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Re: Genetic Variability Could Be Confusing SNP Studies in AS

Dear Editor,

It is with great interest that I read the study entitled "A novel gene variation of TNF associated with ankylosing spondylitis: a reconfirmed study" by Zhu et al(1). In treating AS in Los Angeles, California, I see many Hispanic patients especially from Mexican descent. This is very different from the Chinese population presented in this article and may explain some of the difficulties in looking for SNP's in AS.

Zhu et al reported on 79 ankylosing spondylitis (AS) patients and 132 unrelated healthy blood donors. He found that specific haplotype covering TNF gene mutant was strongly associated with AS and speculated that a recombination between HLA-B27 and TNF might have taken place. In studies on Mexican patients, Vargas-Alarcon et al stated that TNF-alpha - 308 polymorphism appears to be associated with the genetic susceptibility U-SpA. which was independent of the susceptibility conferred by HLA- B27(2). Previously this group had also reported that a significant association of HLA-DR1 and HLA-B15 with SpA existed in Mexicans which is independent of B27(3). Others have looked onto the role of HSP-2 and B60 as well with varying results.

In my practice, I find that spondylarthropathy patients of varying genetic backgrounds share many clinic features. Even amongst Latino's, there is much genetic variability based on their country of origin. In searching for a molecular basis for the etiology of AS and other rheumaticdisorders it may be necessary to investigate different ethnic populations more accurately to look for SNP associations.

References

1. Xiaoquan Zhu, Yawen Wang, Liang Sun, Yuguo Song, Fei Sun, Lei Tang, Zhenghao Huo, Jianxin Li, and Ze Yang A novel gene variation of TNF associated with ankylosing spondylitis: a reconfirmed study. Ann Rheum Dis 2007;66:1419-1422.

2. Vargas-Alarcón G, Casasola-Vargas J, Rodríguez-Pérez JM, Huerta-Sil G, Pérez-Hernández N, Londoño J, Pacheco-Tena C, Cardiel MH, Granados J, Burgos-Vargas R. Tumor necrosis factor-alpha promoter polymorphisms in Mexican patients with spondyloarthritis. Hum Immunol 2006;6710):826-32.

Send letter to journal:
Re: TNFA variants and Ankylosing Spondylitis

Dear Editor,

In a study recently published in Annals of the Rheumatic Diseases involving 79 ankylosing spondylitis (AS) patients and 132 unrelated healthy blood donors, Zhu and colleagues report association of a TNFA SNP, rs1799724, with AS [1]. I have many concerns with this paper and its conclusions.

The authors state that only SNP rs1799724 is not in Hardy-Weinberg equilibrium. However, inspection of the genotypes presented in table 2 reveals that rs2284178 is also not in Hardy-Weinberg equilibrium in the controls (P=1.3x10-5). This can occur because of hidden relationship between genotyped individuals, disease-association or selection, but most commonly occurs because of genotyping error.

The two SNPs not in Hardy-Weinberg equilibrium are critical to the findings of the study. The authors suggest that as no linkage disequilibrium was observed between HLA-B27 and rs2284178, which lies between rs1799724 and HLA-B27, or between rs2284178 and rs1799724, that the association of rs1799724 with AS is not due to linkage disequilibrium with HLA-B27. There are three concerns regarding this assertion.

Firstly, the SNP involved, rs2284178, is itself not in Hardy-Weinberg equilibrium, raising the concerns outlined above.

Secondly, the argument assumes that linkage disequilibrium is continuous, which is known not to be the case. It is quite possible for strong linkage disequilibrium to exist between to markers, where an intervening marker shows no linkage disequilibrium with either. For example, using the HapMap data for Chinese (build 35, www.hapmap.org), marker rs2284178 is not in linkage disequilibrium with either markers rs11752262 (r2=0.06) or rs11757602 (r2=0.017) which lie on either side of rs2284178. However, rs11752262 and rs11757602 are themselves in quite strong linkage disequilibrium (r2=1).

Lastly, to calculate Lewontin’s D’ statistic (used by Zhu et al) requires that the marker genotypes are known, rather than simply carriage of an allele, to determine the haplotypic phase of the alleles of interest [2]. Although it is not absolutely clear, the HLA-B27 genotyping method employed by Zhu and colleagues for the control samples does not appear to provide the requisite data [3], appearing only to determine if samples carry HLA-B27 rather than the HLA-B genotype i.e. it does not distinguish between HLA-B27 heterozygotes and homozygotes. It is also not clear, but likely, that the case genotyping is similarly limited. If so, then how did the authors calculate D’? Indeed the markedly different frequency of rs1799724 in B27-positive and -negative AS cases (47.9% vs 62.5%) suggests significant linkage disequilibrium between HLA-B27 and this SNP. However, as only 9 HLA-B27 positive cases were studied, the sample size is inadequate to make any meaningful conclusion about linkage disequilibrium with HLA-B27.

The association findings are also reported uncorrected for the number of SNPs studied, and none of the SNPs remain associated following appropriate Bonferroni-correction.

Whilst there is strong evidence of the existence on non-B27 MHC genes in AS, notably of HLA-B60 [4-6], but also probably others [7], identification of those genes will require much larger studies with complete control for linkage disequilibrium with HLA-B27. Linkage disequilibrium across the MHC is extreme and complex [8, 9], and failure to control for it properly leads to an inability to differentiate true association from linkage disequilibrium.

References

[1] Zhu X, Wang Y, Sun L, Song Y, Sun F, Tang L, et al. A novel gene variation of TNFalpha associated with ankylosing spondylitis: a reconfirmed study. Ann Rheum Dis 2007;66:1419-22.

[2] Lewontin RC. The Interaction of Selection and Linkage. I. General Considerations; Heterotic Models. Genetics 1964;49(1):49-67.

[3] Steffens-Nakken HM, Zwart G, van den Bergh FA. Validation of allele- specific polymerase chain reaction for DNA typing of HLA-B27. Clin Chem 1995;41(5):687-92.

[4] Robinson WP, van der Linden SM, Khan MA, Rentsch HU, Cats A, Russell A, et al. HLA-Bw60 increases susceptibility to ankylosing spondylitis in HLA-B27+ patients. Arthritis Rheum 1989;32:1135-41.

[5] Wei JC, Tsai WC, Lin HS, Tsai CY, Chou CT. HLA-B60 and B61 are strongly associated with ankylosing spondylitis in HLA-B27-negative Taiwan Chinese patients. Rheumatology (Oxford) 2004;43:839-42.

[6] Brown MA, Pile KD, Kennedy LG, Calin A, Darke C, Bell J, et al. HLA class I associations of ankylosing spondylitis in the white population in the United Kingdom. Ann Rheum Dis 1996;55(4):268-70.

[7] Sims AM, Barnardo M, Herzberg I, Bradbury L, Calin A, Wordsworth BP, et al. Non-B27 MHC associations of ankylosing spondylitis. Genes Immun 2007;8(2):115-23.

[8] de Bakker PI, McVean G, Sabeti PC, Miretti MM, Green T, Marchini J, et al. A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC. Nat Genet 2006;38(10):1166-72.