Institute of Human Genetics

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) have gained interest as a pharmacological modelbut their immaturity leads to uncertainty regarding translation. We studied the key player in maintaining ionic homeostasis, the Na+/K+-ATPase (NKA), in hiPSC-CM. Atrial and ventricular engineered heart tissues (EHTs) were produced from hiPSC-CMs. For comparison, adult atrial and ventricular tissues were obtained from patients undergoing open heart surgery. We measured NKA gene expression, NKA pump currents (INKA) and ouabain effects on action potentials and contractility. Computational modelling was used to further investigate the direct and indirect impacts of NKA inhibition. The mRNA abundance of the major NKA isoforms was higher in ventricular than atrial cardiomyocytes (adult and hiPSC-CM). Consistently, INKA was also higher in ventricular than in atrial hiPSC-CM and higher in ventricular than in atrial adult cardiomyocytes. Ouabain potency to block INKA did not differ between hiPSC-derived and adult cardiomyocytes. Ouabain shortened plateau phase in EHT and adult tissue. However, lower ouabain concentrations depolarized diastolic potential and depressed force more in EHTs, demonstrating a higher integrated sensitivity to NKA inhibition. Computational modelling indicated that weaker IK1 increases the susceptibility to depolarisation by NKA block. HiPSC-EHTs express NKA with biophysical characteristics and ouabain-sensitivity that are not different from human tissue. Action potential duration responses in EHT reproduces chamber-specific response pattern as seen in human heart. The higher sensitivity of EHTs to depolarize under ouabain-induced block of NKA needs to be considered when employing hiPSC-CM in drug research.

Zusammenfassung Das Feld der klinischen und molekularen Genetik bei an Epilepsie erkrankten Personen entwickelt sich rasant weiter. In dieser Übersichtsarbeit bietet das Expertenpanel „Epilepsie und Genetik“ der Deutschen Gesellschaft für Epileptologie (DGfE) einen aktualisierten Überblick über die neuen Entwicklungen, sowohl klinisch etabliert als auch derzeit nur im Forschungskontext durchgeführt. Diese bauen auf der vorherigen Version von 2023 der allgemeinen Empfehlungen auf und ergänzen diese mit speziellen Aspekten. Hervorgehoben werden dabei moderne Ansätze in der Interpretation von Varianten unklarer Signifikanz, das Vorgehen bei ungelösten Fällen anhand von neuartigen Sequenziertechnologien und Omics-Ansätzen, die zunehmende Rolle der prä- und postoperativen Sequenzierung im Rahmen der Epilepsiechirurgie und die Risikostratifizierung anhand von polygenen Scores. Aus diesen Aspekten ergeben sich teils unmittelbar klinisch anwendbare Konsequenzen für alle Teilbereiche der Epileptologie.

During defined developmental windows in Drosophila, controlled re-replication generates physiological gene amplification. Although gene amplification has also been observed during human stem cell differentiation, re-replication in human cells has largely been linked to tumor-associated genome instability. Here, we demonstrate that re-replication likewise operates as a physiological mechanism in human stem cells. Using Rerep-Seq and DNA fiber-combing, we identify distinct phases of re-replication during the differentiation of human myoblasts into myotubes and during the lineage commitment of mesenchymal stem cells toward adipogenic, osteogenic, chondrogenic, and neuronal fates. In all differentiation systems examined, re-replication occurred within defined temporal windows. FACS-isolated re-replicating cells exhibited elevated gene expression using RNA-Seq specifically within re-replicated genomic regions. Moreover, re-replicated DNA was detected as extranuclear DNA. These findings support a model in which cells that do not undergo re-replication, and thus avoid increased chromosomal instability, may nonetheless boost the expression of differentiation-relevant genes by acquiring re-replicated DNA released from neighboring re-replicating cells. We propose that human stem cells exploit an evolutionarily conserved re-replication mechanism to transiently increase gene copy number and thereby meet the heightened protein demands associated with differentiation.

Molecular characterization of balanced complex chromosomal rearrangements (CCR) aids in understanding the pathophysiological mechanism and corresponding genotype-phenotype correlations. The present case describes a male child with intellectual disability, developmental delay, and dysmorphism. A thorough and sequential genetic evaluation using karyotyping, fluorescence insitu hybridization (FISH), chromosomal microarray (CMA), and long read sequencing (LRS) identified a genomically balanced CCR. The CCR involved eight chromosomes, the largest to be documented till date for chromoanagenesis and being balanced despite the high level of complex chromosomal involvement. Translocations accounted for the majority of the rearrangements along with an insertion, inversion, and a small deletion likely driven by chromoplexy. Although the CCR was genomically balanced, it may still result in functionally significant genomic consequences including gene disruptions, gene fusions, and position effects. Long read whole genome sequencing using PacBio was used for breakpoint characterization that revealed three protein-coding genes to be disrupted, namely, NLGN4X, LAMA4, and ALG6. Of these, a candidate association was observed for the NLGN4X gene with the intellectual disability phenotype reported in the proband, which is likely due to disruption of transcription and nonsense mediated decay. We show combinatorial application of advanced genomic technologies with orthogonal cytogenetic techniques in delineating balanced CCRs and understanding the biological and potential clinical implications of balanced yet functionally disruptive CCRs.

SummaryNewborn screening (NBS) for spinal muscular atrophy (SMA) enables rapid diagnosis and pre-symptomatic treatment of infants with bi-allelic SMN1 deletions. Standard PCR-based assays detect ∼95% of cases by identifying the absence of SMN1 exon 7; however, rare sequence variants can escape detection. We describe two newborns (in Germany and Australia) identified by NBS as lacking SMN1 but subsequently shown to carry a single SMN1 copy—with no SMN2 in P1 and one SMN2 copy in P2. Gene-specific long-range PCR and Sanger sequencing revealed two distinct 4-bp deletions in SMN1 exon 7 (c.855_858delAGAA [p.Arg288AlafsTer5] in P1 and c.861_864delAAGG [p.Arg288AlafsTer5] in P2). Both variants disrupt the reverse primer-binding site used in NBS assays and cause the same frameshift p.Arg288AlafsTer5, predicted to be deleterious. A plethora of assays demonstrated preserved exon 7 splicing, markedly reduced SMN protein abundance, and wild-type-like protein thermostability. In vivo, expression of the p.Arg288AlafsTer5 protein in zebrafish fully rescued the progressive motor and survival defects of smn1-deficient mutants. These findings raise the possibility that this novel SMN isoform has enhanced functional efficiency relative to the wild type. Population data (gnomAD) suggest that ∼800 individuals of European ancestry may carry these variants in trans with an SMN1 deletion, yet none have been reported with SMA. Based on our data, no therapy was initiated. Both children remain healthy at 24 months of age, avoiding >US$4 million in potential treatment costs. These findings challenge the assumption that complete loss of full-length SMN invariably causes SMA and suggest that very low levels of this novel SMN isoform can sustain normal motor development.

Background: Neutralising antibody titres are widely used as key immunogenicity endpoints in Ebola virus (EBOV) vaccine and monoclonal antibody clinical trials. However, direct comparison of results across studies remains challenging due to the use of heterogeneous neutralisation platforms, ranging from pseudotyped viruses to live EBOV assays. These limitations restrict assay standardisation, validation, scalability, and compliance with good clinical laboratory practice (GCLP), particularly in outbreak-prone and resource-limited settings. There is an unmet need for neutralisation assays that combine biological authenticity with clinical-trial compatibility. Methods: We developed and optimised a fluorescence-based microneutralisation assay using a biologically contained EBOV lacking the essential VP30 gene (EBOVΔVP30), enabling multi-cycle viral replication under containment level 2 conditions. Using a defined panel of serum samples from Ebola virus disease survivors and EBOV-negative controls, we benchmarked EBOVΔVP30 neutralisation titres against previously generated data obtained with wild-type EBOV and pseudotyped virus platforms. Assay performance was evaluated in terms of sensitivity, reproducibility, discrimination between positive and negative samples, and correlation with live virus neutralisation. Calibration was performed using the WHO International Standard for anti-EBOV immunoglobulin. Findings: The EBOVΔVP30 microneutralisation assay robustly distinguished EBOV survivor sera from negative controls (p < 0.0001) and demonstrated a strong correlation with live EBOV neutralisation titres (Spearman ρ = 0.8725). This correlation exceeded that observed for HIV-1-based pseudotyped assays and for the vesicular stomatitis virus-based platforms. The fluorescence-based read-out showed comparable sensitivity to conventional immunostaining, supporting its suitability for high-throughput and standardised implementation. Importantly, assay conditions were compatible with BSL-2 laboratories and GCLP-aligned workflows. Interpretation: Biologically contained EBOVΔVP30 provides a clinically relevant and scalable alternative to existing neutralisation platforms, bridging the gap between pseudotyped assays and wild-type virus testing. By improving biological relevance while maintaining accessibility and standardisation, this assay has the potential to enhance comparability of immunogenicity data across EBOV vaccine and therapeutic antibody (pre-)clinical trials, aligning with global outbreak preparedness and trial harmonisation objectives.

BACKGROUND:Sex and atherosclerotic plaque histology are intertwined, with fibrous plaques being more prevalent in women. Plaque erosion, a significant contributor to acute coronary syndromes, is linked to fibrous plaques and is more prevalent in women than men. We hypothesize that the molecular drivers of histologically determined fibrous plaques differ between men and women.METHODS:Human end-stage atherosclerotic plaques were isolated from carotid endarterectomy patients included in the Athero-Express Biobank. Fibrous plaques were histologically assessed, linked to clinical characteristics, and processed for protein, bulk RNA, single-cell RNA, and DNA methylation data. We leveraged sex-differential gene expression and deconvolution analyses to uncover sex-biased molecular and cellular mechanisms. Spatial transcriptomics localized gene expression patterns in plaques. Furthermore, we studied the female-biased processes in human plaque endothelial cells and vascular smooth muscle cells stimulated with TGF-β (transforming growth factor-β), with or without SMAD3 (SMAD family member 3) inhibition.RESULTS:Of 1889 atherosclerotic plaques (1309 male and 580 female), fibrous lesions were observed in 50% of female and 31% of male patients. Compared with patients with atheromatous plaques (n=494), women with fibrous plaques exhibited a high prevalence of smoking, while men with fibrous plaques presented more often with diabetes. Female fibrous plaques were characterized by smooth muscle cell–driven ECM (extracellular matrix) remodeling, TGF-β response, and endothelial-to-mesenchymal transition, localized to the fibrous cap. Conversely, male plaques were linked to macrophage-mediated inflammation proximal to the core, dependent on diabetes. Finally, we experimentally confirmed these female-biased mechanisms, showing that TGF-β induced endothelial-to-mesenchymal transition in endothelial cells and ECM remodeling in vascular smooth muscle cells, both partly reversed by SMAD3 inhibition.CONCLUSIONS:Women and men with end-stage fibrous atherosclerotic plaques exhibit distinct clinical and molecular profiles. These mechanisms might be candidate pathways to understand plaque erosion from a molecular point of view and may provide promising targets for atherosclerosis therapies, as they account for both sex and plaque phenotype.

Abstract Background The diagnostic accuracy of HbA 1C for prediabetes has been questioned due to its discordance with fasting plasma glucose (FPG) and 2 h oral glucose tolerance test (OGTT) glucose in non-white populations. This study aims to estimate concordance in the diagnosis of prediabetes using HbA 1C FPG, and OGTT in a Filipino-American cohort. Methods Cross-sectional data from 149 Filipino-Americans without known diabetes living in the San Francisco Bay Area were used to compare prevalence of prediabetes as diagnosed by HbA 1C, versus diagnosis by FPG and OGTT. Results Thirty nine percent of subjects met the diagnosis of prediabetes using any one of the measures. Overall agreement between HbA 1C , FPG and OGTT was low. Prevalence was 8.1% by FPG, 8.7% by OGTT and 35% by HbA 1C . BMI, waist-hip ratio, insulin, HOMA-IR, blood pressure, and triglycerides were significantly higher in those with prediabetes by HbA 1C versus normal HbA 1C . Conclusions There is significant discordance between HbA 1C , FPG, and OGTT in diagnosing prediabetes in a Filipino-American cohort. HbA 1C detected four times as many individuals with prediabetes than FPG or OGTT. Individuals classified with prediabetes by HbA 1C had indicators of more insulin resistance compared to individuals with normal HbA 1C suggesting that HbA 1C appears to detect true metabolic abnormalities on the path to diabetes as opposed to detecting false positives. These results have important implications for diabetes and prediabetes screening in Filipinos.

Background and ObjectivesLeigh syndrome is an important manifestation of childhood-onset primary mitochondrial disease. Panel sequencing and whole exome sequencing are cost-effective for diagnosing mitochondrial diseases; however, more than half of mitochondrial disease cases remain genetically undiagnosed. This study aimed to demonstrate that combining whole genome sequencing (WGS) and RNA sequencing (RNA-seq) analyses can identify disease-causing variants that would otherwise be missed.MethodsWe performed WGS and RNA-seq on a patient with Leigh syndrome. Chromosomal phasing using Sanger sequencing of parental and patient blood samples was conducted to confirm compound heterozygous variants. RNA-seq data were analyzed for splicing abnormalities. Overexpression studies of wild-type NDUFA3 in patient-derived fibroblasts were performed to assess restoration of mitochondrial function.ResultsWe discovered compound heterozygous intronic variants (c.86-16_86-15del in intron2 and c.164-362G>A in intron3) of the NDUFA3 gene. RNA-seq data analysis revealed intron retention and exonization in NDUFA3. Exonization was related to a variant involving the mobile element Alu that resulted in complex abnormal splicing events. Overexpression of wild-type NDUFA3 restored mitochondrial dysfunction in patient-derived fibroblasts, confirming NDUFA3 as a Leigh syndrome causative gene.DiscussionThis study highlights the importance of combining WGS and RNA-seq and provides new insights into detecting abnormalities in deep intronic regions, particularly those involving mobile elements, such as Alu. This approach can play a crucial role in identifying genetic variations and elucidating transcriptional control mechanisms that are not readily achieved by conventional methods, especially in the context of mobile element-induced complexities.