Prognostic significance of germline BRCA mutations in patients with HER2-POSITIVE breast cancer.

Background: HER2-positive breast cancers are rare amongst BRCA mutation carriers. No data exist regarding clinicopathological characteristics and prognosis of this subgroup of patients. Materials and methods: Using a retrospective matched cohort design, we collected data from 700 women who were diagnosed with operable invasive breast cancer from January 2006 to December 2016 and were screened for germline BRCA mutations. Clinicopathological features and survival rates were analyzed by BRCA and HER2 status. Results: One hundred and fifteen HER2-positive/BRCA mutated cases were evaluated in comparison to the three control groups: HER2-positive/BRCA wild type (n = 129), HER2-negative/BRCA mutated (n = 222), HER2-negative/BRCA wild type (n = 234). HER2-positive breast cancers were more likely to have high histologic grade and high proliferation rate than HER2-negative neoplasms, regardless of BRCA mutation status. An interaction between BRCA mutations and HER2-positive status was found to correlate with worse survival after adjusting for prognostic variables (HR = 3.4; 95% CI: 1.3-16.7). Conclusions: Co-occurrence of BRCA mutations and HER2-positive status is a poor prognostic factor in patients with early or locally advanced breast cancer. This finding may be a proof of concept that a combined pharmacological intervention directed to these targets could be synergistic

Phase II study of eribulin in combination with gemcitabine for the treatment of patients with locally advanced or metastatic triple negative breast cancer (ERIGE Trial). Clinical and pharmacogenetic results on behalf of the Gruppo Oncologico Italiano di Ricerca Clinica (GOIRC)

Background: There are no well-established chemotherapy regimens for metastatic triple negative breast cancer. The combination of a microtubule inhibitor (eribulin) with a nucleoside analog (gemcitabine) may synergistically induce tumor cell death, especially in tumors like triple negative breast cancers (TNBC) characterized by high cell proliferation, aggressive tumor behavior, and chemo-resistance. Materials and Methods:This is an open-label, national multicenter phase II study evaluating the combination of eribulin (0.88 mg/m2) plus gemcitabine (1000 mg/m2) on day 1 and 8, q21 as either first- or second-line treatment of locally advanced or metastatic TNBC.The primary endpoint was the objective response rate (ORR) for evaluable patients (pts). The study was designed according to the Simon's two stage optimal design. We chose the lower activity (p0) of 0.20 and target activity level (p1) of 0.35. A prospective, molecular correlative study has been being carried out on germinal DNA of study population to assess the role of BRCA mutations and single nucleotide polymorphisms (SNPs) in predicting efficacy and toxicity of the combination regimen. Results: From July 2013 to September 2016, 83 evaluable pts (37 in the first stage, 46 in the second one) were enrolled. They received a median number of 6 cycles of treatment (range 1-24). The ORR (CR+PR) was 37.35% (90% CI: 28.47-46.93) and the clinical benefit rate (CR+PR+SD 65 24wks) was 48.78% (90% CI: 39.24%-58.39%). The most common grade 3-4 adverse events (> 10% of patients) were neutropenia and liver toxicity. With a median follow-up of 28.8 months, the median progression-free survival (PFS) and overall survival (OS) were 5.1 months (95% CI: 4.2-7.0) and 14.7 months (95% CI: 10.2-20.0), respectively. BRCA1/2 deleterious mutations were observed in 15 (22%) out of 68 genotyped pts. Women with BRCA1/2 mutations were associated with worse ORR, PFS and OS than those with BRCA1/2 wild-type. A panel of SNPs in genes of study drug metabolism pathways was evaluated. Among these, CYP3A4 392A >G and FGD4 2044236G>A SNPs were associated with greater liver toxicity by logistic regression analysis. Furthermore, CDA*2 79A>C, RRM1 2455 A>G, and CYP2C8 416G>A SNPs were associated with poorer overall survival by Cox proportional hazards model. Conclusions:The combination of eribulin and gemcitabine shows promising activity and a moderate toxicity profile in metastatic TNBC. BRCA status and pharmacogenetics tests may help identify pts with high probability of response with negligible toxicity

Viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco

Background: Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10. Results: Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T1 plants were 10.8 and 37.0 \u3bcg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells. Conclusion: Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein

Modulating signaling networks by CRISPR/Cas9-mediated transposable element insertion

In a recent past, transposable elements (TEs) were referred to as selfish genetic components only capable of copying themselves with the aim of increasing the odds of being inherited. Nonetheless, TEs have been initially proposed as positive control elements acting in synergy with the host. Nowadays, it is well known that TE movement into host genome comprises an important evolutionary mechanism capable of increasing the adaptive fitness. As insights into TE functioning are increasing day to day, the manipulation of transposition has raised an interesting possibility of setting the host functions, although the lack of appropriate genome engineering tools has unpaved it. Fortunately, the emergence of genome editing technologies based on programmable nucleases, and especially the arrival of a multipurpose RNA-guided Cas9 endonuclease system, has made it possible to reconsider this challenge. For such purpose, a particular type of transposons referred to as miniature inverted-repeat transposable elements (MITEs) has shown a series of interesting characteristics for designing functional drivers. Here, recent insights into MITE elements and versatile RNA-guided CRISPR/Cas9 genome engineering system are given to understand how to deploy the potential of TEs for control of the host transcriptional activity.Fil: Vaschetto, Luis Maria Benjamin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Cátedra de Diversidad Animal I; Argentin

Improvement of regeneration in pepper: a recalcitrant species

[EN] Organogenesis is influenced by factors like genotype, type of explant, culture medium components, and incubation conditions. The influence of ethylene, which can be produced in the culture process, can also be a limiting factor in recalcitrant species like pepper. In this work, bud induction was achieved from cotyledons and hypocotyls-from eight pepper cultivars-on Murashige and Skoog (MS) medium supplemented with 22.2 mu M 6-benzyladenine (6BA) and 5.71 mu M indole-3-acetic acid (IAA), in media with or without silver nitrate (SN) (58.86 mu M), a suppressor of ethylene action. In the SN-supplemented medium, the frequencies of explants with buds and with callus formation were lower in both kinds of explant, but higher numbers of developed shoots were isolated from explants cultured on SN. Bud elongation was better in medium with gibberellic acid (GA(3)) (2.88 mu M) than in medium free of growth regulators or supplemented with 1-aminocyclopropane-1-carboxylic acid (ACC) at 34.5 mu M. However, isolation of shoots was difficult and few plants were recovered. The effect of adding SN following bud induction (at 7 d) and that of dark incubation (the first 7 d of culture) was also assessed in order to improve the previous results. When SN was added after bud induction, similar percentages of bud induction were found for cotyledons (average frequency 89.37% without SN and 94.37% with SN) whereas they doubled in hypocotyls (50% without SN and 87.7% with SN). In addition, in both kinds of explant, the number of developed plants able to be transferred to soil (developed and rooted) was greatly increased by SN. Dark incubation does not seem to improve organogenesis in pepper, and hypocotyl explants clearly represent a better explant choice-with respect to cotyledonary explants-for the pepper cultivars assayed.We thank the COMAV germplasm bank at Universitat Politecnica de Valencia and the Arid Lands Institute for pepper seeds and the Tunisian Ministry of Higher Education and Scientific Research who fund N. Gammoudi's stay.Gammoudi, N.; San Pedro-Galan, T.; Ferchichi, A.; Gisbert Domenech, MC. (2018). Improvement of regeneration in pepper: a recalcitrant species. In Vitro Cellular & Developmental Biology - Plant. 54(2):145-153. https://doi.org/10.1007/s11627-017-9838-1S145153542Ashrafuzzaman M, Hossain MM, Razi Ismail M, Shahidul Haque M, Shahidullah SM, Uz Zaman S (2009) Regeneration potential of seedling explants of chilli (Capsicum annuum). Afr J Biotechnol 8:591–596Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166:1292–1297Brown DC, Thorpe TA (1995) Crop improvement through tissue culture. World J Microbiol Biotechnol 11:409–415Carvalho MAF, Paiva R, Stein VC, Herrera RC, Porto JMP, Vargas DP, Alves E (2014) Induction and morpho-ultrastructural analysis of organogenic calli of a wild passion fruit. Braz Arch Biol Technol 57:581–859Christopher T, Rajam MV (1996) Effect of genotype, explant and medium on in vitro regeneration of red pepper. Plant CellTiss Org Cult 46:245–250Dabauza M, Peña L (2001) High efficiency organogenesis in sweet pepper (Capsicum annuum L.) tissues from different seedling explants. Plant Growth Regul 33:221–229De Filippis LF (2014) Crop improvement through tissue culture. In: Ahmad P, Wani MR, Azooz MM, Tran LSP (eds) Improvement of crops in the era of climate changes, vol 1. Springer, New York, pp 289–346Gammoudi N, Ben Yahia L, Lachiheb B, Ferchichi A (2016) Salt response in pepper (Capsicum annuum L.): components of photosynthesis inhibition, proline accumulation and K+/Na+ selectivity. JJ Aridland Agri 2:1–12González A, Arigita L, Majada J, Sánchez Tamés R (1997) Ethylene involvement in in vitro organogenesis and plant growth of Populus tremula L. Plant Growth Regul 22:1–6Grozeva S, Rodeva V, Todorova V (2012) In vitro shoot organogenesis in Bulgarian sweet pepper (Capsicum annuum L.) varieties. EJBio 8:39–44Gunay AL, Rao PS (1978) In vitro plant regeneration from hypocotyls and cotyledon explants of red pepper (Capsicum). Plant Sci Lett 11:365–372Huxter TJ, Thorpe TA, Reid DM (1981) Shoot initiation in light- and dark-grown tobacco callus: the role of ethylene. Physiol Plant 53:319–326Hyde CL, Phillips GC (1996) Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis. In Vitro-Plant 32:72–80Kothari SL, Joshi A, Kachhwaha S, Ochoa-Alejo N (2010) Chilli peppers—a review on tissue culture and transgenesis. Biotechnol Adv 28:35–48Kumar AO, Rupavathi T, Tata SS (2012) Adventitious shoot bud induction in chili pepper (Capsicum annuum L. cv. X-235). In J Sci Nat 3:192–196Kumar PP, Lakshmanan P, Thorpe TA (1998) Regulation of morphogenesis in plant tissue culture by ethylene. In Vitro Cell Dev Biol Plant 34:94–103Liu W, Parrott WA, Hildebrand DF, Collins GB, Williams EG (1990) Agrobacterium induced gall formation in bell pepper (Capsicum annuum L.) and formation of shoot-like structures expressing introduced genes. Plant Cell Rep 9:360–364Maligeppagol M, Manjula R, Navale PM, Babu KP, Kumbar BM, Laxman RH (2016) Genetic transformation of chilli (Capsicum annuum L.) with Dreb1A transcription factor known to impart drought tolerance. Indian J Biotechnol 15:17–24Mantiri FR, Kurdyukov S, Chen SK, Rose RJ (2008) The transcription factor MtSERF1 may function as a nexus between stress and development in somatic embryogenesis in Medicago truncatula. Plant Signal Behav 3:498–500Mezghani N, Jemmali A, Elloumi N, Gargouri-Bouzid R, Kintzios S (2007) Morpho-histological study on shoot bud regeneration in cotyledon cultures of pepper (Capsicum annuum). Biologia 62:704–710Mohamed-Yasseen Y (2001) Influence of agar and activated charcoal on uptake of gibberellin and plant morphogenesis in vitro. In Vitro Cell Dev Biol - Plant 37:204–205Moshkov IE, Novikova GV, Hall MA, George EF (2008) Plant growth regulators III: ethylene. In: George EF, Hall MA, Klerk G-JD (eds) Plant propagation by tissue culture, vol 1, 3rdedn. Springer, Dordrecht, The Netherlands, pp 239–248Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497Nogueira RC, Paiva R, de Oliveira LM, Soares GA, Soares FP, Castro AHF, Paiva PDO (2007) Calli induction from leaf explants of murici-pequeno (Byrsonima intermedia A. Juss.) Ciênc Agrotec 31:366–370Ochoa-Alejo N, Ramirez-Malagon R (2001) In vitro chili pepper biotechnology. In Vitro Cell Devl Biol Plant 37:701–729Orlińska M, Nowaczy P (2015) In vitro plant regeneration of 4 Capsicum spp. genotypes using different explant types. Turk J Biol 39:60–68Reid MS (1995) Ethylene in plant growth, development and senescence. In: Davies PJ (ed) Plant hormones: physiology, biochemistry and molecular biology, 2nd edn. 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Temperature responses of Rubisco from Paniceae grasses provide opportunities for improving C3 photosynthesis.

Enhancing the catalytic properties of the CO2-fixing enzyme Rubisco is a target for improving agricultural crop productivity. Here, we reveal extensive diversity in the kinetic response between 10 and 37 °C by Rubisco from C3 and C4 species within the grass tribe Paniceae. The CO2 fixation rate (kcatc) for Rubisco from the C4 grasses with nicotinamide adenine dinucleotide (NAD) phosphate malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) photosynthetic pathways was twofold greater than the kcatc of Rubisco from NAD-ME species across all temperatures. The declining response of CO2/O2 specificity with increasing temperature was less pronounced for PCK and NADP-ME Rubisco, which would be advantageous in warmer climates relative to the NAD-ME grasses. Modelled variation in the temperature kinetics of Paniceae C3 Rubisco and PCK Rubisco differentially stimulated C3 photosynthesis relative to tobacco above and below 25 °C under current and elevated CO2. Amino acid substitutions in the large subunit that could account for the catalytic variation among Paniceae Rubisco are identified; however, incompatibilities with Paniceae Rubisco biogenesis in tobacco hindered their mutagenic testing by chloroplast transformation. Circumventing these bioengineering limitations is critical to tailoring the properties of crop Rubisco to suit future climates

A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard

[EN] Background: The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell. The engineering principles of standardization and modularity applied to DNA cloning are impacting plant genetic engineering, by increasing multigene assembly efficiency and by fostering the exchange of well-defined physical DNA parts with precise functional information. Results: Here we describe the adaptation of the RNA-guided Cas9 system to GoldenBraid (GB), a modular DNA con¿ struction framework being increasingly used in Plant Synthetic Biology. In this work, the genetic elements required for CRISPRs-based editing and transcriptional regulation were adapted to GB, and a workflow for gRNAs construction was designed and optimized. New software tools specific for CRISPRs assembly were created and incorporated to the public GB resources site. Conclusions: The functionality and the efficiency of gRNA¿Cas9 GB tools were demonstrated in Nicotiana benthamiana using transient expression assays both for gene targeted mutations and for transcriptional regulation. The availability of gRNA¿Cas9 GB toolbox will facilitate the application of CRISPR/Cas9 technology to plant genome engineeringThis work has been funded by Grant BIO2013-42193-R from Plan Nacional I + D of the Spanish Ministry of Economy and Competitiveness. Vazquez-Vilar M. is a recipient of a Junta de Ampliacion de Estudios fellowship. Bernabe-Orts J.M. is a recipient of a FPI fellowship. We want to thank Nicola J. Patron and Mark Youles for kindly providing humanCas9 and U6-26 clones. We also want to thank Eugenio Gomez for providing Arabidopsis thaliana genomic DNA and Concha Domingo for providing rice genomic DNA. We also want to thank the COST Action FA1006 for the support in the development of the software tools.Vázquez-Vilar, M.; Bernabé-Orts, JM.; Fernández Del Carmen, MA.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.; Granell Richart, A.; Orzáez Calatayud, DV. (2016). A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 12. https://doi.org/10.1186/s13007-016-0101-2S12Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. doi: 10.1038/nprot.2013.143 .Yang X. Applications of CRISPR-Cas9 mediated genome engineering. Mil Med Res. 2015;2:11. doi: 10.1186/s40779-015-0038-1 .Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910–8. doi: 10.1016/j.cell.2013.04.025 .Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv. 2015;33(1):41–52. doi: 10.1016/j.biotechadv.2014.12.006 .Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol. 2015;32:76–84. doi: 10.1016/j.copbio.2014.11.007 .Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol. 2013;31(8):686–8. doi: 10.1038/nbt.2650 .Gao J, Wang G, Ma S, Xie X, Wu X, Zhang X, et al. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol. 2015;87(1–2):99–110. doi: 10.1007/s11103-014-0263-0 .Fauser F, Schiml S, Puchta H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J. 2014;79(2):348–59. doi: 10.1111/tpj.12554 .Schiml S, Fauser F, Puchta H. The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J. 2014;80(6):1139–50. doi: 10.1111/tpj.12704 .Piatek A, Ali Z, Baazim H, Li L, Abulfaraj A, Al-Shareef S, et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol J. 2015;13(4):578–89. doi: 10.1111/pbi.12284 .Beerli RR, Barbas CF 3rd. Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol. 2002;20(2):135–41. doi: 10.1038/nbt0202-135 .Bogdanove AJ, Voytas DF. TAL effectors: customizable proteins for DNA targeting. Science. 2011;333(6051):1843–6. doi: 10.1126/science.1204094 .Nielsen AA, Voigt CA. Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol Syst Biol. 2014;10:763. doi: 10.15252/msb.20145735 .Eeckhaut T, Lakshmanan PS, Deryckere D, Van Bockstaele E, Van Huylenbroeck J. Progress in plant protoplast research. Planta. 2013. doi: 10.1007/s00425-013-1936-7 .Mikami M, Toki S, Endo M. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant Mol Biol. 2015. doi: 10.1007/s11103-015-0342-x .Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, et al. Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol. 2015. doi: 10.1111/nph.13532 .Liu W, Stewart CN Jr. Plant synthetic biology. Trends Plant Sci. 2015;20(5):309–17. doi: 10.1016/j.tplants.2015.02.004 .Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J, Castelijns B, Forment J, Ziarsolo P, et al. GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 2013;162(3):1618–31. doi: 10.1104/pp.113.217661 .Vazquez-Vilar M, Sarrion-Perdigones A, Ziarsolo P, Blanca J, Granell A, Orzaez D. Software-assisted stacking of gene modules using GoldenBraid 2.0 DNA-assembly framework. Methods Mol Biol. 2015;1284:399–420. doi: 10.1007/978-1-4939-2444-8_20 .Duportet X, Wroblewska L, Guye P, Li Y, Eyquem J, Rieders J, et al. A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic Acids Res. 2014;42(21):13440–51. doi: 10.1093/nar/gku1082 .Guo Y, Dong J, Zhou T, Auxillos J, Li T, Zhang W, et al. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 2015;43(13):e88. doi: 10.1093/nar/gkv464 .Engler C, Gruetzner R, Kandzia R, Marillonnet S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE. 2009;4(5):e5553. doi: 10.1371/journal.pone.0005553 .Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juarez P, Fernandez-del-Carmen A, Granell A, et al. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS ONE. 2011;6(7):e21622. doi: 10.1371/journal.pone.0021622 .Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL. CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant. 2014;7(9):1494–6. doi: 10.1093/mp/ssu044 .Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. doi: 10.1126/science.1232033 .Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013;31(8):688–91. doi: 10.1038/nbt.2654 .Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41(15):7429–37. doi: 10.1093/nar/gkt520 .Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA. 2015;112(11):3570–5. doi: 10.1073/pnas.1420294112 .Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS ONE. 2011;6(2):e16765. doi: 10.1371/journal.pone.0016765 .Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci Rep. 2014;4:5400. doi: 10.1038/srep05400 .Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. 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Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

Abstract The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared to information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known non-pathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification. This article is protected by copyright. All rights reserved.Peer reviewe