Breakpoint Analysis Research Articles

Abstract B092: Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy

Abstract Background: Focal high copy number oncogene amplification frequently occurs on extrachromosomal DNA (ecDNA), acentric circular DNA derived from chromosomes during neoplastic transformation and tumor progression. The unique properties of ecDNA facilitate a high degree of genomic plasticity and tumor heterogeneity, which enables rapid acquired resistance to targeted therapies and results in poor patient outcomes. Reintegrated ecDNA, also referred to as homogeneously staining regions (HSR), can exist in dynamic equilibrium with their circular form, although their potential adaptive and resistance properties have not been well characterized. Elucidating the genomic plasticity and mechanisms afforded to cancer cells harboring HSR may provide insights into new therapeutic approaches necessary to address this ecDNA-derived focal amplification. Methods: A novel model was developed that recapitulates the genomic plasticity of focal oncogene amplifications involving FGFR2 and EGFR in response to the FGFR inhibitor, infigratinib. Results: KATOIII gastric cancer cells harbor focal amplification of the FGFR2 oncogene (absolute copy number (CN) = 136 based on next-generation sequencing (NGS) that resides on an HSR as determined by FISH. Short- and long-read NGS revealed prior DNA breakpoints consistent with circular structures containing FGFR2, suggestive of reintegrated ecDNA as HSR. Despite initial sensitivity to FGFR2 inhibition (EC50 = 20 nM), cells acquired resistance following infigratinib dose escalation within 12 weeks. Infigratinib-resistant KATOIII cells retained the FGFR2 HSR while further amplifying FGFR2 on ecDNA, which correlated with increased FGFR2 CN, mRNA, and protein expression. Structural breakpoint analysis of the FGFR2 amplicon revealed conservation between baseline and ecDNA-amplified infigratinib resistant cells, suggesting that the HSR gave rise to ecDNA. Removal of infigratinib resulted in near-complete disappearance of FGFR2 ecDNA and return to near-baseline FGFR2 CN, protein expression, and signaling within 3 weeks. Strikingly, at high infigratinib concentrations that effectively inhibit FGFR2 signaling, cells rapidly switched to EGFR amplified on ecDNA, with concomitant reduction of FGFR2 ecDNA (CN = 553 to 238) and signaling and increased EGFR (CN = 3 to 30), mRNA, and EGFR signaling. Following ecDNA switching from FGFR2 to EGFR, KATOIII cells remained insensitive to infigratinib while acquiring sensitivity to the EGFR inhibitor erlotinib. Mutational analysis revealed no new oncogenic mutations including FGFR2 and EGFR, indicating the EGFR overexpression afforded by ecDNA was sufficient to drive acquired resistance to high infigratinib concentrations. Conclusions: These findings indicate that tumor cells with ecDNA-derived chromosomally reintegrated HSR retain the capacity to amplify ecDNA, thus enabling rapid adaptation to therapeutic pressure. Consequently, new treatment strategies that disable adaptive genomic plasticity are needed to address oncogene amplified cancers that leverage ecDNA biology. Citation Format: Kristen Turner, Homa Hemmati, Cory DuPai, John Bibay, Claire Klebs, Joan Chen, Debbie Liao, Christian Hassig, Shailaja Kasibhatla. Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B092.

Mercury deposition to lake sediments near historic gold mines in northern Canada

Mercury (Hg) contamination in aquatic systems can lead to adverse human and environmental health outcomes. Yellowknife, a city in Canada’s Northwest Territories, is a historic mining community, with two large gold mines (Giant Mine and Con Mine) that used Hg amalgamation methods to extract gold between ∼1938 and 1960. We analyzed dated sediment cores from 20 small lakes to investigate the spatial and temporal Hg deposition patterns within 50 km of Giant Mine. Breakpoint analysis of the within-lake z-score normalized anthropogenic Hg flux indicates two significant time periods of changing emission rates. The first is a significant increase in Hg deposition rate (∼1925) during the time of gold exploration in the region and onset of Hg amalgamation (1938) and the second is a significant decrease in deposition rate that begins around the time of the cessation of Hg amalgamation at Giant Mine (∼1959). Sediment Hg concentrations exceeded the Canadian Council for Ministers of the Environment Interim Sediment Quality Guideline (ISQG) for Hg (0.17 mg/kg dw) in 55% of the lakes (n = 11) during mining (1948–1999). All lakes within 5 km of the Giant Mine roaster stack exceeded CCME ISQG during mining (n = 8), with a 4-fold increase in total Hg concentration observed during mining at these near-field (<5 km from stack) sites. We observed evidence of enriched Hg in near-field, mid-field, and far-field sites. The elevated sedimentary Hg concentrations during mining in near-field sites would have posed a hazard to human and wildlife health during the height of emissions, however the significant decrease in Hg concentrations since the closure of mines in the region demonstrate the potential for recovery in these aquatic ecosystems.

AML with t(4;12)(q12;p13): A Detailed Genomic and Transcriptomic Analysis Reveals Genomic Breakpoint Heterogeneity, Absence of Pdgfra Fusion Transcripts and Presence of Pdgfra Overexpression in a Subset of Cases

Background: Gene fusions are a frequent event in acute myeloid leukaemia (AML). It is estimated that about 40% of AML patients harbour a cytogenetic abnormality resulting in a gene fusion. However, AML with t(4;12)(q12;p13) is very rare, but it is often accompanied by an aggressive clinical course and a poor prognosis. As frequently indicated by fluorescence in situ hybridization analyses (FISH), patients are assumed to harbor an ETV6::PDGFRA fusion, which impacts the choice of therapy, as these patients can be treated with tyrosine kinase inhibitors. The genomic region on chromosome 4 involved in ETV6 rearrangements is densly packed with genes in close spatial proximity. Therefore, the resolution of chromosome banding analysis and also FISH is not sufficient to fully elucidate the fusion partners involved. Recent studies have pointed out that many of these patients do not harbor an ETV6::PDGFRA fusion but other rather unknown fusion partners have been elucidated. Aim: (1) Evaluation of the genomic breakpoints in patients with t(4;12)(q12;p13) ETV6-rearranged AML; (2) determine the alteration of PDGFRA expression in patients with t(4;12). Patients and Methods: 10 patients (F: 4, M: 6; median age: 63 [44-82] years) with a t(4;12)(q12;p13) indicated by FISH and chromosome analysis were included. Samples were analysed by whole genome and whole transcriptome sequencing (WGS/WTS). Sequencing was performed on NovaSeq instrument (Illumina). Fusions were called with Arriba, STAR-Fusion and Manta. For gene expression analysis WTS data were log2 transformed and normalized with the trimmed mean of M-values (TMM) method. Results: Based on chromosome analysis and FISH patients showed break events in 4q12 and 12p13. In all cases a FISH signal constellation typical for an ETV6 rearrangement was observed. The mutation spectrum for each patient of the most commonly mutated genes in AML was examined by use of WGS data. The median number of mutations was 3, with mutations in ASXL1 being the most common (6 patients) and none of the patients featuring mutations in the PDGFRA gene. WTS analysis revealed in-frame fusions in 6 of 10 patients involving genes located on chromosomes 4 and 12 (4 patients with ETV6::CHIC2, 1 patients with ETV6::SCFD2, 1 patient with ETV6::ADAMTS3). None of the 10 examined patients showed a gene fusion, or a fusion transcript involving PDGFRA. Based on WTS data, 7 of the 10 patients showed a PDGFRA overexpression. WGS breakpoint analysis showed that genetic breakpoints on chromosome 12 cluster in introns 1 (4 patients) and 2 (6 patients) of the ETV6 gene. In contrast chromosome breaks on chromosome 4 are heterogeneously distributed in the 4q12 region. Breakpoint comparison indicated that patients with chromosome breaks in a range from chr4:54,804,230-54,962,206 (7 patients) display PDGFRA overexpression. In contrast patients with chromosome breaks further downstream chr4:55,021,511-73,409,362 (3 patients) did not exhibit an increased PDGFRA expression. This suggests an ectopic expression of PDGFRA triggered by the translocation and independent of the existence of an in-frame fusion transcript. A chromosome break in the range of chr4:54,804,230-54,962,206 (seen in 7 patients) might result in the inactivation of a CTCF binding site (chr4:54,796,345-54,796,360) upstream of these chromosome breaks that is already known to induce PDGFRA overexpression when disrupted. Conclusions: Analysis of AML with t(4;12)(q12;p13) translocation by WGS and WTS provides detailed information that separates two subsets distinguished by distinct breakpoint cluster regions on 4q12 and PDGFRA gene expression. The clinical impact of increased PDGFRA gene expression on response to TKI and prognosis as well as the presence of additional fusion transcripts has to be evaluated in further studies.

216 Effect of Dietary Standardized Ileal Digestible Lysine Level on Growth Performance of 12.4 to 31.3 kg Pigs Sired by PIC 800 Boars

Abstract A total of 1,156 PIC 800-sired pigs (12.4 kg BW) were used in a 27-day experiment to estimate the SID lysine requirement of pigs ranging from 12.4 to 31.3 kg BW. Forty-eight pens of mixed sex pigs were used. Pens were blocked by location within the barn, with barrow and gilt numbers in pens kept consistent within block. Pens were allotted to one of five dietary treatments in a randomized design with 9 to 10 replicate pens (10 pens for treatments 1, 3, 5 and 9 pens for treatments 2, 4) per treatment. Dietary SID lysine concentrations evaluated were 1.12, 1.19, 1.26, 1.33, and 1.40%. Diets were corn-soybean meal-based and formulated to equal NE concentrations across treatment. Data were analyzed using a general linear model. Orthogonal contrasts with coefficients for equally spaced treatments were used to analyze for linear and quadratic effects. For significant quadratic effects, breakpoint analysis was conducted (two slope regression or quadratic broken line analysis) using nonlinear regression analysis to estimate for SID lysine requirement. A linear (P &amp;lt; 0.05) response was observed for BW to increase as SID lysine level in the diet increased. Increasing SID lysine increased average daily gain (ADG) quadratically (P = 0.05) with a plateau observed above 1.357% SID Lys. Additionally, increased SID lysine yielded a quadratic effect on G:F (P &amp;lt; 0.05) with a plateau above 1.327% SID lysine. There was a quadratic response (P &amp;lt; 0.05) observed for cost of gain (COG), with no benefit to COG when feeding above 1.201% SID lysine. However, there was a linear response (P &amp;lt; 0.05) observed for return over feed cost (ROFC) as SID lysine increased due to increased BW. There was no effect of SID lysine level fed on feed intake or mortality and morbidity. These data indicate that the estimated SID lysine requirement for PIC 800-sired pigs ranging from 12.4 to 31.3 kg to be 1.327 and 1.357 for optimal feed efficiency and ADG, respectively.

Lake water chemistry and its relationship to shoreline residential development and natural landscape features in Algonquin Provincial Park, Ontario, Canada

There is a scarcity of long-term chemical monitoring data for lakes in Algonquin Provincial Park (APP), with minimal understanding of the impacts of cottage-leases (e.g., cottage lots, campgrounds, and commercial leases) on lake water chemistry. We examine spatial patterns in water chemistry and landscape features of 32 reference and 22 cottage-lease lakes in APP. Multivariate techniques were used to examine differences in water chemistry, and to identify the subset of landscape features that best explain this variability. Breakpoint analysis was used to examine the relationship between gradients of water chemistry and specific landscape features. Lakes were separated along a primary gradient of ions and pH and a secondary gradient of nutrients and colour. These gradients were best explained by a combination of six landscape features (wetlands, elevation, lake depth, road length, and coniferous trees). Except for chloride, there was no statistically significant difference in water chemistry between cottage-lease and reference lakes. A roughly west-to-east gradient in catchment vegetation and lake chemistry was related to the location of the Algonquin Dome, a natural geological feature in APP, and the park’s glacial history. These results emphasize the importance of the park’s topography in influencing regional water chemistry.

Effect of CITES Appendix I listing on illegal pangolin trade as gauged from seizure reports in Indonesia

Pangolins, one of the most heavily trafficked animals, have become the poster child for the illegal wildlife trade. Pangolins are protected throughout their range and their international trade has been regulated through the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Assessing the effectiveness of trade regulations, such as the listing of all pangolins on CITES Appendix I in 2016 precluding all international trade, can be done by analysis of seizure reports. I report 81 large seizures within Indonesia over the last 12 years, the equivalent of 18,024 pangolins representing a value of USD9.42 million. In Indonesia, the number of seizures remained stable at around seven a year. There was a significant decrease in the number of seized pangolin equivalents, from 3000 to 4000 in 2011 and 2012 to 400 to 600 in 2021 and 2022. A break point analysis showed that any changes in this trend did not coincide with changes in international trade regulations. Seizures were made in 17/22 provinces where pangolins occur. At the provincial level seizures or the number of confiscated pangolins was not linked to minimum wage, area, human population, or international connectiveness. Despite the international nature of the pangolin trade, where reported, suspects that were arrested were Indonesian nationals. Over time the proportion of seizures comprising solely of scales has increased from ~ 5 to 100%. These observations of seizures of large amounts of pangolin scales, in the absence of bodies, carcasses, or meat, suggest there is a hidden trade in these parts, in Indonesia and/or elsewhere. There is no clear support that the CITES Appendix I listing had a direct effect on the pangolin trade within Indonesia.

The nexus between COVID-19 and finfish export from Sri Lanka to its main trading partners; the United States and the European Union

The Sri Lankan seafood sector has been seriously disturbed during the Covid 19 pandemic due to a bleak demand outlook as well as an array of supply challenges. Identification of the effects of this new challenge to the global fish trade is critical to take actions to sustain the market and manage resilience in such uncertain periods. Study aims to assess the impact of the Covid-19 outbreak on Sri Lanka's finfish exports to its key trading partners, the United States of America (USA) and the European Union (EU). The time series data were gathered from the United Nations commodity trade database between January 2017 and December 2022. Study used structural break point analysis of time series data, followed by time series regression with the Covid-19 impact as a dummy variable using EVIEWS statistical software. Compared to 2019, total finfish exports to the United States dropped by 56.5% in 2020 and 72.5% in 2021 while finfish exports to European market increased by 7% and 28% in 2020 and 2021, respectively. The Covid-19 dummy was statistically significant in the time series regression, with a coefficient value of -71.93 in USA trade flow which implies that Covid-19 has a negative impact on finfish trade to the USA. In contrast, statistically significant impact was not found in finfish trade to the EU during the pandemic and this mainly because European seafood market impacted due to Covid-19 yet imports from developing countries fell slightly and overall demand for seafood has remained largely stable throughout 2020. However, study suggested that future research should focus on the country-specific product sensitivity at the individual product level.

Whole genome characterization, recombination, variation, satellites and phylogeny analyses of Tomato leaf curl Palampur virus infecting courgetti squash (Cucurbita pepo L.) in Iraq

Durning season of 2021-2022, symptoms of leaf curl, yellowing, crumpling and stunting were observed commonly on courgetti crop in Al-Yusufiyah region, Baghdad Province, Iraq. Diseased courgetti leaves were collected randomly and sent for sequencing with Illumina MiSeq. Pairwise nucleotide analysis of the assembled contigs revealed the maximum identity of 98-99% and 95-99% with Tomato leaf curl Palampur virus (ToLCPalV) for DNA-A and DNA-B components, respectively. The DNA-A component (OQ693629.1) contains six main genes encoding six proteins, while DNA-B component (OQ693630.1) comprises only two genes encoding two proteins, with the presence of non-coding regions between genes in both the two segments. Furthermore, the recombination breakpoint analysis shows that ToLCPalV Baghdad-1/Iraq segment DNA-A (OQ693629.1) and ToLCPalV isolate Mosul segment A (OR052249.1) are the parent of the recombinant isolates: ToLCPalV isolate Kufa segment DNA-A (OP810506.1) and ToLCPalV segment A (ON254271.1) from Karbala. However, the ToLCPalV Baghdad-1/Iraq segment DNA-B (OQ693630.1) with ToLCPalV segment DNA-B (OP620406.1) from Kufa are the parent of ToLCPalV isolate Babylon1 segment DNA-B (ON229620.1). As well as, there was no evidence discovered for the association of alpha- and/or beta-satellites with ToLCPalV infecting courgetti crop. Single nucleotide polymorphisms (SNP) analysis indicates a common genetic variation within the current isolate under study and the other Iraqi isolates. Phylogenetic analysis confirms the genetic relationship between the genomics components of Iraqi ToLCPalV and various international isolates of the same virus, especially those distributed in Saudi Arabia and Iran that could be a possible the origin and introduction of ToLCPalV into Iraq.&#x0D;

Trends in Opioid Medication Adherence During the COVID-19 Pandemic: Retrospective Cohort Study.

The recent pandemic had the potential to worsen the opioid crisis through multiple effects on patients' lives, such as the disruption of care. In particular, good levels of adherence with respect to medication for opioid use disorder (MOUD), recognized as being important for positive outcomes, may be disrupted. This study aimed to investigate whether patients on MOUD experienced a drop in medication adherence during the recent COVID-19 pandemic. This retrospective cohort study used Medicaid claims data from 6 US states from 2018 until the start of 2021. We compared medication adherence for people on MOUD before and after the beginning of the COVID-19 pandemic in March 2020. Our main measure was the proportion of days covered (PDC), a score that measures patients' adherence to their MOUD. We carried out a breakpoint analysis on PDC, followed by a patient-level beta regression analysis with PDC as the dependent variable while controlling for a set of covariates. A total of 79,991 PDC scores were calculated for 37,604 patients (age: mean 37.6, SD 9.8 years; sex: n=17,825, 47.4% female) between 2018 and 2021. The coefficient for the effect of COVID-19 on PDC score was -0.076 and was statistically significant (odds ratio 0.925, 95% CI 0.90-0.94). The COVID-19 pandemic was negatively associated with patients' adherence to their medication, which had declined since the beginning of the pandemic.

The Precipitation Concentration in the Cheliff Watershed, Algeria: A Critical Analysis for Sustainable Water Resource Management

In this study, a statistical analysis of historical and projected values of the annual, seasonal, and supra-seasonal precipitation concentration index (PCI) was conducted for different stations in the Cheliff watershed. Statistical methods such as breakpoint and trend analysis using Pettitt and Mann–Kendall tests were used for the PCI values of annual, monthly, and supra-seasonal precipitation data between 1950 and 2014. The PCI values varied between 14.24% and 24.86% annually, between 10.75% and 18.74% seasonally, and between 11.23% and 26.98% supra-seasonally. According to the Pettitt and Mann–Kendall tests, there was an insignificant change in precipitation distribution during the study period. Spatial analysis using the inverse distance weighting (IDW) method confirmed the minor variability in precipitation distribution in the study area. The Pearson correlation coefficient between oscillation indices and PCI values at different scales showed significant values with the Mediterranean Oscillation index (MOi), North Atlantic Oscillation (NAO), and Western Mediterranean Oscillation index (WeMOi), highlighting their potential influence on annual PCI values. For the climate projection scenarios, projected PCI values align with historical PCI values. Application of Pettitt and Mann-Kendall tests to the projected PCI series shows that there will not be a significant change in future precipitation distribution.

Design of a Genetic Algorithm Based Dynamic Learning Method for Improved Channel Modelling in mmWave Radios via Temporal Breakpoint Analysis

Design of a Genetic Algorithm Based Dynamic Learning Method for Improved Channel Modelling in mmWave Radios via Temporal Breakpoint Analysis

Cost-effectiveness analysis of antibiotic prophylaxis versus no antibiotic prophylaxis for acute cholecystectomy

ObjectiveFor acute cholecystitis, the treatment of choice is laparoscopic cholecystectomy. In mild-to-moderate cases, the use of antibiotic prophylaxis for the prevention of postoperative infectious complications (POICs) lacks evidence regarding its...

Relationship between meteorological factors and mortality in patients with coronavirus disease 2019: A cross-sectional study

BackgroundRecent studies on COVID-19 have demonstrated that poverty, comorbidities, race/ethnicity, population density, mobility, hygiene and use of masks are some of the important correlates of COVID-19 outcomes. In fact, weather conditions also play an important role in enhancing or eradicating health issues. Based on Chinese experience, the development of SARS and COVID-19 is partially associated with alterations in climate that align with the seasonal shifts of the “24 solar terms.” However, the applicability of this pattern to other countries, particularly the United States, which has the highest global incidence and mortality rates, remains subject to ongoing investigation. We need to find more evidence to in the U.S. states verify the relationship between meteorological factors and COVID-19 outcomes to provide epidemiological and environmental support for the COVID-19 pandemic prevention and resource preservation. ObjectiveTo evaluate the relationship between meteorological factors and Coronavirus Disease 2019 (COVID-19) mortality. MethodsWe conducted an ecological cross-sectional study to evaluate the relationship between meteorological factors (maximum temperature, minimum temperature, humidity, wind speed, precipitation, atmospheric pressure) and COVID-19 mortality. This retrospective observational study examines mortality rates among COVID-19 patients in the three US states, California, Texas, and New York, with the highest fatality numbers, between March 7, 2020 and March 7, 2021. The study draws upon data sourced from the publicly accessible Dryad database. The daily corresponding meteorological conditions were retrieved from the National Oceanic and Atmospheric Administration Global Meteorological website (https://www.ncei.noaa.gov/maps/hourly/). This study employed multivariate linear regression analysis to assess the correlation between six meteorological factors and COVID-19 mortality. Gaussian distribution models were utilized to generate smooth curves for examining the linear association between maximum or minimum temperature and mortality. Additionally, breakpoint analysis was conducted to evaluate the threshold effect of temperature. ResultsWe found that the death toll of patients with COVID-19 decreased with an increase in the highest and lowest ambient temperatures (p < 0.001). In our study, we observed a seasonal difference in mortality rates, with a higher number of deaths occurring during winter months, particularly in January and February. However, mortality rates decreased significantly in March. Notably, we found no statistically significant correlation between relative humidity, average precipitation, and average wind speed with COVID-19 mortality (all p > 0.05). Daily COVID-19 death was negatively correlated with the maximum temperature (β = −22, 95% CI, −26.2 to −17.79 –, p < 0.01), while the maximum temperature was below 30 °C. Similarly, the number of deaths was negatively correlated with the minimum temperature (β = −27.46, 95% CI, −31.48 to −23.45, p < 0.01), when the minimum temperature was below 8 °C. Our study found a significant association between temperature and COVID-19 mortality, with every 1 °C increase in maximum or minimum temperature resulting in a decrease of 22 and 27 deceased cases, respectively. The relationship between atmospheric pressure and COVID-19 mortality was not fully elucidated due to its complex interaction with maximum temperature. ConclusionsThis empirical study adds to the existing body of research on the impact of climate factors on COVID-19 prevention and resource allocation. Policymakers and health scientists may find these findings useful in conjunction with other social factors when making decisions related to COVID-19 prevention and resource allocation.

Survival rates and mortality risks of Plecturocebus cupreus at the California National Primate Research Center.

This article describes survivorship and explores factors affecting mortality risks in a captive colony of coppery titi monkeys (Plecturocebus cupreus) housed at the California National Primate Research Center (CNPRC), at UC Davis, in Davis, CA. We analyzed data collected on individuals since the colony's creation in the 1960s, with a sample of 600 animals with partially complete information (date of birth, age at death, body mass, parental lineage). We used three methods: (1) Kaplan-Meier regressions followed by a log-rank test to compare survival in male and female titi monkeys, (2) a breakpoint analysis to identify shifts in the survival curves, and (3) Cox regressions to test the effect of body mass change, parental pair tenure, and parental age on mortality risk. We found that males tend to have a longer median lifespan than females (14.9 and 11.4 years; p = 0.094) and that survival decreases earlier in males than in females during adulthood (9.8 and 16.2 years). A body mass loss of 10% from adulthood to the time of death led to a 26% higher risk of dying (p < 0.001) as compared to an individual with stable body mass. We found no evidence of sociobiological factors on mortality risks (parental age, parental pair tenure), but an exploratory analysis suggested that a higher rate of offspring conceptions increases mortality risks. This description of factors influencing survival and mortality in titi monkeys is a first step toward understanding aging in this species to consider titi monkeys as a primate model for socioemotional aging.

Genomic breakpoint-specific monitoring of measurable residual disease in pediatric non-standard-risk acute myeloid leukemia.

Pediatric acute myeloid leukemia (AML) is a highly heterogeneous disease making standardized measurable residual disease (MRD) assessment challenging. Currently, patient-specific DNA-based assays are only rarely applied for MRD assessment in pediatric AML. We tested whether quantification of genomic breakpoint-specific sequences via quantitative polymerase chain reaction (gDNA-PCR) provides a reliable means of MRD quantification in children with non-standardrisk AML and compared its results to those obtained with state-of-the-art ten-color flow cytometry (FCM). Breakpointspecific gDNA-PCR assays were established according to Euro-MRD consortium guidelines. FCM-MRD assessment was performed according to the European Leukemia Network guidelines with adaptations for pediatric AML. Of 77 consecutively recruited non-standard-risk pediatric AML cases, 49 (64%) carried a chromosomal translocation potentially suitable for MRD quantification. Genomic breakpoint analysis returned a specific DNA sequence in 100% (41/41) of the cases submitted for investigation. MRD levels were evaluated using gDNA-PCR in 243 follow-up samples from 36 patients, achieving a quantitative range of at least 10-4 in 231/243 (95%) of samples. Comparing gDNA-PCR with FCM-MRD data for 183 bone marrow follow-up samples at various therapy timepoints showed a high concordance of 90.2%, considering a cut-off of ≥0.1%. Both methodologies outperformed morphological assessment. We conclude that MRD monitoring by gDNA-PCR is feasible in pediatric AML with traceable genetic rearrangements and correlates well with FCM-MRD in the currently applied clinically relevant range, while being more sensitive below that. The methodology should be evaluated in larger cohorts to pave the way for clinical application.

P-721 Nanopore sequencing for detecting reciprocal translocation carrier status in preimplantation genetic testing

Abstract Study question Analyze the concordance between nanopore sequencing and MaReCs to validate the feasibility of nanopore sequencing in distinguishing normal embryos from carrier embryos in PGT-SR cycles. Summary answer Nanopore sequencing is a powerful strategy for accurately distinguishing nontranslocation embryos from translocation carrier embryos and precisely localizing translocation breakpoints. What is known already Balanced reciprocal translocation (BRT) is one of the most common chromosomal abnormalities that causes infertility, recurrent miscarriage, and birth defects. Preimplantation genetic testing (PGT) is widely used to select euploid embryos for BRT carriers to increase the chance of a healthy live birth. Several strategies can be used to distinguish reciprocal translocation carrier embryos from those with a normal karyotype; however, these techniques are time-consuming and difficult to implement in clinical laboratories. Study design, size, duration The Nanopore Sequencing was performed on two pregnant patients conducted on Mapping Allele with Resolved Carrier Status” (MaReCs) in PGT-SR cycles to identify translocation breakpoints for a year. Combined conventional MaReCs ’results, amniocentesis was performed to identify karyotypes at 18–20 weeks of gestation to verify the correctness of the Nanopore Sequencing. Participants/materials, setting, methods A total of 11 embryos from 2 couple1 and 12 embryos from couple 2 were tested simultaneously in PGT-SR cycles with a combination strategy of mate pair sequencing and PCR breakpoint analysis, SNP array-based comprehensive chromosome screening (CCS), NGS following micro-dissecting junction region (Micro-Seq) to distinguish between normal and carrier embryos. The Nanopore Sequencing based on TGS was performed on 2 couples. Lastly, amniocentesis was performed to test karyotypes. Main results and the role of chance The translocation breakpoints in both reciprocal translocation carriers were accurately identifed by nanopore sequencing and were in accordance with the results obtained using MaReCs. More than one euploid non-balanced translocation carrier embryo was identified in both patients. Amniocentesis results revealed normal karyotypes, consistent with the findings by MaReCs and nanopore sequencing. Limitations, reasons for caution One limitation is that it is not possible to identify the translocation breakpoints of Robertsonian translocation carriers or those located in the gap regions of the human genome. In addition, high-molecular-weight genomic DNA and large amounts of peripheral blood samples would be a challenge most PGT centers. Wider implications of the findings The nanopore sequencing accurately detects translocation breakpoints in BRT carriers and distinguishes translocation-free embryos from balanced diploid embryos in clinical PGT-SR cycles. It is superior to short reads (NGS) for the detection of translocation breakpoints, which shows broad prospects for clinical applications in blocking translocation propagation in the population. Trial registration number Not applicable

Long-read genome sequencing identifies cryptic structural variants in congenital aniridia cases

BackgroundHaploinsufficiency of the transcription factor PAX6 is the main cause of congenital aniridia, a genetic disorder characterized by iris and foveal hypoplasia. 11p13 microdeletions altering PAX6 or its downstream regulatory region (DRR) are present in about 25% of patients; however, only a few complex rearrangements have been described to date. Here, we performed nanopore-based whole-genome sequencing to assess the presence of cryptic structural variants (SVs) on the only two unsolved “PAX6-negative” cases from a cohort of 110 patients with congenital aniridia after unsuccessfully short-read sequencing approaches.ResultsLong-read sequencing (LRS) unveiled balanced chromosomal rearrangements affecting the PAX6 locus at 11p13 in these two patients and allowed nucleotide-level breakpoint analysis. First, we identified a cryptic 4.9 Mb de novo inversion disrupting intron 7 of PAX6, further verified by targeted polymerase chain reaction amplification and sequencing and FISH-based cytogenetic analysis. Furthermore, LRS was decisive in correctly mapping a t(6;11) balanced translocation cytogenetically detected in a second proband with congenital aniridia and considered non-causal 15 years ago. LRS resolved that the breakpoint on chromosome 11 was indeed located at 11p13, disrupting the DNase I hypersensitive site 2 enhancer within the DRR of PAX6, 161 Kb from the causal gene. Patient-derived RNA expression analysis demonstrated PAX6 haploinsufficiency, thus supporting that the 11p13 breakpoint led to a positional effect by cleaving crucial enhancers for PAX6 transactivation. LRS analysis was also critical for mapping the exact breakpoint on chromosome 6 to the highly repetitive centromeric region at 6p11.1.ConclusionsIn both cases, the LRS-based identified SVs have been deemed the hidden pathogenic cause of congenital aniridia. Our study underscores the limitations of traditional short-read sequencing in uncovering pathogenic SVs affecting low-complexity regions of the genome and the value of LRS in providing insight into hidden sources of variation in rare genetic diseases.

Exons 1–3 deletion in FLCN is associated with increased risk of pneumothorax in Chinese patients with Birt-Hogg-Dubé syndrome

BackgroundThe pathogenic variants responsible for Birt-Hogg-Dubé syndrome (BHDS) in folliculin (FLCN) gene mostly consist of point mutations. Although large intragenic deletions/duplications have been reported in several case reports, the relationship between large intragenic deletions/duplications and phenotype in BHDS remains unclear.MethodsWe retrospectively identified and reviewed patients with a large intragenic deletion spanning exons 1–3 and analyzed their phenotypic features to compare with those of point mutation carriers in our hospital from January 1, 2017 to August 31, 2022.ResultsTwenty unique point mutations (including 4 novel mutations) were detected in 62 patients from 45 families (90%). Exons 1–3 deletion were identified in 8 patients from 5 families (10%) that resided in the same region, Feidong County of Anhui Province, China. Breakpoint analysis indicated that all the deletion breakpoints were flanked by Alu repeats. The prevalence of exons 1–3 deletion carriers in Feidong County was 8.1-times higher than that for BHDS in Anhui Province, suggesting a clustered phenomenon of exons 1–3 deletion. Significantly increased risk of pneumothorax was observed in those with exons 1–3 deletion compared with point mutations (91% vs. 58%, p value 0.047). The risk of renal cancer may be higher in those with exons 1–3 deletion than for those with point mutations (18% vs. 4%, p > 0.05).ConclusionsLarge intragenic deletion of exons 1–3 in FLCN was identified as a local aggregation phenomenon in Feidong County, China, and was associated with a significantly higher risk of pneumothorax compared to those with point mutations.

An accurate fringe extraction model of small- and medium-sized urban areas using multi-source data

Urban fringes are of great significance to urban development as connecting hubs between urban and rural areas. However, there are many problems in urban fringes, including disorderly spatial layout, waste of social resources, and low quality of human settlements. Rapid and accurate identification of urban fringes has important practical significance for optimizing urban spatial layout, controlling urban unlimited expansion, and protecting land resources. Given the lack of suitable and high-quality fringe extraction models for small- and medium-sized urban areas, this study was based on Gaofen-2 (GF-2) imagery, Suomi National Polar-orbiting Partnership Visible Infrared Imager Radiometer Suite (NPP-VIIRS) imagery, point of interest (POI) data, and WorldPop data, taking the landscape disorder degree, POI kernel density, and night light intensity as urban feature factors and constructing a fringe extraction model of small- and medium-sized urban areas (FEM-SMU). Taking Hantai District in China as the study area, the results of the model were compared to the landscape disorder degree threshold method and POI kernel density breakpoint analysis method, while the generality of the model was further tested in Shangzhou and Hanbin Districts. The results show that the FEM-SMU model has evident improvements over the conventional methods in terms of accuracy, detail, and integrity, and has higher versatility, which can better meet the research needs of small- and medium-sized urban fringes.

Characterization of two novel HIV-1 second-generation recombinants (CRF01_AE/CRF07_BC) identified in Hebei Province, China.

The unique recombinant forms (URFs) of HIV-1 consist of a mixture of subtypes, and each URF has a unique breakpoint. In this study, we identified the near fulllength genome (NFLG) sequences of two novel HIV-1 URFs (Sample ID: BDD034A and BDL060) isolated during HIV-1 molecular surveillance in 2022 in Baoding city, Hebei Province, China. The two sequences were aligned with subtype reference sequences and CRFs from China using MAFFT v7.0, and the alignments were adjusted manually using BioEdit (v7.2.5.0). Phylogenetic and subregion trees were constructed using MEGA11 with the neighbor-joining (N-J) method. Recombination breakpoints were identified by SimPlot (v3.5.1) based on Bootscan analyses. Recombinant breakpoint analysis revealed that the NFLGs of BDD034A and BDL060 were composed of CRF01_AE and CRF07_BC, containing seven segments, respectively. For BDD034A, three CRF01_AE fragments were inserted into the CRF07_BC main framework, whereas for BDL060, three CRF07_BC fragments were inserted into the CRF01_AE main framework. The emergence of the CRF01_AE/CRF07_BC recombinant strains indicates that HIV-1 co-infection is common. The increasing genetic complexity of the HIV-1 epidemic in China warrants continued investigation.