Human Mutation

Background: Short-rib polydactyly syndrome type IV (Beemer-Langer syndrome) is a lethal skeletal ciliopathy that can result from biallelic pathogenic variants in KIAA0586 (TALPID3). Earlier clinical observations suggested an unusually high occurrence of this phenotype in Romani families from the Carpathian Basin. Methods: Whole-exome sequencing was performed in affected individuals from five independent families, and the frequency of KIAA0586 c.1815G>A was assessed in a Hungarian Romani genetic database of 500 individuals. Results: The affected individuals were homozygous for c.1815G>A, a splice-disrupting variant previously shown to cause exon 14 skipping. Four families self-identified as Roma from Hungary and one affected individual was from Turkey. Six heterozygous carriers were identified among 500 individuals in the Hungarian Romani cohort, corresponding to an allele frequency of 0.6%. This was markedly higher than the frequencies cited from ALFA (0.01%) and gnomAD (0.0014%). The Hungarian-Romani and Turkish affected individuals also shared genotypes at informative markers flanking the variant, supporting a shared ancestral haplotype. Conclusions: These observations strengthen evidence that KIAA0586 c.1815G>A is a founder allele enriched in at least some Romani populations. The data support consideration of ancestry-informed carrier testing in appropriately counseled families. Historical admixture may explain the occurrence of the same haplotype in a Turkish case, but this remains a hypothesis requiring broader population-genomic confirmation.

FBXO31 encodes the substrate receptor of an SCF (SKP1-CUL1-F-box) E3 ubiquitin ligase that recognizes C-terminal amide-bearing proteins (CTAPs) and targets them for proteasomal degradation, a function of particular importance in neurons. A recurrent de novo missense variant, c.1000G>A p.(Asp334Asn), reprograms FBXO31 substrate specificity and causes an autosomal-dominant neurodevelopmental disorder with spastic cerebral palsy through a neomorphic gain-of-function mechanism. By contrast, biallelic loss of function has tentatively been linked to a recessive disorder. Here we report a female patient, born to consanguineous parents, with moderate intellectual disability, developmental delay and dysmorphic features, who carries a homozygous 54.3 kb deletion removing exons 2-8 and the coding part of exon 9 of FBXO31. The deletion retains only the exon 1-encoded F-box domain, while removing the coding sequence for the entire C-terminal substrate-recognition region. It is predicted to result in loss of function. Segregation is consistent with autosomal-recessive inheritance, with both parents and three of five unaffected siblings being heterozygous carriers. Together with two previously reported recessive families and the experimentally established neomorphic action of p.(Asp334Asn), the homozygous deletion supports an FBXO31 allelic series comprising dominant gain of function and recessive biallelic loss of function.

Non-coding variants altering mRNA splicing are increasingly recognized as important cause of Mendelian disorders. Deep intronic variants (DIV) that activate cryptic exons (CEs) cause ~20% inherited retinal diseases (IRD) cases, yet it remains challenging to predict intronic variant pathogenicity accurately, thus we used a high throughput splicing assay (HTSA) to measure the effects of rare deep intronic variants in recessive IRDs with one confirmed pathogenic variant. 640 very rare deep intronic variants were chosen in trans-position with known mutation from 76 patients. We tested them with HTSA, which consists of a split-GFP minigene, separated by an SMN1 gene intron into which the 270bp sequences flanking the DIVs were cloned. The plasmid library was transfected into 293HEK cells, and 48 hours later total RNA was extracted from the transfected cells. RNA transcripts produced by HTSA minigene were amplified by RT-PCR and sequenced. The intron sequences spliced between the two GFP exons were identified and quantified. 98 variants activated CE >100 times more in altered oligo compared to reference oligo and were classified as pathogenic in this experimental setting, validated in longer context, 26 variants were classified as VUS. Interestingly, only 6 from 90 variants (6,6%) were predicted to be pathogenic by in silico algorithms (Splice AI) which leaves room for significant improvement of prediction algorithms. The results of this experiment confirmed diagnosis for 50 of 78 patients (64%). So, 19,4% of deep intronic variants were shown to cause CE activation. This can lead to conclusion that significant portion of undiagnosed patients with recessive IRDs carry pathogenic intronic mutation which causes the disease.

CACNA1A-related disorders arise from altered function of the P/Q-type calcium channel Cav2.1 and encompass a broad spectrum of neurological phenotypes, including developmental and epileptic encephalopathy, familial hemiplegic migraine type 1 and episodic ataxia type 2. Although loss-of-function and gain-of-function mechanisms have both been implicated, interpretation of rare CACNA1A missense variants remains challenging because clinical presentation is heterogeneous and many variants identified through diagnostic sequencing remain classified as variants of uncertain significance. To generate clinically interpretable functional evidence, we developed a calibrated high-throughput automated patch-clamp assay to assess the CACNA1A variants. Twenty-six curated benign/likely benign and 30 curated pathogenic/likely pathogenic missense variants were expressed in Flp-In HEK293 cells with CACNB4 and CACNA2D1. We quantified current density, {Lambda}V0.5 activation,{Lambda}V0.5 inactivation and recovery from inactivation (T0.5 RFI), and used benign controls to define parameter-specific normal ranges with {+/-}3 Z-score thresholds. This multi-parameter framework classified all benign controls as functionally normal and 29 of 30 pathogenic controls as abnormal, yielding 100% specificity and 96.7% sensitivity. Current density identified 19 pathogenic controls with loss of function, while gating and recovery from inactivation identified additional abnormal variants with preserved or partially preserved current density. The assay generated likelihood ratios of 0.033 for normal function and 25.13 for abnormal function, supporting BS3 and PS3 functional evidence, respectively. Application to 23 CACNA1A variants identified in Australian patients showed abnormal function in all four variants already classified as pathogenic or likely pathogenic. Among 19 variants of uncertain significance, 11 were functionally abnormal and nine were reclassified as likely pathogenic after integration of PS3 evidence with other ACMG/AMP criteria. These data show that CACNA1A missense variants can produce loss-of-function, gain-of-function or mixed effects, and that multi-parameter functional assessment is required for accurate interpretation. This calibrated automated patch-clamp framework provides clinically interpretable evidence for refining CACNA1A variant classification in diagnostic practice.

CBX1-related syndrome is characterized by developmental delay, hypotonia, autistic features, and mild dysmorphic features. This syndrome is caused by heterozygous missense variants in CBX1, which encodes heterochromatin protein 1 beta (HP1{beta}). These variants are situated within the chromodomain of HP1{beta}, a critical region that mediates the interaction between HP1{beta} and trimethylated H3K9. While a prior study identified three individuals with pathogenic CBX1 variants, the variant and clinical spectrum of CBX1-related syndrome remains largely unknown. Here we report three patients with novel de novo heterozygous variants in CBX1. Patients with CBX1 p.(Leu40Pro) and p.(Cys60Arg) manifested with global developmental delay, while the patient with CBX1 p.(Cys60Gly) primarily exhibited speech delay without concurrent motor developmental delay. All variants reside within chromodomain of the HP1{beta} protein. Interestingly, in vitro cellular assays revealed that CBX1 p.(Cys60Gly) variant showed an intermediate cellular phenotype between the wild type and the CBX1 p.(Leu40Pro) and p.(Cys60Arg) variants, suggesting a possible genotype-phenotype correlation. This report provides further evidence that missense variants within the chromodomain of HP1{beta} cause a syndromic neurodevelopmental disorder, CBX1-related syndrome.

Background: Short-read whole-genome sequencing (sWGS) has limitations in characterizing tandem-repeat expansions and variants in highly homologous or pseudogene-associated regions. Long-read sequencing can address some of these challenges but often benefits from high-molecular-weight DNA. TruPath Genome incorporates spatial proximity information into a short-read sequencing framework. This study evaluated its performance in characterizing selected complex genomic variants using archived clinical DNA specimens. Methods: Ten archived peripheral blood DNA specimens were analyzed, comprising seven cases with disease-associated short tandem repeat (STR) expansions and three cases involving homologous or pseudogene-associated loci (STRC, SMN1/SMN2, and CYP21A2). TruPath results were compared with previously available orthogonal assay results, including triplet repeat-primed PCR (TP-PCR), multiplex ligation-dependent probe amplification (MLPA), quantitative PCR (qPCR), and sWGS-based ExpansionHunter analysis. Results: TruPath generated repeat-length estimates across the evaluated STR loci. For two FMR1 full-mutation alleles, TruPath estimated 284 and 295 CGG repeats, respectively, while TP-PCR reported expansions exceeding 200 repeats. In a DMPK case, TruPath estimated an expanded allele of 1,203 CTG repeats, compared with a TP-PCR result exceeding 100 repeats. At the STRC and SMN1/SMN2 loci, TruPath findings were consistent with available orthogonal results for deletion and copy-number status. At the CYP21A2 locus, TruPath reconstructed the RCCX gene arrangement and assigned the I2G variant and a chimeric allele to different haplotypes. Conclusions: In this small series of archived clinical DNA specimens, TruPath Genome enabled characterization of selected repeat expansions and homologous or pseudogene-associated variants. The findings support further evaluation of proximity-mapped sequencing as a complementary approach for complex genomic regions, particularly when conventional short-read sequencing is limited.

Myotonic dystrophy type 1 (DM1) is caused by a CTG expansion in the 3' untranslated region of the DMPK gene that results in the expression of expanded CUG RNA which trap splicing factors resulting in widespread alternative splicing dysregulation. A panel of these splicing events, or splicing index (SI), is currently used to measure splicing dysregulation in DM1 patients. Despite significant interest in splicing, little is known about its progression over time in DM1. The objective of this study was to evaluate the changes in SI over 3 years in a well-phenotyped cohort of DM1 participants and identify factors contributing to these changes. RNA was extracted from vastus lateralis (VL) biopsies of 20 DM1 participants taken 3 years apart and the SI score was measured using targeted RNA sequencing. Baseline SI scores correlated with percent predicted muscle strength of the ankle dorsiflexors, knee extensors and hand grip. On average, SI scores worsened over 3 years, but the progression varied markedly among participants. Adult/juvenile phenotypes and lower baseline SI scores were among the factors associated with higher progression of SI scores over time. These findings emphasize the importance of integrating clinical phenotype and baseline molecular severity when interpreting longitudinal SI change, particularly in the context of clinical trials.

Background: Dysfunction of the KCND3-encoded Kv4.3 channel, which carries the transient outward potassium current (Ito), has long been suspected to contribute to the pathophysiology of Brugada Syndrome (BrS). Although recent genome-wide association studies (GWAS) have implicated KCND3, the role of rare variation in this gene remains uncertain and clinically unactionable. Methods: Genome sequencing was performed in 983 BrS probands from Europe and Thailand. Putative gain-of-function (GoF) rare variants in KCND3 identified in patients were functionally characterized by patch-clamp analysis of Kv4.3 current properties using a HEK293 cell heterozygous model. Biobank datasets were utilized to evaluate rare-common variant interaction at the KCND3 locus. Results: Eight BrS cases carried six different rare KCND3 variants which clustered at the Kv4.3 C-terminus, with terminal exon rare variants significantly enriched in cases versus study (p=0.004) and population (p=2.0e-6) controls. Four of the five KCND3 case variants studied demonstrated significant GoF effects versus wild-type and putative benign variants, including increased Ito amplitude (p.Leu586Val, p.Ala634Thr) and slower inactivation (p.Gly600Arg, p.Ala654Thr, p.Ala634Thr). Analysis of p.Gly600Arg carriers in the UK Biobank and All of Us revealed a significant enrichment of the protective KCND3 expression-reducing allele of the GWAS lead single-nucleotide polymorphism rs12090194, suggesting a possible epistatic common-rare variant interaction modifies penetrance in carriers. Conclusions: GoF rare variants at the C-terminus of KCND3 are associated with BrS in a small proportion of cases. Determination of the rare and common variant status in BrS patients and their relatives may provide information about disease risk.

Purpose A significant proportion of cases with rare inherited retinal disease (IRD) remain genetically unresolved following short-read whole genome sequencing (WGS). This study aimed to increase the diagnostic yield of two cohorts in a research setting. One cohort was previously screened by short-read WGS (n=120 cases), while the second cohort (n=28 cases) was not included in any previous short-read WGS studies. In contrast to the larger cohort, these cases only underwent either exome sequencing (ES) or single-molecule molecular inversion probe (smMIPs) sequencing as pre-screening in earlier studies. For all cases, we performed a stepwise, case by case reanalysis in which short-read WGS was generated (n=28 cases) and (re)analyzed short-read WGS data using an optimized approach. For a subset of cases (n=20) that remained unresolved, this was followed by long read WGS. Together, these steps aimed to improve the diagnostic yield in these cohorts. Methods Short-read WGS data of 148 IRD-cases were (re)analyzed using updated allele frequency databases, new variant caller and variant predictor tools, as well as updated and extended gene-panels to identify causal single nucleotide variants (SNVs) and structural variants (SVs). For 20 genetically unresolved cases with sufficient high-quality DNA available, long-read WGS was performed. Results The (re)analysis of short-read WGS data in the 120 previously WGS-screened unresolved cases resulted in a diagnostic yield of 15% (18/120). For the 28 individuals without prior WGS, performing WGS analysis within this study provided an additional yield of 32% (9/28). Furthermore, long-read WGS contributed two genetic diagnoses among the 20 long-read WGS-sequenced probands. Amongst other reasons, new genetic diagnoses were attributed to pathogenic variants in genes newly associated with IRDs and in genes that were not included in the gene panels applied in previous WGS studies. The Mobile Element Locator Tool facilitated the identification of two pathogenic Alu insertions in short-read WGS. Long-read WGS identified two additional pathogenic variants. Conclusion These results support periodic reanalysis of existing short-read WGS data as a key component of IRD genetic diagnostics, with selective long-read WGS representing a valuable additional approach for unresolved cases. Together, these complementary strategies offer a practical framework for improving molecular diagnosis and narrowing the remaining diagnostic gap in IRDs.

Background: Recent work has brought large language models (LLMs) into clinical variant interpretation. In most existing approaches, the model is confined to text-level operations, retrieving, extracting, compacting, and summarizing heterogeneous evidence, while the genuine genetic evaluation is delegated to a separate deterministic engine. Other tools employ LLMs to mine functional evidence for a single ACMG criterion, to rerank a classifier's output, or to answer isolated variant questions. Integrative genetic reasoning, by contrast, demands multi-source evidence weighing for each variant, reconciliation of inheritance with observed zygosity and parental genotypes, joint interpretation of variants in the same gene, and the derivation of a case-level conclusion. Moreover, to protect patient privacy, this must be done on hospital premises. Methods: o build an end-to-end variant interpreter that runs on local infrastructure, we benchmarked 13 small language models (SLMs) on 150 multiple-choice genetics questions and selected Qwen3.5-9B. In IVA (Intelligent Variant Analyser), a four-stage pipeline, the model assigns ACMG/AMP criteria, applies de novo, dominant, recessive, and X-linked inheritance logic to trios, and writes a structured report, while deterministic code handles retrieval, phasing, point summation, citation checking, and formatting. We evaluated IVA on synthetic scenarios, 16 solved trios, and 327 solved singleton cases of visual impairment, on a single GPU. Results: On the benchmark, Qwen3.5-9B reached a mean accuracy of 0.81, compared with 0.89 for a 1.6-trillion-parameter cloud model. In the 16 trios, IVA reported every previously identified pathogenic, likely pathogenic, or uncertain-significance variant of interest (sensitivity 100%; precision 39.5%). In the 327 singleton cases, it recovered 93% of the expert-reported causative variants and classified 82% of them as pathogenic or likely pathogenic (precision 43%). Failures fell into four recurrent classes: omission, unverified mechanism, mismatch between reasoning and label, and arithmetic errors. Conclusions: A small language model running on local hardware can serve as the reasoning layer for case-level and trio-based variant interpretation when it is surrounded by deterministic safeguards. Because its outputs are not deterministic, analyst review and prospective multicenter validation remain necessary.

Background: Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neurodegenerative disorders characterized by progressive lower-extremity weakness and spasticity, with or without additional neurological symptoms. Objectives: To characterize the molecular genetic spectrum, inheritance patterns, molecular finding rate, and clinical characteristics of HSP within a multicenter Canadian cohort. Methods: We investigated 671 patients from 423 families who met clinical criteria for HSP identified through the Canadian HSP (CanHSP) registry from 7 medical centers across Canada. Patients were evaluated using known HSP and HSP mimic gene panels, whereas unsolved and more recent patients underwent whole-exome sequencing (WES). 435 individuals underwent gene-panel testing and 485 underwent WES. Copy-number variants (CNVs) were assessed using ExomeDepth, and candidate CNVs using multiplex ligation-dependent probe amplification (MLPA). Results: Molecular findings were identified in 426 of 671 individuals (63.5%): 351 (52.3%) had HSP-associated findings, 70 (10.4%) HSP-mimicking disorders, and five (0.7%) other molecular findings; 245 (36.5%) remained unresolved. Candidate variants were found in 80 relevant genes, 36 of which were HSP-associated genes. The most frequently implicated genes were SPAST (22.5%), SPG7 (5.7%), ATL1 (5.2%), SPG11 (3.6%), and SPG8 (3.3%). Phenotypically, SPAST, ATL1, and SPG8 w[zg1.1]ere predominantly associated with pure HSP, whereas SPG7 and SPG11 more often showed complicated phenotypes, with prominent cerebellar involvement in SPG7 and cognitive and developmental features in SPG11. Conclusion: Patients in CanHSP present with genetic and phenotypic heterogeneity. Reanalysis and complementary approaches, including whole-genome, RNA, and long-read sequencing, may increase diagnostic yield.

Genetic association analyses of exome sequencing data pose distinct methodological challenges in small cohorts, which are common in rare disease research. Data filtering prior to association testing requires particular care, especially when analyzing datasets generated under different study designs. Most of current genetic association tools need to be integrated into complex data processing pipelines, whose implementation requires technical expertise and is prone to error. To alleviate mentioned issues we built a comprehensive, high performance Nextflow workflow for genetic association testing, which includes all commonly performed data filtering and preparation steps, controlled by a rich set of modifiable parameters. We compared our software against existing tools incorporating quality control and aggregated association testing steps and found our solution to offer the most complete suite of data preparation, association testing and reporting modules, making small-cohort association analyses accessible to teams with limited technical expertise.

Several polyglutamine-encoding repeat expansions cause spinocerebellar ataxias (SCAs) and other movement disorders. SCAs are genetically heterogeneous with a substantial diagnostic gap. We used genome sequencing data from 266 individuals of European and Asian ancestry and screened genome-wide for repeat expansions using STRling, a dedicated repeat expansion caller. The cohort comprised patients with movement disorders, predominantly dystonia and ataxia, as well as unaffected participants. Further, we screened another 436 individuals with ataxia and 427 ethnically matched controls through fragment analysis of the most promising candidate locus in EP400. We applied targeted long-read sequencing to expansion carriers and one control individual to resolve the sequence structure of the repeat locus. Finally, we used microsatellite markers in chromosome 12q24.32-33 to test for a common founder. A genome-wide outlier analysis in STRling revealed a polyglutamine-encoding repeat expansion (GRCh38 chr12:132,062,525-132,062,590) in the E1A binding protein p400 (EP400) gene in two patients with ataxia. Through screening of additional ataxia patients, we identified in total 18 patients from 14 families with heterozygous repeat expansions in EP400. All expansion carriers were from Serbia. The repeat number in affected individuals ranged from 37 to 55, whereas the longest control allele had 32 repeats. We demonstrated that non-expanded alleles carried a CAA interruption within the 5-prime CAG tract; this interruption was lost in expanded alleles. Expansion carriers have pure SCA with onset mainly in their 40s and 50s. There is an inverse correlation between the number of repeats and age at onset. The EP400-encoded protein plays an essential role in chromatin remodeling, epigenetic regulation and DNA repair. Pathogenic sequence variants in EP400 have previously been implicated in a neurodevelopmental disorder. Our study identifies repeat expansions in EP400 as a novel and frequent cause of ataxia in Serbian patients. This finding further increases the diagnostic yield of ataxia testing and warrants investigation in ataxia patients worldwide. We add EP400 to the growing list of ataxia-associated repeat loci, which warrants inclusion in diagnostic ataxia panels.

Background: Current Lynch syndrome (LS) prediction models do not account for detailed family structure and tumour mismatch repair (MMR) status. We developed LynchRisk, a Bayesian model that estimates individualized LS probability from pedigree, cancer history and tumour MMR test results. Methods: LynchRisk combines Mendelian inheritance, age- and sex-specific cancer hazards, affected and unaffected relatives, and detailed MMR status. Parameters were derived from the literature. Results: In two simulated cases of probands with LS-associated cancers, family structure and MMR status markedly changed LS probability estimates, with implications for germline testing decisions. LynchRisk also identified situations in which a negative germline result might be considered sufficient to rule out LS or, conversely, prompt further investigations such as somatic MMR or long-read sequencing. Discussion: LynchRisk gives individualized LS probability by combining clinical, family, and tumour data at different stages of the diagnostic process. Further work will focus on recalibrating cancer hazard functions, validating model performance through simulations, and developing an R package for use by interested research groups.

Fragile X Syndrome (FXS) is a neuro-developmental disorder caused by a CGG expansion in FMR1, leading to transcriptional silencing and loss of the encoded protein FMRP. Surprisingly, ~70% of FXS individuals express FMR1, but the RNA is mis-spliced to isoform FMR1-217, composed of exon 1 spliced to a pseudo-exon in intron 1 and cannot produce FMRP. Splice-switching ASOs rescue proper FMR1 splicing and restore FMRP. FMR1-217 mis-splicing increases with CGG repeat length and is negatively correlated with patient IQ. FMR1-217 is associated with ribosome footprints, indicating it is translated into a polypeptide that may impair cognition. R-loops form at the FMR1 locus and extend into the pseudo-exon, but splice-switching ASOs reduce FMR1-217 and elevate FMRP independently of R-loop formation. DRB-based transcription analysis identified impaired Pol II elongation at the 5 prime region of FMR1 in FXS cells, indicated by accumulation of hypophosphorylated Pol II at the transcription start site. Consistent with this, camptothecin-induced Pol II stalling increased FMR1-217 pseudo-exon inclusion. The splicing factors PTBP1 and PTBP2 regulate FMR1-217 splicing in a differentiation stage-dependent manner. Together, these findings indicate that FMR1-217 mis-splicing in FXS is associated with CGG repeat expansion, R-loop formation, impaired co-transcriptional Pol II elongation and context-dependent regulation by PTBP1/PTBP2.

Abstract INTRODUCTION Despite major advances in genomic sequencing and analytical pipelines for rare disorders, more than 50% of affected individuals remain without a molecular diagnosis. One challenge is the interpretation of inherited variants exhibiting incomplete penetrance (IP). MATERIALS AND METHODS We developed a pipeline to systematically investigate inherited variants as the cause of rare disease in 4,325 families, consisting of affected probands and unaffected parents, from the Broad Center for Mendelian Genomics, part of the GREGoR Consortium. As a proof-of-concept of further omics follow-up, epigenomic and transcriptomic analyses were performed in one family harboring an inherited disease-causing frameshift variant in CHD8. RESULTS We identified 81 families where the affected proband harbored a putative disease-causing variant inherited from a reportedly unaffected parent. Clinical re-evaluation identified 30 families in which an inherited variant was considered diagnostic, including six previously missed diagnoses. In this cohort, inherited IP variants account for 1.6% of the diagnosed cases, with an overall diagnostic yield of 0.4% in the cohort. Methylation analysis of the CHD8 family confirmed a positive CHD8 clinical episignature in the male proband, but also in the reportedly unaffected mother. Transcriptomic analysis showed that despite similar CHD8 haploinsufficiency, the proband and mother displayed divergent downstream transcriptional effects. DISCUSSION This study highlights the importance of considering parentally inherited IP variants in rare disease, with at least 1 in 60 diagnostic variants inherited from a reportedly unaffected parent, and demonstrates the potential of integrating omics evaluation to improve variant interpretation.

Human genetics needs benchmarks for evaluating variant scores against observed phenotypic consequences. We therefore present UKBBGym, which evaluates coding and non-coding scores against plasma protein abundance and quantitative traits observed in carriers of ultra-rare variants in the UK Biobank. This design avoids ascertainment biases of clinical labels, potential physiological mismatch of experimental assays, and confounding pertaining to common-variant associations. UKBBGym recapitulates relative performance on pathogenicity prediction while enabling comparison across variant classes and molecular mechanisms. This shows that scores designed for coding and splicing variants correlate more strongly with protein abundance and quantitative traits than scores modelling transcriptional regulation. Moreover, experimental assays do not consistently outperform computational scores for predicting missense effects. Comparing captured to detectable variance indicates scope for improvement, particularly for indels and loss-of-function variants. Finally, for coding variants, UKBBGym can be reproduced from public summary statistics, providing an accessible population-phenotype benchmark complementary to clinical labels and experimental assays.

To evaluate the clinical implementation of long-read genome sequencing (lrGS) and establish the population reference resources needed to support its use in diagnostics, we integrate two complementary initiatives: Genome of Sweden (GoS), Sweden's contribution to Genome of Europe (GoE), and the Genomic Medicine Sweden - Rare Diseases (GMS-RD) long-read sequencing pilot. The initial GoS release of 576 individuals provides population-scale maps of genetic and epigenetic variation. Among 485 GMS-RD probands, 161 (33.2%) receive a molecular diagnosis, with lrGS providing added clinical value in 40 (24.8%). As a first-line test, lrGS could have avoided at least 42 follow-up analyses and an additional testing cycle in 7.2% of probands, reducing costs and the burden on patients. By integrating population and clinical long-read genomics, Sweden establishes a national ecosystem for genomic research and clinical diagnostics. Our findings support the clinical implementation of lrGS and offer a scalable model for national collaboration.

Objective: Kabuki syndrome type 2 (KS2) is a rare X-linked disorder caused by pathogenic variants in KDM6A and is frequently associated with hearing impairment. Because auditory phenotypes include sensorineural, conductive, and mixed hearing loss, we examined whether Kdm6a deficiency directly impairs cochlear morphology or auditory function in mice. Methods: We generated Nestin-Cre; Kdm6a conditional mutants and a germline-derived global Kdm6a deletion model using Sox2-Cre-mediated germline recombination. Auditory function and cochlear morphology were assessed by auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), and histological analyses. Results: Whole-body hemizygous or homozygous Kdm6a deletion resulted in embryonic lethality. Analyses therefore focused on Nestin-Cre; Kdm6aflox/Y males and females carrying a germline-derived heterozygous Kdm6a deletion. In Nestin-Cre mutants, ABR thresholds, wave I amplitudes and latencies, and DPOAE measures at 8, 16, and 32 kHz were comparable with littermate controls at 4 and 8 weeks. In heterozygous deletion females, ABR thresholds, wave I amplitudes and latencies at 4-64 kHz and DPOAE measures at 8, 16, and 32 kHz were comparable with male and female wild-type controls. No overt cochlear pathology was observed in either model. Conclusion: Kdm6a deficiency in the viable mouse models examined did not produce overt cochlear pathology or baseline auditory dysfunction. These findings suggest that partial Kdm6a loss alone is insufficient to produce a robust primary cochlear phenotype in young mice and highlight the potential contributions of non-cochlear, developmental, central, and later-onset mechanisms to hearing impairment in KS2. Clinically, these findings support careful evaluation for potentially treatable middle-ear disease.

Pathogenic variants in POLR1C, which encodes a shared subunit of RNA Polymerases (Pols) I and III, cause Treacher Collins syndrome (TCS) and POLR3-related leukodystrophy. While Pol I and Tp53-dependent mechanisms have been implicated in the pathogenesis of TCS, the basis of hypomyelination in POLR1C-associated POLR3-related leukodystrophy remains incompletely understood. Here, we show that polr1c mutant zebrafish exhibit reduced myelination in addition to previously described craniofacial anomalies. Oligodendrocyte precursor cells exhibit increased activation of the Tp53 pathway; however, these cells do not undergo apoptosis. Consistent with this finding, tp53 inhibition reduces cell death in polr1c mutants but fails to restore myelination, indicating that myelination deficits are not driven by Tp53-dependent progenitor loss in this model. polr1c mutants also exhibit reduced rRNA transcription by Pol I and reduced expression of some Pol III-transcribed tRNAs. Altogether, these data indicate distinct tissue-specific responses to polr1c deficiency and suggest persistent impairment of rRNA transcription contributes to deficient myelin development. These findings expand the developmental consequences of polr1c loss and advance our understanding of the molecular basis of POLR1C-associated diseases.