{"paper_id":"10fdc231-4069-4618-8b05-19817759dca1","body_text":"Human Organoids for Rapid Validation of Gene Variants Linked to Cochlear Malformations | 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 Research Article Human Organoids for Rapid Validation of Gene Variants Linked to Cochlear Malformations Mohammad Faraz Zafeer, Memoona Ramzan, Duygu Duman, Ahmet Mutlu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4474071/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jan, 2025 Read the published version in Human Genetics → Version 1 posted 13 You are reading this latest preprint version Abstract Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital deafness. Most patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1 (p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed significant developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a tool to rapidly validate the pathogenicity of DNA variants associated with cochlear malformations. Candidate gene Cochlear malformation CRISPR/Cas9 Deafness Inner ear anomaly iPSC Organoids Variants of uncertain significance Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Approximately one in 500 newborns are diagnosed with permanent hearing loss (HL) (Bainbridge & Wallhagen 2014 ; Petit et al. 2023 ). Inner ear anomalies (IEAs) affecting the cochlea are reported in about 25% of these children (Bamiou et al. 2000 ; Brotto et al. 2021 ; Ocak et al. 2019 ). Studies performed in various animal models shed light on fundamental mechanisms governing vertebrate inner ear development (Chen et al. 2017 ; Torii et al. 2016 ; Whitfield et al. 2002 ). However, mutations in relatively few of the genes recognized in animal model systems have been shown to cause cochlear malformations in humans. Interpretation of the identified variants through genetic testing in HL requires collating and analyzing the available literature for supporting evidence, followed by a formal classification based on this evidence (Patel et al. 2021 ). A recent study shows that 70% of the identified missense variants in people with HL are classified as variants of uncertain significance (VUS) (Tollefson et al. 2023 ). Functional studies can help establish causality in these cases, which are often lacked in published studies. It is difficult to directly examine the molecular and cellular processes leading to the establishment of the human inner ear. It is located deep within the skull surrounded by bone and other tissues (Nomura et al. 2014 ). There is also a paucity of biopsy material appropriate for molecular analysis, particularly in the early stages of development. Finally, the range of testing that can be carried out on human embryos and fetuses is limited by ethical considerations, further complicating research (Plomer 2013 ). The recent advancements in stem cell technology have enabled us to create three-dimensional organoids similar to developing human ears using human induced pluripotent stem cells (iPSCs) (Doda et al. 2023 ; Koehler et al. 2017 ; Qi et al. 2024 ; Steinhart et al. 2022 ; van der Valk et al. 2023 ). These iPSC-derived structures imitate the basic cartography of the developing ear and can, therefore, be used to investigate how development at the cellular stage happens in this organ (Doda et al. 2023 ; Koehler et al. 2017 ; Romano et al. 2022 ; Steinhart et al. 2022 ; van der Valk et al. 2023 ). Generating inner ear organoids (IEOs) involves differentiating pluripotent stem cells into otic placode-like cells. These cells later form cavities resembling otic vesicles that contain hair cells, supporting cells, and neurons like those seen in the inner ear after maturation (Koehler et al. 2017 ; Moore et al. 2023 ; Nie & Hashino 2020 ; Romano et al. 2022 ; Tang et al. 2020 ). Organoids hold several advantages over animal models, including the ability to study human-specific aspects of inner ear development and disease with much shorter timelines than animal models (Qi et al. 2024 ). These lines retain the genetic architecture of the cells from whom the lines were derived and are amenable to genomic engineering approaches, including CRISPR/Cas9-based methods (Rivron et al. 2023 ). In this study, we investigate the impact of DNA variants in known cochlear malformation genes FGF3 (Fibroblast Growth Factor 3) and GREB1L (GREB1 Like Retinoic Acid Receptor Coactivator), along with a candidate gene, PBXIP1 (PBX Homeobox Interacting Protein 1), in inner ear development via IEOs. FGF3 participates in the early development of the inner ear (Tekin et al. 2007 ). Pathogenic variants in FGF3 cause deafness with LAMM (Labyrinthine Aplasia, Microtia, and Microphthalmia; OMIM 610706), an autosomal recessively-inherited syndrome characterized by missing inner ear structures as well as small external ears and teeth. GREB1L has a role in neural crest development and retinoic acid pathway (Brophy et al. 2017 ; Vega-Lopez et al. 2018 ). Several reports have associated GREB1L variants with autosomal dominant urogenital or cochlear anomalies (Adadey et al. 2022 ; De Tomasi et al. 2017 ; Herlin et al. 2019 ; Jacquinet et al. 2020 ; Kim et al. 2022 ; Schrauwen et al. 2018a ). In two patients with cochlear malformations, we detected previously unreported missense variants in FGF3 and GREB1L , interpreted as VUS. In another patient, we detected a nonsense variant in PBXIP1 , a gene not previously associated with a human phenotype. By establishing monoclonal IEOs for knockout and patient-specific variants of these genes, we show differences in organoid size, number of luminal spaces (otic vesicles), and lower expression of otic vesicle markers in both knockout and variant-bearing organoids compared to controls. Methods Enrollment of subjects and exome sequencing of probands The study was approved by the Institutional Review Board (Protocol no. 20081138) at the University of Miami (USA) and Ethics Committee (Protocol no. 012413) at Ankara University Medical School (Türkiye). Written informed consents were obtained from all participants and in the case of minors, it was obtained from parents. Audiometry was performed to measure average hearing thresholds for all participants under standard conditions and guidelines. All affected individuals were examined by a clinical geneticist and otolaryngologist. For exome sequencing (ES), we followed a recently published protocol (Ramzan et al. 2024 ). Briefly, single nucleotide, indel, and copy number variants (CNVs) in all known deafness genes were analyzed. Variants were retained for further evaluation if they had an allele frequency of less than 0.01. The variants in all known genes for HL were analyzed using a larger list retrieved from hereditary hearing loss homepage ( https://hereditaryhearingloss.org/ ) and OMIM. If there was no candidate variant identified in known deafness genes, ES data were re-examined for all genes containing variants less than 0.01 allele frequency. CNV analysis with ES data used CoNIFER v.02.2 with default parameters. It uses a singular value decomposition method to correct systematic biases and identifies a CNV call if the corrected signal reaches a predefined threshold at no less than three consecutive exons (Krumm et al. 2012 ). We performed Sanger sequencing to confirm candidate variants and evaluate segregation within respective families. Candidate variants were classified according to ACMG and ClinGen Hearing Loss Expert Panel (HL-EP) specifications, aligned with the ACMG/AMP Variant Interpretation Guidelines (Oza et al. 2018 ; Richards et al. 2015 ; Tavtigian et al. 2020 ). AlphaMissense was included in the prediction of pathogenicity as described (Zhuo et al. 2024 ). iPSC maintenance and validation Cells from a validated male European lymphocyte-derived iPSC line (ASE9203) were cultured on Vitronectin (Thermo Scientific, USA, Cat# A2858501) coated culture ware and maintained in E8-Flex (Thermo Scientific, USA, Cat# A2858501) media supplemented with 100 µg/mL Normocin (Invivogen, USA, Cat# ant-nr-05), following the manufacturer's guidelines. Routine passages utilized 1x Revitacell (Thermo Scientific, USA, Cat# A2644501). We regularly assessed pluripotency markers OCT4 (Cell Signaling Technology, USA, Cat# 2840) and TRA-1-60 (Cell Signaling Technology, USA, Cat# 4746) through Immunocytochemistry (ICC). Generation of CRISPR/Cas9-edited variants We generated monoclonal cell lines for FGF3 KO , FGF3 c.493A>G , GREB1L KO , GREB1L c.556T>C , and PBXIP1 c.1722G>A as previously described (Ramzan et al. 2024 ). Custom sgRNA and HDR donor blocks were designed and procured using the Alt-R-CRISPR/Cas9 platform (IDT); sequences are available in supplementary Table 1. Cas9-gRNA ribonucleoprotein complexes were assembled under sterile conditions using 140 pmol sgRNA and 40 pmol EnGen® Spy Cas9 HF1 (New England Biolabs, USA, Cat# M0667M) and incubated for 10 mins at room temperature. Following the incubation, 8x10 5 cells were suspended in the RNP complex and premixed 3 µM HDR donor block in 80 µL of nucleofector solution. The final volume of the nucleofection mix was 100 µL. Nucleofections are performed using the P3-primary Cell 4D-Nucleofector Kit (Lonza, USA, Cat# V4XP-3024) and preprogrammed pulse protocol CA-137 in the 4D-Nucleofector system (Lonza). Post-nucleofections, iPSCs were cultured in E8-flex media supplemented with CloneR™2 (Stem Cell Technologies, Canada, Cat# 100–0691) and 1 µM of the HDR enhancer-V2 (IDT). DNA isolation and confirmation of CRISPR/Cas9-edited cell pools and off-targets DNA isolation was done using QuickExtract DNA Extraction Solution (Biosearch Technologies, USA, Cat# QE09050); the resulting DNA was then PCR amplified using Phusion® High-Fidelity PCR Master Mix (New England Biolabs, USA, Cat# M0531S) and sequenced with primers flanking (Supplementary Table S1 ) the guide region and Sanger sequencing at Genewiz (Azenta Life Sciences). Outcomes of CRISPR/Cas9 knock-ins were assessed using ICE Analysis (Conant et al. 2022 ). For every guide sequence, off-target analysis was done using bioinformatic tools such as Cas-OFFinder (Bae et al. 2014 ) and the sgRNA guide analysis tool from IDT in Coralville, Iowa, USA. The top five off-target sites were Sanger sequenced for flanking primers and analyzed for all the clonal lines used in this study (Supplementary Table S2). Isolation of monoclonal lines Single-cell monoclonal lines were isolated using Poisson distribution (Sanjurjo-Soriano et al. 2022 ). CRISPR/Cas9 pools were briefly dissociated using StemPro accutase (Thermo Scientific, USA, Cat# A1110501). The resulting cell suspension was collected in E8-Flex media (Thermo Scientific, USA, Cat# A2858501) containing CloneR™2 (Stem Cell Technologies, Canada, Cat# 100–0691) and centrifuged at 300 rpm. After centrifugation, the single-cell suspension was passed through 70 µm strainers (SP Bel-Art, USA, Cat# H13680-0070), and the cells were then counted using a Countess II cell counter (Thermo Scientific, USA). Depending on the cell concentrations, serial dilutions were performed to achieve a typical concentration of 1 cell per 100 µL of media. The cells were then plated in 96-well plates precoated with rhLaminin (Thermo Scientific, USA, Cat# A29249), and media changes were done with 1x CloneR™2-containing E8 media every other day for the first week and then with 0.5x CloneR™2 containing E8-Flex media till the single cell colonies reached a passaging confluence. Clonal lines were identified using Sanger sequencing, and from the same experiments, clonal lines bearing large frameshift deletions disrupting the open reading frame were used as knockouts for FGF3 and GREB1L (Supplementary Figure S1 ). Generation of inner ear organoids IEOs were generated using the previously described protocol by (Moore et al. 2023 ). Briefly, on the first day of differentiation (day − 2), cells were dissociated with StemPro accutase (Thermo Scientific, USA, A1110501). The cells were then passed through 70 µm strainers (SP Bel-Art, USA, Cat# H13680-0070), and 3500 cells/well were plated in 96-well Nunc-Sphera U-bottom plates (Thermo Scientific, USA, Cat# 174925) in 100 µL of E8-Flex media containing 20 µM Y-27632 (Stem Cell Technologies, Canada, Cat# 72304). After 4 hours of initial plating, 100 µL of fresh E8-Flex media was added to each well, bringing the final concentration of Y-27632 to 10 µM. All the aggregates were collected on day 0, washed thrice with E6-Medium (Thermo Scientific, USA, Cat# A1516401) and IEO differentiation media (E6-Medium, 2% GFR-Matrigel (Corning, Sigma Aldrich, Cat# 354230) 10 µM SB431542 (Stem Cell Technologies, Canada, Cat# 72232), 4 ng/mL FGF2 (Peprotech, Thermo, USA, Cat# 100-18B) and 2.5 ng/mL BMP4 (Stemgent, USA, Cat# 03–0007)). Cell aggregates were transferred to a new 96-well Nunc-Sphera U-bottom plate in 100 µL of differentiation media. On day 3, 25 µL E6-Medium containing 100 µg/mL Normocin, 250 ng/mL FGF-2, and 200 nM LDN193189 (Stem Cell Technologies, Canada, Cat# 72147) were added, bringing the volume to 125 µL. On days 6 and 9 of differentiation, cell aggregates were washed thrice with E6 media and then thrice with E6 media containing 100 µg/mL Normocin, 3 mM CHIR99021 (Stemgent, USA, Cat# 04-0004-02), 200 nM LDN193189, and 50 ng/mL FGF2. The cell aggregates were transferred to fresh 96-well U-bottom plates in 250 µL of new media. Cell aggregates were washed thrice on the 11th day of differentiation with Advanced DMEM/F12 (Thermo Scientific, USA, Cat# 12634010). Then they were transferred to 90 mm Nunc low-attachment plates (Thermo Scientific, USA, Cat# 174945) in 10 mL of organoid maturation media (OMM) comprising Advanced DMEM/F12, Neurobasal medium (Thermo Scientific, USA, Cat# 21103049), 1X Glutamax, 0.5X B-27 supplement without Vitamin A, 0.5X N-2 supplement, 0.1 mM 2-Mercaptoethanol, 100 µg/mL Normocin, and supplemented with 1% GFR-Matrigel and 3 µM CHIR99021. On days 13 and 15, culture media was changed to OMM + 3 mM CHIR99021 + 1 mM Purmorphamine (Stem Cell Technologies, Canada, Cat# 72202). On day 18, aggregates were washed to eliminate CHIR99021, and the media was changed to OMM + 3 mM IWP-2 (Stem Cell Technologies, Canada, Cat# 72122) + 1 mM Purmorphamine. The media was changed on day 20, with fresh media used on day 18. On day 22, cultures were washed and transferred to an anti-adhere solution coated with a low attachment 100 mm culture dish in OMM-only. The cultures received half media change every 3rd day and complete media change every 7th day until day 60. Samples were collected on day 25 and day 35 for RNA sequencing and on day 25, day 35, and day 60 for immunohistochemistry. Immunohistochemistry and sectioning IEOs on day 25 and day 60 were collected, washed with PBS twice, fixed with 4% paraformaldehyde, and processed at the Cancer Modeling Shared Resource core, Sylvester Cancer Center, University of Miami. Serial sections of 5 µm thickness were antigen-retrieved using 10 mM citrate buffer pH6. For immunohistochemistry, sections were permeabilized with 0.4% triton-X for 10 mins and blocked with 5% BSA + 0.01% Tween20. Primary antibody incubations for MYO7A 1:50 ((MYO7A 138-1, deposited to the DSHB by Orten, D.J. (DSHB Hybridoma Product MYO7A 138-1)), and SOX2 1:100 (Cell Signaling Technology, USA, Cat# 3579) was done in the blocking buffer for overnight at 4 o C. The following sections were washed thrice with PBS the following day, and secondary antibody incubations at 1:500 dilutions for Anti-Mouse AlexaFluor-647 (Thermo Scientific, USA, Cat# A32728TR) and anti-Rabbit AlexaFluor-488 (Thermo Scientific, USA, Cat# A32787TR) were done for 1hr at room temperature, respectively. Slides were mounted with ProLong™ Glass Antifade (Thermo, USA, Cat# P36980) and images were acquired using Zeiss LSM 980 with AiryScan 2 (Zeiss, Germany) at Flow Cytometry Shared Resource (FCSR), University of Miami. All the images were analyzed using the Fiji Image Analysis Tool or ImageJ (Schindelin et al. 2012 ). Western blot analysis Cells were harvested in RIPA buffer supplemented with 1x HALT protease and phosphatase inhibitor (Thermo Scientific, USA, Cat# 78441). Protein quantification was performed using a Thermo Scientific™ Pierce™ BCA kit (Thermo Scientific, USA, Cat# 23227). Equal amounts of protein were then reduced and loaded onto a 4–20% Tris-Glycine gradient gel for separation, following the method described by Laemmli (Laemmli 1970 ). Subsequently, proteins were transferred onto a 0.22 µm PVDF membrane using the Turbo-trans Blot system (Biorad, USA). The membranes were then blocked in 5% BSA for 1.5 hours and incubated overnight at 4°C with primary PBXIP1 antibody (Proteintech, Thermo Scientific, USA, Cat# 12102-1-AP) diluted at 1:1000 in 5% BSA + TBST (TBS with 0.5% Tween). After washing with TBST, the blots were incubated with HRP-conjugated anti-rabbit goat secondary antibody (1:3000) diluted in 5% BSA + TBST for 1.5 hours at room temperature. Following the termination of antibody reactions, the blots were washed three times with TBST and developed using the West Pico Super-Signal ECL substrate (Thermo Scientific, USA, 37069). Finally, visualization was performed using FluorChemE (ProteinSimple, USA). mRNA expression analysis The expression of GREB1L, FGF3 , and PBXIP1 mRNA in monoclonal lines was analyzed using qRT-PCR. Total RNA was isolated with TRIzol Reagent (Thermo, USA, Cat# 15596026) according to the manufacturer’s instructions. cDNA was synthesized using qScript XLT cDNA SuperMix (Quanta Biosciences, USA, Cat# 9516-025). The primers amplifying the transcript were GREB1L sense 5’-CAGTTTCCTGGCATCACATTTC-3’ antisense 5’-GTAACCACACTGTCTCCTCTTC-3’; FGF3 sense 5’-ATTGCTCCTGGGTGGAAATTA-3’ antisense 5’-AGAGAGAAAGAGAGGGAGAGTG-3’; PBXIP1 sense 5’-GGCCTCTCTGCTAAGAACATAC-3’ antisense 5’-GATGCCATCCTCACCAAAGA-3’. Quantification and statistical analysis All the statistical analyses are performed using GraphPad Prism 10. Paired analyses were done using the student’s t-test. Multiple comparisons were performed using one-way ANOVA with Tukey’s multiple comparisons test. The results are expressed as Mean ± SEM; a statistical difference of p ≤ 0.05 was considered significant. The significant differences are marked with (*) whenever comparisons were made between GREB1L c.556T>C , FGF3c .493A>G , FGF3 KO , GREB1L KO , PBXIP1 c.1722G>A and their respective controls GREB1L WT , FGF3 WT , PBXIP1 WT . Details about the number of replicates and significance notation are provided in the figure legends. Results Identification of candidate genes and variants In our ongoing studies on HL, we identified an individual homozygous for an FGF3 variant (c.493A > G; p.Arg165Gly) and another individual who is heterozygous for a GREB1L variant (c.556T > C; p.Cys186Arg) (Fig. 1 a-b and Supplementary Table S3). The proband with the FGF3 variant is a 6-year-old male with bilateral congenital profound deafness whose temporal bone CT scan showed bilateral labyrinthine aplasia. Physical examination revealed normal-sized but prominent external ears and widely spaced lower incisor teeth. The parents were first cousins without HL. The proband with the GREB1L variant is a 5-year-old male with congenital profound sensorineural HL in the left ear associated with common cavity malformation (Supplementary Figure S2A). Hearing and imaging studies in the right ear are normal. An ultrasound examination for kidney and urinary system anomalies is unremarkable. His developmental history is normal. Parents have normal hearing and do not have the variant detected in the proband. Although both variants are highly conserved among different vertebrate species (Fig. 1 b), following ACMG guidelines, both variants are interpreted as VUS (Supplementary Table S3). Thus, increasing the certainty of their pathogenicity depends on functional abnormalities that additional studies can demonstrate. In the same cohort, we identified a candidate gene, PBXIP1 , for bilateral cochlear aplasia. The proband is an 8-year-old female who was born with bilateral profound sensorineural HL without additional abnormalities. CT scans of the temporal bone showed bilateral cochlear aplasia (Supplementary Figure S2B). Initially, the search for variants in known deafness genes ended with no variant of interest that could be associated with HL in this family. Parents were consanguineous and there were 11 regions of homozygosity greater than 2Mb in the proband (Supplementary Table S4). After filtering of variants and Sanger sequencing of family members, only one variant co-segregated with the phenotype: the proband is homozygous for the nonsense variant c.1722G > A (p.Trp574*) in PBXIP1 and parents are heterozygous (Fig. 1 a and Supplementary Table S3). Variants in this gene have not been previously associated with human phenotypes. PBXIP1 is present in the nucleoplasm and cytoplasm of cells in most tissues ( https://www.proteinatlas.org/ ); the gEAR database ( https://www.umgear.org/ ) shows its highest expression in prosensory duct floor and lateral duct floor in developing human cochlea along with significant expression in all other parts of cochlea, such as roof, periotic mesenchymal cells, and medial duct floor (Supplementary Figures S3 and S4) (van der Valk et al. 2023 ). Generation of isogenic knockout and variant-bearing iPSCs via CRISPR/Cas9 The GREB1L c.556T>C , GREB1L KO , FGF3 c.493A>G , FGF3 KO , and PBXIP1 c.1722G>A variants introduced into a control iPSC cell line were confirmed by Sanger sequencing (Supplementary Figure S1 ). Sanger sequencing of the in silico predicted off-target genomic loci showed no unintended mutation had been introduced into the monoclonal iPSC lines (Supplementary Table S2). All the monoclonal iPSC lines generated in the study retained their pluripotency as assessed by immunocytochemical staining for the pluripotency markers OCT4 and TRA1-60 (Fig. 1 c). Effects of knockout and variant incorporation on gene expression Analysis of gene and protein levels for the missense variant-bearing monoclonal iPSC lines GREB1L c.556T>C (Figs. 2 a, d, and g) and FGF3 c.493A>G (Figs. 2 b, e, and h) showed no significant differences in expression compared to their respective wild-type (WT) parental iPSC line (i.e. GREB1L WT and FGF3 WT ). In contrast, the monoclonal iPSC lines bearing the CRISPR/Cas9-derived knockout of GREB1L ( GREB1L KO ) (Figs. 2 a, d, and g) and FGF3 ( FGF3 KO ) (Figs. 2 b, e, and h) showed significantly abrogated expression of these genes compared to their respective parental lines. The PBXIP1 c.1722G>A nonsense variant is predicted to produce a premature termination codon, potentially resulting in a truncated protein. Nonsense-mediated decay (NMD) is an mRNA quality control mechanism eukaryotic cells use to degrade mRNAs that harbor premature termination codons. The PBXIP1 c.1722G > A variant is located 375 nucleotides away from the last intron, which makes NMD likely. A significant decrease in PBXIP1 mRNA expression in monoclonal line bearing c.1722G > A was observed (p ≤ 0.001) consistent with the mRNA undergoing NMD (Fig. 2 c). Immunofluorescent analysis of PBXIP1 showed a reduction of PBXIP1 in the mutant cells compared to WT cells (Figs. 2 f and i). Immunoblot analysis of whole cell lysates from PBXIP1 WT and PBXIP1 c.1722G>A lines show that PBXIP1 c.1722G>A variant produced truncated protein as indicated by the detection of a smaller band of around 62.83 kilodaltons consistent with the predicted size of the truncated protein ( https://www.bioinformatics.org/sms/prot_mw.html ) (Fig. 2 j). In addition to the production of a truncated protein, the amount of PBXIP1 protein was decreased in the PBXIP1c.1722G > A cells compared to the WT parental line. These results suggest that c.1722G > A leads to NMD with a reduced amount of truncated PBXIP1 protein being produced. Inner ear organoids from variant-bearing iPSCs show size reduction during development To determine if candidate variants from patients with cochlear malformations altered the development of the inner ear, IEOs were derived from the monoclonal variant bearing iPSC lines and the isogenic control iPSC lines. Our initial analysis focused on the morphometric characteristics of the IEOs. IEOs were produced using an aggregation approach in 3D suspension culture. All organoids were initiated by seeding 3,500 iPSCs per well of a 96-well low adherence plate. The cross-sectional area of the resulting organoids (variant bearing compared to the WT isogenic control lines) was assessed on day 25 (Fig. 3 c and f) and 35 (Fig. 3 d and f) after the initiation of IEO differentiation. On day 25, we found that the IEOs derived from all the isogenic controls – GREB1L WT , FGF3 WT , and PBXIP1 WT have similar cross-sectional areas. However, all variants of interest show growth restrictions compared to their respective control counterparts - GREB1L c.556T>C and GREB1L KO compared to GREB1L WT ; FGF3 c.493A>G and FGF3 KO compared to FGF3 WT ; PBXIP1 c.1722G>A compared to PBXIP1 WT (Fig. 3 c and f). Consistently, the cross-sectional area analysis on day 35 IEOs showed that the variant bearing lines had decreased area compared to their respective isogenic WT controls (Fig. 3 d and f). Of note, FGF3 c.493A>G shows the smallest cross-sectional area of all IEOs (p ≤ 0.001). This is not unexpected since FGF3 has been shown to work for very early otic structure development (Jeong et al. 2018 ). In addition to the decrease in cross-sectional area, the HL variant-bearing IEOs had reduced cell confluence in otic vesicles and significantly reduced number of otic vesicles/IEO compared to their isogenic control IEOs (Supplementary Figure S5). Inner ear organoids from variant-bearing iPSCs show a lower abundance of otic progenitor markers PAX2/PAX8 are essential markers for early otic vesicle development and have been previously reported to be associated with IEO development (Li et al. 2024 ). We have examined PAX2/PAX8 abundance (Figs. 3 a, 3 b, and 3 e) in the otic vesicular area and found that variant-bearing iPSC-derived IEOs showed a lower abundance of early otic progenitor markers (PAX8/PAX2) in GREB1L c.556T>C (p ≤ 0.01), FGF3 c.493A>G (p ≤ 0.01), PBXIP1 c.1722G>A (p ≤ 0.01), GREB1L KO (p ≤ 0.01), and FGF3 KO (p ≤ 0.001). PBXIP1 c.1722G>A showed a significant reduction in the abundance of otic progenitor markers (PAX8/PAX2) and overall size reduction, suggesting its pathogenicity in early otic development. Inner ear organoids from variant-bearing iPSCs lack hair cell-like populations from mature organoids MYO7A and SOX2-positive cells in IEOs are crucial for mimicking the development of the inner ear. These cells define prosensory cell populations that give rise to hair cells and supporting cells essential for hearing and balance functions. In IEOs, MYO7A + and SOX2 + hair cell-like cells indicate the development of functional sensory epithelia resembling that found in the inner ear. Organoids derived from GREB1L c.556T>C , FGF3 c.493A>G , PBXIP1 c.1722G>A , GREB1L KO , FGF3 KO , and PBXIP1 c.1722G>A show significantly reduced (p ≤ 0.001) MYO7A + population in comparison to their isogenic controls GREB1L WT , FGF3 WT , and PBXIP1 WT , respectively (Fig. 4 a, 4 b). In the inner ear, MYO7A is restricted to the sensory cells of the vestibular and cochlear organs (hair cells). The significant decrease in MYO7A in the HL-variant bearing IEOs suggests a defect in the early stages of hair cell development. Additionally, all the organoids derived from variant-bearing iPSC lines show low levels of SOX2, suggesting the presence of a rudimentary supporting cell population (Fig. 4 a, 4 c). Discussion Here, we present our proof of principle study for the use of human IEOs to rapidly validate uncertain DNA variants detected in patients with cochlear malformations. Moreover, we generated isogenic cell lines from control iPSCs, eliminating the need to obtain patient cells, which can be difficult and time-consuming. From the start of our experiments, the study takes approximately 90 days, making this approach applicable in clinical diagnostics and gene discovery for cochlear malformations. FGF3 is a small protein (Fig. 1 b) serving as a signaling molecule released from the hindbrain during early development. It is implicated in forming prospective sensory tissues along with FGF10. In mice, Fgf3 is expressed in the otic vesicle during otic placode induction and subsequently at the early stage of inner ear morphogenesis (Hatch et al. 2007 ; Wilkinson et al. 1988 ). Models with loss of function variants in FGF3 demonstrated perturbed expression of WNT-induced genes and ultimate patterning defects in the dorsal otocyst. However, the otic genes expressed in ventral otocyst for cochlea development were not critically influenced (Hatch et al. 2007 ). To the best of our knowledge, our study demonstrates, for the first time, early developmental anomalies of the inner ear in human IEOs and provides functional proof of the role of FGF3 variants detected in affected individuals in driving impairment in early developmental stages of the inner ear. GREB1L encodes a GREB1-like retinoic acid receptor coactivator (Fig. 1 b). Several de novo and inherited variants in this gene have recently been shown to cause bilateral HL with malformed cochleae (Schrauwen et al. 2018a ; Schrauwen et al. 2020a ; Schrauwen et al. 2020b ). However, no prominent hearing or vestibular defects were observed in zebrafish models for this gene (Schrauwen et al. 2018b ). Greb1l knockout mice die embryonically, while heterozygous or compound heterozygous mice showed no significant hearing phenotype ( www.mousephenotype.org ) (De Tomasi et al. 2017 ). In contrast with previously reported individuals with GREB1L variants, our proband has unilateral cochlear aplasia with normal hearing in the other ear. GREB1L is a neural crest regulatory molecule implicated in the embryonic development of many tissues, including the cochlea (Plouhinec et al. 2014 ). As mutations in other genes involved in neural crest cell migration, such as PAX3 and KITL , have also been shown to cause unilateral HL, the observed clinical phenotype in our proband is not completely surprising (Lee et al. 2023 ; Zazo et al. 2015). In this study, we show that the mutant and knockout GREB1L organoids show decreased expression of otic progenitors and the absence of sensory cells (MYO7A + cells) at the mature stage tested compared to isogenic controls organoids, providing a clarified role of this gene in inner ear development. PBXIP1 encodes the PBX homeobox-interacting protein 1 (Fig. 1 b), which is involved in cell differentiation through the PI3K/AKT pathway (Manavathi et al. 2012 ). Previously no evidence has existed that this gene causes inner ear anomalies and deafness in mice or humans. Detection of a loss of function variant, its expression in the cochlea, and role in cell differentiation, made this gene a candidate for inner ear anomalies. The phenotypic and expression data from the organoids establish PBXIP1`s role in the development of the inner ear. It is important to point out that the observed abnormalities are identical to those caused by variants in FGF3 and GREB1L , two established genes for cochlear malformations. Additional families with PBXIP1 variants will secure the establishment of this gene as causing HL. In summary, we demonstrate the role of PBXIP1 , FGF3 , and GREB1L in developing otic cells and their differentiation into sensory cells in human organoids. As a study model for HL, organoids may serve as a fast and reliable system for investigating genes involved in cochlear malformations. Declarations Author contributions Conceptualization: MFZ and MT; Data collection: MFZ, MR, DD, SS, AM, TK, SF; Formal analysis: MFZ, MR, DD; Funding acquisition: MT; Writing original draft: MFZ, MR and MT; Writing-review and editing: MFZ, MR, DD, AM, SS, TK, SF, BAD, SG, DMD and MT. Acknowledgements The authors are grateful to the participating families and clinical team for participation and cooperation. We are also thankful to iPSC core at University of Miami for providing the cell lines and facilities for use. Funding This work was supported by grant NIH R01DC009645 and R01DC012836 to MT. Declaration of interest None to declare. Supplementary information The supplementary information contains 4 tables and five figures. References Adadey SM, Aboagye ET, Esoh K, Acharya A, Bharadwaj T, Lin NS, Amenga-Etego L, Awandare GA, Schrauwen I, Leal SM, Wonkam A (2022) A novel autosomal dominant GREB1L variant associated with non-syndromic hearing impairment in Ghana. BMC Med Genomics, 15, 237. Bae S, Park J, Kim J-S (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics, 30, 1473-1475. 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Supplementary Files Graphicalabstract.jpeg SupplementarymaterialHG05242024.docx Cite Share Download PDF Status: Published Journal Publication published 09 Jan, 2025 Read the published version in Human Genetics → Version 1 posted Editorial decision: Revision requested 09 Jul, 2024 Reviews received at journal 07 Jul, 2024 Reviews received at journal 25 Jun, 2024 Reviewers agreed at journal 20 Jun, 2024 Reviews received at journal 11 Jun, 2024 Reviews received at journal 06 Jun, 2024 Reviewers agreed at journal 02 Jun, 2024 Reviewers agreed at journal 30 May, 2024 Reviewers agreed at journal 28 May, 2024 Reviewers invited by journal 27 May, 2024 Submission checks completed at journal 25 May, 2024 Editor assigned by journal 25 May, 2024 First submitted to journal 24 May, 2024 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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(a)\\u003c/strong\\u003e Graphical representation of participating families, segregating variants, and their location on respective gene and protein. Squares represent males, and circles indicate females. Filled symbols show affected individuals. Arrows point to the probands (p) of the respective families. The mutated residue is mentioned with an arrow in each chromatogram as well as in the protein schematic. \\u003cstrong\\u003e(b) \\u003c/strong\\u003eGraphical representation of mutated genes, proteins, and localization of variants.\\u003cstrong\\u003e (c)\\u003c/strong\\u003e Immunostaining of OCT4 and TRA-1-60 in monoclonal lines derived after CRISPR/Cas9 editing in \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003csup\\u003e \\u003c/sup\\u003e\\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e \\u003c/em\\u003eand\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e, respectively\\u003cem\\u003e.\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSP:\\u003c/strong\\u003e Signal Peptide, \\u003cstrong\\u003eMBD:\\u003c/strong\\u003e Microtubule Binding Domain, \\u003cstrong\\u003eCC1:\\u003c/strong\\u003e Coil-coiled Domain 1, \\u003cstrong\\u003eCC2:\\u003c/strong\\u003e Coil-coiled Domain 2, \\u003cstrong\\u003eNLS:\\u003c/strong\\u003e Nuclear Localization Sequence, \\u003cstrong\\u003ePID:\\u003c/strong\\u003e PBX1 interacting Domain, \\u003cstrong\\u003eERID:\\u003c/strong\\u003e Erα interacting Domain, \\u003cstrong\\u003eNES:\\u003c/strong\\u003e Nuclear Export Sequence.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1600dpi.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4474071/v1/160da5bc6370b8af7a0e95af.jpeg\"},{\"id\":58150755,\"identity\":\"8b32834f-af7b-4b45-b448-db22f509d514\",\"added_by\":\"auto\",\"created_at\":\"2024-06-11 20:08:34\",\"extension\":\"jpeg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":18490225,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eExpression of \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eGREB1L, FGF3,\\u003c/strong\\u003e\\u003c/em\\u003e\\u003csup\\u003e\\u003cstrong\\u003e \\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cem\\u003e\\u003cstrong\\u003eand PBXIP1\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e in WT and edited lines. (a)\\u003c/strong\\u003e qRT-PCR analysis of \\u003cem\\u003eGREB1L\\u003c/em\\u003e expression levels in \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e, and\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e.\\u003cem\\u003e \\u003c/em\\u003e\\u003cstrong\\u003e(b) \\u003c/strong\\u003eqRT-PCR analysis of \\u003cem\\u003eFGF3 \\u003c/em\\u003eexpression levels in \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003ec\\u003c/sup\\u003e\\u003csup\\u003e\\u003cem\\u003e.493A\\u0026gt;G \\u003c/em\\u003e\\u003c/sup\\u003eand\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e.\\u003cem\\u003e \\u003c/em\\u003e\\u003cstrong\\u003e(c)\\u003c/strong\\u003e qRT-PCR analysis of \\u003cem\\u003ePBXIP1 \\u003c/em\\u003eexpression levels in\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e and PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e. \\u003csup\\u003e\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003e(d, g)\\u003c/strong\\u003e Effect of missense p.Cys186Arg variant on GREB1L in iPSCs. \\u003cstrong\\u003e(e, h) \\u003c/strong\\u003eEffect of missense p.Arg165Gly variant on FGF3 in iPSCs. \\u003cstrong\\u003e(f, i) \\u003c/strong\\u003eEffect of nonsense variant p.Trp574* showing slight change in signal for PBXIP1 \\u003cstrong\\u003e(j)\\u003c/strong\\u003e Western blot showing the smaller sized band in mutated PBXIP1. The results are expressed as Mean ± SEM (n=3), and the statistical difference of p ≤0.05 was considered significant. The significant differences are marked with (*) whenever comparisons were made between edited lines\\u003csup\\u003e\\u003cem\\u003e \\u003c/em\\u003e\\u003c/sup\\u003eand their respective controls\\u003cem\\u003e.\\u003c/em\\u003e *: p\\u0026lt;0.05; \\u003cem\\u003e**\\u003c/em\\u003e:\\u003cem\\u003e p\\u0026lt;0.01\\u003c/em\\u003e;\\u003cem\\u003e ***\\u003c/em\\u003e:\\u003cem\\u003e p\\u0026lt;0.001\\u003c/em\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4474071/v1/2b4f7fe00b6dc25c95b3da50.jpeg\"},{\"id\":58150757,\"identity\":\"10c8dcf0-b873-49cd-b4dc-32e79660b305\",\"added_by\":\"auto\",\"created_at\":\"2024-06-11 20:08:35\",\"extension\":\"jpeg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":20944802,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eDifferences of early otic lineage markers and cross-sectional area in WT and edited lines. (a, b)\\u003c/strong\\u003e PAX2 and PAX8 quantification in inner ear organoids (IEOs) (n=3), respectively. The results are expressed as Mean ± SD, and the statistical difference of p ≤0.05 was considered significant. The significant differences are marked with (*) whenever comparisons were made between \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003csup\\u003e\\u003cem\\u003e \\u003c/em\\u003e\\u003c/sup\\u003eand their respective controls \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e.\\u003cem\\u003e \\u003c/em\\u003e\\u003cstrong\\u003e(c, d)\\u003c/strong\\u003e Cross-sectional area analysis of day 25 and day 35 IEO (n=3), respectively. Area quantifications were done using ImageJ; results are expressed as Mean ±SD, and the statistical difference of p ≤0.05 was considered significant. The significant differences are marked with (*) whenever comparisons were made between \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003csup\\u003e\\u003cem\\u003e \\u003c/em\\u003e\\u003c/sup\\u003eand their respective controls \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e.\\u003cem\\u003e \\u003c/em\\u003e\\u003cstrong\\u003e(e)\\u003c/strong\\u003e Representative images with PAX2/PAX8 immunostaining in \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e \\u003c/em\\u003eand\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e. \\u003cstrong\\u003e(f)\\u003c/strong\\u003e Representative H\\u0026amp;E images of \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e \\u003c/em\\u003eand\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A \\u003c/em\\u003e\\u003c/sup\\u003eIEOs on day 25 and day35. *: p\\u0026lt;0.05; **: p\\u0026lt;0.01; ***: p\\u0026lt;0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4474071/v1/8999066c2a36f2aa58917923.jpeg\"},{\"id\":58150756,\"identity\":\"4f23fb1b-c5a2-4f94-a784-86fd15eaa6c9\",\"added_by\":\"auto\",\"created_at\":\"2024-06-11 20:08:35\",\"extension\":\"jpeg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":22225306,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnalysis of the presence of mature hair cell-like populations. (a)\\u003c/strong\\u003e MYO7A and SOX2 localization in \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e \\u003c/em\\u003eand\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e. \\u003cstrong\\u003e(b-d)\\u003c/strong\\u003e Quantification of MYO7A and SOX2 signals in \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e \\u003c/em\\u003eand\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e, respectively (n=3). The results are expressed as Mean ±SEM, and the statistical difference of p ≤0.05 was considered significant. The significant differences are marked with (*) whenever comparisons were made between \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e GREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003csup\\u003e\\u003cem\\u003e \\u003c/em\\u003e\\u003c/sup\\u003eand their respective controls \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e FGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e,\\u003cem\\u003e PBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e.\\u003cem\\u003e \\u003c/em\\u003e*: p\\u0026lt;0.05; **: p\\u0026lt;0.01; ***: p\\u0026lt;0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4474071/v1/884b4cc212a3a67b809ab8e9.jpeg\"},{\"id\":73693971,\"identity\":\"26de7af6-7aa6-4075-a908-e4ec507633d6\",\"added_by\":\"auto\",\"created_at\":\"2025-01-13 16:10:09\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":77670825,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4474071/v1/fc665494-34a5-4b56-b613-d17e7d075d3f.pdf\"},{\"id\":58150751,\"identity\":\"660c87ef-5968-4ac3-baa6-6ebabb2e8b2e\",\"added_by\":\"auto\",\"created_at\":\"2024-06-11 20:08:34\",\"extension\":\"jpeg\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1315691,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Graphicalabstract.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4474071/v1/1acf8d9c0cdf9ddf550e77ad.jpeg\"},{\"id\":58150753,\"identity\":\"c6e0c63c-f99b-4345-a1f4-32efc8241896\",\"added_by\":\"auto\",\"created_at\":\"2024-06-11 20:08:34\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":6864071,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementarymaterialHG05242024.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4474071/v1/8354143264bec159ed81bdec.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Human Organoids for Rapid Validation of Gene Variants Linked to Cochlear Malformations\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eApproximately one in 500 newborns are diagnosed with permanent hearing loss (HL) (Bainbridge \\u0026amp; Wallhagen \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Petit et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Inner ear anomalies (IEAs) affecting the cochlea are reported in about 25% of these children (Bamiou et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e; Brotto et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Ocak et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Studies performed in various animal models shed light on fundamental mechanisms governing vertebrate inner ear development (Chen et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Torii et al. \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Whitfield et al. \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e). However, mutations in relatively few of the genes recognized in animal model systems have been shown to cause cochlear malformations in humans.\\u003c/p\\u003e \\u003cp\\u003eInterpretation of the identified variants through genetic testing in HL requires collating and analyzing the available literature for supporting evidence, followed by a formal classification based on this evidence (Patel et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). A recent study shows that 70% of the identified missense variants in people with HL are classified as variants of uncertain significance (VUS) (Tollefson et al. \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Functional studies can help establish causality in these cases, which are often lacked in published studies.\\u003c/p\\u003e \\u003cp\\u003eIt is difficult to directly examine the molecular and cellular processes leading to the establishment of the human inner ear. It is located deep within the skull surrounded by bone and other tissues (Nomura et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). There is also a paucity of biopsy material appropriate for molecular analysis, particularly in the early stages of development. Finally, the range of testing that can be carried out on human embryos and fetuses is limited by ethical considerations, further complicating research (Plomer \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe recent advancements in stem cell technology have enabled us to create three-dimensional organoids similar to developing human ears using human induced pluripotent stem cells (iPSCs) (Doda et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Koehler et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Qi et al. \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Steinhart et al. \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; van der Valk et al. \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). These iPSC-derived structures imitate the basic cartography of the developing ear and can, therefore, be used to investigate how development at the cellular stage happens in this organ (Doda et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Koehler et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Romano et al. \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Steinhart et al. \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; van der Valk et al. \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Generating inner ear organoids (IEOs) involves differentiating pluripotent stem cells into otic placode-like cells. These cells later form cavities resembling otic vesicles that contain hair cells, supporting cells, and neurons like those seen in the inner ear after maturation (Koehler et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Moore et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Nie \\u0026amp; Hashino \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Romano et al. \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Tang et al. \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Organoids hold several advantages over animal models, including the ability to study human-specific aspects of inner ear development and disease with much shorter timelines than animal models (Qi et al. \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). These lines retain the genetic architecture of the cells from whom the lines were derived and are amenable to genomic engineering approaches, including CRISPR/Cas9-based methods (Rivron et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eIn this study, we investigate the impact of DNA variants in known cochlear malformation genes \\u003cem\\u003eFGF3\\u003c/em\\u003e (Fibroblast Growth Factor 3) and \\u003cem\\u003eGREB1L\\u003c/em\\u003e (GREB1 Like Retinoic Acid Receptor Coactivator), along with a candidate gene, \\u003cem\\u003ePBXIP1\\u003c/em\\u003e (PBX Homeobox Interacting Protein 1), in inner ear development via IEOs. \\u003cem\\u003eFGF3\\u003c/em\\u003e participates in the early development of the inner ear (Tekin et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e). Pathogenic variants in \\u003cem\\u003eFGF3\\u003c/em\\u003e cause deafness with LAMM (Labyrinthine Aplasia, Microtia, and Microphthalmia; OMIM 610706), an autosomal recessively-inherited syndrome characterized by missing inner ear structures as well as small external ears and teeth. \\u003cem\\u003eGREB1L\\u003c/em\\u003e has a role in neural crest development and retinoic acid pathway (Brophy et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Vega-Lopez et al. \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Several reports have associated \\u003cem\\u003eGREB1L\\u003c/em\\u003e variants with autosomal dominant urogenital or cochlear anomalies (Adadey et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; De Tomasi et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Herlin et al. \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Jacquinet et al. \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Kim et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Schrauwen et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2018a\\u003c/span\\u003e). In two patients with cochlear malformations, we detected previously unreported missense variants in \\u003cem\\u003eFGF3\\u003c/em\\u003e and \\u003cem\\u003eGREB1L\\u003c/em\\u003e, interpreted as VUS. In another patient, we detected a nonsense variant in \\u003cem\\u003ePBXIP1\\u003c/em\\u003e, a gene not previously associated with a human phenotype. By establishing monoclonal IEOs for knockout and patient-specific variants of these genes, we show differences in organoid size, number of luminal spaces (otic vesicles), and lower expression of otic vesicle markers in both knockout and variant-bearing organoids compared to controls.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEnrollment of subjects and exome sequencing of probands\\u003c/h2\\u003e \\u003cp\\u003eThe study was approved by the Institutional Review Board (Protocol no. 20081138) at the University of Miami (USA) and Ethics Committee (Protocol no. 012413) at Ankara University Medical School (T\\u0026uuml;rkiye). Written informed consents were obtained from all participants and in the case of minors, it was obtained from parents. Audiometry was performed to measure average hearing thresholds for all participants under standard conditions and guidelines. All affected individuals were examined by a clinical geneticist and otolaryngologist.\\u003c/p\\u003e \\u003cp\\u003eFor exome sequencing (ES), we followed a recently published protocol (Ramzan et al. \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Briefly, single nucleotide, indel, and copy number variants (CNVs) in all known deafness genes were analyzed. Variants were retained for further evaluation if they had an allele frequency of less than 0.01. The variants in all known genes for HL were analyzed using a larger list retrieved from hereditary hearing loss homepage (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://hereditaryhearingloss.org/\\u003c/span\\u003e\\u003cspan address=\\\"https://hereditaryhearingloss.org/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) and OMIM. If there was no candidate variant identified in known deafness genes, ES data were re-examined for all genes containing variants less than 0.01 allele frequency. CNV analysis with ES data used CoNIFER v.02.2 with default parameters. It uses a singular value decomposition method to correct systematic biases and identifies a CNV call if the corrected signal reaches a predefined threshold at no less than three consecutive exons (Krumm et al. \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). We performed Sanger sequencing to confirm candidate variants and evaluate segregation within respective families.\\u003c/p\\u003e \\u003cp\\u003eCandidate variants were classified according to ACMG and ClinGen Hearing Loss Expert Panel (HL-EP) specifications, aligned with the ACMG/AMP Variant Interpretation Guidelines (Oza et al. \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Richards et al. \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Tavtigian et al. \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). AlphaMissense was included in the prediction of pathogenicity as described (Zhuo et al. \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eiPSC maintenance and validation\\u003c/h2\\u003e \\u003cp\\u003eCells from a validated male European lymphocyte-derived iPSC line (ASE9203) were cultured on Vitronectin (Thermo Scientific, USA, Cat# A2858501) coated culture ware and maintained in E8-Flex (Thermo Scientific, USA, Cat# A2858501) media supplemented with 100 \\u0026micro;g/mL Normocin (Invivogen, USA, Cat# ant-nr-05), following the manufacturer's guidelines. Routine passages utilized 1x Revitacell (Thermo Scientific, USA, Cat# A2644501). We regularly assessed pluripotency markers OCT4 (Cell Signaling Technology, USA, Cat# 2840) and TRA-1-60 (Cell Signaling Technology, USA, Cat# 4746) through Immunocytochemistry (ICC).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGeneration of CRISPR/Cas9-edited variants\\u003c/h2\\u003e \\u003cp\\u003eWe generated monoclonal cell lines for \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eGREB1L\\u003c/em\\u003e \\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e, and \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e as previously described (Ramzan et al. \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Custom sgRNA and HDR donor blocks were designed and procured using the Alt-R-CRISPR/Cas9 platform (IDT); sequences are available in supplementary Table\\u0026nbsp;1. Cas9-gRNA ribonucleoprotein complexes were assembled under sterile conditions using 140 pmol sgRNA and 40 pmol EnGen\\u0026reg; Spy Cas9 HF1 (New England Biolabs, USA, Cat# M0667M) and incubated for 10 mins at room temperature. Following the incubation, 8x10\\u003csup\\u003e5\\u003c/sup\\u003e cells were suspended in the RNP complex and premixed 3 \\u0026micro;M HDR donor block in 80 \\u0026micro;L of nucleofector solution. The final volume of the nucleofection mix was 100 \\u0026micro;L. Nucleofections are performed using the P3-primary Cell 4D-Nucleofector Kit (Lonza, USA, Cat# V4XP-3024) and preprogrammed pulse protocol CA-137 in the 4D-Nucleofector system (Lonza). Post-nucleofections, iPSCs were cultured in E8-flex media supplemented with CloneR\\u0026trade;2 (Stem Cell Technologies, Canada, Cat# 100\\u0026ndash;0691) and 1 \\u0026micro;M of the HDR enhancer-V2 (IDT).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDNA isolation and confirmation of CRISPR/Cas9-edited cell pools and off-targets\\u003c/h2\\u003e \\u003cp\\u003eDNA isolation was done using QuickExtract DNA Extraction Solution (Biosearch Technologies, USA, Cat# QE09050); the resulting DNA was then PCR amplified using Phusion\\u0026reg; High-Fidelity PCR Master Mix (New England Biolabs, USA, Cat# M0531S) and sequenced with primers flanking (Supplementary Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e) the guide region and Sanger sequencing at Genewiz (Azenta Life Sciences). Outcomes of CRISPR/Cas9 knock-ins were assessed using ICE Analysis (Conant et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). For every guide sequence, off-target analysis was done using bioinformatic tools such as Cas-OFFinder (Bae et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e) and the sgRNA guide analysis tool from IDT in Coralville, Iowa, USA. The top five off-target sites were Sanger sequenced for flanking primers and analyzed for all the clonal lines used in this study (Supplementary Table S2).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIsolation of monoclonal lines\\u003c/h2\\u003e \\u003cp\\u003eSingle-cell monoclonal lines were isolated using Poisson distribution (Sanjurjo-Soriano et al. \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). CRISPR/Cas9 pools were briefly dissociated using StemPro accutase (Thermo Scientific, USA, Cat# A1110501). The resulting cell suspension was collected in E8-Flex media (Thermo Scientific, USA, Cat# A2858501) containing CloneR\\u0026trade;2 (Stem Cell Technologies, Canada, Cat# 100\\u0026ndash;0691) and centrifuged at 300 rpm. After centrifugation, the single-cell suspension was passed through 70 \\u0026micro;m strainers (SP Bel-Art, USA, Cat# H13680-0070), and the cells were then counted using a Countess II cell counter (Thermo Scientific, USA). Depending on the cell concentrations, serial dilutions were performed to achieve a typical concentration of 1 cell per 100 \\u0026micro;L of media. The cells were then plated in 96-well plates precoated with rhLaminin (Thermo Scientific, USA, Cat# A29249), and media changes were done with 1x CloneR\\u0026trade;2-containing E8 media every other day for the first week and then with 0.5x CloneR\\u0026trade;2 containing E8-Flex media till the single cell colonies reached a passaging confluence. Clonal lines were identified using Sanger sequencing, and from the same experiments, clonal lines bearing large frameshift deletions disrupting the open reading frame were used as knockouts for \\u003cem\\u003eFGF3\\u003c/em\\u003e and \\u003cem\\u003eGREB1L\\u003c/em\\u003e (Supplementary Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGeneration of inner ear organoids\\u003c/h2\\u003e \\u003cp\\u003eIEOs were generated using the previously described protocol by (Moore et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Briefly, on the first day of differentiation (day \\u0026minus;\\u0026thinsp;2), cells were dissociated with StemPro accutase (Thermo Scientific, USA, A1110501). The cells were then passed through 70 \\u0026micro;m strainers (SP Bel-Art, USA, Cat# H13680-0070), and 3500 cells/well were plated in 96-well Nunc-Sphera U-bottom plates (Thermo Scientific, USA, Cat# 174925) in 100 \\u0026micro;L of E8-Flex media containing 20 \\u0026micro;M Y-27632 (Stem Cell Technologies, Canada, Cat# 72304). After 4 hours of initial plating, 100 \\u0026micro;L of fresh E8-Flex media was added to each well, bringing the final concentration of Y-27632 to 10 \\u0026micro;M. All the aggregates were collected on day 0, washed thrice with E6-Medium (Thermo Scientific, USA, Cat# A1516401) and IEO differentiation media (E6-Medium, 2% GFR-Matrigel (Corning, Sigma Aldrich, Cat# 354230) 10 \\u0026micro;M SB431542 (Stem Cell Technologies, Canada, Cat# 72232), 4 ng/mL FGF2 (Peprotech, Thermo, USA, Cat# 100-18B) and 2.5 ng/mL BMP4 (Stemgent, USA, Cat# 03\\u0026ndash;0007)). Cell aggregates were transferred to a new 96-well Nunc-Sphera U-bottom plate in 100 \\u0026micro;L of differentiation media. On day 3, 25 \\u0026micro;L E6-Medium containing 100 \\u0026micro;g/mL Normocin, 250 ng/mL FGF-2, and 200 nM LDN193189 (Stem Cell Technologies, Canada, Cat# 72147) were added, bringing the volume to 125 \\u0026micro;L. On days 6 and 9 of differentiation, cell aggregates were washed thrice with E6 media and then thrice with E6 media containing 100 \\u0026micro;g/mL Normocin, 3 mM CHIR99021 (Stemgent, USA, Cat# 04-0004-02), 200 nM LDN193189, and 50 ng/mL FGF2. The cell aggregates were transferred to fresh 96-well U-bottom plates in 250 \\u0026micro;L of new media.\\u003c/p\\u003e \\u003cp\\u003eCell aggregates were washed thrice on the 11th day of differentiation with Advanced DMEM/F12 (Thermo Scientific, USA, Cat# 12634010). Then they were transferred to 90 mm Nunc low-attachment plates (Thermo Scientific, USA, Cat# 174945) in 10 mL of organoid maturation media (OMM) comprising Advanced DMEM/F12, Neurobasal medium (Thermo Scientific, USA, Cat# 21103049), 1X Glutamax, 0.5X B-27 supplement without Vitamin A, 0.5X N-2 supplement, 0.1 mM 2-Mercaptoethanol, 100 \\u0026micro;g/mL Normocin, and supplemented with 1% GFR-Matrigel and 3 \\u0026micro;M CHIR99021. On days 13 and 15, culture media was changed to OMM\\u0026thinsp;+\\u0026thinsp;3 mM CHIR99021\\u0026thinsp;+\\u0026thinsp;1 mM Purmorphamine (Stem Cell Technologies, Canada, Cat# 72202). On day 18, aggregates were washed to eliminate CHIR99021, and the media was changed to OMM\\u0026thinsp;+\\u0026thinsp;3 mM IWP-2 (Stem Cell Technologies, Canada, Cat# 72122)\\u0026thinsp;+\\u0026thinsp;1 mM Purmorphamine. The media was changed on day 20, with fresh media used on day 18. On day 22, cultures were washed and transferred to an anti-adhere solution coated with a low attachment 100 mm culture dish in OMM-only. The cultures received half media change every 3rd day and complete media change every 7th day until day 60. Samples were collected on day 25 and day 35 for RNA sequencing and on day 25, day 35, and day 60 for immunohistochemistry.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunohistochemistry and sectioning\\u003c/h2\\u003e \\u003cp\\u003eIEOs on day 25 and day 60 were collected, washed with PBS twice, fixed with 4% paraformaldehyde, and processed at the Cancer Modeling Shared Resource core, Sylvester Cancer Center, University of Miami. Serial sections of 5 \\u0026micro;m thickness were antigen-retrieved using 10 mM citrate buffer pH6. For immunohistochemistry, sections were permeabilized with 0.4% triton-X for 10 mins and blocked with 5% BSA\\u0026thinsp;+\\u0026thinsp;0.01% Tween20. Primary antibody incubations for MYO7A 1:50 ((MYO7A 138-1, deposited to the DSHB by Orten, D.J. (DSHB Hybridoma Product MYO7A 138-1)), and SOX2 1:100 (Cell Signaling Technology, USA, Cat# 3579) was done in the blocking buffer for overnight at 4\\u003csup\\u003eo\\u003c/sup\\u003eC. The following sections were washed thrice with PBS the following day, and secondary antibody incubations at 1:500 dilutions for Anti-Mouse AlexaFluor-647 (Thermo Scientific, USA, Cat# A32728TR) and anti-Rabbit AlexaFluor-488 (Thermo Scientific, USA, Cat# A32787TR) were done for 1hr at room temperature, respectively. Slides were mounted with ProLong\\u0026trade; Glass Antifade (Thermo, USA, Cat# P36980) and images were acquired using Zeiss LSM 980 with AiryScan 2 (Zeiss, Germany) at Flow Cytometry Shared Resource (FCSR), University of Miami. All the images were analyzed using the Fiji Image Analysis Tool or ImageJ (Schindelin et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern blot analysis\\u003c/h2\\u003e \\u003cp\\u003eCells were harvested in RIPA buffer supplemented with 1x HALT protease and phosphatase inhibitor (Thermo Scientific, USA, Cat# 78441). Protein quantification was performed using a Thermo Scientific\\u0026trade; Pierce\\u0026trade; BCA kit (Thermo Scientific, USA, Cat# 23227). Equal amounts of protein were then reduced and loaded onto a 4\\u0026ndash;20% Tris-Glycine gradient gel for separation, following the method described by Laemmli (Laemmli \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e1970\\u003c/span\\u003e). Subsequently, proteins were transferred onto a 0.22 \\u0026micro;m PVDF membrane using the Turbo-trans Blot system (Biorad, USA). The membranes were then blocked in 5% BSA for 1.5 hours and incubated overnight at 4\\u0026deg;C with primary PBXIP1 antibody (Proteintech, Thermo Scientific, USA, Cat# 12102-1-AP) diluted at 1:1000 in 5% BSA\\u0026thinsp;+\\u0026thinsp;TBST (TBS with 0.5% Tween). After washing with TBST, the blots were incubated with HRP-conjugated anti-rabbit goat secondary antibody (1:3000) diluted in 5% BSA\\u0026thinsp;+\\u0026thinsp;TBST for 1.5 hours at room temperature. Following the termination of antibody reactions, the blots were washed three times with TBST and developed using the West Pico Super-Signal ECL substrate (Thermo Scientific, USA, 37069). Finally, visualization was performed using FluorChemE (ProteinSimple, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003emRNA expression analysis\\u003c/h2\\u003e \\u003cp\\u003eThe expression of \\u003cem\\u003eGREB1L, FGF3\\u003c/em\\u003e, and \\u003cem\\u003ePBXIP1\\u003c/em\\u003e mRNA in monoclonal lines was analyzed using qRT-PCR. Total RNA was isolated with TRIzol Reagent (Thermo, USA, Cat# 15596026) according to the manufacturer\\u0026rsquo;s instructions. cDNA was synthesized using qScript XLT cDNA SuperMix (Quanta Biosciences, USA, Cat# 9516-025). The primers amplifying the transcript were \\u003cem\\u003eGREB1L\\u003c/em\\u003e sense 5\\u0026rsquo;-CAGTTTCCTGGCATCACATTTC-3\\u0026rsquo; antisense 5\\u0026rsquo;-GTAACCACACTGTCTCCTCTTC-3\\u0026rsquo;; \\u003cem\\u003eFGF3\\u003c/em\\u003e sense 5\\u0026rsquo;-ATTGCTCCTGGGTGGAAATTA-3\\u0026rsquo; antisense 5\\u0026rsquo;-AGAGAGAAAGAGAGGGAGAGTG-3\\u0026rsquo;; \\u003cem\\u003ePBXIP1\\u003c/em\\u003e sense 5\\u0026rsquo;-GGCCTCTCTGCTAAGAACATAC-3\\u0026rsquo; antisense 5\\u0026rsquo;-GATGCCATCCTCACCAAAGA-3\\u0026rsquo;.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eQuantification and statistical analysis\\u003c/h2\\u003e \\u003cp\\u003eAll the statistical analyses are performed using GraphPad Prism 10. Paired analyses were done using the student\\u0026rsquo;s t-test. Multiple comparisons were performed using one-way ANOVA with Tukey\\u0026rsquo;s multiple comparisons test. The results are expressed as Mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SEM; a statistical difference of p\\u0026thinsp;\\u0026le;\\u0026thinsp;0.05 was considered significant. The significant differences are marked with (*) whenever comparisons were made between \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eFGF3c\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e and their respective controls \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e. Details about the number of replicates and significance notation are provided in the figure legends.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIdentification of candidate genes and variants\\u003c/h2\\u003e \\u003cp\\u003eIn our ongoing studies on HL, we identified an individual homozygous for an \\u003cem\\u003eFGF3\\u003c/em\\u003e variant (c.493A\\u0026thinsp;\\u0026gt;\\u0026thinsp;G; p.Arg165Gly) and another individual who is heterozygous for a \\u003cem\\u003eGREB1L\\u003c/em\\u003e variant (c.556T\\u0026thinsp;\\u0026gt;\\u0026thinsp;C; p.Cys186Arg) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea-b and Supplementary Table S3). The proband with the \\u003cem\\u003eFGF3\\u003c/em\\u003e variant is a 6-year-old male with bilateral congenital profound deafness whose temporal bone CT scan showed bilateral labyrinthine aplasia. Physical examination revealed normal-sized but prominent external ears and widely spaced lower incisor teeth. The parents were first cousins without HL. The proband with the \\u003cem\\u003eGREB1L\\u003c/em\\u003e variant is a 5-year-old male with congenital profound sensorineural HL in the left ear associated with common cavity malformation (Supplementary Figure S2A). Hearing and imaging studies in the right ear are normal. An ultrasound examination for kidney and urinary system anomalies is unremarkable. His developmental history is normal. Parents have normal hearing and do not have the variant detected in the proband. Although both variants are highly conserved among different vertebrate species (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb), following ACMG guidelines, both variants are interpreted as VUS (Supplementary Table S3). Thus, increasing the certainty of their pathogenicity depends on functional abnormalities that additional studies can demonstrate.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn the same cohort, we identified a candidate gene, \\u003cem\\u003ePBXIP1\\u003c/em\\u003e, for bilateral cochlear aplasia. The proband is an 8-year-old female who was born with bilateral profound sensorineural HL without additional abnormalities. CT scans of the temporal bone showed bilateral cochlear aplasia (Supplementary Figure S2B). Initially, the search for variants in known deafness genes ended with no variant of interest that could be associated with HL in this family. Parents were consanguineous and there were 11 regions of homozygosity greater than 2Mb in the proband (Supplementary Table S4). After filtering of variants and Sanger sequencing of family members, only one variant co-segregated with the phenotype: the proband is homozygous for the nonsense variant c.1722G\\u0026thinsp;\\u0026gt;\\u0026thinsp;A (p.Trp574*) in \\u003cem\\u003ePBXIP1\\u003c/em\\u003e and parents are heterozygous (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea and Supplementary Table S3). Variants in this gene have not been previously associated with human phenotypes. PBXIP1 is present in the nucleoplasm and cytoplasm of cells in most tissues (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.proteinatlas.org/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.proteinatlas.org/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e); the gEAR database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.umgear.org/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.umgear.org/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) shows its highest expression in prosensory duct floor and lateral duct floor in developing human cochlea along with significant expression in all other parts of cochlea, such as roof, periotic mesenchymal cells, and medial duct floor (Supplementary Figures S3 and S4) (van der Valk et al. \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGeneration of isogenic knockout and variant-bearing iPSCs via CRISPR/Cas9\\u003c/h2\\u003e \\u003cp\\u003eThe \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003ec.556T\\u0026gt;C\\u003c/sup\\u003e, \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eFGF3\\u003c/em\\u003e \\u003csup\\u003ec.493A\\u0026gt;G\\u003c/sup\\u003e, \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e, and \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e variants introduced into a control iPSC cell line were confirmed by Sanger sequencing (Supplementary Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e). Sanger sequencing of the \\u003cem\\u003ein silico\\u003c/em\\u003e predicted off-target genomic loci showed no unintended mutation had been introduced into the monoclonal iPSC lines (Supplementary Table S2). All the monoclonal iPSC lines generated in the study retained their pluripotency as assessed by immunocytochemical staining for the pluripotency markers OCT4 and TRA1-60 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffects of knockout and variant incorporation on gene expression\\u003c/h2\\u003e \\u003cp\\u003eAnalysis of gene and protein levels for the missense variant-bearing monoclonal iPSC lines \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea, d, and g) and \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb, e, and h) showed no significant differences in expression compared to their respective wild-type (WT) parental iPSC line (i.e. \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e and \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e). In contrast, the monoclonal iPSC lines bearing the CRISPR/Cas9-derived knockout of \\u003cem\\u003eGREB1L\\u003c/em\\u003e (\\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e) (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea, d, and g) and \\u003cem\\u003eFGF3\\u003c/em\\u003e (\\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e) (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb, e, and h) showed significantly abrogated expression of these genes compared to their respective parental lines. The \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e nonsense variant is predicted to produce a premature termination codon, potentially resulting in a truncated protein. Nonsense-mediated decay (NMD) is an mRNA quality control mechanism eukaryotic cells use to degrade mRNAs that harbor premature termination codons. The \\u003cem\\u003ePBXIP1\\u003c/em\\u003ec.1722G\\u0026thinsp;\\u0026gt;\\u0026thinsp;A variant is located 375 nucleotides away from the last intron, which makes NMD likely. A significant decrease in \\u003cem\\u003ePBXIP1\\u003c/em\\u003e mRNA expression in monoclonal line bearing c.1722G\\u0026thinsp;\\u0026gt;\\u0026thinsp;A was observed (p\\u0026thinsp;\\u0026le;\\u0026thinsp;0.001) consistent with the mRNA undergoing NMD (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec). Immunofluorescent analysis of PBXIP1 showed a reduction of PBXIP1 in the mutant cells compared to WT cells (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ef and i). Immunoblot analysis of whole cell lysates from \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e and \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e lines show that \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e variant produced truncated protein as indicated by the detection of a smaller band of around 62.83 kilodaltons consistent with the predicted size of the truncated protein (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.bioinformatics.org/sms/prot_mw.html\\u003c/span\\u003e\\u003cspan address=\\\"https://www.bioinformatics.org/sms/prot_mw.html\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ej). In addition to the production of a truncated protein, the amount of PBXIP1 protein was decreased in the PBXIP1c.1722G\\u0026thinsp;\\u0026gt;\\u0026thinsp;A cells compared to the WT parental line. These results suggest that c.1722G\\u0026thinsp;\\u0026gt;\\u0026thinsp;A leads to NMD with a reduced amount of truncated PBXIP1 protein being produced.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eInner ear organoids from variant-bearing iPSCs show size reduction during development\\u003c/h2\\u003e \\u003cp\\u003eTo determine if candidate variants from patients with cochlear malformations altered the development of the inner ear, IEOs were derived from the monoclonal variant bearing iPSC lines and the isogenic control iPSC lines. Our initial analysis focused on the morphometric characteristics of the IEOs. IEOs were produced using an aggregation approach in 3D suspension culture. All organoids were initiated by seeding 3,500 iPSCs per well of a 96-well low adherence plate. The cross-sectional area of the resulting organoids (variant bearing compared to the WT isogenic control lines) was assessed on day 25 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec and f) and 35 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed and f) after the initiation of IEO differentiation. On day 25, we found that the IEOs derived from all the isogenic controls \\u0026ndash; \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e, and \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e have similar cross-sectional areas. However, all variants of interest show growth restrictions compared to their respective control counterparts -\\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e and \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e compared to \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e; \\u003cem\\u003eFGF3\\u003c/em\\u003e \\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e and \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e compared to \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e; \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e compared to \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec and f). Consistently, the cross-sectional area analysis on day 35 IEOs showed that the variant bearing lines had decreased area compared to their respective isogenic WT controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed and f). Of note, \\u003cem\\u003eFGF3\\u003c/em\\u003e \\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e shows the smallest cross-sectional area of all IEOs (p\\u0026thinsp;\\u0026le;\\u0026thinsp;0.001). This is not unexpected since \\u003cem\\u003eFGF3\\u003c/em\\u003e has been shown to work for very early otic structure development (Jeong et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). In addition to the decrease in cross-sectional area, the HL variant-bearing IEOs had reduced cell confluence in otic vesicles and significantly reduced number of otic vesicles/IEO compared to their isogenic control IEOs (Supplementary Figure S5).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eInner ear organoids from variant-bearing iPSCs show a lower abundance of otic progenitor markers\\u003c/h2\\u003e \\u003cp\\u003ePAX2/PAX8 are essential markers for early otic vesicle development and have been previously reported to be associated with IEO development (Li et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). We have examined PAX2/PAX8 abundance (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb, and \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ee) in the otic vesicular area and found that variant-bearing iPSC-derived IEOs showed a lower abundance of early otic progenitor markers (PAX8/PAX2) in \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e (p\\u0026thinsp;\\u0026le;\\u0026thinsp;0.01), \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e (p\\u0026thinsp;\\u0026le;\\u0026thinsp;0.01), \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e (p\\u0026thinsp;\\u0026le;\\u0026thinsp;0.01), \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e (p\\u0026thinsp;\\u0026le;\\u0026thinsp;0.01), and \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e (p\\u0026thinsp;\\u0026le;\\u0026thinsp;0.001). \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e showed a significant reduction in the abundance of otic progenitor markers (PAX8/PAX2) and overall size reduction, suggesting its pathogenicity in early otic development.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eInner ear organoids from variant-bearing iPSCs lack hair cell-like populations from mature organoids\\u003c/h2\\u003e \\u003cp\\u003eMYO7A and SOX2-positive cells in IEOs are crucial for mimicking the development of the inner ear. These cells define prosensory cell populations that give rise to hair cells and supporting cells essential for hearing and balance functions. In IEOs, MYO7A\\u003csup\\u003e+\\u003c/sup\\u003e and SOX2\\u003csup\\u003e+\\u003c/sup\\u003e hair cell-like cells indicate the development of functional sensory epithelia resembling that found in the inner ear. Organoids derived from \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.556T\\u0026gt;C\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.493A\\u0026gt;G\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eKO\\u003c/em\\u003e\\u003c/sup\\u003e, and \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ec.1722G\\u0026gt;A\\u003c/em\\u003e\\u003c/sup\\u003e show significantly reduced (p\\u0026thinsp;\\u0026le;\\u0026thinsp;0.001) MYO7A\\u003csup\\u003e+\\u003c/sup\\u003e population in comparison to their isogenic controls \\u003cem\\u003eGREB1L\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eFGF3\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e, and \\u003cem\\u003ePBXIP1\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eWT\\u003c/em\\u003e\\u003c/sup\\u003e, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea, \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb). In the inner ear, MYO7A is restricted to the sensory cells of the vestibular and cochlear organs (hair cells). The significant decrease in MYO7A in the HL-variant bearing IEOs suggests a defect in the early stages of hair cell development. Additionally, all the organoids derived from variant-bearing iPSC lines show low levels of SOX2, suggesting the presence of a rudimentary supporting cell population (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea, \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eHere, we present our proof of principle study for the use of human IEOs to rapidly validate uncertain DNA variants detected in patients with cochlear malformations. Moreover, we generated isogenic cell lines from control iPSCs, eliminating the need to obtain patient cells, which can be difficult and time-consuming. From the start of our experiments, the study takes approximately 90 days, making this approach applicable in clinical diagnostics and gene discovery for cochlear malformations.\\u003c/p\\u003e \\u003cp\\u003eFGF3 is a small protein (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb) serving as a signaling molecule released from the hindbrain during early development. It is implicated in forming prospective sensory tissues along with FGF10. In mice, \\u003cem\\u003eFgf3\\u003c/em\\u003e is expressed in the otic vesicle during otic placode induction and subsequently at the early stage of inner ear morphogenesis (Hatch et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e; Wilkinson et al. \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e1988\\u003c/span\\u003e). Models with loss of function variants in \\u003cem\\u003eFGF3\\u003c/em\\u003e demonstrated perturbed expression of WNT-induced genes and ultimate patterning defects in the dorsal otocyst. However, the otic genes expressed in ventral otocyst for cochlea development were not critically influenced (Hatch et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e). To the best of our knowledge, our study demonstrates, for the first time, early developmental anomalies of the inner ear in human IEOs and provides functional proof of the role of \\u003cem\\u003eFGF3\\u003c/em\\u003e variants detected in affected individuals in driving impairment in early developmental stages of the inner ear.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eGREB1L\\u003c/em\\u003e encodes a GREB1-like retinoic acid receptor coactivator (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb). Several \\u003cem\\u003ede novo\\u003c/em\\u003e and inherited variants in this gene have recently been shown to cause bilateral HL with malformed cochleae (Schrauwen et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2018a\\u003c/span\\u003e; Schrauwen et al. \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2020a\\u003c/span\\u003e; Schrauwen et al. \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e2020b\\u003c/span\\u003e). However, no prominent hearing or vestibular defects were observed in zebrafish models for this gene (Schrauwen et al. \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2018b\\u003c/span\\u003e). \\u003cem\\u003eGreb1l\\u003c/em\\u003e knockout mice die embryonically, while heterozygous or compound heterozygous mice showed no significant hearing phenotype (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ewww.mousephenotype.org\\u003c/span\\u003e\\u003cspan address=\\\"http://www.mousephenotype.org\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) (De Tomasi et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). In contrast with previously reported individuals with \\u003cem\\u003eGREB1L\\u003c/em\\u003e variants, our proband has unilateral cochlear aplasia with normal hearing in the other ear. GREB1L is a neural crest regulatory molecule implicated in the embryonic development of many tissues, including the cochlea (Plouhinec et al. \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). As mutations in other genes involved in neural crest cell migration, such as \\u003cem\\u003ePAX3\\u003c/em\\u003e and \\u003cem\\u003eKITL\\u003c/em\\u003e, have also been shown to cause unilateral HL, the observed clinical phenotype in our proband is not completely surprising (Lee et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Zazo et al. 2015). In this study, we show that the mutant and knockout \\u003cem\\u003eGREB1L\\u003c/em\\u003e organoids show decreased expression of otic progenitors and the absence of sensory cells (MYO7A\\u0026thinsp;+\\u0026thinsp;cells) at the mature stage tested compared to isogenic controls organoids, providing a clarified role of this gene in inner ear development.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003ePBXIP1\\u003c/em\\u003e encodes the PBX homeobox-interacting protein 1 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb), which is involved in cell differentiation through the PI3K/AKT pathway (Manavathi et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). Previously no evidence has existed that this gene causes inner ear anomalies and deafness in mice or humans. Detection of a loss of function variant, its expression in the cochlea, and role in cell differentiation, made this gene a candidate for inner ear anomalies. The phenotypic and expression data from the organoids establish \\u003cem\\u003ePBXIP1`s\\u003c/em\\u003e role in the development of the inner ear. It is important to point out that the observed abnormalities are identical to those caused by variants in \\u003cem\\u003eFGF3\\u003c/em\\u003e and \\u003cem\\u003eGREB1L\\u003c/em\\u003e, two established genes for cochlear malformations. Additional families with \\u003cem\\u003ePBXIP1\\u003c/em\\u003e variants will secure the establishment of this gene as causing HL.\\u003c/p\\u003e \\u003cp\\u003eIn summary, we demonstrate the role of \\u003cem\\u003ePBXIP1\\u003c/em\\u003e, \\u003cem\\u003eFGF3\\u003c/em\\u003e, and \\u003cem\\u003eGREB1L\\u003c/em\\u003e in developing otic cells and their differentiation into sensory cells in human organoids. As a study model for HL, organoids may serve as a fast and reliable system for investigating genes involved in cochlear malformations.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization: MFZ and MT; Data collection: MFZ, MR, DD, SS, AM, TK, SF; Formal analysis: MFZ, MR, DD; Funding acquisition: MT; Writing original draft: MFZ, MR and MT; Writing-review and editing: MFZ, MR, DD, AM, SS, TK, SF, BAD, SG, DMD and MT.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors are grateful to the participating families and clinical team for participation and cooperation. We are also thankful to iPSC core at University of Miami for providing the cell lines and facilities for use.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by grant NIH R01DC009645 and R01DC012836 to MT.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDeclaration of interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNone to declare.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSupplementary information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe supplementary information contains 4 tables and five figures.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eAdadey SM, Aboagye ET, Esoh K, Acharya A, Bharadwaj T, Lin NS, Amenga-Etego L, Awandare GA, Schrauwen I, Leal SM, Wonkam A (2022) A novel autosomal dominant GREB1L variant associated with non-syndromic hearing impairment in Ghana. 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Genet Med Open,\\u003cem\\u003e \\u003c/em\\u003e2, 101547.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"human-genetics\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"huge\",\"sideBox\":\"Learn more about [Human Genetics](https://www.springer.com/journal/439)\",\"snPcode\":\"439\",\"submissionUrl\":\"https://submission.nature.com/new-submission/439/3\",\"title\":\"Human Genetics\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Candidate gene, Cochlear malformation, CRISPR/Cas9, Deafness, Inner ear anomaly, iPSC, Organoids, Variants of uncertain significance\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4474071/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4474071/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eDevelopmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital deafness. Most patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected \\u003cem\\u003eFGF3\\u003c/em\\u003e (p.Arg165Gly) and \\u003cem\\u003eGREB1L\\u003c/em\\u003e (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and \\u003cem\\u003ePBXIP1\\u003c/em\\u003e(p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed significant developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a tool to rapidly validate the pathogenicity of DNA variants associated with cochlear malformations.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Human Organoids for Rapid Validation of Gene Variants Linked to Cochlear Malformations\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-06-11 20:08:29\",\"doi\":\"10.21203/rs.3.rs-4474071/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-07-09T19:07:46+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-07T09:19:23+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-06-25T17:20:34+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"282097083166515928785970895046874816486\",\"date\":\"2024-06-20T05:30:04+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-06-11T21:40:32+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-06-06T17:54:23+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"139455840599602132078050558177398762181\",\"date\":\"2024-06-02T10:00:19+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"159779571121149968401651097451986760144\",\"date\":\"2024-05-30T17:08:07+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"193068304744299346171610019547507520055\",\"date\":\"2024-05-28T17:42:11+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-05-27T13:14:55+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-05-25T08:36:20+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-05-25T08:36:20+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Human Genetics\",\"date\":\"2024-05-24T18:55:02+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"human-genetics\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"huge\",\"sideBox\":\"Learn more about [Human Genetics](https://www.springer.com/journal/439)\",\"snPcode\":\"439\",\"submissionUrl\":\"https://submission.nature.com/new-submission/439/3\",\"title\":\"Human Genetics\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"4d95f563-03ec-4cfc-82dc-a741af5fa332\",\"owner\":[],\"postedDate\":\"June 11th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-01-13T16:02:52+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4474071\",\"link\":\"https://doi.org/10.1007/s00439-024-02723-9\",\"journal\":{\"identity\":\"human-genetics\",\"isVorOnly\":false,\"title\":\"Human Genetics\"},\"publishedOn\":\"2025-01-09 15:57:38\",\"publishedOnDateReadable\":\"January 9th, 2025\"},\"versionCreatedAt\":\"2024-06-11 20:08:29\",\"video\":\"\",\"vorDoi\":\"10.1007/s00439-024-02723-9\",\"vorDoiUrl\":\"https://doi.org/10.1007/s00439-024-02723-9\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4474071\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4474071\",\"identity\":\"rs-4474071\",\"version\":[\"v1\"]},\"buildId\":\"cBFmMYwuxLRRLfASyISRj\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}