Reply to Reeves et al.: No evidence for rare ERAP1 haplotypes and haplotype combinations in ankylosing spondylitis

The comments on our recent paper (1) by Reeves et al. (2) concentrate on the functional effects of novel ERAP1 (endoplasmic reticulum aminopeptidase 1) haplotypes and haplotype combinations. The very nice in vitro functional experiments, which Reeves et al. have previously published (3) and highlight in their letter (2), clearly show that various combinations of different amino acid substitutions in synthetic ERAP1 molecules can have profound functional effects. This is unsurprising, but the relevance of many of the “polymorphisms” that these authors have described and their contribution to “real life” haplotypic combinations and their involvement in the pathophysiology of ankylosing spondylitis (AS) is doubtful. Most of the “new” rare variants described by Reeves et al. in patients and healthy controls have not been confirmed by others and are not recorded in standard DNA databases (1, 4). We suggest that repeat sequencing of the relevant exons of genomic DNA from the original 19 AS cases and 17 controls (rather than from cDNA) might be the best way to resolve this issue.

Reeves et al.’s elegant functional studies (2, 3) compare many ERAP1 combinations that are either exceptionally rare or that we do not recognize at all in the general population or in people with AS in much larger studies (1, 4). For example, there is no corroborating evidence to suggest that the “*003,” “*004,” “*006,” “*007,” “*009,” “*012,” or “*013” ERAP1 haplotypes actually exist. Our data also suggest that “*005” is exceptionally rare (allele frequency ∼0.001) (1), in contrast to the previous suggestion by Reeves et al. that it has an allele frequency of 0.29 in AS cases (3). Many of their comparisons in the in vitro functional studies are between artificial haplotype combinations that include these spurious variants. However, the functional data also include valuable comparisons of variants that are known to exist, such as *001 and *002. It is quite clear that when ERAP1-deficient cells are reconstituted with the combination of *001 + *002 (common in the general population and also in AS), there is restoration of peptide trimming activity and HLA class I cell surface expression. In contrast, reconstitution with the *001 + *001 combination has little effect. However, this is most likely explained simply by the presence of the hypofunctional R528 in *001 compared with the AS-predisposing normally functioning 528K in *002. One does not have to invoke an influence from other residues necessarily to explain this. We note that this reconstituting effect is apparent for all six haplotype combinations studied by Reeves et al. (3) containing *002, except *002 + *006 (which we have never actually observed in more than 28,000 individuals). The relevance of functional effects arising from artificial ERAP1 haplotypes or combinations of haplotypes seems highly doubtful. We believe that the onus is now on Reeves et al. to provide additional genomic sequencing data to support the concept that these exceptionally rare unusual ERAP1 haplotypes and haplotype combinations actually exist.