Disease modeling of ADAMTS9-related nephropathy using kidney organoids reveals its roles in tubular cells and podocytes.

Introduction: Mutations in ADAMTS9 cause nephronophthisis-related ciliopathies (NPHP-RC), which are characterized by multiple developmental defects and kidney diseases. Patients with NPHP-RC usually have normal glomeruli and negligible or no proteinuria. Herein, we identified novel compound-heterozygous ADAMTS9 variants in two siblings with NPHP-RC who had glomerular manifestations, including proteinuria. Methods: To investigate whether ADAMTS9 dysfunction causes NPHP and glomerulopathy, we differentiated ADAMTS9 knockout human induced pluripotent stem cells (hiPSCs) into kidney organoids. Single-cell RNA sequencing was utilized to elucidate the gene expression profiles from the ADAMTS9 knockout kidney organoids. Results: ADAMTS9 knockout had no effect on nephron differentiation; however, it reduced the number of primary cilia, thereby recapitulating renal ciliopathy. Single-cell transcriptomics revealed that podocyte clusters express the highest levels of ADAMTS9, followed by the proximal tubules. Loss of ADAMTS9 increased the activity of multiple signaling pathways, including the Wnt/PCP signaling pathway, in podocyte clusters. Conclusions: Mutations in ADMATS9 cause a glomerulotubular nephropathy in kidney and our study provides insights into the functional roles of ADMATS9 in glomeruli and tubules.

Clinical implication by differential analytical performances of serum free light chain quantitation analysis using fully automated analyzers.

OBJECTIVES: Free light chain (FLC) is used for the diagnosis and prediction with regard to the progression risk of plasma cell disorders and Freelite reagent using the SPAplus analyzer (The Binding Site) has been one of the widely used option. However, N Latex FLC reagent with the Atellica CH 930 analyzer (Siemens Healthineers) has shown the advantages of automation and high throughput. We aimed to evaluated clinical implication by differential analytical performances of two assays. METHODS: A total of 322 serum samples were collected from 193 patients requested for FLC analysis including 131 multiple myeloma patients. The precision, linearity, dilution recovery of N Latex FLC assay was evaluated. We compared the two assays and analyzed the monomer-dimer pattern for discrepant results. RESULTS: The precision, linearity, and dilution recovery performance was appropriate for the routine use in clinical laboratories. Despite the good correlation within normal range, proportional bias up-to 170% was observed in samples with high concentrations especially for lambda. The higher value samples with N Latex FLC assay contained more monomer forms than controls. All opposite changes of FLC burden by the N Latex FLC assay proved to present concordant dynamic changes when assessed by serum protein electrophoresis. CONCLUSIONS: Clinical laboratories should be aware of the inter-assay variability of FLC quantitative measurements using different platforms, especially for high concentrations of both kappa and lambda measurements, possibly due to monomer/dimer ratio diversity. Clinical interpretations for multiple myeloma disease status might not be dramatically affected only when the same assay is utilized during follow-up periods.

Associations of LDL Cholesterol, Non-HDL Cholesterol, and Apolipoprotein B With Cardiovascular Disease Occurrence in Adults: Korean Genome and Epidemiology Study.

Background: Despite the superiority of non-HDL cholesterol (non-HDL-C) and apolipoprotein B (ApoB) as lipid markers for atherosclerotic cardiovascular disease (ASCVD), these are only suitable as secondary markers. We compared LDL cholesterol (LDL-C), non-HDL-C, and ApoB concentrations with respect to the occurrence of cardiovascular disease in adults enrolled in the Korean Genome and Epidemiology Study (KoGES). Methods: We used information on age; sex; medical history; family history of ASCVD; current lipid-lowering therapy; current smoking status; and creatinine, total cholesterol, HDL-C, LDL-C, triglyceride, and ApoB concentrations from 5,872 KoGES participants without ASCVD. New ASCVD development was monitored during the 8-year follow-up period. Adjusted hazard ratios (aHRs) for ASCVD of LDL-C, non-HDL-C, and ApoB concentrations were calculated based on the multivariate Cox regression analyses. The participants were also grouped as low and high according to the median values for each lipid marker, and calculated aHRs of each group combined by two lipid makers. Results: ApoB showed the highest aHR per 1-SD for ASCVD (1.26; 95% confidence interval [CI], 1.11-1.43), followed by non-HDL-C (1.25; 95% CI, 1.11-1.41) and LDL-C (1.20; 95% CI, 1.06-1.37). The group with low LDL-C and high ApoB concentrations had a significantly higher aHR for ASCVD (1.61; 95% CI, 1.05-2.48) compared to the reference group values (low LDL-C and low ApoB concentrations). The aHR for the group with high LDL-C and low ApoB concentrations was not significant (1.30; 95% CI, 0.79-2.16). Conclusions: ApoB, non-HDL-C, and LDL-C are independent risk factors for ASCVD. Increases in the aHR per 1-SD for ASCVD were more strongly affected by ApoB, followed by non-HDL-C and LDL-C. Participants with low LDL-C and high ApoB concentrations showed increased ASCVD risk. For individuals with ASCVD risk factors, even those presenting normal LDL-C concentrations, measuring ApoB concentrations can provide useful information for better evaluation of ASCVD risk.

Performance Evaluation of the DxC 700 AU Chemistry Analyzer in Hemoglobin A1c Measurement

Background: Accurate measurement of glycated hemoglobin (HbA1c) is crucial for a diabetes diagnosis and subsequent patient management. The detection method and presence of variant Hb can interfere with HbA1c measurements. We evaluated the HbA1c-measuring performance of the DxC 700 AU (Beckman Coulter, Brea, CA, USA) immunoassay-based device in comparison with another immunoassay device and the reference method. Methods: A total of 120 normal and 14 variant Hb samples were analyzed using the Cobas c 513 (Roche Diagnostics, Mannheim, Germany) and DxC 700 AU analyzers. Variant Hb samples were also analyzed using the reference method, along with 20 normal samples. The accuracy, precision, linearity, and carryover were determined. Results: DxC 700 AU results strongly correlated with those of Cobas c 513 and exhibited accuracy in comparison with the reference method. The within-run, between-run, between-day, and total imprecision (%CV) values for the low- and high-concentration control materials were below 2%. The results of DxC 700 AU were linear over a wide HbA1c range (3.39%-18.30%). Although DxC 700 AU performed well in the presence of variant Hb, the HbA1c concentration was underestimated in the presence of fetal Hb. The possibility of interference from a high HbH proportion could not be ruled out. Conclusions: The overall analytical performance of DxC 700 AU was acceptable. The device is accurate, precise, and linear over a wide HbA1c concentration range. Although DxC 700 AU results highly correlated with those of Cobas c 513, caution should be exercised in cases of high HbF and HbH concentrations.

The role of Jagged1 as a dynamic switch of cancer cell plasticity in PDAC assembloids.

Background: Pancreatic ductal adenocarcinoma (PDAC), which commonly relapses due to chemotherapy resistance, has a poor 5-year survival rate (< 10%). The ability of PDAC to dynamically switch between cancer-initiating cell (CIC) and non-CIC states, which is influenced by both internal and external events, has been suggested as a reason for the low drug efficacy. However, cancer cell plasticity using patient-derived PDAC organoids remains poorly understood. Methods: First, we successfully differentiated CICs, which were the main components of PDAC organoids, toward epithelial ductal carcinomas. We further established PDAC assembloids of organoid-derived differentiated ductal cancer cells with endothelial cells (ECs) and autologous immune cells. To investigate the mechanism for PDAC plasticity, we performed single-cell RNA sequencing analysis after culturing the assembloids for 7 days. Results: In the PDAC assembloids, the ECs and immune cells acted as tumor-supporting cells and induced plasticity in the differentiated ductal carcinomas. We also observed that the transcriptome dynamics during PDAC re-programming were related to the WNT/beta-catenin pathway and apoptotic process. Interestingly, we found that WNT5B in the ECs was highly expressed by trans interaction with a JAG1. Furthermore, JAG1 was highly expressed on PDAC during differentiation, and NOTCH1/NOTCH2 were expressed on the ECs at the same time. The WNT5B expression level correlated positively with those of JAG1, NOTCH1, and NOTCH2, and high JAG1 expression correlated with poor survival. Additionally, we experimentally demonstrated that neutralizing JAG1 inhibited cancer cell plasticity. Conclusions: Our results indicate that JAG1 on PDAC plays a critical role in cancer cell plasticity and maintenance of tumor heterogeneity.

Comparison of blood collection tubes for 29 biochemical analytes in pediatric patients with central venous catheters.

BACKGROUND: Pediatric cancer patients undergoing chemotherapy or radiation therapy generally require a central venous catheter (CVC). However, serum drawn from CVCs has several drawbacks for use in routine chemistry tests. Biochemical analytes were evaluated using heparin plasma instead of serum to maintain turnaround time and to prevent problems caused by micro-clot formation or delayed clotting time. METHODS: Venous blood samples from 52 pediatric oncology patients with chemoports or Hickman catheters were collected in serum separating tubes (SSTs) and lithium heparin tubes (LHTs). A total of 29 parameters were analyzed on a Cobas c702 (Roche Diagnostics, Mannheim, Germany). Passing-Bablok regression and Bland-Altman difference plots were used for statistical analyses. RESULTS: When the mean value of each analyte measured from LHT was compared with those from SST, percentage bias was within the desirable bias limit in most of the analytes. However, albumin, potassium, and inorganic phosphorus showed a negative constant bias of -3.0%, -5.3%, and -1.6%, respectively, and total protein showed a positive constant bias of + 3.8%. CONCLUSIONS: The use of LHTs for sample collection from pediatric patients with CVCs could be helpful for routine chemistry analyses. The results of potassium and total protein should be interpreted with consideration of the difference between serum and plasma samples.

OSBPL2 mutations impair autophagy and lead to hearing loss, potentially remedied by rapamycin.

Intracellular accumulation of mutant proteins causes proteinopathies, which lack targeted therapies. Autosomal dominant hearing loss (DFNA67) is caused by frameshift mutations in OSBPL2. Here, we show that DFNA67 is a toxic proteinopathy. Mutant OSBPL2 accumulated intracellularly and bound to macroautophagy/autophagy proteins. Consequently, its accumulation led to defective endolysosomal homeostasis and impaired autophagy. Transgenic mice expressing mutant OSBPL2 exhibited hearing loss, but osbpl2 knockout mice or transgenic mice expressing wild-type OSBPL2 did not. Rapamycin decreased the accumulation of mutant OSBPL2 and partially rescued hearing loss in mice. Rapamycin also partially improved hearing loss and tinnitus in individuals with DFNA67. Our findings indicate that dysfunctional autophagy is caused by mutant proteins in DFNA67; hence, we recommend rapamycin for DFNA67 treatment.Abbreviations: ABR: auditory brainstem response; ACTB: actin beta; CTSD: cathepsin D; dB: decibel; DFNA67: deafness non-syndromic autosomal dominant 67; DPOAE: distortion product otoacoustic emission; fs: frameshift; GFP: green fluorescent protein; HsQ53R-TG: human p.Q53Rfs*100-transgenic: HEK 293: human embryonic kidney 293; HFD: high-fat diet; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NSHL: non-syndromic hearing loss; OHC: outer hair cells; OSBPL2: oxysterol binding protein-like 2; SEM: scanning electron microscopy; SGN: spiral ganglion neuron; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: transgenic; WES: whole-exome sequencing; YUHL: Yonsei University Hearing Loss; WT: wild-type.

In vivo outer hair cell gene editing ameliorates progressive hearing loss in dominant-negative Kcnq4 murine model.

Outer hair cell (OHC) degeneration is a major cause of progressive hearing loss and presbycusis. Despite the high prevalence of these disorders, targeted therapy is currently not available. Methods: We generated a mouse model harboring Kcnq4W276S/+ to recapitulate DFNA2, a common genetic form of progressive hearing loss accompanied by OHC degeneration. After comprehensive optimization of guide RNAs, Cas9s, vehicles, and delivery routes, we applied in vivo gene editing strategy to disrupt the dominant-negative allele in Kcnq4 and prevent progressive hearing loss. Results:In vivo gene editing using a dual adeno-associated virus package targeting OHCs significantly improved auditory thresholds in auditory brainstem response and distortion-product otoacoustic emission. In addition, we developed a new live-cell imaging technique using thallium ions to investigate the membrane potential of OHCs and successfully demonstrated that mutant allele disruption resulted in more hyperpolarized OHCs, indicating elevated KCNQ4 channel activity. Conclusion: These findings can facilitate the development of targeted therapies for DFNA2 and support the use of CRISPR-based gene therapy to rectify defects in OHCs.

Differential genetic diagnoses of adult post-lingual hearing loss according to the audiogram pattern and novel candidate gene evaluation.

Ski-slope hearing loss (HL), which refers to increased auditory threshold at high frequencies, is common in adults. However, genetic contributions to this post-lingual HL remain largely unknown. Here, we prospectively investigated deafness-associated and novel candidate genes causing ski-slope HL. We analyzed 192 families with post-lingual HL via gene panel and/or exome sequencing. With an overall molecular diagnostic rate of 35.4% (68/192) in post-lingual HL, ski-slope HL showed a lower diagnostic rate (30.7%) compared with other conditions (40.7%). In patients who showed HL onset before the age of 40, genetic diagnostic probability was significantly lower for ski-slope HL than for other conditions. Further analysis of 51 genetically undiagnosed patients in the ski-slope HL group identified three variants in delta-like ligand 1 (DLL1), a Notch ligand, which presented in vitro gain-of-function effects on Notch downstream signaling. In conclusion, genetic diagnostic rates in post-lingual HL varied according to audiogram patterns with age-of-onset as a confounding factor. DLL1 was identified as a candidate gene causing ski-slope HL.

COCH-related autosomal dominant nonsyndromic hearing loss: a phenotype-genotype study.

This phenotype-genotype study aimed to investigate the extent of audioprofile variability related to cochlin major domains and to identify potential ethnic-specific differences associated with COCH-related hearing loss. Eight Korean families (26 cases) were diagnosed with COCH-related hearing loss by exome sequencing. Audiometric test results were combined with those from nine published East Asian families (20 cases) and compared with those from 38 European-descent families (277 cases). Audioprofiles were created by grouping audiometric test results into age ranges by age at testing and then averaging hearing loss thresholds by frequency within age ranges. The functional impact of the identified variants was assessed in vitro by examining the intracellular trafficking, secretion, and cleavage of cochlin. In both East Asian and European-descent families segregating COCH-related hearing loss, deafness-associated variants in non-LCCL domains of cochlin were associated with hearing loss that was more severe earlier in life than hearing loss caused by variants in the LCCL domain. Consistent with this phenotypic difference, functional studies demonstrated distinct pathogenic mechanisms for COCH variants in a domain-dependent manner; specifically, a cytotoxic effect was observed for the p.Phe230Leu variant, which is located in the vWFA1 domain. No ethnic-specific differences in hearing loss progression were observed, except for those attributable to an overrepresentation of presymptomatic cases in the European-descent cohort.

Association of circulating metabolites with incident type 2 diabetes in an obese population from a national cohort.

AIMS: Obesity is the most common risk factor for type 2 diabetes. However, not all obese individuals develop diabetes. In the era of precision medicine, metabolomics may reveal the fundamental metabolic status of an individual. Our aim was to assess the association of metabolites with incident type 2 diabetes in obese individuals using Korean Genome and Epidemiology Cohort Study. METHODS: Using 12 years of metabolomic data from 2,580 individuals, we performed a metabolomic study to define metabolically healthy obesity in an obese population (n = 704) with incident type 2 diabetes. Cox proportional hazards regression model and survival analysis were performed adjusted for the traditional risk factors of type 2 diabetes. RESULTS: Our study revealed that spermine, acyl-alkyl phosphatidylcholines (C34:3, C36:3, C42:1), hydroxy sphingomyelin (C22:2, C14:1), and sphingomyelin (C16:0) were associated with incident type 2 diabetes in obese individuals after the adjustment for risk factors and correction of multiple comparisons by Bonferroni method. Five metabolites (except hydroxy sphingomyelin C14:1 and sphingomyelin C16:0) were also significantly associated with incident type 2 diabetes in lean individuals. CONCLUSIONS: This study highlights the need for defining metabolically healthy obesity based on serum metabolites and elucidates potential biomarkers for type 2 diabetes in an obese population.

Association of vancomycin trough concentration on the treatment outcome of patients with bacteremia caused by Enterococcus species.

BACKGROUND: Pharmacokinetic-pharmacodynamic (PK/PD) targets of vancomycin therapy have been recognized for methicillin-resistant Staphylococcus aureus infections but not for other gram-positive bacterial infections. Therefore, we investigated whether vancomycin concentration targets such as the trough level and ratio of the area under the curve to minimum inhibitory concentration (AUC/MIC) are associated with the treatment outcome in enterococcal bacteremia. METHODS: A retrospective cohort analysis enrolled patients with bacteremia caused by vancomycin-susceptible Enterococcus faecium and Enterococcus faecalis who were treated with vancomycin from January 2007 to December 2017 at a tertiary hospital located in Seoul, South Korea. Patients without vancomycin concentrations were excluded from the study. The primary outcome was 28-day all-cause mortality. RESULTS: A total of 37 patients were enrolled-26 with E. faecium infection and 11 with E. faecalis infection. The 28-day all-cause mortality rate was 21.6 %. In univariate analysis, vancomycin trough level (≤ 15 µg/mL; p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality but not AUC24/MIC (> 389; p = 0.479). In multivariate analysis, vancomycin trough concentration (≤ 15 µg/mL; p = 0.041) and younger age (p = 0.031) were associated with 28-day mortality in patients with enterococcal bacteremia. CONCLUSIONS: In this study, a vancomycin trough level of 15 µg/mL or lower was associated with 28-day mortality in enterococcal bacteremia. However, relatively large prospective studies are needed to examine the efficacy of vancomycin PK/PD parameters in patients with enterococcal bacteremia.

Activation of KCNQ4 as a Therapeutic Strategy to Treat Hearing Loss.

Potassium voltage-gated channel subfamily q member 4 (KCNQ4) is a voltage-gated potassium channel that plays essential roles in maintaining ion homeostasis and regulating hair cell membrane potential. Reduction of the activity of the KCNQ4 channel owing to genetic mutations is responsible for nonsyndromic hearing loss, a typically late-onset, initially high-frequency loss progressing over time. In addition, variants of KCNQ4 have also been associated with noise-induced hearing loss and age-related hearing loss. Therefore, the discovery of small compounds activating or potentiating KCNQ4 is an important strategy for the curative treatment of hearing loss. In this review, we updated the current concept of the physiological role of KCNQ4 in the inner ear and the pathologic mechanism underlying the role of KCNQ4 variants with regard to hearing loss. Finally, we focused on currently developed KCNQ4 activators and their pros and cons, paving the way for the future development of specific KCNQ4 activators as a remedy for hearing loss.

Heterogeneity of MYO15A variants significantly determine the feasibility of acoustic stimulation with hearing aid and cochlear implant.

Autosomal recessive nonsyndromic hearing loss 3 (DFNB3) mainly leads to congenital and severe-to-profound hearing impairment, which is caused by variants in MYO15A. However, audiological heterogeneity in patients with DFNB3 hinders precision medicine in hearing rehabilitation. Here, we aimed to elucidate the heterogeneity of the auditory phenotypes of MYO15A variants according to the affected domain and the feasibilities for acoustic stimulation. We conducted whole-exome sequencing for 10 unrelated individuals from seven multiplex families with DFNB3; 11 MYO15A variants, including the novel frameshift c.900delT (p.Pro301Argfs*143) and nonsense c.4879G > T (p.Glu1627*) variants, were identified. In seven probands, residual hearing at low frequencies was significantly higher in the groups with one or two N-terminal frameshift variants in trans conformation compared to that in the group without these variants. This is consistent with the 56 individuals from the previously published reports that carried a varying number of N-terminal truncating variants in MYO15A. In addition, patients with missense variants in the second FERM domain had better hearing at low frequencies than patients without these variants. Subsequently, acoustic stimulation provided by devices such as hearing aids or cochlear implants was feasible in patients with one or two N-terminal truncating variants or a second FERM missense variant. In conclusion, N-terminal or second FERM variants in MYO15A allow the practical use of acoustic stimulation through hearing aids or electroacoustic stimulation for aural rehabilitation.

Dynamic Chronological Changes in Serum Triglycerides Are Associated With the Time Point for Non-alcoholic Fatty Liver Disease Development in the Nationwide Korean Population Cohort.

Background: We investigated the effects of anthropometric, laboratory, and lifestyle factors on the development of non-alcoholic fatty liver disease (NAFLD) in a nationwide, population-based, 4-year retrospective cohort. Methods: The propensity score-matched study and control groups contained 1,474 subjects who had data in the Korean National Health Insurance Service-National Sample Cohort in 2009, 2011, and 2013. NAFLD was defined using medical records of a diagnosis confirmed by primary clinicians and meeting two previously validated fatty liver prediction models. Chronological changes in anthropometric variables, laboratory results, and lifestyle factors during two periods were compared between patient and control groups in order to find out parameters with consistent dynamics in pre-NAFLD stage which was defined as period just before the NAFLD development. Results: Among the 5 anthropometric, 10 laboratory, and 3 lifestyle factors, prominent chronological decremental changes in serum triglycerides were consistently observed during the pre-NAFLD stage, although the degrees of changes were more predominant in men (-9.46 mg/dL) than women (-5.98 mg/dL). Furthermore, weight and waist circumference changes during the pre-NAFLD stage were noticeable only in women (+0.36 kg and +0.9 cm for weight and waist circumference, respectively), which suggest gender difference in NAFLD. Conclusion: Early screening strategies for people with abrupt chronological changes in serum triglycerides to predict NAFLD development before the progression is recommended.

Gut microbiota-derived metabolite trimethylamine N-oxide as a biomarker in early Parkinson's disease.

OBJECTIVES: This study aimed to investigate the potential of using changes in the plasma levels of trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, as a biomarker in early Parkinson's disease (PD). METHODS: Plasma TMAO levels were measured in 85 patients with drug-naïve early stage PD and 20 healthy controls. A linear mixed model was used to assess longitudinal changes in levodopa-equivalent dose (LED) during follow-up (>2 y) in three tertile PD groups according to plasma TMAO levels. Additionally, a Cox regression analysis was performed to assess the effect of plasma TMAO levels on dementia conversion. RESULTS: Plasma TMAO levels of patients with PD were lower than those of healthy controls. A linear mixed model demonstrated that patients with PD and lower levels of TMAO (<4.75 µmol/L; i.e., lowest tertile group) exhibited faster increases in LED over time. The Cox regression model did not reveal that plasma TMAO level was associated with the risk for dementia conversion (P = 0.488). However, when we divided patients with PD into two subgroups according to bet cutoff TMAO level to maximize the log-rank statistics, the PD group with a low plasma TMAO level (<6.92 µmol/L) had a higher risk (with borderline statistical significance) for PD-dementia conversion than the group with a high TMAO level (hazard ratio: 7.565; 95% confidence interval, 1.004-57.019; P = 0.050). CONCLUSIONS: The results demonstrate that lower baseline plasma TMAO levels are associated with faster increases in LED and tend to increase the risk for PD-dementia conversion, suggesting the prognostic implications of TMAO in early stage PD.

Incidence of malignancy and related mortality after kidney transplantation: a nationwide, population-based cohort study in Korea.

Post-transplant malignancy (PTM) is a leading cause of premature mortality among kidney transplantation recipients. However, population-based cohort studies that cover incidence, mortality, and risk factors for PTM are rarely reported, especially in East Asia. We designed a retrospective cohort study using a national population-based database. A total of 9915 kidney recipients between 2003 and 2016 were included. During this period, 598 cases (6.0%) of de novo PTM occurred. The most common PTM was thyroid cancer (14.2%), followed by colorectal (11.2%), kidney (10.7%), and stomach cancers (8.9%). The standardised incidence ratio for all-site cancer was 3.9. The risks of Kaposi sarcoma (192.9) and kidney cancer (21.1) were more than 10 times those of the general population. Cancer-related deaths were 89 (14.9%) with liver cancer being the highest (14.6%), followed by lung cancer (13.5%), non-Hodgkin lymphoma (NHL) (12.4%), stomach cancer (9.0%), and colorectal cancer (7.9%). The standardised mortality ratio (SMR) was slightly elevated (1.4). A notable increase in SMR was observed for lymphoma (9.3 for Hodgkin lymphoma and 5.5 for NHL). Older age and graft failure were significantly related to PTM. These findings reflecting geographical variation have implications for the development of strategies for fatal cancers to prevent premature deaths from PTM.