Biallelic structural variants in three patients with ERCC8-related Cockayne syndrome and a potential pitfall of copy number variation analysis.

Cockayne syndrome (CS) is a rare autosomal recessive disorder caused by mutations in ERCC8 or ERCC6. Most pathogenic variants in ERCC8 are single nucleotide substitutions. Structural variants (SVs) have been reported in patients with ERCC8-related CS. However, comprehensive molecular detection, including SVs of ERCC8, in CS patients remains problematic. Herein, we present three Japanese patients with ERCC8-related CS in whom causative SVs were identified using whole-exome-based copy number variation (CNV) detection tools. One patient showed compound heterozygosity for a 259-kb deletion and a deletion of exon 4 which has previously been reported as an Asia-specific variant. The other two patients were homozygous for the same exon 4 deletion. The exon 4 deletion was detected only by the ExomeDepth software. Intrigued by the discrepancy in the detection capability of various tools for the SVs, we evaluated the analytic performance of four whole-exome-based CNV detection tools using an exome data set from 337 healthy individuals. A total of 1,278,141 exons were predicted as being affected by the 4 CNV tools. Interestingly 95.1% of these affected exons were detected by one tool alone. Thus, we expect that the use of multiple tools may improve the detection rate of SVs from aligned exome data.

A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function.

Cockayne syndrome (CS) is a premature ageing condition characterized by microcephaly, growth failure, and neurodegeneration. It is caused by mutations in ERCC6 or ERCC8 encoding for Cockayne syndrome B (CSB) and A (CSA) proteins, respectively. CSA and CSB have well-characterized roles in transcription-coupled nucleotide excision repair, responsible for removing bulky DNA lesions, including those caused by UV irradiation. Here, we report that CSA dysfunction causes defects in the nuclear envelope (NE) integrity. NE dysfunction is characteristic of progeroid disorders caused by a mutation in NE proteins, such as Hutchinson-Gilford progeria syndrome. However, it has never been reported in Cockayne syndrome. We observed CSA dysfunction affected LEMD2 incorporation at the NE and increased actin stress fibers that contributed to enhanced mechanical stress to the NE. Altogether, these led to NE abnormalities associated with the activation of the cGAS/STING pathway. Targeting the linker of the nucleoskeleton and cytoskeleton complex was sufficient to rescue these phenotypes. This work reveals NE dysfunction in a progeroid syndrome caused by mutations in a DNA damage repair protein, reinforcing the connection between NE deregulation and ageing.

HiPSC-derived 3D neural models reveal neurodevelopmental pathomechanisms of the Cockayne Syndrome B.

Cockayne Syndrome B (CSB) is a hereditary multiorgan syndrome which-through largely unknown mechanisms-can affect the brain where it clinically presents with microcephaly, intellectual disability and demyelination. Using human induced pluripotent stem cell (hiPSC)-derived neural 3D models generated from CSB patient-derived and isogenic control lines, we here provide explanations for these three major neuropathological phenotypes. In our models, CSB deficiency is associated with (i) impaired cellular migration due to defective autophagy as an explanation for clinical microcephaly; (ii) altered neuronal network functionality and neurotransmitter GABA levels, which is suggestive of a disturbed GABA switch that likely impairs brain circuit formation and ultimately causes intellectual disability; and (iii) impaired oligodendrocyte maturation as a possible cause of the demyelination observed in children with CSB. Of note, the impaired migration and oligodendrocyte maturation could both be partially rescued by pharmacological HDAC inhibition.

Differential processing of RNA polymerase II at DNA damage correlates with transcription-coupled repair syndrome severity.

DNA damage severely impedes gene transcription by RNA polymerase II (Pol II), causing cellular dysfunction. Transcription-Coupled Nucleotide Excision Repair (TC-NER) specifically removes such transcription-blocking damage. TC-NER initiation relies on the CSB, CSA and UVSSA proteins; loss of any results in complete TC-NER deficiency. Strikingly, UVSSA deficiency results in UV-Sensitive Syndrome (UVSS), with mild cutaneous symptoms, while loss of CSA or CSB activity results in the severe Cockayne Syndrome (CS), characterized by neurodegeneration and premature aging. Thus far the underlying mechanism for these contrasting phenotypes remains unclear. Live-cell imaging approaches reveal that in TC-NER proficient cells, lesion-stalled Pol II is swiftly resolved, while in CSA and CSB knockout (KO) cells, elongating Pol II remains damage-bound, likely obstructing other DNA transacting processes and shielding the damage from alternative repair pathways. In contrast, in UVSSA KO cells, Pol II is cleared from the damage via VCP-mediated proteasomal degradation which is fully dependent on the CRL4CSA ubiquitin ligase activity. This Pol II degradation might provide access for alternative repair mechanisms, such as GG-NER, to remove the damage. Collectively, our data indicate that the inability to clear lesion-stalled Pol II from the chromatin, rather than TC-NER deficiency, causes the severe phenotypes observed in CS.

Cockayne Syndrome Linked to Elevated R-Loops Induced by Stalled RNA Polymerase II during Transcription Elongation.

Mutations in the Cockayne Syndrome group B (CSB) gene cause cancer in mice, but premature aging and severe neurodevelopmental defects in humans. CSB, a member of the SWI/SNF family of chromatin remodelers, plays diverse roles in regulating gene expression and transcription-coupled nucleotide excision repair (TC-NER); however, these functions do not explain the distinct phenotypic differences observed between CSB-deficient mice and humans. During investigating Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism that involves elongating RNA polymerase II (RNAPII) undergoing transient pauses at internal T-runs where CSB is required to propel RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled with G-rich sequences upstream, exacerbates genome instability by promoting R-loop formation. These R-loop prone motifs are notably abundant in relatively long genes related to neuronal functions in the human genome, but less prevalent in the mouse genome. These findings provide mechanistic insights into differential impacts of CSB deficiency on mice versus humans and suggest that the manifestation of the Cockayne Syndrome phenotype in humans results from the progressive evolution of mammalian genomes.

The ARK2N-CK2 complex initiates transcription-coupled repair through enhancing the interaction of CSB with lesion-stalled RNAPII.

Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.

Cockayne Syndrome Patient iPSC-Derived Brain Organoids and Neurospheres Show Early Transcriptional Dysregulation of Biological Processes Associated with Brain Development and Metabolism.

Cockayne syndrome (CS) is a rare hereditary autosomal recessive disorder primarily caused by mutations in Cockayne syndrome protein A (CSA) or B (CSB). While many of the functions of CSB have been at least partially elucidated, little is known about the actual developmental dysregulation in this devasting disorder. Of particular interest is the regulation of cerebral development as the most debilitating symptoms are of neurological nature. We generated neurospheres and cerebral organoids utilizing Cockayne syndrome B protein (CSB)-deficient induced pluripotent stem cells derived from two patients with distinct severity levels of CS and healthy controls. The transcriptome of both developmental timepoints was explored using RNA-Seq and bioinformatic analysis to identify dysregulated biological processes common to both patients with CS in comparison to the control. CSB-deficient neurospheres displayed upregulation of the VEGFA-VEGFR2 signalling pathway, vesicle-mediated transport and head development. CSB-deficient cerebral organoids exhibited downregulation of brain development, neuron projection development and synaptic signalling. We further identified the upregulation of steroid biosynthesis as common to both timepoints, in particular the upregulation of the cholesterol biosynthesis branch. Our results provide insights into the neurodevelopmental dysregulation in patients with CS and strengthen the theory that CS is not only a neurodegenerative but also a neurodevelopmental disorder.

Transcription-coupled repair of DNA-protein cross-links depends on CSA and CSB.

Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.

Spectrum of ERCC6-Related Cockayne Syndrome (Type B): From Mild to Severe Forms.

(1) Background: Cockayne syndrome (CS) is an ultra-rare multisystem disorder, classically subdivided into three forms and characterized by a clinical spectrum without a clear genotype-phenotype correlation for both the two causative genes ERCC6 (CS type B) and ERCC8 (CS type A). We assessed this, presenting a series of patients with genetically confirmed CSB. (2) Materials and Methods: We retrospectively collected demographic, clinical, genetic, neuroimaging, and serum neurofilament light-chain (sNFL) data about CSB patients; diagnostic and severity scores were also determined. (3) Results: Data of eight ERCC6/CSB patients are presented. Four patients had CS I, three patients CS II, and one patient CS III. Various degrees of ataxia and spasticity were cardinal neurologic features, with variably combined systemic characteristics. Mean age at diagnosis was lower in the type II form, in which classic CS signs were more evident. Interestingly, sNFL determination appeared to reflect clinical classification. Two novel premature stop codon and one novel missense variants were identified. All CS I subjects harbored the p.Arg735Ter variant; the milder CS III subject carried the p.Leu764Ser missense change. (4) Conclusion: Our work confirms clinical variability also in the ERCC6/CSB type, where manifestations may range from severe involvement with prenatal or neonatal onset to normal psychomotor development followed by progressive ataxia. We propose, for the first time in CS, sNFL as a useful peripheral biomarker, with increased levels compared to currently available reference values and with the potential ability to reflect disease severity.

DNA repair deficiencies and neurodegeneration.

Neurodegenerative diseases are the second most prevalent cause of death in industrialized countries. Alzheimer's Disease is the most widespread and also most acknowledged form of dementia today. Together with Parkinson's Disease they account for over 90 % cases of neurodegenerative disorders caused by proteopathies. Far less known are the neurodegenerative pathologies in DNA repair deficiency syndromes. Such diseases like Cockayne - or Werner Syndrome are described as progeroid syndromes - diseases that cause the premature ageing of the affected persons, and there are clear implications of such diseases in neurologic dysfunction and degeneration. In this review, we aim to draw the attention on commonalities between proteopathy-associated neurodegeneration and neurodegeneration caused by DNA repair defects and discuss how mitochondria are implicated in the development of both disorder classes. Furthermore, we highlight how nematodes are a valuable and indispensable model organism to study conserved neurodegenerative processes in a fast-forward manner.

A compound heterozygous mutation of ERCC8 is responsible for a family with Cockayne syndrome.

BACKGROUND: Cockayne syndrome is an inherited heterogeneous defect in transcription-coupled DNA repair (TCR) cause severe clinical syndromes, which may affect the nervous system development of infants and even lead to premature death in some cases. ERCC8 diverse critical roles in the nucleotide excision repair (NER) complex, which is one of the disease-causing genes of Cockayne syndrome. METHODS AND RESULTS: The mutation of ERCC8 in the patient was identified and validated using WES and Sanger sequencing. Specifically, a compound heterozygous mutation (c.454_460dupGTCTCCA p. T154Sfs*13 and c.755_759delGTTTT p.C252Yfs*3) of ERCC8 (CSA) was found, which could potentially be the genetic cause of Cockayne syndrome in the proband. CONCLUSION: In this study, we identified a novel heterozygous mutation of ERCC8 in a Chinese family with Cockayne syndrome, which enlarging the genetic spectrum of the disease.

Senescence, regulators of alternative splicing and effects of trametinib treatment in progeroid syndromes.

Progeroid syndromes such as Hutchinson Gilford Progeroid syndrome (HGPS), Werner syndrome (WS) and Cockayne syndrome (CS), result in severely reduced lifespans and premature ageing. Normal senescent cells show splicing factor dysregulation, which has not yet been investigated in syndromic senescent cells. We sought to investigate the senescence characteristics and splicing factor expression profiles of progeroid dermal fibroblasts. Natural cellular senescence can be reversed by application of the senomorphic drug, trametinib, so we also investigated its ability to reverse senescence characteristics in syndromic cells. We found that progeroid cultures had a higher senescence burden, but did not always have differences in levels of proliferation, DNA damage repair and apoptosis. Splicing factor gene expression appeared dysregulated across the three syndromes. 10 µM trametinib reduced senescent cell load and affected other aspects of the senescence phenotype (including splicing factor expression) in HGPS and Cockayne syndromes. Werner syndrome cells did not demonstrate changes in in senescence following treatment. Splicing factor dysregulation in progeroid cells provides further evidence to support this mechanism as a hallmark of cellular ageing and highlights the use of progeroid syndrome cells in the research of ageing and age-related disease. This study suggests that senomorphic drugs such as trametinib could be a useful adjunct to therapy for progeroid diseases.

Characterisation of a novel missense mutation in the ERCC5 gene leading to group G xeroderma pigmentosum/Cockayne syndrome overlap.

Xeroderma pigmentosum-Cockayne syndrome complex (XP-CS) is exceedingly rare, with 43 cases described over the past five decades; 21 of these cases exhibited mutations in the ERCC5 endonuclease associated with xeroderma pigmentosum, group G.We report the first known phenotypic characterisation of the homozygous chromosome 13 ERCC5, Exon 11, c.2413G>A (p.Gly805Arg) missense mutation in a female toddler presenting with findings of both XP and CS.Her severe presentation also questions previous hypotheses that only truncating mutations and early missense mutations of XPG are capable of producing the dire findings of XP-CS.

Epigenomic signature of accelerated ageing in progeroid Cockayne syndrome.

Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS) are rare genetic disorders caused by mutation of the DNA repair and multifunctional CSA or CSB protein, but only CS patients display a progeroid and neurodegenerative phenotype, providing a unique conceptual and experimental paradigm. As DNA methylation (DNAm) remodelling is a major ageing marker, we performed genome-wide analysis of DNAm of fibroblasts from healthy, UVSS and CS individuals. Differential analysis highlighted a CS-specific epigenomic signature (progeroid-related; not present in UVSS) enriched in three categories: developmental transcription factors, ion/neurotransmitter membrane transporters and synaptic neuro-developmental genes. A large fraction of CS-specific DNAm changes were associated with expression changes in CS samples, including in previously reported post-mortem cerebella. The progeroid phenotype of CS was further supported by epigenomic hallmarks of ageing: the prediction of DNAm of repetitive elements suggested an hypomethylation of Alu sequences in CS, and the epigenetic clock returned a marked increase in CS biological age respect to healthy and UVSS cells. The epigenomic remodelling of accelerated ageing in CS displayed both commonalities and differences with other progeroid diseases and regular ageing. CS shared DNAm changes with normal ageing more than other progeroid diseases do, and included genes functionally validated for regular ageing. Collectively, our results support the existence of an epigenomic basis of accelerated ageing in CS and unveil new genes and pathways that are potentially associated with the progeroid/degenerative phenotype.

[Progeroid syndromes : Aging, skin aging, and mechanisms of progeroid syndromes]. / Progeroide Syndrome : Alterung, Hautalterung und Mechanismen progeroider Erkrankungen.

Progeroid syndromes (PSs) are characterized by the premature onset of age-related pathologies. PSs display a wide range of heterogeneous pathological symptoms that also manifest during natural aging, including vision and hearing loss, atrophy, hair loss, progressive neurodegeneration, and cardiovascular defects. Recent advances in molecular pathology have led to a better understanding of the underlying mechanisms of these diseases. The genetic mutations underlying PSs are functionally linked to genome maintenance and repair, supporting the causative role of DNA damage accumulation in aging. While some of those genes encode proteins with a direct involvement in a DNA repair machinery, such as nucleotide excision repair (NER), others destabilize the genome by compromising the stability of the nuclear envelope, when lamin A is dysfunctional in Hutchinson-Gilford progeria syndrome (HGPS) or regulate the DNA damage response (DDR) such as the ataxia telangiectasia-mutated (ATM) gene. Understanding the molecular pathology of progeroid diseases is crucial in developing potential treatments to manage and prevent the onset of symptoms. This knowledge provides insight into the underlying mechanisms of premature aging and could lead to improved quality of life for individuals affected by progeroid diseases.

Next-generation sequencing through multi-gene panel testing for the diagnosis of a Chinese patient with atypical Cockayne syndrome.

BACKGROUND: Cockayne syndrome (CS, OMIM #133540, #216400) is a rare autosomal recessive disease involving multiple systems, typically characterized by microcephaly, premature aging, growth retardation, neurosensory abnormalities, and photosensitivity. The age of onset is related to the severity of the clinical phenotype, which may lead to fatal outcomes. METHODS: We report a 3-year-old girl who presented with photosensitivity, gait abnormalities, stunting, and microcephaly and showed atypical clinical classification due to mild clinical manifestations at an early onset age. RESULTS: Next-generation sequencing reveals the frameshift mutation (c.394_398del, p.Leu132Asnfs*6) and a novel microdeletion of ERCC8 (exon4del, p.Arg92fs). CONCLUSION: Therefore, it is still necessary to carry out next-generation sequencing for CS patients with atypical clinical manifestations, which is essential for diagnosis and accurate genetic counseling.

Siblings with Cockayne Syndrome B Type III Presenting with Slowly Progressive Cerebellar Ataxia.

Two patients, 48- and 50-year-old sisters, presented with a characteristic facial appearance with slowly progressive deafness and cerebellar ataxia starting in their 30s. Genetic testing identified compound heterozygous pathogenic variants in the ERCC6 gene: c.1583G>A (p.G528E) and c.1873T>G (p.Y625D). A diagnosis of Cockayne syndrome (CS) B type III was made. CS is usually diagnosed in childhood with well-defined facial characteristics and photosensitivity. This case report describes rare cases of adulthood CS with a primary presentation of slowly progressing deafness and cerebellar ataxia. CS should be considered in adults with characteristic facial and skin findings, deafness, and cerebellar ataxia.

Cockayne syndrome group A protein localizes at centrosomes during mitosis and regulates Cyclin B1 ubiquitination.

Mutations in CSA and CSB proteins cause Cockayne syndrome, a rare genetic neurodevelopment disorder. Alongside their demonstrated roles in DNA repair and transcription, these two proteins have recently been discovered to regulate cytokinesis, the final stage of the cell division. This last finding allowed, for the first time, to highlight an extranuclear localization of CS proteins, beyond the one already known at mitochondria. In this study, we demonstrated an additional role for CSA protein being recruited at centrosomes in a strictly determined step of mitosis, which ranges from pro-metaphase until metaphase exit. Centrosomal CSA exerts its function in specifically targeting the pool of centrosomal Cyclin B1 for ubiquitination and proteasomal degradation. Interestingly, a lack of CSA recruitment at centrosomes does not affect Cyclin B1 centrosomal localization but, instead, it causes its lasting centrosomal permanence, thus inducing Caspase 3 activation and apoptosis. The discovery of this unveiled before CSA recruitment at centrosomes opens a new and promising scenario for the understanding of some of the complex and different clinical aspects of Cockayne Syndrome.