Biology subjects

Kremer, H.

Publications and source records attributed to Kremer, H..

9 recordsLinked to original sources

bioRxiv2026

Genome sequencing reveals novel pathogenic deep-intronic PCDH15 variants, amenable to antisense oligonucleotide-based splice correction

Despite substantial advances in diagnostic testing, 10-15% of Usher syndrome patients remain without a genetic diagnosis, having significant implications for genetic counseling and potential future therapeutic interventions. In this study, genome sequencing data from probands clinically presenting with Usher syndrome were analyzed. Two novel deep-intronic variants were identified in PCDH15, c.3983+3635A>G and c.3123-1728A>G, in two independent patients. Both deep-intronic variants were classified as likely pathogenic and predicted to alter PCDH15 pre-mRNA splicing. Using a minigene splice assay and iPSC-derived photoreceptor precursor cells from patients, we confirmed that both variants lead to the inclusion of a pseudoexon in the PCDH15 transcript introducing a stop codon and subsequent premature termination of protein translation. We designed and evaluated antisense oligonucleotides (ASOs) with the purpose of redirecting aberrant pre-mRNA splicing caused by both deep-intronic variants. For both variants, designed ASOs were successful in restoring normal splicing patterns, highlighting their potential as a future therapeutic intervention strategy to halt the progression of retinitis pigmentosa caused by these novel variants. Overall, these findings contribute to the understanding of Usher syndrome caused by deep-intronic pathogenic variants in PCDH15 and describe for the first time the use of an ASO-mediated splice correction strategy for individuals diagnosed with these variants.

genetics↗

bioRxiv2026

Patient-Derived Inner Ear Organoids as a Disease Modeling and Therapy Validation Platform For Hereditary Inner Ear Disorders

BackgroundHereditary inner ear disorders comprise a highly heterogenous group of disorders and are a major cause of hearing and vestibular dysfunction. Despite advances in genetic diagnosis, the development of precision therapies has been limited by the lack of relevant and scalable human model systems that can accommodate the wide spectrum of disease-causing variants and support the evaluation of therapeutic interventions. We established patient-derived inner ear organoids (IEOs) as a platform to assess antisense oligonucleotide (ASO)-based therapeutic strategies for hereditary hearing loss. MethodsTwo representative genetic models were selected: recessive syndromic Usher syndrome type IIa (USH2A) and dominant non-syndromic DFNA9 (COCH). Human induced pluripotent stem cells (iPSCs) were generated from a patient carrying a homozygous pathogenic USH2A variant and a patient carrying a frequently occurring pathogenic COCH variant. In parallel, isogenic iPSC lines were created by introducing the same disease-causing variants into a healthy donor background. Following differentiation into IEOs, disease-associated transcript expression was evaluated. Splice-switching and RNase H1-mediated gapmer ASOs were assessed for target engagement. ASO biodistribution and cellular uptake was also examined in both IEOs and adult human vestibular tissue. ResultsPatient-derived and isogenic iPSCs were successfully differentiated into IEOs that recapitulated disease-associated transcript expression. ASOs showed efficient uptake into disease-relevant cell populations in both IEOs and adult human vestibular tissue. In USH2A-variant IEOs, splice-switching ASO treatment corrected aberrant splicing. In COCH-variant IEOs, gapmer ASO treatment reduced total COCH transcript levels, achieving up to 75% knockdown in patient-derived IEOs. ConclusionsPatient-derived and isogenic variant IEOs provide a versatile and scalable human platform for evaluating ASO therapies for hereditary hearing loss. Their adaptability to diverse genetic variants, inheritance patterns, and ASO modalities makes them well suited to address the genetic heterogeneity of hereditary inner ear diseases and establishes IEOs as a broadly applicable preclinical model for rare hereditary inner ear diseases.

neuroscience↗

bioRxiv2025

Integrin-TGFβ axis induces partial EMT in basal-like cells to lead collective invasion

Collective invasion is the predominant mode of cancer cell dissemination in breast cancer and represents the initial step of metastatic spread. Basal-like leader cells drive this process by maintaining cell-cell junctions with the follower cells while extending actin-rich protrusions and remodeling the collagen I-rich peritumoral stroma. These features resemble those of individually-invading cells following epithelial-to-mesenchymal transition (EMT). However, how leader cells acquire these traits while preserving cohesion within the collective remains unclear. Here, we identify a collagen I-responsive subset of basal-like cells that coexpress cytoskeletal, extracellular matrix (ECM)-remodeling, and epithelial junction genes. We show that integrin 2 (Itg2) links collagen I engagement to mesenchymal reprogramming by inducing inhibin beta A (INHBA) expression and activating tumor growth factor {beta} (TGF{beta}) signaling. This, in turn, upregulates vimentin while preserving epithelial junction gene expression. In parallel, Itg2 promotes ECM degradation through a TGF{beta}-independent mechanism. This study identifies Itg2-TGF{beta} axis as a key regulator of partial EMT and leader cell function, highlighting it as a potential therapeutic target in aggressive breast cancers.

cell biology↗

bioRxiv2024

Pushing the limits of single molecule transcript sequencing to uncover the largest disease-associated transcript isoforms in the human neural retina

Sequencing technologies have long limited the comprehensive investigation of large transcripts associated with inherited retinal diseases (IRDs) like Usher syndrome, which involves 11 associated genes with transcripts up to 19.6 kb. To address this, we used PacBio long-read mRNA isoform sequencing (Iso-Seq) following standard library preparation and an optimized workflow to enrich for long transcripts in the human neural retina. While our workflow achieved sequencing of transcripts up to 15 kb, this was insufficient for Usher syndrome-associated genes USH2A and ADGRV1, with transcripts of 18.9 kb and 19.6 kb, respectively. To overcome this, we employed the Samplix Xdrop System for indirect target enrichment of cDNA, a technique typically used for genomic DNA capture. This method facilitated the successful capture and sequencing of ADGRV1 transcripts as well as the full-length 18.9 kb USH2A transcripts. By combining algorithmic analysis with detailed manual curation of sequenced reads, we identified novel isoforms and alternative splicing events across the 11 Usher syndrome-associated genes, with implications for diagnostics and therapy development. Our findings demonstrate the Xdrop systems adaptability for cDNA capture and the advantages of integrating computational and manual transcript analyses. The full neural retina sequencing dataset is available via EGA under identifier EGAD50000000720.

genetics↗

bioRxiv2020

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations

Mutations in USH2A, encoding usherin, are the most common cause of syndromic and non-syndromic retinitis pigmentosa (RP). The two founder mutations in exon 13 (c.2299delG and c.2276G>T) collectively account for ~34% of USH2A-associated RP cases. Skipping of exon 13 from the USH2A transcript during pre-mRNA splicing presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in larvae of the previously published ush2a exon 13 zebrafish mutant resulted in the production of usherin{Delta}exon13 and completely restored retinal function. RNA antisense oligonucleotides were investigated for their potential to specifically induce human USH2A exon 13 skipping. Lead candidate QR-421a induced dose-dependent exon 13 skipping in iPSC-derived photoreceptor precursors from a patient homozygous for the USH2A c.2299delG mutation. Intravitreal delivery of QR-421a in non-human primates showed that QR-421a penetrates the retinal outer nuclear layer and induces detectable levels of exon 13 skipping until at least 3 months post injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment for RP caused by mutations in exon 13 of the USH2A gene.

genetics↗

bioRxiv2020

Allele-specific antisense oligonucleotide therapy for dominantly inherited hearing impairment DFNA9.

The c.151C>T founder mutation in COCH is a frequent cause of late onset, dominantly inherited hearing impairment and vestibular dysfunction (DFNA9) in the Dutch/Belgian population. The initial clinical symptoms only manifest between the 3rd and 5th decade of life, which leaves ample time for therapeutic intervention. The dominant inheritance pattern and established non-haploinsufficiency disease mechanism indicate that suppressing translation of mutant COCH transcripts has high therapeutic potential. Single-Molecule Real-Time (SMRT) sequencing resulted in the identification of 11 variants with a low population-frequency (< 10%), that are specific to the c.151C>T mutant COCH allele. Proof of concept was obtained that gapmer antisense oligonucleotides (AONs), directed against the c.151C>T mutation or mutant allele-specific intronic variants, are able to specifically induce mutant COCH transcript degradation when delivered to transgenic cells expressing COCH minigenes. Sequence optimization of the AONs against the c.151C>T mutation resulted in a lead molecule that reduced the levels of mutant COCH transcripts by ~60% in a transgenic cell model, without affecting wildtype COCH transcript levels. With the proven safety of AONs in humans, and rapid advancements in inner ear drug delivery, our in-vitro studies indicate that AONs offer a promising treatment modality for DFNA9.

genetics↗

bioRxiv2019

A RIPOR2 in-frame deletion is a frequent and highly penetrant cause of adult-onset hearing loss

Hearing loss is one of the most prevalent disabilities worldwide, and has a significant impact on quality of life. The adult-onset type of the condition is highly heritable but the genetic causes are largely unknown, which is in contrast to childhood-onset hearing loss. We identified an in-frame deletion of 12 nucleotides in RIPOR2 as a highly penetrant cause of adult-onset progressive hearing loss that segregated as an autosomal dominant trait in 12 families from the Netherlands. Hearing loss associated with the deletion in 63 subjects displayed variable audiometric characteristics and an average age of onset of 30.6 years (SD 14.9 years, range 0-70 years). A functional effect of the RIPOR2 variant was demonstrated by aberrant localization of the mutant RIPOR2 in the stereocilia of cochlear hair cells and failure to rescue morphological defects in RIPOR2-deficient hair cells, in contrast to the wildtype protein. Strikingly, the RIPOR2 variant is present in 18 of 22,952 individuals not selected for hearing loss in the Southeast Netherlands. Collectively, these data demonstrate that an inherited form of adult-onset hearing loss is relatively common, with potentially thousands of individuals at risk in the Netherlands and beyond, which makes it an attractive target for developing a (genetic) therapy.

genetics↗

bioRxiv2018

Expert Specification of the ACMG/AMP Variant Interpretation Guidelines for Genetic Hearing Loss

Due to the high genetic heterogeneity of hearing loss, current clinical testing includes sequencing large numbers of genes, which often yields a significant number of novel variants. Therefore, the standardization of variant interpretation is crucial to provide consistent and accurate diagnoses. The Hearing Loss Variant Curation Expert Panel was created within the Clinical Genome Resource to provide expert guidance for standardized genomic interpretation in the context of hearing loss. As one of its major tasks, our Expert Panel has adapted the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) standards and guidelines for the interpretation of sequence variants in hearing loss genes. Here, we provide a comprehensive illustration of the newly specified ACMG/AMP hearing loss rules. Three rules remained unchanged, four rules were removed, and the remaining twenty-one rules were specified. Of the specified rules, four had general recommendations, seven were gene/disease considerations, seven had strength-level specifications, and three rules had both gene/disease and strength-level specifications. These rules were further validated and refined using a pilot set of 51 variants assessed by curators. These hearing loss-specific ACMG/AMP rules will help standardize variant interpretation, ultimately leading to better care for individuals with hearing loss.\n\nGRANT NUMBERSResearch reported in this publication was supported by the National Human Genome Research Institute (NHGRI) under award number U41HG006834.

genetics↗

bioRxiv2017

CiliaCarta: An Integrated And Validated Compendium Of Ciliary Genes

The cilium is an essential organelle at the surface of most mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We found experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins. In addition, we show that OSCP1, which has previously been implicated in two distinct non-ciliary functions, causes a cilium dysfunction phenotype when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 836 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/.

bioinformatics↗