Lack of the human choline transporter-like protein SLC44A2 causes hearing impairment and a rare red blood phenotype.

Blood phenotypes are defined by the presence or absence of specific blood group antigens at the red blood cell (RBC) surface, due to genetic polymorphisms among individuals. The recent development of genomic and proteomic approaches enabled the characterization of several enigmatic antigens. The choline transporter-like protein CTL2 encoded by the SLC44A2 gene plays an important role in platelet aggregation and neutrophil activation. By investigating alloantibodies to a high-prevalence antigen of unknown specificity, found in patients with a rare blood type, we showed that SLC44A2 is also expressed in RBCs and carries a new blood group system. Furthermore, we identified three siblings homozygous for a large deletion in SLC44A2, resulting in complete SLC44A2 deficiency. Interestingly, the first-ever reported SLC44A2-deficient individuals suffer from progressive hearing impairment, recurrent arterial aneurysms, and epilepsy. Furthermore, SLC44A2null individuals showed no significant platelet aggregation changes and do not suffer from any apparent hematological disorders. Overall, our findings confirm the function of SLC44A2 in hearing preservation and provide new insights into the possible role of this protein in maintaining cerebrovascular homeostasis.

Prevalence and determinants of oral and cervicogenital HPV infection: Baseline analysis of the Michigan HPV and Oropharyngeal Cancer (MHOC) cohort study.

We determined baseline oral and cervicogenital human papillomavirus (HPV) prevalence and determinants of infection in the Michigan HPV and Oropharyngeal Cancer (MHOC) study. We enrolled 394 college-age and older participants of both sexes in Ann Arbor, Michigan and the surrounding area. All participants provided an oral sample at baseline, and 130 females provided a cervicogenital sample. Samples were tested for 18 HPV genotypes using polymerase chain reaction (PCR) MassArray. Participants filled out sociodemographic and behavioral questionnaires. Prevalence ratios for HPV oral or cervicogenital prevalence by predictor variables were estimated in univariable log-binomial models. Analysis was conducted 2018-20. In the full cohort, baseline oral HPV prevalence was 10.0% for any detected genotype (among the 338 valid oral tests at baseline) and 6.5% for high-risk types, and cervicogenital prevalence was 20.0% and 10.8%, respectively (among the 130 first valid cervicogenital tests). Oral HPV prevalence did not vary by sex, with 10.5% of women and 9.0% of men having an infection. We found a high prevalence of oral and cervicogenital HPV infection in college-age participants reporting no lifetime sexual partners. Reporting a single recent partner was associated with a lower oral HPV prevalence (PR 0.39, 95% CI: 0.16, 0.96) than reporting no recent (but at least one ever) partner. No similar protective effect was seen for cervicogenital HPV. Both oral and cervicogenital prevalence increased with the number of recent partners for most sexual behaviors. We observed an ecological fallacy masking the direction of impact of vaccination on HPV prevalence in the full cohort compared to the college-aged and the age 23+ populations considered separately. Substance use was not significantly associated with oral or cervicogenital HPV infection. Many studies report substantially higher oral HPV infection prevalence in men than in women. That difference may not be uniform across populations in the US.

Incidence and clearance of oral and cervicogenital HPV infection: longitudinal analysis of the MHOC cohort study.

OBJECTIVES: The Michigan HPV and Oropharyngeal Cancer study aimed to evaluate patterns of oral and cervicogenital human papillomavirus (HPV) infection prevalence, incidence, and clearance as well as their relationship to sexual behaviours. DESIGN: Cohort SETTING: General public in and around Ann Arbor, Michigan. PARTICIPANTS: 394 college-age and older-adult participants of both sexes provided oral samples, and 325 completed at least 2 visits. 130 who provided a cervicogenital samples, and 127 completed at least 2 visits. OUTCOMES: Incidence and clearance rates as well as HRs for oral and cervicogenital HPV. RESULTS: Oral HPV infections were transient, with only 16% of genotypes persisting to the next visit. The mean time to clearance of a genotype was 46 days (95% CI 37 to 58). In contrast, cervicogenital infections were more persistent, with 56% of genotypes persisting to the next visit. The mean time to clearance of a genotype was 87 days (95% CI 74 to 102). HPV vaccination was associated with reduced incidence of cervicogenital HPV infection (HR 0.63; 95% CI 0.47 to 0.83) but not oral HPV infection. Incidence of oral HPV infection was associated with 2+ recent deep kissing partners (HR 2.00; 95% CI 1.13 to 3.56). Incidence of both oral (HR: 1.70; 95% CI 1.08 to 2.68) and cervicogenital (HR 2.46; 95% CI 1.69 to 3.59) was associated with 2+ recent sexual partners. CONCLUSIONS: Detection of oral HPV was highly transient, but incidence was associated with recent deep kissing and sexual partners. Detection of cervicogenital HPV was more persistent, and incidence was positively associated with recent sexual partners and negatively associated with HPV vaccination.

Slc44a2 deletion alters tetraspanin and N-cadherin expression: Reduced adhesion and enhanced proliferation in cultured mesenchymal lung cells.

Slc44a2 is reported to interact with tetraspanins CD9 and CD81. To investigate how Slc44a2 affects adhesion protein expression, cells from wild-type (WT) Slc44a2+/+, heterozygous (HET) Slc44a2+/-, and knockout (KO) Slc44a2-/- mice were cultured from lung tissue. The cultured cells expressed vimentin, N-cadherin, p120 catenin, beta-catenin, actin, CD9, and CD81, but not E-cadherin. Vimentin expression with lack of E-cadherin indicated that the cultured cells were of mesenchymal origin. Slc44a2 KO cells and HET cells demonstrated lower adherence and faster proliferation than the WT cells. All three groups displayed dramatically altered intracellular distribution of N-cadherin, CD9, and CD81. The CD9 membrane foci observed in WT cell membranes were less frequent and diminished in size in HET cells and KO cells. N-cadherin was dispersed throughout both the cytoplasm and membrane in WT cells, with similar yet weaker distribution in HET cells; however, in KO cells, N-cadherin was densely aggregated in the perinuclear cytoplasm. CD81 had a distribution pattern in WT, HET, and KO cells similar to that of N-cadherin with dense cytoplasmic clusters in the cells. KO cells also exhibited reduced filamentous actin as compared to WT cells. These results suggest that Slc44a2 is necessary for proper cellular localization of adhesion proteins and growth regulation that may be related to altered adhesion signals.

Characteristics of head and neck squamous cell carcinoma cell Lines reflect human tumor biology independent of primary etiologies and HPV status.

Explanations for the differences in clinical outcomes in head and neck squamous cell carcinomas (HNSCCs) when compared by similar tumor location, stage, nodal status, human papillomavirus (HPV) status, and patient history remain elusive. Cell lines are an excellent tool of study for understanding the in vitro properties of cancers. However, HNSCC cell lines from progression-free and/or HPV-positive tumors are very rare. Here we studied HPV-positive and HPV-negative University of Michigan squamous cell carcinoma cell lines (2 HPV-, 2 HPV16+, 1 HPV18+) coming from donors with nonoropharyngeal sites and variant clinical outcomes. Cell morphology and proliferation were assessed, and immunofluorescence and Western blotting evaluated tumor biomarkers (TP53, RB1, p16, HPV E6 and E7, EGFR, Cyclin D1, Ki-67, and beta-catenin). Slow in vitro proliferation, long lag phase before exponential proliferation, lower maximal cell density, and higher wild-type TP53 expression were common to cell lines from patients who experienced long-term disease-free survival. In contrast, shorter lag phases, rapid proliferation, and high maximal cell density were observed in cell lines from patients who experienced aggressive tumor progression leading to death. Membrane-bound beta-catenin was present in all cell lines, but nuclear beta-catenin was associated with the more lethal cancers. In summary, the HNSCC cell lines present key characteristics, independent of primary etiologies and HPV infection, that mirror the behavior of the tumors from which they were derived.

Characterization of hemostasis in mice lacking the novel thrombosis susceptibility gene Slc44a2.

INTRODUCTION: Recent genome wide association studies (GWAS) identified a novel susceptibility locus for thrombosis, harbouring the SLC44A2 gene which encodes the Solute Carrier Family 44 Member 2 protein (SLC44A2). Thus far, SLC44A2 has not been studied in the context of thrombosis, and may be a unique contributor to thrombotic disease. Here we utilize mice lacking SLC44A2 (Slc44a2-/-) to evaluate a possible role of SLC44A2 in hemostasis. METHODS: Slc44a2-/- mice were evaluated in key aspects of normal hemostasis including a challenge of vascular damage by applying laser induced injury to the cremaster muscle arteriole. RESULTS: Slc44a2-/- mice had comparable levels of thrombin generation and gene expression of coagulation related genes, as compared to littermate wild type controls. Lower levels of circulating plasma Von Willebrand factor (VWF) were measured in Slc44a2-/- mice, while no difference in VWF multimerization or vascular localization was detected. Upon in vivo laser injury of the cremaster arterioles, we detected an impairment of clot formation for Slc44a2-/- mice. CONCLUSIONS: Although mice lacking SLC44A2 are normal for several hemostasis parameters, we do observe a reduction of plasma VWF levels and an altered response upon vascular damage, which suggests that SLC44A2 contributes to hemostasis upon injury. These findings are in line with the reported GWAS data and support further research on SLC44A2 in thrombosis.

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

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.

Hair Cell Loss, Spiral Ganglion Degeneration, and Progressive Sensorineural Hearing Loss in Mice with Targeted Deletion of Slc44a2/Ctl2.

SLC44A2 (solute carrier 44a2), also known as CTL2 (choline transporter-like protein 2), is expressed in many supporting cell types in the cochlea and is implicated in hair cell survival and antibody-induced hearing loss. In mice with the mixed C57BL/6-129 background, homozygous deletion of Slc44a2 exons 3­10 (Slc44a2(Δ/Δ)resulted in high-frequency hearing loss and hair cell death. To reduce effects associated with age-related hearing loss (ARHL) in these strains, mice carrying the Slc44a2Δ allele were backcrossed to the ARHL-resistant FVB/NJ strain and evaluated after backcross seven(N7) (99 % FVB). Slc44a2(Δ/Δ) mice produced abnormally spliced Slc44a2 transcripts that contain a frame shift and premature stop codons. Neither full-length SLC44A2 nor a putative truncated protein could be detected in Slc44a2(Δ/Δ) mice, suggesting a likely null allele. Auditory brain stem responses (ABRs) of mice carrying the Slc44a2Δ allele on an FVB/NJ genetic background were tested longitudinally between the ages of 2 and 10 months. By 6 months of age,Slc44a2(Δ/Δ) mice exhibited hearing loss at 32 kHz,but at 12 and 24 kHz had sound thresholds similar to those of wild-type Slc44a2(+/+) and heterozygous +/Slc44a2Δ mice. After 6 months of age, Slc44a2(Δ/Δ) mutants exhibited progressive hearing loss at all frequencies and +/Slc44a2(Δ) mice exhibited moderate threshold elevations at high frequency. Histologic evaluation of Slc44a2(Δ/Δ) mice revealed extensive hair cell and spiral ganglion cell loss, especially in the basal turn of the cochlea. We conclude that Slc44a2 function is required for long-term hair cell survival and maintenance of hearing.

Age-related changes in expression of CTL2/SLC44A2 and its isoforms in the mouse inner ear.

The membrane glycoprotein CTL2/SLC44A2 is expressed by supporting cells in the inner ear and has been identified as a target of antibodies that may induce auto-immune hearing loss. To determine if CTL2/SLC44A2 also has roles in inner ear development and to distinguish between isoform-specific roles, we assessed age-related changes in expression of CTL2/SLC44A2 isoforms and protein in the developing murine inner ear. We determined that both isoform p1 and isoform p2 (named for the upstream p1 and proximal p2 promoters that control alternate exons 1a and 1b) were robustly expressed as early as E14 and persisted during embryonic development, but after birth the p1 isoform fell to barely detectable levels while isoform p2 levels were maintained. This trend continued and became even more apparent later in post-natal development and remained in mature ears until at least 6 weeks of age. In aged (18 mo old) mice, the level of isoform p1 transcripts rose again to levels similar to the p2 isoform like that seen early in development. At the earliest stage examined, CTL2/SLC44A2 protein was expressed in both immature supporting cells and immature sensory cells, but after birth expression in the sensory cells declined in both the utricle and cochlea and by day P1 expression of CTL2/SLC44A2 was restricted to supporting cells. The changes we observed in isoform distribution are indicative of differential developmental roles and age related changes between the two isoforms of CTL2/SLC44A2 in the inner ear.

Autoantibodies to recombinant human CTL2 in autoimmune hearing loss.

OBJECTIVES/HYPOTHESIS: Choline transporter-like protein 2 (CTL2), a 68-72 kDa inner-ear membrane glycoprotein, is a candidate target antigen in autoimmune hearing loss (AIHL). The objective of this study was to test recombinant human CTL2 as a potential target for the detection of human autoantibodies in patients with AIHL. STUDY DESIGN: In vitro assay development. METHODS: Human inner ear CTL2 mRNA was cloned into baculovirus and used to infect insect cells. Immunofluorescence and western blotting were used to determine optimal expression of recombinant human CTL2 (rHuCTL2) in insect cells. AIHL patient sera of known reactivity with guinea pig inner ear were tested for antibodies to purified rHuCTL2 on western blots. Sera from normal hearing donors were used as controls. RESULTS: The rHuCTL2 protein migrated as three bands: a core protein of 62 kDa and two N-glycosylated bands at 66 and 70 kDa. Sera from 6/12 (50%) of AIHL patients with antibody to the 68-72 kDa inner-ear protein or to supporting cells also have antibody to rHuCTL2. Four of the four patients with antibody to rHuCTL2 responded to corticosteroids, whereas 4/8 that lacked antibody to rHuCTL2 did not. Among normal human sera, 80% were negative; binding was barely detectable in 3/15 (20%). CONCLUSIONS: The rHuCTL2 protein can be produced efficiently and used as a substrate for testing human sera. Antibodies to rHuCTL2 were detected in 50% of inner-ear-reactive AIHL sera. Additionally, circulating antibody to rHuCTL2 is with associated response to corticosteroids in some AIHL patients.

Galanin receptor subtype 2 suppresses cell proliferation and induces apoptosis in p53 mutant head and neck cancer cells.

PURPOSE: Galanin and its three receptors (GALR1-3) are expressed in many normal tissues, but silenced in some tumors. Contradictory roles for galanin and its receptors in various tumors have been reported. To understand their function, investigations of individual galanin receptors are necessary. In head and neck squamous carcinoma cells (HNSCC) with silenced GALR1 and GALR2, we showed that reexpressed GALR1 suppresses tumor cell proliferation via Erk1/2-mediated effects on cdk inhibitors and cyclin D1. Others showed that GALR2 could induce apoptosis in neuroblastoma cells with wild-type p53, whereas GALR2 stimulated proliferation in small cell lung cancer. In this study, we investigated the role of GALR2 in HNSCC cells that have mutant p53 and do not express GALR1. EXPERIMENTAL DESIGN: UM-SCC-1, a human oral carcinoma cell line with a splice site mutation causing a 46-bp p53 off-frame deletion, was stably transfected to express GALR2 (UM-SCC-1-GALR2). RESULTS: Galanin treatment of UM-SCC-1-GALR2 caused morphologic changes and a marked decrease in cell number that were not observed in UM-SCC-1-mock cells. Galanin and GALR2 resulted in decreased bromodeoxyuridine incorporation, p27(Kip1) and p57(Kip2) up-regulation, and decreased cyclin D1 expression. These effects were similar to GALR1 signaling in HNSCC, but GALR2 also induced caspase-3-dependent apoptosis, which was confirmed by Annexin-V staining and DNA fragmentation analysis. These were not observed with GALR1. CONCLUSION: This study shows that GALR2 reexpression can inhibit cell proliferation and induce apoptosis in HNSCC cells with mutant p53. GALR2 may be a feasible target for HNSCC therapy.

Corticosteroid response and supporting cell antibody in autoimmune hearing loss.

OBJECTIVE: To determine whether antibodies to supporting cells are associated with response to corticosteroids in patients with autoimmune sensorineural hearing loss. DESIGN: Prospective analysis of antibody to inner-ear antigens. SETTING: Collaborating otology practices in Pennsylvania, Michigan, and Indiana. PATIENTS: Sixty-three patients with rapidly progressive unilateral or bilateral sensorineural hearing loss of unknown cause suggestive of autoimmune sensorineural hearing loss. INTERVENTIONS: Pretreatment audiometry, serum analysis by Western blot (WB) and immunofluorescence (IF) tests, corticosteroid therapy, and follow-up audiometry. MAIN OUTCOME MEASURES: Antibody reactivity and audiogram changes were analyzed for association with response to treatment. RESULTS: More than half of the patients (37/63) had antibodies to both a 68- to 72-kDa protein and to inner-ear supporting cells, 16 patients had positive results on one assay only, and 10 had negative results on both. Twenty-eight patients improved and 35 did not. The WB findings did not correlate with response. Of the WB-positive patients, 49% (21/43) improved, as did 35% (7/20) of the WB-negative patients (P = .30). In contrast, 53% (25/47) of IF-positive patients improved, compared with only 19% (3/16) in the IF-negative group (P = .02). Of those who improved, 89% (25/28) were IF positive. CONCLUSIONS: Antibody to an inner-ear supporting cell antigen was significantly associated with hearing improvement after corticosteroid therapy (relative rate, 2.8). Patients with IF-positive serum are nearly 3 times more likely to experience improved hearing with corticosteroid treatment than those who are IF negative. Antibodies to inner-ear supporting cell antigen may have value in diagnosis and treatment of patients with autoimmune sensorineural hearing loss.

Identification and characterization of choline transporter-like protein 2, an inner ear glycoprotein of 68 and 72 kDa that is the target of antibody-induced hearing loss.

The Kresge Hearing Research Institute-3 (KHRI-3) antibody binds to a guinea pig inner ear supporting cell antigen (IESCA) and causes hearing loss. To gain insight into the mechanism of antibody-induced hearing loss, we used antibody immunoaffinity purification to isolate the IESCA, which was then sequenced by mass spectroscopy, revealing 10 guinea pig peptides identical to sequences in human choline transporter-like protein 2 (CTL2). Full-length CTL2 cDNA sequenced from guinea pig inner ear has 85.9% identity with the human cDNA. Consistent with its expression on the surface of supporting cells in the inner ear, CTL2 contains 10 predicted membrane-spanning regions with multiple N-glycosylation sites. The 68 and 72 kDa molecular forms of inner ear CTL2 are distinguished by sialic acid modification of the carbohydrate. The KHRI-3 antibody binds to an N-linked carbohydrate on CTL2 and presumably damages the organ of Corti by blocking the transporter function of this molecule. CTL2 mRNA and protein are abundantly expressed in human inner ear. Sera from patients with autoimmune hearing loss bind to guinea pig inner ear with the same pattern as CTL2 antibodies. Thus, CTL2 is a possible target of autoimmune hearing loss in humans.