Sex differences in symptom presentation and their impact on diagnostic accuracy in Werner syndrome.

AIM: Whether sex differences exist in hereditary progeroid syndromes remains unclear. In this study, we investigated sex differences in patients with Werner syndrome (WS), a model of human aging, using patient data at the time of diagnosis. METHODS: The presence of six cardinal signs in the diagnostic criteria was retrospectively evaluated. RESULTS: We found that the percentage of patients with all cardinal signs was higher in males than in females (54.2% vs. 21.2%). By the age of 40 years, 57.1% of male patients with WS presented with all the cardinal signs, whereas none of the female patients developed all of them. In particular, the frequency of having a high-pitched, hoarse voice, a characteristic of WS, was lower in female patients. The positive and negative predictive values for clinical diagnosis were 100% for males and females, indicating the helpfulness of diagnostic criteria regardless of sex. More female patients than male (86.7% vs. 64%) required genetic testing for their diagnosis because their clinical symptoms were insufficient, suggesting the importance of genetic testing for females even if they do not show typical symptoms of WS. Finally, the frequency of abnormal voice was lower in patients with WS harboring the c.3139-1G > C homozygous mutation. CONCLUSION: These results indicate, for the first time, that there are sex differences in the phenotypes of hereditary progeroid syndromes. The analysis of this mechanism in this human model of aging may lead to the elucidation of sex differences in the various symptoms of normal human aging. Geriatr Gerontol Int 2024; 24: 161-167.

Senescence-associated inflammation and inhibition of adipogenesis in subcutaneous fat in Werner syndrome.

Werner syndrome (WS) is a hereditary premature aging disorder characterized by visceral fat accumulation and subcutaneous lipoatrophy, resulting in severe insulin resistance. However, its underlying mechanism remains unclear. In this study, we show that senescence-associated inflammation and suppressed adipogenesis play a role in subcutaneous adipose tissue reduction and dysfunction in WS. Clinical data from four Japanese patients with WS revealed significant associations between the decrease of areas of subcutaneous fat and increased insulin resistance measured by the glucose clamp. Adipose-derived stem cells from the stromal vascular fraction derived from WS subcutaneous adipose tissues (WSVF) showed early replicative senescence and a significant increase in the expression of senescence-associated secretory phenotype (SASP) markers. Additionally, adipogenesis and insulin signaling were suppressed in WSVF, and the expression of adipogenesis suppressor genes and SASP-related genes was increased. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), alleviated premature cellular senescence, rescued the decrease in insulin signaling, and extended the lifespan of WS model of C. elegans. To the best of our knowledge, this study is the first to reveal the critical role of cellular senescence in subcutaneous lipoatrophy and severe insulin resistance in WS, highlighting the therapeutic potential of rapamycin for this disease.

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.

Generation of disease-specific and CRISPR/Cas9-mediated gene-corrected iPS cells from a patient with adult progeria Werner syndrome.

Adult progeria Werner syndrome (WS), a rare autosomal recessive disorder, is characterized by accelerated aging symptoms after puberty. The causative gene, WRN, is a member of the RecQ DNA helicase family and is predominantly involved in DNA replication, repair, and telomere maintenance. Here, we report the generation of iPS cells from a patient with WS and correction of the WRN gene by the CRISPR/Cas9-mediated method. These iPSC lines would be a valuable resource for deciphering the pathogenesis of WS.

Fibroblasts from different body parts exhibit distinct phenotypes in adult progeria Werner syndrome.

Werner syndrome (WS), also known as adult progeria, is characterized by accelerated aging symptoms from a young age. Patients with WS experience painful intractable skin ulcers with calcifications in their extremities, subcutaneous lipoatrophy, and sarcopenia. However, there is no significant abnormality in the trunk skin, where the subcutaneous fat relatively accumulates. The cause of such differences between the limbs and trunk is unknown. To investigate the underlying mechanism behind these phenomena, we established and analyzed dermal fibroblasts from the foot and trunk of two WS patients. As a result, WS foot-derived fibroblasts showed decreased proliferative potential compared to that from the trunk, which correlated with the telomere shortening. Transcriptome analysis showed increased expression of genes involved in osteogenesis in the foot fibroblasts, while adipogenic and chondrogenic genes were downregulated in comparison with the trunk. Consistent with these findings, the adipogenic and chondrogenic differentiation capacity was significantly decreased in the foot fibroblasts in vitro. On the other hand, the osteogenic potential was mutually maintained and comparable in the foot and trunk fibroblasts. These distinct phenotypes in the foot and trunk fibroblasts are consistent with the clinical symptoms of WS and may partially explain the underlying mechanism of this disease phenotype.

A novel podocyte protein, R3h domain containing-like, inhibits TGF-ß-induced p38 MAPK and regulates the structure of podocytes and glomerular basement membrane.

Not only in kidney glomerular physiological function but also glomerular pathology especially in diabetic condition, glomerular podocytes play pivotal roles. Therefore, it is important to increase our knowledge about the genes and proteins expressed in podocytes. Recently, we have identified a novel podocyte-expressed gene, R3h domain containing-like (R3hdml) and analyzed its function in vivo as well as in vitro. Transforming growth factor-ß (TGF-ß) signaling regulated the expression of R3hdml. And R3hdml inhibited p38 mitogen-activated protein kinase phosphorylation, which was induced by TGF-ß, leading to the amelioration of podocyte apoptosis. Furthermore, a lack of R3hdml in mice significantly worsened glomerular function in streptozotocin (STZ)-induced diabetes, while overexpression of R3hdml ameliorated albuminuria in STZ-induced diabetes. Our results surmise that the functional analyses of R3hdml may lead to the development of novel therapeutic strategies for diabetic nephropathy in the future. KEY MESSAGES: • A novel podocyte expressed protein R3h domain containing-like was identified. • R3HDML inhibits podocyte apoptosis by inhibiting TGF-ß-mediated p38 MAPK signaling. • Overexpression of R3HDML ameliorates albuminuria in STZ-induced diabetes mice. • R3HDML may prove to be a novel therapeutic strategy for diabetic nephropathy.

R3hdml regulates satellite cell proliferation and differentiation.

In this study, we identified a previously uncharacterized skeletal satellite cell-secreted protein, R3h domain containing-like (R3hdml). Expression of R3hdml increases during skeletal muscle development and differentiation in mice. Body weight and skeletal muscle mass of R3hdml knockout (KO) mice are lower compared to control mice. Expression levels of cell cycle-related markers, phosphorylation of Akt, and expression of insulin-like growth factor within the skeletal muscle are reduced in R3hdml KO mice compared to control mice. Expression of R3hdml increases during muscle regeneration in response to cardiotoxin (CTX)-induced muscle injury. Recovery of handgrip strength after CTX injection was significantly impaired in R3hdml KO mice, which is rescued by R3hdml. Our results indicate that R3hdml is required for skeletal muscle development, regeneration, and, in particular, satellite cell proliferation and differentiation.

Recent Trends in WRN Gene Mutation Patterns in Individuals with Werner Syndrome.

OBJECTIVES: To determine recent trends in mutation patterns in the WRN gene, which cause Werner syndrome (WS), a rare, inheritable progeroid syndrome in Japan. DESIGN: Retrospective cohort. SETTING: Longitudinal survey of WS and literature search for case reports. PARTICIPANTS: Individuals whose genetic testing their facilities had requested between 2009 and October 2016 (N = 67). MEASUREMENTS: A nationwide epidemiological study was conducted from 2009 to 2011 to improve understanding of the pathology of WS and develop therapeutic guidelines. Since 2009, Chiba University Hospital consecutively evaluated the WRN gene in 67 individuals throughout Japan who had requested genetic testing. A literature search was also conducted for case reports on Japanese WS reported since 1997. RESULTS: A definitive diagnosis of WS was confirmed genetically in 50 of 67 participants. Through the literature search, 16 individuals diagnosed genetically with WS were identified. Of these 66 individuals with WS, 42 were homozygous for a WRN mutation, and 21 were compound heterozygotes. One novel mutant allele was identified in an individual with the compound heterozygous genotype. The proportion of compound heterozygotes (31.8%) was significantly greater than reported previously (14.2%), indicating that the incidence of consanguineous marriage of parents has decreased. CONCLUSION: The increased frequency of individuals with WS with the compound heterozygous genotype is a recent trend in Japan. A long-term follow-up study on WRN homozygotes and compound heterozygotes will allow the relationship between WRN genotype and clinical severity of WS to be evaluated in the future.

A case of Werner syndrome without metabolic abnormality: implications for the early pathophysiology.

Werner syndrome (WS) is an autosomal recessive progeroid disorder caused by mutations in the WRN DNA helicase. It is characterized by the graying and loss of hair, juvenile cataracts, sclerosis and ulceration of skin, insulin-resistant diabetes mellitus, dyslipidemia, abdominal adiposity, osteoporosis, atherosclerosis, and malignant neoplasm. Patients are usually diagnosed in their 30s or 40s, but the early pathophysiology of the syndrome is still not fully understood. Here we report a 29-year-old female patient who displayed cataracts, hair graying, and tendinous calcinosis. Her parents were first cousins. Interestingly, the patient lacked the metabolic signs typical for WS, including glucose intolerance, dyslipidemia, and visceral fat accumulation. A hyperinsulinemic response at 30 min was observed in an oral glucose tolerance test. Mutational analysis for the WRN gene revealed a homozygous nucleotide substitution 3190C>T in exon 24, resulting in a protein product with replacement of an arginine residue at position 573 by termination codon (Arg987Ter). The mutated WRN protein was unable to translocate into the nucleus in an in vitro cell assay. A WS patient with an Arg987Ter mutation has been previously reported in Switzerland, the present case is the first to be identified in Asia. This case demonstrates the early clinical features of WS and suggests that metabolic abnormality, including insulin resistance, is not an essential component of WS at disease onset. Moreover, a follow-up study of such case would be useful to understand how the various clinical symptoms in WS develop and progress over the years.