Jowett et al. (1) have examined the association of single nucleotide polymorphism (SNP) rs8050136 in intron 1 of the FTO (fat mass– and obesity-associated) gene with levels of transcripts up to 5 Mb in either direction of this SNP in lymphocytes from 1,240 individuals. This is an important study because it has been a matter of debate through which gene the obesity-associated SNPs in intron 1 of the FTO gene affect body weight regulation. The authors did not find an association of these SNPs with FTO gene expression but found a strong association with transcript levels of the RBL2 (retinoblastoma-like 2) gene located 270 kb upstream of FTO. We note that the findings have not been replicated in an independent cohort.
Studies on cis-regulatory effects on gene transcription in humans are hampered by the fact that the tested individuals unavoidably differ in genetic background, age, life events, and environment. These problems can be circumvented by determining the ratio of allelic transcript levels in heterozygous individuals so that each allele serves as an internal control for the other (2). For this approach, only a few subjects are needed.
Using fluorescence-tagged SNP analysis, we have determined allelic transcript levels of RBL2 in blood samples from six individuals heterozygous for SNP rs3929 (C/G) in the 3′ untranslated region of the gene. Three of these individuals were also heterozygous for the FTO SNP rs8050136 (C/A), and three were homozygous (two AA and one CC). If FTO variants affected RBL2 gene expression in cis, we would expect severely skewed RBL2 allelic expression in the FTO heterozygous individuals, but not in the FTO homozygous individuals.
The forward primers used for amplification of genomic DNA (gDNA) and cDNA fragments spanning rs3929 were 5′-TGAGCTATGTGCATTTGCATT-3′ and 5′-CCTTATTACGCCGTCTCCAA-3′, respectively. The reverse primer for both gDNA and cDNA was 5′-TCACCAAAATGTCCCCTCAT-3′. For primer extension, we used the reverse primer 5′-CCTCATGTTACTAACAGGCTGTAACT-3′ and the SNaPshot kit (Applied Biosystems, Foster City, CA). Allelic gDNA ratios were used to normalize the cDNA ratios. Means and SD were calculated with JMP7 (SAS, Cary, NC). For a more detailed description of the assay, see ref. 2.
In FTO heterozygous and homozygous individuals, we observed allelic RBL2 transcript ratios of 0.88 ± 0.02 (mean ± SD) and 0.90 ± 0.02, respectively. Our results show that allelic expression of the RBL2 gene is slightly skewed but independent of the FTO genotype. The skewing is probably due to a cis-regulatory RBL2 SNP that is in linkage disequilibrium with rs3929.
In summary, we have used a direct experimental approach to investigate whether RBL2 gene expression is regulated by SNPs in the FTO gene. Our findings do not support the claim of Jowett et al. that variants at FTO influence RBL2 gene expression at large genetic distances.
Note added in proof:
Recently, we found that the SNPs in intron 1 of the FTO gene affect allelic transcript levels of the FTO gene (3).
This work was supported by a grant from the Bundesministerium für Bildung und Forschung (NGFN plus 01GS0820).
No potential conflicts of interest relevant to this article were reported.