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High‐resolution HLA class II sequencing of Swedish multiple sclerosis patients

Abstract Multiple sclerosis (MS) is a chronic neurological disease believed to be caused by autoimmune pathogenesis. The aetiology is likely explained by a complex interplay between inherited and environmental factors. Genetic investigations into MS have been conducted for over 50 years, yielding >100 associations to date. Globally, the strongest linkage is with the human leukocyte antigen (HLA) HLA‐DRB5*01:01:01‐DRB1*15:01:01‐DQA1*01:02:01‐DQB1*06:02:01 haplotype. Here, high‐resolution sequencing of HLA was used to determine the alleles of DRB3, DRB4, DRB5, DRB1, DQA1, DQB1, DPA1 and DPB1 as well as their extended haplotypes and genotypes in 100 Swedish MS patients. Results were compared to 636 population controls. The heterogeneity in HLA associations with MS was demonstrated; among 100 patients, 69 extended HLA‐DR‐DQ genotypes were found. Three extended HLA‐DR‐DQ genotypes were found to be correlated to MS; HLA‐DRB5*01:01:01‐DRB1*15:01:01‐DQA1*01:02:01‐DQB1*06:02:01 haplotype together with (A) HLA‐DRB4*01:01:01//DRB4*01:01:01:01‐DRB1*07:01:01‐DQA1*02:01//02:01:01‐DQB1*02:02:01, (B) HLA‐DRBX*null‐DRB1*08:01:01‐DQA1*04:01:01‐DQB1*04:02:01, and (C) HLA‐DRB3*01:01:02‐DRB1*03:01:01‐DQA1*05:01:01‐DQB1*02:01:01. At the allelic level, HLA‐DRB3*01:01:02 was considered protective against MS. However, when combined with HLA‐DRB3*01:01:02‐DRB1*03:01:01‐DQA1*05:01:01‐DQB1*02:01:01, this extended haplotype was considered a predisposing risk factor. This highlights the limitations as included with investigations of single alleles relative to those of extended haplotypes/genotypes. In conclusion, with 69 genotypes presented among 100 patients, high‐resolution sequencing was conducted to underscore the wide polymorphisms present among MS patients. Additional studies in larger cohorts will be of importance to define MS among the patient group not associated with HLA‐DRB5*01:01:01‐DRB1*15:01:01‐DQA1*01:02:01‐DQB1*06:02:01.

Blood samples were collected in cell preparation tubes (CPTs). Controls (n = 636) included in the HLA Next Generation Sequencing (NGS) analysis were obtained as previously described (Lind et al., 2019). These controls were obtained as two different study groups: (A) Swedish general population (GP) controls (n = 448) and (B) Life-Gene controls (n = 188; https://www.lifegene.se/For-scientists/About-LifeGene) (Lind et al., 2019;Zhao et al., 2016). Sample collection and HLA typing was performed at different time-points; the two different control groups were verified to be harmonize (no statistical differences for any HLA alleles, haplotypes or genotypes) before put together as one larger group of general population controls. General population controls refer to a large enough and randomly selected group of individuals, which represent the HLA genotypes present in the Swedish population.
The study was approved by the regional ethics board in Lund, Sweden (dnr 2015/257 with amendment 2017/670).

HLA high-resolution typing
DNA was extracted using Qiagen Blood Maxi Kit according to manufacturer's protocol and as previously described (Lind et al., 2019).
In brief, blood clots were lysed through addition of protease (Qiagen protease) and lysis buffer (AL buffer, Qiagen). Next, incubation of sample-mixtures was at 70 • C for 10 min. Addition of 99.
The significance threshold for all hypothesis testing was unadjusted and set to .05. Correction for multiple testing was applied using the Benjamini-Hochberg Procedure (Benjamini & Hochberg, 1995).

HLA-DRB3, DRB4 and DRB5-DRB1-DQA1-DQB1 genotypes
A total of 95 HLA-DRB3, DRB4 and DRB5-DRB1-DQA1-DQB1 genotypes were present among MS patients or GP (Supplementary Table   S1). Among 100 MS patients, 69 different genotypes were found. Relative to GP controls, a higher frequency of three genotypes were found in MS patients. Notably, all these three genotypes included a single

DISCUSSION
For simplified reading of the discussion section, significant haplotypes are presented as the following abbreviations: A   (Barcellos et al., 2006). Although a dose effect may explain the data (Barcellos et al., 2006;Barcellos et al., 2003) (Sawcer et al., 2014). The high-resolution HLA typing makes it possible to further dissect the possible contribution of other alleles, perhaps haplotypes, to the risk of MS.
A weakness of our study was the fact that the HLA-DPA1 and HLA-DPB1 alleles could not be assigned to extended haplotypes and genotypes. This was largely attributed to the very high frequency of HLA-DPA1*01:03:01 (>80%) among both patients and controls, which precluded computation of the other rare allele variants among the families that were used as templates (Lind et al., 2019). Another limitation was the missing information of age at first symptom and family history of MS, these parameters would have been of importance especially among non-HLA-DR15-DQ6 patients.
It appears overly simplistic to characterize MS as having a single aetiology and pathogenesis. The heterogeneous clinical presentation, variable clinical course, inconsistency in genetic markers, unpredictable therapeutic response and diverse histopathological findings may be indicative of divergence in the demyelination pathways (Lucchinetti et al., 2000;McFarland & Martin, 2007). The highresolution HLA as presented here would represent an important approach to better delineate MS aetiology and pathogenesis in the future.
In summary, we have confirmed previous HLA associations to MS, while also offering plausible explanations to help guide future research.
Using high-resolution sequencing, we have highlighted the widespread polymorphisms as 69 distinct genotypes present among 100 Swedish MS patients and further studies in a larger number of patients will be needed to further delineate MS not associated with HLA-DR15-DQ6.2.