Raymond T. O'KEefe

The ClpXP ATP-dependent unfoldase-protease complex plays important roles in maintaining protein homeostasis in the mitochondria. Pathogenic variants of CLPP have been identified in patients with Perrault syndrome type 3 (PRLTS3), a rare recessive mitochondrial disease characterized by sensorineural hearing loss, ovarian dysfunction, and neurological abnormalities. However, it remains unclear how these ClpP variants cause this disease. Here we identify three cellular substrates of ClpXP using proteomic analysis of PRLTS3 patient-derived fibroblasts, with follow up studies using induced pluripotent stem cells (iPSCs) and iPSC-derived neural progenitor cells, alongside in vitro biochemical assays. These substrates are: CHCHD2, a bi-organellar regulator of oxidative phosphorylation and mitochondrial stress responses; ALAS1, which catalyzes the rate-limiting step in heme biosynthesis; and TFAM, an essential mitochondrial transcription factor. Efficient degradation of these substrates by ClpXP requires the adaptor protein PDIP38. We propose that dysregulated cellular levels of these substrates might contribute to the etiology of PRLTS3.

Genetic disorders impacting vision affect millions of individuals worldwide, including age-related macular degeneration (common) and inherited retinal disorders (rare). There is an incomplete understanding of the impact of genetic variation on gene expression in the human retina and its role in genetic disorders. Through the generation of whole genome sequencing and bulk RNA-sequencing of neurosensory retina and retinal pigment epithelium from 201 post-mortem eyes, we uncover common and rare genomic variants shaping retinal expression profiles. This includes 1,483,595 significant cis-expression quantitative trait loci impacting 9,959 and 3,699 genes in neurosensory retina and retinal pigment epithelium, respectively, with associated genomic variants enriched to cis-candidate regulatory elements and notable shared eGenes between both tissues. We also detect 1051 expression outliers and prioritise 299 rare non-coding single-nucleotide, structural variants or copy number variants as plausible drivers for 28% of outlier events. This study increases understanding of gene expression regulation in the human retina.

Neurodevelopmental disorders (NDDs) affect 2–4% of the population, are predominantly genetic and remain unsolved in ~50% of individuals. We show that rare biallelic variants in RNU2-2 are enriched and over-transmitted in individuals with unresolved NDDs. We define a recessive RNU2-2 syndrome, delineate its unique genetic architecture and show that it manifests clinically as a severe developmental and epileptic encephalopathy. We find that candidate biallelic variants are significantly correlated with reduced U2-2 abundance, implicating compromised transcript stability as a probable pathomechanism. We identify a decreased ratio of U2-2 to its paralog U2-1 as a potential diagnostic biomarker for this condition. We show that the recessive RNU2-2 syndrome is genetically, clinically and mechanistically distinct from the dominant RNU2-2 disorder. Within our cohort, the recessive RNU2-2 syndrome emerges as by far the most frequent recessive NDD, greatly disproportionate to the small genomic footprint of this non-protein-coding gene.

Biallelic hypomorphic variants in PRORP cause the rare autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a clinical spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy with developmental delay and epilepsy. Here, we report two new affected individuals with biallelic PRORP variants with clinical features consistent with COXPD54. One individual was homozygous for c.1505G > A p.Arg502Gln, whereas the other was compound heterozygous for c.1510C > T, p.His504Tyr and c.893C > A, p.Ser298Ter (NM_014672.4). In vitro tRNA processing assays revealed decreased mitochondrial 5′ tRNA leader cleavage by human RNase P complex with the two novel missense PRORP metallonuclease domain variants. These data provide further evidence that biallelic PRORP variants disrupt 5’ tRNA leader cleavage and are associated with a pleiotropic phenotype of COXPD54.

R-loops are DNA–RNA hybrid structures that may promote mutagenesis. However, their contribution to human Mendelian disorders is unexplored. Here we show excess de novo variants in genomic regions that form R-loops (henceforth, ‘R-loop regions’) and demonstrate enrichment of R-loop region variants (RRVs) in ribozyme, snoRNA and snRNA genes, specifically in rare disease cohorts. Using this insight, we report neurodevelopmental disorders (NDDs) caused by rare variants in two major spliceosomal RNA encoding genes, RNU2-2 and RNU5B-1. These, along with the recently described RNU4-2-related ReNU syndrome, provide a genetic explanation for a substantial proportion of individuals with NDDs.

Precursor mRNA (pre-mRNA) must undergo splicing to remove intron sequences and join exons. This splicing process is catalysed by an RNA/protein complex called the spliceosome. At the centre of the catalytic spliceosome is the U5 small nuclear ribonucleoprotein (snRNP). Pathogenic variants in U5 snRNP core proteins are associated with various diseases commonly known as spliceosomopathies. Variants in TXNL4A and EFTUD2 manifest in craniofacial malformations while variants in PRPF8 and SNRNP200 manifest in retinitis pigmentosa. This perspective highlights research addressing how these specific manifestations come about as the spliceosome is required in all cells and at all developmental stages. Cell and animal models can replicate the human clinical specificity providing explanations for the specificity of the disorders. We propose that future research could benefit from models originating from patient-derived induced pluripotent stem cells (iPSCs) and isogenic controls to compare the coding and non-coding transcriptomic perturbations. Analysis of spliceosomal protein complexes and their interactome could also uncover novel insights on molecular pathogenesis. Finally, as studies highlight changes in metabolic processes, metabolomic studies could become a new venture in studying the consequences of U5 snRNP variants.

Purpose: To quantify the impact of non-canonical FBN1 splice site variants in undiagnosed Marfan syndrome (MFS), a connective tissue disorder associated with skeletal abnormalities and Familial Thoracic Aortic Aneurysm Disease (FTAAD). Methods: A systematic analysis of ultra-rare FBN1 variants was performed using genome sequencing data from the 100,000 Genomes Project. Variants were annotated with SpliceAI and the significance of enrichment among individuals with FTAAD was assessed using Fisher’s exact test. Experimental validation utilised RNAseq, RT-PCR, minigene constructs and replication analysis was with data from UK Biobank. Results: Using aggregate data for 78,195 individuals, we identified 13,864 singleton SNVs in FBN1 of which 21 were predicted to impact splicing (SpliceAI >0.5). Incidence of candidate splice variants in individuals recruited with FTAAD (9/703) was significantly elevated compared with that seen in non-FTAAD participants (12/77,492; OR=84, p =9.7×10 ⁻¹⁴ ). Additional analysis uncovered a further 14 families harbouring 11 different FBN1 splice variants. A total of 20 candidate splice variants in 23 families were identified, of which 70% lay beyond the ±8 splice regions. RNA testing confirmed the predicted splice aberration in 14/20 and for 9/20, pseudoexonization was the likely splicing-anomaly. Conclusion: Our findings indicate that non-canonical splice variants may account for ~3% of families with undiagnosed FTAAD, highlighting the importance of incorporating analysis of introns and confirmatory RNA testing into genetic testing for MFS.