature Red List in the category of vulnerable species. We examined the genetic variability and population structure of three populations represented by 64 individuals sampled from Reserva Federal de Abufari, Tapauá, Amazonas State; Reserva de Desenvolvimento Sustentável Mamirauá, Tefé, Amazonas State, and Terra Santa, Pará State. All of these are over 1000 km from each other. A partial 415-bp sequence of the mitochondrial gene ND1 was utilized as a molecular marker. Seven haplotypes were observed; the most common haplotype was shared by all the areas sampled, while the rarest haplotypes were represented by a single individual and were thus restricted to a single locality. The sharing of the most common haplotype, the high number of migrants (Nm) and the AMOVA results indicate a lack of genetic structure among the sampling localities. The levels of genetic variability observed were homogeneous among the sampling localities. These results (O{middle tilde} ST and Nm) are compatible with what is known about the ecology of this species, which has a great migratory capacity. © FUNPEC-RP.

Knowledge about the distribution of mutational fitness effects (DMFE) is essential for many evolutionary models. In recent years, the properties of the DMFE have been carefully described for some microorganisms. In most cases, however, this information has been obtained only for a single environment, and very few studies have explored the effect that environmental variation may have on the DMFE. Environmental effects are particularly relevant for the evolution of multihost parasites and thus for the emergence of new pathogens. Here we characterize the DMFE for a collection of twenty single-nucleotide substitution mutants of Tobacco etch potyvirus (TEV) across a set of eight host environments. Five of these host species were naturally infected by TEV, all belonging to family Solanaceae, whereas the other three were partially susceptible hosts belonging to three other plant families. First, we found a significant virus genotype-by-host species interaction, which was sustained by differences in genetic variance for fitness and the pleiotropic effect of mutations among hosts. Second, we found that the DMFEs were markedly different between Solanaceae and non-Solanaceae hosts. Exposure of TEV genotypes to non-Solanaceae hosts led to a large reduction of mean viral fitness, while the variance remained constant and skewness increased towards the right tail. Within the Solanaceae hosts, the distribution contained an excess of deleterious mutations, whereas for the non-Solanaceae the fraction of beneficial mutations was significantly larger. All together, this result suggests that TEV may easily broaden its host range and improve fitness in new hosts, and that knowledge about the DMFE in the natural host does not allow for making predictions about its properties in an alternative host., This research was supported by the Spanish Ministry of Science and Innovation grant BFU2009-06993 to SFE. JL and JMC were supported by the JAE program from CSIC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

KiSS-1 is ametastasis suppressor gene, its inactivation linked to advanced tumor stage and dismal prognosis. We studied its mutational status ,transcription and protein expression in human cancer cell lines and patients with early breast cancer.Tumor tissue DNA and messenger RNA (mRNA) of KiSS1 exons III and IV from the human cancer cell lines Hela, Jurkat, A549, W138t, MCF-7 and from formalin-fixed resected breast adenocarcinomas from 50 women were analysed by means of PCR-SSCP, RT-PCR and sequencing. Tumor tissue was stained for KiSS1 protein expression by means of the streptavidin-biotin complex immunoperoxidase assay. Presence of KiSS1 mutation, mRNA levels and protein staining were examined for correlations with patient/tumor characteristics. A transversion in exon IVa replacing cytosine with guanine was identified 242 base pairs from the translation start site (242C>G) in the cell lines MCF-7, A549 and in 5/50 tumors (10%), resulting in substitution of proline by arginine (P81R) and alteration of the protein tertiary structure. As the substitution was present in germ-line DNA in 3/5 breast cancer patients harbouring the polymorphism in their tumor, the incidence of tumour-specific somatic mutation was 4% among the 50 patiens with early breast cancer. Although the P81R substitution was associated with reduced KiSS1 protein immunoreactivity (56% in wild-type tumors versus 20% in KiSS1-variant tumours) and with axillary nodal involvement (55% in wild-type versus 80% in KiSS1-variant tumors), the correlations did not reach statistical significance. KiSS1 mRNA was detected in only 15/48 tumours (31%) and showed no correlation with mutation or protein expression. Twenty-six tumors stained for KiSS1 protein, in contrast to the universal strong staining seen in normal breast parenchyma and placental tissues. At amedian follow-up of 38 months, relapses occurred in 20% of women with non wild-type tumors versus 13% of women with wild-type KiSS1 tumors (p=0.7). Presence of KiSS1 mutation, mRNA levels and protein expression did not have prognostic significance for relapse-free survival.In conclusion, altered nucleotide sequence and repression of transcription are two potential mechanisms of suppression of the anti-metastatic effects of KiSS1 in early breast cancer: Confirmation in larger cohorts and study of functional effects of the 242C>G exon IVa mutation are warranted. Keywords: KiSS1, metastasis-suppressor gene, breast cancer, mutation, transcription. Neoplasma

In a previous issue of AJHB, Fernandes et al. (2008. Am J Hum Biol 20:185-190.) describe instances of identity by state at multiple short tandem repeat loci between human Y chromosomes belonging to different E-M35 sub-haplogroups. They interpret these findings as evidence for multiple mutational events in at least two loci (M78 and M81). Here, we introduce a novel polymorphic marker (V68), potentially useful to investigate the issue. This marker and sequence data, reported here for the first time, reinforce our previous interpretations on the phylogenetic structure of the E3b haplogroup. We discuss these results in the frame of general approaches to attain robust phylogenetic inferences based on biallelic polymorphism data.

Background: A partial deficiency in Protoporphyrinogen oxidase (PPOX) produces the mixed disorder Variegate Porphyria (VP), the second acute porphyria more frequent in Argentina. Identification of patients with an overt VP is absolutely important because treatment depends on an accurate diagnosis but more critical is the identification of asymptomatic relatives to avoid acute attacks which may progress to death. Methods: We have studied at molecular level 18 new Argentinean patients biochemically diagnosed as VP. PPOX gene was amplified in one or in twelve PCR reactions. All coding exons, flanking intronic and promoter regions were manual or automatically sequenced. For RT-PCR studies RNA was retrotranscripted, amplified and sequenced. PPOX activity in those families carrying a new and uncharacterized mutation was performed. Results: All affected individuals harboured mutations in heterozygous state. Nine novel mutations and 3 already reported mutations were identified. Six of the novel mutations were single nucleotide substitutions, 2 were small deletions and one a small insertion. Three single nucleotide substitutions and the insertion were at exon-intron boundaries. Two of the single nucleotide substitutions, c.471G>A and c.807G>A and the insertion (c.388+3insT) were close to the splice donor sites in exons 5, 7 and intron 4 respectively. The other single nucleotide substitution was a transversion in the last base of intron 7, g.3912G>C (c.808-1G>C) so altering the consensus acceptor splice site. However, only in the first case the abnormal band showing the skipping of exon 5 was detected. The other single nucleotide substitutions were transversions: c.101A>T, c.995G>C and c.670 T>G that result in p.E34V, p.G332A and W224G aminoacid substitutions in exons 3, 10 and 7 respectively. Activity measurements indicate that these mutations reduced about 50% PPOX activity and also that they co-segregate with this reduced activity value. Two frameshift mutations, c.133delT and c.925delA, were detected in exons 3 and 9 respectively. The first leads to an early termination signal 22 codons downstream (p.S45fsX67) and the second leads to a stop codon 5 codons downstream (p.I309fsX314). One reported mutation was a missense mutation (p.G232R) and 2 were frameshift mutations: c.1082insC and 1043insT. The last mutation was detected in six new apparently unrelated Argentinean families. Conclusion: Molecular analysis in available family members revealed 14 individuals who were silent carriers of VP. Molecular techniques represent the most accurate approach to identify unaffected carriers and to provide accurate genetic counselling for asymptomatic individuals. The initial screening includes the insertion search. © 2008 Rossetti et al; licensee BioMed Central Ltd. Fil:Rossetti, M.V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Granata, B.X. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Giudice, J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Parera, V.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Batlle, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Missense PTPN11 mutations cause Noonan and LEOPARD syndromes (NS and LS), two developmental disorders with pleiomorphic phenotypes. PTPN11 encodes SHP2, an SH2 domain-containing protein tyrosine phosphatase functioning as a signal transducer. Generally, different substitutions of a particular amino acid residue are observed in these diseases, indicating that the crucial factor is the residue being replaced. For a few codons, only one substitution is observed, suggesting the possibility of specific roles for the residue introduced. We analyzed the biochemical behavior and ligand-binding properties of all possible substitutions arising from single-base changes affecting codons 42, 139, 279, 282 and 468 to investigate the mechanisms underlying the invariant occurrence of the T42A, E139D and I282V substitutions in NS and the Y279C and T468M changes in LS. Our data demonstrate that the isoleucine-to-valine change at codon 282 is the only substitution at that position perturbing the stability of SHP2's closed conformation without impairing catalysis, while the threonine-to-alanine change at codon 42, but not other substitutions of that residue, promotes increased phosphopeptide-binding affinity. The recognition specificity of the C-SH2 domain bearing the E139D substitution differed substantially from its wild-type counterpart acquiring binding properties similar to those observed for the N-SH2 domain, revealing a novel mechanism of SHP2's functional dysregulation. Finally, while functional selection does not seem to occur for the substitutions at codons 279 and 468, we point to deamination of the methylated cytosine at nucleotide 1403 as the driving factor leading to the high prevalence of the T468M change in LS.

We investigated whether smoking is associated with mutations in the Von Hippel-Lindau (VHL) gene in 337 cases of sporadic renal cell carcinoma (RCC) among 120 852 people followed for 11.3 years; the findings suggest that smoking causes RCC independently of VHL gene mutations. © 2006 Cancer Research. Chemicals / CAS: adenine, 22177-51-1, 2922-28-3, 73-24-5; cytosine, 71-30-7; guanine, 69257-39-2, 73-40-5; thymine, 65-71-4; Von Hippel-Lindau Tumor Suppressor Protein, EC 6.3.2.19

We investigated whether smoking is associated with mutations in the Von Hippel-Lindau (VHL) gene in 337 cases of sporadic renal cell carcinoma (RCC) among 120 852 people followed for 11.3 years; the findings suggest that smoking causes RCC independently of VHL gene mutations. © 2006 Cancer Research. Chemicals / CAS: adenine, 22177-51-1, 2922-28-3, 73-24-5; cytosine, 71-30-7; guanine, 69257-39-2, 73-40-5; thymine, 65-71-4; Von Hippel-Lindau Tumor Suppressor Protein, EC 6.3.2.19

Associations between dietary intake of various fats and specific K-ras mutations in colorectal cancer (CRC) were investigated within the framework of The Netherlands Cohort Study on diet and cancer (NLCS). After 7.3 years of follow-up and with exclusion of the first 2.3 years, 448 colon and 160 rectal cancer patients and 2948 subcohort members (55-69 years at baseline) were available for data-analyses. Mutation analysis of the K-ras gene was performed on all archival colon and rectal adenocarcinoma specimens. Case-cohort analyses were used to compute adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) for colon and rectal cancer cases and for K-ras mutation subgroups. The intake of total, saturated and monounsaturated fat was not significantly associated with colon or rectal cancer. High intake of dietary polyunsaturated fat and, specifically, linoleic acid is associated with an increased risk of mutated K-ras colon tumours. The RRs for 1 SD of increase of polyunsaturated fat and linoleic acid were 1.21 (95% CI 1.05-1.41) and 1.22 (95% CI 1.05-1.42), respectively, and similar associations were observed for both G > A transitions and G >T or G > C transversions in the colon. In contrast, no significant associations were observed with rectal cancer risk, overall nor with specific K-ras mutation status. A high intake of polyunsaturated fat, in particular linoleic acid, may be an important dietary risk factor for K-ras mutated colon tumours, possibly by generating G > A transitions or G > T or G > C transversions in the K-ras oncogene. © Oxford University Press 2004; all rights reserved.

36 758 760 Cited By :5

Associations between dietary intake of various fats and specific K-ras mutations in colorectal cancer (CRC) were investigated within the framework of The Netherlands Cohort Study on diet and cancer (NLCS). After 7.3 years of follow-up and with exclusion of the first 2.3 years, 448 colon and 160 rectal cancer patients and 2948 subcohort members (55-69 years at baseline) were available for data-analyses. Mutation analysis of the K-ras gene was performed on all archival colon and rectal adenocarcinoma specimens. Case-cohort analyses were used to compute adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) for colon and rectal cancer cases and for K-ras mutation subgroups. The intake of total, saturated and monounsaturated fat was not significantly associated with colon or rectal cancer. High intake of dietary polyunsaturated fat and, specifically, linoleic acid is associated with an increased risk of mutated K-ras colon tumours. The RRs for 1 SD of increase of polyunsaturated fat and linoleic acid were 1.21 (95% CI 1.05-1.41) and 1.22 (95% CI 1.05-1.42), respectively, and similar associations were observed for both G > A transitions and G >T or G > C transversions in the colon. In contrast, no significant associations were observed with rectal cancer risk, overall nor with specific K-ras mutation status. A high intake of polyunsaturated fat, in particular linoleic acid, may be an important dietary risk factor for K-ras mutated colon tumours, possibly by generating G > A transitions or G > T or G > C transversions in the K-ras oncogene. © Oxford University Press 2004; all rights reserved.

Activation of K-ras oncogene has been implicated in colorectal carcinogenesis, being mutated in 30-60% of the adenocarcinomas. In this study, 737 incident colorectal cancer (CRC) patients, originating from 120 852 men and women (55-69 years at baseline) participating in the Netherlands Cohort Study (NLCS), were studied in order to evaluate subgroups with respect to K-ras mutation status. Mutation analysis of the exon 1 fragment of the K-ras oncogene, spanning codons 8-29, was performed on archival colorectal adenocarcinoma samples of all patients using macrodissection, nested PCR and direct sequencing of purified fragments. The method of mutation detection was validated by the confirmation of reported K-ras status in CRC cell lines, a good correlation between fresh-frozen and routinely fixed, paraffin-embedded tissue, a detection limit of 5% mutated DNA and a good reproducibility. Various types of K-ras mutations were evaluated with respect to tumour sub-localization, Dukes' stage and tumour differentiation. In 37% (271/737) of the patients the exon 1 fragment of K-ras gene was found to be mutated The predominant mutations are G>A transitions and G>T transversions, and codons 12 and 13 are the most frequently affected codons. Patients with a rectal tumour were found to have the highest frequency of G>T transversions as compared with patients with a colon or rectosigmoid tumour. This difference appeared to be confined to women with a rectal tumour harbouring G>T transversions. No significant differences were observed for Dukes' stage with respect to types of K-ras mutation, which does not support direct involvement of the K-ras oncogene in adenocarcinoma progression. The equal distribution of K-ras mutations among cases with or without a family history of colorectal cancer argues against an important role for this mutation in familial colorectal cancer, and could imply that K-ras mutations are more probably involved in environmental mechanisms of colorectal carcinogenesis.

K-ras oncogene mutations in sporadic colorectal cancer in The Netherlands Cohort Study.Brink M, de Goeij AF, Weijenberg MP, Roemen GM, Lentjes MH, Pachen MM, Smits KM, de Bruine AP, Goldbohm RA, van den Brandt PA.Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Epidemiology, Maastricht University, PO Box 616, The Netherlands. m.brink@epid.unimaas.nlActivation of K-ras oncogene has been implicated in colorectal carcinogenesis, being mutated in 30-60% of the adenocarcinomas. In this study, 737 incident colorectal cancer (CRC) patients, originating from 120 852 men and women (55-69 years at baseline) participating in the Netherlands Cohort Study (NLCS), were studied in order to evaluate subgroups with respect to K-ras mutation status. Mutation analysis of the exon 1 fragment of the K-ras oncogene, spanning codons 8-29, was performed on archival colorectal adenocarcinoma samples of all patients using macrodissection, nested PCR and direct sequencing of purified fragments. The method of mutation detection was validated by the confirmation of reported K-ras status in CRC cell lines, a good correlation between fresh-frozen and routinely fixed, paraffin-embedded tissue, a detection limit of 5% mutated DNA and a good reproducibility. Various types of K-ras mutations were evaluated with respect to tumour sub-localization, Dukes' stage and tumour differentiation. In 37% (271/737) of the patients, the exon 1 fragment of K-ras gene was found to be mutated. The predominant mutations are G>A transitions and G>T transversions, and codons 12 and 13 are the most frequently affected codons. Patients with a rectal tumour were found to have the highest frequency of G>T transversions as compared with patients with a colon or rectosigmoid tumour. This difference appeared to be confined to women with a rectal tumour harbouring G>T transversions. No significant differences were observed for Dukes' stage with respect to types of K-ras mutation, which does not support direct involvement of the K-ras oncogene in adenocarcinoma progression. The equal distribution of K-ras mutations among cases with or without a family history of colorectal cancer argues against an important role for this mutation in familial colorectal cancer, and could imply that K-ras mutations are more probably involved in environmental mechanisms of colorectal carcinogenesis.

Activated protein C resistance (APCR), the most common risk factor for venous thrombosis, is the result of a G to A base substitution at nucleotide 1691 (R506Q) in the factor V gene. Current techniques to detect the factor V Leiden mutation, such as determination of restriction length polymorphisms, do not have the capacity to screen large numbers of samples in a rapid, cost- effective test. The aim of this study was to apply the first nucleotide change (FNC) technology, to the detection of the factor V Leiden mutation. After preliminary amplification of genomic DNA by polymerase chain reaction (PCR), an allele-specific primer was hybridised to the PCR product and extended using fluorescent terminating dideoxynucleotides which were detected by colorimetric assay. Using this ELISA-based assay, the prevalence of the factor V Leiden mutation was determined in an Australian blood donor population (n = 500). A total of 18 heterozygotes were identified (3.6%) and all of these were confirmed with conventional MnlI restriction digest. No homozygotes for the variant allele were detected. We conclude from this study that the frequency of 3.6% is compatible with others published for Caucasian populations. In addition, the FNC technology shows promise as the basis for a rapid, automated DNA based test for factor V Leiden.

Molecular investigations on a young patient and her family were undertaken to identify the molecular defect responsible for a mild form of osteogenesis imperfecta (OI) with blue sclerae, dentinogenesis imperfecta and joint laxity. Analysis of collagenous proteins from the proband's fibroblasts showed the presence of two populations of α2(I) chains, one normal and one migrating faster on SDS gels, thereby suggesting deletion of amino acid sequences. The faster migrating chains were retained mainly in the cell layer and not found in the extracellular matrix deposited by cultured fibroblasts. Chemical cleavage of mismatch (CCM) analysis on the patient's proα2(I) mRNA:normal cDNA heteroduplexes localized the molecular defect. cDNA sequencing revealed a deletion of exon 20 (54 bp) in about half of the molecules. Genomic DNA sequencing revealed heterozygosity for a G-to-C transversion of the last nucleotide of intron 19, which changed the 3′ consensus splicing site. As a consequence proα2(I)mRNA was abnormally spliced from the last codon of exon 19 to the first codon of exon 21. To our knowledge, this is the first acceptor site mutation so far described in an OI patient. Restriction analysis indicated that the mutation was present also in three other affected family members. The full sequence of COL1A2 introns 19 and 20 are reported. © 1994 Springer-Verlag. 93 681 687 Cited By :11

The molecular defect in the abnormal fibrinogen Dusart (Paris V) that is associated with thrombophilia was determined by sequence analysis of genomic DNA that had been amplified using the polymerase chain reaction. The propositus was heterozygous for a single base change (C → T) in the Aα- chain gene, resulting in the amino acid substitution Aα 554 Arg → Cys. Restriction analysis of the amplified DNA derived from the family members showed that his father and his two sons were also heterozygous. Electron microscopic studies on fibrin formed from purified fibrinogen Dusart demonstrated fibers that were much thinner than in normal fibrin. In contrast to the previously observed defective binding of plasminogen, the binding of thrombospondin to immobilized fibrinogen Dusart was similar to that of normal fibrinogen. Immunoblot analysis of plasma fibrinogen demonstrated that a substantial part of the fibrinogen Dusart molecules were disulfide-linked to albumin. The plasma of the affected family members also contained fibrinogen- albumin complexes. Furthermore, small amounts of high molecular weight complexes containing fibrinogen were detected in all the heterozygous individuals. These data indicate that the molecular abnormality in fibrinogen Dusart (Aα 554 Arg → Cys) results in defective lateral association of the fibrin fibers and disulfide-linked complex formation with albumin, and is associated with a family history of recurrent thrombosis in the affected individuals. Chemicals/CAS: antithrombin III, 90170-80-2; fibrinogen, 9001-32-5; plasminogen, 9001-91-6; protein C, 60202-16-6; thrombin, 9002-04-4; Fibrin, 9001-31-4; fibrinogen Dusard; Fibrinogen, 9001-32-5; Fibrinogens, Abnormal; Platelet Membrane Glycoproteins; Sulfhydryl Compounds; Thrombospondins

The molecular defect in the abnormal fibrinogen Dusart (Paris V) that is associated with thrombophilia was determined by sequence analysis of genomic DNA that had been amplified using the polymerase chain reaction. The propositus was heterozygous for a single base change (C → T) in the Aα- chain gene, resulting in the amino acid substitution Aα 554 Arg → Cys. Restriction analysis of the amplified DNA derived from the family members showed that his father and his two sons were also heterozygous. Electron microscopic studies on fibrin formed from purified fibrinogen Dusart demonstrated fibers that were much thinner than in normal fibrin. In contrast to the previously observed defective binding of plasminogen, the binding of thrombospondin to immobilized fibrinogen Dusart was similar to that of normal fibrinogen. Immunoblot analysis of plasma fibrinogen demonstrated that a substantial part of the fibrinogen Dusart molecules were disulfide-linked to albumin. The plasma of the affected family members also contained fibrinogen- albumin complexes. Furthermore, small amounts of high molecular weight complexes containing fibrinogen were detected in all the heterozygous individuals. These data indicate that the molecular abnormality in fibrinogen Dusart (Aα 554 Arg → Cys) results in defective lateral association of the fibrin fibers and disulfide-linked complex formation with albumin, and is associated with a family history of recurrent thrombosis in the affected individuals. Chemicals/CAS: antithrombin III, 90170-80-2; fibrinogen, 9001-32-5; plasminogen, 9001-91-6; protein C, 60202-16-6; thrombin, 9002-04-4; Fibrin, 9001-31-4; fibrinogen Dusard; Fibrinogen, 9001-32-5; Fibrinogens, Abnormal; Platelet Membrane Glycoproteins; Sulfhydryl Compounds; Thrombospondins

Skin fibroblast from a proband with lethal osteogenesis imperfecta synthesized a type I procollagen containing a cysteine residue in the α1(I) helical domain. Assay of thermal stability of the triple helix by proteinase digestion demonstrated a decreased temperature for thermal unfolding of the protein. Of special importance was the observation that assays of thermal stability by proteinase digestion revealed two bands present in a 2:1 ratio of about 140 and 70 kDa the 140 kDa band was reducible to a 70 kDa band. Further analysis of the fragments demonstrated that the cysteine mutation produced a local unfolding of the triple helix around residue 700 and apparently exposed the arginine residue at position 704 in both the α1(I) and α2(I) chains. Analysis of cDNAs and genomic DNAs demonstrated a single-base mutation that changed the GGT codon for glycine-691 of the α1(I) chain to a TGT codon for cysteine. The mutation was not found in DNA from either of the proband's parents. Since the proteinase assay of helical stability generated a fragment of 700 residues that retained disulphide-bonded cysteine residues at α1-691, the results provide one of the first indications that glycine substitutions in type I procollagen can alter the conformation of the triple helix at a site that is C-terminal to the site of the substitution. 279 747 752 Cited By :9

A fraction of the proα1(I) and proα2(I) chains in type I procollagen synthesized by the fibroblasts from a proband with a lethal variant of osteogenesis imperfecta were overmodified by posttranslational reactions. After digestion with pepsin, some of the α1(I) chains were recovered as disulfide-linked dimers. Mapping of cyanogen bromide peptides indicated that the disulfide link was contained in α1-CB6, the cyanogen bromide fragment containing amino acid residues 823-1014 of the α1(I) chain. Nucleotide sequencing of cDNA clones demonstrated a substitution of T for G that converted glycine 904 of the α1(I) chain to cysteine. A large fraction of the type I procollagen synthesized by the proband's fibroblasts, had a thermostability that was 3-4°C lower than the normal type I procollagen as assayed by brief proteinase digestion. In addition, the type I procollagen synthesized by the proband's fibroblasts was secreted with an abnormal kinetic pattern in that there was a lag period of about 30 min in pulse-chase experiments. The mutation of glycine to cysteine was not found in type I procollagen synthesized by fibroblasts from the proband's parents. Therefore, the mutation was a sporadic one. However, the mother's fibroblasts synthesized a type I procollagen in which part of the proα chains were overmodified and had a lower thermostability. Therefore, the proband may have inherited a mutated allele for type I procollagen from her mother that contributed to the lethal phenotype. The mother was assymptomatic. She was somewhat short and had slightly blue sclerae but no definitive signs of a connective tissue abnormality. The observations on the mother indicated, therefore, that a mutation that causes synthesis of a type I procollagen with a lowered thermal stability does not necessarily produce a heritable disorder of connective tissue. 83 574 584 Cited By :24

Background More than 90% of Congenital Adrenal Hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the HLA class III area on the short arm of chromosome 6p21.3. In this region, a 30 kb deletion produces a non functional chimeric gene with its 5' and 3' ends corresponding to CYP21A1P pseudogene and CYP21A2, respectively. To date, five different CYP21A1P/CYP21A2 chimeric genes have been found and characterized in recent studies. In this paper, we describe a new CYP21A1P/CYP21A2 chimera (CH-6) found in an Italian CAH patient. Methods Southern blot analysis and CYP21A2 sequencing were performed on the patient. In addition, in order to isolate the new CH-6 chimeric gene, two different strategies were used. Results The CYP21A2 sequencing analysis showed that the patient was homozygote for the g.655C/A>G mutation and heterozygote for the p.P30L missense mutation. In addition, the promoter sequence revealed the presence, in heterozygosis, of 13 SNPs generally produced by microconversion events between gene and pseudogene. Southern blot analysis showed that the woman was heterozygote for the classic 30-kb deletion producing a new CYP21A1P/CYP21A2 chimeric gene (CH-6). The hybrid junction site was located between the end of intron 2 pseudogene, after the g.656C/A>G mutation, and the beginning of exon 3, before the 8 bp deletion. Consequently, CH-6 carries three mutations: the weak pseudogene promoter region, the p.P30L and the g.655C/A>G splice mutation. Conclusion We describe a new CYP21A1P/CYP21A2 chimera (CH-6), associated with the HLA-B15, DR13 haplotype, in a young Italian CAH patient.