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

Lack of the human choline transporter‐like protein SLC44A2 causes hearing impairment and a rare red blood phenotype – By Koehl et al., 2023.

Background

The presence or absence of specific antigens at the red blood cell surface determines blood phenotypes. These characteristics are inherited and result from genetic polymorphism at the blood group loci. A null phenotype in which all antigens in one system are absent is seen in most blood group systems. Individuals with null phenotypes are usually healthy, but in some cases, they may develop hematologic or nonhematologic conditions. The choline transporter-like protein CTL2 encoded by the SLC44A2 gene is present in various human tissues, including the kidney, lung, inner ear, endothelial, and blood cells. Studies suggest that this protein is essential in platelet aggregation and neutrophil activation. In this study, Koehl and colleagues show that SLC44A2 is expressed in red blood cells and carries new blood group antigens called RIF and VER, establishing the CTL2 blood group system.


Methods

Flow neutrophil adhesion assays

This experiment involved growing HMEC‐1 (Human Microvascular Endothelial Cell line) monolayers in Vena8 Endothelial+ Biochips. The cells were activated with TNFα (10 ng/ml) for 24 hours before the adhesion assay. The researchers measured adhesion under flow conditions using Vena8 Endothelial +  biochips and ExiGo Pumps. Thirty minutes before cell perfusion, they isolated neutrophils from healthy donors and incubated them with anti‐RIF, anti‐VER, or irrelevant IgG1 antibodies. The next step was to perfuse the samples for 45 min at 1 dyn/cm2 through the biochip channels containing HMEC‐1 monolayers. Then, they labeled adherent neutrophils with anti‐CD16 alexa 488‐conjugated mouse monoclonal antibody for 15 min and monitored neutrophil adhesion to endothelial cells using a microscope and software. They quantified adhesion levels by measuring the surface area of fluorescent patches using the ImageJ Software.

Results

Neutrophil adhesion to endothelial cells.

The main findings of this experiment were:

  • Both anti‐VER and anti‐RIF alloantibodies significantly increase neutrophil adhesion to endothelial cells and neutrophil aggregate formation compared with nonimmune human IgG1 (Figure 6 D).

This result suggests that anti‐VER and anti‐RIF antibodies may induce aggregates of neutrophils, like that caused by anti‐HNA‐3 antibodies.

Figure 6. Anti‐SLC44A2 red cell alloantibodies activate neutrophil adhesion to endothelial cells. Effect of anti‐VER and anti‐RIF on the adhesion of neutrophils from healthy controls to endothelial cells (HMEC‐1). Adherent neutrophils were quantified, and means ± standard error of the median of three independent experiments are shown. **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical analyses were performed using GraphPad Prism 7. Statistical test was performed using paired nonparametric t‐test (Wilcoxon t‐test).

Other experiments in this study

To identify the genes encoding high-prevalence blood group antigens with an unknown molecular basis, the group focused their research on the serum of a pregnant woman, proband 1, born in Morocco (Rif coast area), who developed a red blood cell antibody during her pregnancy. They called this antibody anti-RIF. The researchers performed whole-exome sequencing of seven RIF individuals to identify the gene underlying the RIF- antigen. SLC44A2 was considered the most prominent candidate because this gene encodes the choline transporter‐like 2 (CTL2), a membrane protein expressed in blood cells.The researchers also performed whole-exome sequencing of the RIF− European proband 2, which indicated a mutation in the SLC44A2 gene despite her red blood cells not reacting with anti‐RIF antibody. They also performed a serological investigation to characterize the blood phenotype of this proband. Flow cytometry experiments showed that this alloantibody was not an anti‐RIF because it reacted with RIF- red blood cells. They called this antibody anti-VER after the city of birth of proband 2 (Verona, Italy).


Other findings in this study:

  • SLC44A2‐deficient individuals have progressive hearing impairment.

  • The absence of SLC44A2 from platelets and red blood cells does not cause any apparent hematological disorder.

  • Human SLC44A2 is dispensable for ex vivo platelet aggregation.

Conclusion

These findings confirm the function of SLC44A2 in hearing preservation and provide new insights into the possible role of this protein in maintaining cerebrovascular homeostasis.

You can see more details of the experiments here.


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