The fragility of a structurally diverse duplication block triggers recurrent genomic amplification.

The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic analyses. Structural diversity in the human population suggests that these blocks are unstable in the germline; however, whether or not these blocks are also unstable in the cancer genome remains elusive. Here we report that the 500 kb block called KRTAP_region_1 (KRTAP-1) on 17q12-21 recurrently demarcates the amplicon of the ERBB2 (HER2) oncogene in breast tumors. KRTAP-1 carries numerous tandemly-duplicated segments that exhibit diversity within the human population. We evaluated the fragility of the block by cytogenetically measuring the distances between the flanking regions and found that spontaneous distance outliers (i.e DNA breaks) appear more frequently at KRTAP-1 than at the representative common fragile site (CFS) FRA16D. Unlike CFSs, KRTAP-1 is not sensitive to aphidicolin. The exonuclease activity of DNA repair protein Mre11 protects KRTAP-1 from breaks, whereas CtIP does not. Breaks at KRTAP-1 lead to the palindromic duplication of the ERBB2 locus and trigger Breakage-Fusion-Bridge cycles. Our results indicate that an insufficiently investigated area of the human genome is fragile and could play a crucial role in cancer genome evolution.

Impact of Physical Activity on Dialysis and Nondialysis Days and Clinical Outcomes Among Patients on Hemodialysis.

OBJECTIVE: Patients undergoing hemodialysis (HD) have different physical activity (PA) patterns on HD and non-HD days. Nonetheless, whether these differences are associated with clinical outcomes remains unclear. We examined the association of PA levels on HD and non-HD days with cardiovascular (CV) hospitalizations and mortality. METHODS: Outpatients undergoing HD from 2002 to 2019 were retrospectively enrolled. The number of steps performed over 3 HD days and 4 non-HD days was recorded via accelerometry. Outcomes were all-cause mortality and a composite of CV hospitalizations and mortality. Patients were divided into two groups, each according to the median number of steps performed on HD (2371 steps/day) and non-HD days (3752 steps/day). Further, we categorized them into 4 groups according to each median values: "more active on HD/more active on non-HD (MM)," "more active on HD/less active on non-HD (ML)," "less active on HD/more active on non-HD (LM)," and "less active on HD/less active on non-HD (LL)." Cox and mixed-effects Poisson regression models were used for these outcomes. RESULTS: We analyzed 512 patients (median follow-up, 3.4 years). Higher PA on HD (hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.54-0.65), and non-HD (HR, 0.84; 95% CI, 0.80-0.88) was associated with lower mortality risk, respectively. Further, the ML group (HR, 1.20; 95% CI, 1.13-1.28), LM group (HR, 1.82; 95% CI, 1.53-2.17), and LL group (HR, 1.83; 95% CI, 1.65-2.02) had higher mortality risks than the MM group. Associations of PA with multiple CV hospitalizations and mortality were similar to those between PA and mortality. CONCLUSIONS: Higher PA on HD and non-HD days was associated with lower risks of CV hospitalizations and mortality. However, higher PA levels on either HD or non-HD days alone did not improve clinical outcomes.

Mechanisms Underlying Recurrent Genomic Amplification in Human Cancers.

Focal copy-number increases (genomic amplification) pinpoint oncogenic driver genes and therapeutic targets in cancer genomes. With the advent of genomic technologies, recurrent genomic amplification has been mapped throughout the genome. Recurrent amplification could be solely due to positive selection for the tumor-promoting effects of amplified gene products. Alternatively, recurrence could result from the susceptibility of the loci to amplification. Distinguishing between these possibilities requires a full understanding of the amplification mechanisms. Two mechanisms, the formation of double minute (DM) chromosomes and breakage-fusion-bridge (BFB) cycles, have been repeatedly linked to genomic amplification, and the impact of both mechanisms has been confirmed in cancer genomics data. We review the details of these mechanisms and discuss the mechanisms underlying recurrence.

Association between frailty and bone loss in patients undergoing maintenance hemodialysis.

Frailty is significantly associated with bone loss in the general population. However, it is unclear whether this association also exists in patients undergoing hemodialysis who have chronic kidney disease-mineral and bone disorder (CKD-MBD). This study aimed to assess the association between frailty and bone loss in patients undergoing hemodialysis. This cross-sectional study included 214 (90 women, 124 men) Japanese outpatients undergoing maintenance hemodialysis three times per week, with a mean age of 67.1 years (women) and 66.8 years (men). Frailty was defined based on criteria set forth by the Cardiovascular Health Study (CHS)-19 (21.1%) women and 47 (37.9%) men were robust, 41 (45.6%) women and 43 (34.7%) men were pre-frail, and 30 (33.3%) women and 34 (27.4%) men were frail. For bone mass, quantitative ultrasound (QUS) parameters (speed of sound, broadband ultrasound attenuation, stiffness index) of the calcaneus were measured. The association between frailty and QUS parameters was determined separately for women and men using multivariate analysis of covariance (ANCOVA), with adjustments for clinical characteristics including age, body mass index, hemodialysis vintage, diabetes, current smoking, serum albumin, phosphate, corrected calcium, intact parathyroid hormone, and medication for CKD-MBD (vitamin D receptor activator, calcimimetics). ANCOVA revealed that all QUS parameters declined significantly with increasing levels of frailty in both sexes (P < 0.05). In conclusion, frailty (as defined by CHS criteria) should be considered a risk factor for bone loss in patients undergoing hemodialysis.

Physical Activity Dose for Hemodialysis Patients: Where to Begin? Results from a Prospective Cohort Study.

OBJECTIVE: Greater physical activity is associated with lower risk of mortality in persons with kidney disease; however, little is known about the appropriate dose of physical activity among hemodialysis patients. Here detected the minimum level of habitual physical activity to help inform interventions aimed at improving outcomes in the dialysis population. DESIGN: The design was prospective cohort study. SUBJECTS: Clinically stable outpatients in a hemodialysis unit from October 2002 to March 2014 were assessed for their eligibility to be included in this 7-year prospective cohort study. We used the Youden index to determine the optimal cutoff points for physical activity. The prognostic effect of physical activity on survival was estimated by Cox proportional hazards regression analysis. The number of steps per nondialysis day was recorded by accelerometer at study entry. MAIN OUTCOME MEASURE: The main outcome measure was all-cause mortality. RESULTS: There were 282 participants who had a mean age of 65 ± 11 years and 45% were female. A total of 56 deaths occurred during the follow-up period (56 months [interquartile range: 29-84 months]). The cutoff value for the physical activity discriminating those at high risk of mortality was 3,752 steps. After adjustment for the effect of confounders, the hazard ratio in the group of <4,000 steps was 2.37 (95% confidence interval: 1.22-4.60, P = .01) compared with the others. CONCLUSIONS: Engaging in physical activity is associated with decreased mortality risk among hemodialysis patients. Our findings of a substantial mortality benefit among those who engage in at least 4,000 steps provide a basis for as a minimum initial recommendation kidney health providers can provide for mobility disability-free hemodialysis patients.

Impediment of Replication Forks by Long Non-coding RNA Provokes Chromosomal Rearrangements by Error-Prone Restart.

Naturally stalled replication forks are considered to cause structurally abnormal chromosomes in tumor cells. However, underlying mechanisms remain speculative, as capturing naturally stalled forks has been a challenge. Here, we captured naturally stalled forks in tumor cells and delineated molecular processes underlying the structural evolution of circular mini-chromosomes (double-minute chromosomes; DMs). Replication forks stalled on the DM by the co-directional collision with the transcription machinery for long non-coding RNA. RPA, BRCA2, and DNA polymerase eta (Polη) were recruited to the stalled forks. The recruitment of Polη was critical for replication to continue, as Polη knockdown resulted in DM loss. Rescued stalled forks were error-prone and switched replication templates repeatedly to create complex fusions of multiple short genomic segments. In mice, such complex fusions circularized the genomic region surrounding MYC to create a DM during tumorigenesis. Our results define a molecular path that guides stalled replication forks to complex chromosomal rearrangements.

Palindromic amplification of the ERBB2 oncogene in primary HER2-positive breast tumors.

Oncogene amplification confers a growth advantage to tumor cells for clonal expansion. There are several, recurrently amplified oncogenes throughout the human genome. However, it remains unclear whether this recurrent amplification is solely a manifestation of increased fitness resulting from random amplification mechanisms, or if a genomic locus-specific amplification mechanism plays a role. Here we show that the ERBB2 oncogene at 17q12 is susceptible to palindromic gene amplification, a mechanism characterized by the inverted (palindromic) duplication of genomic segments, in HER2-positive breast tumors. We applied two genomic approaches to investigate amplification mechanisms: sequencing of DNA libraries enriched with tumor-derived palindromic DNA (Genome-wide Analysis of Palindrome Formation) and whole genome sequencing (WGS). We observed significant enrichment of palindromic DNA within amplified ERBB2 genomic segments. Palindromic DNA was particularly enriched at amplification peaks and at boundaries between amplified and normal copy-number regions. Thus, palindromic gene amplification shaped the amplified ERBB2 locus. The enrichment of palindromic DNA throughout the amplified segments leads us to propose that the ERBB2 locus is amplified through the mechanism that repeatedly generates palindromic DNA, such as Breakage-Fusion-Bridge cycles. The genomic architecture surrounding ERBB2 in the normal genome, such as segmental duplications, could promote the locus-specific mechanism.

Sex-related differences in long-term pulmonary outcomes of neonatal hyperoxia in mice.

AIM: Premature infants are often exposed to hyperoxia to maintain adequate oxygenation, which may lead to the development of bronchopulmonary dysplasia (BPD). Sex-specific differences exist in the development and severity of BPD. Only a few studies have examined the mechanisms underlying these sex-related differences. The aim of the present study is to examine the sex-related long-term effects of neonatal hyperoxia on the lungs of adult mice. MATERIALS AND METHODS: Newborn mice were exposed to 95% oxygen (hyperoxia) for 96 hours and were allowed to recover in room air to adulthood (8 weeks of age). Lung tissues were excised at 4 days, 14 days, or 8 weeks of age. Short-term effects of neonatal hyperoxia on the mouse lung and sex-related differences in pulmonary function, airway hyper-responsiveness, and lung structure in adult mice were assessed. RESULTS: Neonatal hyperoxia was found to have no differential effect on body weight, muscarinic acetylcholine receptor gene expression, or bronchiolar epithelial thickness in adult mice. Respiratory resistance was increased and sensitivity to methacholine was decreased in male adult mice following exposure to neonatal hyperoxia, whereas delayed alveolarization was observed in female adult mice following exposure to neonatal hyperoxia. CONCLUSIONS: The findings of the present study demonstrate that neonatal hyperoxia differentially affects pulmonary outcome in female and male adult mice.

[Laparoscopic Gastrectomy for Gastric Adenocarcinoma of the Fundic Gland Type - Report of a Case].

A 69-year-old man underwent esophagogastroduodenoscopy, which showed a slightly depressed lesion at the greater curvature of the gastric body. We diagnosed gastric adenocarcinoma of the fundic gland type(GA-FG)from examination of the biopsy specimen. Endoscopic submucosal dissection(ESD)was performed for curative resection. The pathological examination revealed a positive vertical margin. Consequently, laparoscopic gastrectomy was additionally performed. GA-FG has recently been proposed as a new entity of gastric adenocarcinoma. GA-FG mostly develops without Helicobacter pylori infection and often invades the submucosa, regardless of size. However, GA-FG rarely demonstrates lymphatic and venous invasion despite deep submucosal invasion. Since most GA-FG cases undergo ESD, few reports of surgical resection exist. Here, we report our experience of laparoscopic gastrectomy for GA-FG.

Replication fork integrity and intra-S phase checkpoint suppress gene amplification.

Gene amplification is a phenotype-causing form of chromosome instability and is initiated by DNA double-strand breaks (DSBs). Cells with mutant p53 lose G1/S checkpoint and are permissive to gene amplification. In this study we show that mammalian cells become proficient for spontaneous gene amplification when the function of the DSB repair protein complex MRN (Mre11/Rad50/Nbs1) is impaired. Cells with impaired MRN complex experienced severe replication stress and gained substrates for gene amplification during replication, as evidenced by the increase of replication-associated single-stranded breaks that were converted to DSBs most likely through replication fork reversal. Impaired MRN complex directly compromised ATM/ATR-mediated checkpoints and allowed cells to progress through cell cycle in the presence of DSBs. Such compromised intra-S phase checkpoints promoted gene amplification independently from mutant p53. Finally, cells adapted to endogenous replication stress by globally suppressing genes for DNA replication and cell cycle progression. Our results indicate that the MRN complex suppresses gene amplification by stabilizing replication forks and by securing DNA damage response to replication-associated DSBs.

Homology-mediated end-capping as a primary step of sister chromatid fusion in the breakage-fusion-bridge cycles.

Breakage-fusion-bridge (BFB) cycle is a series of chromosome breaks and duplications that could lead to the increased copy number of a genomic segment (gene amplification). A critical step of BFB cycles leading to gene amplification is a palindromic fusion of sister chromatids following the rupture of a dicentric chromosome during mitosis. It is currently unknown how sister chromatid fusion is produced from a mitotic break. To delineate the process, we took an integrated genomic, cytogenetic and molecular approach for the recurrent MCL1 amplicon at chromosome 1 in human tumor cells. A newly developed next-generation sequencing-based approach identified a cluster of palindromic fusions within the amplicon at ∼50-kb intervals, indicating a series of breaks and fusions by BFB cycles. The physical location of the amplicon (at the end of a broken chromosome) further indicated BFB cycles as underlying processes. Three palindromic fusions were mediated by the homologies between two nearby inverted Alu repeats, whereas the other two fusions exhibited microhomology-mediated events. Such breakpoint sequences indicate that homology-mediated fold-back capping of broken ends followed by DNA replication is an underlying mechanism of sister chromatid fusion. Our results elucidate nucleotide-level events during BFB cycles and end processing for naturally occurring mitotic breaks.

Gene amplification system based on double rolling-circle replication as a model for oncogene-type amplification.

Gene amplification contributes to a variety of biological phenomena, including malignant progression and drug resistance. However, details of the molecular mechanisms remain to be determined. Here, we have developed a gene amplification system in yeast and mammalian cells that is based on double rolling-circle replication (DRCR). Cre-lox system is used to efficiently induce DRCR utilizing a recombinational process coupled with replication. This system shows distinctive features seen in amplification of oncogenes and drug-resistance genes: (i) intra- and extrachromosomal amplification, (ii) intensive chromosome rearrangement and (iii) scattered-type amplification resembling those seen in cancer cells. This system can serve as a model for amplification of oncogenes and drug-resistance genes, and improve amplification systems used for making pharmaceutical proteins in mammalian cells.

A novel gene amplification system in yeast based on double rolling-circle replication.

Gene amplification is involved in various biological phenomena such as cancer development and drug resistance. However, the mechanism is largely unknown because of the complexity of the amplification process. We describe a gene amplification system in Saccharomyces cerevisiae that is based on double rolling-circle replication utilizing break-induced replication. This system produced three types of amplification products. Type-1 products contain 5-7 inverted copies of the amplification marker, leu2d. Type-2 products contain 13 to approximately 100 copies of leu2d (up to approximately 730 kb increase) with a novel arrangement present as randomly oriented sequences flanked by inverted leu2d copies. Type-3 products are acentric multicopy minichromosomes carrying leu2d. Structures of type-2 and -3 products resemble those of homogeneously staining region and double minutes of higher eukaryotes, respectively. Interestingly, products analogous to these were generated at low frequency without deliberate DNA cleavage. These features strongly suggest that the processes described here may contribute to natural gene amplification in higher eukaryotes.