Maternal histone methyltransferases antagonistically regulate monoallelic expression in C. elegans.

Undefined epigenetic programs act to probabilistically silence individual autosomal alleles, generating unique individuals, even from genetic clones. This sort of random monoallelic expression can explain variation in traits and diseases that differences in genes and environments cannot. Here, we developed the nematode Caenorhabditis elegans to study monoallelic expression in whole tissues, and defined a developmental genetic regulation pathway. We found maternal H3K9 histone methyltransferase (HMT) SET-25/SUV39/G9a works with HPL-2/HP1 and LIN-61/L3MBTL2 to randomly silence alleles in the intestinal progenitor E-cell of 8-cell embryos to cause monoallelic expression. SET-25 was antagonized by another maternal H3K9 HMT, MET-2/SETDB1, which works with LIN-65/ATF7ZIP and ARLE-14/ARL14EP to prevent monoallelic expression. The HMT-catalytic SET domains of both MET-2 and SET-25 were required for regulating monoallelic expression. Our data support a model wherein SET-25 and MET-2 regulate histones during development to generate patterns of somatic monoallelic expression that are persistent but not heritable.

Caspase 5 depletion is linked to hyper-inflammatory response and progeroid syndrome.

A progeroid family was found to harbor a pathogenic variant in the CASP5 gene that encodes inflammatory caspase 5. Caspase 5-depleted fibroblasts exhibited hyper-activation of inflammatory cytokines in response to pro-inflammatory stimuli. Long-term intermittent hyper-inflammatory response is likely the cause of the accelerated aging phenotype comprised of earlier onset of common aging diseases, supporting inflammaging as a potential common disease mechanism of progeroid syndromes and possibly normative aging.

Woodhouse-Sakati syndrome in an Indian patient with a novel pathogenic variant.

Woodhouse-Sakati syndrome consists of hypogonadism, diabetes mellitus, alopecia, ECG abnormalities, and dystonia. This condition is caused by the loss of function of the DCAF17 gene. Most of the patients have been reported from Greater Middle Eastern countries. We report a 38 male from southern India who presented with syncope and massive hemoptysis due to ruptured bronchopulmonary collaterals. He also had alopecia, cataracts, recently diagnosed diabetes and hypogonadism. Whole exome sequencing showed a novel homozygous truncating variant in the DCAF17 gene. Despite embolization of the aortopulmonary collaterals, the patient died of recurrent hemoptysis.

Renal dysfunction, malignant neoplasms, atherosclerotic cardiovascular diseases, and sarcopenia as key outcomes observed in a three-year follow-up study using the Werner Syndrome Registry.

Werner syndrome is an adult-onset progeria syndrome that results in various complications. This study aimed to clarify the profile and secular variation of the disease. Fifty-one patients were enrolled and registered in the Werner Syndrome Registry. Their data were collected annually following registration. A cross-sectional analysis at registration and a longitudinal analysis between the baseline and each subsequent year was performed. Pearson's chi-squared and Wilcoxon signed-rank tests were used. Malignant neoplasms were observed from the fifth decade of life (mean onset: 49.7 years) and were observed in approximately 30% of patients during the 3-year survey period. Regarding renal function, the mean estimated glomerular filtration rate calculated from serum creatinine (eGFRcre) and eGFRcys, which were calculated from cystatin C in the first year, were 98.3 and 83.2 mL/min/1.73 m2, respectively, and differed depending on the index used. In longitudinal analysis, the average eGFRcre for the first and fourth years was 74.8 and 63.4 mL/min/1.73 m2, showing a rapid decline. Secular changes in Werner syndrome in multiple patients were identified. The prevalence of malignant neoplasms is high, and renal function may decline rapidly. It is, therefore, necessary to carry out active and detailed examinations and pay attention to the type and dose of the drugs used.

Key elements of cellular senescence involve transcriptional repression of mitotic and DNA repair genes through the p53-p16/RB-E2F-DREAM complex.

Cellular senescence is a dynamic stress response process that contributes to aging. From initiation to maintenance, senescent cells continuously undergo complex molecular changes and develop an altered transcriptome. Understanding how the molecular architecture of these cells evolve to sustain their non-proliferative state will open new therapeutic avenues to alleviate or delay the consequences of aging. Seeking to understand these molecular changes, we studied the transcriptomic profiles of endothelial replication-induced senescence and senescence induced by the inflammatory cytokine, TNF-α. We previously reported gene expressional pattern, pathways, and the mechanisms associated with upregulated genes during TNF-α induced senescence. Here, we extend our work and find downregulated gene signatures of both replicative and TNF-α senescence were highly overlapped, involving the decreased expression of several genes associated with cell cycle regulation, DNA replication, recombination, repair, chromatin structure, cellular assembly, and organization. We identified multiple targets of p53/p16-RB-E2F-DREAM that are essential for proliferation, mitotic progression, resolving DNA damage, maintaining chromatin integrity, and DNA synthesis that were repressed in senescent cells. We show that repression of multiple target genes in the p53/p16-RB-E2F-DREAM pathway collectively contributes to the stability of the senescent arrest. Our findings show that the regulatory connection between DREAM and cellular senescence may play a potential role in the aging process.

Accelerated epigenetic aging and DNA methylation alterations in Berardinelli-Seip congenital lipodystrophy.

Berardinelli-Seip congenital lipodystrophy type 2 (CGL2) is a very rare human genetic disorder with potential significance to the understanding of the pathobiology of aging. CGL2 patients display characteristic progeroid features and suffer from type 2 diabetes, insulin resistance and fatty liver. In this study, we profiled genome-wide DNA methylation levels in CGL2 patients with BSCL2 mutations to study epigenetic age acceleration and DNA methylation alterations. This analysis revealed significant age acceleration in blood DNA of CGL2 patients using both first- and second-generation epigenetic clocks. We also observed a shortened lifespan of Caenorhabditis elegans following knockdown of the BSCL2 homolog seip-1 on a daf-16/forkhead box, class O mutant background. DNA methylation analysis revealed significant differentially methylated sites enriched for lyase activity, kinase regulator activity, protein kinase regulator activity and kinase activator activity. We could also observe significant hypomethylation in the promoter of the dual specificity phosphatase 22 gene when comparing CGL2 patients versus controls. We conclude that in line with the observed progeroid features, CGL2 patients exhibit significant epigenetic age acceleration and DNA methylation alterations that might affect pathways/genes of potential relevance to the disease.

Rapid emergence of transcriptional heterogeneity upon molecular stress predisposes cells to two distinct states of senescence.

Slowing aging can reduce the risk of chronic diseases. In particular, eliminating senescent cells is a promising approach to slow aging. Previous studies found that both cells from older animals and senescent cells have noisy gene expression. Here, we performed a large-scale single-cell RNA-sequencing time course to understand how transcriptional heterogeneity develops among senescent cells. We found that cells experiencing senescence-inducing oxidative stress rapidly adopt one of two major transcriptional states. One senescent cell state is associated with stress response, and the other is associated with tissue remodeling. We did not observe increased stochastic gene expression. This data is consistent with the idea that reproducible, limited, distinct, and coherent transcriptional states exist in senescent cell populations. These physiologically distinct senescent cell subtypes may each affect the aging process in unique ways and constitute a source of heterogeneity in aging.

TNF-α/IFN-γ synergy amplifies senescence-associated inflammation and SARS-CoV-2 receptor expression via hyper-activated JAK/STAT1.

Older age and underlying conditions such as diabetes/obesity or immunosuppression are leading host risk factors for developing severe complications from COVID-19 infection. The pathogenesis of COVID-19-related cytokine storm, tissue damage, and fibrosis may be interconnected with fundamental aging processes, including dysregulated immune responses and cellular senescence. Here, we examined effects of key cytokines linked to cellular senescence on expression of SARS-CoV-2 viral entry receptors. We found exposure of human umbilical vein endothelial cells (HUVECs) to the inflammatory cytokines, TNF-α + IFN-γ or a cocktail of TNF-α + IFN-γ + IL-6, increased expression of ACE2/DPP4, accentuated the pro-inflammatory senescence-associated secretory phenotype (SASP), and decreased cellular proliferative capacity, consistent with progression towards a cellular senescence-like state. IL-6 by itself failed to induce substantial effects on viral entry receptors or SASP-related genes, while synergy between TNF-α and IFN-γ initiated a positive feedback loop via hyper-activation of the JAK/STAT1 pathway, causing SASP amplification. Breaking the interactive loop between senescence and cytokine secretion with JAK inhibitor ruxolitinib or antiviral drug remdesivir prevented hyper-inflammation, normalized SARS-CoV-2 entry receptor expression, and restored HUVECs proliferative capacity. This loop appears to underlie cytokine-mediated viral entry receptor activation and links with senescence and hyper-inflammation.

Targeted long-read sequencing identifies missing pathogenic variants in unsolved Werner syndrome cases.

BACKGROUND: Werner syndrome (WS) is an autosomal recessive progeroid syndrome caused by variants in WRN. The International Registry of Werner Syndrome has identified biallelic pathogenic variants in 179/188 cases of classical WS. In the remaining nine cases, only one heterozygous pathogenic variant has been identified. METHODS: Targeted long-read sequencing (T-LRS) on an Oxford Nanopore platform was used to search for a second pathogenic variant in WRN. Previously, T-LRS was successfully used to identify missing variants and analyse complex rearrangements. RESULTS: We identified a second pathogenic variant in eight of nine unsolved WS cases. In five cases, T-LRS identified intronic splice variants that were confirmed by either RT-PCR or exon trapping to affect splicing; in one case, T-LRS identified a 339 kbp deletion, and in two cases, pathogenic missense variants. Phasing of long reads predicted all newly identified variants were on a different haplotype than the previously known variant. Finally, in one case, RT-PCR previously identified skipping of exon 20; however, T-LRS did not detect a pathogenic DNA sequence variant. CONCLUSION: T-LRS is an effective method for identifying missing pathogenic variants. Although limitations with computational prediction algorithms can hinder the interpretation of variants, T-LRS is particularly effective in identifying intronic variants.

Werner syndrome in a Lebanese family.

Werner syndrome (WS) is an extremely rare, autosomal recessive segmental progeroid disorder caused by biallelic pathogenic variants in the WRN, which encodes a multifunctional nuclear protein that belongs to the RecQ family of DNA helicases. Despite extensive research on WS in the last years, the population-specific mutational spectrum still needs to be elucidated. Moreover, there is an evident lack of detailed clinical descriptions accompanied with photographs of affected individuals. Here, we report a consanguineous Lebanese family in whom we identified a pathogenic homozygous nonsense variant c.1111G>T, p.Glu371* in the WRN. The index individual, at the age of 54 years, was suspected to have WS due to a history of early-onset cataracts, premature hair loss and graying, chronic nonhealing leg ulcers, Achilles' tendon calcifications, type 2 diabetes mellitus, dyslipidemia, hypothyroidism, and premature coronary artery disease. His four sisters, three of which deceased in the fifth decade, had clinical signs suggestive of WS. Moreover, his daughter, aged 23 years, had short stature, hair loss and flat feet. Taken together, we report a detailed clinical course of disease in several affected members of a consanguineous family, which is additionally documented by photographs.

DNA methylation signatures in Blood DNA of Hutchinson-Gilford Progeria syndrome.

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder caused by mutations in the LMNA gene and characterized by premature and accelerated aging beginning in childhood. In this study, we performed the first genome-wide methylation analysis on blood DNA of 15 patients with progeroid laminopathies using Infinium Methylation EPIC arrays including 8 patients with classical HGPS. We could observe DNA methylation alterations at 61 CpG sites as well as 32 significant regions following a 5 Kb tiling analysis. Differentially methylated probes were enriched for phosphatidylinositol biosynthetic process, phospholipid biosynthetic process, sarcoplasm, sarcoplasmic reticulum, phosphatase regulator activity, glycerolipid biosynthetic process, glycerophospholipid biosynthetic process, and phosphatidylinositol metabolic process. Differential methylation analysis at the level of promoters and CpG islands revealed no significant methylation changes in blood DNA of progeroid laminopathy patients. Nevertheless, we could observe significant methylation differences in classic HGPS when specifically looking at probes overlapping solo-WCGW partially methylated domains. Comparing aberrantly methylated sites in progeroid laminopathies, classic Werner syndrome, and Down syndrome revealed a common significantly hypermethylated region in close vicinity to the transcription start site of a long non-coding RNA located anti-sense to the Catenin Beta Interacting Protein 1 gene (CTNNBIP1). By characterizing epigenetically altered sites, we identify possible pathways/mechanisms that might have a role in the accelerated aging of progeroid laminopathies.

SMAD4 mutations and cross-talk between TGF-ß/IFNγ signaling accelerate rates of DNA damage and cellular senescence, resulting in a segmental progeroid syndrome-the Myhre syndrome.

SMAD4 encodes a member of the SMAD family of proteins involved in the TGF-ß signaling pathway. Potentially heritable, autosomal dominant, gain-of-function heterozygous variants of SMAD4 cause a rare developmental disorder, the Myhre syndrome, which is associated with a wide range of developmental and post-developmental phenotypes that we now characterize as a novel segmental progeroid syndrome. Whole-exome sequencing of a patient referred to our International Registry of Werner Syndrome revealed a heterozygous p.Arg496Cys variant of the SMAD4 gene. To investigate the role of SMAD4 mutations in accelerated senescence, we generated cellular models overexpressing either wild-type SMAD4 or mutant SMAD4-R496C in normal skin fibroblasts. We found that cells expressing the SMAD4-R496C mutant exhibited decreased proliferation and elevated expression of cellular senescence and inflammatory markers, including IL-6, IFNγ, and a TGF-ß target gene, PAI-1. Here we show that transient exposure to TGF-ß, an inflammatory cytokine, followed by chronic IFNγ stimulation, accelerated rates of senescence that were associated with increased DNA damage foci and SMAD4 expression. TGF-ß, IFNγ, or combinations of both were not sufficient to reduce proliferation rates of fibroblasts. In contrast, TGF-ß alone was able to induce preadipocyte senescence via induction of the mTOR protein. The mTOR inhibitor rapamycin mitigated TGF-ß-induced expression of p21, p16, and DNA damage foci and improved replicative potential of preadipocytes, supporting the cell-specific response to this cytokine. These findings collectively suggest that persistent DNA damage and cross-talk between TGF-ß/IFNγ pathways contribute to a series of molecular events leading to cellular senescence and a segmental progeroid syndrome.

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.

Novel LMNA mutations in Greek and Myanmar Patients with Progeroid Features and Cardiac Manifestations.

Segmental progeroid syndromes are groups of genetic disorders with multiple features resembling accelerated aging. The International Registry of Werner Syndrome (Seattle, WA) recruits pedigrees of progeroid syndromes from all over the world. We identified two novel LMNA mutations, p.Asp300Gly in a patient from Myanmar, and p.Asn466Lys, in a patient from Greece. Both were referred to our Registry for the genetic diagnosis because of the accelerated aged-appearance and cardiac complications. LMNA mutations are the second most common genetic cause of progeroid syndromes after WRN mutations in our Registry. As the next generation sequencing becomes readily available, we expect to identify more cases of rare genetic diseases in the developing countries.

Multisystem Progeroid Syndrome With Lipodystrophy, Cardiomyopathy, and Nephropathy Due to an LMNA p.R349W Variant.

BACKGROUND: Pathogenic variants in lamin A/C (LMNA) cause a variety of progeroid disorders including Hutchinson-Gilford progeria syndrome, mandibuloacral dysplasia, and atypical progeroid syndrome. Six families with 11 patients harboring a pathogenic heterozygous LMNA c.1045C>T; p.R349W variant have been previously reported to have partial lipodystrophy, cardiomyopathy, and focal segmental glomerulosclerosis (FSGS), suggesting a distinct progeroid syndrome. METHODS: We report 6 new patients with a heterozygous LMNA p.R349W variant and review the phenotype of previously reported patients to define their unique characteristics. We also performed functional studies on the skin fibroblasts of a patient to seek the underlying mechanisms of various clinical manifestations. RESULTS: Of the total 17 patients, all 14 adults with the heterozygous LMNA p.R349W variant had peculiar lipodystrophy affecting the face, extremities, palms, and soles with variable gain of subcutaneous truncal fat. All of them had proteinuric nephropathy with FSGS documented in 7 of them. Ten developed cardiomyopathy, and 2 of them died early at ages 33 and 45 years. Other common features included premature graying, alopecia, high-pitched voice, micrognathia, hearing loss, and scoliosis. Metabolic complications, including diabetes mellitus, hypertriglyceridemia, and hepatomegaly, were highly prevalent. This variant did not show any abnormal splicing, and no abnormal nuclear morphology was noted in the affected fibroblasts. CONCLUSIONS: The heterozygous LMNA p.R349W variant in affected individuals has several distinct phenotypic features, and these patients should be classified as having multisystem progeroid syndrome (MSPS). MSPS patients should undergo careful assessment at symptom onset and yearly metabolic, renal, and cardiac evaluation because hyperglycemia, hypertriglyceridemia, FSGS, and cardiomyopathy cause major morbidity and mortality.

A Case Report of Werner's Syndrome With a Novel Mutation From India.

Werner's syndrome (WS) or progeria adultorum is a heritable autosomal recessive disease in which the aging process is accelerated, just after puberty. It is caused by mutations in the WRN gene, which encodes a member of the RECQ family of DNA helicases and has a role in DNA repair. WS is being more appropriately recognized as a condition in which the lack of WRN protein results in an overall decline in the normal physiological functions of various organs rather than premature aging. Here, we describe a rare case of WS with a novel mutation from India. Our patient was an adult male with a history of growth arrest since puberty and other clinical features such as sclerodermatous skin changes, premature graying and thinning of hair, bilateral cataract, a single non-healing ulcer, hypothyroidism, underdeveloped secondary sexual characters with hypogonadism, infertility, squeaky voice, and early signs of arteriosclerosis. On genetic analysis, he was found to have a homozygous pathogenic variant c.3190C>T in exon 26 of the WRN gene, which has never been reported in WS.

Cell-to-Cell Variation in Gene Expression for Cultured Human Cells Is Controlled in Trans by Diverse Genes: Implications for the Pathobiology of Aging.

Cell-to-cell variation in gene expression increases among homologous cells within multiple tissues during aging. We call this phenomenon variegated gene expression (VGE). Long, healthy life requires robust and coordinated gene expression. We posit that nature may have evolved VGE as a bet-hedging mechanism to protect reproductively active populations. The price we may pay is accelerated aging. That hypothesis will require the demonstration that genetic loci are capable of modulating degrees of VGE. While loci controlling VGE in yeast and genes controlling interindividual variation in gene expression in Caenorhabditis elegans have been identified, there has been no compelling evidence for the role of specific genetic loci in modulations of VGE of specific targets in humans. With the assistance of a core facility, we used a customized library of siRNA constructs to screen 1,195 human genes to identify loci contributing to the control of VGE of a gene with relevance to the biology of aging. We identified approximately 50 loci controlling VGE of the prolongevity gene, SIRT1. Because of its partial homology to FOXO3A, a variant of which is enriched in centenarians, our laboratory independently confirmed that the knockdown of FOXF2 greatly diminished VGE of SIRT1 but had little impact upon the VGE of WRN. While the role of these VGE-altering genes on aging in vivo remains to be determined, we hypothesize that some of these genes can be targeted to increase functionality during aging.

Inactivating Mutations in Exonuclease and Polymerase Domains in DNA Polymerase Delta Alter Sensitivities to Inhibitors of dNTP Synthesis.

POLD1 encodes the catalytic subunit of DNA polymerase delta (Polδ), the major lagging strand polymerase, which also participates in DNA repair. Mutations affecting the exonuclease domain increase the risk of various cancers, while mutations that change the polymerase active site cause a progeroid syndrome called mandibular hypoplasia, deafness, progeroid features, and lipodystrophy (MDPL) syndrome. We generated a set of catalytic subunit of human telomerase (hTERT)-immortalized human fibroblasts expressing wild-type or mutant POLD1 using the retroviral LXSN vector system. In the resulting cell lines, expression of endogenous POLD1 was suppressed in favor of the recombinant POLD1. The siRNA screening of DNA damage-related genes revealed that fibroblasts expressing D316H and S605del POLD1 were more sensitive to knockdowns of ribonuclease reductase (RNR) components, RRM1 and RRM2 in the presence of hydroxyurea (HU), an RNR inhibitor. On the contrary, SAMHD1 siRNA, which increases the concentration of dNTPs, increased growth of wild type, D316H, and S605del POLD1 fibroblasts. Hypersensitivity to dNTP synthesis inhibition in POLD1 mutant lines was confirmed using gemcitabine. Our finding is consistent with the notion that reduced dNTP concentration negatively affects the cell growth of hTERT fibroblasts expressing exonuclease and polymerase mutant POLD1.