Minor Transcripts Research Articles

Development and evaluation of a rapid one-step high sensitivity real-time quantitative PCR system for minor BCR-ABL (e1a2) test in Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL).

Minimal residual disease assessment of BCR-ABL messenger ribonucleic acid levels is crucial in Philadelphia chromosome-positive acute lymphoblastic leukemia for prognosis and treatment planning. However, accurately quantifying minor BCR-ABL transcripts, which comprise 70% of Philadelphia chromosome-positive acute lymphoblastic leukemia cases, lacks a national-approved method. We developed the "Otsuka" minor BCR-ABLmessenger ribonucleic acid assay kit with exceptional precision (0.00151%). Minor BCR-ABL messenger ribonucleic acid levels were analyzed in 175 adults, 36 children with acute lymphoblastic leukemia and 25 healthy individuals to evaluate the kit's performance. The "Otsuka" kit showed high concordance with a commonly used chimeric gene screening method, indicating reliable detection of positive cases. Quantitative results demonstrated a robust correlation with both a laboratory-developed test and a diagnostic research product. The "Otsuka" kit performs comparably or even surpass to conventional products, providing valuable insights into Philadelphia chromosome-positive acute lymphoblastic leukemia pathology. The 'Otsuka" minor BCR-ABL messenger ribonucleic acid assay kit exhibits excellent performance in quantifying minor BCR-ABL transcripts in Philadelphia chromosome-positive acute lymphoblastic leukemia patients. Our results align well with established screening methods and show a strong correlation with laboratory-developed tests and diagnostic research products. The "Otsuka" kit holds great promise as a valuable tool for understanding Philadelphia chromosome-positive acute lymphoblastic leukemia pathology and guiding effective treatment strategies.

Genetic predisposition to lung adenocarcinoma outcome is a feature already present in patients' noninvolved lung tissue.

Emerging evidence suggests that the prognosis of patients with lung adenocarcinoma can be determined from germline variants and transcript levels in nontumoral lung tissue. Gene expression data from noninvolved lung tissue of 483 lung adenocarcinoma patients were tested for correlation with overall survival using multivariable Cox proportional hazard and multivariate machine learning models. For genes whose transcript levels are associated with survival, we used genotype data from 414 patients to identify germline variants acting as cis-expression quantitative trait loci (eQTLs). Associations of eQTL variant genotypes with gene expression and survival were tested. Levels of four transcripts were inversely associated with survival by Cox analysis (CLCF1, hazard ratio [HR]=1.53; CNTNAP1, HR=2.17; DUSP14, HR=1.78; and MT1F: HR=1.40). Machine learning analysis identified a signature of transcripts associated with lung adenocarcinoma outcome that was largely overlapping with the transcripts identified by Cox analysis, including the three most significant genes (CLCF1, CNTNAP1, and DUSP14). Pathway analysis indicated that the signature is enriched for ECM components. We identified 32 cis-eQTLs for CNTNAP1, including 6 with an inverse correlation and 26 with a direct correlation between the number of minor alleles and transcript levels. Of these, all but one were prognostic: the six with an inverse correlation were associated with better prognosis (HR < 1) while the others were associated with worse prognosis. Our findings provide supportive evidence that genetic predisposition to lung adenocarcinoma outcome is a feature already present in patients' noninvolved lung tissue.

Identification and functional characterization of the first deep intronic GLA mutation (IVS4+1326C>T) causing renal variant of Fabry disease

BackgroundFabry disease (FD, OMIM #301500) is an X-linked lysosomal disorder caused by the deficiency of α-galactosidase A (α-GalA), encoded by the GLA gene. Among more than 1100 reported GLA mutations, few were deep intronic mutations which have been linked to classic and cardiac variants of FD.Methods and resultsWe report a novel hemizygous deep intronic GLA mutation (IVS4+1326C>T) in a 33-year-old Chinese man with a mild α-GalA deficiency phenotype involving isolated proteinuria and predominant globotriaosylceramide deposits in podocytes. IVS4+1326C>T, which appears to be the first deep intronic GLA mutation associated with renal variant of FD, was identified by Sanger sequencing the entire GLA genomic DNA sequence of the patient’s peripheral mononuclear blood lymphocytes (PBMCs). Further sequencing of cDNA from PBMCs of the patient revealed a minor full-length GLA transcript accounting for about 25% of total GLA transcript, along with two major aberrantly spliced GLA transcripts encoding mutant forms of α-GalA with little enzyme activity characterized by in vitro α-GalA overexpression system in the HEK293T cells. Thus, the combined clinical phenotype, genetic analysis and functional studies verified the pathogenicity of IVS4+1326C>T.ConclusionsThe identification of IVS4+1326C>T establishes a link between deep intronic GLA mutation and the renal variant of FD, which extends the mutation spectrum in GLA gene and justifies further study of how IVS4+1326C>T and potentially other deep intronic GLA mutations contribute to Fabry podocytopathy through aberrant splicing. Future studies should also assess the true incidence of IVS4+1326C>T in patients with different variants of FD, which may improve early genetic diagnosis to allow timely treatment that can prevent disease progression and improve survival.

Loss of centromeric RNA activates the spindle assembly checkpoint in mammalian female meiosis I.

The repetitive sequences of DNA centromeric regions form the structural basis for kinetochore assembly. Recently they were found to be transcriptionally active in mitosis, with their RNAs providing noncoding functions. Here we explore the role, in mouse oocytes, of transcripts generated from within the minor satellite repeats. Depletion of minor satellite transcripts delayed progression through meiosis I by activation of the spindle assembly checkpoint. Arrested oocytes had poorly congressed chromosomes, and centromeres were frequently split by microtubules. Thus, we have demonstrated that the centromeric RNA plays a specific role in female meiosis I compared with mitosis and is required for maintaining the structural integrity of centromeres. This may contribute to the high aneuploidy rates observed in female meiosis.

Abstract 1534: Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors

Abstract Mesothelin (MSLN) is a tumor-associated antigen over-expressed on the cell surface of various malignant tumor cells, including majority of mesothelioma and ovarian cancer. MSLN-directed therapy has been intensively studied in preclinical and clinical settings. However, the clinical efficacy and safety of mesothelin-targeted therapy remains to be demonstrated. To identify a safe and effective target for CAR-T cell therapy in solid tumors, we used artificial intelligent platform developed with public and private RNA transcription database to identify cancer-associated antigens caused by alternative spicing. One of interesting targets is an alternative spicing isoform from MSLN. Human MSLN transcript has at least three isoforms. The isoform 1 is the predominant transcript detected in normal and tumor tissues and has been a promising target for cancer immunotherapy. The isoform 2 is the minor transcript using alternatively spliced exons producing 8 additional amino acids insertion compared to isoform 1. The isoform 3 produces a truncated and soluble MSLN. Our data demonstrated that MSLN isoform 2 is specifically expressed in ovarian cancers but not normal tissues, confirmed by Q-PCR studies. Furthermore, we generated mouse hybridoma antibodies specifically targeting MSLN isoform 2. The antibody specificity was screened and confirmed by ELISA-based reaction to MSLN isoform 2-specific peptide and flow cytometry-based binding to 293T cells overexpressing MSLN isoform 2 but not isoform 1 proteins. We further detected the endogenous MSLN isoform 2 expression in human mesothelioma cell line NCI H226 and further confirmed MSLN isoform 2 expression on human primary mesothelioma and ovarian cancer tissue but not normal tissues by immunohistochemistry staining. To study if targeting MSLN isoform 2 with chimeric antigen receptor (CAR) can control the tumor growth, we made CAR retrovirus construct and showed that genetically modified allogenic gamma-delta T cells expressing CAR for MSLN isoform 2 can kill human mesothelioma cells in vitro and in vivo. In summary, we have demonstrated that MSLN isoform 2 is a tumor-specific antigen, which can be targeted for CAR-T cell therapy. Citation Format: Xiaohong Wang, Yibin Chen, Anupama Gopisetty, Leonardo Mirandola, Lucia Piccotti, Quynh Nguyen, Maurizio Chiriva-Internati. Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1534.

Molecular Responses after Switching from a Standard-Dose Twice-Daily Nilotinib Regimen to a Reduced-Dose Once-Daily Schedule in Patients with Chronic Myeloid Leukemia: A Real Life Observational Study (NILO-RED)

IntroductionStandard dosing regimen for the BCR-ABL1 tyrosine kinase inhibitor (TKI) nilotinib in chronic myeloid leukemia (CML) is 300mg twice daily (BID) in the frontline setting and 400mg BID in the 2nd line setting or beyond. Patients (pts) are instructed to fast within 2 hours before and 1 hour after medication intake due to significant high-fat food effects on drug bioavailability. On-label dose adjustments to a lower-dose once-daily (QD) regimen are recommended solely in case of significant toxicity.Study goalThe primary aim of our observational two-center study was to ask whether nilotinib-treated adult CML pts were able to maintain major molecular responses (MMR) within 1 year after conversion to a more convenient nilotinib QD regimen at reduced doses, regardless of reasons for tapering nilotinib. Pts with minor transcripts or a history of blast crisis or allogeneic stem cell transplantation were excluded.PatientsEighty one pts in chronic phase were identified and included and results from the first 67 pts with a follow-up of at least 12 months after the switch to the once-daily dose-reduced nilotinib regimen are presented. Median age at study entry was 52 years (range: 19-82). There were 25 males (37.3%) and the Sokal score was low, intermediate, high or unknown in 43.3%, 37.3%, 13.4% and 6% of pts, respectively. All pts had major-type BCR-ABL1 transcripts. Nilotinib was started as 1st line TKI at 300mg BID in 68.6% of pts and as 2nd or subsequent line TKI at 400 or 300 mg BID in 31.4% of pts due to intolerance, warning, resistance or absence of a deep molecular response on prior therapy. Median duration of nilotinib prior switching to a reduced QD dosing was 29 months (range: 1-94) and median duration of MMR was 25 months (range: 3-212). Primary reason for the switch to a reduced QD nilotinib dosing was non-serious adverse events in 37.3% of pts and improvement in pts convenience in 62.7% of pts. Nilotinib was reduced down to 450mg QD, 400mg QD or 300mg QD in 86.6%, 10.4% and 3% of pts, respectively. A 2nd dose reduction from 450mg to 300mg QD was performed in 30 pts after a median time of 18 months (range: 4-37). The median duration of low-dose nilotinib QD was 24 months (5-54). No severe tolerance issues were encountered and no ischemic cardiovascular events occurred. Eleven pts stopped nilotinib due to treatment-free remission attempt (n=10) or persistent adverse event (n=1). All pts were alive at last follow-up.Evolution of molecular responsesAt baseline, the number of pts in MMR, MR4 and ≥MR4.5 was 9 (13.4%), 17 (25.4%) and 41 (61.2%), respectively and 32 pts (47.8%) had undetectable BCR-ABL1 transcripts. Median BCR-ABL1/ABL1 % IS was 0.006% (range: 0.0007-0.063) and median ABL1 copy number in pts with undetectable transcripts was 87259 (range: 36018-921339). Only 2 pts treated with 1st line nilotinib lost MMR 4 and 6 months after QD dose reduction of nilotinib with BCR-ABL1/ABL1 % IS at 0.13 and 0.11%, respectively. Both pts spontaneously regained MMR 4 months later without any treatment modification. Kaplan-Meier analysis of survival without unconfirmed MMR loss was 97% (95% CI: 92.9-100) at 12 months and analysis by molecular response category showed that none of the pts who were at least in MR4 at baseline lost MMR (Figure 1). At last follow-up on low-dose QD nilotinib, the number of pts in MMR, MR4 and ≥MR4.5 was 4 (6.3%), 11 (16.4%) and 52 (77.6%), respectively and 37 pts had undetectable transcripts (55.2%). Median BCR-ABL1/ABL1 % IS was 0.0035% (range: 0.00025-0.058) and median ABL1 copy number in pts with undetectable transcripts was 96000 (range: 23285-472000).Ten pts in long-lasting deep molecular response stopped nilotinib without further treatment after a median duration of TKI and of nilotinib of 60 months (range: 50-149) and 56 months (range: 42-87), respectively. Their follow-up after nilotinib discontinuation was 18 months (range: 5-33). Only 1 of them lost MMR and restarted therapy 5 months later while the others remained treatment-free.ConclusionSwitching to a nilotinib QD regimen at reduced doses as maintenance therapy after achievement of a MMR on standard-dose nilotinib BID schedule is feasible and safe in chronic phase CML pts regardless of prior treatment history and does not compromise the anti-leukemic efficacy of nilotinib. Our results may pave the way for clinical trials aiming at systematic avoidance of overtreatment and improvement of pts convenience in treatment with nilotinib. [Display omitted] DisclosuresRea:Pfizer: Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Honoraria. Cayuela:BMS: Honoraria; Novartis: Honoraria. Etienne:BMS: Speakers Bureau; Incyte: Speakers Bureau; Novartis: Consultancy, Research Funding.

Symmetrically dimethylated histone H3R2 promotes global transcription during minor zygotic genome activation in mouse pronuclei

Paternal genome reprogramming, such as protamine–histone exchange and global DNA demethylation, is crucial for the development of fertilised embryos. Previously, our study showed that one of histone arginine methylation, asymmetrically dimethylated histone H3R17 (H3R17me2a), is necessary for epigenetic reprogramming in the mouse paternal genome. However, roles of histone arginine methylation in reprogramming after fertilisation are still poorly understood. Here, we report that H3R2me2s promotes global transcription at the 1-cell stage, referred to as minor zygotic genome activation (ZGA). The inhibition of H3R2me2s by expressing a histone H3.3 mutant H3.3R2A prevented embryonic development from the 2-cell to 4-cell stages and significantly reduced global RNA synthesis and RNA polymerase II (Pol II) activity. Consistent with this result, the expression levels of MuERV-L as minor ZGA transcripts were decreased by forced expression of H3.3R2A. Furthermore, treatment with an inhibitor and co-injection of siRNA to PRMT5 and PRMT7 also resulted in the attenuation of transcriptional activities with reduction of H3R2me2s in the pronuclei of zygotes. Interestingly, impairment of H3K4 methylation by expression of H3.3K4M resulted in a decrease of H3R2me2s in male pronuclei. Our findings suggest that H3R2me2s together with H3K4 methylation is involved in global transcription during minor ZGA in mice.

Global network analysis in Schizosaccharomyces pombe reveals three distinct consequences of the common 1-kb deletion causing juvenile CLN3 disease

Juvenile CLN3 disease is a recessively inherited paediatric neurodegenerative disorder, with most patients homozygous for a 1-kb intragenic deletion in CLN3. The btn1 gene is the Schizosaccharomyces pombe orthologue of CLN3. Here, we have extended the use of synthetic genetic array (SGA) analyses to delineate functional signatures for two different disease-causing mutations in addition to complete deletion of btn1. We show that genetic-interaction signatures can differ for mutations in the same gene, which helps to dissect their distinct functional effects. The mutation equivalent to the minor transcript arising from the 1-kb deletion (btn1102–208del) shows a distinct interaction pattern. Taken together, our results imply that the minor 1-kb deletion transcript has three consequences for CLN3: to both lose and retain some inherent functions and to acquire abnormal characteristics. This has particular implications for the therapeutic development of juvenile CLN3 disease. In addition, this proof of concept could be applied to conserved genes for other mendelian disorders or any gene of interest, aiding in the dissection of their functional domains, unpacking the global consequences of disease pathogenesis, and clarifying genotype–phenotype correlations. In doing so, this detail will enhance the goals of personalised medicine to improve treatment outcomes and reduce adverse events.

Minimal residual disease (MRD) positivity at allogeneic hematopoietic cell transplantation, not the quantity of MRD, is a risk factor for relapse of Philadelphia chromosome-positive acute lymphoblastic leukemia

Minimal residual disease (MRD) monitoring by quantitative real-time reverse transcription PCR (qRT-PCR) is the standard of care in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL). We evaluated the impact of MRD status at hematopoietic cell transplantation (HCT) on relapse, as measured by a unified protocol at a central laboratory. Only patients with Ph-positive ALL who had minor transcripts (e1a2) and who underwent allogeneic HCT in first complete remission between 2008 and 2017 were included. First, patients with negative-MRD (n = 196) and positive-MRD (n = 61) at HCT were analyzed. As expected, MRD positivity at HCT was significantly associated with an increased risk of hematological relapse (hazard ratio [HR], 2.91; 95% CI 1.67-5.08; P < 0.001) in the multivariate analysis. Next, patients with positive-MRD were divided into low-MRD (n = 39) and high-MRD (n = 22) groups. In the multivariate analysis, high-MRD at HCT was not significantly associated with an increased risk of hematological relapse compared to the low-MRD group (HR 1.10; 95% CI 0.54-2.83; P = 0.620). These results indicate that the therapeutic decisions should be made based on MRD positivity, rather than on the MRD level, at HCT.

Elicitation of fruit defense response by active edible coatings embedded with phenylalanine to improve quality and storability of avocado fruit

Edible coatings attract high research attention as an effective natural approach for maintaining fresh agricultural produce quality and storability. In this study, a series of polysaccharide-based coatings that contain, for the first time, an elicitor of fruit defense response, phenylalanine, were developed and applied on avocado fruit. After an initial screening of a series of polysaccharides coatings, the chitosan and carboxymethyl cellulose with stearic acid (CMC + StA) were chosen, and phenylalanine elicitor was added then to the polysaccharide matrices. These active edible coatings of either chitosan or CMC + StA that contained phenylalanine significantly reduced the fruit's natural decay caused by the fungal pathogens Colletotrichum and Alternaria. The coated avocado fruit has also demonstrated higher resistance to storage at sub-optimal temperature and showed less pitting, decay, and internal browning. The observed cold resistance correlated with a minor transcript upregulation of several genes as lipoxygenase, heat-shock protein, and several transcripts in the phenylpropanoid pathway. Interestingly, the coated fruit had a better flavor than the control fruit. Taken together, the reported results point to the ability of these new edible coatings to significantly increased fruit resistance to fungal pathogens and cold stress while maintaining the fruit quality and storability and improving their taste.

A model for long-term infection of bovine papillomavirus type 1 in Saccharomyces cerevisiae.

We have previously reported that bovine papillomavirus type 1 (BPV1) can replicate its genome and produces infectious virus-like particles in short-term BPV1 virion-infected Sacharomyces cerevisiae (Zhao and Frazer, 2002). Here, we report viral RNA transcription and L1 capsid protein expression in long-term BPV1 virion-infected S. cerevisiae culture. Northern blot hybridization showed that viral RNA was detected in long-term BPV1-infected S. cerevisiae cultures (82-108 days). The levels of the viral RNA transcription varied significantly over the long time period, which showed active transcription at an early stage (Day 3 to Day 16), weak transcription at a middle stage (Day 23 to Day 45) and stable transcription at the late stage of culture (Day 55 to Day 82/85/95). Three major BPV1 transcripts of 4.3, 2.6 and 1.8Kb were identified, with 4.3Kb a minor transcript and the 1.8Kb the most prominent transcript compared with the 2.6Kb species. Immunoblotting showed that L1 capsid protein was expressed, with its variable amounts corresponding to the levels of RNA transcription over the time period. 35S-methionine/cysteine labeling and immunoprecipitation proved that the detected L1 protein was newly synthesized in BPV1-infected S. cerevisiae cultures. 33.3-54.2% of the cell colonies expressed L1 protein. Thus, the S. cerevisiae system, as a promising model, may be used not only for the study of virus like particle formation of BPV1 in vitro, but also for further functional analysis of individual viral genes in BPV1 life cycle. Keywords: BPV1; viral RNA transcription; expression of L1 capsid protein; virion-infected Saccharomyces cerevisiae.

AF10 Is Split by MLL and HEAB, a Human Homolog to a Putative Caenorhabditis elegans ATP/GTP-Binding Protein in an Invins(10; 11)(p12;q23q12)

Invins(10;11)(p12;q23q12) is one of the rare but recurring chromosome rearrangements seen in acute monoblastic leukemia. We cloned the proximal 10p breakpoint from one patient and showed that the MLL gene at 11q23 was fused to the 3' portion of AF10 at 10p12. In addition, we cloned the telomeric 10p junction and we found that the 5' portion of AF10 was juxtaposed to a previously unidentified gene at 11q12, which we call HEAB (a human homolog to a hypothetical Caenorhabditis elegans ATP/GTP-binding protein). These results indicate that the AF10 gene is split into a 5' AF10 and a 3' AF10 portion by the 11q23q12 chromosome segment and that both breakpoint junctions result in fusion transcripts of 5' AF10/HEAB and MLL/3' AF10. Only the MLL/3' AF10 fusion mRNA results in an in-frame fusion. Northern blot analysis of HEAB expression shows that a 2.0-kb major transcript is expressed ubiquitously in human tissues and is especially abundant in testis and skeletal muscle, whereas a 3.2-kb minor transcript is noted with the highest level of expression in thymus and peripheral blood leukocytes. The HEAB gene encodes a 425-amino acid protein that is rich in valine and leucine. HEAB protein shows high homology in its entire amino acid sequence to a putative C elegans protein and contains an adenosine triphosphate (ATP)/guanosine triphosphate (GTP)-binding motif that has homology to the ATP-binding transporter superfamily or to GTP-binding proteins. Our results could explain the high frequency of complex insertion and other rearrangement events that involve 10p12 and 11q12 and 11q23. The finding that different portions of a single gene are involved in fusions with two independent genes in the same leukemic cell is unique in the analysis of chromosome translocations.

TSC1 intragenic deletion transmitted from a mosaic father to two siblings with cardiac rhabdomyomas: Identification of two aberrant transcripts

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder characterized by non-cancerous tumors in multiple organs including the brain, kidney, lung, heart, and skin. We encountered a Japanese family consisting of two siblings (a four-year-old boy and a one-year-old girl) with multiple cardiac rhabdomyomas conveying a high risk of TSC and apparently unaffected sibling (a two-year-old girl) and parents. Whole exome sequencing and application of Integrative Genomic Viewer revealed an identical intragenic TSC1 deletion with the breakpoints on intron 15 and exon 19 in the affected siblings, but not in the apparently unaffected sibling and parents. Subsequently, PCR-based analyses were performed using primers flanking the deletion, showing that the deletion was also present in the father and that the deletion occurred between chr9:135,777,038 (bp) and chr9:135,780,540 (bp) in association with a one bp overlap. Furthermore, RT-PCR analyses were carried out using lymphoblastoid cell lines, revealing a major in-frame insertion/deletion transcript produced by aberrant splicing using a cryptic ″ag″ splice acceptor motif at intron 15 (r.1998_2438delinsTTCATTAGGTGG) and a minor frameshift transcript generated by aberrant splicing between exon 15 and exon 20 (r.1998_2502del, p.Lys666Asnfs*15) in the affected siblings. These findings imply that the intragenic deletion producing two aberrant transcripts was generated as a somatic pathogenic variant involving the germline in the father and was transmitted to the affected siblings.

Characterization of splice-altering mutations in inherited predisposition to cancer

Mutations responsible for inherited disease may act by disrupting normal transcriptional splicing. Such mutations can be difficult to detect, and their effects difficult to characterize, because many lie deep within exons or introns where they may alter splice enhancers or silencers or introduce new splice acceptors or donors. Multiple mutation-specific and genome-wide approaches have been developed to evaluate these classes of mutations. We introduce a complementary experimental approach, cBROCA, which yields qualitative and quantitative assessments of the effects of genomic mutations on transcriptional splicing of tumor suppressor genes. cBROCA analysis is undertaken by deriving complementary DNA (cDNA) from puromycin-treated patient lymphoblasts, hybridizing the cDNA to the BROCA panel of tumor suppressor genes, and then multiplex sequencing to very high coverage. At each splice junction suggested by split sequencing reads, read depths of test and control samples are compared. Significant Z scores indicate altered transcripts, over and above naturally occurring minor transcripts, and comparisons of read depths indicate relative abundances of mutant and normal transcripts. BROCA analysis of genomic DNA suggested 120 rare mutations from 150 families with cancers of the breast, ovary, uterus, or colon, in >600 informative genotyped relatives. cBROCA analysis of their transcripts revealed a wide variety of consequences of abnormal splicing in tumor suppressor genes, including whole or partial exon skipping, exonification of intronic sequence, loss or gain of exonic and intronic splicing enhancers and silencers, complete intron retention, hypomorphic alleles, and combinations of these alterations. Combined with pedigree analysis, cBROCA sequencing contributes to understanding the clinical consequences of rare inherited mutations.

Development of Reference Control Material for the Evaluation of the Cepheid Prototype * Xpert® BCR-ABL P190 Ultra Assay

Background: Patients with Philadelphia+ Acute Lymphoid Leukemia (Ph+ ALL) respond to the same tyrosine kinase inhibitors (TKIs) that work against Chronic Myeloid Leukemia (CML). However, the dominant BCR-ABL breakpoint is the e1a2 (p190) minor transcript with the e13a2/e14a2 (p210) major transcripts, accounting for 20-30% of the ALL patients, together with &lt;5% of the CML patients. Currently, unlike the p210 major transcripts for CML, there are no International Standards available for the quantitation of the e1a2 p190 transcript for both CML and ALL. This poses a challenge when developing a quantitative assay to monitor the e1a2 p190 transcript levels. Objectives: Implementation of a certified plasmid DNA (pDNA) reference material through copy number (CN) calibration was used to develop a primary control pDNA at Cepheid. The reference pDNA contains ABL and BCR-ABL b3a2-p210 (IRMM_ERM-AD623 BCR-ABL Calibrator) for standardization of ABL and BCR-ABL e1a2-p190 quantifications. This process allows generation of in-house secondary RNA control panel, for calibration and standardization of the Xpert BCR-ABL p190 Ultra assay to the WHO IS (NIBSC-09/138). Method: Titration studies were performed with IRMM's pDNA to derive a CN calibration curve of the ABL control gene relative to ABL Cycle Threshold (Ct), to assign CN to Cepheid's pDNA containing e1a2 target gene and ABL control gene in a 1:1 ratio. The obtained CN information for Cepheid's pDNA was then used to generate corresponding ABL Ct and e1a2 Ct in a simulated biological matrix, followed by the assignment of CN to the in-house RNA control material. A 5-log concentration range of a malignant BCR-ABL e1a2 positive ALL cell line, SUP-B15, is used as an interim in-house control material with an assigned % BCR-ABL e1a2/ABL value to evaluate the assay's linearity/dynamic range performance. Results: Through the ABL calibration curve generated from IRMM reference material, CN of Cepheid's e1a2:ABL pDNA were assigned. The ABL Ct was generated using IRMM reference material with a 6-log dynamic range of ABL CN, slope of -1.41, R2=0.9924 (Figure 1). Using the ABL Ct obtained from the Cepheid's pDNA construct, allowed us to link Cepheid's pDNA ABL CN to IRMM's, thus linking e1a2 CN to ABL CN, demonstrated in a 5-log dynamic range of ABL and e1a2 CN. Similar slopes of -1.491, R2=0.9972 and -1.464, R2=0.9966 were obtained from Cepheid's pDNA between input CN and output Ct for e1a2 and ABL, respectively (Figure 2). Finally, a 5-log dynamic range of ∆Ct (e1a2-ABL) was generated from SUP-B15 cells, yielding slope of -1.427, R2=0.9917 (Figure 3), allowing assignment of %BCR-ABL e1a2/ABL value. Conclusion: In conclusion, implementing a certified pDNA reference material (IRMM) allowed linkage of known ABL CN to Cepheid's pDNA. Preliminary data from SUP-B15 cells suggested that we achieved the goal of generating a control material with assigned e1a2 CN. * Prototype test. Not for use in diagnostic procedures. Not reviewed by any regulatory body. Disclosures No relevant conflicts of interest to declare.

Insertion Mutations of the shaggy Gene, Encoding Protein Kinase GSK3, Extend Drosophila melanogaster Lifespan

The shaggy gene of Drosophila melanogaster encodes highly conserved serine-threonine protein kinase GSK3 (Glycogen Syntase Kinase 3), which plays an important role in different signaling pathways and metabolic processes. This study is the first to demonstrate that shaggy mutations affect lifespan, with an increase in longevity associated with a decrease in synapse activity. The results from this study on changes in shaggy expression in mutants suggest that the observed phenotypic changes are based on an alternation in the expression of minor shaggy transcripts induced by mutational changes in their regulatory region.

Regional fat depot masses are influenced by protein-coding gene variants.

Waist-to-hip ratio (WHR) is a prominent cardiometabolic risk factor that increases cardio-metabolic disease risk independently of BMI and for which multiple genetic loci have been identified. However, WHR is a relatively crude proxy for fat distribution and it does not capture all variation in fat distribution. We here present a study of the role of coding genetic variants on fat mass in 6 distinct regions of the body, based on dual-energy X-ray absorptiometry imaging on more than 17k participants. We find that the missense variant CCDC92S70C, previously associated with WHR, is associated specifically increased leg fat mass and reduced visceral but not subcutaneous central fat. The minor allele-carrying transcript of CCDC92 is constitutively more highly expressed in adipose tissue samples. In addition, we identify two coding variants in SPATA20 and UQCC1 that are associated with arm fat mass. SPATA20K422R is a low-frequency variant with a large effect on arm fat only, and UQCC1R51Q is a common variant reaching significance for arm but showing similar trends in other subcutaneous fat depots. Our findings support the notion that different fat compartments are regulated by distinct genetic factors.

Functional classification of ATM variants in ataxia-telangiectasia patients.

Ataxia-telangiectasia (A-T) is a recessive disorder caused by biallelic pathogenic variants of ataxia-telangiectasia mutated (ATM). This disease is characterized by progressive ataxia, telangiectasia, immune deficiency, predisposition to malignancies, and radiosensitivity. However, hypomorphic variants may be discovered associated with very atypical phenotypes, raising the importance of evaluating their pathogenic effects. In this study, multiple functional analyses were performed on lymphoblastoid cell lines from 36 patients, comprising 49 ATM variants, 24 being of uncertain significance. Thirteen patients with atypical phenotype and presumably hypomorphic variants were of particular interest to test strength of functional analyses and to highlight discrepancies with typical patients. Western-blot combined with transcript analyses allowed the identification of one missing variant, confirmed suspected splice defects and revealed unsuspected minor transcripts. Subcellular localization analyses confirmed the low level and abnormal cytoplasmic localization of ATM for most A-T cell lines. Interestingly, atypical patients had lower kinase defect and less altered cell-cycle distribution after genotoxic stress than typical patients. In conclusion, this study demonstrated the pathogenic effects of the 49 variants, highlighted the strength of KAP1 phosphorylation test for pathogenicity assessment and allowed the establishment of the Ataxia-TeLangiectasia Atypical Score to predict atypical phenotype. Altogether, we propose strategies for ATM variant detection and classification.

Transcriptomic-Proteomic Correlation in the Predation-Evoked Venom of the Cone Snail, Conus imperialis.

Individual variation in animal venom has been linked to geographical location, feeding habit, season, size, and gender. Uniquely, cone snails possess the remarkable ability to change venom composition in response to predatory or defensive stimuli. To date, correlations between the venom gland transcriptome and proteome within and between individual cone snails have not been reported. In this study, we use 454 pyrosequencing and mass spectrometry to decipher the transcriptomes and proteomes of the venom gland and corresponding predation-evoked venom of two specimens of Conus imperialis. Transcriptomic analyses revealed 17 conotoxin gene superfamilies common to both animals, including 5 novel superfamilies and two novel cysteine frameworks. While highly expressed transcripts were common to both specimens, variation of moderately and weakly expressed precursor sequences was surprisingly diverse, with one specimen expressing two unique gene superfamilies and consistently producing more paralogs within each conotoxin gene superfamily. Using a quantitative labelling method, conotoxin variability was compared quantitatively, with highly expressed peptides showing a strong correlation between transcription and translation, whereas peptides expressed at lower levels showed a poor correlation. These results suggest that major transcripts are subject to stabilizing selection, while minor transcripts are subject to diversifying selection.

Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 (IL-4) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

High-throughput sequencing has revealed a tremendous complexity of cellular transcriptomes, which is partly due to the generation of multiple alternative transcripts from a single gene locus. Because alternative transcripts often have low abundance in bulk cells, the functions of most of these transcripts and their relationship with their canonical counterparts remain unclear. Here we applied single-cell RNA-Seq to analyze the transcriptome complexity of in vitro-differentiated, murine type 2 T helper (Th2) cells. We found that cytokine gene transcripts contribute most of the intercellular heterogeneity, with a group of universal cytokines, including interleukins 1a, 2, 3, and 16, being bimodally expressed. At the single-cell level, use of alternative promoters prevalently generated alternative transcripts. For instance, although undetectable in bulk cells, a noncoding RNA isoform of IL-4 (IL4nc), which was driven by an intronic promoter in the IL-4 locus, was predominantly expressed in a subset of Th2 cells. IL4nc displayed distinct temporal expression patterns compared with the canonical IL-4 mRNA and post-transcriptionally promoted the production of IL-4 protein in Th2 cells. In conclusion, our findings reveal a mechanism whereby minor noncanonical transcripts post-transcriptionally regulate expression of their cognate canonical genes.