Evaluation of glucose tolerance and effect of dietary management on increased visceral fat in a patient with Werner syndrome.

Werner syndrome (WS), a type of progeria, is a hereditary condition caused by a mutation in the WRN gene. A 62-year-old Japanese woman was diagnosed with WS at the age of 32 and has been visiting the hospital for follow-up since the last 30 years. The patient developed diabetes at the age of 46, and at the age of 60, her body mass index increased from 20.1 to 22.7 kg/m2 owing to her unhealthy eating habits; her visceral fat area at the age of 61 was 233 cm2. With dietary control, her body weight, including the visceral fat and subcutaneous fat, decreased at the age of 62, and her insulin secretion, obesity, and fatty liver improved. We conducted the oral glucose challenge test four times, including at the prediabetic stage, to evaluate the insulin-secretion ability. The patient's insulin resistance gradually increased for more than 14 years, and her insulin secretion ability began to decrease 14 years after her diabetes diagnosis. Despite a remarkable decrease in body weight and fat mass with dietary management, the psoas muscle index did not decrease significantly in proportion to the body weight or fat mass. However, muscle mass monitoring is important for preventing the progression of sarcopenia. Hence, gradual reduction of visceral fat and weight by dietary management may be useful in treating diabetes in patients with WS, particularly in those whose visceral fat is significantly increased.

Folate modulates guanine-quadruplex frequency and DNA damage in Werner syndrome.

Guanine-quadruplexes (G4) are stable tetra-stranded DNA structures that may cause DNA replication stress and inhibit gene expression. Defects in unwinding these structures by DNA helicases may result in telomere shortening and DNA damage. Furthermore, due to mutations in WRN helicase genes in Werner syndrome, G4 motifs are likely to be key elements in the expression of premature aging phenotypes. The methylation of DNA plays a significant role in the stability and occurrence of G4. Thus, G4 frequency and DNA methylation mechanisms may be affected by excesses or deficiencies in methyl donors such as folate. B-Lymphocytes from Werner patients (n = 5) and healthy individuals (n = 5) were cultured in RPMI medium under condition of folate deficiency (20 nM) or sufficiency (200 nM) for 14 days. Cells were fixed on microscope slides for immunofluorescent staining to measure G4 frequency and γH2AX (a marker of DNA strand breaks) intensity, using automated quantitative imaging fluorescent microscopy. There was a significant increase (p < 0.05) in G4 levels in Werner syndrome patients compared to healthy controls. Werner and control cells grown in 20 nM folate media also showed significant increases in G4 (p < 0.001) and γH2AX (p < 0.01) signals compared with the same cells grown in 200 nM folate. Control cells grown in 20 nM folate also showed a significant reduction in DNA methylation levels (P < 0.05). The results of this study suggest that the occurrence of DNA G4 structures can be modulated in vitro via nutrients with important roles in methylation.

Metabolic and Phenotypic Differences between Mice Producing a Werner Syndrome Helicase Mutant Protein and Wrn Null Mice.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter mean life span. In contrast, mice lacking the entire Wrn protein (i.e. Wrn null mice) do not exhibit a premature aging phenotype. In this study, we used a targeted mass spectrometry-based metabolomic approach to identify serum metabolites that are differentially altered in young Wrn helicase mutant and Wrn null mice. An antibody-based quantification of 43 serum cytokines and markers of cardiovascular disease risk complemented this study. We found that Wrn helicase mutants exhibited elevated and decreased levels, respectively, of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine IL-18. Wrn helicase mutants also exhibited an increase in serum hydroxyproline and plasminogen activator inhibitor-1, markers of extracellular matrix remodeling of the vascular system and inflammation in aging. We also observed an abnormal increase in the ratio of very long chain to short chain lysophosphatidylcholines in the Wrn helicase mutants underlying a peroxisome perturbation in these mice. Remarkably, the Wrn mutant helicase protein was mislocalized to the endoplasmic reticulum and the peroxisomal fractions in liver tissues. Additional analyses with mouse embryonic fibroblasts indicated a severe defect of the autophagy flux in cells derived from Wrn helicase mutants compared to wild type and Wrn null animals. These results indicate that the deleterious effects of the helicase-deficient Wrn protein are mediated by the dysfunction of several cellular organelles.

Werner's syndrome may be lost in the shadow of the scleroderma.

We describe three patients with Werner's syndrome (WS), two of whom had been mistakenly diagnosed as having scleroderma. We would like to discuss briefly the importance of differentiation of these two disorders from each other.

Aging-associated inflammation in healthy Japanese individuals and patients with Werner syndrome.

Minor inflammation-driven aging (inflammaging) has been proposed to explain human aging mechanism. To study the inflammatory condition associated with normal human aging, highly sensitive CRP (hsCRP) was examined in the sera collected from 217 healthy Japanese individuals aged between 1 and 100years and 41 mutation-proven Japanese Werner syndrome (WS) patients. The serum hsCRP was assayed by ELISA. The serum hsCRP level increased significantly (p<0.001) with normal aging from both sexes. The serum hsCRP was significantly elevated in WS (mean±SE: 11.0±1.6µg/ml) compared with age-matched normal population (1.3±0.3µg/ml, p<0.001) and normal elderly population ages between 71 and 100years (4.2±0.7µg/ml, p<0.001). Both normal aging and WS were associated with minor inflammation that can be evaluated by serum hsCRP. WS may be a good candidate to study inflammaging.

Ageing in Werner syndrome.

Oxidative stress markers including pentosidine and homocysteine were examined comparing them with inflammation markers including highly sensitive C-reactive protein (hsCRP) and matrix metalloproteinase-9 (MMP-9) in serum from patients with Werner syndrome (WS) and healthy individuals. Elevation of serum pentosidine correlated significantly with normal aging in healthy individuals (p < 0.0004). Serum pentosidine in WS increased significantly compared with age-matched healthy individuals (p < 0.05). Serum homocysteine levels increased insignificantly with normal aging in healthy individuals and in WS compared with age-matched healthy individuals. As both pentosidine and homocysteine levels did not correlate with hsCRP or MMP-9, both oxidative stress markers may be differentially regulated by inflammation.

Elevation of soluble Fas (APO-1, CD95) ligand in natural aging and Werner syndrome.

The pathophysiological process of natural human aging has not been studied adequately due to the lack of an appropriate human model. Since recent investigations have suggested that inflammation possibly contributes to the pathogenesis of age-related disorders including atherosclerosis, cancer, and diabetes mellitus, the term "inflammaging," a combination of "inflammation" and "aging," has been coined. Werner syndrome (WS), caused by the loss of function of RecQ3 DNA/RNA helicase, is a typical progeroid syndrome mimicking natural aging, although it is extremely rare outside of Japan. We sought to examine WS patients from an immunological/inflammatory perspective. Sera from 14 mutation-proven WS patients (ages: 33-70 years) and 21 healthy Japanese adults ages 15 to 95 years were examined with ELISA for soluble Fas ligand (sFasL) to compare conventional inflammation markers. With natural aging, a statistically significant correlation (p < 0.0001) was observed in the serum level of sFasL. The sFasL in WS, a level comparable to that in healthy elderly ages 83 to 95 years, had significantly increased (p < 0.05) compared to that in young healthy individuals ages 15 to 70 years. A significant correlation was noted between the serum levels of conventional inflammation markers such as CRP (p < 0.025), ESR (p < 0.024), and WBC count (p < 0.0085). In conclusion, an increased level of serum sFasL in natural aging and WS patients may suggest a common pathophysiological mechanism: inflammation. WS may be a good model for analyzing inflammaging.

N-glycomic changes in serum proteins during human aging.

N-glycan profiling of the human serum glycoproteins including immunoglobulin fraction on different age groups of healthy persons shows substantial changes with increasing age in three major N-glycan structures. In individuals more than 40-50 years of age, there is an increase in under-galactosylated glycans and a decrease in the core alpha-1,6-fucosylated bi-galactosylated biantennary structure. These three glycan structures are also substantially changed in a Werner syndrome patient, to a level comparable or even more pronounced than those observed in a healthy Italian centenarian population. These data show that the glycosylation machineries in both liver cells and B-cells are affected in a similar way by the aging process despite their highly different nature. The observed changes in the glycan structures are indicative that biosynthetic processes are at the basis of the changes, possibly together with changes in serum clearing of glycan-altered proteins. Our data suggest that measurement of the N-glycan level changes could provide a noninvasive surrogate marker for general health or for age-related disease progression, and for monitoring the improvement of health after therapy.

[Age dymamics of stable chromosome aberration frequency in humans with natural and pathological senescence]. / Vozrastnaia dinamika chastoty stabil'nykh khromosomnykh aberratsii u cheloveka pri estestvennom i patologicheskom starenii.

The age dynamics of stable chromosome aberration (SCA) frequency was analysed by fluorescent in situ hybridization (FISH) in human blood lymphocytes derived from donors, irradiated by low doses of ionizing radiation (Chernobyl clean-up workers, nuclear weapon testers, etc.) and patients with hereditary premature aging--Werner's syndrome and Hutchinson-Gilford's syndrome. It was found that the level of SCA was age-dependent and increased in irradiated persons. So, the SCA level may be really an index of a so-called "radiation senescence", and may show a real biological age of irradiated persons. The patients with Werner's syndrome demonstrate increased SCA level in blood lymphocytes, corresponding to the premature aging of the organisms. But in the case of another form of premature aging--Hutchinson--Gilford's syndrome-- no rise of SCA level was found. Some possible reasons of such results are discussed.

Hyaluronan is not elevated in urine or serum in Hutchinson-Gilford Progeria Syndrome.

Elevations in urinary hyaluronan have been used as the principal laboratory indicator for diagnosis of Hutchinson-Gilford Progeria Syndrome (HGPS). Previous reports have provided evidence suggesting that children with HGPS have altered hyaluronan metabolism as indicated by a mean 17-fold increase in urinary hyaluronan over normal values. In addition, adults with Werner's syndrome have elevated urinary hyaluronan and even more prominent elevations in serum hyaluronan over age-matched controls. It is not known whether serum hyaluronan is elevated or whether serum hyaluronan levels correlate with urinary hyaluronan levels in children with HGPS. In a large cohort of 19 HGPS patients, we sought to confirm elevations in urinary hyaluronan concentration, to establish whether serum hyaluronan is elevated, to measure the size of urinary hyaluronan, and to determine whether serum or urine hyaluronidase levels are altered. We have analyzed urinary and serum hyaluronan levels in patients with HGPS and control patients (1) by using an enzyme-linked immunosorbent assay (ELISA)-like method in which sample hyaluronan in solution and hyaluronan in solid phase compete for a solution of biotinylated hyaluronan-binding protein, and (2) by fluorophore-assisted carbohydrate electrophoresis. The size of urinary hyaluronan was measured by using Sepharose CL-6B size exclusion chromatography. Serum and urinary hyaluronidases were evaluated quantitatively, by using ELISA, and qualitatively, by using a gel detection method. HGPS patients did not show a significant elevation in either urinary or serum hyaluronan. We detected no difference in the size of urinary hyaluronan between HGPS children and age-matched controls. Serum and urinary hyaluronidase levels were not significantly different in normal and HGPS patients. These studies indicate that neither serum nor urinary hyaluronan concentration is a reliable diagnostic or prognostic marker for HGPS and underscore a difference between adult and childhood progerias.

Elevation of serum hyaluronan level in Werner's syndrome.

BACKGROUND: There is a well-established association between Werner's syndrome (WS) and hyperhyaluronic aciduria; however, to date hyaluronan (HA) in the serum has not been statistically linked with WS. Recently, the gene that causes WS has been defined as a DNA helicase on chromosome 8, and 19 different mutations in WS patients have been identified. It is not known whether the mutation type of the Werner helicase gene affects the levels of serum and urine HA in WS patients. OBJECTIVE: To evaluate the association of serum HA with WS, and the relationship of the serum and urine HA levels to the mutation type. METHODS: HA both in the serum and the urine was measured in 40 patients with WS and 114 normal controls. The serum and urine HA were quantified by sandwich binding protein assay and competitive ELISA-like method, respectively. The muation on WS helicase gene was analyzed by mutant-allele-specific amplification and oligomer ligation assay. RESULTS: WS patients showed significantly higher levels of HA in the serum (mean +/- SD: 115.7 +/- 119.8 ng/ml, p < 0.001) and urine (1,040.8 +/- 777.3 ng/mg creatinine, Cre, p < 0.001) than age-matched controls (serum HA: 15.8 +/- 14.2 ng/ml, urine HA: 379.7 +/- 124.2 ng/mg Cre). The serum and urine HA levels of WS patients are almost equal to those of normal controls over 80 years (serum HA: 118.5 +/- 108.4 ng/ml, urine HA: 914.5 +/- 712.1 ng/mg Cre). There was a significant correlation between serum and urine HA levels in WS patients (r = 0.42, p = 0.007). Analysis of the mutation on the helicase gene in 22 WS patients showed that among 44 chromosomes, 3 (6.8%) chromosomes had type 1 mutation, 22 (50.0%) had type 4 mutation, 14 (31.8%) had type 6 mutation, and the rest had other mutations. The serum and urine levels of HA did not show any significant association with the mutation type. CONCLUSION: The hyperhyaluronic aciduria in WS reflects the high level of serum HA. The serum and urine HA levels are useful biochemical markers for WS irrespective of the muation type of the Werner helicase gene.

Differential regulation of human RecQ family helicases in cell transformation and cell cycle.

Three human RecQ DNA helicases, WRN, BLM and RTS, are involved in the genetic disorders associated with genomic instability and a high incidence of cancer. RecQL1 and RecQL5 also belong to the human RecQ helicase family, but their correlation with genetic disorders, if any, is unknown. We report here that in human B cells transformed by Epstein-Barr virus (EBV), human fibroblasts and umbilical endothelial cells transformed by simian virus 40, the expression of WRN, BLM, RTS and RecQL1 was sharply up-regulated. In B cells this expression was stimulated within 5-40 h by the tumor promoting agent phorbol myristic acetate (PMA). Interestingly, RecQL5beta, an alternative splicing product of RecQL5 with a nuclear localization signal, is expressed in resting B cells without significant modulation of its synthesis by EBV or PMA, suggesting it has a role in resting cells. We also roughly determined the number of copies per cell for the five RecQ helicase in B cells. In addition, levels of the different RecQ helicases are modulated in different ways during the cell cycle of actively proliferating fibroblasts and umbilical endothelial cells. Our results support the view that the levels of WRN, BLM, RTS and RecQL1 are differentially up-regulated to guarantee genomic stability in cells that are transformed or actively proliferating.

Marked decrease in plasma apolipoprotein A-I and high density lipoprotein-cholesterol in a case with Werner syndrome.

The patient was a 39-year-old Japanese male with a body height of 160 cm and weight of 48 kg who was diagnosed as Werner syndrome of homozygote for mutation 4. His plasma total cholesterol (TC), triglycerides (TGs), high density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-I (apo A-I) levels were 7.2, 2.1, 1 mmol/l and 128 mg/dl, respectively. During the clinical course of treatment of this patient, his plasma levels of HDL-C and apo A-I declined drastically to levels of as low as 0.2 mmol/l and 10 mg/dl, respectively, with concurrent reciprocal increase in plasma TG levels. Plasma HDL-C, apo A-I and TG levels gradually returned to original values. Lipoprotein lipase activity and mass in post-heparin plasma were markedly low when the apo A-I and HDL-C levels decreased to 10 mg/dl and 0.21 mmol/l, respectively, and these values improved when the apo A-I and HDL-C levels returned to more normal values of 106 mg/dl and 0.94 mmol/l, respectively. The result of direct sequence of the exon 3 and 4, and the promoter region of the apo A-I gene of the patient revealed no single nucleotide changes. These results suggest that in the present patient, impaired hydrolysis of TGs in TG-rich lipoproteins, is due at least in part to a decreased LPL enzyme level, reduced the formation of nascent HDL, resulting in unusually low plasma levels of HDL-C and apo A-I.

Werner's syndrome T lymphocytes display a normal in vitro life-span.

Werner's syndrome (WS) is an autosomal recessive disorder displaying many features consistent with accelerated ageing. Fibroblasts from WS patients show a distinct mutator phenotype (characterised by the production of large chromosomal deletions) and a profound reduction in proliferative capacity. The disorder results from a mutation in a novel ReqQ helicase. Recently, we demonstrated that the proliferative defect was corrected by the ectopic expression of telomerase. From these data, we propose that mutations in the wrn gene lead to deletions at or near the telomere which reduce the cells replicative life-span. This hypothesis predicts that cell types which retain the ability to upregulate telomerase as part of their response to a proliferative stimulus would fail to show any significant effect of wrn gene mutations upon life-span. Human T lymphocytes represent a well-characterised example of such a cell type. To test the hypothesis, WS T lymphocytes were cultured until they reached replicative senescence. These cultures displayed life-spans which did not differ significantly from those of normal controls. These findings are consistent with the hypothesis that the effects of wrn mutations on replicative life-span are telomere-mediated.

Reconsideration of senescence, immortalization and telomere maintenance of Epstein-Barr virus-transformed human B-lymphoblastoid cell lines.

We review recent data on senescence and immortalization of human B-lymphoblastoid cell lines (LCLs) transformed by the Epstein-Barr virus (EBV). Although EBV-transformed LCLs are generally believed to be immortalized, a series of recent studies, including ours, provided strong evidence that they are mostly mortal and have non-malignant properties, except for a small proportion of LCLs that are immortalized by developing a strong telomerase activity. A large proportion of mortal LCLs have exceptionally long lifespans. Some of them have a lifespan over 150 population-doubling levels, keeping a relatively constant telomere length in spite of the absence of a detectable telomerase activity, suggesting that they maintain telomeres by a pathway other than that using telomerase. Here we propose a model of an alternative pathway to maintain telomeres of such long-lived mortal LCLs by exploiting extra-chromosomal telomere repeat DNA, which was recently found by us.

Increased blood plasminogen activator inhibitor-1 and intercellular adhesion molecule-1 as possible risk factors of atherosclerosis in Werner syndrome.

Werner syndrome is a rare premature aging syndrome accompanied by severe atherosclerosis. The etiology of atherosclerosis is suspected to be due to its complications, namely diabetes mellitus, hyperinsulinemia and hyperlipidemia. But from an autopsy case we found that some other risk factors may be involved in the mechanism of atherosclerosis in this syndrome. Previously we revealed that the plasminogen activator inhibitor-1 (PAI-1) gene was being overexpressed in skin fibroblasts from a patient with this syndrome. PAI-1 is a potent inhibitor of tissue plasminogen activator and a possible risk factor of atherosclerosis. This led us to assess the plasma concentration of PAI-1. Our working hypothesis was that the PAI-1 gene was upregulated or not fully suppressed in cells responsible for the production of PAI-1 in plasma as well as in fibroblasts. The results show a high concentration of plasma PAI-1. One of the well-known physiological substances that induce the PAI-1 gene is tumor necrosis factor-alpha, which also induces other possible risk factors of atherosclerosis, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1. We found the serum concentrations of ICAM-1 to be elevated in patients with this syndrome. We conclude that high concentrations of PAI-1 and ICAM-1 in blood may be one of the potent causes of severe atherosclerosis in Werner syndrome.

Effects of topoisomerase II inhibition in lymphoblasts from patients with progeroid and "chromosome instability" syndromes.

DNA topoisomerase II is involved in DNA topologic changes through the formation of a cleavable complex. This is stabilized by the antitumor drug VP16, which results in DNA breakage, aberrant recombination, and cell death. In this work, we compare the chromosomal damage induced by VP16 with that induced by bleomycin (BLM) in lymphoblasts from patients affected by the chromosome breakage syndromes ataxia telangiectasia (AT), xeroderma pigmentosum (XP), and Bloom syndrome (BS), and by the progeroid syndromes Werner (WS) and Cockayne (CS). Patients affected by AT, XP, BS, and WS have a greatly enhanced risk of developing cancer. The results show that AF and WS cells are hypersensitive to VP16, as revealed in the higher proportion of metaphases showing exchange figures and more than two breaks. All lines except AT and one CS line showed normal sensitivity to BLM. Our data on the sensitivity to VP16 of all these mutant cells underline the fact that VP16 damage is amplified only in cells that have abnormal illegitimate recombination (i.e., AT and WS).