Division of Labor between PCNA Loaders in DNA Replication and Sister Chromatid Cohesion Establishment.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.

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.

ATRX Promotes DNA Repair Synthesis and Sister Chromatid Exchange during Homologous Recombination.

ATRX is a chromatin remodeler that, together with its chaperone DAXX, deposits the histone variant H3.3 in pericentromeric and telomeric regions. Notably, ATRX is frequently mutated in tumors that maintain telomere length by a specific form of homologous recombination (HR). Surprisingly, in this context, we demonstrate that ATRX-deficient cells exhibit a defect in repairing exogenously induced DNA double-strand breaks (DSBs) by HR. ATRX operates downstream of the Rad51 removal step and interacts with PCNA and RFC-1, which are collectively required for DNA repair synthesis during HR. ATRX depletion abolishes DNA repair synthesis and prevents the formation of sister chromatid exchanges at exogenously induced DSBs. DAXX- and H3.3-depleted cells exhibit identical HR defects as ATRX-depleted cells, and both ATRX and DAXX function to deposit H3.3 during DNA repair synthesis. This suggests that ATRX facilitates the chromatin reconstitution required for extended DNA repair synthesis and sister chromatid exchange during HR.

Structural investigation of pathogenic RFC1 AAGGG pentanucleotide repeats reveals a role of G-quadruplex in dysregulated gene expression in CANVAS.

An expansion of AAGGG pentanucleotide repeats in the replication factor C subunit 1 (RFC1) gene is the genetic cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), and it also links to several other neurodegenerative diseases including the Parkinson's disease. However, the pathogenic mechanism of RFC1 AAGGG repeat expansion remains enigmatic. Here, we report that the pathogenic RFC1 AAGGG repeats form DNA and RNA parallel G-quadruplex (G4) structures that play a role in impairing biological processes. We determine the first high-resolution nuclear magnetic resonance (NMR) structure of a bimolecular parallel G4 formed by d(AAGGG)2AA and reveal how AAGGG repeats fold into a higher-order structure composed of three G-tetrad layers, and further demonstrate the formation of intramolecular G4s in longer DNA and RNA repeats. The pathogenic AAGGG repeats, but not the nonpathogenic AAAAG repeats, form G4 structures to stall DNA replication and reduce gene expression via impairing the translation process in a repeat-length-dependent manner. Our results provide an unprecedented structural basis for understanding the pathogenic mechanism of AAGGG repeat expansion associated with CANVAS. In addition, the high-resolution structures resolved in this study will facilitate rational design of small-molecule ligands and helicases targeting G4s formed by AAGGG repeats for therapeutic interventions.

Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures.

CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A2G3)n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the pathogenic (A2G3)n and nonpathogenic (A4G)n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in vitro reveals triplex H-DNA formation by only the pathogenic repeat. Consistently, bioinformatic analysis of S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs. Finally, the pathogenic, but not the nonpathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that the CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.

Replication protein A dynamically re-organizes on primer/template junctions to permit DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.

DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, PCNA, carry out DNA synthesis during lagging strand replication, initiation of leading strand replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process involving the major single strand DNA (ssDNA)-binding protein complex, RPA, the processivity sliding clamp loader, RFC, PCNA and pol δ. During this process, the interactions of RPA, RFC and pol δ with a P/T junction all significantly overlap. A burning issue that has yet to be resolved is how these overlapping interactions are accommodated during this process. To address this, we design and utilize novel, ensemble FRET assays that continuously monitor the interactions of RPA, RFC, PCNA and pol δ with DNA as pol δ holoenzymes are assembled and initiate DNA synthesis. Results from the present study reveal that RPA remains engaged with P/T junctions throughout this process and the RPA•DNA complexes dynamically re-organize to allow successive binding of RFC and pol δ. These results have broad implications as they highlight and distinguish the functional consequences of dynamic RPA•DNA interactions in RPA-dependent DNA metabolic processes.

Differences between bacteria and eukaryotes in clamp loader mechanism, a conserved process underlying DNA replication.

Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader replication factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the Escherichia coli clamp loader at high resolution using cryo-electron microscopy. We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how the clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.

Structural polymorphism of the nucleic acids in pentanucleotide repeats associated with the neurological disorder CANVAS.

Short tandem repeats are inherently unstable during DNA replication depending on repeat length, and the expansion of the repeat length in the human genome is responsible for repeat expansion disorders. Pentanucleotide AAGGG and ACAGG repeat expansions in intron 2 of the gene encoding replication factor C subunit 1 (RFC1) cause cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and other phenotypes of late-onset cerebellar ataxia. Herein, we reveal the structural polymorphism of the RFC1 repeats associated with CANVAS in vitro. Single-stranded AAGGG repeat DNA formed a hybrid-type G-quadruplex, whereas its RNA formed a parallel-type G-quadruplex with three layers. The RNA of the ACAGG repeat formed hairpin structure comprising C-G and G-C base pairs with A:A and GA:AG mismatched repeats. Furthermore, both pathogenic repeat RNAs formed more rigid structures than those of the nonpathogenic repeat RNAs. These findings provide novel insights into the structural polymorphism of the RFC1 repeats, which may be closely related to the disease mechanism of CANVAS.

The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts.

Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors.

Role of the repeat expansion size in predicting age of onset and severity in RFC1 disease.

RFC1 disease, caused by biallelic repeat expansion in RFC1, is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I-V ß = -1.06, P < 0.001; lobules VI-VII ß = -0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion.

Identification of RFC4 as a potential biomarker for pan-cancer involving prognosis, tumour immune microenvironment and drugs.

RFC4 is required for DNA polymerase δ and DNA polymerase ε to initiate DNA template expansion. Downregulated RFC4 inhibits tumour proliferation by causing S-phase arrest and inhibiting mitosis, resulting in the reduction of tumour cells. RFC4 has been implicated that it plays an important role in the initiation and progression of cancers, but a comprehensive analysis of the role of RFC4 in cancer has not been performed. We comprehensively analysed the expression, prognosis, methylation level, splicing level, relationship of RFC4 and immune infiltration, and pan-cancer immunotherapy response used various databases (including TCGA, GTEx, UALCAN, Oncosplicing, TIDE, TISCH, HPA and CAMOIP), and experimented its biological function in HCC. Through pan-cancer analysis, we found that RFC4 is significantly upregulated in most tumours. The tumour patients with high expression of RFC4 have poor prognosis. The methylation level and variable splicing level of RFC4 were abnormal in most tumours compared with the adjacent tissues. Furthermore, RFC4 was closely associated with immune cell infiltration in various cancers. RFC4 was significantly co-expressed with immune checkpoints and other immune-related genes. The expression of RFC4 could indicate the immunotherapy efficacy of some tumours. The RFC4 expression was associated with sensitivity to specific small molecule drugs. Cell experiments have shown that downregulated RFC4 can inhibit cell cycle and tumour cell proliferation. We conducted a systematic pan-cancer analysis of RFC4, and the results showed that RFC4 can serve as a biomarker for cancer diagnosis and prognosis. These findings open new perspectives for precision medicine.

Streamlined two-step fragment analysis PCR and exome sequencing of RFC1 for diagnostic testing of suspected CANVAS patients.

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is caused by biallelic pathogenic expansions, or compound heterozygosity with other pathogenic variants in the RFC1 gene. CANVAS is estimated to be underdiagnosed, both because of the lack of formal diagnostic criteria and molecular challenges that translate to lesser access and high cost of routine testing. Our aim was to address the need for making CANVAS genetic testing routine, by designing a streamlined two-step PCR consisting of a short-allele screening PCR and a confirmatory PCR with fragment capillary electrophoresis detection. Exome sequencing of RFC1 was additionally foreseen to resolve potential compound heterozygosity cases. Specificity of our approach was evaluated using ataxia patients with known non-CANVAS diagnoses, and optimized using Southern blot confirmed CANVAS patients. We evaluated our approach by testing patients consecutively referred for clinically suspected CANVAS using first the two-step PCR, followed by exome sequencing. Our approach was able to accurately identify negative and confirm positive cases in prospectively collected suspected CANVAS patients presenting with at least three typical clinical signs. The proposed testing approach provides an alternative method able to clearly distinguish between CANVAS negative and positive cases and can be easily incorporated into the genetic diagnostic laboratory workflow.

Cognitive Impairment Is Part of the Phenotype of Cerebellar Ataxia, Neuropathy, Vestibular Areflexia Syndrome (CANVAS).

BACKGROUND: Little is known about the impact of the cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) on cognition. OBJECTIVE: Our objective was to determine the frequency and severity of cognitive impairment in RFC1-positive patients and describe the pattern of deficits. METHODS: Participants underwent a comprehensive neuropsychological assessment. Volume of the cerebellum and its lobules was measured in those who underwent a 3 Tesla-magnetic resonance scan. RESULTS: Twenty-one patients underwent a complete assessment, including 71% scoring lower than the cutoff at the Montreal Cognitive assessment and 71% having a definite cerebellar cognitive affective/Schmahmann syndrome. Three patients had dementia and seven met the criteria of mild cognitive impairment. Severity of cognitive impairment did not correlate with severity of clinical manifestations. Performance at memory and visuospatial functions tests negatively correlated with the severity of cerebellar manifestations. CONCLUSION: Cognitive manifestations are frequent in RFC1-related disorders. They should be included in the phenotype and screened systematically. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Comprehensive Analysis of a Japanese Pedigree with Biallelic ACAGG Expansions in RFC1 Manifesting Motor Neuronopathy with Painful Muscle Cramps.

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive multisystem neurologic disorder caused by biallelic intronic repeats in RFC1. Although the phenotype of CANVAS has been expanding via diagnostic case accumulation, there are scant pedigree analyses to reveal disease penetrance, intergenerational fluctuations in repeat length, or clinical phenomena (including heterozygous carriers). We identified biallelic RFC1 ACAGG expansions of 1000 ~ repeats in three affected siblings having sensorimotor neuronopathy with spinocerebellar atrophy initially presenting with painful muscle cramps and paroxysmal dry cough. They exhibit almost homogeneous clinical and histopathological features, indicating motor neuronopathy. Over 10 years of follow-up, painful intractable muscle cramps ascended from legs to trunks and hands, followed by amyotrophy and subsequent leg pyramidal signs. The disease course combined with the electrophysical and imagery data suggest initial and prolonged hyperexcitability and the ensuing spinal motor neuron loss, which may progress from the lumbar to the rostral anterior horns and later expand to the corticospinal tract. Genetically, heterozygous ACAGG expansions of similar length were transmitted in unaffected family members of three successive generations, and some of them experienced muscle cramps. Leukocyte telomere length assays revealed comparatively shorter telomeres in affected individuals. This comprehensive pedigree analysis demonstrated a non-anticipating ACAGG transmission and high penetrance of manifestations with a biallelic state, especially motor neuronopathy in which muscle cramps serve as a prodromal and disease progress marker. CANVAS and RFC1 spectrum disorder should be considered when diagnosing lower dominant motor neuron disease, idiopathic muscle cramps, or neuromuscular hyperexcitability syndromes.

Electrophysiological features of the peripheral neuropathy in patients with pathologic biallelic RFC1 repeat expansions.

INTRODUCTION/AIMS: Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is caused by RFC1 expansions. Sensory neuronopathy, polyneuropathy, and involvement of motor, autonomic, and cranial nerves have all been described with RFC1 expansions. We aimed to describe the electrodiagnostic features of patients with RFC1 expansions through multimodal electrophysiological investigations. METHODS: Thirty-five patients, with a median age of 70 years, and pathologic biallelic repeat expansions in the RFC1 gene, were tested for motor and sensory nerve conduction, flexor carpi radialis (FCR) and soleus H-reflexes, blink reflex, electrochemical skin conductance, sympathetic skin response (SSR), and heart rate variability with deep breathing (HRV). RESULTS: Only 16 patients (46%) exhibited the full clinical CANVAS spectrum. Distal motor amplitudes were normal in 30 patients and reduced in the legs of five patients. Distal sensory amplitudes were bilaterally reduced in a non-length dependent manner in 30 patients. Conduction velocities were normal. Soleus H-reflexes were abnormal in 19/20 patients of whom seven had preserved Achilles reflexes. FCR H-reflexes were absent or decreased in amplitude in 13/14 patients. Blink reflex was abnormal in 4/19 patients: R1 latencies for two patients and R2 latencies for two others. Fourteen out of 31 patients (45%) had abnormal results in at least one autonomic nervous system test, either for ESC (12/31), SSR (5/14), or HRV (6/19). DISCUSSION: Less than half of the patients with RFC1 expansions exhibited the full clinical CANVAS spectrum, but nearly all exhibited typical sensory neuronopathy and abnormal H-reflexes. Involvement of small nerve fibers and brainstem neurons was less common.

Unexpected new insights into DNA clamp loaders: Eukaryotic clamp loaders contain a second DNA site for recessed 5' ends that facilitates repair and signals DNA damage: Eukaryotic clamp loaders contain a second DNA site for recessed 5' ends that facilitates repair and signals DNA damage.

Clamp loaders are pentameric AAA+ assemblies that use ATP to open and close circular DNA sliding clamps around DNA. Clamp loaders show homology in all organisms, from bacteria to human. The eukaryotic PCNA clamp is loaded onto 3' primed DNA by the replication factor C (RFC) hetero-pentameric clamp loader. Eukaryotes also have three alternative RFC-like clamp loaders (RLCs) in which the Rfc1 subunit is substituted by another protein. One of these is the yeast Rad24-RFC (Rad17-RFC in human) that loads a 9-1-1 heterotrimer clamp onto a recessed 5' end of DNA. Recent structural studies of Rad24-RFC have discovered an unexpected 5' DNA binding site on the outside of the clamp loader and reveal how a 5' end can be utilized for loading the 9-1-1 clamp onto DNA. In light of these results, new studies reveal that RFC also contains a 5' DNA binding site, which functions in gap repair. These studies also reveal many new features of clamp loaders. As reviewed herein, these recent studies together have transformed our view of the clamp loader mechanism.

Abnormal activation of RFC3, A YAP1/TEAD downstream target, promotes gastric cancer progression.

BACKGROUND: Gastric cancer (GC) is a malignant tumor with a high mortality rate, and thus, it is necessary to explore molecular mechanisms underlying its progression. While replication factor C subunit 3 (RFC3) has been demonstrated to function as an oncogene in many cancers, its role in GC remains unclear. METHODS: Tumor tissues were collected from clinical GC patients, and the expression of RFC3 was analyzed. NCI-N87 and HGC-27 cells were infected with lentivirus sh-RFC3 to knock down RFC3 expression. RFC3 expression levels were determined, in addition to cell biological behaviors both in vitro and in vivo. The relationship between RFC3 and the YAP1/TEAD signaling pathway was detected by dual luciferase reporter assay. RESULTS: RFC3 was upregulated in GC tumor tissues. RFC3 knockdown inhibited cell proliferation, promoted cell apoptosis of GC cells, and suppressed cell migration and invasion. Moreover, depleted RFC3 suppressed tumor growth and metastasis in vivo. Mechanistically, the YAP1/TEAD axis activated RFC3 expression transcriptionally by binding to the RFC3 promoter. CONCLUSIONS: RFC3 was transcriptional activated by the YAP1/TEAD signaling pathway, thus promoting GC progression. RFC3 may be a promising therapeutic target for GC.

RFC1: Motifs and phenotypes.

Biallelic intronic expansions (AAGGG)exp in intron 2 of the RFC1 gene have been shown to be a common cause of late-onset ataxia. Since their first description, the phenotypes, neurological damage, and pathogenic variants associated with the RFC1 gene have been frequently updated. Here, we review the various motifs, genetic variants, and phenotypes associated with the RFC1 gene. We searched PubMed for scientific articles published between March 1st, 2019, and January 15th, 2024. The motifs and phenotypes associated with the RFC1 gene are highly heterogeneous, making molecular diagnosis and clinical screening and investigation challenging. In this review we will provide clues to give a better understanding of RFC1 disease. We briefly discuss new methods for molecular diagnosis, the origin of cough in RFC1 disease, and research perspectives.

RFC5, regulated by circ_0038985/miR-3614-5p, functions as an oncogene in the progression of colorectal cancer.

Replication factor C 5 (RFC5) is involved in a variety of biological functions of cancer. However, the expression pattern of RFC5 and the underlying mechanisms in colorectal cancer (CRC) remain elusive. Here, we show that RFC5 is significantly upregulated in CRC tissues and cells. Patients with CRC and increased RFC5 levels have an unfavorable prognosis. RFC5 can promote the proliferation, migration, and invasion of CRC cells and inhibit the apoptosis of CRC cells. Additionally, upstream of RFC5, we constructed the competing endogenous RNA network and confirmed that RFC5 in this network was inhibited by miR-3614-5p by directly targeting its 3'-untranslated regions. We verified that circ_0038985, which is positively correlated with RFC5, directly targeted miR-3614-5p. Overexpression of circ_0038985 promoted CRC cell migration and invasion, and these effects were partially reversed by the reintroduction of miR-3614-5p. Moreover, we found that RFC5 may promote the vascular endothelial growth factor A (VEGFa)/vascular endothelial growth factor receptor 2 (VEGFR2)/extracellular signal-regulated protein kinase (ERK) pathway. The knockdown of RFC5 reduced CRC tumorigenesis in vivo. Collectively, these data demonstrate that the circ_0038985/miR-3614-5p/RFC5 axis plays a critical role in the progression of CRC, and RFC5 may promote CRC progression by affecting the VEGFa/VEGFR2/ERK pathway.

New Cerebellar Ataxia, Neuropathy, Vestibular Areflexia Syndrome cases are caused by the presence of a nonsense variant in compound heterozygosity with the pathogenic repeat expansion in the RFC1 gene.

The biallelic pathogenic repeat (AAGGG)400-2000 intronic expansion in the RFC1 gene has been recently described as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and as a major cause of late-onset ataxia. Since then, many heterozygous carriers have been identified, with an estimated allele frequency of 0.7% to 4% in the healthy population. Here, we describe in two affected CANVAS sisters the presence of the nonsense c.724C > T p.(Arg242*) variant in compound heterozygosity with the pathogenic repeat expansion in the RFC1 gene. Further RNA analysis demonstrated a reduced expression of the p.Arg242* allele in patients confirming an efficient nonsense-mediated mRNA decay. We also highlight the importance of considering the sequencing of the RFC1 gene for the diagnosis, especially in patients with CANVAS diagnosis carriers of the AAGGG repeat expansion.