br STAR Methods br Acknowledgments We thank Prof Y Tomari
Acknowledgments We thank Prof. Y. Tomari at the University of Tokyo for experimental advice and K. Hanada for technical assistance. This research was supported by Grants-in-Aid for Scientific Research on Innovative Areas “Nascent Chain Biology” (JP15H01548 and JP17H05677 to T.I. and JP26116002 to H.I.), “Live-Protein Dynamics,” (JP15H01656 and JP17H05897 to H.S.) and “Non-coding RNA Neo-taxonomy” (JP26113007 to H.T.) and Grants-in-Aid for Scientific Research (B) (JP16H04756 to T.I., JP15H04324 to H.I., and JP16KT0068 to H.T.) from the Japan Society for the Promotion of Science (JSPS), Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS) (JP18am0101082) from the Japan Agency for Medical Research and Development (AMED), the Takeda Science Foundation, the RIKEN Pioneering Project “Dynamic Structural Biology,” and the Cooperative Research Program of the Institute for Protein Research, Osaka University (CRa-18-01 to H.T. and T.I.).
Introduction The C-C chemokine receptor type 5 (CCR5) molecule is a G-protein coupled receptor expressed in different cell types, including T 57 9 and macrophages. Among other cellular and physiological functions, CCR5 modulates leukocyte trafficking and immune responses (Alkhatib, 2009). The CCR5Δ32 variant is characterized by a 32 base-pair (bp) deletion in the coding region of the CCR5 gene, resulting in a truncated protein, which is not expressed on the cell surface of homozygous individuals (Alkhatib, 2009; Samson et al., 1996). Among Euro-descendant populations, the Δ32 allele frequency varies around 10% (Galvani and Novembre, 2005). In Brazil, it was estimated at 4% (Silva-Carvalho et al., 2016). The infection by hepatitis C virus (HCV) is strongly influenced by host genetic factors (Ellwanger et al., 2017). Of note, it was suggested that changes in the CCR5 expression due to CCR5Δ32 could impact the susceptibility to HCV infection and progression of HCV-related diseases, once CCR5 signalling is important for the immune response against HCV (Ahlenstiel et al., 2004; Coenen and Nattermann, 2010). Nevertheless, data concerning the influence of CCR5 on hepatitis C virus (HCV) infection susceptibility is still controversial. In this context, our study evaluated the influence of CCR5Δ32 in HCV infection, HCV/HIV co-infection, and HCV-related diseases in a large sample of Brazilian individuals.
Methods CCR5Δ32 (rs333) was genotyped according to Chies and Hutz (2003), with minor adaptations. For the amplifications, the following primers were used: CCR5a 5′-GGTCTTCATTACACCTGC-3′; CCR5b 5′-AGGATTCCCGAGTAGCAGATG-3′. The PCR mix reaction (total volume of 25 μl) was composed of: 1 μl of DNA (0.2–0.5 μg), 2.5 μl of 10× buffer with 30 mM of MgCl2, 0.2 μl Taq DNA polymerase (5 U/μl), 10 mM dNTP, and 10 pmol of each primer. PCR reactions underwent one initial denaturation cycle at 94 °C for 7 min, followed by 40 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, and then a final extension cycle at 72 °C for 7 min. Genotyping of PCR products was performed on a 3% agarose gel with ethidium bromide under UV light. The pattern of bands visualized is: wild-type homozygous (wt/wt) – a single 137 bp band; heterozygous (wt/Δ32) – two bands of 137 and 105 bp respectively; variant homozygous (Δ32/Δ32) – a single 105 bp band. Due to the low number of CCR5Δ32 homozygous, individuals were classified into Δ32 allele carriers and Δ32 allele non-carriers, and the number of carriers and non-carriers were used for comparisons between groups. Initially, to evaluate the potential involvement of the Δ32 allele on the susceptibility to HCV infection or HCV/HIV co-infection, the number of Δ32 allele carriers and non-carriers was compared between groups as follows: HCV+ group vs. Control group; HIV+ group vs. Control group; HCV+ group vs. HIV+ group; HCV+/HIV+ group vs. Control group; HCV+/HIV+ group vs. HCV+ group; HCV+/HIV+ group vs. HIV+ group. Then, to evaluate the potential involvement of the Δ32 allele in HCV-related diseases, individuals from the HCV+ group were stratified according to clinical/histological criteria and compared as follows: HCV+/fibrosis group vs. Control group; HCV+/fibrosis group vs. HCV+/control group; HCV+/cirrhosis group vs. Control group; HCV+/cirrhosis group vs. HCV+/control group; HCV+/HCC group vs. Control group; HCV+/HCC group vs. HCV+/control group. For clarification, “Control group” refers to control/unfected individuals, “HCV+/control group” refers to HCV+ individuals without HCV-related diseases, and “HCC” refers to hepatocarcinoma.