title: The antibody genetics of multiple sclerosis: comparing next-generation sequencing to sanger sequencing.
abstract: We previously identified a distinct mutation pattern in the antibody genes of B cells isolated from cerebrospinal fluid (CSF) that can identify patients who have relapsing-remitting multiple sclerosis (RRMS) and patients with clinically isolated syndromes who will convert to RRMS. This antibody gene signature (AGS) was developed using Sanger sequencing of single B cells. While potentially helpful to patients, Sanger sequencing is not an assay that can be practically deployed in clinical settings. In order to provide AGS evaluations to patients as part of their diagnostic workup, we developed protocols to generate AGS scores using next-generation DNA sequencing (NGS) on CSF-derived cell pellets without the need to isolate single cells. This approach has the potential to increase the coverage of the B-cell population being analyzed, reduce the time needed to generate AGS scores, and may improve the overall performance of the AGS approach as a diagnostic test in the future. However, no investigations have focused on whether NGS-based repertoires will properly reflect antibody gene frequencies and somatic hypermutation patterns defined by Sanger sequencing. To address this issue, we isolated paired CSF samples from eight patients who either had MS or were at risk to develop MS. Here, we present data that antibody gene frequencies and somatic hypermutation patterns are similar in Sanger and NGS-based antibody repertoires from these paired CSF samples. In addition, AGS scores derived from the NGS database correctly identified the patients who initially had or subsequently converted to RRMS, with precision similar to that of the Sanger sequencing approach. Further investigation of the utility of the AGS in predicting conversion to MS using NGS-derived antibody repertoires in a larger cohort of patients is warranted.
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center , Dallas, TX , USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Rounds
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center , Dallas, TX , USA.
authors.forename: Ann J
authors.initials: AJ
authors.lastname: Ligocki
authors.affiliation: Department of Clinical Sciences, University of Texas Southwestern Medical Center , Dallas, TX , USA.
authors.forename: Mikhail K
authors.initials: MK
authors.lastname: Levin
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center , Dallas, TX , USA.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Greenberg
authors.affiliation: DioGenix Inc. , Bethesda, MD , USA.
authors.forename: Douglas W
authors.initials: DW
authors.lastname: Bigwood
authors.affiliation: DioGenix Inc. , Bethesda, MD , USA.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Eastman
authors.affiliation: Department of Clinical Sciences, University of Texas Southwestern Medical Center , Dallas, TX , USA.
authors.forename: Lindsay G
authors.initials: LG
authors.lastname: Cowell
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center , Dallas, TX , USA ; Department of Immunology, University of Texas Southwestern Medical Center , Dallas, TX , USA.
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Monson
keywords: B cell; Roche 454; antibody; multiple sclerosis; next-generation sequencing