Insights into phenotypic differences between humans and mice with p.T721M and other C-terminal variants of the SLC26A4 gene

The knock-in mouse generated in this study, the Slc26a4^T721M/T721M with the Slc26a4 p.T721M variant, demonstrated normal audiovestibular phenotypes and inner ear morphologies, similar to wild-type mice. To investigate whether the p.T721M variant could contribute to SNHI in mice through the haplo-insufficiency mode, we further generated mice with compound heterozygous variants (Slc26a4^{919-2A>G/T721M}) by intercrossing Slc26a4^+/T721M mice with Slc26a4^{919-2A>G/919-2A>G} mice, which segregated the c.919-2A > G variant with abolished function. Compound heterozygous mice for p.T721M and c.919-2A > G (i.e., Slc26a4^{919-2A>G/T721M} mice) also had normal audiovestibular phenotypes, indicating that a single p.T721M allele was sufficient to maintain normal inner ear physiology in the mice.

The SLC26A4 p.T721M variant has been documented sporadically in hearing-impaired families worldwide, including two Mediterranean families¹⁸, two Iranian families¹⁹, two Japanese family^20,21, three Chinese families^22,23, and one Taiwanese family²⁴. Although this variant is widely distributed across different populations, its prevalence is relatively low compared to other SLC26A4 variants, such as c.919-2A > G or p.H723R. According to the American College of Medical Genetics and Genomics (ACMG) guidelines in the Varsome platform²⁵, SLC26A4 p.T721M is classified as “pathogenic” by fulfilling the criteria of PM1, PM2, PP2, PP3, and PP5. It is located in the hotspot region of the SLC26A4 gene, where the majority of pathogenic variants occur (PM1). Its allele frequency is < 0.0001 across various ethnic groups in gnomAD (PM2). The majority of non-VUS missense variants in SLC26A4 have been reported as “pathogenic” in UniProt (PP2). In well-established databases (e.g., ClinVar, DVD²⁶, and UniProt) and in several prediction algorithms (e.g., SIFT, Polyphen2, LRT, FATHMM, Mutation Taster, etc.), p.T721M was categorized as “pathogenic” (PP3 and PP5). Clinically, both homozygosity for p.T721M and compound heterozygosity for p.T721M with another SLC26A4 mutation have been linked to non-syndromic DFNB4 or PS, as characterized by EVA, progressive or fluctuating severe-to-profound SNHI, and/or goiter^18,19,20,21. Specifically, the hearing features have been detailed in two compound heterozygotes, one with symmetric SNHI (right/left: 103.75/110 dBHL)²¹, and the other with asymmetric SNHI (right/left: 112.5/68.75 dBHL)²⁰. These typical clinical manifestations in p.T721M homozygotes and compound heterozygotes suggest that p.T721M is a pathogenic SLC26A4 variant in humans.

In previous cell line studies, a number of SLC26A4 variants, including p.P123S, p.M147V, p.L236P, p.S657N, p.T721M, and p.H723R, have been demonstrated to confer pathogenicity by affecting the trafficking process, rather than the expression level of pendrin^15,17. However, the affected trafficking process can be rescued by certain treatments^15,17. Low-temperature incubation has been reported to rescue the trafficking of pendrin with p.H723R, which originally accumulated in the endoplasmic reticulum, but not the trafficking of pendrin with p.L236P, which originally accumulated in centrosomal regions¹⁷. Similarly, salicylate treatment could rescue the trafficking of pendrin with p.P123S, p.M147V, p.S657N, and p.H723R, and restore the function of pendrin as an anion exchanger, but not that of pendrin with p.T721M¹⁵. These lines of evidence also support the view that the SLC26A4 p.T721M variant is pathogenic and implies that p.T721M is more pathogenic compared to other missense SLC26A4 variants, such as p.H723R.

To our surprise, the pathogenicity of p.T721M as predicted by the ACMG guidelines and evidenced by the cell line studies was not observed in our mouse model with the Slc26a4 p.T721M variant. These findings are consistent with our previous studies in mouse models with Slc26a4 p.H723R¹⁰ and p.C565Y¹² variants. In the cochlea of mice, pendrin is expressed at the spiral prominence and outer sulcus cells²⁷, which is almost the same as its expression in the cochlea of primates²⁸. Therefore, the position of pendrin expression does not appear to be a major factor contributing to the phenotypic discrepancy between species.

Alternatively, the inter-species phenotypic discrepancy may be attributed to the structural differences between mouse and human pendrin.

To date, five mouse models with missense Slc26a4 variants, including p.L236P¹¹, p.S408F⁸, p.C565Y¹², p.T721M (this study), and p.H723R¹⁰, have been documented in the literature (Fig. 5A). Abnormal audiovestibular phenotypes were observed in mice with p.L236P and p.S408F, but not in mice with p.C565Y, p.T721M, and p.H723R. Notably, p.L236P and p.S408F are located in the transmembrane domains of pendrin^27,28. In contrast, p.C565Y, p.T721M, and p.H723R are located in the C-terminus of pendrin comprised of amino acid residues 508–780^27,28 (Fig. 5B). From an evolutionary perspective, the amino acid sequence of the pendrin C-terminus is less conserved, sharing only 86% identity between mice and humans. In contrast, the amino acid sequence of the transmembrane domains shared 92% identity between the two species (https://www.expasy.org/&https://www.uniprot.org/). Accordingly, we speculate that the absence of phenotypes in mice with Slc26a4 p.C565Y, p.T721M, and p.H723R variants could be attributed to different protein structures at the C-terminus of human and mouse pendrin.

Five documented murine missense Slc26a4 variants. Five mouse models with missense Slc26a4 variants, including p.L236P, p.S408F, p.C565Y, p.T721M, and p.H723R, have been documented in the literature. (A) Scheme of the genomic DNA. Squares represent exons and lines represent introns. (B) Scheme of the 12-transmembrane domain model of pendrin. The five missense variants are indicated with stars, with p.L236P indicated in green, p.S408F in purple, p.C565Y in orange, p.T721M in red, and p.H723R in blue. L236P and p.S408F are located in the transmembrane domains, whereas p.C565Y, p.T721M, and p.H723R are located at the C-terminus of pendrin.

Another line of evidence that supports our speculation is the hH723R Tg mouse model¹⁴. Instead of creating the variant on murine genomic DNA, the authors delivered a sequence of human cDNA harboring the p.H723R variant into pronuclear-stage mouse embryos. This humanized transgenic mouse model revealed profound SNHI¹⁰, suggesting that C-terminus SLC26A4 variants identified in humans might require a human peptide backbone to exhibit their pathogenicity.

Rapp et al. analyzed the locations of SLC26A4 mutations and identified a high density of SLC26A4 mutations in the anion-binding transmembrane domains (TMs), including TM1, 3, 8, and 10²⁹. The findings indicate that when present in these transmembrane domains, SLC26A4 variants are more likely to be pathogenic. This could possibly explain the higher evolutionary conservation of amino acid sequences between human and mouse pendrin at the transmembrane domains, as well as the clear phenotypic manifestation of profound SNHI in mice with Slc26a4 variants at the transmembrane domains, such as Slc26a4^L236P/L236P and Slc26a4^loop/loop mice (i.e., mice with p.S408F).

In summary, using a genotype-driven approach, we generated a knock-in mouse model segregating the deafness-associated SLC26A4 p.T721M variant in humans. Surprisingly, mice with the Slc26a4 p.T721M variant exhibited a normal audiovestibular phenotype and inner ear morphology. Because there might be differences in the pathogenicity of specific SLC26A4 variants between humans and mice, caution is needed while extrapolating the results of animal studies to humans.