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.

MSPrecise: A molecular diagnostic test for multiple sclerosis using next generation sequencing

“…19 One modification was to include only unique sequences that had two or more copies after sequence filtering (redundancy 1) in an attempt to increase our confidence that the sequences being analyzed were representative of the B cell pool and not a result of sequence errors generated during either PCR amplification or NGS. We compared the sequence coverage obtained with redundancy filter (Rl) and without (R0) (Table 4).…”

“…19 Of note, only VH4 amplifications were performed for this analysis since the unique SHM accumulation was identified only in this family.…”

“…Mutation analyses were performed as previously published 19 and as detailed in Supplementary Method 1.3.…”

“…Indeed, we have demonstrated and confirmed that CSF-derived B cells from RRMS patients expressing rearranged variable heavy chain family 4 ( VH4 ) genes have an exaggerated accumulation of replacement mutations at 6 codon positions. 18, 19 …”

Peripheral VH4+ plasmablasts demonstrate autoreactive B cell expansion toward brain antigens in early multiple sclerosis patients

“…In fact, antibody genetic studies have led to several key discoveries in MS that show expansion of particular genes, excessive receptor editing, dysregulation in B cell selection [6, 16, 22, 23, 37, 38, 54, 58, 67, 75-77, 79, 80, 83, 84, 96-98, 105], and even a mutational biomarker that identifies patients who will convert to MS with 86-92% accuracy [16, 84, 85]. Here, we demonstrate that VH4 genes are used more extensively by peripheral plasmablasts from CIS-PTM patients in comparison to a previously published healthy donor plasmablasts responding to influenza infection (Fig.…”