The three genotypes for each of the two variants give a total of nine combinations, however, since the haplotype rs43702418CGCA:rs1175550A was not present in any of the samples, only six possible combinations are depicted here. logic of Vel antigen expression, and lengthen the set of markers for genetic Vel blood group typing. Recently, we and two other research groups recognized a previously unreported erythroid gene, Small Integral Membrane Protein 1 (exon 3. While these findings provide a molecular background for the Vel-negative (Vel?) phenotype, they do not explain the recurrent clinical observation that Vel-positive (Vel+) individuals show great variability in antigen expression. Weakly Vel+ erythrocytes (Vel+poor) can be mistyped as Vel? by routine serological phenotyping, creating risk for transfusion reactions in patients with anti-Vel inadvertently transfused with Vel+poor erythrocytes. While some of the variation can be attributed to heterozygosity for the 17-bp deletion1, this mutation Trifloxystrobin does not take into account all the variance. The common SNP rs1175550 (A? ?G, minor Trifloxystrobin allele frequency, MAF?=?0.22 in Caucasians), located in intron 2 of at a higher level than individuals who carry the major allele (rs1175550A)2,5,6. However, it is not known whether rs1175550 itself is usually causal, or a proxy for another (as yet undetected) causal variant. Moreover, the molecular effects of this variant have not been defined, or whether additional variants modulate expression. To address these questions, we fine-mapped a candidate regulatory region in intron 2 made up of rs1175550 and multiple erythroid transcription factor binding sites in Swedish and African American blood donors. We recognized rs1175550 and a previously unreported tri-nucleotide insertion (rs143702418; C? ?CGCA) as correlated with and Vel antigen expression. While these variants, and their effects on expression, were inseparable in the Swedish samples, their effects could be separated in the African American samples where the linkage disequilibrium in intron 2 was not as tight. Our data show that rs1175550 and rs143702418 independently modulate expression, although the effect of rs1175550 dominated that of rs143702418. Using gel shift assays, we found that both variants influence nuclear protein binding, and Rabbit Polyclonal to SLC39A1 that the strong effect of rs1175550 may be mediated by differential binding of TAL1. Results rs1175550 is located in a regulatory region in SMIM1 intron 2 To understand the role of rs1175550, we examined its neighborhood in intron 2 for transcription factor binding sites using ChIP-seq Trifloxystrobin data in the Encyclopedia of DNA Elements compendium (ENCODE)7. This revealed a 500?bp region with increased acetylation of lysine 27 on histone 3, increased DNaseI hypersensitivity, and binding sites for multiple transcription factors, including the erythroid factors GATA-1, TAL1 and ZBTB7A. The region contained eight sequence variants annotated with a MAF of more than 1% in dbSNP 1448 (Fig. 1a). Open in a separate window Physique 1 rs1175550 and rs143702148 association with and Vel antigen expression.(a) Diagrammatic representation of the region around rs1175550 with neighbouring variants and data from your UCSC Genome Browser. Blue: ENCODE H3K27 ChIP-seq data for K562 erythroleukemia cells. Black: ENCODE DNaseI hypersensitivity data. Bottom: Transcription factor binding sites inferred by ChIP-seq in the ENCODE project; greyscale indicates transmission strength, K indicates that the data was produced in K562 cells. Transcription factors with strong signal or known function in erythropoiesis were included. (b) expression in the Swedish samples by RT-qPCR. Expression values were normalised against the sample with the highest expression and plotted individually with indication of the median and inter-quartile range (IQR). Statistical analyses were performed with Kruskal-Wallis one-way analysis of variance with Dunns multiple comparisons test. (c) Vel antigen expression on RBCs measured by circulation cytometry (MFI; median fluorescence intensity) with human anti-Vel serum and PE-conjugated goat-anti-human secondary antibody. All samples were normalised to the sample with the highest expression and plotted together with the median and IQR. Statistical analyses were performed with Kruskal-Wallis one-way analysis of variance with Dunns multiple comparisons test. Association data for the seven other SNPs, including rs143702418, in the region are given in Supplementary Figs S1 and S2. (d) Western blot with polyclonal anti-SMIM1 on a subset of 9 samples from your Swedish collection with the indicated rs1175550 genotype plus a control sample homozygous for the 17-bp deletion. SMIM1 is usually thought to exist as a monomer and dimer in the RBC membrane. (e) Bands in D) quantified by densitometry analysis. Mean values .