The Hellenic type of nondeletional hereditary persistence of fetal hemoglobin results from a novel mutation (g.-109G>T) in the HBG2 gene promoter

Nondeletional hereditary persistence of fetal hemoglobin (nd-HPFH), a rare hereditary condition resulting in elevated levels of fetal hemoglobin (Hb F) in adults, is associated with promoter mutations in the human fetal globin (HBG1 and HBG2) genes. In this paper, we report a novel type of nd-HPFH due to a HBG2 gene promoter mutation (HBG2:g.-109G>T). This mutation, located at the 3′ end of the HBG2 distal CCAAT box, was initially identified in an adult female subject of Central Greek origin and results in elevated Hb F levels (4.1%) and significantly increased Gγ-globin chain production (79.2%). Family studies and DNA analysis revealed that the HBG2:g.-109G>T mutation is also found in the family members in compound heterozygosity with the HBG2:g.-158C>T single nucleotide polymorphism or the silent HBB:g.-101C>T β-thalassemia mutation, resulting in the latter case in significantly elevated Hb F levels (14.3%). Electrophoretic mobility shift analysis revealed that the HBG2:g.-109G>T mutation abolishes a transcription factor binding site, consistent with previous observations using DNA footprinting analysis, suggesting that guanine at position HBG2/1:g.-109 is critical for NF-E3 binding. These data suggest that the HBG2:g-109G>T mutation has a functional role in increasing HBG2 transcription and is responsible for the HPFH phenotype observed in our index cases

Single-cell RNA sequencing of liver fine-needle aspirates captures immune diversity in the blood and liver in chronic hepatitis B patients

Background and Aims: HBV infection is restricted to the liver, where it drives exhaustion of virus-specific T and B cells and pathogenesis through dysregulation of intrahepatic immunity. Our understanding of liver-specific events related to viral control and liver damage has relied almost solely on animal models, and we lack useable peripheral biomarkers to quantify intrahepatic immune activation beyond cytokine measurement. Our objective was to overcome the practical obstacles of liver sampling using fine-needle aspiration and develop an optimized workflow to comprehensively compare the blood and liver compartments within patients with chronic hepatitis B using single-cell RNA sequencing. Approach and Results: We developed a workflow that enabled multi-site international studies and centralized single-cell RNA sequencing. Blood and liver fine-needle aspirations were collected, and cellular and molecular captures were compared between the Seq-Well S3 picowell-based and the 10× Chromium reverse-emulsion droplet–based single-cell RNA sequencing technologies. Both technologies captured the cellular diversity of the liver, but Seq-Well S3 effectively captured neutrophils, which were absent in the 10× dataset. CD8 T cells and neutrophils displayed distinct transcriptional profiles between blood and liver. In addition, liver fine-needle aspirations captured a heterogeneous liver macrophage population. Comparison between untreated patients with chronic hepatitis B and patients treated with nucleoside analogs showed that myeloid cells were highly sensitive to environmental changes while lymphocytes displayed minimal differences. Conclusions: The ability to electively sample and intensively profile the immune landscape of the liver, and generate high-resolution data, will enable multi-site clinical studies to identify biomarkers for intrahepatic immune activity in HBV and beyond.</p

Quantitation of Pretreatment Serum IP-10 Improves the Predictive Value of an IL28B Gene Polymorphism for Hepatitis C Treatment Response

Polymorphisms of IL28B gene are highly associated with sustained virological response (SVR) in patients with chronic hepatitis C treated with peginterferon and ribavirin. Quantitation of Interferon-γ Inducible Protein-10 (IP-10) may also differentiate antiviral response. We evaluated IP-10 levels in pretreatment serum from 115 non-responders and 157 sustained responders in the VIRAHEP-C cohort, including African Americans (AA) and Caucasian Americans (CA). Mean IP-10 was lower in sustained responders compared to non-responders (460 ± 37 pg/ml vs 697 ± 49 pg/ml, p600 pg/ml) was 67%. We assessed the combination of pretreatment IP-10 levels with IL28B genotype as predictors of treatment response. The IL28B polymorphism rs12979860 was tested in 210 participants. CC, CT, or TT genotypes were found in 30%, 49%, and 21%, respectively, with corresponding SVR rates of 87%, 50%, and 39% (p<0.0001). Serum IP-10 levels within the IL28B genotype groups provided additional information regarding the likelihood of SVR (p< 0.0001). CT carriers with low IP-10 had 64% SVR versus 24% with high IP-10. Similarly, a higher SVR rate was identified for TT and CC carriers with low versus high IP-10 (TT: 48% versus 20%, CC: 89% versus 79%). IL28B genotype and baseline IP-10 levels were additive but independent when predicting SVR in both AA and CA

Interview sur La boisson Outox

info:eu-repo/semantics/publishe

A new vector for recombination-based cloning of large DNA fragments from yeast artificial chromosomes.

The functional analysis of genes frequently requires manipulation of large genomic regions embedded in yeast artificial chromosomes (YACs). We have designed a yeast-bacteria shuttle vector, pClasper, that can be used to clone specific regions of interest from YACs by homologous recombination. The important feature of pClasper is the presence of the mini-F factor replicon. This leads to a significant increase in the size of the plasmid inserts that can be maintained in bacteria after cloning by homologous recombination in yeast. The utility of this vector lies in its ability to maintain large fragments in bacteria and yeast, allowing for mutagenesis in yeast and simplified preparation of plasmid DNA in bacteria. Using PCR-generated recombinogenic fragments in pClasper we cloned a 27 kb region from a YAC containing the Hoxc cluster and a 130 kb region containing the entire Hoxb cluster. No rearrangements were seen when the recombinants in the shuttle vector were transferred to bacteria. We outline the potential uses of pClasper for functional studies of large genomic regions by transgenic and other analyses

Properties of a CCAAT box-binding protein.

NF-Y is a sequence-specific DNA-binding protein that interacts with the conserved Y motif of the major histocompatibility complex class II gene, E alpha. Since it is actually a CCAAT box-binding protein, NF-Y also attaches to other promoters bearing CCAAT sequences; yet, it is neither of the previously described transcription factors, CBP or CTF/NF-1. In this report, we document the cell-type distribution and various biochemical properties of NF-Y. The most important findings are that this protein is ubiquitously distributed, that it is probably a metallo-protein, that it has a protease-resistant DNA-binding domain and that the NF-Y/E alpha-olgo complex seems extremely large (greater than 200kD). These data should prove useful in comparisons of NF-Y with other sequence-specific DNA-binding proteins; they have already provided new insights into NF-Y's structure