SLC44A2 single nucleotide polymorphisms, isoforms, and expression: Association with severity of Meniere's disease?

Abstract

SLC44A2 was discovered as the target of an antibody that causes hearing loss. Knockout mice develop age related hearing loss, loss of sensory cells and spiral ganglion neurons. SLC44A2 has polymorphic sites implicated in human disease. Transfusion related acute lung injury (TRALI) is linked to rs2288904 and genome wide association studies link rs2288904 and rs9797861 to venous thromboembolism (VTE), coronary artery disease and stroke. Here we report linkage disequilibrium of rs2288904 with rs3087969 and the association of these SLC44A2 SNPs with Meniere's disease severity. Tissue-specific isoform expression differences suggest that the N-terminal domain is linked to different functions in different cell types. Heterozygosity at rs2288904 CGA/CAA and rs3087969 GAT/GAC showed a trend for association with intractable Meniere's disease compared to less severe disease and to controls. The association of SLC44A2 SNPs with VTE suggests that thrombi affecting cochlear vessels could be a factor in Meniere's disease.

Keywords: Amino acid polymorphisms; Choline transporter-like protein 2: solute carrier protein 44A2; DNA sequence differences; Hearing; Human gene expression; Meniere's disease; Vestibular tissue.

Figure 1

Genotyping strategies. A. SLC44A2 gene located on Chromosome 19p13.1 has 23 exons with alternate exons 1a and 1b driven by an upstream p1 and a proximal p2 promoter respectively. Exon 1a is 22.9 kb upstream of exon 1b. The p1 and p2 SLC44A2 isoforms code for full-length proteins that differ only in exon1 (a or b). The p1 isoform (iso1) has 704 and the p2 isoform (iso2) has 706 amino acids. To examine the full-length expressed sequences the cDNA primers Iso1F and Iso2F were used with a single reverse primer Iso1/2R followed by sequencing with a set of overlapping sequencing primers (as reported in Nair et al. 2004). Focal genotyping of rs2288904 was done initially with DNA primers E5–7F and E5–7R followed by sequencing of the PCR product (Figure 1A). Subsequent genotyping of rs2288904 (c.455CGA/CAA) and rs3087969 (c.198GAT/GAC) was carried out using TaqMan assays with DNA as illustrated in Figure 1B.

Figure 2

RT-PCR results in 11 human vestibular tissue samples and one representative human epithelial cell line (UMSCC 22A) showing relative isoform1 and isoform 2 expression. Human inner tissues are indicated by UMHL- (University of Michigan Hearing Loss) and a 4 digit number (i.e. UMHL1304, UMHL1330 etc.). VS=Vestibular schwannoma, MD=Meniere’s disease. Lanes are labeled iso1, iso2 or actin in the representative samples shown. Note that iso1 is more abundant in all samples except in UMSCC 22A in which iso2 is the more abundant isoform. Actin was equally amplified in all cases.

Figure 3

SLC44A2 iso1 and iso2 expression pattern in blood cells (A) and other human tissues (B). A. Isoform 1 is expressed exclusively human blood cells. Top panel: Proliferating EBV transformed human lymphoblasts from Meniere’s disease patients (MD8, MD10, MD22, MD32, MD33). Middle panel: MD34, MD38 and whole blood samples (Normal 4, and Normal 7 from normal hearing donors). Lower panel: Isoform expression in human endothelial cells, fetal liver, adult liver and adult lung. Human endothelial cells predominantly express iso1 with lower iso2 expression. Fetal and adult liver exhibit weak iso1 and undetectable iso2 expression. In contrast human lung strongly expresses iso1 and iso2 to an equivalent degree. Actin cDNA was used as a control. 1kbL=One Kb Ladder.

Figure 4

CTL2/SLC44A2 isoform expression in squamous carcinoma cell lines by quantitative-RT-PCR. The upper panel shows the Q-RT-PCR results for SLC44A2 isoform 1 and 2 expression in head and neck cancer (UM-SCC-14A, -14C, -22A, -22B), cervical cancer (ME-180) and HPV-transformed human oral keratinocyte (HOK-16B) samples. RPLP0 (Ribosomal Protein Large P0) was used as endogenous control. In all of these epithelial cell lines iso2 expression levels are greater than that of iso1. The lower panel shows agarose gel electrophoresis of the Q-RT-PCR products. For each sample, left lane is iso1, middle lane is iso2 and right lane is RPLP0. This data demonstrates that in squamous cell carcinomas and HPV-transformed mucosal cells SLC44A2 iso2 transcripts are more abundant than iso1 transcripts.