HCV and Cryoglobulin Vasculitis Genome-Wide Consortium Studies

Zignegno et al

7

Supplementary Methods

HCV and cryoglobulin vasculitis genome-wide consortium studies

CharlesSubjects were enrolled at the Rockefeller University Hospital and the NewYork-Presbyterian Hospital in longitudinal studies to investigate immunological control of HCV infection [1]All subjects were HCV antibody positive and had chronic HCV infection, as determined by serial HCV RNA measurements. Cryoglobulin vasculitis was diagnosed clinically and confirmed in the laboratory with cryoprecipitate testing, serum immunofixation, and/or PCR analysis for clonal complementarity-determining region 3. The studies were approved by the Institutional Review Boards at the Rockefeller University and New York Presbyterian Hospitals. Donors gave written informed consent according to the Declaration of Helsinki before enrollment.

RomeCryoCohort. The cohort includes 95 Caucasian patients followed up since 2001 at the regional referral centre for mixed cryoglobulinemia at the Policlinico Umberto I, Sapienza, University of Rome, Italy. All patients had a diagnosis of HCV-associated mixed cryoglobulinemic vasculitis. Patients’ blood samples were processed for DNA extraction according to the QIAamp DNA Blood Midi Kit protocol, after obtaining written informed consent in accordance with the Institutional Review Board and with the Declaration of Helsinki.This study was supported by the Intramural Research Program of the Sapienza University of Rome.

Cacoub, Paris VIREP-C Study. The objective of this study was to investigate the role of regulatory T cells populations in a longitudinal prospective study including HCV infected patients, RNA+, with or without autoimmune complications (vasculitis, cryoglobulinemia, auto-antibodies), followed during and after antiviral therapy. Patients were included between 2004 and 2011. This study included a longitudinal study of Treg cells in HCV infected patients, with or without autoimmune complications follow during and after antiviral therapy; a characterization of Treg with cell-surface antigens CD25, CD62L, CCR7, CD45 RO, and intracellular expression of GITR and CTLA-4; and the expression profile of Foxp3, a transcription factor specific of CD4+ CD25+ Treg will be performed by quantitative RT-PCR. (ii) the correlation of CD4+ CD25high and Reg1+ Treg with the severity of hepatitis (grade of hepatic fibrosis, HCV viremia and genotype, response to antiviral therapy) and with the severity of autoimmune complications. (iii) and functional assay of CD4+ CD25high and Reg1+ cells; the capacity of immuno-magnetically sorted CD4+ CD25high T-cells to suppress the proliferation of autologous responder CD25- T-cells; the assessment of IFN- and IL-10 levels in supernatants from cultured cells. Funding from French National Agency for AIDS and hepatitis research (ANRS).

Center for Systemic Manifestations of Hepatitis Viruses (MASVE).The patients included in this cohort were consecutively recruited at the outpatient clinic of the MASVE Interdepartmental Center (Florence, Italy) from 2003 to 2011. All cases were Caucasian and anti-HCV positive and all had a definite diagnosis of HCV-associated mixed cryoglobulinemic vasculitis; the subjects were regularly followed-up by the clinical staff with a blood drawn at least 2-3 times a year in order to confirm the diagnosis by clinical and biohumoral metachronous evaluations. An informed consent form was signed by each patient according to the Declaration of Helsinki and the study was approved by the local ethics committee.

Boston Area HCV Study: Transmission, Immunity, Outcomes Network (BAHSTION). Subjects were enrolled from outpatient clinics from 1998 to 2007 from three hospitals (Massachusetts General Hospital, Brigham and Women's Hospital and Lemuel Shattuck Hospital). All subjects were HCV antibody positive. Race/ethnicity was self reported and categorized as non-Hispanic Caucasian, non-Hispanic African-American, Hispanic, Asian, Native American, or Other All subjects gave written informed consent for this study, approved by each Institutional Review Board.[2]

Mangia. This cohort is made up of HCV mono-infected patients recruited since 1998 in a longitudinal study panned to investigate the role of host genetics in HCV infection.[3] The cohort includes 479 patients with HCV positive antibody test. Of them, 116 spontaneously cleared the virus, the remaining are chronically HCV infected and underwent histological or non invasive evaluation of severity of liver damage. All the patients are followed up by repeated HCV RNA testing at the Research laboratory of IRCCS “Casa Sollievo della Sofferenza” and confirmed as spontaneous resolvers or chronically infected by assays of current sensitivity of <25 IU/ml. All the subjects are Caucasian. All the patients who cleared the virus and 268 out of 363 HCV chronic patients have been included in the present GWAS. The original study was approved by the Ethic Committee of the above IRCCS in Italy. Patients with different clinical manifestations including those with vasculitis, cryoglobulinemia and autoantibodies were followed-up regardless of they were treated or not and in the latter case during and after treatment during the last 15 years.

REVELL. A case control study was performed on serologically confirmed HCV positive blood donors identified within a network of 17 blood banks in Western and Southern USA. Cases were confirmed HCV antibody–positive, RNA-negative consecutive donors(negative by minipool HCV RNA screening as routinely performed on blood donors as well as individual sample RNA screening, both employing the Gen-Probe Procleix transcription mediated amplification assay). Controls were selected based on age (2 years) and sex matching, from a database of serologically confirmed, viremic donors detected by minipool RNA screening in the same blood bank network. Study design called for a ratio of one case to two controls. Consenting subjects then had additional testing to confirm outcomes. The Committee on Human Research at the University of California, San Francisco, approved the study protocol.

Toulouse cohort. White subjects of French extraction born in the south of France were selected between April 1, 1995 and June 1, 2008. HCV antibody and RNA determinations were made to identify those with chronic persistent infection and spontaneous resolution, as described.[4] Funding from French National Agency for AIDS and hepatitis research (ANRS).

United Kingdom Drug Use cohort. This cohort is based in London and is based in a Hepatology clinic at the Kings College. These subjects were identified with HCV clearance or persistence based on testing done for clinical care and supplemented by hepatologist Salim Khakoo. Most individuals are Caucasian, HIV negative, and injection drug users.[5]

DNA sample preparation

DNA was submitted to the Center for inherited Disease Research (CIDR) at Johns Hopkins University for genotyping. Sample volume was 25 uL in 1X TE (10mMtris, 1mM ECTA, pH 8). Non-WGA DNA was submitted with a concentration between 50ng/uL and 100 ng/uL.WGA DNA was submitted with a minimum concentration of 100ng/uL.

Supplemental Figures and Tables

Supplementary Figure 1A: Distribution of Cases for first two principal components by study site.

Supplementary Figure 1B: Distribution of Control Participants for the first two principal components by study site.

Supplementary Figure 2: Quantile-quantile plot of adjusted GWAS results. Genomic Inflation was estimated to be 0.97.

Zignegno et al

7

Supplementary Table 1: Tissue specific cryoglobulinemia in the discovery and confirmatory studies.

Zignegno et al

7

References

1. Charles ED, Green RM, Marukian S, et al. Clonal expansion of immunoglobulin M+CD27+ B cells in HCV-associated mixed cryoglobulinemia. Blood 2008;111(3):1344-56 doi: 10.1182/blood-2007-07-101717[published Online First: Epub Date]|.

2. Kim AY, Kuntzen T, Timm J, et al. Spontaneous control of HCV is associated with expression of HLA-B 57 and preservation of targeted epitopes. Gastroenterology 2011;140(2):686-96.e1 doi: 10.1053/j.gastro.2010.09.042[published Online First: Epub Date]|.

3. Mangia A, Gentile R, Cascavilla I, et al. HLA class II favors clearance of HCV infection and progression of the chronic liver damage. J Hepatol 1999;30(6):984-9

4. Alric L, Fort M, Izopet J, et al. Genes of the major histocompatibility complex class II influence the outcome of hepatitis C virus infection. Gastroenterology 1997;113(5):1675-81

5. Khakoo SI, Thio CL, Martin MP, et al. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 2004;305(5685):872-4 doi: 10.1126/science.1097670[published Online First: Epub Date]|.