ZNF804A and schizophrenia: An open peer commentary

I have read with interest a recent report on the putative role of ZNF804A variants in conferring susceptibility to schizophrenia [Li et al., 2012]. The authors conclude to a significant risk-enhancing effect of the A allele from a non-coding SNP, rs359895, in Asian populations. It appears, however, that this claim is built on shaky foundations. For rs359895 and one third of all 28 SNPs genotyped, it is impossible to determine whether the alleles listed correspond to theminor or themajor alleles. As can be shownusing reference data forAsian populations, for example from the 1000Genomes Project, genotyping of 19 non-canonical substitutions was performed on alternating DNA strands (Fig. 1). This implies that the strand used for genotyping the remaining nine canonical A:T or G:C substitutions cannot be imputed and the respective data for theYuxi sample are non-informative. Striking deviations from reference frequencies canbeobserved for rs7557843 and rs17584522.As for rs722385, alleles have obviously been muddled. Missing strand information equally applies to the BIOX and Singapore samples. Even ifwedisregardall concerns regardingambiguous alleles, the nominal allelic association for rs359895 does not reach a P-value of 0.00078. Using 330 cases and 612 controls supposedly carrying the ‘‘A’’ allele, Fisher’s Exact test or a Chi-square test give a P1⁄4 0.006, that is a value which does not survive corrections for multiple testing (P corrected1⁄4 0.006 281⁄4 0.168). Primer sequences, reference sequence specifications and the genotype raw data are missing. As the authors used a variety of different protocols in genotyping their various samples, and have relaxed parameters that may indicate stratification artifacts by 1:50 of the usual stringency (see the Supplemental material), the missing data are essential for assessing the validity of these results. The number of genotypes actually called is also cryptic. To judge by the contents of Tables II and S1, 404 out of 555 Kunming cases and 607 out of 1143 Kunming controls were used in the analysis of rs359895. This corresponds to call rates of 0.72 and 0.53, respectively. For the Beijing sample in Table S1, which is divided into Beijing and Shanxi subsamples in Table II, genotype counts are inconsistent, that is the two subsamples give higher totals when they are added up. Moreover, 974 Yuxi subjects are missing from Table S1. The difference in gene expression levels induced by rs359895 alleles is a further concern. Assuming that the error bars shown in Figure 2 correspond to standard deviations, it is evident that they overlap, precluding a significance at the 5% level, let alone a P1⁄4 0.004. No numerical data are shown and no correction is being offered for the confounding effects of medication taken by the subjects investigated. Regarding evidence by the same authors of a putative gene regulatory activity of rs359895 [Li et al., 2011], more questions arise. Again, information is lacking on the primers used for cloning. Hence, there is no telling which DNA strand, that is which allele, was amplified and then transfected into HeLa and HEK293T cells. According to the protocol published, carrier status for neighboring variantswas also not controlled in blood donors. In otherwords, effects observed cannot be ascribed to rs359895—even if theDNA strand and allele were identifiable—because of the likely presence of additional sequence variation in the constructs generated and assayed. In summary, the data published so far offer little insight into the functionality and the prevalence of the two rs359895 alleles in schizophrenia. Pending the disclosure of key experimental details, the claim of association advanced by the authors cannot be replicated.