BACKGROUND:Chromatinopathies represent a genetically and clinically heterogeneous group of neurodevelopmental disorders (NDDs) caused by pathogenic variants in genes regulating chromatin structure and function. The phenotypic overlap and genetic complexity of these conditions pose significant diagnostic challenges, often resulting in unresolved variants of uncertain significance (VUS). RESULTS:We investigated a cohort of 400 routine diagnostic individuals with NDD using whole-exome sequencing and genome-wide DNA methylation profiling via the clinically validated EpiSign assay. Episignature classification was used to aid in variant interpretation and to define molecular subtypes of chromatinopathy. Seventeen percent of individuals (67/400) harbored variants in chromatinopathy-associated genes, including 55 novel variants. DNA methylation profiling was performed in 60 individuals with 62 variants in chromatin regulator genes. Of these, 26 individuals (43%) exhibited disorder-specific episignatures consistent with the associated clinical diagnosis. Importantly, methylation profiles supported the pathogenicity of several variants previously classified as VUS and demonstrated diagnostic concordance with known disease-associated genes including ANKRD11, SETD5, KMT2A, KDM5C, CHD8, and others. CONCLUSION:Our study highlights the improved diagnostic yield and clinical utility of combining genomic and epigenomic profiling in patients with clinically and/or genetically suspected chromatinopathies. Integration of EpiSign analysis facilitated variant reclassification, delineated genotype-epigenotype-phenotype correlations, and expanded the episignature atlas for rare neurodevelopmental disorders.

EPS8L2, encoding EPS8 signaling adaptor L2 protein, is critical for stereocilia maintenance in cochlear hair cells. Four previously published families have replicated autosomal recessive non-syndromic hearing loss (DFNB106), yet the mutational and clinical spectrum remains poorly described. This study expands the mutational and clinical spectrum of EPS8L2-associated hearing impairment by identifying five additional individuals in four families, more than doubling the number of reported cases to date, while going deeper into clinical aspects. These include two frameshift variants c.818_827dup, p.(Ala279Glyfs*36) and c.1430dup, p.(Val478Serfs*25), the first nonsense variant c.1878C > A, p.(Tyr626Ter), and the first missense variant with a spliceogenic effect c.767C > G, p.(Thr256Arg). Functional analysis confirmed that the c.767C > G variant causes exon 9 skipping, leading to a frameshift r.701_768del, p.(Gly234Alafs*55). A shared 4.17 Mb homozygous region among two unrelated Iranian families with this variant suggested a founder effect. These individuals uniquely showed U-shaped audiograms as a new association of more severe middle frequency hearing loss. Clinically, affected individuals presented moderate to moderately severe hearing loss that was mostly progressive, with an onset starting between the prelingual stage, extending postlingually. A younger sibling of the first European proband diagnosed with DFNB106 passed newborn hearing screening but was identified with compound heterozygous variants, underscoring the importance of early genetic testing. The progressive nature of EPS8L2-related hearing loss is unusual for recessive non-syndromic forms and highlights the need for early monitoring and intervention. This work emphasizes the need for early molecular diagnosis and long-term clinical follow-up, while describing the natural history of EPS8L2 that will be important for future therapeutic development.

BACKGROUND: Hearing loss (HL) is one of the most common congenital conditions and exhibits substantial clinical and genetic heterogeneity. More than 150 genes are associated with non-syndromic hearing loss (NSHL), while over 600 genes are linked to syndromic hearing loss (SHL). Importantly, the absence of additional clinical symptoms at the time of diagnosis does not necessarily exclude SHL. An increasing number of functionally disruptive variants in a growing number of genes have been shown to initially present as isolated HL, only later revealing syndromic features. METHODS: We analyzed clinical data from 111 patients across 102 unrelated families, selected from over 600 individuals negative for GJB2 and STRC variants. Molecular inversion probe panel, exome, or genome sequencing was performed, and patients were retrospectively divided into three subgroups following variant interpretation. Molecular docking was performed on select non-synonymous substitutions. RESULTS: Subgroup 1 included 30 patients with variants in neurodevelopmental disorder (NDD)-associated genes. HL was the first clinical manifestation in 80% of patients, with it being the sole first symptom in half. Subgroup 2 was comprised of 52 patients with variants in SHL-associated genes unrelated to NDD, while subgroup 3 included 29 patients with variants in genes associated with both NSHL and SHL, such as SLC26A4 and USH1C. In subgroups 2 and 3, HL was the sole initial symptom for nearly all patients (92% and 100%, respectively). Across the cohort, 99 variants in 44 genes were identified, including 36 novel variants. CONCLUSION: The frequent absence of syndromic features at presentation may lead to genetic testing or analysis restricted to NSHL-associated genes. Our findings highlight the critical role of comprehensive genomic testing in the diagnostic workup of HL, enabling earlier identification of syndromic forms and facilitating timely medical management, genetic counseling, and anticipatory care.

Identifying new genes responsible for non-syndromic hearing loss remains a critical goal as many patients still lack a molecular diagnosis despite comprehensive genetic testing. The tectorial membrane (TM) is a specialized acellular matrix of the inner ear, essential for stimulating mechanosensitive hair cell stereocilia bundles and maintaining frequency tuning and auditory sensitivity. Although mutations in genes encoding several non-collagenous proteins found in the TM (TECTA, CEACAM16, OTOG, OTOGL) have been identified as deafness genes, definitive evidence implicating β-tectorin (TECTB) has been lacking. Here, we present multiple lines of genetic and experimental evidence linking heterozygous missense variants in TECTB (NM_058222.3:c.674G>A, p.Cys225Tyr and NM_058222.3:c.853C>T, p.Arg285Cys), with hearing loss. Each variant affects highly conserved residues within or directly flanking the zona pellucida domain. Using a Tectb-C225Y knock-in mouse model, we show that homozygous animals exhibit severe hearing loss and profound disruption of TM morphology, while heterozygous animals display decreased staining density within the TM and increased susceptibility to noise-induced hearing loss, despite normal auditory thresholds. These findings identify TECTB as a novel human deafness gene, further elucidate its contribution to maintaining TM integrity and resilience against environmentally-related auditory decline.

BACKGROUND:Proteasome inhibitors (PIs) are indispensable for the treatment of multiple myeloma (MM), the second most common hematologic malignancy. Although primary resistance to PIs is rare, most patients eventually relapse and develop acquired resistance, with underlying mechanisms that remain incompletely understood and appear to be drug-specific. In the case of bortezomib, resistance is often associated with PSMB5 mutations. In contrast, resistance to carfilzomib (CFZ) is mediated by overexpression of the drug efflux transporter ABCB1. However, the regulatory mechanisms driving ABCB1 upregulation in CFZ-resistant MM remain unclear. METHODS:An integrative multi-omics analysis was conducted using paired samples from a CFZ-sensitive and -resistant MM patient, alongside resistant cell line models. Whole-genome sequencing (WGS), whole-genome bisulfite sequencing (WGBS), and RNA sequencing (RNA-seq) were used to assess the genotype (structural variants, single nucleotide variants, and copy number variations), methylation status, and the expression of the ABCB1 locus. ABCB1 promoter methylation levels and expression levels in an independent MM subcohort were analyzed to determine clinical relevance. Functional validation was performed using dual-luciferase reporter assays, DNMT1 knockdown, and treatment with DNA methyltransferase inhibitors (DNMTis) to evaluate methylation-dependent regulation of ABCB1 expression. RESULTS:Significant hypomethylation of the ABCB1 downstream promoter region was identified (GH07J087598) in a CFZ-resistant patient sample, which correlated with elevated ABCB1 expression. Consistent with the paired CFZ-resistant case, the independent MM subcohort showed a significant inverse association between ABCB1 promoter methylation and ABCB1 expression. These findings align with results obtained from CFZ-resistant MM cell line models, which demonstrated reduced promoter methylation and elevated ABCB1 expression compared to their wild-type counterparts. Furthermore, treatment with DNA methyltransferase inhibitors as well as DNMT1 knockdown enhanced ABCB1 expression while demethylating the promoter, thereby validating the functional significance of promoter hypomethylation in ABCB1 overexpression. CONCLUSIONS:Our findings highlight ABCB1 promoter hypomethylation as a potential epigenetic driver of CFZ resistance in MM. These results underscore the clinical relevance of epigenetic regulation in drug resistance and the potential of targeting DNA methylation as a therapeutic strategy to overcome resistance in MM.

Hereditary hearing loss is highly genetically heterogeneous, with emerging overlap between genes implicated in early-onset and age-related hearing loss. We report a consanguineous family with autosomal recessive, non-syndromic hearing loss in which the proband harbors a homozygous splice-site variant in PALM3 NM_001145028.2:c.314+1G>A and a homozygous missense variant in OTOA NM_144672.4:c.1939G>C p.(Gly647Arg). A minigene assay for the PALM3 variant demonstrated aberrant splicing with exon skipping, resulting in either a frameshift or a large in-frame deletion, both consistent with loss of function and impacting all known transcripts. While the organ of Corti from 12-month-old heterozygous Palm3 mice showed preserved overall architecture, a study describing Palm3 knockout mice exhibit auditory dysfunction, supporting a possible auditory phenotype with loss of function. Although a dual molecular diagnosis cannot be excluded, the combined genetic, functional, and comparative data support PALM3 as a candidate gene potentially contributing to autosomal recessive hearing loss.

IntroductionProstate Cancer-3 (PC-3) cells, commonly used as a model for aggressive, androgen-independent prostate cancer, display numerous genetic alterations that contribute to advanced disease, including the loss of tumor suppressors and dysregulated inflammatory signaling. Recent evidence has highlighted the pleiotropic roles of lipocalin 2 (LCN2) in promoting tumor cell proliferation, adhesion, and stress resistance. This study aimed to investigate the functional and molecular effects of LCN2 depletion in PC-3 cells. MethodsWe conducted a genetic analysis of both the parental PC-3 cell line and a newly created LCN2-deficient PC-3 clone #1 (PC-3 LCN2-KO#1), developed using CRISPR/Cas9 technology. Short tandem repeat (STR) analyses confirmed the authenticity and lineage of each cell line, while next-generation sequencing coupled with RT-qPCR validation was used to identify differentially expressed genes and any potential genomic changes resulting from the CRISPR/Cas9 editing process.Results and DiscussionOur analysis aligned with our previous findings showing that LCN2 is involved in inflammation, endoplasmic reticulum stress responses, and cytoskeletal organization. Previously we have shown that LCN2-deficient cells exhibited decreased invasiveness, disrupted F-actin dynamics, and increased sensitivity to stress-inducing conditions. Consistent with these observations, spectral karyotyping (SKY) and analysis of spontaneously occurring micronuclei revealed an elevated level of chromosomal aberrations in the LCN2-deficient cell line. These results emphasize the significance of LCN2 in driving prostate cancer aggressiveness and provide a foundation for exploring targeted interventions that disrupt LCN2-mediated pathways in advanced disease.

Background:Understanding the phenotypic spectrum of disease-associated genes is essential for accurate diagnosis and targeted therapy. FRMPD4 (FERM and PDZ Domain Containing 4) has previously been associated with intellectual disability and epilepsy. However, its potential role in non-syndromic hearing loss has not been explored. Methods:We performed genetic analysis in two unrelated families presenting with non-syndromic sensorineural hearing loss, identifying maternally inherited missense variants in FRMPD4. Clinical phenotyping included audiological assessment and evaluation for neurodevelopmental involvement. Cross-species expression analyses were conducted in Drosophila, zebrafish, and mouse. Functional characterization included quantitative evaluation of sound-evoked responses in Drosophila nicht gut hörend (ngh) mutants, assessment of neuronal development and acoustic startle responses in zebrafish loss of function models, and morphological cochlear analyses with auditory brainstem response measurements in knockout mice. Results:Three affected males from two unrelated families presented with prelingual, bilaterally symmetrical sensorineural hearing loss, with confirmed congenital onset in one individual and no evidence of neurodevelopmental abnormalities. Cross-species analyses demonstrated evolutionarily conserved expression of FRMPD4 in auditory structures. In Drosophila, quantitative analysis of sound-evoked responses in ngh mutants revealed impaired auditory function. Zebrafish loss of function models exhibited reduced neuronal populations in the otic vesicle and posterior lateral line, abnormal neuromast development, and diminished acoustic startle responses. In mice, Frmpd4 knockout resulted in high-frequency hearing loss and cochlear abnormalities consistent with the human phenotype. Conclusions:Our findings expand the phenotypic spectrum of FRMPD4 to include non-syndromic sensorineural hearing loss and establish its evolutionarily conserved role in auditory function. These results have direct implications for genetic diagnosis and variant interpretation in patients with hearing loss.

Purpose:While heterozygous de novo missense variants in the microtubule-binding GAR domain of Microtubule-actin cross-linking factor 1 (MACF1) cause Lissencephaly 9 with Complex Brainstem Malformations [MIM #618325], the phenotypic impact of variants outside this domain remains unclear. Methods:Through collaborative efforts, we assembled a cohort of 10 affected individuals from 8 unrelated families with either biallelic or monoallelic non-GAR domain MACF1 variants who exhibit partially overlapping yet unique phenotypic traits. Combined with previously reported cases, we analyzed genotype and phenotype data from 29 individuals using Human Phenotype Ontology (HPO)-based unsupervised hierarchical clustering. Results:Clustering revealed two distinct phenotypic signatures, suggesting domain-specific effects. Variants outside the GAR domain associate with broader neurodevelopmental phenotypes and variable craniofacial and skeletal expressivity. Additionally, enrichment analysis (p < 0.001) using OMIM HPO sets supported these findings. In contrast to the GAR domain's strong correlation with lissencephaly and brainstem malformations, biallelic non-GAR domain MACF1 variants were linked to diverse developmental anomalies. Conclusion:These results expand the phenotypic spectrum of MACF1-related disorders and highlight the relevance of domain-specific variant effects. Comprehensive genetic and phenotypic assessments are essential for understanding the role of MACF1 in development, informing diagnosis, and guiding future research on cytoskeletal regulation in neurodevelopment.

Ribosomal DNA transcription is essential for ribosome biogenesis and the production of proteins. Using a combination of droplet digital PCR and deep bisulfite sequencing, we have quantified both the absolute number as well as the methylation level of individual rDNA transcription units (TU) in blood samples of 139 young healthy individuals and 141 sex- and age-matched individuals with unsolved syndromal developmental delay (DD), ranging from 0.02 to 18.4 years in age. There were no between-group differences in average promoter methylation, absolute copy number (CN), extreme CN, and hypomethylated (0-10%) presumably active CN. This largely excludes rDNA CN as a modulating factor in DD. The absolute CN in all 280 samples was 423.7 ± 108.4 (median 410, range 153 to 1,000) and the active CN was 175.0 ± 36.4 (median 174, range 70 to 376). Similar to adults, the absolute CN did not change from birth to sexual maturity but was strongly (Pearson ρ = 0.64, P < 0.001) correlated with promoter methylation. In contrast to adults, there was no significant correlation between age and promoter methylation and no age-related loss of active copies from birth to < 20 years. The number of completely unmethylated copies even significantly (Pearson ρ = 0.15; P = 0.01) increased during childhood and adolescence. Our results suggest that rDNA promoter methylation and the age-related loss of active rDNA TU, which are a hallmark of the aging process, start only after reaching sexual maturity.

Quality check of DBS assays with artificially methylated standard DNAs. DNA standards of 0%, 25%, 50%, 75% and 100% methylation were sequenced with the PSMB5, PSMC2, PSMC5, PSMC6, PSMD1 and PSMD5 DBS assays to confirm assay sensitivity. Results for all investigated DBS amplicons correlated well with the expected curves (P < 0.001) (A). The sequencing depths of the PSMD5 DBS assay was 10-3 (B).

Genetic variants in the Würzburg dataset.

Cell survival of DNMTi pretreated KMS11 cells under BTZ exposure. AlamarBlue assay after 72 hours of incubation with varying concentrations of BTZ (0.5 nM to 4 nM) on DNMTi pretreated cells (48 h 500 nM Aza, 72 h 100 nM Dec) and PI naïve KMS11. No resensitizing effect to PI treatment was observed.

DNA promoter methylation of a subset of proteasome subunit genes. Heatmap comparing methylation pattern of 35 MM patients and 30 PBMC samples. The methylation degree is given by a color code: red – methylated state, blue – unmethylated state. Each line represents an individual sample, each column a single CpG. The methylation degree is encoded by color with blue for an unmethylated and red for a methylated state. Artificially methylated controls were included. For PSMD5, hypermethylation was only present in MM cells, not in PBMCs. For PSMC2, hypermethylation occurred in both, MM cells and PBMCs, indicating a physiological state probably independent of disease development and progression. In the remaining proteasome subunit genes, no aberrant promoter methylation was detected.

In this study, droplet digital PCR and deep bisulfite sequencing were used to study the absolute and active rDNA copy number (CN) and the effect of paternal age on human and mouse sperm. The absolute CN ranged from 98 to 404 (219 ± 47) in human and from 98 to 177 (133 ± 14) in mouse sperm. Methylation of the human upstream control element/core promoter (UCE/CP) region and the 5' external transcribed spacer, as well as that of the mouse CP, the spacer promoter, and 28S rDNA, significantly increased with donor age and absolute CN. Overall, rDNA hypomethylation was much more pronounced in mouse sperm, with 101.7 ± 11.4 copies showing a completely (0%) unmethylated and 11.3 ± 2.8 (8.5%) a slightly methylated (1-10%) CP region, compared to humans with 25.7 ± 9.5 (12%) completely unmethylated and 83.0 ± 19.8 slightly methylated UCE/CP regions. Although the absolute CN was much higher in human sperm, the number of copies with a hypomethylated (0-10%) promoter was comparable in humans (108.7 ± 28.3) and mice (113.0 ± 12.2). However, in mice, the majority (77%) of all copies were completely unmethylated, whereas in humans a high percentage (38%) showed one or two single CpG methylation errors. These different germline methylation dynamics may be due to species differences in reproductive strategies and lifespan. Complete demethylation of the sperm rDNA promoter in mice may be essential for embryonic genome activation, which already occurs at the 2-cell stage in mice and at the 4-8-cell stage in humans. The paternal age effect has been conserved between humans and mice with some notable differences. In humans, the number of hypomethylated (0-10%) copies decreased with age, whereas in mice only the completely unmethylated copies decreased with age. The number of methylated rDNA copies (>1% in mice and >10% in humans) significantly increased with age.

Patient-derived mutations impair PI response to Carfilzomib and Ixazomib. Cytotoxicity assay and IC50 values of RPMI-8226 MM cell lines harboring two different stably expressed patient-derived mutations in proteasome subunits. The mutants were more resistant to the proteasome inhibitors Carfilzomib and Ixazomib compared to the controls (*) harboring lentivirally-encoded WT sequence of PSMC6/PSMD1. Shown are the means and standard deviations of three independent experiments (biological replicates) and three technical replicates in each experiment. The PSMC6 and PSMD1 expression levels were equivalent for the mutants and the controls.

Hearing loss (HL) is one of the most common congenital disorders, affecting 1-2 in 1,000 newborns. Modern genetic diagnostics using large gene panels and/or whole exome analysis (WES) can identify disease-causing mutations in 25-50 ∼ 43

The absolute number of rDNA transcription units (TU) can vary by about one order of ten among individuals. Apart from extensive rDNA copy number (CN) variation and instability in many cancers, there is little information on the extent of intraindividual CN variation between normal tissues. Here we used droplet digital PCR and deep bisulfite sequencing to determine both the absolute rDNA CN and the number of presumably active CN with a hypomethylated (≤10 %) promoter region in up to six different tissues of 13 autopsy probands. In general, the absolute rDNA CN as well as the frequency of the minor A variant were highly similar between tissues (cerebellum, cerebrum, colon, heart, intestine, kidney, liver, and spleen) of the same individual. However, in some probands absolute CN in one or multiple tissues was much higher than in the other tissues, indicative of relaxation/breakdown of the CN control system. The amplified copies were inactivated by promoter methylation and, thus, the number of active CN was largely independent from absolute CN. Collectively, our data suggest that with some notable exceptions absolute and even more active rDNA CN are maintained during development and differentiation in different tissues of the same individual. Despite the low intraindividual variation active CN appeared to systematically vary between tissues. Cerebellum and cerebrum consistently exhibited lower active CN than the other analyzed tissues. We estimate that >50 active rDNA TU are required for normal tissue/organ function.

Raw data measurements of the dual-luciferase reporter assay in the L363 cell line. All measurements were performed in duplicates on 20.000 cells.

Both absolute and presumably active rDNA (with a hypomethylated promoter region) copy number (CN) in the haploid human sperm genome are highly variable among individuals. Using a combination of droplet digital PCR and deep bisulfite sequencing, we have quantified absolute and presumably active rDNA CN in sperm samples (N = 190) with normal (NSPs) vs. abnormal semen parameters (ASPs), as well as in samples leading or not leading to a clinical pregnancy. ASP samples had a significantly lower presumably active CN (104 ± 31) than normozoospermic samples (115 ± 31). The loss of presumably active rDNA copies is explained by an increased promoter methylation (13.9% in ASP vs. 12.1% in NSP). When correcting for confounding factors, most importantly semen quality, samples not leading to a clinical pregnancy after IVF/ICSI displayed a significantly lower absolute (225 ± 51) and presumably active CN (103 ± 30) than samples with pregnancy (249 ± 62 and 115 ± 31, respectively). This between-group difference was most noticeable in normozoospermic males: absolute CN 220 ± 54; presumably active CN 107 ± 32 in samples without pregnancy and absolute CN 246 ± 63; presumably active CN 120 ± 28 in samples with pregnancy. We propose that absolute/active rDNA CN in sperm is a modulating factor contributing to idiopathic male infertility. In NSP samples, presumably active CN increases with absolute CN, which may have a positive impact on fertility and ART outcome. Our results suggest that approximately 60 active sperm rDNA copies are sufficient to establish a pregnancy.