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1.
PLoS One ; 10(4): e0123525, 2015.
Article in English | MEDLINE | ID: mdl-25874872

ABSTRACT

Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01 allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic assays have been developed for HLA-B*57:01 screening, each with specific sensitivity, turnaround time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing. All three assays had a maximum sensitivity of >99. However, differences in specificity were recorded, i.e. 84.3%, 97.2% and >99% for flow cytometry, qPCR and SSP PCR CE respectively. Our data indicate that the most specific and sensitive of the compared methods is the SSP PCR CE. Flow cytometry pre-screening can substantially decrease the number of genetic tests for HLA-B*57:01 typing in a clinical setting.


Subject(s)
Dideoxynucleosides/administration & dosage , Drug Hypersensitivity/prevention & control , HIV Infections/drug therapy , HLA-B Antigens/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Inhibitors/administration & dosage , Alleles , Antiretroviral Therapy, Highly Active , DNA Primers/chemical synthesis , DNA Primers/genetics , Dideoxynucleosides/adverse effects , Drug Hypersensitivity/etiology , Drug Hypersensitivity/genetics , Drug Hypersensitivity/immunology , Electrophoresis, Capillary , Flow Cytometry , Gene Expression , Genetic Testing/methods , HIV Infections/genetics , HIV Infections/immunology , HIV Infections/virology , HIV-1/drug effects , HIV-1/enzymology , HIV-1/genetics , HLA-B Antigens/immunology , Humans , Nucleic Acid Hybridization/methods , Reverse Transcriptase Inhibitors/adverse effects , Sensitivity and Specificity
2.
PLoS One ; 7(11): e50204, 2012.
Article in English | MEDLINE | ID: mdl-23226247

ABSTRACT

BACKGROUND: As Lens epithelium-derived growth factor (LEDGF/p75) is an important co-factor involved in HIV-1 integration, the LEDGF/p75-IN interaction is a promising target for the new class of allosteric HIV integrase inhibitors (LEDGINs). Few data are available on the genetic variability of LEDGF/p75 and the influence on HIV disease in vivo. This study evaluated the relation between LEDGF/p75 genetic variation, mRNA expression and HIV-1 disease progression in order to guide future clinical use of LEDGINs. METHODS: Samples were derived from a therapy-naïve cohort at Ghent University Hospital and a Spanish long-term-non-progressor cohort. High-resolution melting curve analysis and Sanger sequencing were used to identify all single nucleotide polymorphisms (SNPs) in the coding region, flanking intronic regions and full 3'UTR of LEDGF/p75. In addition, two intronic tagSNPs were screened based on previous indication of influencing HIV disease. LEDGF/p75 mRNA was quantified in patient peripheral blood mononuclear cells (PBMC) using RT-qPCR. RESULTS: 325 samples were investigated from patients of Caucasian (n = 291) and African (n = 34) origin, including Elite (n = 49) and Viremic controllers (n = 62). 21 SNPs were identified, comprising five in the coding region and 16 in the non-coding regions and 3'UTR. The variants in the coding region were infrequent and had no major impact on protein structure according to SIFT and PolyPhen score. One intronic SNP (rs2737828) was significantly under-represented in Caucasian patients (P<0.0001) compared to healthy controls (HapMap). Two SNPs showed a non-significant trend towards association with slower disease progression but not with LEDGF/p75 expression. The observed variation in LEDGF/p75 expression was not correlated with disease progression. CONCLUSIONS: LEDGF/p75 is a highly conserved protein. Two non-coding polymorphisms were identified indicating a correlation with disease outcome, but further research is needed to clarify phenotypic impact. The conserved coding region and the observed variation in LEDGF/p75 expression are important characteristics for clinical use of LEDGINs.


Subject(s)
Disease Progression , HIV Infections/genetics , HIV Infections/pathology , HIV-1 , Intercellular Signaling Peptides and Proteins/genetics , Mutation , Polymorphism, Single Nucleotide , 3' Untranslated Regions , Adult , Amino Acid Sequence , Conserved Sequence , Exons , Female , HIV Infections/diagnosis , HIV Infections/virology , Humans , Introns , Leukocytes, Mononuclear/metabolism , Male , Molecular Sequence Data , Open Reading Frames , Prognosis , RNA, Messenger/genetics , Sequence Analysis, DNA
3.
Hum Mutat ; 29(1): 205, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18157818

ABSTRACT

Pseudoxanthoma elasticum (PXE) is a heritable connective tissue disorder characterized by ocular, cutaneous and cardiovascular manifestations. It is caused by mutations in the ABCC6 gene (chr. 16p13.1), encoding a transmembrane transporter protein, the substrate and biological function of which are currently unknown. A comprehensive clinical and molecular study of 38 Belgian PXE probands and 21 relatives (4 affected and 17 carriers) was performed. An extensive clinical evaluation protocol was implemented with serial fundus, skin and cardiovascular evaluation. We report on 14 novel mutations in the ABCC6 gene. We observed extensive variability in severity of both cutaneous and ocular lesions. The type of skin lesion however usually remained identical throughout the evolution of the disorder, while ophthalmological progression was mainly due to functional decline. Peripheral artery disease (53%) and stroke (15%) were significantly more prevalent than in the general population (10-30% and 0.3-0.5% respectively). Interestingly, we also observed a relatively high incidence of subclinical peripheral artery disease (41%) in our carrier population. We highlight the significance of peripheral artery disease and stroke in PXE patients as well as the subclinical manifestations in carriers. Through follow-up data we gained insight into the natural history of PXE. We propose a cost- and time-efficient two-step method of ABCC6 analysis which can be used in different populations. Additionally, we created a diagnostic flowchart and attempted to define the role of molecular analysis of ABCC6 in the work-up of a PXE patient.


Subject(s)
Genetic Testing , Pseudoxanthoma Elasticum/diagnosis , Adolescent , Adult , Cohort Studies , DNA/blood , DNA Mutational Analysis , Female , Humans , Male , Middle Aged , Multidrug Resistance-Associated Proteins/genetics , Pedigree , Peripheral Vascular Diseases/diagnosis , Pseudoxanthoma Elasticum/genetics , Stroke/diagnosis
4.
Methods Mol Med ; 126: 81-95, 2006.
Article in English | MEDLINE | ID: mdl-16930007

ABSTRACT

Marfan syndrome is an autosomal-dominant connective tissue disorder characterized by pleiotropic manifestations involving the skeletal, ocular, and cardiovascular systems and resulting from mutations in the gene for fibrillin, FBN1. The clinical diagnosis is based on a set of well-defined clinical criteria (Ghent nosology). Nevertheless, the age-related nature of some clinical manifestations and the variable phenotypic expression of the disorder may hamper the diagnosis. In those instances, molecular analysis of the FBN1 gene is helpful to identify at-risk individuals. Mutations are spread over the entire FBNJ gene and there are no particular hot spots. Different standard methodologies are available to identify these mutations, however, one of the most sensitive techniques is denaturing high-performance liquid chromatography. This approach allows the performance of the analysis in a semi-automated manner and has a mutation detection rate of approx 95%.


Subject(s)
DNA Mutational Analysis/methods , Marfan Syndrome/genetics , Microfilament Proteins/genetics , Cells, Cultured , Chromatography, High Pressure Liquid , DNA/genetics , DNA/isolation & purification , DNA, Complementary/biosynthesis , Fibrillin-1 , Fibrillins , Genome, Human/genetics , Humans , Nucleic Acid Amplification Techniques , Nucleic Acid Denaturation , RNA, Messenger/isolation & purification
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