Biallelic variants in CRIPT cause a Rothmund-Thomson-like syndrome with increased cellular senescence.

PURPOSE: Rothmund-Thomson syndrome (RTS) is characterized by poikiloderma, sparse hair, small stature, skeletal defects, cancer, and cataracts, resembling features of premature aging. RECQL4 and ANAPC1 are the 2 known disease genes associated with RTS in >70% of cases. We describe RTS-like features in 5 individuals with biallelic variants in CRIPT (OMIM 615789). METHODS: Two newly identified and 4 published individuals with CRIPT variants were systematically compared with those with RTS using clinical data, computational analysis of photographs, histologic analysis of skin, and cellular studies on fibroblasts. RESULTS: All CRIPT individuals fulfilled the diagnostic criteria for RTS and additionally had neurodevelopmental delay and seizures. Using computational gestalt analysis, CRIPT individuals showed greatest facial similarity with individuals with RTS. Skin biopsies revealed a high expression of senescence markers (p53/p16/p21) and the senescence-associated ß-galactosidase activity was elevated in CRIPT-deficient fibroblasts. RECQL4- and CRIPT-deficient fibroblasts showed an unremarkable mitotic progression and unremarkable number of mitotic errors and no or only mild sensitivity to genotoxic stress by ionizing radiation, mitomycin C, hydroxyurea, etoposide, and potassium bromate. CONCLUSION: CRIPT causes an RTS-like syndrome associated with neurodevelopmental delay and epilepsy. At the cellular level, RECQL4- and CRIPT-deficient cells display increased senescence, suggesting shared molecular mechanisms leading to the clinical phenotypes.

In vivo evidence for GDP-fucose transport in the absence of transporter SLC35C1 and putative transporter SLC35C2.

Slc35c1 encodes an antiporter that transports GDP-fucose into the Golgi and returns GMP to the cytoplasm. The closely related gene Slc35c2 encodes a putative GDP-fucose transporter and promotes Notch fucosylation and Notch signaling in cultured cells. Here, we show that HEK293T cells lacking SLC35C1 transferred reduced amounts of O-fucose to secreted epidermal growth factor-like repeats from NOTCH1 or secreted thrombospondin type I repeats from thrombospondin 1. However, cells lacking SLC35C2 did not exhibit reduced fucosylation of these epidermal growth factor-like repeats or thrombospondin type I repeats. To investigate SLC35C2 functions in vivo, WW6 embryonic stem cells were targeted for Slc35c2. Slc35c2[-/-] mice were viable and fertile and exhibited no evidence of defective Notch signaling during skeletal or T cell development. By contrast, mice with inactivated Slc35c1 exhibited perinatal lethality and marked skeletal defects in late embryogenesis, typical of defective Notch signaling. Compound Slc35c1[-/-]Slc35c2[-/-] mutants were indistinguishable in skeletal phenotype from Slc35c1[-/-] embryos and neonates. Double mutants did not exhibit the exacerbated skeletal defects predicted if SLC35C2 was functionally important for Notch signaling in vivo. In addition, NOTCH1 immunoprecipitated from Slc35c1[-/-]Slc35c2[-/-] neonatal lung carried fucose detected by binding of Aleuria aurantia lectin. Given that the absence of both SLC35C1, a known GDP-fucose transporter, and SLC35C2, a putative GDP-fucose transporter, did not lead to afucosylated NOTCH1 nor to the severe Notch signaling defects and embryonic lethality expected if all GDP-fucose transport were abrogated, at least one more mechanism of GDP-fucose transport into the secretory pathway must exist in mammals.

Patient-derived iPSCs link elevated mitochondrial respiratory complex I function to osteosarcoma in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma, small stature, skeletal anomalies, sparse brows/lashes, cataracts, and predisposition to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have multiple skeletal anomalies and a significantly increased incidence of osteosarcoma. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts validated decreased bone morphogenesis while revealing aberrantly upregulated mitochondrial respiratory complex I gene expression. RTS osteoblast metabolic assays demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation (OXPHOS), and increased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell proliferation of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes in RTS osteoblasts revealed that chemical inhibition of mitochondrial respiratory complex I impaired cell proliferation, induced senescence, and decreased MAPK signaling and cell cycle associated genes, but increased H19 and ribosomal protein genes. In summary, our study suggests that mitochondrial respiratory complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies.

RECQ DNA Helicases and Osteosarcoma.

The RECQ family of DNA helicases is a conserved group of enzymes that plays an important role in maintaining genomic stability. Humans possess five RECQ helicase genes, and mutations in three of them - BLM, WRN, and RECQL4 - are associated with the genetic disorders Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome (RTS), respectively. These syndromes share overlapping clinical features, and importantly they are all associated with an increased risk of cancer. Patients with RTS have the highest specific risk of developing osteosarcoma compared to all other cancer predisposition syndromes; therefore, RTS serves as a relevant model to study the pathogenesis and molecular genetics of osteosarcoma. The "tumor suppressor" function of the RECQ helicases continues to be an area of active investigation. This chapter will focus primarily on the known cellular functions of RECQL4 and how these may relate to tumorigenesis, as well as ongoing efforts to understand RECQL4's functions in vivo using animal models. Understanding the RECQ pathways will provide insight into avenues for novel cancer therapies in the future.

[Release Mechanisms of Iron and Manganese from Sediments in Jinpen Reservoir].

In response to the annual hypolimnetic anoxia in stratified reservoirs, water-lifting aerators (WLAs) were used in Jinpen Reservoir to supplement the dissolved oxygen in the bottom water and suppress the release of reduced pollutants from sediment. However, due to the influence of geomorphic characteristics at the bottom of the reservoir, there were some differences in the efficiency of artificial mixing and aeration. After the deactivation of WLAs, the dissolved oxygen in the bottom water of some deeper areas was rapidly depleted, resulting in the re-release of pollutants. To explore the release mechanisms and diffusion intensity of iron and manganese during this period, the representative samples in the main reservoir area were collected to measure the distribution of dissolved iron and manganese in the pore water and overlying water and calculate the diffusive flux of dissolved iron and manganese across the sediment-water interface. The results showed that the bottom water of the lower terrain rapidly entered the anaerobic condition after the system was deactivated, resulting in the release of a large amount of dissolved manganese into the overlying water, the maximum concentration of which was 0.42 mg·L-1. However, the bottom water of the higher terrain briefly entered a state of hypoxia, after which the dissolved oxygen concentration increased rapidly, so the dissolved manganese concentration increased moderately to 0.17 mg·L-1. The distribution of iron and manganese in the pore-water-overlying water showed that the dissolved manganese was released more easily into the overlying water than the iron under anaerobic conditions and constant accumulation in the upper sediments and overlying water. However, the release of dissolved iron was not only suppressed by dissolved oxygen but also by other oxidants such as manganese oxide. The diffusion flux of dissolved manganese declined after the system was deactivated. A mass balance calculation demonstrated that the accumulation of dissolved manganese in the anaerobic layer was not only related to the diffusion flux but also to the sedimentation flux and the thickness of the anaerobic layer. Therefore, the biogeochemical cycle of iron and manganese in the anaerobic layer requires further study.

Mutations in ANAPC1, Encoding a Scaffold Subunit of the Anaphase-Promoting Complex, Cause Rothmund-Thomson Syndrome Type 1.

Rothmund-Thomson syndrome (RTS) is an autosomal-recessive disorder characterized by poikiloderma, sparse hair, short stature, and skeletal anomalies. Type 2 RTS, which is defined by the presence of bi-allelic mutations in RECQL4, is characterized by increased cancer susceptibility and skeletal anomalies, whereas the genetic basis of RTS type 1, which is associated with juvenile cataracts, is unknown. We studied ten individuals, from seven families, who had RTS type 1 and identified a deep intronic splicing mutation of the ANAPC1 gene, a component of the anaphase-promoting complex/cyclosome (APC/C), in all affected individuals, either in the homozygous state or in trans with another mutation. Fibroblast studies showed that the intronic mutation causes the activation of a 95 bp pseudoexon, leading to mRNAs with premature termination codons and nonsense-mediated decay, decreased ANAPC1 protein levels, and prolongation of interphase. Interestingly, mice that were heterozygous for a knockout mutation have an increased incidence of cataracts. Our results demonstrate that deficiency in the APC/C is a cause of RTS type 1 and suggest a possible link between the APC/C and RECQL4 helicase because both proteins are involved in DNA repair and replication.

Generation of an induced pluripotent stem cell line from an individual with a heterozygous RECQL4 mutation.

The DNA helicase RECQL4 is known for its roles in DNA replication and repair. RECQL4 mutations cause several genetic disorders including Rothmund-Thomson syndrome (RTS), characterized by developmental defects and predisposition to osteosarcoma. Here we reprogrammed fibroblasts with a heterozygous RECQL4 mutation (c.1878 + 32_1878 + 55del24) to induced pluripotent stem cells (iPSCs). These iPSCs are pluripotent and are able to be differentiated into all three germ layers, providing a novel tool to further interrogate the role of RECQL4 DNA helicase in vitro.

Osteosarcoma: Molecular Pathogenesis and iPSC Modeling.

Rare hereditary disorders provide unequivocal evidence of the importance of genes in human disease pathogenesis. Familial syndromes that predispose to osteosarcomagenesis are invaluable in understanding the underlying genetics of this malignancy. Recently, patient-derived induced pluripotent stem cells (iPSCs) have been successfully utilized to model Li-Fraumeni syndrome (LFS)-associated bone malignancy, demonstrating that iPSCs can serve as an in vitro disease model to elucidate osteosarcoma etiology. We provide here an overview of osteosarcoma predisposition syndromes and review recently established iPSC disease models for these familial syndromes. Merging molecular information gathered from these models with the current knowledge of osteosarcoma biology will help us to gain a deeper understanding of the pathological mechanisms underlying osteosarcomagenesis and will potentially aid in the development of future patient therapies.

Generalized metabolic bone disease and fracture risk in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by poikiloderma, small stature, sparse hair, skeletal abnormalities, increased risk of osteosarcoma, and decreased bone mass. To date, there has not been a comprehensive evaluation of the prevalence and extent of metabolic bone disease in RTS. Furthermore, the mechanisms that result in this phenotype are largely unknown. In this report, we provide a detailed evaluation of 29 individuals with RTS with respect to their metabolic bone status including bone mineral density, calcium kinetics studies, and markers of bone remodeling. We show that individuals with RTS have decreased areal bone mineral density. Additionally, we demonstrate that the presence of pathogenic variants in RECQL4 and low bone mineral density correlate with the history of increased risk of fractures. Using a RECQL4-deficient mouse model that recapitulates skeletal abnormalities seen in individuals with RTS, we demonstrate that generalized skeletal involvement is likely due to decreased osteogenesis. Our findings are clinically relevant as they may help in the risk stratification of patients with RTS and also in the identification of individuals who may benefit from additional surveillance and management of metabolic bone disease.

Aging in Rothmund-Thomson syndrome and related RECQL4 genetic disorders.

Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disease which manifests several clinical features of accelerated aging. These findings include atrophic skin and pigment changes, alopecia, osteopenia, cataracts, and an increased incidence of cancer for patients carrying RECQL4 germline mutations. Mutations in RECQL4 are responsible for the majority of cases of RTS. RECQL4 belongs to RECQ DNA helicase family which has been shown to participate in many aspects of DNA metabolism. In the past several years, accumulated evidence indicates that RECQL4 is important not only in cancer development but also in the aging process. In this review, based on recent research data, we summarize the common aging findings in RTS patients and propose possible mechanisms to explain the aging features in these patients.

RECQL4 Regulates p53 Function In Vivo During Skeletogenesis.

RECQ DNA helicases play critical roles in maintaining genomic stability, but their role in development has been less well studied. Rothmund-Thomson syndrome, RAPADILINO, and Baller-Gerold syndrome are rare genetic disorders caused by mutations in the RECQL4 gene. These patients have significant skeletal developmental abnormalities including radial ray, limb and craniofacial defects. To investigate the role of Recql4 in the developing skeletal system, we generated Recql4 conditional knockout mice targeting the skeletal lineage. Inactivation of Recql4 using the Prx1-Cre transgene led to limb abnormalities and craniosynostosis mimicking the major bone findings in human RECQL4 patients. These Prx1-Cre(+) ;Recql4(fl/fl) mice as well as Col2a1-Cre(+) ;Recql4(fl/fl) mice exhibited growth plate defects and an increased p53 response in affected tissues. Inactivation of Trp53 in these Recql4 mutants resulted in genetic rescue of the skeletal phenotypes, indicating an in vivo interaction between Recql4 and Trp53, and p53 activation as an underlying mechanism for the developmental bone abnormalities in RECQL4 disorders. Our findings show that RECQL4 is critical for skeletal development by modulating p53 activity in vivo.

RECQ DNA helicases and osteosarcoma.

The RECQ family of DNA helicases is a conserved group of enzymes that are important for maintaining genomic integrity. In humans, there are five RECQ helicase genes, and mutations in three of them-BLM, WRN, and RECQL4-are associated with the genetic disorders Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome (RTS), respectively. Importantly all three diseases are cancer predisposition syndromes. Patients with RTS are highly and uniquely susceptible to developing osteosarcoma; thus, RTS provides a good model to study the pathogenesis of osteosarcoma. The "tumor suppressor" role of RECQL4 and the other RECQ helicases is an area of active investigation. This chapter reviews what is currently known about the cellular functions of RECQL4 and how these may relate to tumorigenesis, as well as ongoing efforts to understand RECQL4's functions in vivo using animal models. Understanding the RECQ pathways may provide insight into avenues for novel cancer therapies in the future.

Slc35c2 promotes Notch1 fucosylation and is required for optimal Notch signaling in mammalian cells.

Mammalian Notch receptors require modification by fucose on epidermal growth factor-like (EGF) repeats of their extracellular domain to respond optimally to signal induction by canonical Notch ligands. Inactivation of the Golgi GDP-fucose transporter Slc35c1 in mouse or human does not cause marked defects in Notch signaling during development, and shows milder fucosylation defects than those observed in mice unable to synthesize GDP-fucose, indicating the existence of another mechanism for GDP-fucose transport into the secretory pathway. We show here that fibroblasts from mice or humans lacking Slc35c1 exhibit robust Notch signaling in co-culture signaling assays. A potential candidate for a second GDP-fucose transporter is the related gene Slc35c2. Overexpression of Slc35c2 reduces expression of the fucosylated epitopes Lewis X and sialylated Lewis X in CHO cells, indicating competition with Slc35c1. The fucosylation of a Notch1 EGF repeat fragment that occurs in the endoplasmic reticulum was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1- and Jagged1-induced Notch signaling were reduced, and the fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy of rat intestinal epithelial cells and HeLa cells, and analysis of rat liver membrane fractions showed that Slc35c2 is primarily colocalized with markers of the cis-Golgi network and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The combined results suggest that Slc35c2 is either a GDP-fucose transporter that competes with Slc35c1 for GDP-fucose, or a factor that otherwise enhances the fucosylation of Notch and is required for optimal Notch signaling in mammalian cells.