A Novel SLC26A4 Frameshift Variant Is Associated with Reduced Mutant-Allele RNA Abundance in Blood Leukocytes

A Novel SLC26A4 Frameshift Variant Is Associated with Reduced Mutant-Allele RNA Abundance in Blood Leukocytes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Novel SLC26A4 Frameshift Variant Is Associated with Reduced Mutant-Allele RNA Abundance in Blood Leukocytes Yuan Zhan, Hongyan Jiang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8954141/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A novel SLC26A4 mutation, c.997_998insA, was identified in a 4-year-old child presenting with classic enlarged vestibular aqueduct (EVA). This single-nucleotide insertion causes a frameshift and introduces a premature termination codon (PTC), suggesting a potential role for nonsense-mediated mRNA decay (NMD). Although AlphaGenome predicted no appreciable reduction in RNA abundance, pyrosequencing demonstrated a significant decrease in the transcript level of the mutant allele compared with the wild-type allele in peripheral blood from the proband’s mother. These results indicate that the pathogenic mechanism of this SLC26A4 variant may arise primarily at the post-transcriptional (RNA regulation) level, rather than solely through altered protein function. Biological sciences/Genetics Biological sciences/Molecular biology Nonsense-mediated mRNA decay SLC26A4 hereditary hearing loss Figures Figure 1 Figure 2 Figure 3 Introduction A significant proportion of the proteins encoded by deafness-related genes are ion channels, e.g. Pendrin, also known as sodium-independent chloride/iodide transporter, that is encoded by the SLC26A4 gene. 1 – 3 SLC26A4 is the second most frequent gene involved in the hereditary hearing loss population which mainly expresses in inner ear, thyroid and proximal renal tubule. 4 It has been considered as the gold standard for deciding the pathogenicity of the SLC26A4 mutations, as well as other membrane protein encoding genes, that constructing a mutation-specific cDNA clone in 293T or other cell strains, then observing whether the expressed protein is capable to exactly arrive at the cell membrane or keep the permeability completeness of the ion channel that protein form. 5–7 However, strictly speaking, it is possible that although the protein encoded by the gene is inherently functional, issues at the RNA level may prevent the DNA—rather than the cDNA—from being fully expressed as the corresponding protein. Nonsense mediated mRNA decay (NMD) is a post-transcript regulation mechanism. 8 According to current understanding of NMD, if a mutation introduces a premature termination codon (PTC) in a mRNA locating more than 55 nucleotides upstream of the final splice junction, the presence of a downstream exon junction complex (EJC)— combined to mRNA while splice —will trigger NMD during the pioneer round of translation. 9 By NMD, the PTC-containing mRNA is eliminated and the truncated protein can't be translation. 10 So, if a mutation introduces a PTC, the cDNA constructed by site directed mutagenesis can't simulate the real condition in vivo. Compared to abnormal protein function, NMD is also an important pathogenic mechanism. Here, a significant reduction in RNA abundance from the mutant allele was observed in leukocytes from a heterozygous carrier with a novel insertion mutation. Results Clinical phenotype of the proband Examinations showed that the proband, the 4-year-old girl, had no external or middle malformations and no other systemic malformations. Auditory steady-state evoked response (ASSR) and auditory brainstem response (ABR) indicated no obvious potential was evoked until the largest sound intensity. Standard temporal CT was performed with contiguous 1.0 mm scans and revealed both Mondini malformation and enlarged vestibular aqueduct (EVA) which is a hallmark of SLC26A4 mutation 11 . (Fig. 1). Thyroid function tests and thyroid ultrasonography are within normal ranges, except for a 0.2 cm × 0.2 cm cyst detected in the right lobe. The thyroid function results are as follows: free T3: 5.480 pmol/L (normal range: 2.630–5.700), free T4: 15.490 pmol/L (normal range: 9.010–19.050), and TSH: 2.212 mIU/L (normal range: 0.350–4.940). Identification of a Novel SLC26A4 Variant and Segregation Analysis The genomic DNA analysis of the girl's peripheral blood confirmed the presence of an SLC26A4 mutation. This included a compound heterozygosity of c.997_998insA and c.919-2A > G, the latter being the most frequent mutation in the Chinese SLC26A4 mutation population 12 . We searched the databases TGP (The 1000 Genomes Project), dbSNP (Single Nucleotide Polymorphism Database), HGMD (Human Gene Mutation Database), and the Deafness Variation Database (DVD). No record of c.997_998insA was found, indicating that it is a novel mutation. The two mutations were verified by DNA analysis of her parents’ leukocytes, revealing that c.919-2A > G originated from her father and c.997_998insA from her mother. The Sanger sequencing chromatogram of the mother's DNA is shown in Fig. 2A. Mutant-Allele RNA Abundance in the Proband’s Mother AlphaGenome was used to predict the effect of the c.997_998insA variant. 13 Because cochlear-relevant tissues are not included in the AlphaGenome ontology datasets, thyroid tissue and venous blood were selected as proxy tissues for the prediction. Figure 3 shows that this variant is predicted to have no effect on SLC26A4 transcript expression in these tissues. The analysis code is provided in the Supplementary File. However, the Sanger sequencing of the RT-PCR fragment spanning exons 8 and 9, which include the 997 and 998 loci, showed that the mutant transcript peaks were lower compared with those of the normal transcript (Fig. 2B). Pyrosequencing of the same RT-PCR amplicon further indicated that mutant transcripts were 40.0 ± 5.9% lower than the normal transcripts (Fig. 2C and Fig. 2D). Discussion In this study, we show that a single-base insertion in SLC26A4 leads to a marked decline in mutant-allele transcript abundance, an effect that was not predicted by the AI-based tool AlphaGenome. A likely reason for this prediction miss is that AlphaGenome’s training data and modeling focus are enriched for noncoding regulatory variation, which may limit sensitivity to coding variants that reduce mRNA abundance 13 . Given that this frameshift variant introduces a downstream premature termination codon (PTC), nonsense-mediated mRNA decay (NMD) is the most likely explanation 14 , 15 . In other studies on NMD, disease-related cells such as fibroblasts and cartilage cells derived from skin biopsies or surgeries were cultured and treated with cycloheximide, a translation inhibitor that binds to the E-site of the 60S ribosomal subunit. Cycloheximide severely disrupts the translocation step, thereby suppressing NMD during the pioneer round of translation. Based on these studies, two main observations were made: (1) in cells treated with cycloheximide, NMD activity is restricted; (2) mutant transcripts in leukocytes undergo less efficient NMD compared to disease-related cells, indicating that NMD exhibits tissue specificity across different diseases 16 – 18 . Given that the SLC26A4 gene primarily functions in the inner ear, thyroid, and proximal renal tubule, we cannot directly apply the same experimental approaches as described above. However, drawing on these findings, we speculate that NMD may function more effectively in the patient’s inner-ear-derived cells, resulting in a complete degradation of mutant transcripts. Currently, the mainstream approach for gene therapy in hearing loss involves delivering target genes into the inner ear using viral vectors, most commonly adeno-associated viruses (AAV), enabling the expression of functional proteins to restore hearing 19 – 21 . However, the neutralizing antibodies acquired from the wild-type AAV transfection can act against recombinant AAV in the circulatory system, and the cytotoxic T lymphocyte response induced by CD8 T cells can destroy successfully transfected target cells 22 , 23 . These effects can potentially reduce the efficacy of AAV-mediated gene therapy. Therefore, many clinical studies exclude individuals with high serum levels of AAV-specific antibodies 21 , 24 . Here, the translational readthrough-inducing drugs (TRIDs) provide another potential option for individuals with nonsense-mediated mRNA decay (NMD) 25 . At present, TRIDs mainly include aminoglycosides and ataluren. Aminoglycosides, which are interestingly regarded as ototoxic drugs, promote readthrough by increasing near-cognate transfer RNA mispairing during translation 26 , 27 . Ataluren, identified through high-throughput screening, stimulates readthrough by inhibiting release factor activity, which is essential for termination of protein synthesis 28 , 29 . These two drugs and their derivatives, especially the latter, have demonstrated remarkable ability to restore the expression of functional proteins in Duchenne muscular dystrophy (DMD) and cystic fibrosis nonsense mutation models 30 – 32 . To summarize, by analyzing a novel mutation, we demonstrate for the first time that NMD occurs in a SLC26A4 mutant carrier's leukocytes. Considering nonsense and frame shift mutation account for about 40% in GJB2 and 20% in SLC26A4 mutations and most of them could give rise to PTCs, it is attractive to explore what is the role played by NMD in the molecular mechanism of hereditary hearing loss. Methods Case ascertainment and clinical examination A family of three visit our clinic for genetic consulting because their first 4-year-old daughter was born with severe sensorineural hearing loss. The girl received a cochlear implant on one side a year ago. Before the cochlear implantation, she received a complete physical examination along with an audiological examination, including otoscopy, middle ear analysis, Auditory Brainstem Response (ABR), and Auditory Steady-State Response (ASSR). Standard temporal CT was performed with contiguous 1.0 mm scans before cochlear implantation. This time, her family history of hearing loss, including her parents, was also recorded. Since SLC26A4 mutation potentially involves abnormal thyroid function (Pendred syndrome, OMIM#: 274600), thyroid function, thyroid ultrasonography was measured. This study was reviewed and approved by the Institutional Review Board of The First Affiliated Hospital, Sun Yat-sen University. Written informed consent was obtained from the child’s parents, and the parents also provided consent for their own participation. All study procedures were carried out in accordance with the principles of the Declaration of Helsinki. DNA test The girl's peripheral blood DNA was extracted to test the GJB2, SLC26A4, and mitochondrial genes by Sanger sequencing. Her mother and father's peripheral blood DNA were also collected to verify the SLC26A4 mutation by Sanger sequencing. RNA test After the heterozygous carrier, the proband’s mother, had her peripheral blood drawn, the RNA it contained was immediately extracted to prevent possible degradation. blood RNA was obtained by acid guanidinium thiocyanate–phenol–chloroform extraction. 33 Then, the RNA was reversed to cDNA by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). The cDNA region containing the mutation site was then amplified by Polymerase chain reaction (PCR). To achieve more precise quantification and comparison of normal and mutant transcripts, the RT-PCR fragment was quantified using pyrosequencing on the PyroMark Q96 ID System (Qiagen, Germany). The experiments were performed 3 times independently. Declarations Competing interests The authors declare no competing interests. Funding The study was supported by grants from the National Natural Science Fundation of China (No.81271076) Author Contribution Y.Z. conceived and designed the study, performed the experiments, analyzed and interpreted the data, and drafted the manuscript. H.Y.J. conceived and designed the study, reviewed the manuscript. Acknowledgement We would like to thank the proband and her parents for their participation and for providing samples for this study. Data availability All relevant data are included in the article and its Supplementary Materials. The variant has been submitted to ClinVar under accession number SCV000115341.2. Author Information Yuan Zhan Hongyan Jiang Corresponding author Corresponding author: Yuan Zhan Email: [email protected] References Kelsell, D. P. et al. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387 , 80–83 (1997). Hilgert, N., Smith, R. J. H. & Van Camp, G. Forty-six genes causing nonsyndromic hearing impairment: Which ones should be analyzed in DNA diagnostics? Mutat. Research/Reviews Mutat. Res. 681 , 189–196 (2009). Everett, L. A. et al. Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nat. Genet. 17 , 411–422 (1997). Mount, D. B. & Romero, M. F. The SLC26 gene family of multifunctional anion exchangers. Pflugers Arch. 447 , 710–721 (2004). Dossena, S. et al. Functional characterization of wild-type and mutated pendrin (SLC26A4), the anion transporter involved in Pendred syndrome. J. Mol. Endocrinol. 43 , 93–103 (2009). Yoon, J. S. et al. Heterogeneity in the processing defect of SLC26A4 mutants. J. Med. Genet. 45 , 411–419 (2008). Brownstein, Z. N. et al. A novel SLC26A4 (PDS) deafness mutation retained in the endoplasmic reticulum. Arch. Otolaryngol. Head Neck Surg. 134 , 403–407 (2008). Baker, K. E. & Parker, R. Nonsense-mediated mRNA decay: terminating erroneous gene expression. Curr. Opin. Cell. Biol. 16 , 293–299 (2004). Le Hir, H. & Séraphin, B. EJCs at the heart of translational control. Cell 133 , 213–216 (2008). Maquat, L. E. Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nat. Rev. Mol. Cell. Biol. 5 , 89–99 (2004). Reardon, W., OMahoney, C. F., Trembath, R., Jan, H. & Phelps, P. D. Enlarged vestibular aqueduct: a radiological marker of pendred syndrome, and mutation of the PDS gene. QJM 93 , 99–104 (2000). Hu, H. et al. Molecular analysis of hearing loss associated with enlarged vestibular aqueduct in the mainland Chinese: a unique SLC26A4 mutation spectrum. J. Hum. Genet. 52 , 492–497 (2007). Avsec, Ž. et al. Advancing regulatory variant effect prediction with AlphaGenome. Nature 649 , 1206–1218 (2026). Chang, J. C. & Kan, Y. W. beta 0 thalassemia, a nonsense mutation in man. Proc. Natl. Acad. Sci. U S A . 76 , 2886–2889 (1979). Losson, R. & Lacroute, F. Interference of nonsense mutations with eukaryotic messenger RNA stability. Proc. Natl. Acad. Sci. U S A . 76 , 5134–5137 (1979). Furniss, D., Critchley, P., Giele, H. & Wilkie, A. O. M. Nonsense-mediated decay and the molecular pathogenesis of mutations in SALL1 and GLI3. Am. J. Med. Genet. A . 143A , 3150–3160 (2007). Magyar, I. et al. Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes. Hum. Mutat. 30 , 1355–1364 (2009). Bateman, J. F., Freddi, S., Nattrass, G. & Savarirayan, R. Tissue-specific RNA surveillance? Nonsense-mediated mRNA decay causes collagen X haploinsufficiency in Schmid metaphyseal chondrodysplasia cartilage. Hum. Mol. Genet. 12 , 217–225 (2003). Al-Moyed, H. et al. A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice. EMBO Mol. Med. 11 , e9396 (2019). Akil, O. et al. Dual AAV-mediated gene therapy restores hearing in a DFNB9 mouse model. Proc. Natl. Acad. Sci. U S A . 116 , 4496–4501 (2019). Lv, J. et al. AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial. Lancet 403 , 2317–2325 (2024). Louis Jeune, V., Joergensen, J. A., Hajjar, R. J. & Weber, T. Pre-existing anti-adeno-associated virus antibodies as a challenge in AAV gene therapy. Hum. Gene Ther. Methods . 24 , 59–67 (2013). Mingozzi, F. et al. CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat. Med. 13 , 419–422 (2007). Qi, J. et al. AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness. Adv. Sci. (Weinh) . 11 , e2306788 (2024). Nagel-Wolfrum, K., Möller, F., Penner, I., Baasov, T. & Wolfrum, U. Targeting Nonsense Mutations in Diseases with Translational Read-Through-Inducing Drugs (TRIDs). BioDrugs 30, 49–74 (2016). Bidou, L., Bugaud, O., Belakhov, V., Baasov, T. & Namy, O. Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells. RNA Biol. 14 , 378–388 (2017). Prokhorova, I. et al. Aminoglycoside interactions and impacts on the eukaryotic ribosome. Proc. Natl. Acad. Sci. U S A . 114 , E10899–E10908 (2017). Welch, E. M. et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 447 , 87–91 (2007). Ng, M. Y., Li, H., Ghelfi, M. D., Goldman, Y. E. & Cooperman, B. S. Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms. Proc. Natl. Acad. Sci. U S A . 118 , e2020599118 (2021). Xue, X. et al. Synthetic Aminoglycosides Efficiently Suppress Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations and Are Enhanced by Ivacaftor. Am. J. Respir Cell. Mol. Biol. 50 , 805–816 (2014). Amar-Schwartz, A. et al. Inhibition of nonsense-mediated mRNA decay may improve stop codon read-through therapy for Duchenne muscular dystrophy. Hum. Mol. Genet. 32 , 2455–2463 (2023). Fiduccia, I. et al. Promoting readthrough of nonsense mutations in CF mouse model: Biodistribution and efficacy of NV848 in rescuing CFTR protein expression. Mol. Ther. 32 , 4514–4523 (2024). Chomczynski, P. & Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162 , 156–159 (1987). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8954141","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":597011349,"identity":"55e23313-3db3-49bf-bb8b-9520896c3a33","order_by":0,"name":"Yuan Zhan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYDACCRDxw4aBD0QnwER5CGlh7EljYCNNCwPbYYgWOMCnRX5287PHPDzn5dkkstMePGCoS5w/I4Hxwds2BnlzHFoY5xwzN+axuG3YJpG73SCB4XDihhsJzIZz2xgMdzZg18IskWAmzcNzmxGoZZtEAsOBxA0SCWzSvG0MCQYHsGthk0j/Js3Dds4eqgXsMPbf+LTwSOQAbWE7kAjVwpzYcCOBjRmfFgmJnDLJuT3JyW08b4FaDA4bbzjzsFlyzjkJww04tMjPSN8m8eaHnW0/e+42yR8VdbLz25MPfnhTZiOPyxYQYELEggGDYwMDYwMDNL5wAsYfSBx7vEpHwSgYBaNgRAIAA2FUGkG/u5oAAAAASUVORK5CYII=","orcid":"","institution":"First Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":true,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Zhan","suffix":""},{"id":597011350,"identity":"6cc21951-350d-4a1b-83c5-95b75a9569e8","order_by":1,"name":"Hongyan Jiang","email":"","orcid":"","institution":"Hainan General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongyan","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2026-02-24 07:39:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8954141/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8954141/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103602398,"identity":"524b6e97-43f3-4331-b9ef-5fddcf61cacd","added_by":"auto","created_at":"2026-02-27 14:14:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":679267,"visible":true,"origin":"","legend":"\u003cp\u003eLeft temporal bone scan of 4-year-old girl with hearing loss. (A)Axial view shows the enlarged vestibular adequate(arrow). (B)Coronal section, level of cochlear apex, indicate classic Mondini deformity, that absence of the osseous spiral lamina and confluence of the apical and middle turns of the cochlear(arrow).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8954141/v1/176b0b92058962a152f4b684.png"},{"id":103602395,"identity":"bd391982-84dc-4e7f-b3c4-aa8a19795759","added_by":"auto","created_at":"2026-02-27 14:14:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4617421,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing analysis of the heterozygous SLC26A4 mutation which the mother carries(A)heterozygous c.997_998insA/R333Kfs7X was indentified in DNA level by Sanger sequencing. (B) Heterozygous c.997_998insA allele-specific cDNA was show lower peak height than the normal one and a stop codon (TGA) generates downstream. The top row in the sequencing chart shows the normal coding sequence, while the bottom row shows the frameshift mutation coding sequence. The sequence in the shadow is analyzed by pyrosequencing. (C)The pyrogram of the cDNA pyrosequencing. In pyrosequencing, the designed base sequences indicated at the bottom of the pyrogram are artificially added to the text library one by one. In the yellow box, the mutant allele-specific peaks are on the right of the vertical line and the wild-type allele-specific is on the left. The first high G peak, the first high T peak, and the subsequent G peak represent the normal coding sequence GGGGGTTTTTG on one allele, while the following A peak, G peak, T peak, and G peak represent the frameshift mutation sequence AGGGGGTTTTTG on the other allele. The G peak with arrow is used for quantification. The final C peak, T peak, and C peak represent the common CCTCC sequence in both alleles. (D)The pyrosequencing result of the control cDNA. (wild-type SLC26A4)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8954141/v1/14c1769eb091d9a4a4c296b6.png"},{"id":103602397,"identity":"88e53885-c871-4ee6-824b-d98e50932bf0","added_by":"auto","created_at":"2026-02-27 14:14:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1261599,"visible":true,"origin":"","legend":"\u003cp\u003eAlphaGenome-predicted impact of SLC26A4 c.997_998insA on transcript expression in thyroid and whole blood (A) Thyroid tissue. The alternative (ALT) allele corresponding to SLC26A4 c.997_998insA (an insertion of base 'A' at chr7:107,683,532, hg38) is located on the positive strand of chromosome 7. The predicted RNA signal for the ALT allele largely overlaps with that of the reference (REF) allele, predicating no reduction in transcript abundance and no exon-skipping events. (B) Whole blood. Similarly, the predicted RNA profiles of the ALT and REF alleles overlap, predicting no change in SLC26A4 transcript abundance in whole blood. Only polyadenylated (polyA+) mRNA is represented in the prediction.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8954141/v1/2ff1fbfdef05e596fceba591.png"},{"id":104399724,"identity":"a87e18a5-145c-4ada-a702-98a9b550c613","added_by":"auto","created_at":"2026-03-11 12:07:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6820161,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8954141/v1/7c0e6645-d2f9-4b09-8254-10f765de928e.pdf"},{"id":103602396,"identity":"a9a0fc0d-74a9-4244-877b-00bb25a89c8f","added_by":"auto","created_at":"2026-02-27 14:14:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":111865,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8954141/v1/f210ddeacda7750fedb08a5a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel SLC26A4 Frameshift Variant Is Associated with Reduced Mutant-Allele RNA Abundance in Blood Leukocytes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA significant proportion of the proteins encoded by deafness-related genes are ion channels, e.g. Pendrin, also known as sodium-independent chloride/iodide transporter, that is encoded by the SLC26A4 gene.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e SLC26A4 is the second most frequent gene involved in the hereditary hearing loss population which mainly expresses in inner ear, thyroid and proximal renal tubule.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e It has been considered as the gold standard for deciding the pathogenicity of the SLC26A4 mutations, as well as other membrane protein encoding genes, that constructing a mutation-specific cDNA clone in 293T or other cell strains, then observing whether the expressed protein is capable to exactly arrive at the cell membrane or keep the permeability completeness of the ion channel that protein form. \u003csup\u003e5\u0026ndash;7\u003c/sup\u003e However, strictly speaking, it is possible that although the protein encoded by the gene is inherently functional, issues at the RNA level may prevent the DNA\u0026mdash;rather than the cDNA\u0026mdash;from being fully expressed as the corresponding protein.\u003c/p\u003e\u003cp\u003eNonsense mediated mRNA decay (NMD) is a post-transcript regulation mechanism.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e According to current understanding of NMD, if a mutation introduces a premature termination codon (PTC) in a mRNA locating more than 55 nucleotides upstream of the final splice junction, the presence of a downstream exon junction complex (EJC)\u0026mdash; combined to mRNA while splice \u0026mdash;will trigger NMD during the pioneer round of translation.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e By NMD, the PTC-containing mRNA is eliminated and the truncated protein can't be translation.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e So, if a mutation introduces a PTC, the cDNA constructed by site directed mutagenesis can't simulate the real condition in vivo. Compared to abnormal protein function, NMD is also an important pathogenic mechanism.\u003c/p\u003e\u003cp\u003eHere, a significant reduction in RNA abundance from the mutant allele was observed in leukocytes from a heterozygous carrier with a novel insertion mutation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eClinical phenotype of the proband\u003c/h2\u003e \u003cp\u003eExaminations showed that the proband, the 4-year-old girl, had no external or middle malformations and no other systemic malformations. Auditory steady-state evoked response (ASSR) and auditory brainstem response (ABR) indicated no obvious potential was evoked until the largest sound intensity. Standard temporal CT was performed with contiguous 1.0 mm scans and revealed both Mondini malformation and enlarged vestibular aqueduct (EVA) which is a hallmark of SLC26A4 mutation \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. (Fig.\u0026nbsp;1). Thyroid function tests and thyroid ultrasonography are within normal ranges, except for a 0.2 cm \u0026times; 0.2 cm cyst detected in the right lobe. The thyroid function results are as follows: free T3: 5.480 pmol/L (normal range: 2.630\u0026ndash;5.700), free T4: 15.490 pmol/L (normal range: 9.010\u0026ndash;19.050), and TSH: 2.212 mIU/L (normal range: 0.350\u0026ndash;4.940).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of a Novel SLC26A4 Variant and Segregation Analysis\u003c/h2\u003e \u003cp\u003eThe genomic DNA analysis of the girl's peripheral blood confirmed the presence of an SLC26A4 mutation. This included a compound heterozygosity of c.997_998insA and c.919-2A\u0026thinsp;\u0026gt;\u0026thinsp;G, the latter being the most frequent mutation in the Chinese SLC26A4 mutation population \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. We searched the databases TGP (The 1000 Genomes Project), dbSNP (Single Nucleotide Polymorphism Database), HGMD (Human Gene Mutation Database), and the Deafness Variation Database (DVD). No record of c.997_998insA was found, indicating that it is a novel mutation. The two mutations were verified by DNA analysis of her parents\u0026rsquo; leukocytes, revealing that c.919-2A\u0026thinsp;\u0026gt;\u0026thinsp;G originated from her father and c.997_998insA from her mother. The Sanger sequencing chromatogram of the mother's DNA is shown in Fig.\u0026nbsp;2A.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMutant-Allele RNA Abundance in the Proband’s Mother\u003c/h3\u003e\n\u003cp\u003eAlphaGenome was used to predict the effect of the c.997_998insA variant.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Because cochlear-relevant tissues are not included in the AlphaGenome ontology datasets, thyroid tissue and venous blood were selected as proxy tissues for the prediction. Figure\u0026nbsp;3 shows that this variant is predicted to have no effect on SLC26A4 transcript expression in these tissues. The analysis code is provided in the Supplementary File. However, the Sanger sequencing of the RT-PCR fragment spanning exons 8 and 9, which include the 997 and 998 loci, showed that the mutant transcript peaks were lower compared with those of the normal transcript (Fig.\u0026nbsp;2B).\u003c/p\u003e \u003cp\u003ePyrosequencing of the same RT-PCR amplicon further indicated that mutant transcripts were 40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9% lower than the normal transcripts (Fig.\u0026nbsp;2C and Fig.\u0026nbsp;2D).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we show that a single-base insertion in SLC26A4 leads to a marked decline in mutant-allele transcript abundance, an effect that was not predicted by the AI-based tool AlphaGenome. A likely reason for this prediction miss is that AlphaGenome\u0026rsquo;s training data and modeling focus are enriched for noncoding regulatory variation, which may limit sensitivity to coding variants that reduce mRNA abundance\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Given that this frameshift variant introduces a downstream premature termination codon (PTC), nonsense-mediated mRNA decay (NMD) is the most likely explanation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn other studies on NMD, disease-related cells such as fibroblasts and cartilage cells derived from skin biopsies or surgeries were cultured and treated with cycloheximide, a translation inhibitor that binds to the E-site of the 60S ribosomal subunit. Cycloheximide severely disrupts the translocation step, thereby suppressing NMD during the pioneer round of translation. Based on these studies, two main observations were made: (1) in cells treated with cycloheximide, NMD activity is restricted; (2) mutant transcripts in leukocytes undergo less efficient NMD compared to disease-related cells, indicating that NMD exhibits tissue specificity across different diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven that the SLC26A4 gene primarily functions in the inner ear, thyroid, and proximal renal tubule, we cannot directly apply the same experimental approaches as described above. However, drawing on these findings, we speculate that NMD may function more effectively in the patient\u0026rsquo;s inner-ear-derived cells, resulting in a complete degradation of mutant transcripts.\u003c/p\u003e \u003cp\u003eCurrently, the mainstream approach for gene therapy in hearing loss involves delivering target genes into the inner ear using viral vectors, most commonly adeno-associated viruses (AAV), enabling the expression of functional proteins to restore hearing\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, the neutralizing antibodies acquired from the wild-type AAV transfection can act against recombinant AAV in the circulatory system, and the cytotoxic T lymphocyte response induced by CD8 T cells can destroy successfully transfected target cells\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These effects can potentially reduce the efficacy of AAV-mediated gene therapy. Therefore, many clinical studies exclude individuals with high serum levels of AAV-specific antibodies\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Here, the translational readthrough-inducing drugs (TRIDs) provide another potential option for individuals with nonsense-mediated mRNA decay (NMD)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. At present, TRIDs mainly include aminoglycosides and ataluren. Aminoglycosides, which are interestingly regarded as ototoxic drugs, promote readthrough by increasing near-cognate transfer RNA mispairing during translation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Ataluren, identified through high-throughput screening, stimulates readthrough by inhibiting release factor activity, which is essential for termination of protein synthesis\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. These two drugs and their derivatives, especially the latter, have demonstrated remarkable ability to restore the expression of functional proteins in Duchenne muscular dystrophy (DMD) and cystic fibrosis nonsense mutation models\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo summarize, by analyzing a novel mutation, we demonstrate for the first time that NMD occurs in a SLC26A4 mutant carrier's leukocytes. Considering nonsense and frame shift mutation account for about 40% in GJB2 and 20% in SLC26A4 mutations and most of them could give rise to PTCs, it is attractive to explore what is the role played by NMD in the molecular mechanism of hereditary hearing loss.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCase ascertainment and clinical examination\u003c/h2\u003e \u003cp\u003eA family of three visit our clinic for genetic consulting because their first 4-year-old\u003c/p\u003e \u003cp\u003edaughter was born with severe sensorineural hearing loss. The girl received a cochlear implant on one side a year ago. Before the cochlear implantation, she received a complete physical examination along with an audiological examination, including otoscopy, middle ear analysis, Auditory Brainstem Response (ABR), and Auditory Steady-State Response (ASSR). Standard temporal CT was performed with contiguous 1.0 mm scans before cochlear implantation. This time, her family history of hearing loss, including her parents, was also recorded. Since SLC26A4 mutation potentially involves abnormal thyroid function (Pendred syndrome, OMIM#: 274600), thyroid function, thyroid ultrasonography was measured. This study was reviewed and approved by the Institutional Review Board of The First Affiliated Hospital, Sun Yat-sen University. Written informed consent was obtained from the child\u0026rsquo;s parents, and the parents also provided consent for their own participation. All study procedures were carried out in accordance with the principles of the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDNA test\u003c/h2\u003e \u003cp\u003eThe girl's peripheral blood DNA was extracted to test the GJB2, SLC26A4, and mitochondrial genes by Sanger sequencing. Her mother and father's peripheral blood DNA were also collected to verify the SLC26A4 mutation by Sanger sequencing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA test\u003c/h3\u003e\n\u003cp\u003eAfter the heterozygous carrier, the proband\u0026rsquo;s mother, had her peripheral blood drawn, the RNA it contained was immediately extracted to prevent possible degradation. blood RNA was obtained by acid guanidinium thiocyanate\u0026ndash;phenol\u0026ndash;chloroform extraction.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Then, the RNA was reversed to cDNA by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). The cDNA region containing the mutation site was then amplified by Polymerase chain reaction (PCR). To achieve more precise quantification and comparison of normal and mutant transcripts, the RT-PCR fragment was quantified using pyrosequencing on the PyroMark Q96 ID System (Qiagen, Germany). The experiments were performed 3 times independently.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was supported by grants from the National Natural Science Fundation of China (No.81271076)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.Z. conceived and designed the study, performed the experiments, analyzed and interpreted the data, and drafted the manuscript. H.Y.J. conceived and designed the study, reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank the proband and her parents for their participation and for providing samples for this study.\u003c/p\u003e\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eAll relevant data are included in the article and its Supplementary Materials. The variant has been submitted to ClinVar under accession number SCV000115341.2.\u003c/p\u003e \n\u003cp\u003e\u003cstrong\u003eAuthor Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuan Zhan \u0026nbsp; Hongyan Jiang\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorresponding author: Yuan Zhan Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKelsell, D. P. et al. 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Biochem.\u003c/em\u003e \u003cb\u003e162\u003c/b\u003e, 156\u0026ndash;159 (1987).\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nonsense-mediated mRNA decay, SLC26A4, hereditary hearing loss","lastPublishedDoi":"10.21203/rs.3.rs-8954141/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8954141/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel SLC26A4 mutation, c.997_998insA, was identified in a 4-year-old child presenting with classic enlarged vestibular aqueduct (EVA). This single-nucleotide insertion causes a frameshift and introduces a premature termination codon (PTC), suggesting a potential role for nonsense-mediated mRNA decay (NMD). Although AlphaGenome predicted no appreciable reduction in RNA abundance, pyrosequencing demonstrated a significant decrease in the transcript level of the mutant allele compared with the wild-type allele in peripheral blood from the proband\u0026rsquo;s mother. These results indicate that the pathogenic mechanism of this SLC26A4 variant may arise primarily at the post-transcriptional (RNA regulation) level, rather than solely through altered protein function.\u003c/p\u003e","manuscriptTitle":"A Novel SLC26A4 Frameshift Variant Is Associated with Reduced Mutant-Allele RNA Abundance in Blood Leukocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 14:14:41","doi":"10.21203/rs.3.rs-8954141/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fdda9628-d199-470b-ab61-820d12df424c","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63528540,"name":"Biological sciences/Genetics"},{"id":63528541,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-02-28T16:09:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 14:14:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8954141","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8954141","identity":"rs-8954141","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}