{"paper_id":"04b0e353-366f-4f63-8074-8baddc5c9259","body_text":"Manuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n1 \n \nHaploinsufficiency of KPNA7 causes otosclerosis, likely due to the 1 \nrelease of import inhibition of PTHrP and the reactivation of 2 \nchondrogenesis in the globuli interossei 3 \n 4 \nTammy Benteau,1† Nelly Abdelfatah,1† Anne Griffin,1 Cindy Penney,1 Pingzhao Hu,2 Susan G. 5 \nStanton,3 Guangju Zhai1, Maxime Maheu4, Terry-Lynn Young1,3.  6 \n 7 \n1Division of Biomedical Sciences, Faculty of Medicine, Memorial University, St. John’s, NL, 8 \nCanada, A1B 3V6; 2Department of Biochemistry and Computer Science, Western University, 9 \nLondon, ON, Canada, N6G 2V4; 3National Centre for Audiology & School of Communication 10 \nSciences and Disorders, Faculty of Health Sciences, Western University, London, ON, Canada 11 \nN6G 1H1, 4École d’Orthophonie et d’Audiologie, Université de Montréal, Montréal, Québec, 12 \nCanada, H3C 3J7.  13 \n 14 \n 15 \n†These authors contributed equally to this work. 16 \n*Correspondence:  tlyoung@mun.ca, Orcid ID 0000-0003-4673-6470 17 \n  18 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n2 \n \nAbstract  19 \n 20 \nOtosclerosis is a genetic bone disorder restricted to the otic capsule and a common cause of 21 \nconductive hearing loss with both familial and sporadic cases. To date, 14 genomic loci (OTSC) 22 \nand four underlying OTSC genes (MEPE, SERPINF1, FOXL1, SMARCA4) have been identified 23 \nin autosomal dominant families. A combined genetic/genomics approach on five affected 24 \nsiblings of Northern European ancestry from the island of Newfoundland, Canada identified a 25 \npremature stop mutation in Karyopherin subunit α7 (KPNA7, c.49C>T, p.R17X). KPNA7 maps 26 \nto OTSC2 (7q22.1) and encodes the newest of the seven-member importin-α family of nuclear 27 \ntransporters and plays a critical role in early embryonic cleavage events and zygotic genome 28 \nactivation. Previous studies reveal that recessive KPNA7 variants cause skeletal abnormalities, 29 \nincluding scoliosis and ocular hypertelorism in two sisters with Partial Corpus Callosum 30 \nAgenesis-Cerebellar Vermis Hypoplasia With Posterior Fosa Cysts Syndrome and more 31 \nrecently, have been implicated in preimplantation embryo arrest (PREMBA) (OMIM 614107). 32 \nInterestingly, KPNA7 is also a maternal factor with an exclusively embryonic role and likely 33 \ninhibits non-classical NLS transport of PTHrP, a known activator of chondrogenesis. We 34 \npropose that KPNA7 haploinsufficiency causes a failure in nuclear transport inhibition of PTHrP 35 \nin the quiescent embryonic cells of the globuli interossei in the otic capsule and re-activates 36 \nchondrogenesis. The KPNA7 discovery provides new insights into the pathogenesis of 37 \notosclerosis and potential for targeted therapies. 38 \n39 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n3 \n \nAuthor Summary 40 \nOtosclerosis is a distinctly human genetic bone disorder of the otic capsule and a major cause of 41 \nprogressive hearing loss in young adults, particularly in females. Even though otosclerosis has 42 \nbeen recognized as a distinct entity for a long time, both its pathogenesis and restriction to the 43 \notic capsule remains a mystery. Here, we use a combined genetic/genomics approach to identify 44 \na premature stop mutation in five affected siblings of Northern European ancestry from the 45 \nisland of Newfoundland, Canada. KPNA7 encodes the newest of the seven-member importin-α 46 \nfamily of nuclear transporters and plays a critical role in early embryonic cleavage events and 47 \nzygotic genome activation. Based on the unique features of the otic capsule, we hypothesize that 48 \nthe premature stop mutation in KPNA7 leads to haploinsufficiency causing a failure in nuclear 49 \ntransport inhibition of PTHrP and reactivates chondrogenesis in the otherwise quiescent 50 \nembryonic cells within the otic capsule. The KPNA7 discovery provides new insights into the 51 \npathogenesis of otosclerosis and potential for targeted therapies. 52 \nIntroduction 53 \n 54 \nOtosclerosis is a uniquely human skeletal disorder restricted to the otic capsule of the temporal 55 \nbone and a common cause of progressive conductive hearing loss (HL) in young adults. An 56 \nautosomal dominant (AD) disease with environmental triggers, otosclerosis is clinically 57 \ncharacterized by abnormal bone deposition in the middle ear, distorting the fine structures of the 58 \nossicular chain and limiting the movement of the stapes bone against the oval window. The 59 \nimmobilization of the stapes bone results in conductive HL as well as sensorineural HL in some 60 \npatients due to abnormal bone growth extending into the fluid-filled inner ear (1). The location 61 \nof the inner ear within the osseous labyrinth greatly impacts perilymph sampling for diagnostic 62 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n4 \n \npurposes and local drug delivery. Visualization during surgery to replace or repair stapes fixation 63 \ndue to otosclerosis validates the diagnosis, and in most cases, restores the conductive component 64 \nof hearing to those able to access stapes surgical prosthesis replacement. Otosclerosis risk factors 65 \ninclude positive family history, sex (female), measles and pregnancy (1, 2). Recognised as a 66 \nmedical entity some 125 years ago (3), this restricted bony disorder cannot be predicted, stopped 67 \nor medically treated, and its pathogenesis remains a mystery. 68 \n 69 \nDespite several decades of research efforts, the otosclerosis (OTSC) genes have been recalcitrant 70 \nto discovery because of the genetically heterogeneous nature of otosclerosis and the rarity of AD 71 \nfamilies under study (2). So far, 14 distinct (OTSC1-14) loci have been mapped in AD families 72 \nand four causative OTSC genes (MEPE, SERPINF1, FOXL1, SMARCA4) identified (Hereditary 73 \nHearing Loss Homepage). A recent search for susceptibility factors involving 3504 otosclerosis 74 \ncases from three biobank studies revealed 23 novel loci linked to genes whose dysregulation in 75 \nbone remodeling and mineralization causes rare monogenic skeletal disorders (4). These near 76 \nprotein associations provide insight into the nature of the highly penetrant OTSC genes but not 77 \ntheir identity, as GWAS studies exclude rare variants by design. Given the high genetic 78 \nheterogeneity underlying skeletal dysplasias, with 461 genes known to cause monogenic forms, 79 \nmore OTSC genes are anticipated (5). 80 \n 81 \nThe otic capsule has a highly complex anatomy, and its embryonic development is one of the 82 \nmost complicated examples of cellular morphogenesis in any biologic system (6). In temporal 83 \nbone, the inner ear tissues and spaces are enclosed within the bony otic capsule, the hardest bone 84 \nin the body, a critical feature essential to maintaining hearing integrity (7). The otic capsule 85 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n5 \n \nforms through endochondral ossification, one of two essential pathways of bone formation that 86 \nuses cartilage as a bone template during fetal development. Mesenchymal stem cells differentiate 87 \ninto chondrocytes (cartilage cells) which proliferate rapidly, hypertrophy and secrete the 88 \nextracellular matrix that undergoes mineralization. Eventually the hypertrophic chondrocytes die 89 \nthrough apoptosis and are replaced by osteocytes that become trapped in bony matrix. Although 90 \nthe otic capsule is fully formed by the fifth fetal month (8), islands of embryonic tissue 91 \ncontaining quiescent chondrocytes and osteocytes, known as the globuli interossei, are uniquely 92 \nretained by the otic capsule and persist throughout life. These embryonic remnants are 93 \nincreasingly implicated as the site of otosclerosis in the temporal bone (9-11). 94 \n 95 \nThe human skeleton continues to grow and repair (remodel) postnatally via the well-studied 96 \nRANK/RANKL/OPG pathway. In contrast, the otic capsule and the ossicular chain are fully 97 \nformed in utero and remodeling is virtually absent due to the overproduction of OPG 98 \n(osteoprotegerin) (12). In the bony lacunae of the otic capsule, osteocytes communicate via an 99 \nintercellular canalicular network that provides nutrient transport and bathes the perilacunar 100 \nmatrix with OPG. As a normal part of aging, osteocytes die but are not replaced as OPG prevents 101 \nosteoclasts from maturing and beginning the remodeling process (13). How bone remodeling in 102 \nthe otic capsule occurs in the presence of overproduction of OPG close to the inner ear is a 103 \nmystery, and suggests other parallel protective mechanisms must play a role (13, 14). 104 \n 105 \nHerein we identify a stop mutation in KPNA7 (Karyopherin subunit α7), the newest member of 106 \nthe Importin-α (Imp-α) transport factors involved in nucleocytoplasmic trafficking, in a white 107 \nfamily of Northern European descent. Nucleocytoplasmic trafficking is a highly efficient and 108 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n6 \n \nregulated system comprising of 60 proteins of the nuclear transport system where dysregulation 109 \nis linked to major diseases such as cancer, viral infections, inflammation and neurodegenerative 110 \ndiseases, making them prime targets for therapies (15). Expression of KPNA7 is highly restricted 111 \nto oocytes and early embryogenesis where recessive mutations cause congenital skeletal 112 \nabnormalities (16) and preimplantation embryo arrest (PREMBA) (17). Perhaps most significant 113 \nto otosclerosis, Imp-αs are known to inhibit the nuclear import of parathyroid related protein 114 \n(PTHrP), a major regulator of chondrogenesis (18). We explore how embryonically expressed 115 \ngenes such as KPNA7 may cause adult-onset HL and garner insights into the pathobiology of 116 \notosclerosis and why the rest of the human skeleton is likely spared from abnormal bone 117 \ndeposition and disease. 118 \nResults 119 \nClinical recruitment, pedigree structure and classification of hearing loss 120 \nThe proband (PID III-1) was diagnosed at 42 with severe HL due to otosclerosis in both ears, 121 \nwhich began in teenage years. Pre-surgery audiogram revealed bilateral, conductive HL with 122 \nborderline cochlear (sensorineural) loss. Hearing improved bilaterally after two successive 123 \nstapedectomies (Fig 1). The diagnosis of otosclerosis was confirmed upon surgical visualization 124 \nof stapes fixation. Based on the medical questionnaires, HL started in the teens for all but one 125 \nsibling who noticed HL in adulthood, and hearing was restored in all siblings after stapedectomy 126 \nsurgery. The pedigree structure is consistent with both AD and autosomal recessive (AR) 127 \ninheritance, but X-linked inheritance can be ruled out as otosclerosis is not more severe in males. 128 \nHaplotype sharing on chromosome 7q and targeted gene sequencing 129 \nWhen testing for linkage to mapped OTSC loci and recapitulating disease-associated haplotypes, 130 \nwe would expect, under an AD model, to observe a single otosclerosis-associated haplotype that 131 \nis shared exclusively among affected family members. Conversely, under an AR model, we 132 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n7 \n \nwould expect all affected to share the same maternal and paternal disease haplotypes. Although 133 \nwe have limited clinical data on the paternal side, under the AD model, we observed a shared 134 \npaternal disease haplotype encompassing OTSC2 (7q) (Fig 2). Conversely, no sharing was 135 \nobserved for OTSC1 (15q), OTSC3 (6p), OTSC4 (16q), OTSC5 (3q), OTSC7 (6q), OTSC8 (9p) 136 \nor the COL1A1 (17q) and NOG (17q) genomic regions. Subsequent targeted gene sequencing of 137 \n11 positional candidate genes within the disease interval on 7q did not identify the otosclerosis 138 \ngene. The proband also screened negative for the 15-base pair (bp) deletion in FOXL1 139 \n(rs764026385; OTSC11) that we previously identified in a Newfoundland family, and for rare 140 \notosclerosis variants in SERPINF1. 141 \nGenome wide analysis under AR linked regions 142 \nAlthough there was evidence for haplotype sharing on 7q, targeted gene sequencing did not 143 \nidentify the gene. Under an AR model, linkage simulation derived the theoretical maximum 144 \nLOD score (LODmax=2.5) given the pedigree structure. SNP genotyping and multipoint linkage 145 \nanalysis yielded LODobs=2.5 at chr17q25.1-q25.3, spanning a region of 5.9 Mb. Exome 146 \nsequencing of the 141 positional candidate genes revealed zero homozygous variants and 49 147 \nheterozygous variants. Only two genes, TEN1 and EVPL, had two or more variants consistent 148 \nwith AR inheritance however, these were filtered out due to high population frequencies and/or 149 \nbenign functional predictions (19).  150 \nGenotype wide analysis under AD linked regions 151 \nAssuming AD inheritance, the LODmax (1.73) was not obtained but suggestive LOD (LOD > 1) 152 \nscores were observed at five distinct genomic loci (7q, 10p, 10q, 16q, 17q). Several of these 153 \noverlapped with OTSC disease intervals, specifically OTSC2 (7q), OTSC4 (16q), NOG ( 17q). 154 \nWe identified 542 positional candidate genes under the five suggestive loci. Exome sequencing 155 \nyielded 301 variants and of these,153 variants were filtered out as they were not identified in all 156 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n8 \n \naffected. We could remove 37 variants as they were also identified in \"solved\" FOXL1 cases. Of 157 \nthe remaining variants, 69 had MAF >2%, reducing the variants of interest to 23 silent, 15 158 \nmissense, one intronic and one nonsense. The 15 missense mutations were all predicted to be 159 \nbenign, and 16 (15 silent, one intronic) were predicted to have no effect on splicing. Only the 160 \nnonsense mutation in KPNA7 gene on 7q (OTSC2) remained after variant filtering.  161 \nSanger validation, cascade sequencing and in silico analyses 162 \nKPNA7, c.49 C>T (NM_001145715.3) is rare (0.015%, gnomAD, rs746784660) and has been 163 \nreported in ClinVar as a VUS (ID: 652650). We found that KPNA7, c.49 C>T is absent in 187 164 \nHL probands and in controls. Cascade sequencing confirmed co-segregated with otosclerosis 165 \n(Fig 2). KPNA7, c.49 C>T (NM_001145715.3) is located near the 5' end of KPNA7 and is 166 \npredicted to cause premature stop mutation, p. R17X, subjecting the truncated RNA transcript to 167 \nnonsense mediated decay and resulting in haploinsufficiency (Alamut Visual Plus, version 1.13, 168 \nSophia Genetics (2024)). Previously, recessive mutations in KPNA7 cause congenital skeletal 169 \nabnormalities (16) and preimplantation embryo arrest (PREMBA) (17). We conclude that 170 \nKPNA7, c.49 C>T, p. R17X is pathogenic according to HL ACMG criteria PVS1 and PM2 (20, 171 \n21). 172 \nDiscussion 173 \n 174 \nSummary of findings 175 \nWe identify KPNA7, the newest of the human Imp-α transport factors, as the first of the nuclear 176 \ntransport system of proteins to cause AD otosclerosis. Surgical reports in a white multiplex 177 \nfamily of Northern European extraction confirmed the diagnosis and affected siblings reported 178 \nHL as young adults. Using comprehensive genetic and genomics analyses, we identified a 179 \ndisease-associated haplotype on chromosome 7q22.1 overlapping the OTSC2 locus (22). The 180 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n9 \n \nabsence of paternal DNA was overcome by recruiting maternal siblings, confirming paternal 181 \ntransmission of a premature stop mutation in KPNA7 [(NM_001145715.3), c.49 C>T, p. R17X]. 182 \nKPNA7 is located within the OTSC2 locus and resides in the vicinity of RELN (OMIM 605727), 183 \na gene whose intronic variants have been validated in GWAS otosclerosis case studies (4, 23). 184 \nAlthough not normally expressed in adult tissues, KPNA7 is reactivated in cancer cell lines, with 185 \nthe highest expression detected in pancreatic cell lines harbouring an amplification of the 7q21-186 \n22 genomic locus where KPNA7 resides (24). 187 \nOtosclerosis may be the failure of two or more parallel protective mechanisms  188 \nBloch and colleagues have eloquently modeled how age-dependent microdamage accumulates in 189 \nthe human perilabyrinthine bone where bone remodeling is essentially absent (13, 25-29). 190 \nClusters of dead osteocytes (cellular voids) lose connection with each other as the perilacunar 191 \nmatrix breaks down, rendering OPG and other nutrients beyond the reach of viable osteocytes 192 \ntrapped within these voids (26). In their model of aging, quiescent cells within the globuli 193 \ninterossei may be able to break free of suppressive actions of OPG and go on to complete the 194 \nprocess of endochondral ossification. However, the accumulation of voids alone is unlikely to be 195 \ncausative as clinical otosclerosis is not prevalent (0.30–0.38% in Europeans) and histologic 196 \notosclerosis has been observed in under 3% of temporal bone autopsy specimens (30). Tissue 197 \nfrom patients with labyrinthitis ossificans, a rare reaction to infection where the membranous 198 \nlabyrinth turns to bone, essentially halting OPG production, show the persistence of cartilage 199 \nremnants and the absence of bone remodeling, suggesting parallel mechanisms, in addition to 200 \nOPG, protect the otic capsule from remodeling (27). Our exploration into the function of 201 \nKPNA7 in embryonic cells suggests that KPNA7 represents a parallel protective mechanism. 202 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n10 \n \nThe structure of Imp-αs and their many roles in nuclear transport  203 \nCells require an active transport machinery to shuttle RNA, protein and other macromolecules to 204 \ntheir correct subcellular localizations to maintain homeostasis and carry out normal functions. 205 \nFor proteins, the most utilized nuclear import pathway is mediated by Imp-αs (31). KPNA7 206 \nencodes the newest of the seven-member imp-α karyopherins, a group of highly conserved 207 \nproteins that share a common structure consisting of a body of ten helical Armadillo (ARM) 208 \nrepeats, a short C-terminal region of acidic amino acids and an N-terminal, Importin- β (Imp-β) 209 \nbinding (IBB) domain. Most proteins targeted for transport into the nucleus contain a nuclear 210 \nlocalization signal (NLS) motif, a lysine-rich stretch of basic amino acids containing one 211 \n(monopartite) or two (bipartite) basic regions separated by a linker region in their amino acid 212 \nsequence (31). The first NLS motif to be recognized and best characterized is the classical NLS 213 \n(cNLS). Imp-αs function as adaptors that recognize and bind to cNLS in their cargo proteins. 214 \nProteins with non-classical NLS (ncNLS) motifs can also be bound and transported directly by 215 \nImp-βs without the need for Imp-α adaptors.  216 \n 217 \nExcept for KPNA7, Imp-αs are maintained in a closed state (autoinhibited) in the cytoplasm with 218 \nthe highly flexible IBB domain folding back onto itself blocking the NLS binding groove from 219 \nbinding NLS-containing cargo (32, 33). In cNLS transport, the IBB domain binds Imp-β, 220 \nexposing the NLS binding groove for specific cargo protein binding. The cargo-Imp-α-β 221 \ntricomplex, once formed, is rapidly transported to the nucleus via Imp-β interactions with select 222 \nnucleoporins lining the central channel of the nuclear pore complex. In ncNLS transport, Imp-β 223 \nbinds and transports NLS-containing cargo in the absence of Imp-α. Inside the nucleus, Imp-β 224 \nbinds RanGTP and releases Imp-α and cargo protein and then Imp-α and Imp-β are recycled 225 \nback to the cytoplasm by export receptors. 226 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n11 \n \nImp-αs can also act as negative regulators for the nuclear import of certain proteins by 227 \ncompeting with Imp-β for NLS binding to cargo, or by forming a transport-incompetent complex 228 \nin the cytoplasm, preventing cargo from entering the nucleus. For example, in the presence of 229 \nImp-α, TRF1 forms a complex with Imp-α-β but this complex remains in the cytoplasm. In 230 \ncontrast, Imp-α competes with the binding of imp-β to Snail zinc finger domain, resulting in 231 \nineffective nuclear accumulation of Snail, leading to a decrease in its cellular protein level 232 \nthrough subsequent degradation by the protease system, with implications for the prevention of 233 \ntumor cell invasion by inhibiting Snail localization (34). Perhaps most significant to otosclerosis, 234 \nImp-αs are known inhibitors of PTHrP, a multifunctional cytokine and a major regulator of 235 \nchondrogenesis in the human skeleton sharing structural similarities with, and the same receptor 236 \n(PTH1R) as parathyroid hormone (PTH) (35). Interestingly, PTH1R mRNA expression in 237 \notosclerotic stapes led Grayeli and colleagues to hypothesize that abnormal cellular response to 238 \nPTH played a role in abnormal remodeling in otosclerosis (36).  239 \nCharacteristics of KPNA7, the newest nuclear import factor 240 \nKPNA7 is the most recent and divergent of the seven human Imp-αs and an intriguing OTSC 241 \ngene as it is not normally expressed in adult tissues. KPNA7 is recognised as a maternal factor 242 \nessential for embryogenesis and fertility (37) and plays a critical role in protein transport in 243 \noocytes and early embryos and is critical to early embryonic cleavage events and zygotic 244 \ngenome activation (38). KPNA7 is known to have many cargo proteins, up to 377 have been 245 \nreported (24). KPNA7 has a unique ability among the Imp-αs to maintain an open state and has 246 \nthe strongest IBB domain capacity for Imp-β, likely critical to producing pre-formed Imp-α-β 247 \nheterodimers to increase the transport rate of cargo proteins in the early stages of embryogenesis 248 \n(38). KPNA7 is the most abundant Imp-α in germinal vesicle and metaphase II-stage oocytes 249 \n(39) and is nearly absent from the eight-cell embryo onward (38), being rapidly degraded during 250 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n12 \n \nzygotic genome activation and barely detectable in morula- and blastocyst-stage embryos (40). 251 \nRecessive KPNA7 mutations result in low protein expression levels, interfering with the nuclear 252 \nimport of RSL1D1 (also known as cellular senescence-inhibited gene protein: CSIG) (17). 253 \nRSL1D1 negatively regulates PTEN via translational suppression causing increased cell 254 \nproliferation, is significantly elevated in the tumors of colorectal cancer patients predicting 255 \npoorer survival outcomes and is a potential new target for cancer therapies (41). Imp-α has been 256 \nshown to inhibit ncNLS transport of certain proteins including PTHrP (42). As the N-terminal 257 \ndomain of Imp-β binds PTHrP at HEAT repeats 2-11, but also binds Imp-α (IBB domain) at 258 \nHEAT repeats 7-19, this partial overlap of binding sites may explain ncNLS transport inhibition 259 \nof PTHrP (43). 260 \nNuclear import of PTHrP drives developmental pathways in a context-specific manner 261 \nThe otic capsule forms through a series of molecular and cellular signaling processes where 262 \nmesenchymal progenitor cells undergo condensation and differentiation into chondrocytes which 263 \nsubsequently proliferate and hypertrophy, followed by mineralization of the extracellular matrix 264 \nand apoptosis of the chondrocytes (44) (Fig 3). PTHrP is necessary for endochondral 265 \nossification, regulating chondrocyte maturation, proliferation and differentiation (44). In fact, 266 \nPTHrP is essential to development. PTHrP null mice (Pthrp -/-) die early in the postnatal period 267 \nand display severe chondrodysplasia with reduced endochondral development and excessive 268 \nmineralization (45). Experiments testing the effect of mechanical strain on chondrocytes showed 269 \nthat PTHrP expression increased during the proliferation and matrix forming stages under 270 \nconditions of cyclical strain due to Indian Hedgehog (Ihh) signaling in chondrocytes (46). The 271 \nIhh signaling pathway also works with other signaling molecules and pathways to promote 272 \nchondrocyte proliferation and inhibit hypertrophy and forms a negative feedback loop with 273 \nPTHrP pivotal in regulating cartilage development (47). Upon activation of Ihh expression from 274 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n13 \n \nan external trigger such as mechanical stress, Ihh binds to the transmembrane protein Patched 275 \n(Ptc), releasing inhibition of Smoothened (Smo) at the cell surface, which then activates the 276 \nexpression of the downstream signaling molecule Gli. Gli enters the nucleus and regulates the 277 \nexpression of downstream signaling factors SOX9, RUNX2 and PTHrP (48) (Fig 3).   278 \nHow KPNA7 haploinsufficiency may cause otosclerosis  279 \nAs PTHrP nuclear import is integral to its function in cells, strategies to block PTHrP nuclear 280 \nimport could have important effects on target cell function (18). Endochondral ossification is 281 \ninitiated by nuclear import of PTHrP and quiescent chondrocytes are known to express PTHrP 282 \n(49). Upon entering the nucleus by ncNLS transport chondrocyte regulation is achieved through 283 \nthe Ihh-PTHrP pathway (44), as well as the WNT/ β-catenin pathway (50) via negative feedback 284 \nloops. PTHrP is both fast acting and fast to be removed (51). In a scenario where the KPNA7-285 \nImp-β complex is pre-formed, potentially KPNA7 would be able to prevent binding of PTHrP by 286 \nbinding a different cargo or outcompete PTHrP and enter the nucleus in a cargo-free state, 287 \nthereby inhibiting nuclear transport of PTHrP, as suggested by Oostdyk and colleagues for other 288 \ncargo proteins (38). While KPNA7 has a time-limited role in the nuclear import of factors 289 \nrequired for maternal-to-zygotic transition and early embryogenesis, it is logical to think that 290 \nKPNA7 also inhibits endochondral ossification by preventing nuclear import of PTHrP in the 291 \nunique and peculiar case of the embryonic cells of the globuli interossei.  292 \n 293 \nAlthough not normally expressed in adult tissues, KPNA7 is reactivated in cancer cell lines 294 \npromoting carcinogenesis by increasing the rate of import of cell cycle factors due to genomic 295 \namplification on 7q21-22. Unlike amplification in pancreatic cancer, the effect of a heterozygous 296 \nKPNA7 stop mutation identified in otosclerosis patients is predicted to cause its phenotypic 297 \neffects via haploinsufficiency. Therefore, it is unlikely that otosclerosis is due to increased 298 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n14 \n \nimport of KPNA7 cargo proteins. The quiescent chondrocytes of the globuli interossei, unique to 299 \nthe otic capsule, would provide a likely substrate for KPNA7 (10, 11). While a mutation in an 300 \nImp-α exclusively expressed in early development would not be expected to impact the rest of 301 \nthe human skeleton, we hypothesize that KPNA7 plays a key role in preventing PTHrP nuclear 302 \nimport in quiescent chondrocytes. If this is indeed the case, KPNA7 haploinsufficiency may 303 \nactivate chondrogenesis leading to the deposition of spongy, disorganized bone characteristic of 304 \nosteosclerotic specimens, and specifically, the mineralization stage of the extracellular matrix in 305 \nhypertrophic chondrocytes. If this speculation is true, otosclerosis is not the result of pathologic 306 \nbone resorption and deposition, but one of re-activation of chondrogenesis, providing an 307 \nalternative mechanism for its pathogenesis.  308 \nOTSC genes and their roles in endochondral ossification  309 \nA closer look at the OTSC genes MEPE, FOXL1 and SMARCA4 reveal they are involved in 310 \nendochondral ossification signaling pathways (Fig 3). In addition, the OTSC gene SERPINF1 311 \nencodes for PEDF, which is involved in many biological processes including bone formation, 312 \nbinding to extracellular matrix proteins including collagen and glycosaminoglycan, and is 313 \ninvolved in the mineralization of bone matrix (52). Rämö and colleagues identified otosclerosis 314 \nsusceptibility genes BMP-2,-3,-4,-7 (involved in bone development) and SOX9 and RUNX2 315 \n(4). MEPE normally serves as a decoy receptor for pre-osteoclasts inhibiting osteoclast 316 \nmaturation and its ASARM motif, upon proteolytic cleavage by β-cathepsin, inhibits 317 \nmineralization by binding to hydroxyapatite crystals (53). In zebrafish, foxl1 (OTSC11) regulates 318 \nthe expression of collagen genes such as col1a1 and col11a2, and results in a delay in jawbone 319 \nmineralization (54). SMARCA4 (also known as BRG1) plays a role in ossicle formation during 320 \nembryogenesis and may be important for regulation of osteoblast differentiation and 321 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n15 \n \nmaintenance of postnatal bone homeostasis in the otic capsule (55). WNT/ β-catenin is also 322 \ninvolved in proliferation and differentiation through a negative feedback loop with PTHrP (50). 323 \nRe-activation of endochondral ossification pathways in the globuli interossei, a new 324 \nmechanistic model for otosclerosis  325 \nOtosclerosis is likely due to germline susceptibility factors interacting with environmental 326 \ntriggers (measles infection, pregnancy, etc.) to generate the pathological phenotype (2). Perhaps 327 \ndeficient KPNA7 causes the reactivation of chondrogenesis and the resumption of endochondral 328 \nossification after loss of repression by OPG in aging otic capsules. From this perspective, the 329 \npathologic spongy bone deposition seen in otosclerosis may be the result of dysregulation caused 330 \nby mutations in OTSC genes regulating endochondral ossification. Endochondral ossification 331 \ninvolves multiple signaling pathways including the Ihh pathway, which regulates chondrocyte 332 \nmaturation and bone formation, the PTHrP pathways and bone morphogenetic proteins (BMPs). 333 \nIhh regulates chondrocyte maturation and bone formation, and bone morphogenetic proteins 334 \n(BMPS) induce chondrocyte differentiation via regulating the expression of SOX9 and 335 \nstimulating endochondral ossification via transcriptional regulation of RUNX2.  336 \nCould dysregulation of PTHrP due to KPNA7 cause otosclerosis in women.  337 \nIn a literature review on otosclerosis and pregnancy, Fabbris and colleagues found that the only 338 \nsignificant correlation was between pregnancy and disease onset, noting that cases with hearing 339 \nimpairment increased with number of pregnancies (56). We know that PTHrP levels are higher 340 \nin pregnant women, and are significantly higher in lactating women, being detected in breast 341 \nmilk at levels exceeding 10,000 times those found in the blood of hypercalcemia of malignancy 342 \npatients or normal controls (57). Otosclerosis during pregnancy may be the result of increased 343 \nbone remodeling (RANK/RANKL/OPG) (57-59) or due to susceptibility variants in KPNA7 or 344 \nother Imp-αs involved in regulating the actions of PTHrP.  345 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n16 \n \nOverlapping therapeutic targets in skeletal disorders and cancer  346 \nOTSC genes FOXL1, SMARCA4 and KPNA7 are increasingly recognised for their role in 347 \ncarcinogenesis (60-64). By extension, therapeutic targets for cancer, otosclerosis and other 348 \nskeletal disorders increasingly overlap because the development, proliferation and migration of 349 \ncancer cells mimic critical pathways of embryonic development. This includes deregulation in 350 \nnucleocytoplasmic transport. For example, expression of KPNA7 in adult tissues is almost non-351 \nexistent, except in pancreatic cancer cell lines where KPNA7 causes a significant decrease in cell 352 \ngrowth due to G1 arrest, accompanied by an increased expression of p21, a key regulator of the 353 \ncell cycle (65). As we have seen, low KPNA7 levels causes PREMBA through dysregulated 354 \nimport of RSL1D1, which in turn, negatively regulates PTEN and is a potential new target for 355 \ncolorectal cancer (41). Imp-αs also facilitate the nuclear import of Smad proteins in the TGF- 356 \nβ/Smad3 pathway and show promise as therapeutic targets in rotator cuff injuries (66). There is 357 \nan epidemic of colorectal cancer in young people (67) and of adult-onset skeletal disorders 358 \ncausing a public health issue in the global North. An investment in therapeutic options for cancer 359 \nand for the repair of cartilage for aging knees and hips may benefit patients with otosclerosis by 360 \nproviding target drugs for off-label clinical trials. 361 \nLimitations of this study 362 \nAlthough KPNA7 maps to the OTSC2 locus (22), we did not have access to the family used to 363 \nmap OTSC2, therefore we cannot confirm or deny that KPNA7 is OTSC2. Association between 364 \notosclerosis and RELN may be due to the close physical proximity of RELN to disease-variants 365 \nin KPNA7. We assume equal expression of maternal and paternal KPNA7 alleles in the globuli 366 \ninterossei however we do not have patient-derived disease tissue to examine the functional 367 \nconsequences of KPNA7, c.49C>T, p.R17X. Otosclerosis due to KPNA7 haploinsufficiency may 368 \nbe related to the dysregulated import of RSL1D1 or there may be alternative mechanistic 369 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n17 \n \nexplanations for the pathogenesis of KPNA7 as the specific binding affinities and cargo 370 \npreferences of KPNA7, being the most recent member of the Imp-αs, have not been fully 371 \nexplored. Evidence from this study suggests that reactivation of endochondral ossification, not the 372 \nde-repression of bone remodeling, underlies otosclerosis due to KPNA7 mutation. Future 373 \nresearch is warranted along this direction to describe the modulators of KPNA7 function and to 374 \ndesign potential therapies to modulate the PTHrP- Imp-α interactions for therapeutic purposes. 375 \nFuture directions 376 \nWe used strict clinical criteria and a combination of genetic and genomic analyses to 377 \nsuccessfully identify a novel OTSC gene, KPNA7, the second OTSC gene identified in the white 378 \nsettler population of the island of Newfoundland, Canada. KPNA7 is the first member of the 379 \nnucleocytoplasmic trafficking system to cause otosclerosis. We propose that normally, KPNA7 380 \nplays a critical role of inhibiting the nuclear import of PTHrP in the globuli interossei and that 381 \nKPNA7 haploinsufficiency results in reactivation of endochondral ossification, providing an 382 \nadditional or alternative paradigm to the central hypothesis that dysregulation of bone 383 \nremodeling repression causes otosclerosis. As the molecular signaling pathways involved in 384 \nembryogenesis also play a role in the fate of neoplastic cells, investigating the shared cellular 385 \nand molecular signaling pathways and molecules that cause other monogenic skeletal disorders 386 \nand carcinomas may provide unique insights into novel therapeutic targets for common maladies 387 \nincluding otosclerosis, osteoarthritis and cancer. Difficulties with accessing the complex, fluid-388 \nfilled cavity of the inner ear within the dense temporal bone, currently hampering diagnosis and 389 \ntargeted drug delivery, may be behind us with the advent of microneedle technologies for safe 390 \nsampling of inner ear fluids and local treatments, as microlitres of perilymph fluid can be 391 \nextracted this way (68). The class of molecules that do not encode for proteins but regulate gene 392 \nexpression, non-coding RNA, are revealing an increasing role in cellular process, including 393 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n18 \n \ndifferentiation and maturation, and their dysregulation can cause cancer. Perhaps renewed 394 \ninterest in solving the OTSC loci should include looking for non-coding RNA targets. 395 \nMaterials and Methods 396 \nEthics Statement 397 \nPrior approval to study live research participants with HL and their blood relatives was granted 398 \nby the regional ethics authority (Hearing Loss Project #01.186, Human Research Ethics Board, 399 \nSt John’s, NL, Canada). Informed consent from research participants was by written consent. 400 \nClinical recruitment, pedigree structure and classification of HL 401 \nResearch participants underwent audiological and medical examinations, permitted access to 402 \ntheir medical records, and completed a medical questionnaire (19).  HL was classified based on 403 \nthe pure-tone threshold averages of 0.5, 1.0 and 2.0 kHz, as defined by the American Speech and 404 \nHearing Association (AHSA). Family members were assessed by our clinical team to update 405 \naudiograms and confirm middle ear status. A difference of >10 dB HL between air and bone 406 \nconduction sensitivity represented a significant conductive component associated with impaired 407 \nsound transfer through the middle ear. Conservative clinical criteria were used to assign 408 \notosclerosis affection status: affected were blood relatives with surgically confirmed otosclerosis 409 \nat any age; unaffected were blood relatives ≥ 55 years of age with normal bilateral hearing 410 \nthresholds. Of nine siblings in generation III, five have otosclerosis (Fig 2). The father (PID II-7) 411 \nreportedly had normal hearing but was \"very sensitive to noise\" and the mother (PID II-8) had 412 \nHL (uncategorized) along with two of her nine siblings (PID II-16, PID II-17) who are reported 413 \nto have age-related HL. X-linked inheritance could be ruled out as otosclerosis is not more 414 \nsevere in males; however, without further clinical information, the inheritance pattern is 415 \nconsistent with both AD and AR inheritance. 416 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n19 \n \nTest for linkage to mapped OTSC loci-haplotype sharing and targeted sequencing  417 \nGenomic DNA was extracted from peripheral blood (69). Informative microsatellite markers (4-418 \n10 per locus) spanning seven OTSC loci and two otosclerosis-suceptibility genes including 419 \nOTSC1 (15q), OTSC2 (7q), OTSC3 (6p), OTSC4 (16q), OTSC5 (3q), OTSC7 (6q), OTSC8 (9p) 420 \nand genes COL1A1 (17q) and NOG (17q) were genotyped and shared alleles noted among 421 \naffected siblings. Primers were labelled (6-FAM), amplified using touchdown PCR, size 422 \nfractionated (ABI PRISM model 3130xl) and analyzed with Gene Mapper software (v4.0). 423 \nHaplotypes were recapitulated according to “least recombination rules” and paternal haplotypes 424 \nwere inferred due to absence of DNA. Full gene sequencing (Sanger) was performed on three 425 \naffected (PIDs III-1, III-3, III-9) and one unaffected (PID III-7) for select annotated genes 426 \n[March 2006 assembly (NCBI build 36.1)] within OTSC loci with disease-associated haplotypes. 427 \nGenes were selected for sequencing if they had a functional role in bone remodeling or immune 428 \nresponse. Primers were designed to amplify the longest isoform, including all intron/exon 429 \nboundaries and UTRs and purified PCR products were bidirectionally sequenced (Big Dye 430 \nTerminator V3.1 kit; ABI PRISM 3130xl DNA Analyzer). We used Mutation Surveyor software 431 \n(version 4.07, SoftGenetics LLC State College, PA 16803) to select quality reads and analyze 432 \nDNA sequences. Rare sequencing variants (<2 %) were subjected to in silico tools (SIFT, 433 \nPolyPhen, Human Splicing Finder (HSF), MaxEntScan, NNSLICE, GeneSplicer, Known 434 \nconstitutive signals) to predict pathogenicity. Variants were filtered out if they were absent in 435 \naffected or present in unaffected relatives. The proband was also screened for the 15-bp coding 436 \ndeletion in FOXL1 (rs764026385; OTSC11) identified in an NL family (70) and for the rare 437 \notosclerosis variants in SERPINF1 (71). All remaining variants were subjected to cascade 438 \nsequencing to verify co-segregation with otosclerosis in the family. 439 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n20 \n \nGenome wide SNP genotyping and multipoint linkage analysis 440 \nAs a targeted genetic analysis did not identify the otosclerosis gene and the inheritance pattern is 441 \nnot clear, genome wide SNP genotyping and multipoint linkage analysis were performed under 442 \nboth AR and AD models with Merlin (version 1.1.2) (72, 73), assuming complete (100%) 443 \npenetrance. LOD scores were calculated at recombination fractions of 0.000 to 0.5000. We used 444 \nthe 610K Illumina SNP array (Genome Centre, McGill University, QC, Canada) on samples 445 \nfrom five affected siblings (PIDs III-1, III-3, III-6, III-8, III-9), an unaffected sibling (PID III-7), 446 \na maternal sibling with HL (PID II-17) and a maternal sibling with normal hearing (PID II-15). 447 \nAs well, nine population control samples with normal hearing were used to estimate minor allele 448 \nfrequencies of unavailable family members. Genotypes were analyzed at The Centre for Applied 449 \nGenomics (TCAG, University of Toronto, ON, Canada) and exported from GenomeStudio 450 \nsoftware (v2010.3).   451 \nSequencing, variant filtering and cascade sequencing under linked regions 452 \nExome sequencing was carried out on four affected siblings (PIDs III-1, III-3, III-6, and III-9) 453 \nand two older controls (55, 60 yrs old) with normal hearing thresholds. Library preparation was 454 \ndone with TrueSeq Prep Kit and samples run on the Illumina Hiseq 2000, generating 50-150 455 \nmillion 100-bp paired end reads. Reads >32-bp long were aligned to the 1000 genome reference 456 \nusing Burrows-Wheeler Aligner (BWA) and merged with Picard software (Broad Institute). 457 \nWhere multiple base mismatches and false positive variant calls were recorded, insertions and 458 \ndeletions were realigned using GATK software (73, 74). The percentage of aligned region 459 \ncoverage was detected using the Genome Centre's in-house database. The regions were identified 460 \nas high coverage (>400X), low coverage (<50X), low mean mapq MQ (<20X) and no data. Rare 461 \nvariants were considered at a higher frequency (MAF<2%) to account for potential founder 462 \neffects. Rare variants, absent in one or more affected, present in one or more controls or also 463 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n21 \n \nidentified in solved otosclerosis patients (e.g. FOXL1) were filtered out. Variants with a 464 \nminimum of 20X coverage were analysed with in silico tools including samtools mpileup 465 \nalgorithm (75), SnpSift (76), SnpEff (76), SIFT, PolyPhen-2, PANTHER and ClustalW. In 466 \naddition, the functional consequence of KPNA7, c.49 C>T, p. R17X was analysed using Alamut 467 \nVisual Plus by Sophia Genetics, version 1.13 (2024). At this stage, allele frequencies of genetic 468 \nvariants were checked in two research cohorts, the NL Osteoarthritis Study (NFOAS) consisting 469 \nof 1,000 total joint (knee and/or hip) replacement patients and the NL Colorectal Cancer 470 \nRegistry, where samples were genotyped by Illumina microarray platforms and then imputed 471 \nwith 1000 genome project data as reference panels. All remaining variants underwent cascade 472 \nscreening and were tested against 38 otosclerosis probands (from ON Canada; Western 473 \nUniversity ethics #103,679), 149 uncategorized HL probands (from NL) and 727 population 474 \ncontrols.  475 \nWeb Resources 476 \n 477 \nASHA (Amer Speech-Language Hearing Assoc), https://www.asha.org/practice-portal/clinical-478 \ntopics/hearing-loss/ 479 \nBurrows-Wheeler Aligner BWA, http://bio-bwa.sourceforge.net/ 480 \nClinVar, https://www.ncbi.nlm.nih.gov/clinvar/ 481 \nClustalW, https://www.genome.jp/tools-bin/clustalw 482 \ndbSNP, https://www.ncbi.nlm.nih.gov/snp/ 483 \ngnomAD, https://gnomad.broadinstitute.org/ 484 \nHereditary Hearing Loss homepage, http://hereditaryhearingloss.org/ 485 \nHuman Splicing Finder, www.umd.be/HSF3/ 486 \n 487 \n1000 genomes, https://www.internationalgenome.org/home 488 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n22 \n \nOnline Mendelian Inheritance in Man, http://omim.org 489 \nPrimer3, https://bioinfo.ut.ee/primer3-0.4.0/ 490 \nPANTHER, http://www.pantherdb.org/ 491 \nPolyPhen-2, http://genetics.bwh.harvard.edu/pph2/  492 \nRefSeq, https://www.ncbi.nlm.nih.gov/refseq/ 493 \nSIFT, https://sift.bii.a-star.edu.sg/ 494 \nSNP database, http://www.ncbi.nlm.nih.gov/projects/SNP/ 495 \nThe MathWorks, Inc. https://www.mathworks.com 496 \nVarsome, http://varsome.com 497 \nUCSC Genome Browser, https://genome.ucsc.edu/ 498 \nAcknowledgements 499 \nThis study was funded by the Canadian Institutes of Health Research (#222294), Canadian 500 \nFoundation for Innovation (#9384, #13120), and Genome Canada/Genome Atlantic (AMGGI) to 501 \nT.L.Y. Support was also provided by Memorial University, Town of Grand Falls-Windsor 502 \n(Excite Corporation) and the Government of Newfoundland and Labrador. One of the first 503 \nauthors, N.A., is a recipient of a CIHR Fellowship and this paper includes contributions from 504 \ntheir PhD research.   505 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n23 \n \nReferences 506 \n 507 \n1. Sönmez S, Orhan KS, Baumgartner W-D, Zarowski A, Özgirgin NO, Barbara M, et al., 508 \neditors. the Fifth International Symposium on Otosclerosis and Stapes Surgery. 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A 683 \npathogenic deletion in Forkhead Box L1 (FOXL1) identifies the first otosclerosis (OTSC) gene. 684 \nHuman genetics. 2022;141(3):965-79. 685 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n27 \n \n71. Ziff JL, Crompton M, Powell HR, Lavy JA, Aldren CP, Steel KP, et al. Mutations and 686 \naltered expression of SERPINF1 in patients with familial otosclerosis. Hum Mol Genet. 687 \n2016;25(12):2393-403. 688 \n72. Abecasis GR, Cherny SS, Cookson WO, Cardon LR. Merlin--rapid analysis of dense 689 \ngenetic maps using sparse gene flow trees. Nat Genet. 2002;30(1):97-101. 690 \n73. 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Fly (Austin). 2012;6(2):80-92. 701 \n702 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n28 \n \n 703 \n 704 \nFig 1. Pre- and post-stapedectomy audiograms reveal successful surgical treatment of 705 \nbilateral otosclerosis in the proband (PID III-1). 706 \nPre-surgery audiogram revealed a conductive loss with borderline cochlear (sensorineural) HL. 707 \nPost stapedectomy of the right ear improved hearing and the air-bone gap was mainly resolved 708 \nbut some HL remained, especially in the low frequencies. After stapedectomy of the left ear at 709 \nage 44, hearing improved across all frequencies with only a mild sensorineural HL remaining in 710 \nthe mid to high frequencies. At age 51, the proband experienced mild hearing loss in the low and 711 \nmid frequencies of the left ear but high frequencies showed moderate to severe loss at 4000 Hz 712 \nand 8000 Hz, likely due to disease processes on the cochlear side of the round window. Pre = 713 \npre-stapedectomy audiogram, age 42; post RT = post-stapedectomy audiogram, right ear, age 42; 714 \npost bil 1 = first post-bilateral stapedectomy audiogram, age 44; post bil 2 = second post-715 \nbilateral stapedectomy audiogram, age 51;  = unmasked bone conduction; □ = masked bone 716 \nconduction. Generated by MathWorks. (2020). MATLAB (Version R2020a)[Computer software].  717 \n  718 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n29 \n \n 719 \n 720 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n30 \n \nFig 2. Pedigree and otosclerosis-associated haplotype of a multiplex family from NL with 721 \nclinically confirmed otosclerosis.  722 \n(A) Pedigree of NL family with AD otosclerosis co-segregating with the KPNA7, c.49C>T 723 \npremature stop mutation and transmitted from the paternal side (orange haplotype). KPNA7 724 \nmaps to the OTSC2 locus in the vicinity of RELN. (B) Sequencing electropherogram generated 725 \nfrom Mutation Surveyor software showing the heterozygous nonsense mutation. We used 726 \ndiamonds instead of squares (males) and circles (females) to protect the identity of research 727 \nparticipants. 728 \n  729 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint \n\nManuscript: KPNA7 causes otosclerosis                   Benteau et al., 2025 \n31 \n \n 730 \nFig 3. New mechanistic model for otosclerosis due to pathogenic mutations in KPNA7 and 731 \nother OTSC genes within the aging otic capsule. 732 \nOtosclerosis genes (*) and otosclerosis susceptibility genes (green circles) are involved in 733 \nendochondral ossification signaling pathways. We hypothesize that KPNA7 haploinsufficiency 734 \nallows the nuclear import of PTHrP into the quiescent cells of globuli interossei trapped within 735 \ncellular voids of the aging otic capsule, re-activating endochondral ossification pathways in these 736 \nembryonic tissues. Created in BioRender. BENTEAU, T. 737 \n(2025) https://BioRender.com/mfhqhzv. 738 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.22.671726doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}