Expanding the genetic and phenotypic landscape of replication factor C complex-related disorders: RFC4 deficiency is linked to a multisystemic disorder.

The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.

Robinow syndrome in an extremely preterm infant: Novel homozygous ROR2 variant detected by rapid exome sequencing.

An extremely preterm infant presented with clinical and radiological features of Robinow syndrome including butterfly vertebrae, posterior rib fusion, brachydactyly, nail hypoplasia, and retromicrognathia resulting in difficult endotracheal intubation in the intensive care setting. Rapid trio exome sequencing detected a novel homozygous likely pathogenic missense variant in the ROR2 gene, NM_004560.3:c.950A>G, p.(Tyr317Cys), for which both parents were heterozygous carriers. In-silico protein modeling predicted a deleterious effect on the function of the protein. We report an extreme premature infant with novel homozygous likely pathogenic variant in the ROR2 gene consistent with autosomal recessive Robinow syndrome. This case expands the phenotypic and genotypic spectrum of this disorder and highlights the benefit of performing rapid exome sequencing early during evaluation to aid in patient management and providing accurate genetic counseling to families.

Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome.

PURPOSE: Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort. METHODS: We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. RESULTS: Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. CONCLUSION: Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome.

Compromised paraspeckle formation as a pathogenic factor in FUSopathies.

Paraspeckles are nuclear bodies formed by a set of specialized proteins assembled on the long non-coding RNA NEAT1; they have a role in nuclear retention of hyperedited transcripts and are associated with response to cellular stress. Fused in sarcoma (FUS) protein, linked to a number of neurodegenerative disorders, is an essential paraspeckle component. We have shown that its recruitment to these nuclear structures is mediated by the N-terminal region and requires prion-like activity. FUS interacts with p54nrb/NONO, a major constituent of paraspeckles, in an RNA-dependent manner and responds in the same way as other paraspeckle proteins to alterations in cellular homeostasis such as changes in transcription rates or levels of protein methylation. FUS also regulates NEAT1 levels and paraspeckle formation in cultured cells, and FUS deficiency leads to loss of paraspeckles. Pathological gain-of-function FUS mutations might be expected to affect paraspeckle function in human diseases because mislocalized amyotrophic lateral sclerosis (ALS)-linked FUS variants sequester other paraspeckle proteins into aggregates formed in cultured cells and into neuronal inclusions in a transgenic mouse model of FUSopathy. Furthermore, we detected abundant p54nrb/NONO-positive inclusions in motor neurons of patients with familial forms of ALS caused by FUS mutations, but not in other ALS cases. Our results suggest that both loss and gain of FUS function can trigger disruption of paraspeckle assembly, which may impair protective responses in neurons and thereby contribute to the pathogenesis of FUSopathies.

Multistep process of FUS aggregation in the cell cytoplasm involves RNA-dependent and RNA-independent mechanisms.

Fused in sarcoma (FUS) is an RNA-binding protein involved in pathogenesis of several neurodegenerative diseases. Aggregation of mislocalized FUS into non-amyloid inclusions is believed to be pivotal in the development of cell dysfunction, but the mechanism of their formation is unclear. Using transient expression of a panel of deletion and chimeric FUS variants in various cultured cells, we demonstrated that FUS accumulating in the cytoplasm nucleates a novel type of RNA granules, FUS granules (FGs), that are structurally similar but not identical to physiological RNA transport granules. Formation of FGs requires FUS N-terminal prion-like domain and the ability to bind specific RNAs. Clustering of FGs coupled with further recruitment of RNA and proteins produce larger structures, FUS aggregates (FAs), that resemble but are clearly distinct from stress granules. In conditions of attenuated transcription, FAs lose RNA and dissociate into RNA-free FUS complexes that become precursors of large aggresome-like structures. We propose a model of multistep FUS aggregation involving RNA-dependent and RNA-independent stages. This model can be extrapolated to formation of pathological inclusions in human FUSopathies.

Novel compound heterozygous TTN variants as a cause of severe neonatal congenital contracture syndrome without cardiac involvement diagnosed with rapid trio exome sequencing.

This report focuses on a case of severe congenital myopathy with arthrogryposis without cardiac involvement due to compound heterozygous variants in the TTN gene. The proband presented with severe axial hypotonia, arthrogryposis and severe respiratory insufficiency with ventilator dependence. Electromyogram was abnormal with absent motor responses but preserved sensory nerve responses. Rapid gene-agnostic trio exome sequencing detected novel compound heterozygous variants in the TTN gene. One variant is a truncating frameshift located in the meta-transcript only exon 195. The other variant is a nonsense variant in exon 327 which affects all recognised post-natal transcripts apart from one. This case presents with a severe phenotype and adds to the expanding known variants associated with autosomal recessive titinopathy. It also demonstrates the utility of rapid trio exome sequencing when used early in the clinical course.

Early lethality and neuronal proteinopathy in mice expressing cytoplasm-targeted FUS that lacks the RNA recognition motif.

Mutations to the RNA binding protein, fused in sarcoma (FUS) occur in ∼5% of familial ALS and FUS-positive cytoplasmic inclusions are commonly observed in these patients. Altered RNA metabolism is increasingly implicated in ALS, yet it is not understood how the specificity with which FUS interacts with RNA in the cytoplasm can affect its aggregation in vivo. To further understand this, we expressed, in mice, a form of FUS (FUS ΔRRMcyt) that lacked the RNA recognition motif (RRM), thought to impart specificity to FUS-RNA interactions, and carried an ALS-associated point mutation, R522G, retaining the protein in the cytoplasm. Here we report the phenotype and results of histological assessment of the brain of transgenic mice expressing this isoform of FUS. Results demonstrated that neuronal expression of FUS ΔRRMcyt caused early lethality often preceded by severe tremor. Large FUS-positive cytoplasmic inclusions were found in many brain neurons; however, neither neuronal loss nor neuroinflammatory response was observed. In conclusion, the extensive FUS proteinopathy and severe phenotype of these mice suggests that affecting the interactions of FUS with RNA in vivo may augment its aggregation in the neuronal cytoplasm and the severity of disease processes.

Recruitment into stress granules prevents irreversible aggregation of FUS protein mislocalized to the cytoplasm.

Fused in sarcoma (FUS) belongs to the group of RNA-binding proteins implicated as underlying factors in amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. Multiple FUS gene mutations have been linked to hereditary forms, and aggregation of FUS protein is believed to play an important role in pathogenesis of these diseases. In cultured cells, FUS variants with disease-associated amino acid substitutions or short deletions affecting nuclear localization signal (NLS) and causing cytoplasmic mislocalization can be sequestered into stress granules (SGs). We demonstrated that disruption of motifs responsible for RNA recognition and binding not only prevents SG recruitment, but also dramatically increases the protein propensity to aggregate in the cell cytoplasm with formation of juxtanuclear structures displaying typical features of aggresomes. Functional RNA-binding domains from TAR DNA-binding protein of 43 kDa (TDP-43) fused to highly aggregation-prone C-terminally truncated FUS protein restored the ability to enter SGs and prevented aggregation of the chimeric protein. Truncated FUS was also able to trap endogenous FUS molecules in the cytoplasmic aggregates. Our data indicate that RNA binding and recruitment to SGs protect cytoplasmic FUS from aggregation, and loss of this protection may trigger its pathological aggregation in vivo.