Compared Copy Number Research Articles

Characterization of the Prenatal Ultrasound Phenotype Associated With 7q11.23 Microduplication Syndrome and Williams-Beuren Syndrome.

This study aimed to characterize the intrauterine phenotype of fetuses with 7q11.23 microduplication syndrome and Williams-Beuren syndrome (WBS) to provide insight into prenatal genotype and phenotype correlations in the 7q11.23 region. Seven fetuses with 7q11.23 microduplication syndrome and sixteen with WBS were diagnosed via array comparative genomic hybridization (array CGH) or copy number variation sequencing (CNV-seq) at our center. Clinical data were also systematically collected and analyzed, including intrauterine phenotype, pregnancy outcome, and inheritance. In our cases, the most common prenatal ultrasound feature of 7q11.23 microduplication syndrome was cardiovascular defects; less frequent features included choroid plexus cysts, anencephaly, bilateral pyelectasis, and cervical lymphatic hygroma. On the other hand, WBS was mainly associated with cardiovascular defects and intrauterine growth retardation. Other clinical phenotypes included hypoechoic frontal horn of the right lateral ventricle, crossed fused renal ectopia, hyperechogenic bowel, hyperechogenic right thoracic cavity, and hyperechogenic hepatic parenchyma/intrahepatic duct wall. Our study describes a series of new ultrasound features identified prenatally in fetuses with 7q11.23 microduplications and microdeletions with the intent of expanding the prenatal phenotype associated with copy number variants in this chromosomal region. Additional studies are needed to clearly delineate specific prenatal features associated with these rare genetic entities.

Comparison of Rodent Infectious Agent Detection by Exhaust Dust Testing and Traditional Sentinel Testing Using Quantitative Polymerase Chain Reaction.

Improved diagnostic capabilities and a desire to reduce or refine the use of animals as soiled bedding sentinels (SBS) have driven interest in developing the use of PCR-based testing methods, such as exhaust dust testing (EDT), for routine rodent health surveillance. We compared the absolute and quantitative PCR results from EDT filters with SBS mice by routine screening via a panel of 19 infectious agents including agents known to be excluded or present in the colony. In this study, EDT and SBS were compared at days 0, 90, and 180 in 3 facilities (n = 12 rooms) with animals housed on IVC racks (n = 19 double-sided and 23 single-sided racks). All racks were negative for excluded agents (n = 15 agents) during the study. The bacterial agent Helicobacter spp. was consistently detected on EDT filters while less consistently detected via SBS. EDT filters detected Corynebacterium bovis better than SBS in areas where the agent was present. EDT filters and SBS mice tested for murine norovirus (MNV) demonstrated agreement for positive tests by both PCR and serology. For rodent chaphamaparvovirus-1 (RCHPV-1) we compared EDT to urine and feces from SBS. Six cages of SBS were positive for RCHPV-1 by fecal PCR with 5 out of 6 testing positive on urine, while only 3 out of 6 EDT filters tested positive. Since real-time fluorogenic PCR was used for testing, relative PCR copy numbers for each positive finding were evaluated to estimate organism load at the rack level. Copy numbers allowed for further characterization of agent presence within a colony. Furthermore, we compared copy numbers with cage census for MNV and Helicobacter spp., which was positively correlated for EDT testing but not for SBS. Overall, our results demonstrate that EDT's ability to detect many commonly excluded agents is comparable to or better than SBS.

Evaluating clinically translatable conditioning for platelet gene therapy in murine hemophilia A with inhibitors

BackgroundPlatelet gene therapy is effective in hemophilia A (HA) mice even with inhibitors. Fludarabine (Flu), along with busulfan (Bu) or melphalan (Mel), preconditioning has been shown to be highly effective for hematopoietic stem cell transplantation in the clinic. ObjectivesTo evaluate the efficacy of Bu–Flu and Mel–Flu preconditioning in platelet gene therapy of HA with inhibitors. MethodsBu–Flu and Mel–Flu were used to condition HA mice preimmunized with recombinant human factor (F)VIII. An optimal 660 centigray total body irradiation was used as a control regimen in parallel. Platelet–FVIII expression was introduced by transplantation of 2bF8 lentivirus (LV)-transduced hematopoietic stem cells. Animals were analyzed by fluorescence-activated cell sorting, quantitative polymerase chain reaction, FVIII assays, and tail bleeding tests. ResultsBu–Flu, but not Mel–Flu, enabled successful 2bF8 gene therapy. All recipients achieved >55% chimerism post hematopoietic stem cell transplantation in both Bu–Flu and 660 centigray groups, with comparable copy numbers of 2bF8 cassette and the platelet–FVIII levels. The bleeding phenotype was rescued in 2bF8LV-transduced recipients. FVIII inhibitor titers declined with time, with comparable disappearance time of inhibitors between the 2 groups. When animals were rechallenged with recombinant human FVIII after the titers dropped to undetectable levels, no inhibitors were detected in 2bF8LV-transduced recipients. In contrast, all untransduced transplanted control mice produced inhibitors. These data demonstrate that immune tolerance was established in 2bF8LV-transduced primed HA mice under Bu–Flu conditioning. ConclusionBu–Flu preconditioning allows for successfully introducing platelet–FVIII expression to restore hemostasis and induce immune tolerance in primed HA mice, suggesting that this approach is a promising clinically translatable strategy for gene therapy of HA with inhibitors.

Intratumor genetic heterogeneity and head and neck cancer relapse

BackgroundHead and neck squamous cell carcinomas are treated by surgery, radiotherapy (RT), chemoradiotherapy (CRT) or combinations thereof, but locoregional recurrences (LRs) occur in 30–40% of treated patients. We have previously shown that in approximately half of the LRs after CRT, cancer driver mutations are not shared with the index tumor. AimTo investigate two possible explanations for these genetically unrelated relapses, treatment-induced genetic changes and intratumor genetic heterogeneity. MethodsTo investigate treatment-induced clonal DNA changes, we compared copy number alterations (CNAs) and mutations between primary and recurrent xenografted tumors after treatment with (C)RT. Intratumor genetic heterogeneity was studied by multi-region sequencing on DNA from 31 biopsies of 11 surgically treated tumors. ResultsInduction of clonal DNA changes by (C)RT was not observed in the xenograft models. Multi-region sequencing demonstrated variations in CNA profiles between paired biopsies of individual tumors, with copy number heterogeneity scores varying from 0.027 to 0.333. In total, 32 cancer driver mutations could be identified and were shared in all biopsies of each tumor. Remarkably, multi-clonal mutations in these same cancer driver genes were observed in 6 of 11 tumors. Genetically distinct heterogeneous cell cultures could also be established from single tumors, with different biomarker profiles and drug sensitivities. ConclusionIntratumor genetic heterogeneity at the level of the cancer driver mutations might explain the discordant mutational profiles in LRs after CRT, while there are no indications in xenograft models that these changes are induced by CRT.

Comparative Copy Number Variation Profiling of GL01, an Immortalized Non-small Cell Lung Cancer Cell Line Derived from a Filipino Patient, and A549 Lung Adenocarcinoma Cells

Comparative Copy Number Variation Profiling of GL01, an Immortalized Non-small Cell Lung Cancer Cell Line Derived from a Filipino Patient, and A549 Lung Adenocarcinoma Cells

Heterogeneity of DNA methylation profiles and copy number alterations in 10782 adult-type glioblastomas, IDH-wildtype.

The morphological patterns leading to the diagnosis of glioblastoma may also commonly be observed in several other distinct tumor entities, which can result in a mixed bag of tumors subsumed under this diagnosis. The 2021 WHO Classification of CNS Tumors has separated several of these entities from the diagnosis of glioblastoma, IDH-wildtype. This study determines the DNA methylation classes most likely receiving the diagnosis glioblastoma, IDH wildtype according to the definition by the WHO 2021 Classification and provides comparative copy number analyses. We identified 10782 methylome datasets uploaded to the web page www.molecularneuropathology.org with a calibrated score of ≥0.9 by the Heidelberg Brain Tumor Classifier version v12.8. These methylation classes were characterized by the diagnosis glioblastoma being the most frequent classification encountered in each of the classes according to the WHO 2021 definition. Further, methylation classes selected for this study predominantly contained adult patients. Unsupervised clustering confirmed the presence of nine methylation classes containing tumors most likely receiving the diagnosis glioblastoma, IDH-wildtype according to the WHO 2021 definition. Copy number analysis and a focus on genes with typical numerical alterations in glioblastoma revealed clear differences between the nine methylation classes. Although great progress in diagnostic precision has been achieved over the last decade, our data clearly demonstrate that glioblastoma, IDH-wildtype still is a heterogeneous group in need of further stratification.

Whole-Genome Comparative Copy Number Alteration Profiling between Malignant Pleural Mesothelioma and Asbestos-Induced Chronic Pleuritis.

Malignant pleural mesothelioma (MPM) is rare and aggressive cancer. The most important risk factor for MPM is exposure to asbestos. In this study, we scanned the genomes of individuals MPM and asbestos-induced chronic pleuritis (AICP) to compare and determine copy number alterations (CNAs) between two asbestos-related diseases. We used high-resolution SNP arrays to compare CNA profiles between MPM (n = 55) and AICP (n = 18). DNAs extracted from pleural tissues in both groups. SNP array analysis revealed common losses at 1p, 3p, 6q, 9p, 13q, 14q, 15q, 16q, 22q and frequent gains at chromosomes 1, 3, 5, 7, 8, and 6p, 12q, 15q, 17p, 20q in MPMs (frequencies max 67%-min 30%; these alterations were not detected in AICPs. Besides detecting well-known MPM-associated CNAs, our high -resolution copy number profiling also detected comparatively rare CNAs for MPMs including losses like 9q33.3, 16q and gains of 1p, 1q, 3p, 3q, 6p, 7q, 15q, 12q, 17p, 20q at significant frequencies in the MPM cohort. We also observed Copy Number gains clustered on the NF2 locus in AICPs, whereas this region was commonly deleted in MPMs. According to this distinct genomic profiles between the two groups, AICPs genomes can be clearly distinguished from highly altered MPM genomes. Hence, we can suggest that SNP arrays can be used as a supporting diagnostic tool in terms of discriminating asbestos-related malignant disease such as MPM and benign pleural lesions, which can be challenging in most instances.

A Novel Approach: Nanopore Sequencing of Native Cell-Free DNA in Diffuse-Large B-Cell Lymphoma Patients

Introduction: Nanopore sequencing (NS), a third generation long-read sequencing technology, is increasingly being used to study cancer genomes. NS has many advantages over next-generation sequencing, including the capability to simultaneously extract base sequence as well as base modifications in the same run without any further library modification. Notably, its portability and ability to provide real-time data make it a potentially powerful tool for clinical applications. This study explores the use of NS to directly sequence native cell free DNA (cfDNA) from patients with diffuse-large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma. Methods: For this exploratory study, 20 patients with either a new diagnosis or relapsed DLBCL were enrolled. We adapted NS methods for cfDNA and sequenced cfDNA before each chemotherapy cycle using both an Illumina platform (as a control) and the Nanopore platform. For differential methylation, we also sequenced plasma cfDNA from five healthy donors on the Nanopore. Results: Our findings demonstrate that we can directly sequence cfDNA on the Nanopore platform, obtaining a whole genome coverage (average 1.2x) with deeper coverage in some regions and also observe ~60% global DNA methylation level in CG context. From this data we are now analyzing regions that are differentially methylated (DMRs) between normal and DLBCL, determining SNV/Indel profiles, and comparing copy number variants (CNVs). These results will be available and presented at the time of the meeting. Conclusions: NS is capable of detecting cfDNA and measuring methylation levels, offering a promising and potentially clinically applicable new approach for the study of cfDNA. Future directions: As we continue this research, our vision is to leverage the portability and real-time data capabilities of NS for clinical applications. By simultaneously combining multiple genomic features with machine learning, we aim to develop a method for measuring lymphoma cfDNA-based MRD in DLBCL patients to be used in clinical settings.

Cladribine and Cyclophosphamide Lymphodepletion Prior to Axicabtagene Ciloleucel in Relapsed or Refractory Large B-Cell Lymphoma

Introduction : Chemotherapy-based lymphodepletion (LD) with fludarabine and cyclophosphamide (Flu/Cy) is a standard preparatory step for chimeric antigen T-cell (CAR T) therapy and its importance for optimal CAR T expansion and efficacy is well-established. An international Flu shortage required our center and others to utilize alternative agents for LD or risk interruption in patient therapy. When unable to utilize Flu as part of the LD regimen for patients with relapsed/refractory large B-cell lymphoma (RR LBCL) that required CD19-directed CAR T as a standard of care therapy, our center adopted a standard operating procedure to substitute Flu with Cladribine (Clad), an alternate purine analogue with similar chemical structure, as part of Clad/Cy LD prior to axicabtagene ciloleucel (axi-cel). Here we report outcomes in these patients. Methods : We performed a retrospective, single-center analysis of consecutive RR LBCL patients who received Clad/Cy LD prior to axi-cel at Moffitt Cancer Center between 8/2022 and 1/2023, and compared outcomes to a historical cohort, consisting of 144 Flu/Cy axi-cel LBCL patients treated between 11/2017 and 2/2021. The Clad/Cy LD regimen, given as standard care, maintained Cy at 500 mg/m 2 and substituted Flu 30 mg/m 2 with Clad 5 mg/m 2/day with both agents given on Days -5, -4, and -3. For efficacy we compared best overall response (BOR) at 90 days and progression-free survival (PFS) comparing the two LD regimens. For safety we compared the rates of cytokine release syndrome (CRS) and neurotoxicity as graded by ASTCT criteria. We also performed correlative analyses exploring absolute lymphocyte count (ALC) kinetics, axi-cel expansion by quantitative PCR, and changes in the cytokine milieu as assessed by the enzyme-linked immunosorbent Ella™ system (Bio-Techne, Oxford, UK) at serial time points following infusion. Results : 23 patients received Clad/Cy LD followed by axi-cel for R/R DLBCL. Baseline characteristics of the study cohort based on LD are shown in the Table. Compared to the historical cohort, Clad/Cy patients received axi-cel after fewer lines of treatment (median 2 [range, 1-3] vs 2 [range, 1-7]; Rank-Sum test p<0.01) and had a greater proportion of relapsed (not refractory) disease (52% vs 22%, p<0.01). The 90-day BOR did not differ between the Clad/Cy and Flu/Cy groups (79% vs 84%; P=0.54) with a similar distribution of complete (22% vs 24%) and partial responders (57% vs 60%). With a median follow-up of 4.6 (95% CI, 3.2-6.2) months for Clad/Cy and 24.2 (95% CI, 21.5-25.8) months for Flu/Cy cohorts, PFS was similar (Figure 1B, Log-Rank p=0.84). Rates of CRS grade ≥ 2 were higher in the Clad/Cy cohort (70% vs 44%; p=0.02), but no CRS grade ≥ 3 was observed in the Clad/Cy compared to 13 (9%) of Flu/Cy patients, including three grade 5 events. Neurotoxicity ≥ grade 2 (44% vs 43%; p=0.77) and peak C-reactive protein levels (15 vs Flu/Cy 12; p=0.3) did not differ for the Clad/Cy and Flu/Cy cohorts. Both regimens had comparable peak axi-cel copy numbers (Clad/Cy 7.9 × 10 6 vs Flu/Cy 5.1 × 10 6 copies/µg). Clad/Cy resulted in a predictable decrease in the ALC from Day -5 to Day 0, but a less profound degree of lymphopenia on Day 0 (0.04 vs 0.02; p<0.01) with ALC 0 achieved later (Day +2 [IQR, +1 to +4] vs Day +1 [IQR, -1 to +2] for Flu/Cy). The median magnitude of change of cytokine levels (Delta = Day -5 - Day 0 concentration) differed between Clad/Cy and Flu/Cy for all tested analytes except IL-6 (3.4 vs 4.6 pg/mL for Flu/Cy; p=0.29). Clad/Cy resulted in greater increases in IL-2 (0.5 vs 0.2 for Flu/Cy; p=0.01) and GM-CSF (2.0 vs 0.9 for Flu/Cy; p=0.03), whereas Flu/Cy had higher IL-15 (31.4 vs 16.8 for Clad/Cy; p<0.01). Interestingly, IFN-γ tended to increase in Clad/Cy and to decrease in Flu/Cy (1.2 vs 0.0; p=0.01). Conclusions : Clad/Cy LD is feasible for axi-cel conditioning in the treatment of RR LBCL. Although it resulted in less profound LD on the day of CAR T infusion, a later ALC nadir, and a different cytokine milieu compared to Flu/Cy, it was associated with similar peak axi-cel expansion and, at short term follow-up, there appears to be similar initial efficacy and toxicity. Future analyses focusing on response durability and long-term toxicities such as infections and post-CAR T cytopenias are warranted.

Can Whole Genome and Whole Transcriptome Sequencing Replace Standard Procedures in CLL Diagnostics?

Background: The advantages of genome-wide sequencing approaches over conventional methods in CLL diagnostics are a matter of debate and exact guidelines for the application of next-generation sequencing in a diagnostic context are currently missing. Aim: Compare the accuracy of whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) in determining the presence of point mutations, IGHV mutational status (IGHVms), and chromosomal aberrations to conventional procedures in a clinical setting. Patients and Methods: The cohort comprised 317 CLL patients. Diagnosis was established following WHO guidelines. WGS (100x, 2x151bp) and WTS (5x10 7 reads, 2x101bp) data were generated on NovaSeq instruments. For routine diagnostics, sequencing was carried out by Sanger or targeted panel sequencing (1500x, 2x101bp). Chromosome banding analysis (CBA) and FISH were available for 231 patients. WGS data were analyzed for chromosomal aberrations and copy number alterations using Manta and GATK. IGHVms was determined by Sanger sequencing and/or fragment analysis for 248 patients. Analysis of IGHVms was carried out using IgCaller (WGS) and MiXCR (WTS). Results: Comparing copy number variants (CNV) and balanced structural variants (SV) between CBA and WGS revealed a high concordance. In detail, 297 CNV were detected by both techniques while 32 and 52 were detected only by CBA (small subclones) or by WGS, respectively. 33 balanced SV were detected by both while 8 and 3 were detected only by CBA and WGS, respectively. A complex karyotype (≥ 3 abnormalities) was observed by both techniques in 55 cases, while 5 and 10 cases were categorized as complex by CBA only or WGS only, respectively. Survival analysis of cases with 3 to 5 chromosomal aberrations predicted by routine or WGS revealed similar hazard ratios that were positively correlated with the number of aberrations. For comparing SNV calls from routine and WGS, we investigated genes with validated mutations in at least ten patients in routine diagnostics. This criterion was met by ATM, MYD88, NOTCH1, SF3B1, and TP53. We observed high concordance between routine and WGS data ( ATM 99.4%, MYD88 100%, NOTCH1 97.8%, SF3B1 100%, TP53 99.1%). Five mutations were detected in routine only and three in WGS only (routine only/WGS only: ATM 1/0, MYD88 0/0, NOTCH1 2/2, SF3B1 0/0, TP53 2/1) and found to be caused by low variant allele frequency (5 miscalls, VAF < 5%) and/or filtering out of low quality variants (5 cases). IGHV rearrangements analyzed by Sanger (248 patients), WGS (242/248 patients), and WTS (236/248 patients) data were compared for matches in the rearranged IGHV. IGHVms calling employed 98% of sequence identity as cutoff. Calling the rearranged IGHV was concordant in 93%, 95%, and 92% when comparing Sanger/MiXCR, Sanger/IgCaller, and MiXCR/IgCaller calls, respectively. For IGHVms, the corresponding percentages of concordance were 91%, 86%, and 83%. Mismatches in calling the IGHVms were found in borderline cases. Detection of stereotyped IGHV was fully concordant. In addition to reliably replicating routine results, we find that WGS/WTS data can also outperform routine procedures. In our hands, the highest hazard ratio (4.94) in survival analysis distinguishing mutated CLL (mCLL) from unmutated CLL (uCLL) is observed using WTS for IGHVms determination and 97% sequence identity as cutoff (208 patients, up to 30 years follow up). Survival for borderline cases (97.00-97.99% sequence identity) was statistically indistinguishable from uCLL in routine diagnostics as well as WGS/WTS. Furthermore, using WGS, CN-LOH regions and deletions below the detection limit of CBA (10 MB) can be detected. We observed such regions in samples with mutated TP53 (5 CN-LOH), ATM (2 CN-LOH, 4 deletions < 10MB), and deletions of minimally overlapping region chr8:21,700,000-23,600,000, whose loss was associated with poor survival (3/29 deletions < 3.1 MB). Conclusions: WGS/WTS data reliably replicate results obtained by standard procedures in a diagnostic setting. Sequencing offers advantages such as a unified workflow, better sensitivity for small deletions, CN-LOH detection, genome-wide analyses of coding and non-coding mutations, among others. Challenges of short read based sequencing regard the detection limit for point mutations and specificity/sensitivity in detecting chromosomal aberrations.

GeneToCN: an alignment-free method for gene copy number estimation directly from next-generation sequencing reads

Genomes exhibit large regions with segmental copy number variation, many of which include entire genes and are multiallelic. We have developed a computational method GeneToCN that counts the frequencies of gene-specific k-mers in FASTQ files and uses this information to infer copy number of the gene. We validated the copy number predictions for amylase genes (AMY1, AMY2A, AMY2B) using experimental data from digital droplet PCR (ddPCR) on 39 individuals and observed a strong correlation (R = 0.99) between GeneToCN predictions and experimentally determined copy numbers. An additional validation on FCGR3 genes showed a higher concordance for FCGR3A compared to two other methods, but reduced accuracy for FCGR3B. We further tested the method on three different genomic regions (SMN, NPY4R, and LPA Kringle IV-2 domain). Predicted copy number distributions of these genes in a set of 500 individuals from the Estonian Biobank were in good agreement with the previously published studies. In addition, we investigated the possibility to use GeneToCN on sequencing data generated by different technologies by comparing copy number predictions from Illumina, PacBio, and Oxford Nanopore data of the same sample. Despite the differences in variability of k-mer frequencies, all three sequencing technologies give similar predictions with GeneToCN.

Genome-Wide Detection of Copy Number Variations Associated with Miniature Features in Horses.

Copy number variations (CNVs) are crucial structural genomic variants affecting complex traits in humans and livestock animals. The current study was designed to conduct a comprehensive comparative copy number variation analysis among three breeds, Debao (DB), Baise (BS), and Warmblood (WB), with a specific focus on identifying genomic regions associated with miniature features in horses. Using whole-genome next-generation resequencing data, we identified 18,974 CNVs across 31 autosomes. Among the breeds, we found 4279 breed-specific CNV regions (CNVRs). Baise, Debao, and Warmblood displayed 2978, 986, and 895 distinct CNVRs, respectively, with 202 CNVRs shared across all three breeds. After removing duplicates, we obtained 1545 CNVRs from 26 horse genomes. Functional annotation reveals enrichment in biological functions, including antigen processing, cell metabolism, olfactory conduction, and nervous system development. Debao horses have 970 genes overlapping with CNVRs, possibly causing their small size and mountainous adaptations. We also found that the genes GHR, SOX9, and SOX11 may be responsible for the miniature features of the Debao horse by analyzing their overlapping CNVRs. Overall, this study offers valuable insights into the widespread presence of CNVs in the horse genome. The findings contribute to mapping horse CNVs and advance research on unique miniature traits observed in the Debao horse.

Fusion of Rabies Virus Glycoprotein or gh625 to Iduronate-2-Sulfatase for the Treatment of Mucopolysaccharidosis Type II.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a mutation in the IDS gene, resulting in deficiency of the enzyme iduronate-2-sulfatase (IDS) causing heparan sulfate (HS) and dermatan sulfate (DS) accumulation in all cells. This leads to skeletal and cardiorespiratory disease with severe neurodegeneration in two thirds of sufferers. Enzyme replacement therapy is ineffective at treating neurological disease, as intravenously delivered IDS is unable to cross the blood-brain barrier (BBB). Hematopoietic stem cell transplant is also unsuccessful, presumably due to insufficient IDS enzyme production from transplanted cells engrafting in the brain. We used two different peptide sequences (rabies virus glycoprotein [RVG] and gh625), both previously published as BBB-crossing peptides, fused to IDS and delivered via hematopoietic stem cell gene therapy (HSCGT). HSCGT with LV.IDS.RVG and LV.IDS.gh625 was compared with LV.IDS.ApoEII and LV.IDS in MPS II mice at 6 months post-transplant. Levels of IDS enzyme activity in the brain and peripheral tissues were lower in LV.IDS.RVG- and LV.IDS.gh625-treated mice than in LV.IDS.ApoEII- and LV.IDS-treated mice, despite comparable vector copy numbers. Microgliosis, astrocytosis, and lysosomal swelling were partially normalized in MPS II mice treated with LV.IDS.RVG and LV.IDS.gh625. Skeletal thickening was normalized by both treatments to wild-type levels. Although reductions in skeletal abnormalities and neuropathology are encouraging, given the low levels of enzyme activity compared with control tissue from LV.IDS- and LV.IDS.ApoEII-transplanted mice, the RVG and gh625 peptides are unlikely to be ideal candidates for HSCGT in MPS II and are inferior to the ApoEII peptide that we have previously demonstrated to be more effective at correcting MPS II disease than IDS alone.

TERT Promoter Mutation c.-124C>T Commonly Occurs in Low-Grade Fibromatosis-like Metaplastic Breast Carcinoma.

Low-grade fibromatosis-like metaplastic carcinoma (FLMC) is a very rare subtype of triple-negative metaplastic (spindle cell) breast carcinoma. It is characterized by the proliferation of spindle cells closely resembling fibromatosis, which represents a benign fibroblastic/myofibroblastic breast proliferation. Unlike most triple-negative and basal-like breast cancers, FLMC has a very low potential for metastases, but demonstrates frequent local recurrences. To genetically characterize FLMC. To this end, we analyzed 7 cases by targeted next-generation sequencing for 315 cancer-related genes and performed comparative microarray copy number analysis in 5 of these cases. All cases shared TERT alterations (6 patients with recurrent c.-124C>T TERT promoter mutation and 1 patient with copy number gain encompassing the TERT locus), had oncogenic PIK3CA/PIK3R1 mutations (activation of the PI3K/AKT/mTOR pathway), and lacked mutations in TP53. TERT was overexpressed in all FLMCs. CDKN2A/B loss or mutation was observed in 4 of 7 cases (57%). Furthermore, tumors displayed chromosomal stability, with only few copy number variations and a low tumor mutational burden. We conclude that FLMCs typically show the recurrent TERT promoter mutation c.-124C>T, activation of the PI3K/AKT/mTOR pathway, low genomic instability, and wild-type TP53. In conjunction with previous data of metaplastic (spindle cell) carcinoma with and without fibromatosis-like morphology, FLMC is most likely distinguished by TERT promoter mutation. Thus, our data support the notion of a distinct subgroup within low-grade metaplastic breast cancer with spindle cell morphology and associated TERT mutations.

Investigation on the applicability of a long-range reverse-transcription quantitative polymerase chain reaction assay for the rapid detection of active viruses

BackgroundAlthough conventional polymerase chain reaction (PCR) methods are widely used in diagnosis, the titer of the pathogenic virus is difficult to determine based on the PCR. In our prior report, a long-range reverse-transcription quantitative PCR (LR-RT-qPCR) assay was developed to assess the titer of UV-irradiated influenza A virus (IAV) rapidly. In this research, we focused on whether the LR-RT-qPCR assay could evaluate the titer of IAV inactivated by other methods.MethodsIAV was inactivated by: heating at 100 °C for periods ranging from 1 to 15 min, treating with 0.12% sodium hypochlorite for periods ranging from 3 to 30 min, or treating with 70% ethanol for periods ranging from 10 to 30 min. Fifty percent tissue culture infectious dose (TCID50) assay was performed to confirm the efficacy of the inactivation methods, followed by LR-RT-qPCR to investigate the correlation between infectivity and copy number.ResultsOne minute heating, 3 min sodium hypochlorite treatment, or 10 min ethanol treatment was sufficient to deactivate IAV. Changes before and after the inactivations in the copy numbers on LR-RT-qPCR were significantly different among the inactivation methods. Heat-inactivation drastically decreased the copy number to below the cutoff value around 5 copies/μL after 5 min treatment. The inactivation time of heating estimated using LR-RT-qPCR was marginally higher than that determined using TCID50. However, the treatments with sodium hypochlorite or ethanol moderately and minimally affected the copy numbers obtained using LR-RT-qPCR (~ 1 digit or no copy number decrease), respectively.ConclusionsIn addition to good applicability in UV-irradiation previously reported, the LR-RT-qPCR method is suitable for evaluating the effect of heat-inactivation on IAV infectivity. However, minor modifications may be made and investigated in the future to reduce the time intervals with TCID50. Although this method is not applicable for the ethanol inactivation, rapid evaluation of the effects of chlorination on IAV can be determined by comparing copy numbers before and after treatment using the LR-RT-qPCR method.

Clinical and genetic analysis of three children with 22q13 deletion syndrome

To explore the clinical and genetic characteristics of three children with 22q13 deletion syndrome. Clinical data were collected and copy number variations in the patients and their parents were detected by using array-based comparative genomic hybridization (aCGH) and copy number variation sequencing (CNV-seq). The DECIPHER, ClinGen, OMIM, PubMed and Gene Review databases were retrieved for pathogenicity analysis. The common phenotypes of the three children have included variable global developmental delay, among which speech delay was the most obvious. Patient 1 had abnormalities of corpus callosum shown by magnetic resonance imaging. Patient 2 had dental crowding, pale skin, thick palms, hypotonia, and other facial features. Patient 3 had the mildest symptoms including language dysfunction, which has caught up with the development and improved significantly. All of the three children had harbored de novo deletions of 22q13.33q13.33 region, which spanned 0.84 Mb, 8.70 Mb and 0.90 Mb and involved 37, 126, and 34 genes, respectively. Above finding has enriched the clinical and genetic characteristics of 22q13 deletion syndrome and laid a foundation for genetic counseling and prenatal diagnosis.

Abstract 6058: The serine synthesis pathway contributes to the radiation-induced metabolic plasticity in glioblastoma multiforme

Abstract Purpose: Glioblastoma multiforme (GBM) remains a fatal disease despite aggressive treatment approaches. Post-surgical radiation therapy (RT) is the only treatment that significantly improves survival of GBM patients but recurrence is inevitable. Elucidating the mechanisms of resistance to RT could transform GBM patient outcomes. The purpose of this study was to elucidate the effect of radiation on the serine synthesis pathway (SSP) and the role of SSP in boosting antioxidant defenses in irradiated GBM cells and promoting radioresistance. Methods: We compared copy number alterations (CNA) and amplification of SSP enzymes (PHGDH, PSAT1, PSPH, SHMT1/2) in low-grade gliomas (LGG, n = 511) and GBM (n = 575) via TCGA pan-cancer analysis. In vitro HPLC/MS-based metabolomics assays were used to study the changes in overall metabolite levels induced by radiation in patient-derived GBM specimens. Targeted metabolomics with 13C-labeled glucose was used for tracking the fate of glucose towards SSP intermediates and antioxidant species. Gene expression levels after radiation were measured via molecular biology approaches. De novo SSP was targeted via pharmacological inhibition of PHGDH. Modified clonogenic, sphere forming assays (SFA) on gliomasphere cultures were used to determine radiosensitivity. Dependence on extracellular serine/glycine was determined via serine/glycine depletion experiments. Results: The TCGA analysis revealed increased CNAs in all the de novo SSP enzymes in GBM tumors relative to the LGGs. 82% of GBM tumors had CNAs or amplification of the PSPH gene, which codes for the ultimate enzyme in serine synthesis. In vitro metabolomics assays revealed that radiation increases the levels of serine, glycine and cystine in gliomaspheres, and this is accompanied by elevated levels of reduced glutathione (GSH). Targeted glucose metabolomics revealed radiation-induced upregulation of the de novoSSP, with a concomitant increase in gene expression of SSP enzymes. Downstream of the SSP, metabolomics data show that irradiated GBM cells elevate nucleotide and precursor levels, likely to support DNA damage repair mechanisms after radiation. The inhibition of PHGDH, the first rate-limiting enzyme in de novo SSP, significantly reduced GBM cell survival in vitro. Extracellular serine/glycine appeared to also be important contributors to radiation survival. Gene expression of SLC1A4, an amino acid transporter, was upregulated by radiation, while depletion of extracellular serine/glycine led to significant decreased survival in in vitro SFAs. Conclusion: Here we provide evidence that the de novo serine synthesis pathway is an important contributor to the metabolic reprogramming of GBM cells after radiation that promotes radiation resistance. These data call for further investigations to evaluate the radiosensitizing potential of targeting de novo SSP in GBM. Citation Format: Justine Bailleul, Yangjingyi Ruan, Erina Vlashi. The serine synthesis pathway contributes to the radiation-induced metabolic plasticity in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6058.

Array Comparative Genomic Hybridisation and Droplet Digital PCR Uncover Recurrent Copy Number Variation of the TTN Segmental Duplication Region.

Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain such segmental duplication regions. Using our custom Comparative Genomic Hybridisation array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses.

Copy number variations across the blood–brain barrier in multiple sclerosis

ObjectiveMultiple sclerosis (MS) is a neuroinflammatory disease where immune cells cross the blood–brain barrier (BBB) into the central nervous system (CNS). What predisposes these immune cells to cross the BBB is still unknown. Here, we examine the possibility that genomic rearrangements could predisposespecific immune cells in the peripheral blood to cross the BBB and form sub‐populations of cells involved in the inflammatory process in the CNS.MethodsWe compared copy number variations in paired peripheral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF) cells from MS patients. Thereafter, using next generation sequencing, we studied the T‐cell receptor beta (TRB) locus rearrangements and profiled the αβ T cell repertoire in peripheral CD4+ and CD8+ T cells and in the CSF.ResultsWe identified deletions in the T‐cell receptor alpha/delta (TRA/D), gamma (TRG), and TRB loci in CSF cells compared to PBMCs. Further characterization revealed diversity of the TRB locus which was used to describe the character and clonal expansion of T cells in the CNS. T‐cell repertoire profiling from either side of the BBB concluded that the most frequent clones in the CSF samples are unique to an individual. Furthermore, we observed a difference in the proportion of expanded T‐cell clones when comparing samples from MS patients in relapse and remission with opposite trends in CSF and peripheral blood.InterpretationThis study provides a characterization of the T cells in the CSF and might indicate a role of expanded clones in MS pathogenicity.

Comparing Copy Number Variations in a Danish Case Cohort of Individuals With Psychiatric Disorders

Although the association between several recurrent genomic copy number variants (CNVs) and mental disorders has been studied for more than a decade, unbiased, population-based estimates of the prevalence, disease risks and trajectories, fertility, and mortality to contrast chromosomal abnormalities and advance precision health care are lacking. To generate unbiased, population-based estimates of prevalence, disease risks and trajectories, fertility, and mortality of CNVs implicated in neuropsychiatric disorders. In a population-based case-cohort study, using the Lundbeck Foundation Initiative for Integrative Psychiatric Research (iPSYCH) 2012 database, individuals born between May 1, 1981, and December 31, 2005, and followed up until December 31, 2012, were analyzed. All individuals (n = 57 377) with attention-deficit/hyperactivity disorder (ADHD), major depressive disorder (MDD), schizophrenia (SCZ), autism spectrum disorder (ASD), or bipolar disorder (BPD) were included, as well as 30 000 individuals randomly drawn from the database. Data analysis was conducted from July 1, 2017, to September 7, 2021. Copy number variants at 6 genomic loci (1q21.1, 15q11.2, 15q13.3, 16p11.2, 17p12, and 17q12). Population-unbiased hazard ratio (HR) and survival estimates of CNV associations with the 5 ascertained psychiatric disorders, epilepsy, intellectual disability, selected somatic disorders, fertility, and mortality. Participants' age ranged from 1 to 32 years (mean, 12.0 [IQR, 6.9] years) during follow-up, and 38 662 were male (52.3%). Copy number variants broadly associated with an increased risk of autism spectrum disorder and ADHD, whereas risk estimates of SCZ for most CNVs were lower than previously reported. Comparison with previous studies suggests that the lower risk estimates are associated with a higher CNV prevalence in the general population than in control samples of most case-control studies. Significant risk of major depressive disorder (HR, 5.8; 95% CI, 1.5-22.2) and sex-specific risk of bipolar disorder (HR, 17; 95% CI, 1.5-189.3, in men only) were noted for the 1q21.1 deletion. Although CNVs at 1q21.1 and 15q13.3 were associated with increased risk across most diagnoses, the 17p12 deletion consistently conferred less risk of psychiatric disorders (HR 0.4-0.8), although none of the estimates differed significantly from the general population. Trajectory analyses noted that, although diagnostic risk profiles differed across loci, they were similar for deletions and duplications within each locus. Sex-stratified analyses suggest that pathogenicity of many CNVs may be modulated by sex. The findings of this study suggest that the iPSYCH population case cohort reveals broad disease risk for some of the studied CNVs and narrower risk for others, in addition to sex differential liability. This finding on genomic risk variants at the level of a population may be important for health care planning and clinical decision making, and thus the advancement of precision health care.