Comparison of methods for in-house screening of HLA*B57:01 to prevent abacavir hypersensitivity in HIV-1 care

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

Comprehensive characterization of LEDGF/p75 in a HIV-1-infected patient cohort

BACKGROUND: Lens epithelium derived growth factor/transcriptional co-activator p75 (LEDGF/p75) is an important cellular co-factor for the HIV enzyme integrase. In the present study, we evaluated if genetic variation in the LEDGF/p75 gene and mRNA expression levels might explain differences in HIV disease progression. METHODS: Samples were derived from a therapy-naïve patient cohort from the Ghent University Hospital and from the long-term-non-progressor patient Spanish RIS cohort. A comprehensive genomic scan of the coding region and 3’UTR of LEDGF/p75 was performed using high resolution melting curve analysis and Sanger sequencing to identify single nucleotide polymorphisms (SNPS). In addition, LEDGF/p75 mRNA expression levels were determined from patient PBMCs using RT-qPCR with validated reference genes for normalization. RESULTS: In total 325 patient samples were investigated, of which 291 (90%) of Caucasian and 34 (10%) of African origin, and among which a large group of Elite controllers (n=49) and Viremic controllers (n=62). In these samples, 24 SNPs were analyzed, including 5 in the coding region (2 synonymous and 3 non-synonymous), 17 in the flanking non-coding regions and in the 3’UTR, and two additional tagSNPs as described by Madlala et al. (Aids, 2011) in two South African cohorts. One SNP in the 3’UTR region (rs2737835, n=46) had a higher representation in Caucasian Elite controllers and was correlated with lower LEDGF/p75 mRNA levels (P=0.047) and with a slower CD4 decline (P= 0.042). rs2737828 (n=13) was under-represented in Caucasian HIV patients and linked to lower LEDGF/p75 expression (P=0.013). The presence of intron SNP (rs16933270, n=6) was associated with a slower CD4 decline in African patients (P=0.017), and this CD4 decline was comparable with that of African slow disease progressors. Interestingly, the presence of one tagSNP (rs12339417, n=95) was significantly correlated with a decreased viral load, but in contrast to the results of Madlala et al. (Aids, 2011), this SNP was not correlated with the CD4 slope and neither with LEDGF/p75 mRNA levels. CONCLUSIONS: Although the data of the present investigation was not entirely comparable with the results of Madlala et al. (Aids, 2011), our data supports their hypothesis that host factors influence HIV disease progression. The observed differences between the European and South African cohorts may be of ethnical origin, or due to different infection phases. In the investigated cohorts, two SNPs were associated with lower LEDGF/p75 mRNA expression in Caucasians, and one SNP was associated with slower disease progression in Africans. The significant correlation with the tagSNP (rs12339417) and the decreased viral load is surprising as this was not correlated with a delayed CD4 decline, nor with LEDGF/p75 expression. This might indicate that either conformational changes or factors upstream of mRNA transcription might influence the action of LEDGF/p75 in these HIV patients

Characterization of LEDGF/p75 genetic variants and association with HIV-1 disease progression

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.This work was partly supported by the Flemish Agency for Innovation by Science and Technology (CellCoVir - IWT file nr 60813). Linos Vandekerckhove is supported by the National Fund for Scientific Research – Belgium as Principal Investigator. The Spanish RIS cohort and HIV BioBank are integrated in the Spanish AIDS Research Network supported by Instituto de Salud Carlos III (Grant RD06/0006/0035) and Fundación para la Investigación y Prevención del SIDA en España (FIPSE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Characterization of mutations leading to recessive dystrophic epidermolysis bullosa and Marfan syndrome in a single patient.

Dystrophic epidermolysis bullosa (DEB) is a rare genetic skin disorder. In this report we have investigated an Italian child affected with recessive DEB (RDEB) and demonstrated that he was homozygous for the mutation R226X in the type VII collagen gene (COL7A1), leading to absence of type VIT collagen at the dermal-epidermal junction. There was no family history of inherited skin blistering but the child's father was affected by Marfan syndrome, an autosomal dominant connective tissue disorder that results from mutations in the fibrillin-1 gene (FBN1). Analysis of this gene showed that the RDEB patient and his father were both heterozygous for a novel FBN1 mutation, C1971Y. This mutation affects one of the six obligate cysteine residues within one of the calcium-binding epidermal growth factor-like regions of the protein. At the age of 2-years the RDEB patient showed signs of early aortic dilatation, suggesting that he is likely to develop a Marfan syndrome phenotype in the future. This is a unique case of these two coexisting inherited disorders

Comprehensive molecular screening of the FBN1 gene favors locus homogeneity of classical Marfan syndrome

In order to estimate the contribution of mutations at the fibrillin,1 locus (FBN1) to classical Marfan syndrome (MFS) and to study possible phenotypic differences between patients with an FBN1 mutation vs. without, a comprehensive molecular study of the FBN1 gene in a cohort of 93 MFS patients fulfilling the clinical diagnosis of MFS according to the Ghent nosology was performed. The initial mutation screening by CSGE/SSCP allowed identification of an FBN1-mutation in 73 patients. Next, sequencing of all FBN1-exons was performed in 11 mutation-negative patients, while in nine others, DHPLC was used. This allowed identification of seven and five additional mutations, respectively. Southern blot analysis revealed an abnormal hybridization pattern in one more patient. A total of 23 out of the 85 mutations identified here are reported for the first time. Phenotypic comparison of MFS patients with cysteine-involving mutations vs. premature termination mutations revealed significant differences in ocular and skeletal involvement. The phenotype of the eight patients without proven FBN1 mutation did not differ from the others With respect to the presence of major cardiac, ocular, and skeletal manifestations or positive familial history. Most likely, a portion of FBN1-mutations remains undetected because of technical limitations. In conclusion, the involvement of the FBN1-gene could be demonstrated in at least 91% of all MFS patients (85/93), which strongly suggests that this gene is the predominant, if not the sole, locus for MFS