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.


INTRODUCTION
Werner syndrome is an autosomal recessive adult-onset progeroid disorder that affects approximately 700-2,000 individuals in Japan [1][2][3][4]. Patients with Werner syndrome present with various aging phenotypes from a young age. Graying and/or loss of hair presents in their third decade of life; bilateral cataracts and diabetes, around their fourth decade of life; and atherosclerotic diseases and malignant neoplasms, around their fifth decade of life [5]. These patients also develop a high proportion of skin ulcers, often requiring amputation of the lower extremities. Although it was previously reported that patients with Werner syndrome die at 46 years of age on average [6], recent evidence indicates the average age of death is 59 years [7]. In Werner syndrome, quality of life (QOL) and activities of daily living (ADL) decline due to the various symptoms [8].
Since it is a rare disease, there is often a long period between the disease onset and the diagnosis [9]. Therefore, early detection and therapeutic intervention are important. Recently, the number of long-term survivors has increased due to therapeutic advances; however, new complications have also been observed. Although detailed and long-term involvement in medical care is essential for maintaining QOL and ADL, few reports have followed the changes over time in patients with Werner syndrome.
The Werner Syndrome Registry was established in 2017 to investigate the disease, recruit participants for clinical trials, and provide information to patients and physicians. In this report, the updated cross-sectional and longitudinal analyses of the Werner Syndrome Registry database were performed to reveal the current status and natural course in patients with Werner syndrome. AGING Regarding the blood examination, the gamma-glutamyl transpeptidase (γGTP) level was twice higher than the upper normal limit (for males 13-64, females 9-32 U/L). As for lipid profile, the mean low-density lipoprotein cholesterol (LDL-C) level was 121 ± 28 mg/dL for men and the mean triglyceride (TG) level was 161 ± 96 mg/dL, which were both slightly higher values than the normal range (for patients with diabetes, LDL-C AGING 120 mg/dL, TG 150 mg/dL). Concerning the glucose profile, the mean glycated hemoglobin (HbA1c) was 6.42 ± 1.25%, the mean fasting plasma glucose was 118 ± 29 mg/dL, and the mean postprandial plasma glucose was 142 ± 55 mg/dL, which were all values within the target range (HbA1c 6.5%, fasting plasma glucose 126 mg/dL, postprandial plasma glucose 200 mg/dL). As for renal function, while the estimated glomerular filtration rate calculated from serum creatinine (eGFRcre) was 98.3 ± 36.3 mL/min/1.73 m 2 , the body surface area (BSA)-uncorrected eGFRcre was 77.8 ± 27.0 mL/min. The GFR calculated from cystatin C (eGFRcys) was 83.2 ± 29.5 mL/min/1.73 m 2 . Dissociation was observed between eGFRcre, BSA-uncorrected eGFRcre, and eGFRcys (P = 0.0017).  The blood examination showed that glucose and lipid metabolism were controlled within the target range, and T-Bil decreased (0.55 mg/dL vs. 0.34 mg/dL, P = 0.039), while a new increase in the mean serum potassium was observed (4.33 mEq/L vs. 5.48 mEq/L, P = 0.022). Regarding therapeutic drugs, metformin tended to increase, while thiazolidine tended to decrease in association with diabetes, and the use of ARBs as antihypertensive drugs were discontinued in two patients using them. Table 4 shows the comparison between the point of registration and three years later. A significant decrease in renal function was observed over time. At the point of registration, the mean eGFRcre was 74.8 mL/min/1.73/m 2 , and the mean BSA-uncorrected eGFRcre was 59.3 mL/min, whereas three years later, the mean eGFRcre was 63.4 mL/min/1.73 m 2 (P = 0.078) and the mean BSA-uncorrected eGFRcre was 50.2 mL/min (P = 0.047), which correlated to a decrease of approximately 3 mL/min (/1.73 m 2 ) per a year on average.

Werner Syndrome Registry longitudinal analysis
The results of the survey covering the entire period regarding malignant neoplasms, renal function, and aspiration pneumonia are described below.
Regarding malignant neoplasms, the morbidity due to malignant neoplasms at baseline was 25.5% (out of 51 patients) and 31.4% in the entire survey period. Table 5 shows the type of malignant neoplasms and the age of onset. The breakdown of the malignant neoplasm types revealed epithelial neoplasms in eight patients (15.7%), non-epithelial in four patients (7.8%), and both in three patients (5.9%). Multiple neoplasms were found in 3/51 (5.9%) of all the patients enrolled, and 3/16 (18.8%) of the patients with malignant neoplasms.
Regarding renal function, Figure 1 shows the age group and the mean renal function during the entire survey period. Of the three indices of renal function, there was a discrepancy between eGFRcre and BSA-uncorrected eGFRcre/eGFRcys. The mean eGFRcre, BSA-uncorrected eGFRcre, and eGFRcys for each age decile were as follows: 30s, 129.9/108. 5

DISCUSSION
Data from the registry described in this study revealed the occurrence of changes over time for up to four years after registration in patients with Werner syndrome in Japan. Notably, a novel finding was that the index of renal function may greatly deviate from the actual state of renal function and that the rate of decline in renal function may be rapid. Although there were case reports regarding declining renal function in a patient with Werner syndrome [12][13][14][15], there was no report with a large sample size and long-term follow-up. Moreover, it was clarified that the morbidity due to malignant neoplasms is higher than that of the general population, the age of onset is younger, and both epithelial and nonepithelial neoplasms exist in similar ratios. In addition, aspiration pneumonia was observed in patients in their sixties.
The life expectancy of patients with Werner syndrome is extending [7] due to improvements in understanding the pathology of Werner syndrome and the development of medical treatments for diabetes and dyslipidemia [9]. Therefore, longer-term, more detailed follow-ups and medical interventions are needed. Atherosclerotic cardiovascular diseases (ASCVD), malignant neoplasms, sarcopenia, and leg ulcers were mentioned as the main pathological conditions affecting the prognosis of patients with Werner syndrome. The medical intervention seems to be effective for improving life quality [16][17][18]. Each important comorbidity is now discussed as follows.

Malignant neoplasms
In this study, the morbidity due to malignant neoplasms at baseline was 25.5% (13/51 patients). The morbidity due to malignant neoplasms in patients with Werner syndrome is increasing, and aging is a possible contributing factor [19]. In other words, patients with Werner syndrome are no longer dying from cardiovascular diseases at a young age; therefore, the morbidity due to malignant neoplasms may be increasing as a result.
Moreover, the ratio of epithelial neoplasms to nonepithelial neoplasms is generally approximately 10:1, while patients with Werner syndrome have very high morbidity associated with non-epithelial neoplasms [19,20]. Similarly, the ratio of epithelial neoplasms to nonepithelial neoplasms was 12:7 for the entire study period.
Although the prevalence of epithelial neoplasms has been reported to be significantly higher in patients with diabetes compared to patients without diabetes [19], Lauper et al. reported no association between diabetes and epithelial or non-epithelial neoplasms [21]. In our study, the prevalence of epithelial neoplasms in patients with diabetes was 18.9%, and that in patients without diabetes was 28.6%. Furthermore, reportedly, the age of onset for malignant neoplasm was low in patients with Werner syndrome, with the predominant onset age for malignant neoplasms being 25-65 years. In particular, the morbidity due to non-epithelial neoplasms, in other words, those of mesenchymal origin (sarcoma), is particularly high (20%) in patients with Werner syndrome before the age of 41 years [22]. In other reports, the average age of malignant neoplasm onset was 47.2 years for epithelial neoplasms, 45.2 years for non-epithelial neoplasms, and 45.8 years for all malignant neoplasms [19]. Figure 2 shows the percentage of malignant neoplasms by onset in each age group. Epithelial neoplasms developed at a younger age, and the incidence of non-epithelial neoplasms increased during the late 40s. The average age of onset was 48.4 years for epithelial neoplasms, 52.5 years for non-epithelial neoplasms, and 49.7 years for all malignant neoplasms. Therefore, the age of onset of non-epithelial neoplasms is higher than in previous reports as well. This may be due to the fact that the patients' lifespan has been prolonged by cardiovascular disease prevention and epithelial neoplasm treatment.
The table in Figure 2 shows the age-specific incidence risk of malignant neoplasms in the general population in Japan. Since the probability of malignant neoplasms in the 30s and 60s of the general population is 1.2-21.6%, the morbidity due to malignant neoplasms is higher in the same age group of patients with Werner syndrome. Malignant neoplasms onset is approximately 10 years earlier in patients with Werner syndrome than in the general population [23].
A high percentage of multiple neoplasms is also characteristic of patients with Werner syndrome. In other reports, there were multiple neoplasms in 5.3% of all patients and 15-20% of patients with malignant neoplasms [19,24]. A similar tendency was observed in this study.
Reportedly, two-thirds of neoplasms in the Werner syndrome population were thyroid neoplasms, malignant melanomas, meningiomas, soft tissue sarcomas, leukemia and preleukemic conditions, and osteosarcoma and bone neoplasms; in our study, half of the patients with malignant neoplasms had these neoplasms, and the incidence of breast and lung cancers in the epithelial neoplasm group was also high [25]. Regarding osteosarcoma, X-ray screening of long bones may be useful. Therefore, screening of these neoplasms should be prioritized.
Currently, the main cause of death in patients with Werner syndrome is malignant neoplasms development, and malignant neoplasms greatly influence prognosis; therefore, early detection and treatment by regular screening from a younger age are both very important to ameliorate their prognosis.
There are some cautions to be considered regarding cancer treatment for patients with Werner syndrome.
Reportedly, a patient with a heterozygous mutation in the WRN gene and a retroperitoneal liposarcoma had dramatic renal and hematological toxicity after cytotoxic chemotherapy [26]. For patients with a WRN mutation, close monitoring of the hematologic profile and renal function is needed to avoid severe toxicities. Furthermore, it was reported that radiotherapy is contraindicated in most homozygous patients with recessive radiosensitivity syndromes, including Werner syndrome [27]. Therefore, safer and more effective new cancer treatments for patients with Werner syndrome are needed.
Recently, the mechanism of high morbidity and treatment strategies for malignant neoplasm in patients with Werner syndrome have been getting clear. The Werner syndrome ATP-dependent helicase (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. WRN is associated with Werner syndrome and a predisposition to multiple cancers, such as multiple myeloma [28], myelodysplastic syndrome/ acute myeloid leukemia [29], colorectal cancer [30], breast cancer [31], and ovarian cancer [32]. WRN contributes to chromosomal stability for survival in both normal and cancer cells [33]. WRN is also required for DNA damage repair and the survival of cancer cells with microsatellite instability (MSI) [34]. WRN depletion induces double-strand DNA breaks and promotes apoptosis and cell cycle arrest selectively in MSI models. Loss of WRN leads to synthetic lethality in mismatch repair-deficient/high MSI cells [35].
Genome-wide screening studies have reported that WRN inhibition induces massive chromosome disruption [36] and synthetic lethality in cancer cells with high MSI [37,38]. The development possibility of novel therapeutic agents that target WRN for MSIassociated cancers by pharmacological inhibition of WRN helicase function has been reported [35,36,38]. Reportedly, exposure to small-molecule compounds, shows the percentage of total malignant neoplasms in each age group. The green bar shows the percentage of epithelial neoplasms in each age group. The red bar shows the percentage of non-epithelial neoplasms in each age group. Patients with thyroid follicular cancer, osteosarcoma, lung cancer/undifferentiated polymorphic sarcoma, and soft tissue sarcoma were excluded because the exact age of onset was unknown. such as NSC 19630 (N-(1-(4-((4-methoxyphenyl) amino) quinazolin-6-yl) ethyl)-3-(pyridin-3-yl) acrylamide) and NSC 617145 (5-(4-chlorophenyl)-4-(4methylphenyl)-4,5-dihydro-1H-imidazol-2-amine N-(4methylphenyl) sulfonylacetamide), which inhibits WRN helicase activity, sensitizes cancer cells to DNAdamaging agents [39] and DNA cross-linking agents [40]. Sublethal dosage of small-molecule compounds and the chemotherapy drug act synergistically to inhibit cell proliferation and induce DNA damage in cancer cells. These studies are still in progress and thus the results are highly awaited.

Renal function
According to the renal function index, the discrepancy was observed in patients with Werner syndrome. It may be because patients with Werner syndrome have a smaller body size and less muscle mass than those without Werner syndrome.
The calculation of eGFRcre (mL/min/1.73 m 2 ) requires the serum creatinine level, age, and sex. The calculation of BSA-uncorrected eGFRcre (mL/min) requires five items: serum creatinine level, age, sex, height, and weight. In other words, it considers the physique. The calculation of eGFRcys (mL/min/1.73 m 2 ) requires three items: serum cystatin C level, age, and sex. Therefore, it is not affected by muscle mass.
Since creatinine is easily affected by muscle mass, diet, and exercise, eGFRcre is often higher in older adults with less muscle mass, and discrepancies are likely to be observed. The situation is similar to that in patients with Werner syndrome. Therefore, in the patients with Werner syndrome, it is considered that eGFRcre dissociates from BSA-uncorrected eGFRcre and eGFRcys.
It can be observed from Figure 1 that the eGFRcys/eGFRcre ratios are <1.0. Reportedly, the presence of low eGFRcys/eGFRcre ratios (<1.0) is associated with sarcopenia [41]. In Figure 1, the eGFRcys/eGFRcre ratio is also low in non-elderly people, which may reflect that patients with Werner syndrome are small and have low muscle mass even at a young age. Therefore, there are concerns due to the dissociation of the renal function indices. Side effects may likely occur when using medicines such as antibiotics and NSAIDs for patients with poor renal function. For patients with extremely small body size and decreased renal function, the use of BSAuncorrected eGFRcre or eGFRcys is preferable to eGFRcre. It is important to carefully consider which renal function index to use before deciding the amount of medicine to be used.
The other problem is that when there is a worse eGFRcys than eGFRcre, it is associated with a higher risk of death and end-stage renal disease [42,43]. Therefore, it is necessary to carefully follow the course of a patient's renal function.
Renal function (eGFRcre, BSA-uncorrected eGFRcre) tended to decrease over three years, and BSA-uncorrected eGFRcre showed a significant decrease. The average rate of decline in BSA-uncorrected eGFRcre in the patients with Werner syndrome was 9.1 mL/min for three years, that is, approximately 3 mL/min in a year. In addition, as Figure 1 shows, the decrease in renal function is remarkable in the 30s and after the 50s. The average rate of decline in eGFR for general Japanese people aged 40 and above is 0.  [44]. Therefore, this suggests that the rate of renal function decline was faster than that of the general population of the same age.
While the management of diabetes and hypertension was appropriate, the use of analgesics such as NSAIDs was considered one of the reasons that the rate of renal function decline was rapid. Approximately 70% of patients with Werner syndrome develop painful intractable ulcers, and NSAIDs are frequently used. Although there was no relationship between leg ulcers or NSAIDs and renal function in this three-year follow-up, various factors, such as the presence of peripheral angiopathy, also affect the results; therefore, it may be necessary to pay attention to the dose of NSAIDs in patients with painful intractable ulcers. The use of NSAIDs in older adults is often noted, and similar caution is needed in patients with Werner syndrome, even the non-elderly, whose renal dysfunction cannot be predicted by eGFRcre alone. For other medicines, such as antibiotics, anti-cancer drugs, and ARBs, it is also necessary to care about drug-induced renal damage or side effects due to overdose.

Atherosclerotic cardiovascular diseases and metabolic diseases
ASCVDs, such as myocardial infarction, angina pectoris, and atherothrombotic cerebral infarction, were not common during the study period. The reduction of ASCVD appears to be extending the life expectancy of patients with Werner syndrome. This is presumed to be associated with the improved management of metabolic diseases such as diabetes, dyslipidemia, and hypertension. Regarding diabetes treatments, similar to findings in a previous report [9], pioglitazone, metformin, and insulin sensitizers were often used. While, longitudinally, the percentage of metformin use has increased more than that of pioglitazone, suggesting that the negative effects of pioglitazone on osteoporosis may be considered. Among those with dyslipidemia, statins were used in 58.8% of patients. Although the baseline data were generally within the control target range over four years, the LDL-C level decreased more from baseline, suggesting suitable interventions took place. Hypertriglyceridemia should also be noted. It was reported that a 29-year-old patient with Werner syndrome and hypertriglyceridemia (triglyceride level of 3900 mg/dL) had advanced three-vessel disease requiring coronary artery bypass graft surgery [45]. Therefore, in addition to statins, management of hypertriglyceridemia may also be needed. Regarding antihypertensive medications, the use of ARBs decreased in patients over the four years, and eventually, there were no patients using ARBs. Hyperkalemia was cited as one of the reasons for medication discontinuation, suggesting a relationship with impaired renal function. Although metabolic diseases were sufficiently controlled overall, as life expectancy prolongs, appropriate medications must be selected to address potential side effects. Moreover, patients with severe aortic stenosis [46,47] and heart failure due to impaired coronary microcirculation with no coronary artery stenosis have been reported [48]. Therefore, a comprehensive evaluation of arteriosclerosis in patients with Werner syndrome is still needed.

Sarcopenia
The sarcopenia diagnostic criteria by the Asian Working Group for Sarcopenia 2019 [49] has three indicators, of them, the following three indicators were used in this survey: 1) grip strength (male/female) <28 kg/<18 kg, 2) walking speed <1.0 m/s, and 3) SMI dualenergy X-ray absorptiometry <7.0 kg/m 2 (male) and <5.4 kg/m 2 (female), SMI bioimpedance, <7.0 kg/m 2 (male) and <5.7 kg/m 2 (female). As Table 1 shows, the mean values of grip strength and SMI in the present registry met the criteria of 1) and 3), and sarcopenia was suspected. The patients with Werner syndrome tended to have sarcopenia obesity (visceral fat increase despite low BMI). It was reported that sarcopenia tended to appear before visceral fat accumulation [50]. Moreover, over the years, body weight, BMI, and grip strength tended to decrease in this study. The adipogenic and chondrogenic differentiation capacity was significantly decreased in the foot fibroblasts of patients when Werner syndrome in vitro. It may partially explain the underlying mechanism of sarcopenia in patients with Werner syndrome [51]. The presence of sarcopenia may be related not only to QOL reduction but also to renal function evaluation, as aforementioned, suggesting that sarcopenia prevention is needed. Measures such as leucine intake [52,53] and strength training [54,55], which have general preventive effects on sarcopenia, should be considered. Ghrelin receptor agonist has been shown to increase body weight, muscle mass, and appetite in patients with cancer cachexia [56,57]. These findings suggest that ghrelin receptor agonist also may be effective in patients with Werner syndrome, in whom skeletal muscle mass is significantly reduced.

Other complications associated with aging
As lifespans prolong, complications such as aspiration pneumonia, which are often seen in late older adults, are increasing. Two patients with aspiration pneumonia were reported in this survey. They were aged in their 60s and were thus younger than the average age group that generally experienced aspiration pneumonia. Vaccinations to prevent pneumonia in those aged in their early 60s and oral care may be useful preventive measures. Further, the use of benzodiazepine should be avoided.
This study is limited because the evaluated registry does not cover all patients with Werner syndrome in Japan. However, almost half of the patients confirmed in a previous national survey registered; therefore, the clinical evidence was established based on a highly universal (and the largest) database of Werner syndrome in Japan.
Although the overall prognosis of patients with Werner syndrome is improving, new opportunities for early diagnosis and treatment intervention are still needed. It has been suggested that the inclusion of the WRN gene in genetic analyses for early-onset diabetes, lipodystrophy, or dyslipidemia may offer an opportunity to diagnose patients with Werner syndrome long before the presentation of the full spectrum of symptoms and complications, enabling earlier interventions, including malignant neoplasm screenings and prevention of diseases listed in the clinical criteria [58]. Recently, novel senescent markers, ATP6V0D1 and RTN4, were shown to be increased in cells derived from patients with Werner syndrome, raising a possibility that they may serve as markers of disease progression [59]. Similarly, ribonuclease H2 subunit A (RNaseH2A) was shown to be downregulated in Werner syndrome cells as well as selected cancer cells [60]. Therefore, it may also be used as a diagnosis and severity marker. It was reported that patients with Werner syndrome have thinning of the retinal nerve fiber layer, ganglion cell complex, and choroidal thickness and the loss of visual field [61]. It may be useful for early diagnosis to have retinal and choroidal check-ups with the optical coherence tomography images when patients present with juvenile cataracts of unknown cause. For early detection of complications, genetic testing for complications, such as malignant neoplasms, may also be useful, when Werner syndrome is diagnosed. Early detection makes it possible to reduce risk factors such as smoking and to do early prevention and treatment.
Regarding renal protection, renin-angiotensin system inhibitors are hard to use, since many patients stopped their usage due to hyperkalemia. Additionally, sodiumglucose cotransporter 2 inhibitors are also difficult to use due to concern of sarcopenia in patients with Werner syndrome. However, a mineralocorticoid receptor antagonist, finerenone, may be a good candidate, since it is less likely to cause hyperkalemia.
Regarding intractable skin ulcers, treatment with a functional peptide SR-0379 was found to be safe, welltolerated, and effective for leg ulcers in patients with Werner syndrome in clinical trials [62,63].
Reportedly, p38 inhibitors reduced the accelerated cell senescence in primary fibroblasts from patients with Werner syndrome [64]. It is believed that in the near future, p38 inhibitors may be used in in vivo studies. Nicotinamide adenine dinucleotide (NAD) is important for the activation of sirtuin, a type of protein involved in regulating cellular processes including the aging and death of cells. NAD levels decrease in patients with Werner syndrome [65], therefore supplementation with NAD precursors may be effective. A clinical trial with nicotinamide riboside, a precursor of NAD, which may be effective for intractable ulcers, sarcopenia, and ASCVD, is investigating the safety and efficacy in patients with Werner syndrome [66]. Using state-of-theart technologies, such as genome, transcriptome, and epigenome analyses [67], study of the generation of iPS cells from patients with Werner syndrome and the correction of the WRN gene by the CRISPR/Cas9mediated methods [68] will create new treatments for patients with Werner syndrome.
In conclusion, this study clarified secular changes in multiple patients with Werner syndrome. The morbidity due to malignant neoplasms is high even in those of young age; therefore, it is necessary to carry out active and detailed screening examinations for malignant neoplasms. Renal function declines rapidly; therefore, evaluation based on BSA-uncorrected eGFRcre or eGFRcys and attention to the type and amount of drugs used are needed.

Werner Syndrome Registry (patient registration system)
To reveal the disease profile and prognosis and to seek suitable medical intervention methods, the Werner Syndrome Registry was established. Based on the results of a nationwide primary survey, the patients were recruited to the registry from facilities with patients definitively diagnosed with Werner syndrome according to the diagnostic criteria [20].
The study complied with the ethical rules for human experimentation as specified in the Declaration of Helsinki. The study received approval from the Ethics Board of Chiba University on 27th July 2016 (approval number: 278) and from the Ethics Board of Kyoto University on 29th January 2020 (approval number: R2370). The study was registered at the UMIN Clinical Trial Registry (https://upload.umin.ac.jp/cgi-open-bin/ctr _e/ctr_view.cgi?recptno=R000034058) on 3rd November 2017 (ID: UMIN000029812). Written informed consent was obtained from patients before registration.
The key inclusion criteria for the registry were as follows: 1) patients with confirmed Werner syndrome based on the diagnostic criteria [20], and 2) patients who provided written informed consent prior to their participation in the registry. There were no exclusion criteria. The registry data included patient background (age at the time of onset, diagnosis, and registration; height, weight, BMI, abdominal circumference, and presence of consanguineous marriage), characteristic major signs and clinical symptoms, comorbidities (diabetes mellitus, impaired glucose tolerance, dyslipidemia, hypertension, fatty liver, cerebrovascular diseases, cardiovascular diseases, peripheral artery disease, foot amputation, and malignant neoplasms), the pattern of gene mutation, blood examination (blood cell count, biochemistry, liver and renal functions, and glucose and lipid profiles), visceral fat area, SMI, physical function (grip strength and walking speed), and treatment content. Renal function was evaluated using the following three indices: eGFRcre, BSA-uncorrected eGFRcre, and eGFRcys.
The calculation formula of each eGFR index was as follows:  The datasheets were collected from the facilities annually. The collected data were subsequently inputted into the registration system Fountayn (previously named DATATRACK ONE) (NTT DATA, Tokyo, Japan) [9].

Cross-sectional analysis and longitudinal analysis
The data were extracted on May 12, 2022. Data obtained at the registration time of each patient were analyzed through a cross-sectional analysis study model. For longitudinal analysis of the data in the Werner Syndrome Registry, the data at the time of initial registration and after one, two, and three years were compared in the patients with such data. For continuous variables, the paired t-test was used for items with a normal distribution, and the Wilcoxon signed-rank test was used for items with a non-normal distribution. For binary variables, Pearson's chi-squared test was used for items with 20 patients and more, and Fisher's exact test was used for items with less than 20 patients included. All data were analyzed with JMP pro 15 (SAS Institute, Cary, NC, USA). A p-value less than 0.05 was considered statistically significant.
The prevalence of malignant neoplasms and aspiration pneumonia was also investigated for the entire period from registration to three years later. Regarding malignant neoplasms, the presence or absence of malignant neoplasms, categorization of epithelial or non-epithelial neoplasms, and type of neoplasms were investigated.
Moreover, the association between renal function and leg ulcers/NSAIDs was also investigated. The relationship between renal function (eGFRcre, BSAuncorrected eGFRcre, and eGFRcys) and both the presence or absence of leg ulcers and the use of NSAIDs were investigated using the baseline data.