Detection of human immunodeficiency virus type 1 antiretroviral resistance mutations by high-density DNA probe arrays.

Genotypic resistance testing has become an important tool in the clinical management of patients infected with human immunodeficiency virus type 1 (HIV-1). Standard sequencing methodology and hybridization-based technology are the two principal methods used for HIV-1 genotyping. This report describes an evaluation of a new hybridization-based HIV-1 genotypic test of 99 clinical samples from patients infected mostly with HIV-1 subtype B and receiving treatment. This test combines RNA extraction with magnetic silica particles, amplification by nested reverse transcriptase PCR, and detection with high-density probe arrays designed to detect 204 antiretroviral resistance mutations simultaneously in Gag cleavage sites, protease, reverse transcriptase, integrase, and gp41. The nested reverse transcriptase PCR success rates at viral loads exceeding 1,000 copies/ml were 98% for the 2.1-kb amplicon that covers the Gag cleavage sites and the protease and reverse transcriptase genes, 92% for the gp41 amplicon, and 100% for the integrase amplicon. We analyzed 4,465 relevant codons with the HIV-1 DNA chip genotyping assay and the classic sequence-based method. Key resistance mutations in protease and reverse transcriptase were identified correctly 95 and 92% of the time, respectively. This test should be a valuable alternative to the standard sequence-based system for HIV-1 drug resistance monitoring and a useful diagnostic tool for simultaneous multiple genetic analyses.

Use of a high-density DNA probe array for detecting mutations involved in rifampicin resistance in Mycobacterium tuberculosis.

A Mycobacterium high-density DNA probe array designed to detect rpoB mutations conferring rifampicin resistance in Mycobacterium tuberculosis was evaluated. The rpoB hybridisation patterns produced by 41 susceptible (RifS) and 59 rifampicin-resistant (RifR) clinical isolates of M. tuberculosis were compared with the results of conventional dideoxynucleotide sequencing of the rpoB gene. For all the RifR isolates, the rpoB hybridisation patterns correlated with the rpoB sequencing results. Among the 59 isolates, 11 distinct amino-acid changes were detected by the DNA probe array. Of these, 36 (61%) corresponded to replacement of the serine residue found in position 531 (S531L in 34 isolates and S531W in two isolates), 16 (27%) affected histidine 526 (five H526D, five H526Y, four H526L, one H526N and one H526R), four (6.8%) replaced aspartate 516 with a valine, and one (1.7%) replaced glutamine 513 with a leucine. Deletion of the asparagine residue at position 519 was detected in one isolate susceptible to rifampicin, but yielding c. 0.1% resistant colonies on rifampicin-containing medium. No mutation was detected in the rpoB region from one isolate yielding c. 5% of resistant colonies on rifampicin-containing medium. Finally, a D516Y substitution was detected in association with an unexpected mutation, G523W, not tiled on the DNA probe array, but which could be detected by analysing the hybridisation pattern obtained with the wild-type probes covering codon 523. In conclusion, the Mycobacterium probe array is a promising approach to rapid detection of mutations involved in rifampicin resistance in M. tuberculosis.

Labeling during cleavage of nucleic acids for their detection on DNA chips.

A new and efficient strategy for labeling of nucleic acids prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the terminal phosphates of cleaved DNA and RNA fragments with a reporter molecule bearing aryldiazomethane group.

Universal labeling chemistry for nucleic acid detection on DNA-arrays.

We show here a new and efficient aqueous chemistry for labeling of any class of nucleic acids for their detection on DNA chip. The labels contain a diazo function as reactive moiety and biotin as detectable unit. The highly selective reaction of diazo group on the phosphate does not disrupt base pairing recognition and hybridization specificity.

Polymorphisms of the endothelin-A and -B receptor genes in relation to blood pressure and myocardial infarction: the Etude Cas-Témoins sur l'Infarctus du Myocarde (ECTIM) Study.

Endothelin-1 is a potent vasoconstrictor that has also mitogenic properties, stimulating the synthesis and secretion of several vasoactive molecules. There is much evidence to suggest that endothelin-1 might be involved in the pathogenesis of hypertension, atherosclerosis, and ischemic heart disease. Endothelin-1 exerts its effects through at least two receptors, ET(A) and ET(B), which are encoded by different genes and have separate tissue distributions and biologic properties. The objective of this study was to identify polymorphisms of the ET(A) and ET(B) receptor genes and to study their association with myocardial infarction (MI) and blood pressure. The coding regions and 1.3 kb upstream of the ET(A) and ET(B) receptor genes were explored by polymerase chain reaction/single strand conformation polymorphism. Six polymorphisms were found in the ET(A) receptor gene and three in the ET(B) receptor gene. Most of these polymorphisms were frequent. Associations between the detected polymorphisms, blood pressure, and MI were examined in the ECTIM study, a multicenter study comparing 652 patients having survived an MI and 773 controls from Belfast (Northern Ireland) and France. Alleles at the different polymorphic sites were similarly distributed in patients with MI and controls. Allele frequencies were similar in both countries, except for the ET(A)/-231 G allele, which appeared more frequently in France than in Belfast (P < .01). The mean systolic and diastolic blood pressure levels did not significantly differ between genotypes. However, a C/T substitution located in the nontranslated part of exon 8 of the ET(A) receptor gene (ET(A)/EX8nt1363) was associated with pulse pressure (P < .005). These results do not support an involvement of the endothelin receptor genes in a predisposition to MI or the determination of blood pressure levels, but suggest that a polymorphism of the ET(A) receptor gene might influence the pulse pressure. This result will have to be confirmed in other studies.

Mycobacterium species identification and rifampin resistance testing with high-density DNA probe arrays.

Species identification within the genus Mycobacterium and subsequent antibiotic susceptibility testing still rely on time-consuming, culture-based methods. Despite the recent development of DNA probes, which greatly reduce assay time, there is a need for a single platform assay capable of answering the multitude of diagnostic questions associated with this genus. We describe the use of a DNA probe array based on two sequence databases: one for the species identification of mycobacteria (82 unique 16S rRNA sequences corresponding to 54 phenotypical species) and the other for detecting Mycobacterium tuberculosis rifampin resistance (rpoB alleles). Species identification or rifampin resistance was determined by hybridizing fluorescently labeled, amplified genetic material generated from bacterial colonies to the array. Seventy mycobacterial isolates from 27 different species and 15 rifampin-resistant M. tuberculosis strains were tested. A total of 26 of 27 species were correctly identified as well as all of the rpoB mutants. This parallel testing format opens new perspectives in terms of patient management for bacterial diseases by allowing a number of genetic tests to be simultaneously run.

Strong clustering and stereotyped nature of Notch3 mutations in CADASIL patients.

BACKGROUND: CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy) is commonly overlooked or misdiagnosed owing to its recent identification and its variable mode of presentation. The defective gene in CADASIL is Notch3, which encodes a large transmembrane receptor. To set up a diagnostic test and to delineate the Notch3 domains involved in CADASIL., we undertook mutations analysis in this gene in a group of CADASIL patients. METHODS: 50 unrelated patients with CADASIL and 100 healthy controls were screened for mutations along the entire Notch3 sequence, by means of single-strand conformation polymorphism, heteroduplex, and sequence analysis. FINDINGS: Strongly stereotyped mis-sense mutations, located within the epidermal-growth-factor-like (EGF-like) repeats, in the extracellular domain of Notch3, were detected in 45 patients. Clustering of mutations within the two exons encoding the first five EGF-like repeats was observed (32 patients). All these mutations lead to loss or gain of a cysteine residue and therefore to an unpaired number of cysteine residues within a given EGF domain. None of these mutations was found in the 100 controls. INTERPRETATION: Because of the strong clustering and highly stereotyped nature of the pathogenetic mutations detected in CADASIL patients, and easy and reliable diagnostic test for CADASIL is feasible. The findings suggest that aberrant dimerisation of Notch3, due to abnormal disulphide bridging with another Notch3 molecule or with another protein, may be involved in the pathogenesis of this disorder.

Polymorphisms of the transforming growth factor-beta 1 gene in relation to myocardial infarction and blood pressure. The Etude Cas-Témoin de l'Infarctus du Myocarde (ECTIM) Study.

Transforming growth factor-beta 1 (TGF-beta 1) plays an important role in the modulation of cellular growth and differentiation and the production and degradation of the extracellular matrix. A number of experimental results suggest that TGF-beta 1 may be involved in cardiovascular physiopathology. In the present study, we assessed whether the TGF-beta 1 gene is a candidate gene for coronary heart disease or hypertension. We screened the coding region and 2181 bp upstream of the TGF-beta gene for polymorphisms and identified seven polymorphisms: 3 in the upstream region of the gene at positions -988, -800, and -509 from the first transcribed nucleotide; 1 in a nontranslated region at position +72; 2 in the signal peptide sequence Leu10-->Pro, Arg25-->Pro; and 1 in the region of the gene coding for the precursor part of the protein not present in the active form, Thr263-->Ile. We analyzed these TGF-beta 1 polymorphisms in 563 patients with myocardial infarction and 629 control subjects from four regions in Northern Ireland and France. The Pro25 allele was more frequent in patients than in control subjects in Belfast (P < .01) and Strasbourg (P < .05). The TGF-beta 1 polymorphisms were not associated with the degree of angiographically assessed coronary artery disease in patients. The presence of a Pro25 allele was associated with a lower systolic pressure in the four control groups (P < .002), and a history of hypertension was significantly less frequent in homozygotes or heterozygotes for Pro25 than in hormozygotes for Arg25 (odds ratio, 0.43, 95% confidence interval, 0.19 to 0.92; P < .03). Since the Pro25 allele was associated with an increased risk of myocardial infarction and a reduced risk of hypertension, we favor a cautious interpretation of these apparently inconsistent results. Other studies will need to verify whether these associations are real.

Preparation of conjugates between oligonucleotides and N-vinylpyrrolidone/N-acryloxysuccinimide copolymers and applications in nucleic acid assays to improve sensitivity.

A simple approach has been developed to prepare conjugates between oligodeoxyribonucleotides (ODNs) and linear copolymers of N-vinylpyrrolidone (NVP)/N-acryloxysuccinimide (NAS). These conjugates were of higher than expected molecular weight, as determined by light scattering. Side reactions between amino groups on heterocyclic bases, especially of cytosine, are proposed to explain the formation of aggregates. ODN-NVP/NAS conjugates used in both capture and detection steps in sandwich hybridization increased assay sensitivity. Sensitivity to 10(7) DNA molecules/mL was reached for a 2.3 kb PCR fragment of hepatitis B virus (HBV) genomic DNA.

Comparative study of the glycosylation of platelet glycoprotein GPIIb/IIIa and the vitronectin receptor. Differential processing of their beta-subunit.

The platelet glycoprotein GPIIb/IIIa and the vitronectin receptor (VNR) are alpha beta-heterodimeric proteins and share the same beta-subunit. By performing swainsonine treatment and digestion with endoglycosidase H (Endo H), we showed that the heavy chains of GPIIb and VNR alpha are glycosylated by complex-type oligosaccharide chains, and provided the first evidence for the presence of one complex carbohydrate residue on their light chains. The proteolytic cleavage of pro-GPIIb and the acquisition of Endo H-resistance are independent events occurring in the same Golgi compartment. We demonstrated the Endo H-sensitivity of GPIIIa and VNR beta in all cellular systems tested. In addition, this beta-subunit is differently glycosylated according to whether it is associated with GPIIb or VNR alpha, one carbohydrate chain being processed to the complex type on GPIIIa, but not on VNR beta.

Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes: evidence that assembly between pro-GPIIb and GPIIIa is a prerequisite for expression of the complex on the cell surface.

The platelet membrane glycoproteins GPIIb and GPIIIa form a calcium-dependent heterodimer that functions as a receptor for adhesive proteins on stimulated platelets. In this study, we have investigated the kinetics of the assembly reaction that result in GPIIb-IIIa dimerization. Pulse-chase experiments analysis performed on human megakaryocytes obtained from liquid cultures of chronic myelogenous leukemic patients with antibodies specific for GPIIIa or GPIIb demonstrated the existence of a pro-GPIIb-GPIIIa complex and of a large pool (60%) of unassociated GPIIIa; nearly all the GPIIb and the pro-GPIIb molecules were found associated with GPIIIa. This free GPIIIa was not exposed on the cell surface. Pulse-chase experiments on a subclone of the human megakaryocytic cell line LAMA-84 revealed that the cells from this subclone produced only the pro-GPIIb, which was neither processed into mature GPIIb nor expressed on the cell surface. The expression of GPIIIa in PMA treated cells resulted in the production of the mature GPIIb form and the expression of the GPIIb-IIIa complex on the cell surface. These results indicate that assembly between the early forms of pro-GPIIb and GPIIIa is an obligatory step for the maturation of the heterodimer and its expression on the cell surface.

Cryopreservation of human megakaryocytic progenitor cells (CFU-MK): influence of culture conditions.

The survival of human megakaryocytic progenitor cells (CFU-MK) after freezing using a two-step cooling technique was studied in a methylcellulose culture system, using different stimulating activities and their combinations. The best growth stimulating activity for CFU-MK was found in plasma from aplastic patients (PAP). PAP was roughly three times more potent than optimal doses of human recombinant interleukin 3 (IL3), itself two times more active than our batch of phytohemagglutinin stimulated leukocyte conditioned medium (PHA-LCM). The addition of erythropoietin (EPO) to PHA-LCM doubled the number of megakaryocytic colonies, but had no effect on the number of CFU-MK stimulated by IL3. Of the two batches of PAP used, one was optimal and the other was significantly improved by the addition of PHA-LCM. The percentage recovery of CFU-MK after freezing reached 70 to 80% in cultures stimulated by PHA-LCM and IL3 +/- EPO, and 58% in PAP-supplemented cultures. However, this last value was not significantly different from the previous ones. The percentage recovery of CFU-MK in each condition tested was similar to that obtained with the other myeloid progenitors, CFU-GM and BFU-E. However, the high CFU-MK stimulating activity of PAP is hindered by its high variability and shortage in supply. Thus it seems more appropriate to recommend the use of recombinant human IL3 because of its easily standardizable and consistent CFU-MK stimulating activity.

Biogenesis of the vitronectin receptor in human endothelial cell: evidence that the vitronectin receptor and GPIIb-IIIa are synthesized by a common mechanism.

Human endothelial cells express a membrane glycoprotein alpha beta heterodimer similar to the human platelet glycoprotein IIb-IIIa complex (GPIIb-IIIa). This noncovalently associated complex is the vitronectin receptor (VNR). These two receptors belong to the cytoadhesin family and share the same beta subunit. They express different recognition specificities: platelet GPIIb-IIIa is a receptor for fibrinogen, fibronectin, and von Willebrand factor (vWF), whereas VNR is a receptor for vitronectin, and is possibly a receptor for fibrinogen and vWF. We analyzed the biosynthesis of the endothelial cell VNR. Our data show that VNR alpha is a two-chain protein which is biosynthesized as a single-chain precursor: the pro-VNR alpha. Pro-VNR alpha forms a complex with VNR beta, and this association occurs prior to the Golgi-mediated processing of the oligosaccharide side chains. Mature VNR beta is glycosylated by not fully processed oligosaccharide side chains because it remains endoglycosidase H (endo H) sensitive, even when the complex is expressed on the cell surface. This characteristic appears as a common feature for the members of the cytoadhesin family. These results indicate that although VNR and GPIIb-IIIa are biosynthesized in different cells, their expression is controlled by similar mechanisms, providing further support for the concept that the cytoadhesin family constitutes a distinct group of adhesion receptors.

Immunophenotype of blast cells in chronic myeloid leukemia.

The immunophenotype of peripheral blood blast cells from 14 patients in the chronic phase of chronic myeloid leukemia (CML) was studied using a panel of monoclonal antibodies (McAb) directed against megakaryocytic, granulomonocytic, erythroid and lymphoid antigenic determinants. The blast cells were enriched by a simple bovine serum albumin (BSA) density-cut separation and cooled in liquid nitrogen. The study was done using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique on the thawed blast cells. A consistent pattern of reactivity with McAb was found in all patients, showing that blast cells were heterogeneous. A minor component of the blast cells react with platelet antibodies, most of them being labelled with anti-GPIIb-IIIa McAb. Anti-GPIb and Von Willebrand factor McAb detected 4 times fewer megakaryocytic blast cells, suggesting that these cells are located very early in the differentiation scheme. Two major blast cell compartments were labelled with early myelomonocytic (anti-CD13: MY7) and early erythroid (anti-CD36: FA6-152) McAb. The CD34 (My10) and DR antigens which are expressed by immature blast cells and myeloid progenitors of human bone marrow (BM) were present on more than 50% of the CML blast cells. Thus, the blast cells of chronic phase CML patients, showed the same cellular diversity as the increased progenitor cell compartment observed in this disease, and their differentiation stages seemed to be very closely related.