MNX1-HNF1B Axis Is Indispensable for Intraductal Papillary Mucinous Neoplasm Lineages.

BACKGROUND & AIMS: Chromatin architecture governs cell lineages by regulating the specific gene expression; however, its role in the diversity of cancer development remains unknown. Among pancreatic cancers, pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMN) with an associated invasive carcinoma (IPMNinv) arise from 2 distinct precursors, and their fundamental differences remain obscure. Here, we aimed to assess the difference of chromatin architecture regulating the transcriptional signatures or biological features in pancreatic cancers. METHODS: We established 28 human organoids from distinct subtypes of pancreatic tumors, including IPMN, IPMNinv, and PDAC. We performed exome sequencing (seq), RNA-seq, assay for transposase-accessible chromatin-seq, chromatin immunoprecipitation-seq, high-throughput chromosome conformation capture, and phenotypic analyses with short hairpin RNA or clustered regularly interspaced short palindromic repeats interference. RESULTS: Established organoids successfully reproduced the histology of primary tumors. IPMN and IPMNinv organoids harbored GNAS, RNF43, or KLF4 mutations and showed the distinct expression profiles compared with PDAC. Chromatin accessibility profiles revealed the gain of stomach-specific open regions in IPMN and the pattern of diverse gastrointestinal tissues in IPMNinv. In contrast, PDAC presented an impressive loss of accessible regions compared with normal pancreatic ducts. Transcription factor footprint analysis and functional assays identified that MNX1 and HNF1B were biologically indispensable for IPMN lineages. The upregulation of MNX1 was specifically marked in the human IPMN lineage tissues. The MNX1-HNF1B axis governed a set of genes, including MYC, SOX9, and OLFM4, which are known to be essential for gastrointestinal stem cells. High-throughput chromosome conformation capture analysis suggested the HNF1B target genes to be 3-dimensionally connected in the genome of IPMNinv. CONCLUSIONS: Our organoid analyses identified the MNX1-HNF1B axis to be biologically significant in IPMN lineages.

The arginine transporter Can1 acts as a transceptor for regulation of proline utilization in the yeast Saccharomyces cerevisiae.

Proline is the most abundant amino acid in wine and beer, because the yeast Saccharomyces cerevisiae hardly assimilates proline during fermentation processes. Our previous studies showed that arginine induces endocytosis of the proline transporter Put4, resulting in inhibition of proline utilization. We here report a possible role of arginine sensing in the inhibition of proline utilization. We first found that two basic amino acids, ornithine, and lysine, inhibit proline utilization by inducing Put4 endocytosis in a manner similar to arginine, but citrulline does not. Our genetic screening revealed that the arginine transporter Can1 is involved in the inhibition of proline utilization by arginine. Intriguingly, the arginine uptake activity of Can1 was not required for the arginine-dependent inhibition of proline utilization, suggesting that Can1 has a function beyond its commonly known function of transporting arginine. More importantly, our biochemical analyses revealed that Can1 activates signaling cascades of protein kinase A in response to extracellular arginine. Hence, we proposed that Can1 regulates proline utilization by functioning as a transceptor possessing the activity of both a transporter and receptor of arginine.

Recompleting the Caenorhabditis elegans genome.

Caenorhabditis elegans was the first multicellular eukaryotic genome sequenced to apparent completion. Although this assembly employed a standard C. elegans strain (N2), it used sequence data from several laboratories, with DNA propagated in bacteria and yeast. Thus, the N2 assembly has many differences from any C. elegans available today. To provide a more accurate C. elegans genome, we performed long-read assembly of VC2010, a modern strain derived from N2. Our VC2010 assembly has 99.98% identity to N2 but with an additional 1.8 Mb including tandem repeat expansions and genome duplications. For 116 structural discrepancies between N2 and VC2010, 97 structures matching VC2010 (84%) were also found in two outgroup strains, implying deficiencies in N2. Over 98% of N2 genes encoded unchanged products in VC2010; moreover, we predicted ≥53 new genes in VC2010. The recompleted genome of C. elegans should be a valuable resource for genetics, genomics, and systems biology.

Finding long tandem repeats in long noisy reads.

MOTIVATION: Long tandem repeat expansions of more than 1000 nt have been suggested to be associated with diseases, but remain largely unexplored in individual human genomes because read lengths have been too short. However, new long-read sequencing technologies can produce single reads of 10 000 nt or more that can span such repeat expansions, although these long reads have high error rates, of 10-20%, which complicates the detection of repetitive elements. Moreover, most traditional algorithms for finding tandem repeats are designed to find short tandem repeats (<1000 nt) and cannot effectively handle the high error rate of long reads in a reasonable amount of time. RESULTS: Here, we report an efficient algorithm for solving this problem that takes advantage of the length of the repeat. Namely, a long tandem repeat has hundreds or thousands of approximate copies of the repeated unit, so despite the error rate, many short k-mers will be error-free in many copies of the unit. We exploited this characteristic to develop a method for first estimating regions that could contain a tandem repeat, by analyzing the k-mer frequency distributions of fixed-size windows across the target read, followed by an algorithm that assembles the k-mers of a putative region into the consensus repeat unit by greedily traversing a de Bruijn graph. Experimental results indicated that the proposed algorithm largely outperformed Tandem Repeats Finder, a widely used program for finding tandem repeats, in terms of sensitivity. AVAILABILITY AND IMPLEMENTATION: https://github.com/morisUtokyo/mTR.

The Cdc25/Ras/cAMP-dependent protein kinase A signaling pathway regulates proline utilization in wine yeast Saccharomyces cerevisiae under a wine fermentation model.

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. In this study, we clarified that a glucose response is involved in an inhibitory mechanism of proline utilization in yeast. Our genetic screen showed that strains with a loss-of-function mutation on the CDC25 gene can utilize proline even under fermentation conditions. Cdc25 is a regulator of the glucose response consisting of the Ras/cAMP-dependent protein kinase A (PKA) pathway. Moreover, we found that activation of the Ras/PKA pathway is necessary for the inhibitory mechanism of proline utilization. The present data revealed that crosstalk exists between the carbon and proline metabolisms. Our study could hold promise for the development of wine yeast strains that can efficiently assimilate proline during the fermentation processes.

First-line pembrolizumab for patients with early relapsing urothelial carcinoma after perioperative chemotherapy: a retrospective analysis of bladder cancer and upper urinary tract cancer.

BACKGROUND: First-line pembrolizumab is available for recurrent disease within 12 months after the receipt of platinum-based perioperative chemotherapy. However, the benefit of first-line pembrolizumab is unclear. This study evaluated the oncological outcome of patients treated with pembrolizumab compared with chemotherapy as first-line therapy for early relapsing disease after the receipt of platinum-based perioperative chemotherapy. METHODS: Data from a multicenter study included 454 patients diagnosed with unresectable or metastatic UC from November 2006 to July 2021. We identified patients with early and non-early relapsing disease. Oncological outcomes were evaluated using progression-free survival, overall survival, and survival with disease control. RESULTS: Fifty-three patients with early relapsing disease and 15 patients with non-early relapsing disease were identified. Of 53 patients with early relapsing disease, 26 (49.1%) were treated with pembrolizumab and 27 (50.9%) were treated with chemotherapy as first-line therapy. Fifteen patients with non-early relapsing disease were treated with chemotherapy. Early relapsing disease was associated with shorter progression-free survival and overall survival than non-early relapsing disease. Pembrolizumab was associated with longer progression-free survival and survival with disease control than chemotherapy in patients with early relapsing disease. There was no significant difference in overall survival between pembrolizumab and chemotherapy, but overall survival plateau with a long tail was observed in pembrolizumab. CONCLUSIONS: First-line pembrolizumab in earlier clinical settings for highly malignant tumors might improve the prognosis of patients with early relapsing disease after the receipt of platinum-based perioperative chemotherapy.

[A Case of Refractory Intermittent Hematuria that Occurred during Paclitaxel/Ramucirumab Combination Therapy for Metastatic Gastric Cancer].

A 76-year-old male patient developed right hydronephrosis due to peritoneal and retroperitoneal dissemination after surgery for gastric cancer. A ureteral stent was inserted, and systemic chemotherapy was introduced for metastatic gastric cancer. Disease progression was observed, and paclitaxel/ramucirumab combination therapy was started as the second-line treatment. After seven courses, severe gross hematuria appeared intermittently, and refractory epistaxis was observed concurrently. No hemorrhagic lesion was found in the imaging test and urethrocystoscopy. The patient received conservative treatment, such as blood transfusion, and further examination was planned. However, hematuria and epistaxis resolved spontaneously during the course of treatment. From the clinical course, it was thought to be a hemorrhagic adverse event due to ramucirumab, and the patient's treatment was therefore changed to another drug. The patient recovered without recurrence of gross hematuria.

A new diagnostic indication device of a biomarker growth differentiation factor 15 for mitochondrial diseases: From laboratory to automated inspection.

Mitochondrial diseases (MDs) are occasionally difficult to diagnose. Growth differentiation factor 15 (GDF15) has been reported as a biomarker useful for not only diagnosing MDs, but also evaluating disease severity and therapeutic efficacy. To enable the measurement of serum GDF15 concentrations at medical institutions, we developed a new latex-enhanced turbidimetric immunoassay (LTIA) as an automated diagnostic indication test for MDs. We also examined the equivalency of specificity and sensitivity in measuring serum GDF15 concentrations between a commercially available enzyme-linked immunosorbent assay (ELISA) kit and a novel LTIA device in patients with MDs, disease controls, and healthy controls. A clinical performance study used a newly developed LTIA device and an existing ELISA kit to measure the concentrations of GDF15 in 35 MD patients, 111 disease controls, and 86 healthy controls. The median (first quartile-third quartile) of serum GDF15 concentrations measured with the LTIA device was significantly higher (P < .001) in MD patients (1389.0 U/mL [869.5-1776.0 U/mL]) than in healthy controls (380.5 U/mL [330.2-471.8 U/mL]); the interquartile ranges did not overlap between MD patients and healthy controls. The areas under the curve in disease and healthy controls were 0.812 (95% confidence interval [CI]: 0.734-0.886) and 0.951 (95% CI: 0.910-0.992), respectively. The automated, high-throughput technology-based LTIA device has definite advantages over the ELISA kit in shorter processing time and lower estimated cost per sample measurement. The LTIA device of GDF15 may be a sufficiently reliable, frontline, diagnostic indicator of individuals with suspected MDs in the general population.

Trends in risk classification at diagnosis and choice of primary therapy for prostate cancer: An analysis of 10 839 patients from the Nara Urological Research and Treatment Group registry between 2004 and 2015.

OBJECTIVE: To evaluate trends in risk classification at diagnosis and choice of primary therapy in patients diagnosed with prostate cancer. METHODS: This retrospective study included 10 839 patients who were newly diagnosed with prostate cancer between 2004 and 2015 at 23 Japanese institutions. Risk classification and primary therapies between 2004 and 2015 were evaluated. The trends in risk classification and primary therapy were evaluated using chi-squared tests for trend during four periods (2004-2006; 2007-2009; 2010-2012; and 2013-2015). Binary logistic analysis was used to evaluate the extent to which factors such as age, risk classification, and institution influenced primary therapy choice in the 2013-2015 cohort. RESULTS: The number of patients with very-low or low-risk classification (P < 0.001) and metastasis (P = 0.04) decreased and the number with intermediate-risk classification (P < 0.001) increased during the four periods. A tendency to choose radical prostatectomy as primary therapy for prostate cancer was not observed during the four periods (P = 0.90). The number of patients who chose radiation therapy (P < 0.001) and active surveillance/watchful waiting (P < 0.001) as primary therapies increased during the four periods and the number of patients who chose androgen deprivation therapy (P < 0.001) decreased. Age, institution, and risk classification significantly influenced primary therapy choice. CONCLUSIONS: We have shown the trends in risk classification of prostate cancer and primary therapy choices between 2004 and 2015 in Japan. Age, institution, and risk classification significantly influenced the decision on primary therapy for prostate cancer.

Appropriate Number of Docetaxel Cycles in Castration-Resistant Prostate Cancer Patients Considering Peripheral Neuropathy and Oncological Control.

BACKGROUND: The number of cycles of docetaxel required for castration-resistant prostate cancer (CRPC) is unclear. This study estimated peripheral neuropathy (PN) incidence and the optimal number of treatment cycles in patients receiving docetaxel for CRPC. PATIENTS AND METHODS: The study retrospectively reviewed 82 patients receiving docetaxel for CRPC at an institution between January 2005 and January 2017. Docetaxel (70 or 75 mg/m2) was administered every 3 weeks, and prednisone 5 mg or dexamethasone 0.5 mg was administered twice a day. RESULTS: PN (grade ≥2) was noted in 32 (39.0%) patients. The median cumulative dose of docetaxel associated with PN was 675 mg/m2. No factor significantly predicted the occurrence of PN. The prostate-specific antigen progression rate, prostate cancer-specific survival, and overall survival were significantly better with ≥8 cycles of docetaxel than with <8 cycles (p < 0.05). CONCLUSION: The incidence of PN is high, and 8 treatment cycles are optimal for patients receiving docetaxel for CRPC.

Unexpected presentation and surgical salvage of transplant renal artery dissection caused by vascular clamping: a case report.

BACKGROUND: Transplant renal artery dissection is a rare and serious event that can cause allograft dysfunction and activation of the renin-mediated renovascular hypertension. Most cases are induced by percutaneous transluminal angioplasty, arteriosclerotic disease, or fibromuscular dysplasia. We observed a case of transplant renal artery dissection induced by unusual causes during kidney transplantation. CASE PRESENTATION: A 35-year-old woman, whose mother donated a kidney to her, underwent ABO-incompatible living kidney transplantation. The allograft had one renal artery and vein that were anastomosed to the internal iliac artery and external iliac vein, respectively. Although careful handling was performed in all procedures including vascular clamping, Doppler ultrasonography (US) immediately after reperfusion showed an increase in the systolic blood velocity and urine output was not observed. Arterial anastomotic stenosis was suspected, but upon exploration, a renal artery dissection was detected in the middle portion of the donor artery. The part of the transplant renal artery was resected, and cold reflux was started again. At the resected part of transplant renal artery, dissection was identified. After re-anastomosis, Doppler US revealed that the blood flow of the renal artery was adequate without an increase in the systolic blood velocity, and sufficient blood flow was observed throughout the allograft. Urine output was also observed as soon as blood flow returned, and serum creatinine level decreased to 0.95 mg/dL after surgery. The cause of injury might have been vascular clamping in order to drain the air and check bleeding at the anastomosis. CONCLUSIONS: Our case reaffirmed that careful handling is needed in all procedures, including donor nephrectomy, cannulation for transplant perfusion, vascular clamping, and anastomosis, even without any evidence of arteriosclerosis. Kidney transplant recipients commonly have atherosclerosis and hypertension, which are risk factors for arterial dissection. Early diagnosis and intervention can lead to the prevention of allograft dysfunction. Therefore, close monitoring of allograft blood flow by Doppler US during surgery should be considered.

Trends in treatment outcomes of hydrocele in Japanese children: A single-institute experience.

OBJECTIVES: To investigate trends in treatment outcomes of surgical intervention versus observation for pediatric hydrocele. METHODS: This retrospective study included 175 patients diagnosed with hydrocele at our institution. Hydrocele was diagnosed based on medical history, physical examination and ultrasonography findings. The treatment for these patients was divided into two options: surgical intervention or careful follow up; the outcomes were investigated. RESULTS: The median age at diagnosis was 3 months, and a total of 11 patients (6%) were premature at birth. Hydrocele was diagnosed on the right side, the left side and bilaterally in 106 (61%), 46 (26%) and 23 (13%) patients, respectively. A total of 136 patients showed spontaneous improvement at the median 7 months after diagnosis, and 54 patients underwent surgical intervention. The rate of spontaneous resolution deceased with age, but spontaneous resolution was observed in patients aged >2 years. CONCLUSIONS: Our findings suggest that spontaneous resolution can be observed in patients aged >2 years, and surgical intervention can be carried out effectively and safely. Infant hydrocele should be followed up carefully for at least 1 year without surgical intervention since diagnosis. Investigation of the optimal timing of and appropriate reason for surgical intervention can lead to better management and outcomes in patients with hydrocele. Further research is warranted to support the current clinical practice.

Clinical benefit of early treatment with bone-modifying agents for preventing skeletal-related events in patients with genitourinary cancer with bone metastasis: A multi-institutional retrospective study.

OBJECTIVES: To evaluate the clinical benefit of bone-modifying agents and identify the risk factors of skeletal-related events in patients with genitourinary cancer with newly diagnosed bone metastasis. METHODS: This was a multicenter retrospective study including a total of 650 patients with bone metastasis of the following cancer types: hormone-sensitive prostate cancer (n = 443), castration-resistant prostate cancer (n = 50), renal cell carcinoma (n = 80) and urothelial carcinoma (n = 77). Clinical factors at the time of diagnosis of bone metastasis were analyzed. Early treatment with bone-modifying agents was defined as follows: administration of bone-modifying agents before the development of skeletal-related events and within 6 months from the diagnosis of bone metastasis. RESULTS: During the follow-up period (median 19.0 months, interquartile range 6.0-43.8 months), skeletal-related events were reported in 88 (20%) patients with hormone-sensitive prostate cancer, 17 (34%) patients with castration-resistant prostate cancer, 58 (73%) patients with renal cell carcinoma and 34 (44%) patients with urothelial carcinoma. Early treatment with bone-modifying agents significantly prolonged the time to the first skeletal-related event in castration-resistant prostate cancer, renal cell carcinoma and urothelial carcinoma, but not in hormone-sensitive prostate cancer. Bone pain and elevated alkaline phosphatase levels were independent predictive risk factors of the first skeletal-related event. The subgroup analysis showed that early treatment with bone-modifying agents was associated with prolonged time to the first skeletal-related events in patients with bone pain or elevated alkaline phosphatase levels. CONCLUSIONS: Early treatment with bone-modifying agents should be considered, especially for patients with bone pain and elevated alkaline phosphatase levels, to prevent skeletal-related events in patients with genitourinary cancer with bone metastasis.

[A Case of Small Cell Carcinoma of the Renal Pelvis].

A 84-year-old female visited our hospital with the chief complaint of asymptomatic gross hematuria. The computed tomography and magnetic resonance imaging revealed a large invasive tumor in the right renal pelvis. Metastatic workup was negative. On imaging studies and voided urine cytology, diagnosis of right renal pelvic cancer (cT3N0M0) was made. Laparoscopic right nephroureterectomy was performed. Histopathological examinations revealed a small cell carcinoma of the renal pelvis. The patient declined adjuvant chemotherapy and died 5 months after surgery. Primary small cell carcinoma of the renal pelvis is a rare disease. To our knowledge, this is the 29th case of primary small cell carcinoma of the renal pelvis in the world literature.

[Spontaneous Tumor Lysis Syndrome in a Patient with Testicular Malignant Lymphoma : A Case Report].

A 75-year-old man, highly suspected as having malignant lymphoma originating from his left testis, underwent exploratory orchiectomy for a definitive diagnosis. Laboratory examinations before the surgery showed high lactate dehydrogenase (652 IU/l), elevated serum creatinine level (1.85 mg/dl) and sIL-2R level (8,930 U/ml). As the postoperative course passed uneventfully, the patient was discharged from the hospital on the eighth day after the surgery. Ten days after the surgery the patient was transferred to the emergency room of the hospital complaining of severe abdominal pain and malaise. Laboratory examinations revealed highly elevated lactate dehydrogenase (2,807 IL/l), uric acid (24.9 mg/dl) and serum creatinine (5.31 mg/dl). Computed tomography demonstrated rapid growth of the retroperitoneal mass and occurrence of bilateral hydronephroses. Under the diagnosis of spontaneous tumor lysis syndrome, the patient was urgently treated with hemodialysis, steroidal pulse and rituximab following percutaneous nephrostomy for the right kidney. After improvement of the laboratory data, the patient was transferred to another hospital for the treatment of malignant lymphoma.

[Misdirection of a Catheter for Clean Intermittent Catheterization into the Upper Ureter in a Female Patient with Neurogenic Bladder Due to Myelomeningocele : A Case Report].

We report a case of misdirection of a catheter for clean intermittent catheterization (CIC) into the ureter. A four-year-old girl with neurogenic bladder due to myelomeningocele had been managed with CIC by her parents for several years. From about a month before her visit, macroscopic hematuria appeared intermittently followed by abdominal pain and fever-up for a short time which ceased spontaneously. As cystography demonstrated bilateral vesicoureteral reflux, we performed endoscopic intraureteral injection of Deflux[TM] and during the operation we confirmed influx of bloody urine from the right ureteral orifice but not from the left one. As computed tomography revealed a tubular foreign body located in the upper portion of the right ureter, another endoscopic operation was performed and it was removed successfully. The removed foreign body was identified as a disposable catheter that was used for CIC.

[Predictive Factors of Biochemical Recurrence in Positive Surgical Margin Patients by Radical Prostatectomy].

The predictive factors for biochemical recurrence (BCR) were investigated in patients with positive surgical margin of the extirpated prostate by radical retropubic prostatectomy (RRP). The records of 365 patients who underwent RRP in our hospital between January 2002 and December 2014 were retrospectively analyzed. Patients who had received additional therapy before or after RRP, who had not been followed up for more than a year after surgery, and who had pN1 lesions were excluded from the study. Positive surgical margin was observed in 112 cases. Prostate specific antigen (PSA) before surgery ≥20 ng/ml, biopsy positive core ratio ≥40%, Gleason score of the surgical specimen ≥8, and postoperative PSA nadir ≥0.01 ng/ml were identified as significant predictors of BCR.

[Predictive Factors of Positive Surgical Margin in Radical Prostatectomy].

To identify the predictive factors for positive surgical margin after radical prostatectomy, we retrospectively analyzed the records of 381 patients who underwent radical prostatectomy in our hospital between January 2002 and December 2014. Patients who had received hormonal therapy before surgery were excluded from the study. Positive surgical marginwas observed in121 cases (31.8%), and prostate specific antigen (PSA) before surgery ≧10 ng/ml (HR1.89 : 95%CI 1.17-3. 07) and BMI≧25 kg/m2 (HR2.73 : 95%CI 1.60-4. 68) were identified as significant predictors of positive surgical margin. The existence of PSM significantly correlated to the operation time of 240 minutes or longer (HR2.27 : 95%CI 1. 35-3.79), pT2c or higher local stage (HR2.08 : 95%CI 1.17-3.72) and 7 or higher Gleason score of the resected specimen(HR1.63 : 95%CI 1.03-2.59).

Large hypomethylated domains serve as strong repressive machinery for key developmental genes in vertebrates.

DNA methylation is a fundamental epigenetic modification in vertebrate genomes and a small fraction of genomic regions is hypomethylated. Previous studies have implicated hypomethylated regions in gene regulation, but their functions in vertebrate development remain elusive. To address this issue, we generated epigenomic profiles that include base-resolution DNA methylomes and histone modification maps from both pluripotent cells and mature organs of medaka fish and compared the profiles with those of human ES cells. We found that a subset of hypomethylated domains harbor H3K27me3 (K27HMDs) and their size positively correlates with the accumulation of H3K27me3. Large K27HMDs are conserved between medaka and human pluripotent cells and predominantly contain promoters of developmental transcription factor genes. These key genes were found to be under strong transcriptional repression, when compared with other developmental genes with smaller K27HMDs. Furthermore, human-specific K27HMDs show an enrichment of neuronal activity-related genes, which suggests a distinct regulation of these genes in medaka and human. In mature organs, some of the large HMDs become shortened by elevated DNA methylation and associate with sustained gene expression. This study highlights the significance of domain size in epigenetic gene regulation. We propose that large K27HMDs play a crucial role in pluripotent cells by strictly repressing key developmental genes, whereas their shortening consolidates long-term gene expression in adult differentiated cells.

AgIn: measuring the landscape of CpG methylation of individual repetitive elements.

MOTIVATION: Determining the methylation state of regions with high copy numbers is challenging for second-generation sequencing, because the read length is insufficient to map reads uniquely, especially when repetitive regions are long and nearly identical to each other. Single-molecule real-time (SMRT) sequencing is a promising method for observing such regions, because it is not vulnerable to GC bias, it produces long read lengths, and its kinetic information is sensitive to DNA modifications. RESULTS: We propose a novel linear-time algorithm that combines the kinetic information for neighboring CpG sites and increases the confidence in identifying the methylation states of those sites. Using a practical read coverage of ∼30-fold from an inbred strain medaka (Oryzias latipes), we observed that both the sensitivity and precision of our method on individual CpG sites were ∼93.7%. We also observed a high correlation coefficient (R = 0.884) between our method and bisulfite sequencing, and for 92.0% of CpG sites, methylation levels ranging over [0,1] were in concordance within an acceptable difference 0.25. Using this method, we characterized the landscape of the methylation status of repetitive elements, such as LINEs, in the human genome, thereby revealing the strong correlation between CpG density and hypomethylation and detecting hypomethylation hot spots of LTRs and LINEs. We uncovered the methylation states for nearly identical active transposons, two novel LINE insertions of identity ∼99% and length 6050 base pairs (bp) in the human genome, and 16 Tol2 elements of identity >99.8% and length 4682 bp in the medaka genome. AVAILABILITY AND IMPLEMENTATION: AgIn (Aggregate on Intervals) is available at: https://github.com/hacone/AgIn CONTACT: ysuzuki@cb.k.u-tokyo.ac.jp or moris@cb.k.u-tokyo.ac.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.