Rothmund-Thomson Syndrome Patients Research Articles

A Chinese patient with Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder that has been reported in all ethnicities, with several identifiable pathogenic variants. There have been reported cases indicating that RTS may lead to low birth weight in fetuses, but specific data on the fetal period are lacking. Genetic testing for RTS II is currently carried out by identifying pathogenic variants in RECQL4. In order to determine the cause, we performed whole-genome sequencing (WGS) analysis on the patient and his parents. Variants detected by WGS were confirmed by Sanger sequencing and examined in family members. After analyzing the WGS data, we found a heterozygous nonsense mutation c.2752G>T (p.Glu918Ter) and a novel frameshift insertion mutation c.1547dupC (p.Leu517AlafsTer23) of RECQL4, which is a known pathogenic/disease-causing variant of RTS. Further validation indicated these were compound heterozygous mutations from parents. Our study expands the mutational spectrum of the RECQL4 gene and enriches the phenotype spectrum of Chinese RTS patients. Our information can assist the patient's parents in making informed decisions regarding their future pregnancies. This case offers a new perspective for clinicians to consider whether to perform prenatal diagnosis.

Rothmund-Thomson syndrome, a disorder far from solved.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by a range of clinical symptoms, including poikiloderma, juvenile cataracts, short stature, sparse hair, eyebrows/eyelashes, nail dysplasia, and skeletal abnormalities. While classically associated with mutations in the RECQL4 gene, which encodes a DNA helicase involved in DNA replication and repair, three additional genes have been recently identified in RTS: ANAPC1, encoding a subunit of the APC/C complex; DNA2, which encodes a nuclease/helicase involved in DNA repair; and CRIPT, encoding a poorly characterized protein implicated in excitatory synapse formation and splicing. Here, we review the clinical spectrum of RTS patients, analyze the genetic basis of the disease, and discuss molecular functions of the affected genes, drawing some novel genotype-phenotype correlations and proposing avenues for future studies into this enigmatic disorder.

Hyperubiquitylation of DNA helicase RECQL4 by E3 ligase MITOL prevents mitochondrial entry and potentiates mitophagy

Mutations in the DNA helicase RECQL4 lead to Rothmund-Thomson Syndrome (RTS), a disorder characterized by mitochondrial dysfunctions, premature aging, and genomic instability. However, the mechanisms by which these mutations lead to pathology are unclear. Here we report that RECQL4 is ubiquitylated by a mitochondrial E3 ligase, MITOL, at two lysine residues (K1101, K1154) via K6 linkage. This ubiquitylation hampers the interaction of RECQL4 with mitochondrial importer Tom20, thereby restricting its own entry into mitochondria. We show the RECQL4 2K mutant (where both K1101 and K1154 are mutated) has increased entry into mitochondria and demonstrates enhanced mtDNA replication. We observed that the three tested RTS patient mutants were unable to enter the mitochondria and showed decreased mtDNA replication. Furthermore, we found that RECQL4 in RTS patient mutants are hyper-ubiquitylated by MITOL and form insoluble aggregate-like structures on the outer mitochondrial surface. However, depletion of MITOL allows RECQL4 expressed in these RTS mutants to enter mitochondria and rescue mtDNA replication. Finally, we show increased accumulation of hyper-ubiquitylated RECQL4 outside the mitochondria leads to the cells being potentiated to increased mitophagy. Hence, we conclude regulating the turnover of RECQL4 by MITOL may have a therapeutic effect in RTS patients.

De novo myelodysplastic syndrome in a Rothmund-Thomson Syndrome patient with novel pathogenic RECQL4 variants.

Rothmund-Thomson syndrome (RTS) is a rare autosomal-recessive disorder with clinical features consisting of rash, poikiloderma, sparse hair, short stature, juvenile cataracts, skeletal abnormalities, and cancer predisposition. Genetic studies involving detection of pathogenic RECQL4 variants provide the diagnostic certitude. Osteosarcoma was found in two-thirds RECQL4-mutated RTS patients, while hematological malignancies were rarely reported. The variant diversity of RECQL4 gene has not been fully identified and mutations associated with hematologic malignancies are not well described. In this study, we presented a pedigree of RTS from a Chinese family, among which the proband was diagnosed with de novo myelodysplastic syndrome (MDS). Comprehensive medical examination and chromosome karyotyping were performed on the proband. Whole exome sequencing (WES) was performed on the proband, his sister and his mother. The familial cosegregation of sequence variants derived from WES was conducted by polymerase chain reaction-based Sanger sequencing. Structures of candidate RECQL4 mutants were done by in silico analysis to assess pathogenicity. Three novel RECQL4 germline variants, including c.T274C, c.G3014A, and c.G801C, were identified by WES and validated by Sanger sequencing. Prediction of conformation indicated that the structural stability of human RECQL4 protein was largely affected with these variants. The co-occurring U2AF1 p.S34F and TP53 p.Y220C mutations might contribute to the development of MDS. Our study expands the mutational spectrum of RECQL4 and provides underlying molecular mechanism for the development of MDS in RTS patients.

Molecular and Cellular Responses to Ionization Radiation in Untransformed Fibroblasts from the Rothmund–Thomson Syndrome: Influence of the Nucleo-Shuttling of the ATM Protein Kinase

The Rothmund–Thomson syndrome (RTS) is a rare autosomal recessive disease associated with poikiloderma, telangiectasias, sun-sensitive rash, hair growth problems, juvenile cataracts and, for a subset of some RTS patients, a high risk of cancer, especially osteosarcoma. Most of the RTS cases are caused by biallelic mutations of the RECQL4 gene, coding for the RECQL4 DNA helicase that belongs to the RecQ family. Cellular and post-radiotherapy radiosensitivity was reported in RTS cells and patients since the 1980s. However, the molecular basis of this particular phenotype has not been documented to reliably link the biological and clinical responses to the ionizing radiation (IR) of cells from RTS patients. The aim of this study was therefore to document the specificities of the radiosensitivity associated with RTS by examining the radiation-induced nucleo-shuttling of ATM (RIANS) and the recognition and repair of the DNA double-strand breaks (DSB) in three skin fibroblasts cell lines derived from RTS patients and two derived from RTS patients’ parents. The results showed that the RTS fibroblasts tested were associated with moderate but significant radiosensitivity, a high yield of micronuclei, and impaired DSB recognition but normal DSB repair at 24 h likely caused by a delayed RIANS, supported by the sequestration of ATM by some RTS proteins overexpressed in the cytoplasm. To our knowledge, this report is the first radiobiological characterization of cells from RTS patients at both molecular and cellular scales.

Abstract 3779: Patient-derived iPSCs reveal pharmacologic targeting mitochondrial respiratory complex I for treating Rothmund-Thomson syndrome associated osteosarcoma

Abstract Background: Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by a rash that progresses to poikiloderma; small stature; skeletal anomalies; sparse hair, eyelashes, and/or eyebrows, juvenile cataracts, and an increased risk to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have a significantly increased incidence of osteosarcoma. However, there is currently no available RTS model that recapitulates the bone malignancy phenotype in this disease, severely limiting the ability of researchers to explore new treatment avenues. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. Methods: iPSCs were reprogrammed from fibroblasts of four individuals from two RTS families, each with one affected RTS proband with RECQL4 mutations and one unaffected parent. These patient iPSCs were differentiated to mesenchymal stem cells (MSCs) and then to osteoblasts, the cell-of-origin of osteosarcoma. All of these MSCs and osteoblasts were subjected to transcriptome analysis by RNA-seq. Based on enriched mitochondrial respiratory gene signatures, we also systematically analyzed the enzymatic activities of the major mitochondrial respiratory protein complexes including complex I/II/III and citrate synthase enzyme activity in RTS osteoblasts by Seahorse assay. Finally, we tested the potential therapeutic effect of clinical oxidative phosphorylation complex I inhibitor IACS-010759 to treat RTS cells and dissect the pharmacological mechanisms involved. Results: RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and a gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts revealed decreased bone morphogenesis gene expression but aberrantly upregulated mitochondrial complex I gene expression. Metabolic assays of RTS osteoblasts demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation, but decreased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell viability of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes to RTS osteoblasts suggested that inhibition of mitochondrial complex I in RTS osteoblasts leads to enhanced cell senescence, decreased MAPK signaling and cell cycle associated genes. Conclusion: In summary, we established an RTS iPSC disease platform to dissect the pathological mechanisms involved in RTS-associated osteosarcoma. Our data suggested that mitochondrial complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies. Citation Format: An Xu, Brittany E. Jewell, Dandan Zhu, Mo-Fan Huang, Yi-Hung Chen, Linchao Lu, Ruiying Zhao, Lisa L. Wang, Dung-Fang Lee. Patient-derived iPSCs reveal pharmacologic targeting mitochondrial respiratory complex I for treating Rothmund-Thomson syndrome associated osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3779.

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.

Novel pathogenic variants in the RECQL4 gene causing Rothmund‐Thomson syndrome in three Chinese patients

Rothmund-Thomson syndrome (RTS) is a rare autosomal-recessive disorder characterized by poikiloderma, short stature, sparse hair, skeletal abnormalities, and cancer predisposition. Mutations in ANAPC1 or RECQL4 have been identified to underlie RTS. Either Sanger sequencing or next-generation sequencing (NGS) was performed for three Chinese RTS patients. Copy number variants were called by the eXome-Hidden Markov Model using read-depth data of NGS, and the putative heterozygous deletion was confirmed by PCR with multiple primers. The breakpoints were identified by Sanger sequencing. All patients presented with characteristic features of poikiloderma, short stature, and sparse hair, eyelashes, and eyebrows. In addition, patient 1 had intellectual disability and speech delay, and patient 2 developed osteosarcoma when she was 13years old. Biallelic RECQL4 variants were identified in all three patients. Five of the six variants were novel, including c.119-1G>A, c.2886-1G>A, c.2290C>T (p.Gln764*), and c.3552dupG (p.Arg1185Glufs*42), and a gross deletion encompassing exons 6 to 10. Our study expands the genetic and clinical spectrums of RTS. Furthermore, we reported the first heterozygous gross deletion in RECQL4.

Abstract 6150: The role of oxidative phosphorylation in a novel iPSC <->derived model of osteosarcomagenesis

Abstract Osteosarcoma is the most common bone malignancy in childhood and early adolescence. Thirty percent of patients with the rare genetic disorder Type II Rothmund- Thomson Syndrome (RTS) develop osteosarcoma. Patients with Type II RTS have biallelic mutations in the RECQL4 gene, which encodes an ATP dependent DNA helicase. Unfortunately, many attempts to model RTS associated osteosarcoma have not been successful. Here, we describe a patient-derived induced pluripotent stem cell (iPSC) model capable of recapitulating the process of osteosarcomagenesis. The main goal of our inquiry is to develop a new model to study osteosarcoma in order to elucidate cellular pathways important in osteosarcomagenesis that are potential therapeutic targets. RTS patient fibroblasts were reprogrammed to iPSCs, then subsequently differentiated to mesenchymal stem cells. These cells were validated as pluripotent and then further differentiated to early and late osteoblasts, the potential precursors of osteosarcoma. This model of osteoblast development provides an unlimited resource for inquiry into the molecular mechanism of osteosarcomagenesis. Clear advantages of the iPSC derived osteoblasts include the isogenic nature of the cells, as these iPSC-derived osteoblasts retain the genetic fidelity of the patient from which they were derived. We have also isolated matched parental cells and similarly reprogrammed and differentiated them in order to provide ideal matched controls with clinical relevance, as the parents are heterozygous for the same RECQL4 mutation, yet do not develop osteosarcoma. We believe these cells are the ideal model to study the molecular mechanism associated with RTS-associated osteosarcoma. In order to gain better insight into osteosarcoma pathophysiology, we utilized the iPSC-derived osteoblasts to perform RNAseq and subsequent GSEA and DEseq2 analyses. From these transcriptional studies, we have found dysregulation of oxidative phosphorylation in the RTS patient samples compared to their parental controls. We specifically observe differences in complexes I, III, and IV of the electron transport chain. Interestingly, we also observe changes in vacuolar ATPases that are function to increase proton concentration in the cell and within the extracellular environment. This is consistent with phenomena observed in other cancers, which have been observed to have a similar increase in v-ATPases on the plasma membrane. Inhibitors of these complexes provide us with a starting point to address new therapeutic options for patients. Citation Format: Brittany Ellis Jewell, An Xu, Dandan Zhu, Linchao Lu, Ruiying Zhao, Lisa L. Wang, Dung-Fang Lee. The role of oxidative phosphorylation in a novel iPSC <->derived model of osteosarcomagenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6150.

3059 – PROTEIN TRUNCATING, BUT NOT HELICASE INACTIVATING, RECQL4 MUTATIONS CAUSE BONE MARROW FAILURE

Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disorder characterized by skin rash, skeletal dysplasias, premature ageing, and caused by mutations in RECQL4. RTS is a familial cancer syndrome, with hematological cancers amongst the most common reported malignancies. We previously reported that mice with somatic deletion of Recql4, resulting in null alleles, developed a fully penetrant bone marrow failure syndrome. RTS patients, however, have truncating mutations that result in hypomorphic alleles, not null alleles. To understand the impact of Recql4 mutations on haematopoiesis, we established mice with three distinct point mutations: K525A, which results in an ATP-dependent helicase-inactive but full-length protein, and G522EfsX43 and R347X, which result in truncated protein products. We crossed the point mutant mice to the R26-CreER Recql4 fl/fl line, and at 8-10 weeks of age fed tamoxifen containing food for 30 days to delete the wild-type Recql4 floxed allele leaving only the mutation expressed. Analysis of peripheral blood and bone marrow by FACS showed that only mice carrying truncating mutations developed bone marrow failure. To validate these results in vitro, we immortalized R26-CreER Recql4 point mutant cells with Hoxb8 retrovirus and treated them with tamoxifen for 14 days. In this setting, we also found that truncating mutations caused a marked decrease in cell proliferation, while the helicase-inactive mutation did not. Finally, we performed in vitro complementation assays to determine the capacity of human RECQL4 mutations, similar to our murine alleles, to rescue Hoxb8 R26-CreER Recql4-depleted myeloid cells. Results showed that the helicase-inactive mutant successfully rescued the RECQL4-depleted cells, while the truncating human mutants did not. Collectively, these data demonstrate that the helicase function of RECQL4 is dispensable for hematopoiesis but that the deletion of both the helicase and the C-terminal domains in truncating mutations has deleterious effects on hematopoietic cells.

Abstract B36: A novel model of osteosarcomagenesis reveals dysregulation of oxidative phosphorylation

Abstract The purpose of this study is to develop a new model to study osteosarcoma in order to elucidate cellular pathways important in osteosarcomagenesis that are potential therapeutic targets. Osteosarcoma is the most common bone malignancy in childhood and early adolescence. Thirty percent of patients with the rare genetic disorder Type II Rothmund-Thomson syndrome (RTS) develop osteosarcoma. Patients with RTS have biallelic mutations in the RECQL4 gene, which encodes an ATP-dependent DNA helicase. Unfortunately, many attempts to model RTS-associated osteosarcoma have not been successful. Here, we describe a patient-derived induced pluripotent stem cell (iPSC) model capable of recapitulating the process of osteosarcomagenesis. Briefly, RTS patient fibroblasts are reprogrammed to iPSCs, then subsequently differentiated to mesenchymal stem cells. These cells are validated and further differentiated to early and late osteoblasts, the potential precursors of osteosarcoma. Because the iPSC-derived osteoblasts retain the genetic fidelity of the patient from which they were derived, we believe these cells are the ideal model to study the molecular mechanism associated with RTS-associated osteosarcoma. Utilizing the patient-derived osteoblasts, we have performed RNAseq and subsequent GSEA analyses to explore the transcriptome of RTS osteoblasts and found the dysregulation of oxidative phosphorylation in RTS osteoblasts. Interestingly, it has been reported that specific vacuolar ATPases are upregulated in patient cells. It has been reported that vacuolar ATPases function to increase proton concentration within the mitochondria and also within the extracellular environment. Understanding key mechanisms of osteosarcomagenesis is critical to making steps toward novel therapeutics. Citation Format: Brittany E. Jewell, An Xu, Ruoji Zhou, Dandan Zhu, Linchao Lu, Ruying Zhao, Lisa L. Wang, Dung-Fang Lee. A novel model of osteosarcomagenesis reveals dysregulation of oxidative phosphorylation [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B36.

IPSC reprogramming of fibroblasts from a patient with a Rothmund-Thomson syndrome RTS

Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disease that 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. Mutations in RECQL4 gene are responsible for cases of RTS. RECQL4 belongs to the RECQ DNA helicase family which has been shown to participate in many aspects of DNA metabolism. To be able to study the cellular defects related to the pathology, we derived an induced pluripotent cell line from RTS patient fibroblasts, with the ability to re-differentiate into the three embryonic germ layers.

Abstract 2559: Functional characterization of a RECQL4 mutation in Rothmund Thomson Syndrome

Abstract Osteosarcoma (OS) is the most common malignancy of the bone, typically diagnosed during the adolescent years. Despite early advances in treatment, OS survival rates have not improved in recent times. Targeted treatment remains elusive because OS is a genetically complex and heterogeneous disease and the low incidence of sporadic OS presents a challenge in dissecting the causes and drivers of tumor development. Type II Rothmund-Thomson Syndrome (RTS), a cancer predisposition disorder, presents a potential model for studying OS pathogenesis as roughly 30% of Type II RTS patients are eventually diagnosed with OS. Type II RTS is associated with germline mutations of the DNA helicase RECQL4 that exclusively target the C-terminal helicase domain thought to be involved in various cellular mechanisms of DNA repair. The N-terminal SLD2-homology domain, required for the assembly of the CMG complex at origins of replication, is spared. We used CRISPR/Cas9 gene editing to generate cell lines containing a common RECQL4 mutation identified in Type II RTS patients to further study its effects. Unexpectedly, a mutation previously reported as a nonsense mutation instead upregulates the activation of a known cryptic splice site in exon 14. This splice variant results in the in-frame deletion of the final 66 amino acids of exon 14. Our cell lines exhibit a mildly increased sensitivity to the DNA double-strand break inducing agent neocarzinostatin consistent with RECQL4’s reported functions in DNA damage repair. We will use next generation sequencing techniques to further characterize DNA damage repair signatures in RTS RECQL4 mutant cell lines. Citation Format: Tianyi Wu, Robert L. Walker, Marbin Pineda, MiYoung Lee, Yuelin Zhu, Ashok R. Venkitaraman, Paul S. Meltzer. Functional characterization of a RECQL4 mutation in Rothmund Thomson Syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2559.

RecQL4-Aurora B kinase axis is essential for cellular proliferation, cell cycle progression, and mitotic integrity

Human RecQL4 helicase plays critical roles in the maintenance of genomic stability. Mutations in RecQL4 helicase results in three clinically related autosomal recessive disorders: Rothmund–Thomson syndrome (RTS), RAPADILINO, and Baller–Gerold syndrome. In addition to several premature aging features, RTS patients are characterized by aneuploidy involving either loss or gain of a single chromosome. Chromosome mosaicism and isochromosomes involving chromosomes 2, 7, and 8 have been reported in RecQL4-deficient RTS patients, but the precise role of RecQL4 in chromosome segregation/stability remains to be elucidated. Here, we demonstrate that RecQL4 physically and functionally interacts with Aurora B kinase (AURKB) and stabilizes its expression by inhibiting its ubiquitination process. Our study indicates that the N-terminus of RecQL4 interacts with the catalytic domain of AURKB. Strikingly, RecQL4 suppression reduces the expression of AURKB leading to mitotic irregularities and apoptotic cell death. RecQL4 suppression increases the proportion of cells at the G2/M phase followed by an extensive cell death, presumably owing to the accumulation of mitotic irregularities. Both these defects (accumulation of cells at G2/M phase and an improper mitotic exit to sub-G1) are complemented by the ectopic expression of AURKB. Finally, evidence is provided for the requirement of both human telomerase reverse transcriptase and RecQL4 for stable immortalization and longevity of RTS fibroblasts. Collectively, our study suggests that the RecQL4–AURKB axis is essential for cellular proliferation, cell cycle progression, and mitotic stability in human cells.

DNA replication timing alterations identify common markers between distinct progeroid diseases

Progeroid syndromes are rare genetic disorders that phenotypically resemble natural aging. Different causal mutations have been identified, but no molecular alterations have been identified that are in common to these diseases. DNA replication timing (RT) is a robust cell type-specific epigenetic feature highly conserved in the same cell types from different individuals but altered in disease. Here, we characterized DNA RT program alterations in Hutchinson-Gilford progeria syndrome (HGPS) and Rothmund-Thomson syndrome (RTS) patients compared with natural aging and cellular senescence. Our results identified a progeroid-specific RT signature that is common to cells from three HGPS and three RTS patients and distinguishes them from healthy individuals across a wide range of ages. Among the RT abnormalities, we identified the tumor protein p63 gene (TP63) as a gene marker for progeroid syndromes. By using the redifferentiation of four patient-derived induced pluripotent stem cells as a model for the onset of progeroid syndromes, we tracked the progression of RT abnormalities during development, revealing altered RT of the TP63 gene as an early event in disease progression of both HGPS and RTS. Moreover, the RT abnormalities in progeroid patients were associated with altered isoform expression of TP63 Our findings demonstrate the value of RT studies to identify biomarkers not detected by other methods, reveal abnormal TP63 RT as an early event in progeroid disease progression, and suggest TP63 gene regulation as a potential therapeutic target.

The DNA helicase recql4 is required for normal osteoblast expansion and osteosarcoma formation.

RECQL4 mutations are associated with Rothmund Thomson Syndrome (RTS), RAPADILINO Syndrome and Baller-Gerold Syndrome. These patients display a range of benign skeletal abnormalities such as low bone mass. In addition, RTS patients have a highly increased incidence of osteosarcoma (OS). The role of RECQL4 in normal adult bone development and homeostasis is largely uncharacterized and how mutation of RECQL4 contributes to OS susceptibility is not known. We hypothesised that Recql4 was required for normal skeletal development and both benign and malignant osteoblast function, which we have tested in the mouse. Recql4 deletion in vivo at the osteoblastic progenitor stage of differentiation resulted in mice with shorter bones and reduced bone volume, assessed at 9 weeks of age. This was associated with an osteoblast intrinsic decrease in mineral apposition rate and bone formation rate in the Recql4-deficient cohorts. Deletion of Recql4 in mature osteoblasts/osteocytes in vivo, however, did not cause a detectable phenotype. Acute deletion of Recql4 in primary osteoblasts or shRNA knockdown in an osteoblastic cell line caused failed proliferation, accompanied by cell cycle arrest, induction of apoptosis and impaired differentiation. When cohorts of animals were aged long term, the loss of Recql4 alone was not sufficient to initiate OS. We then crossed the Recql4fl/fl allele to a fully penetrant OS model (Osx-Cre p53fl/fl). Unexpectedly, the Osx-Cre p53fl/flRecql4fl/fl (dKO) animals had a significantly increased OS-free survival compared to Osx-Cre p53fl/fl or Osx-Cre p53fl/flRecql4fl/+ (het) animals. The extended survival was explained when the Recql4 status in the tumors that arose was assessed, and in no case was there complete deletion of Recql4 in the dKO OS. These data provide a mechanism for the benign skeletal phenotypes of RECQL4 mutation syndromes. We propose that tumor suppression and osteosarcoma susceptibility are most likely a function of mutant, not null, alleles of RECQL4.

Senescence induced by RECQL4 dysfunction contributes to Rothmund-Thomson syndrome features in mice.

Cellular senescence refers to irreversible growth arrest of primary eukaryotic cells, a process thought to contribute to aging-related degeneration and disease. Deficiency of RecQ helicase RECQL4 leads to Rothmund–Thomson syndrome (RTS), and we have investigated whether senescence is involved using cellular approaches and a mouse model. We first systematically investigated whether depletion of RECQL4 and the other four human RecQ helicases, BLM, WRN, RECQL1 and RECQL5, impacts the proliferative potential of human primary fibroblasts. BLM-, WRN- and RECQL4-depleted cells display increased staining of senescence-associated β-galactosidase (SA-β-gal), higher expression of p16INK4a or/and p21WAF1 and accumulated persistent DNA damage foci. These features were less frequent in RECQL1- and RECQL5-depleted cells. We have mapped the region in RECQL4 that prevents cellular senescence to its N-terminal region and helicase domain. We further investigated senescence features in an RTS mouse model, Recql4-deficient mice (Recql4HD). Tail fibroblasts from Recql4HD showed increased SA-β-gal staining and increased DNA damage foci. We also identified sparser tail hair and fewer blood cells in Recql4HD mice accompanied with increased senescence in tail hair follicles and in bone marrow cells. In conclusion, dysfunction of RECQL4 increases DNA damage and triggers premature senescence in both human and mouse cells, which may contribute to symptoms in RTS patients.

A Mouse Model Of Rothmund-Thomson Syndrome Reveals An Essential Role For Recql4 In Maintenance Of Hematopoiesis

Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disorder that presents with congenital skeletal malformations, premature ageing and an increased incidence of malignant disease including osteosarcoma and hematologic malignancy. The majority of RTS patients have deleterious germ-line mutations in the RECQL4 helicase. RECQL4 is a member of a family of DNA helicases including Bloom (BLM) and Werner (WRN) syndrome helicases and is thought to play an important role in maintaining chromosome stability. Recql4 deficiency is associated with karyotypic abnormalities and increased rates of aneuploidy. To enable lineage-restricted deletion of Recql4 we generated a mouse Recql4fl/fl line where exon 9 and 10 are flanked by loxP sites. This region encodes the start of the ATP dependent helicase domain and corresponds to human exons 8 and 9, mutations of which are commonly associated with cancers in RTS patients.Germ-line deletion of Recql4 led to embryonic lethality before E10.5. To understand the role of Recql4 in adult hematopoiesis, Rosa26-CreERT2 Recql4+/+, Recql4fl/+ and Recql4fl/fl mice were fed a tamoxifen containing diet (which activates Cre) for up to 4 weeks from 7 weeks of age to somatically delete Recql4. The most striking phenotype observed in these mice was that all R26-CreERT2 Recql4fl/fl animals became anaemic and developed white extremities, which in nearly all cases necessitated euthanasia by 30 days after starting tamoxifen. No differences were observed between R26-CreERT2Recql4+/+ and R26-CreERT2Recql4fl/+. Hematopoietic analyses revealed a severe multi-lineage cytopenia including profound red cell aplasia in the peripheral blood, spleen and bone marrow of Recql4 deficient animals. Effects were observed across all lineages with myeloid, B-lymphoid and T-lymphoid cell numbers severely reduced with a phenotype consistent with acute bone marrow failure. Loss of Recql4 led to a block in B-lymphoid development in the BM at the PrePro-B to Pro-B cell stage. T-lymphoid development was severely interrupted and most populations in the thymus were impacted. Within the stem and progenitor fractions, phenotypic HSCs were preserved in the absence of Recql4 but we found that the erythroid progenitors were nearly completely lost and only CMP/GMP remained. Colony formation was reduced by ∼70% in the deleted bone marrow. Bone marrow transplantation was used to assess the functionality of the Recql4 deleted HSCs. Strikingly, and consistently observed using both R26-CreERT2 and an additional hScl-CreER model, there was a strong selection against deficient cells and a recovery of hematopoiesis by cells with incomplete deletion of Recql4. This result demonstrates an essential role for Recql4 in the maintenance of hematopoiesis. In vitro B and T cell cultures recapitulated the developmental defects observed in the R26-CreERT2 Recql4fl/fl demonstrating a cell intrinsic requirement for Recql4. Loss of Recql4 lead to an accumulation of cells in S-phase and increased levels of DNA damage as measured by gH2Ax staining. Interestingly, the failure in B cell, T cell development and LKS+ CFC formation could be rescued by overexpression of a either WT or a helicase-dead Recql4-K508A mutant.Collectively these data demonstrate that Recql4 is essential for the maintenance of hematopoiesis and acts to maintain the committed progenitor pool. Loss of Recql4 leads to bone marrow failure, demonstrating a unique requirement for Recql4 in the regulation of hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

RECQL4 and p53 potentiate the activity of polymerase γ and maintain the integrity of the human mitochondrial genome

Germline mutations in RECQL4 and p53 lead to cancer predisposition syndromes, Rothmund-Thomson syndrome (RTS) and Li-Fraumeni syndrome (LFS), respectively. RECQL4 is essential for the transport of p53 to the mitochondria under unstressed conditions. Here, we show that both RECQL4 and p53 interact with mitochondrial polymerase (PolγA/B2) and regulate its binding to the mitochondrial DNA (mtDNA) control region (D-loop). Both RECQL4 and p53 bind to the exonuclease and polymerase domains of PolγA. Kinetic constants for interactions between PolγA-RECQL4, PolγA-p53 and PolγB-p53 indicate that RECQL4 and p53 are accessory factors for PolγA-PolγB and PolγA-DNA interactions. RECQL4 enhances the binding of PolγA to DNA, thereby potentiating the exonuclease and polymerization activities of PolγA/B2. To investigate whether lack of RECQL4 and p53 results in increased mitochondrial genome instability, resequencing of the entire mitochondrial genome was undertaken from multiple RTS and LFS patient fibroblasts. We found multiple somatic mutations and polymorphisms in both RTS and LFS patient cells. A significant number of mutations and polymorphisms were common between RTS and LFS patients. These changes are associated with either aging and/or cancer, thereby indicating that the phenotypes associated with these syndromes may be due to deregulation of mitochondrial genome stability caused by the lack of RECQL4 and p53. The biochemical mechanisms by which RECQL4 and p53 affect mtDNA replication have been elucidated. Resequencing of RTS and LFS patients' mitochondrial genome reveals common mutations indicating similar mechanisms of regulation by RECQL4 and p53.

Chronic tibial nonunion in a Rothmund–Thomson syndrome patient

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder caused by biallelic mutations in RECQL4, a helicase involved with chromosomal instability and DNA repair. Patients typically present with a poikilodermatous facial rash, photosensitivity, congenital bony abnormalities, short stature, and have a predilection for osteosarcoma and cutaneous malignancies. We present a 34-year-old male RTS patient, previously diagnosed with osteosarcoma of the right forearm which was successfully treated with resection and chemotherapy, who has had multiple tibial fractures and has suffered from chronic nonunion of the proximal tibias bilaterally for greater than 9 years. The patient subsequently developed generalized lower extremity osteopenia with normal calcium homeostasis and calcitriol levels. As the RTS population continues to reach greater ages we must be mindful of other health concerns that may develop. Bone health is one considerable concern with a large portion of patients having congenital bony abnormalities and many receiving chemotherapy for osteosarcoma. We conclude that screening for bone health and supplementation with calcium and vitamin D may be warranted in RTS patients with a history of fractures and osteosarcoma treatment.