A homozygous missense variant in the WRN gene segregating in a family with progressive pulmonary failure with recurrent spontaneous pneumothorax and interstitial lung disease.

Interstitial lung disease (ILD) is a condition affecting the lung parenchyma by inflammation and fibrosis and can be caused by various exposures, connective tissue diseases (CTD), and genetic disorders. In this report, a family with five patients having progressive respiratory failure that begins with coughing in adolescence, followed by dyspnea and recurrent spontaneous pneumothorax, and death in early adulthood is presented. The patients were diagnosed to have ILD through clinical and radiological evaluations. Molecular genetic analyses of the family provided two homozygous rare variants in the WRN and SFXN5 genes, co-segregating with the phenotype. The network analyses pointed out that the variant in the WRN, rather than that in the SFXN5 gene, could be the main factor in the existence of the ILD phenotype, putatively through the altered DNA repair and telomere maintenance pathways. In silico analyses suggested that the variant could affect the exonuclease activity or the stability of the WRN protein. Moreover, the adolescent-onset pulmonary phenotype described in the case has not been reported in Werner Syndrome, the only disease known to be associated with biallelic WRN pathogenic variants. Thus, the present phenotype could be either a very atypical presentation of Werner syndrome or a new clinical entity associated with the WRN gene.

MUT-7 Provides Molecular Insight into the Werner Syndrome Exonuclease.

Werner syndrome (WS) is a rare recessive genetic disease characterized by premature aging. Individuals with this disorder develop normally during childhood, but their physiological conditions exacerbate the aging process in late adolescence. WS is caused by mutation of the human WS gene (WRN), which encodes two main domains, a 3'-5' exonuclease and a 3'-5' helicase. Caenorhabditis elegans expresses human WRN orthologs as two different proteins: MUT-7, which has a 3'-5' exonuclease domain, and C. elegans WRN-1 (CeWRN-1), which has only helicase domains. These unique proteins dynamically regulate olfactory memory in C. elegans, providing insight into the molecular roles of WRN domains in humans. In this review, we specifically focus on characterizing the function of MUT-7 in small interfering RNA (siRNA) synthesis in the cytoplasm and the roles of siRNA in directing nuclear CeWRN-1 loading onto a heterochromatin complex to induce negative feedback regulation. Further studies on the different contributions of the 3'-5' exonuclease and helicase domains in the molecular mechanism will provide clues to the accelerated aging processes in WS.

Calcification in Werner syndrome associated with lymphatic vessels aging.

In addition to the symptoms of aging, the main symptoms in Werner syndrome (WS), a hereditary premature aging disease, include calcification of subcutaneous tissue with solid pain and refractory skin ulcers. However, the mechanism of calcification in WS remains unclear. In this study, the histological analysis of the skin around the ulcer with calcification revealed an accumulation of calcium phosphate in the lymphatic vessels. Moreover, the morphological comparison with the lymphatic vessels in PAD patients with chronic skin ulcers demonstrated the ongoing lymphatic remodeling in WS patients because of the narrow luminal cross-sectional area (LA) of the lymphatic vessels but the increment of lymphatic microvessels density (MLVD). Additionally, fluorescence immunohistochemical analysis presented the cytoplasmic distribution and the accumulation of WRN proteins in endothelial cells on remodeling lymphatic vessels. In summary, these results point out a relationship between calcification in lymphatic vessels and the remodeling of lymphatic vessels and suggest the significance of the accumulation of WRN mutant proteins as an age-related change in WS patients. Thus, cytoplasmic accumulation of WRN protein can be an indicator of the decreasing drainage function of the lymphatic vessels and the increased risk of skin ulcers and calcification in the lymphatic vessels.

Crosstalk among DNA Damage, Mitochondrial Dysfunction, Impaired Mitophagy, Stem Cell Attrition, and Senescence in the Accelerated Ageing Disorder Werner Syndrome.

Werner syndrome (WS) is an accelerated ageing disease caused by multiple mutations in the gene encoding the Werner DNA helicase (WRN). The major clinical features of WS include wrinkles, grey hair, osteoporosis, and metabolic phenomena such as atherosclerosis, diabetes, and fatty liver, and resemble those seen in normal ageing, but occur earlier, in middle age. Defective DNA repair resulting from mutations in WRN explain the majority of the clinical features of WS, but the underlying mechanisms driving the larger metabolic dysfunction remain elusive. Recent studies in animal models of WS and in WS patient cells and blood samples suggest the involvement of impaired mitophagy, NAD+ depletion, and accumulation of damaged mitochondria in metabolic dysfunction. This mini-review summarizes recent progress in the understanding of the molecular mechanisms of metabolic dysfunction in WS, with the involvement of DNA damage, mitochondrial dysfunction, mitophagy reduction, stem cell impairment, and senescence. Future studies on NAD+ and mitophagy may shed light on potential therapeutic strategies for the WS patients.

Human WRN is an intrinsic inhibitor of progerin, abnormal splicing product of lamin A.

Werner syndrome (WRN) is a rare progressive genetic disorder, caused by functional defects in WRN protein and RecQ4L DNA helicase. Acceleration of the aging process is initiated at puberty and the expected life span is approximately the late 50 s. However, a Wrn-deficient mouse model does not show premature aging phenotypes or a short life span, implying that aging processes differ greatly between humans and mice. Gene expression analysis of WRN cells reveals very similar results to gene expression analysis of Hutchinson Gilford progeria syndrome (HGPS) cells, suggesting that these human progeroid syndromes share a common pathological mechanism. Here we show that WRN cells also express progerin, an abnormal variant of the lamin A protein. In addition, we reveal that duplicated sequences of human WRN (hWRN) from exon 9 to exon 10, which differ from the sequence of mouse WRN (mWRN), are a natural inhibitor of progerin. Overexpression of hWRN reduced progerin expression and aging features in HGPS cells. Furthermore, the elimination of progerin by siRNA or a progerin-inhibitor (SLC-D011 also called progerinin) can ameliorate senescence phenotypes in WRN fibroblasts and cardiomyocytes, derived from WRN-iPSCs. These results suggest that progerin, which easily accumulates under WRN-deficient conditions, can lead to premature aging in WRN and that this effect can be prevented by SLC-D011.

Review of How Genetic Research on Segmental Progeroid Syndromes Has Documented Genomic Instability as a Hallmark of Aging But Let Us Now Pursue Antigeroid Syndromes!

The purpose of this early contribution to the new Fellows Forum of this pioneering journal for what is now called Geroscience is to provide an example of how the author's interest in using the emerging tools of human genetics has led to strong support for one of the hallmarks of aging-Genomic Instability. We shall also briefly review our emerging interests in the genetic analysis of what we have called Antigeroid Syndromes. While there has been significant progress in that direction via genetic studies of centenarians, the search for genetic pathways that make individuals unusually resistant or resilient to the ravages of specific geriatric disorders has been comparatively neglected. We refer to these disorders as Unimodal Antigeroid Syndromes. It is our hope that our young colleagues will consider research efforts in that direction.

The Impact of Vitamin C on Different System Models of Werner Syndrome.

Significance: Werner syndrome (WS) is a rare autosomal recessive malady typified by a pro-oxidant/proinflammatory status, genetic instability, and by the early onset of numerous age-associated illnesses. The protein malfunctioning in WS individuals (WRN) is a helicase/exonuclease implicated in transcription, DNA replication/repair, and telomere maintenance. Recent Advances: In the last two decades, a series of important biological systems were created to comprehend at the molecular level the effect of a defective WRN protein. Such biological tools include mouse and worm (Caenorhabditis elegans) with a mutation in the Wrn helicase ortholog as well as human WS-induced pluripotent stem cells that can ultimately be differentiated into most cell lineages. Such WS models have identified anomalies related to the hallmarks of aging. Most importantly, vitamin C counteracts these age-related cellular phenotypes in these systems. Critical Issues: Vitamin C is the only antioxidant agent capable of reversing the cellular aging-related phenotypes in those biological systems. Since vitamin C is a cofactor for many hydroxylases and mono- or dioxygenase, it adds another level of complexity in deciphering the exact molecular pathways affected by this vitamin. Moreover, it is still unclear whether a short- or long-term vitamin C supplementation in human WS patients who already display aging-related phenotypes will have a beneficial impact. Future Directions: The discovery of new molecular markers specific to the modified biological pathways in WS that can be used for novel imaging techniques or as blood markers will be necessary to assess the favorable effect of vitamin C supplementation in WS. Antioxid. Redox Signal. 34, 856-874.

Meningioma in a patient with Werner syndrome.

Werner syndrome (WS), also known as adult progeria, is extremely rare, with about 1300 known cases in the world, with over 1000 of these in Japan. It occurs due to loss of function mutations in the WRN gene located on chromosome 8p12. WS is characterized by premature aging and increased risk of neoplasms, with meningiomas being the commonest intracranial tumor. We report the case of a 39-year-old male patient, who presented with occasional numbness in right arm for three weeks. The patient had developed signs and symptoms of premature aging which started in his adolescence. MRI brain done was suggestive of left frontal convexity extra-axial lesion, suggestive of meningioma. Genetic analysis performed has identified an autosomal recessive, apparently homozygous c.3383+3A>G mutation, a mutation not previously reported. As per the existing literature, this is the index case of meningioma in Werner syndrome from India. A new mutation has been identified.

Human pluripotent stem cell-based models suggest preadipocyte senescence as a possible cause of metabolic complications of Werner and Bloom Syndromes.

Werner Syndrome (WS) and Bloom Syndrome (BS) are disorders of DNA damage repair caused by biallelic disruption of the WRN or BLM DNA helicases respectively. Both are commonly associated with insulin resistant diabetes, usually accompanied by dyslipidemia and fatty liver, as seen in lipodystrophies. In keeping with this, progressive reduction of subcutaneous adipose tissue is commonly observed. To interrogate the underlying cause of adipose tissue dysfunction in these syndromes, CRISPR/Cas9 genome editing was used to generate human pluripotent stem cell (hPSC) lacking either functional WRN or BLM helicase. No deleterious effects were observed in WRN-/- or BLM-/- embryonic stem cells, however upon their differentiation into adipocyte precursors (AP), premature senescence emerged, impairing later stages of adipogenesis. The resulting adipocytes were also found to be senescent, with increased levels of senescent markers and senescence-associated secretory phenotype (SASP) components. SASP components initiate and reinforce senescence in adjacent cells, which is likely to create a positive feedback loop of cellular senescence within the adipocyte precursor compartment, as demonstrated in normal ageing. Such a scenario could progressively attenuate adipose mass and function, giving rise to "lipodystrophy-like" insulin resistance. Further assessment of pharmacological senolytic strategies are warranted to mitigate this component of Werner and Bloom syndromes.

[Hereditary predispositions to follicular thyroid tumors]. / Prédispositions héréditaires aux tumeurs thyroïdiennes de souche folliculaire.

Familial thyroid cancers of follicular origin are rare and include syndromic and non-syndromic tumours. In familial adenomatous polyposis, the prevalence of papillary thyroid cancer is 2-12% and in 20-40% of cases it is a cribriform-morular papillary thyroid carcinoma. Morules and cribriform pattern are the two main typical criteria, associated with a nuclear and cytoplasmic immunopositivity for beta catenin. DICER1 syndrome is associated with pleuropneumoblastoma, ovarian tumors and thyroid pathology (multinodular goiter and less frequently a well-differentiated thyroid cancer without microscopic particularity). Cowden syndrome is characterized by multiple hamartomas and two-thirds of patients develop thyroid pathology, including multinodular goiter (50-67%) and cancer (35%), the latter being one of the major diagnostic criteria of the syndrome. Classic triad of Carney complex associates lentiginosis, myxoid tumors, and various endocrine abnormalities; thyroid pathology occurs in 10% of cases and may be benign or malignant. In Werner's syndrome, thyroid cancer is present in 18% of cases. McCune-Albright syndrome is characterized by fibrous dysplasia, café-au-lait spots and various endocrinopathies including hyperthyroidism and nodular hyperplasia. Non-syndromic thyroid cancers, which represent the majority of familial cancers, are most often papillary carcinomas. In daily practice, in the presence of multiple benign thyroid nodules and/or thyroid cancer in a young person, or with family thyroid diseases, the pathologist should be aware about hereditary predispositions to propose an oncogenetic consultation.

Studying Werner syndrome to elucidate mechanisms and therapeutics of human aging and age-related diseases.

Aging is a natural and unavoidable part of life. However, aging is also the primary driver of the dominant human diseases, such as cardiovascular disease, cancer, and neurodegenerative diseases, including Alzheimer's disease. Unraveling the sophisticated molecular mechanisms of the human aging process may provide novel strategies to extend 'healthy aging' and the cure of human aging-related diseases. Werner syndrome (WS), is a heritable human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. As a classical premature aging disease, etiological exploration of WS can shed light on the mechanisms of normal human aging and facilitate the development of interventional strategies to improve healthspan. Here, we summarize the latest progress of the molecular understandings of WRN protein, highlight the advantages of using different WS model systems, including Caenorhabditis elegans, Drosophila melanogaster and induced pluripotent stem cell (iPSC) systems. Further studies on WS will propel drug development for WS patients, and possibly also for normal age-related diseases.

ATM pathway activation limits R-loop-associated genomic instability in Werner syndrome cells.

Werner syndrome (WS) is a cancer-prone disease caused by deficiency of Werner protein (WRN). WRN maintains genome integrity by promoting replication-fork stability after various forms of replication stress. Under mild replication stress, WS cells show impaired ATR-mediated CHK1 activation. However, it remains unclear if WS cells elicit other repair pathway. We demonstrate that loss of WRN leads to enhanced ATM phosphorylation upon prolonged exposure to aphidicolin, a specific inhibitor of DNA polymerases, resulting in CHK1 activation. Moreover, we find that loss of WRN sensitises cells to replication-transcription collisions and promotes accumulation of R-loops, which undergo XPG-dependent cleavage responsible for ATM signalling activation. Importantly, we observe that ATM pathway limits chromosomal instability in WS cells. Finally, we prove that, in WS cells, genomic instability enhanced upon chemical inhibition of ATM kinase activity is counteracted by direct or indirect suppression of R-loop formation or by XPG abrogation. Together, these findings suggest a potential role of WRN as regulator of R-loop-associated genomic instability, strengthening the notion that conflicts between replication and transcription can affect DNA replication, leading to human disease and cancer.

CTC1 mutations in a Brazilian family with progeroid features and recurrent bone fractures.

BACKGROUND: Cerebroretinal microangiopathy with calcifications and cysts (CRMCC) is an autosomal recessive disorder caused by pathogenic variants of the conserved telomere maintenance component 1 (CTC1) gene. The CTC1 forms the telomeric capping complex, CST, which functions in telomere homeostasis and replication. METHODS: A Brazilian pedigree and an Australian pedigree were referred to the International Registry of Werner Syndrome (Seattle, WA, USA), with clinical features of accelerated aging and recurrent bone fractures. Whole exome sequencing was performed to identify the genetic causes. RESULTS: Whole exome sequencing of the Brazilian pedigree revealed compound heterozygous pathogenic variants in CTC1: a missense mutation (c.2959C>T, p.Arg987Trp) and a novel stop codon change (c.322C>T, p.Arg108*). The Australian patient carried two novel heterozygous CTC1 variants, c.2916G>T, p.Val972Gly and c.2926G>T, p.Val976Phe within the same allele. Both heterozygous variants were inherited from the unaffected father, excluding the diagnosis of CRMCC in this pedigree. Cell biological studies demonstrated accumulation of double strand break foci in lymphoblastoid cell lines derived from the patients. Increased DSB foci were extended to non-telomeric regions of the genome, in agreement with previous biochemical studies showing a preferential binding of CTC1 protein to GC-rich sequences. CONCLUSION: CTC1 pathogenic variants can present with unusual manifestations of progeria accompanied with recurrent bone fractures. Further studies are needed to elucidate the disease mechanism leading to the clinical presentation with intra-familial variations of CRMCC.

Ectopic hTERT expression facilitates reprograming of fibroblasts derived from patients with Werner syndrome as a WS cellular model.

The induced pluripotent stem cell (iPSC) technology has provided a unique opportunity to develop disease-specific models and personalized treatment for genetic disorders, and is well suitable for the study of Werner syndrome (WS), an autosomal recessive disease with adult onset of premature aging caused by mutations in the RecQ like helicase (WRN) gene. WS-derived fibroblasts were previously shown to be able to generate iPSCs; however, it remains elusive how WS-derived iPSCs behave and whether they are able to mimic the disease-specific phenotype. The present study was designed to address these issues. Unexpectedly, we found that a specific WS fibroblast line of homozygous truncation mutation was difficult to be reprogrammed by using the Yamanaka factors even under hypoxic conditions due to their defect in induction of hTERT, the catalytic unit of telomerase. Ectopic expression of hTERT restores the ability of this WS fibroblast line to form iPSCs, although with a low efficiency. To examine the phenotype of WRN-deficient pluripotent stem cells, we also generated WRN knockout human embryonic stem (ES) cells by using the CRISPR/Cas9 method. The iPSCs derived from WS-hTERT cells and WRN-/- ESCs are fully pluripotent, express pluripotent markers and can differentiate into three germ layer cells; however, WS-iPSCs and WRN-/- ESCs show S phase defect in cell cycle progression. Moreover, WS-iPSCs and WRN-/- ESCs, like WS patient-derived fibroblasts, remain hypersensitive to topoisomerase inhibitors. Collectively, WS-derived iPSCs and WRN-/- ESCs mimic the intrinsic disease phenotype, which may serve as a suitable disease model, whereas not be good for a therapeutic purpose without gene correction.

Everolimus rescues multiple cellular defects in laminopathy-patient fibroblasts.

LMNA encodes the A-type lamins that are part of the nuclear scaffold. Mutations in LMNA can cause a variety of disorders called laminopathies, including Hutchinson-Gilford progeria syndrome (HGPS), atypical Werner syndrome, and Emery-Dreifuss muscular dystrophy. Previous work has shown that treatment of HGPS cells with the mTOR inhibitor rapamycin or with the rapamycin analog everolimus corrects several of the phenotypes seen at the cellular level-at least in part by increasing autophagy and reducing the amount of progerin, the toxic form of lamin A that is overproduced in HGPS patients. Since other laminopathies also result in production of abnormal and potentially toxic lamin proteins, we hypothesized that everolimus would also be beneficial in those disorders. To test this, we applied everolimus to fibroblast cell lines from six laminopathy patients, each with a different mutation in LMNA Everolimus treatment increased proliferative ability and delayed senescence in all cell lines. In several cell lines, we observed that with treatment, there is a significant improvement in nuclear blebbing, which is a cellular hallmark of HGPS and other lamin disorders. These preclinical results suggest that everolimus might have clinical benefit for multiple laminopathy syndromes.

Are there Different Kinds of Aging?

BACKGROUND: Most syndromes of accelerated aging are caused by mutations affecting the integrity of the genetic material. Among them, the most studied is Werner's syndrome, "adult progeria", caused by a recessive autosomal mutation with a frequency of 1 in 10 million, which affects a helicase involved in DNA repair. In Werner syndrome, there is a loss of heterochromatin, though the stability of heterochromatin is also affected in "normal" aging. The Hutchinson-Gilford Progeria Syndrome (HGPS), "child progeria", has an even lower frequency. In most cases, it is caused by a point mutation of a gene coding a protein in the nuclear envelope, lamin A. OBJECTIVES: HGPS may provide valuable insights into the aging process. The symptoms of this condition do not entirely overlap with those of "normal" aging. METHOD: A critical analysis of the accelerated aging syndromes may explain what aging is, and also why some tissues and organs age at accelerated rates as compared to other tissues. RESULTS: In this article, we will discuss the implications of HGPS and other accelerated aging syndromes in the light of the biochemical hypothesis of aging we advanced. According to this hypothesis, some reactions are less stimulated and diminish in time, affecting not only specific biochemical functions, but cellular energy, and therefore its capacity for synthesis. CONCLUSION: Besides, a new vision on aging, possible therapeutic strategies for these conditions and others, with similar mechanisms, are also presented.

Accelerated aging and aging process in the brain.

One of the approaches to the research of the problem of aging is the study of genetic pathologies leading to accelerated aging, such as the Hutchinson-Gilford progeria syndrome, Werner syndrome, and Down syndrome. Probably, this approach can be used in an attempt to understand the neuronal mechanisms underlying normal and pathological brain aging. The analysis of the current state of scientific knowledge about these pathologies shows that in the Hutchinson-Gilford progeria and Werner syndrome, the rate of brain aging is significantly lower than the rate of whole body aging, whereas in Down syndrome, the brain ages faster than other organs due to amyloid-beta accumulation and chronic oxidative stress in the brain tissue. The main point of a previously proposed hypothesis is that the aging of higher animals and humans is associated with an increased level of reactive oxygen species in mitochondria with age, which activates apoptosis, thus reducing the number of functioning cells.

Werner syndrome: a model for sarcopenia due to accelerated aging.

Werner syndrome (WS) is a rare inheritable progeroid syndrome caused by a mutation in the WRN gene. Although WS has been described as a characteristic appearance of very slender extremities with a stocky trunk, few studies have investigated the loss of muscle mass, fat mass distribution (body composition), and mobility according to age and sex. Therefore, the aim of this study was to precisely describe the body composition in WS. Nine Japanese patients with WS (four males and five females; mean age 48±8.8 years) were recruited. Body composition was examined by dual-energy X-ray absorptiometry and computed tomography (CT). The hand grip strength and mobility were evaluated using the two-step test, stand-up test and 25-question geriatric locomotive function scale (GLFS). The mean skeletal muscle index (SMI) was 4.0±0.6 kg/m2. SMI of all patients met the criteria of sarcopenia, even though some patients were aged < 40 years. All patients also showed deceased mobility. In conclusion, these results indicate that all patients with WS, even those aged < 40 years, had already lost muscle mass to the level of sarcopenia. Continued research on sarcopenia in WS might facilitate the discovery of novel mechanisms and development of new treatment strategies for sarcopenia.