SMIM1 underlies the Vel blood group and influences red blood cell traits

Abstract

The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10(-15)). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.

Figure 1. The gene SMIM1 encodes the Vel blood group

a) Red blood cell membrane expression of the Vel antigen measured by flow-cytometry for respectively a Vel-negative, Vel-weak by adsorption/elution, Vel-weak, and a Vel-positive individual. b) Homozygous inheritance of a 17 nucleotide frameshift deletion (hg19, chr1:Δ3691998-3692014:GTCAGCCTAGGGGCTGT/-) in SMIM1 underlies the Vel-negative phenotype. The major allele of the common SNP rs1175550, indicated by the blue circles, was associated with reduced expression of SMIM1 in whole blood (see Fig. 2a) and decreased mean red blood cell hemoglobin concentration (P=8×10⁻¹⁵) in a large meta-analysis of genome-wide association studies (GWAS) of red blood cell parameters (see main text). c) Overexpression of human SMIM1 cDNA in HEK293T cells. Nearly all of the transfected cells showed expression of the Vel antigen as determined with human polyclonal antibodies against Vel and flow-cytometry, confirming SMIM1 as the gene encoding the Vel blood group.

Figure 2. Common SNP rs1175550 is an expression-QTL for SMIM1 and is associated with red blood cell traits

a) rs1175550 is a gene expression-quantitative trait locus (eQTL) for SMIM1 transcript in whole blood in 1420 individuals. b) Open chromatin determined by FAIRE-Seq in erythroblasts, the precursor cell of red blood cells, indicates that the rs1175550 SNP is located in a regulatory element. This is further supported by the binding in the myeloid-erythroid cell line K562 of the subset of transcription factors assayed by the ENCODE Project that are differentially expressed in erythroblasts. c) Expression of the SMIM1 transcript based on RNA-Sequencing in the hematological lineage. SMIM1 is not transcribed in the lymphoid progenitor, but is highly expressed in red blood cell precursor cells, named erythroblasts. FPKM, fragments per kilobase per million d) rs1175550 was also associated with mean corpuscular hemoglobin concentration (MCHC) in red blood cells in the large meta-analysis with P=8.6×10⁻¹⁵ (see ref). Each SNP has both a SMIM1 gene expression-QTL association P-value (black, left axis) and a MCHC association P-value from the meta-analysis of GWAS of red blood cell parameters (red, right axis). The gene expression association signal and the meta-analysis association signal co-localize (i.e., their P-values are correlated), strongly suggesting that changes in SMIM1 expression mediate the GWAS association. A second SNP, rs1184341, which is in strong LD with rs1175550 (r²=0.92) but which was not directly tested in the gene expression study and the GWAS, also showed strong evidence for co-localization (Supplementary Fig. 6), further supporting SMIM1 as the gene underlying the GWAS association peak.

Figure 3. Zebrafish knock down of smim1

Whole mount o-Dianisidine staining for hemoglobin at 3 days post-fertilization showed mild reduction in the total number of mature primitive erythrocytes (black arrow) in the zebrafish smim1 depleted embryos when compared to control. Scalebar, ~100μm