PSMD11 loss-of-function variants correlate with a neurobehavioral phenotype, obesity, and increased interferon response.

Primary proteasomopathies have recently emerged as a new class of rare early-onset neurodevelopmental disorders (NDDs) caused by pathogenic variants in the PSMB1, PSMC1, PSMC3, or PSMD12 proteasome genes. Proteasomes are large multi-subunit protein complexes that maintain cellular protein homeostasis by clearing ubiquitin-tagged damaged, misfolded, or unnecessary proteins. In this study, we have identified PSMD11 as an additional proteasome gene in which pathogenic variation is associated with an NDD-causing proteasomopathy. PSMD11 loss-of-function variants caused early-onset syndromic intellectual disability and neurodevelopmental delay with recurrent obesity in 10 unrelated children. Our findings demonstrate that the cognitive impairment observed in these individuals could be recapitulated in Drosophila melanogaster with depletion of the PMSD11 ortholog Rpn6, which compromised reversal learning. Our investigations in subject samples further revealed that PSMD11 loss of function resulted in impaired 26S proteasome assembly and the acquisition of a persistent type I interferon (IFN) gene signature, mediated by the integrated stress response (ISR) protein kinase R (PKR). In summary, these data identify PSMD11 as an additional member of the growing family of genes associated with neurodevelopmental proteasomopathies and provide insights into proteasomal biology in human health.

Biallelic USP14 variants cause a syndromic neurodevelopmental disorder.

PURPOSE: Imbalances in protein homeostasis affect human brain development, with the ubiquitin-proteasome system (UPS) and autophagy playing crucial roles in neurodevelopmental disorders (NDD). This study explores the impact of biallelic USP14 variants on neurodevelopment, focusing on its role as a key hub connecting UPS and autophagy. METHODS: Here, we identified biallelic USP14 variants in 4 individuals from 3 unrelated families: 1 fetus, a newborn with a syndromic NDD and 2 siblings affected by a progressive neurological disease. Specifically, the 2 siblings from the latter family carried 2 compound heterozygous variants c.8T>C p.(Leu3Pro) and c.988C>T p.(Arg330∗), whereas the fetus had a homozygous frameshift c.899_902del p.(Lys300Serfs∗24) variant, and the newborn patient harbored a homozygous frameshift c.233_236del p.(Leu78Glnfs∗11) variant. Functional studies were conducted using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, western blotting, and mass spectrometry analyses in both patient-derived and CRISPR-Cas9-generated cells. RESULTS: Our investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities. CONCLUSION: Biallelic USP14 variants in NDD patients perturbed protein degradation pathways, potentially contributing to disorder etiology. Altered UPS, autophagy, and mitophagy activities underscore the intricate interplay, elucidating their significance in maintaining proper protein homeostasis during brain development.

Optical Genome Mapping as a Potential Routine Clinical Diagnostic Method.

Chromosome analysis (CA) and chromosomal microarray analysis (CMA) have been successfully used to diagnose genetic disorders. However, many conditions remain undiagnosed due to limitations in resolution (CA) and detection of only unbalanced events (CMA). Optical genome mapping (OGM) has the potential to address these limitations by capturing both structural variants (SVs) resulting in copy number changes and balanced rearrangements with high resolution. In this study, we investigated OGM's concordance using 87 SVs previously identified by CA, CMA, or Southern blot. Overall, OGM was 98% concordant with only three discordant cases: (1) uncalled translocation with one breakpoint in a centromere; (2) uncalled duplication with breakpoints in the pseudoautosomal region 1; and (3) uncalled mosaic triplication originating from a marker chromosome. OGM provided diagnosis for three previously unsolved cases: (1) disruption of the SON gene due to a balanced reciprocal translocation; (2) disruption of the NBEA gene due to an inverted insertion; (3) disruption of the TSC2 gene due to a mosaic deletion. We show that OGM is a valid method for the detection of many types of SVs in a single assay and is highly concordant with legacy cytogenomic methods; however, it has limited SV detection capabilities in centromeric and pseudoautosomal regions.

Transcript capture and ultradeep long-read RNA sequencing (CAPLRseq) to diagnose HNPCC/Lynch syndrome.

PURPOSE: Whereas most human genes encode multiple mRNA isoforms with distinct function, clinical workflows for assessing this heterogeneity are not readily available. This is a substantial shortcoming, considering that up to 25% of disease-causing gene variants are suspected of disrupting mRNA splicing or mRNA abundance. Long-read sequencing can readily portray mRNA isoform diversity, but its sensitivity is relatively low due to insufficient transcriptome penetration. METHODS: We developed and applied capture-based target enrichment from patient RNA samples combined with Oxford Nanopore long-read sequencing for the analysis of 123 hereditary cancer transcripts (capture and ultradeep long-read RNA sequencing (CAPLRseq)). RESULTS: Validating CAPLRseq, we confirmed 17 cases of hereditary non-polyposis colorectal cancer/Lynch syndrome based on the demonstration of splicing defects and loss of allele expression of mismatch repair genes MLH1, PMS2, MSH2 and MSH6. Using CAPLRseq, we reclassified two variants of uncertain significance in MSH6 and PMS2 as either likely pathogenic or benign. CONCLUSION: Our data show that CAPLRseq is an automatable and adaptable workflow for effective transcriptome-based identification of disease variants in a clinical diagnostic setting.

Parallel in-depth analysis of repeat expansions in ataxia patients by long-read sequencing.

Instability of simple DNA repeats has been known as a common cause of hereditary ataxias for over 20 years. Routine genetic diagnostics of these phenotypically similar diseases still rely on an iterative workflow for quantification of repeat units by PCR-based methods of limited precision. We established and validated clinical nanopore Cas9-targeted sequencing, an amplification-free method for simultaneous analysis of 10 repeat loci associated with clinically overlapping hereditary ataxias. The method combines target enrichment by CRISPR-Cas9, Oxford Nanopore long-read sequencing and a bioinformatics pipeline using the tools STRique and Megalodon for parallel detection of length, sequence, methylation and composition of the repeat loci. Clinical nanopore Cas9-targeted sequencing allowed for the precise and parallel analysis of 10 repeat loci associated with adult-onset ataxia and revealed additional parameter such as FMR1 promotor methylation and repeat sequence required for diagnosis at the same time. Using clinical nanopore Cas9-targeted sequencing we analysed 100 clinical samples of undiagnosed ataxia patients and identified causative repeat expansions in 28 patients. Parallel repeat analysis enabled a molecular diagnosis of ataxias independent of preconceptions on the basis of clinical presentation. Biallelic expansions within RFC1 were identified as the most frequent cause of ataxia. We characterized the RFC1 repeat composition of all patients and identified a novel repeat motif, AGGGG. Our results highlight the power of clinical nanopore Cas9-targeted sequencing as a readily expandable workflow for the in-depth analysis and diagnosis of phenotypically overlapping repeat expansion disorders.