Articoli di riviste: "Somatic genetic rescue"

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Revy, Patrick, Caroline Kannengiesser e Alain Fischer. "Somatic genetic rescue in Mendelian haematopoietic diseases". Nature Reviews Genetics 20, n. 10 (11 giugno 2019): 582–98. http://dx.doi.org/10.1038/s41576-019-0139-x.

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Ahmed, Arooj, Luca Guarnera, Jaymeson Gordon, Carlos Bravo-Perez, Arda Durmaz, Yasuo Kubota, Naomi Kawashima et al. "Maladaptive Somatic Rescue in FLT3 Mutations of Suspected Germline Nature". Blood 142, Supplement 1 (28 novembre 2023): 5666. http://dx.doi.org/10.1182/blood-2023-190837.

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Abstract (sommario):

Somatic genetic rescue (SGR) is a very rare process in which the occurrence of a somatic genetic event offsets the consequences of a germline (GL) mutation resulting in genetic mosaicism and, in some cases, in a milder disease phenotype 1. Acquired somatic mutations may be adaptive by countering the negative effects of the primary mutation ( e.g., improving hematopoiesis) or maladaptive by overcorrecting the initial impairment causing a different, possibly opposite, disease phenotype ( e.g., leading to the development of a myeloid malignancy) 2. Exemplary cases of the latter scenario are mainly represented by severe congenital neutropenia (SCN) cases developing AML as consequence of somatic CSF3R mutations 3 and, sporadically, by adaptive SGR in the context of germline GATA2 and secondary acquisition of somatic GATA2 mutation, counterbalancing the germline effect 4. Although SGR has been functionally demonstrated in these two above mentioned cases, given the molecular heterogeneity of myeloid neoplasia (MN) and the rarity of such genetic events, one can suspect that, in various clinical contexts, any mutation in any gene could be the SG rescuer. During our clinical experience, we encountered a case of a 57yo. woman with newly diagnosed aplastic anemia (AA) who was found to harbor a rare germline FLT3R311W mutation (VAF 50%; predicted to be a loss-of-function/hypomorphic alteration; Fig1) who subsequently evolved to MDS with monosomy 7. Coexisting somatic hits included non RUNT-homology domain, RUNX1P398L mutation (VAF 19%). Ultimately, the patient progressed to AML with emergence of a FLT3D825V (VAF 14%) and a NRASG13D (24%) lesions. In this case carrying GL FLT3 variant, biallelic somatic FLT3 mutation may represent maladaptive SGR. Therefore, we reviewed NGS sequencing results of 5,308 patients with MN and found three other suspected cases harboring GL variants in FLT3 gene, which inspired further investigations. In total we identified 3 additional cases (total of 4/5308 screened patients and among 248 somatic FLT3 mutations. Interestingly, the other case GL FLT3A680T occurred in a patient with AA (35yo.) who subsequently progressed to MDS and AML and acquired somatic NPM1L258fs, PTPN11 A72V, WT1 A365fs and most importantly, a somatic CSF3RL619S . The latter may represent an illustrative case of SGR, in analogy to AML developing in the context of SCN. In addition, 2 other cases with MDS or MDS/MPN were identified both presenting with cytopenia. A 53yo. woman diagnosed with MDS/MPN and GL FLT3L262F (gnomAD: 0.00000399) with a compound heterozygous somatic JAK2V617F mutation, again possibly serving as maladaptive clonal SGR event. Finally, we have identified a GL FLT3A291P mutation (VAF 60%, gnomAD frequency: 1.59×10-5) in a 58yo. man who eventually developed AML. In sum, similar to other phosphotyrosine receptor kinases (PTRKs) such as CSF3R, somatic FLT3 mutations may in rare biallelic cases correspond to SGR events of hypomorphic GL mutation or alternatively but not exclusively somatic mutations in other PTRK could evolve to reveal the GL FLT3 deficiency. Indeed, in another abstract by our group (Abstract#187151) we show that somatic FLT3 mutations can accompany GL variants in CSF3R, CSF2RB, and CSF1R. References 1 Revy P, Kannengiesser C, Fischer A. Somatic genetic rescue in Mendelian haematopoietic diseases. Nat Rev Genet. 2019 Oct;20(10):582-598. 2 Kennedy AL, Shimamura A. Genetic predisposition to MDS: clinical features and clonal evolution. Blood. 2019 Mar 7;133(10):1071-1085. 3 Germeshausen M, Kratz CP, Ballmaier M, Welte K. RAS and CSF3R mutations in severe congenital neutropenia. Blood. 2009 Oct 15;114(16):3504-5. 4 Catto LFB, Borges G, Pinto AL, Clé DV, Chahud F, Santana BA, Donaires FS, Calado RT. Somatic genetic rescue in hematopoietic cells in GATA2 deficiency.

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Catto, Luiz Fernando B., Gustavo Borges, André L. Pinto, Diego V. Clé, Fernando Chahud, Barbara A. Santana, Flavia S. Donaires e Rodrigo T. Calado. "Somatic genetic rescue in hematopoietic cells in GATA2 deficiency". Blood 136, n. 8 (20 agosto 2020): 1002–5. http://dx.doi.org/10.1182/blood.2020005538.

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Haploinsufficiency of GATA2 caused by heterozygous loss-of-function mutations is associated with cytopenias and predisposition to myelodysplasia and AML with other variable extrahematopoietic manifestions, including lymphedema, pulmonary alveolar proteinosis, and hearing loss. The authors report on 2 siblings with the disorder whose father was asymptomatic because of an acquired missense mutation in the affected allele that was restricted to hematopoietic cells; surprisingly, he also had no extrahematopoietic complications.

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Orland, Mark, Arda Durmaz, Carlos Bravo-Perez, Carmelo Gurnari, Luca Guarnera, Matteo D'Addona, Aashray Mandala et al. "Elucidating the Somatic Genetic Rescue Underlying Del(20q) Myeloid Neoplasms". Blood 144, Supplement 1 (5 novembre 2024): 4573. https://doi.org/10.1182/blood-2024-208520.

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Deletion of the long arm of chromosome 20 (20q-) is a common lesion in myeloid neoplasia (MN). In Shwachman-Diamond syndrome (DBA), 20q- can evolve as a somatic genetic rescue (SGR) event, but in adult patients with 20q-, germline alterations were not identified. When we previously queried alterations in other inherited BMF genes, no significant hits were identified to explain emergence of this lesion in adult MN as a form of SGR (1). Similarly, 20q- did not appear to represent loss of heterozygosity (LOH) from heterozygous germline mutations. Herein, we aim to examine somatic variants within and outside the 20q- common deleted region (CDR) to narrow the search for culprit genes associated with the pathogenesis and resultant phenotypes of 20q-. This approach will help identify LOH of potential tumor suppressor genes (TSGs) for which deletion would remove the protective allele. Upon review of 4,751 MN patients, we identified 136 cases with del(20q). 79 of the del(20q) patients had sufficient molecular annotation including SNP array, RNASeq and NGS for identification of somatic hits. SNP array was used to create a cartography of the CDR, defined as bands deleted in >50% of patients and deemed at q11.22-q13.13. From the 445 genes within the CDR, the expression of 392 was restricted to hematopoietic cells. Results from log2 fold change showed that while 100 genes were found to have decreased expression vs diploid controls, the clonality of the 20q- inversely correlated with expression of 64 genes allowing for further refining of putative culprits affected by haploinsufficiency. Notably, EIF6, the hallmark gene of SGR in DBA, was haploinsufficient (HI) but did not correlate with increased clonality. There were no genes within the CDR that paradoxically increased with increased clonality suggesting that LOH of a protective allele of oncogenes is not a possibility. From the 64 genes, 7 were known TSGs: RBL1, E2F1, NCOA5, PTPN1, ZMYND8, STK4, and MYBL2. Notably, MYBL2 is a synthetic lethal gene which, if ablated, in mice led to clonal dominance with a resultant MDS phenotype. There were no suspicious, functionally impactful SNPs in 6 of the 7 identified TSGs. In PTPN1, 11 rare SNPs were identified but no allelic imbalance for a minor allele was found. Unsupervised hierarchical clustering of the 64 HI genes correlating with clonality resulted in 2 distinct clusters. There were no clusters resembling 20q- among diploid cases. Further non-linear dimension reduction by UMAP revealed a clear distinction in MDS vs MPN suggesting diverse gene expression patterns associated with each diagnosis (p=.002). To identify potential synthetic lethal targets, we then studied HI genes in the CDR from prior studies to examine for lethality in knockout (KO) murine and cell line models: only L3MBTL1 and MYBL2 were identified with the former not correlating with clonality. Further, we searched for genes on other chromosomes showing consistent compensatory up or down regulation in correlation with 20q. From the 12,583 genes, 172 genes were significantly different between del(20q) vs diploid samples. 143 of the genes were up modulated (17 with known small molecule inhibitors) and 19 were down modulated (none known as synthetic lethal in KO models). We further limited the 172 genes by correlation with clonality: 17 genes had significant correlation (r2 > 0.89). 5 of 17 were known to be important to erythroid differentiation or leukemia: LDB1, RHOA, TCF3, SLC20A1, and HEXIM1. HEXIM1 was identified as a previous synthetic lethal gene with a key role in erythroid non-clonal expansion in a murine model. Hierarchical clustering of non-20q genes with altered expression in 20q- showed 2 clusters: one composed of 3 del(20q) patients with clonality ranging from 40 to 60% and a second with the remaining del(20q) and diploid patients. Further unsupervised subclustering found one cluster (from ten total) with 78% of del(20q), 33% of HR MDS, 27% of LR MDS, and 7% of MPN patients which associates del(20q) more to a diploid MDS than a MPN. In summary, we found 7 TSGs within the CDR and 5 outside of it that may be culprit genes for the del(20q) MN phenotype. There were 2 synthetic lethal target genes (MYBL2, HEXIM1). Experimental analysis of these two will be performed to assess their role as therapeutic targets. Additional gene enrichment and coexpression network analysis of the non-20q genes and diploid MDS samples subclustered with del(20q) is ongoing.

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Starich, Todd, e David Greenstein. "A Limited and Diverse Set of Suppressor Mutations Restore Function to INX-8 Mutant Hemichannels in the Caenorhabditis elegans Somatic Gonad". Biomolecules 10, n. 12 (10 dicembre 2020): 1655. http://dx.doi.org/10.3390/biom10121655.

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In Caenorhabditis elegans, gap junctions couple cells of the somatic gonad with the germline to support germ cell proliferation and gametogenesis. A strong loss-of-function mutation (T239I) affects the second extracellular loop (EL2) of the somatic INX-8 hemichannel subunit. These mutant hemichannels form non-functional gap junctions with germline-expressed innexins. We conducted a genetic screen for suppressor mutations that restore germ cell proliferation in the T239I mutant background and isolated seven intragenic mutations, located in diverse domains of INX-8 but not the EL domains. These second-site mutations compensate for the original channel defect to varying degrees, from nearly complete wild-type rescue, to partial rescue of germline proliferation. One suppressor mutation (E350K) supports the innexin cryo-EM structural model that the channel pore opening is surrounded by a cytoplasmic dome. Two suppressor mutations (S9L and I36N) may form leaky channels that support germline proliferation but cause the demise of somatic sheath cells. Phenotypic analyses of three of the suppressors reveal an equivalency in the rescue of germline proliferation and comparable delays in gametogenesis but a graded rescue of fertility. The mutations described here may be useful for elucidating the biochemical pathways that produce the active biomolecules transiting through soma–germline gap junctions.

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Navrátilová, B., D. Skálová, V. Ondřej, M. Kitner e A. Lebeda. "Biotechnological methods utilized in Cucumis research – A review". Horticultural Science 38, No. 4 (15 novembre 2011): 150–58. http://dx.doi.org/10.17221/143/2010-hortsci.

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Our biotechnological research on selected Cucumis species has encompassed interspecific hybridization via embryo-rescue, in vitro pollination, somatic hybridization and cytogenetics of protoplasts. Embryo-rescue and in vitro pollination are suitable in vitro techniques for production of hybrid embryos. These methods were tested and optimized for cucurbits. Protoplast culture is another valuable tool for biotechnological applications, such as somatic hybridization and genetic transformation. We study protoplast dedifferentiation not only as a biotechnological application in breeding systems, but mainly to describe mechanisms of obtaining totipotency. Protoplasts of cucurbits were studied cytogenetically to observe changes in nuclear architecture during protoplastization and regeneration and for comparison with the expression profile obtained using cDNA-AFLP techniques and reverse transcription for the specific genes involved.

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Gray, N., M. Boals, S. Lewis, M. Yoshida, S. Sahoo e M. Wlodarski. "SIGNATURES OF SOMATIC GENETIC RESCUE IN SAMD9/9L SYNDROMES: DIAGNOSTIC AND PROGNOSTIC UTILITY". Leukemia Research Reports 21 (2024): 100432. http://dx.doi.org/10.1016/j.lrr.2024.100432.

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Venugopal, Parvathy, Peer Arts, Lucy C. Fox, Annet Simons, Devendra K. Hiwase, Peter G. Bardy, Annette Swift et al. "Unraveling facets of MECOM-associated syndrome: somatic genetic rescue, clonal hematopoiesis, and phenotype expansion". Blood Advances 8, n. 13 (9 luglio 2024): 3437–43. http://dx.doi.org/10.1182/bloodadvances.2023012331.

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Xu, Xia, Jiang Lu e O. Lamikanra. "Somatic Embryogenesis in Muscadine Grape". HortScience 30, n. 4 (luglio 1995): 876G—877. http://dx.doi.org/10.21273/hortsci.30.4.876g.

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Low frequency of in vitro regeneration has hampered the adoption of genetic engineering technique for improving the quality of muscadine grape. This study is to develop a straightforward method for high-frequency regeneration of muscadine grapes in vitro. Leaves, petioles, and immature ovules of muscadine grapes were cultured on various media. Embryogenic callus, somatic embryos were formed after 9 weeks inoculated on embryo rescue (ER) medium. The somatic embryos were isolated and subcultured on fresh medium to promote enlargement and increase the number of uniformly sized somatic embryos. Of the medium tested (MS, NN, and ER), the ER medium was the best for somatic embryo growth and multiplication. The somatic embryogenic lines were maintained by transferring the embryos to the fresh ER medium every 4 weeks. Germination was achieved by transferring these embryos to woody plant medium or NN medium. The frequency of somatic embryogenesis of embryo germination appeared to be genotype dependent. The establishment of the somatic embryogenesis system in this study should be a step forward in directly transferring a foreign gene into muscadine grape.

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Xu, Xia, Jiang Lu e O. Lamikanra. "Somatic Embryogenesis in Muscadine Grape". HortScience 30, n. 4 (luglio 1995): 876G—877. http://dx.doi.org/10.21273/hortsci.30.4.876.

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Abstract (sommario):

Low frequency of in vitro regeneration has hampered the adoption of genetic engineering technique for improving the quality of muscadine grape. This study is to develop a straightforward method for high-frequency regeneration of muscadine grapes in vitro. Leaves, petioles, and immature ovules of muscadine grapes were cultured on various media. Embryogenic callus, somatic embryos were formed after 9 weeks inoculated on embryo rescue (ER) medium. The somatic embryos were isolated and subcultured on fresh medium to promote enlargement and increase the number of uniformly sized somatic embryos. Of the medium tested (MS, NN, and ER), the ER medium was the best for somatic embryo growth and multiplication. The somatic embryogenic lines were maintained by transferring the embryos to the fresh ER medium every 4 weeks. Germination was achieved by transferring these embryos to woody plant medium or NN medium. The frequency of somatic embryogenesis of embryo germination appeared to be genotype dependent. The establishment of the somatic embryogenesis system in this study should be a step forward in directly transferring a foreign gene into muscadine grape.

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Unlu, Serhan, Christopher Haddad, Danai Dima, Tariq Kewan, Olisaemeka Ogbue, Waled Bahaj, Carlos Bravo-Perez et al. "Somatic Genetic Rescue Involving CSF3R and Other Novel Phosphothyrosine Kinase Receptor Mutations Occurring in Myeloid Malignancies". Blood 142, Supplement 1 (28 novembre 2023): 3219. http://dx.doi.org/10.1182/blood-2023-187151.

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Somatic gene rescue (SGR), via acquisition and selection of somatic (SM) genetic hits, neutralizes functional defects resulting from hypomorphic germline (GL) mutations. However, this process may be maladaptive and lead to the development of leukemia. Acquisition of SM gain-of-function CSF3R mutations in the context of severe congenital neutropenia (SCN) illustrates such a scenario. Here, we further explored this and similar mechanisms in a cohort of 7121 adults with myeloid neoplasia and bone marrow failure (AML=1473, MDS=2248, other=3400). CSF3R mutations were found in 97 patients. In total, there were 37 and 56 patients with SM and GL mutations, respectively; biallelic GL and SM were 4. Among SM alterations, 18 were pathogenic or likely pathogenic including 8 nonsense truncating variants in class III isoform specific region of the cytoplasmic domain and 10 classical missense mutations in the juxtamembrane region, previously defined in the context of post-SCN AML and CNL. We thus hypothesized that these CSF3R SM mutations may also represent SGR in adult patients harboring hypomorphic GL variants and investigated the presence of SCN-associated alterations ( ELANE, WAS, GFI1) and in other phosphothyrosine kinase receptors (PTKR) such as CSF1R, CSF2RA, CSF2RB, FLT3 and KIT. Indeed, we found biallelic WAS p.D264H GL and CSF3R p.A119T SM in a patient with aplastic anemia (AA). Biallelic CSF3R were present in 4 patients: a) p.Q749X GL withp.M696T SM, b) E835K GL with p.T618I SM, c) p.Y752X GL withp.Q754E SM and d) biallelic p.Y752X GL with p.Q754E GL configuration with T615A SM. Compound heterozygous configuration of CSF3R SM included CSF1R SM variants( CSF3R p.M696T SM & CSF1R p.R294Q GL in a 64 year old. patient with MPN, CSF3R p.M696T SM & CSF1R c.1626+3G>A GL in a 61 year old patient with MDS) and CSF2RB ( CSF3R p.T618I SM & CSF2RB p.V890I GL in a 69 year old female patient with AML with leukopenia). When we analyzed the remaining 66 GL CSF3R variants in 56 carriers (in addition to the aforementioned biallelic combinations), we identified 9 compound heterozygous mutants involving CSF1R SM (n=1), CSF2RA (n=1), CSF2RB (n=3), FLT3(TKD/ITD, n=4/1). Specifically, we found the following configurations and disease associations: a) CSF3R p.V372E GL CSF2RB p.P842L SM in a 49 yo. AA patient who subsequently developed AML with FLT3 TKD, b) MDS patients with CSF3R p.E149 GL CSF1R p.G413S SM, c) CSF3R p.R440Q GL& CSF2RA p.R164Q SM and d) CSF3R p.E405K GL& CSF2RB c.1152+6G>A SM and e) AML patients with CSF3R p.E405K GL& CSF2RB p.R432C SM; f) CSF3R p.L619S GLandg,h)2 AML cases with CSF3R p.E808K GL of which all 3 acquired compound heterozygosity with FLT3 TKD, i) CSF3R p.E808K GL CSF3R p.E835K GL in a 71 year old patient with MDS with FLT3 ITD AML. In addition to FLT3 GOF mutations, the most common SM hits observed in CSF3R GLcarriers were ASXL1 (n=12), TET2 (n=12), and SF3B1 (n=12). Identification of somatic and GL alterations in PTKR genes other than CSF3R, led us to systematically analyze these genes as targets of GL and SM CSF3R alterations. In total, we identified 72 GL and 73 SM CSF1R variants, 7 SM and 19 GL CSF2RA and 47 GL and 69 SM CSFR2B. Among these, we found 3 patients with compound heterozygous CSF2RB SM CSF3R GL, one with CSF2RB GL CSF3R GLas described above. We also identified one patient with CSF2RA SM CSF3R GL . Finally, we identified two patients with CSF1R GL and CSF3R SM, and one with CSF1R SM CSF3R GLmutations. In summary, our study provides evidence for a tremendous complexity of interaction between less penetrant GL alterations of receptor genes and somatic GOF mutations, which may in some instances, correspond to the result of SGR. It is likely that the degree of LOF corresponds to the selection pressure for somatic rescue lesions.

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Sharma, Richa, Sushree S. Sahoo, Masayoshi Honda, Sophie L. Granger, Charnise Goodings, Louis Sanchez, Axel Künstner et al. "Gain-of-function mutations in RPA1 cause a syndrome with short telomeres and somatic genetic rescue". Blood 139, n. 7 (17 febbraio 2022): 1039–51. http://dx.doi.org/10.1182/blood.2021011980.

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Abstract Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

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Oyarbide, Usua, Valentino Bezzerri, Morgan Staton, Christian Boni, Arish Shah, Marco Cipolli, Eliezer Calo e Seth J. Corey. "Eif6 Dosage Alleviates Activation of the Tp53 Pathway in Sbds-Deficient Cells: A Mechanism for Somatic Genetic Rescue in Shwachman-Diamond Syndrome". Blood 144, Supplement 1 (5 novembre 2024): 4090. https://doi.org/10.1182/blood-2024-209812.

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Shwachman-Diamond syndrome (SDS) is characterized by skeletal abnormalities, pancreatic insufficiency, and neutropenia, with an increased risk of developing myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Nearly all cases of SDS are caused by biallelic mutations in the SBDS gene, which is critical for ribosome assembly. SBDS interacts with EFL1 to remove EIF6 from the 60S ribosomal subunit, facilitating ribosome formation in the cytoplasm. Research using mouse and zebrafish models has shown that Sbds deficiency activates the tumor suppressor protein Tp53, leading to developmental abnormalities and tissue-specific defects. TP53 biallelic mutations have been found in SDS patients with MDS/AML, suggesting a survival advantage for these mutations. SBDS deficiency is also linked to acquired deletions of chromosome 20q, where the EIF6 gene is located, or somatic mutations in EIF6. These changes, which have been termed somatic genetic rescue, are thought to compensate for the ribosome assembly defect in SDS and lower the risk of MDS/AML. Yet the mechanism of somatic genetic rescue has not been characterized. We created sbds-null zebrafish that exhibited Eif6 accumulation, changes in levels of ribosome proteins, and activation of Tp53 pathways. We have generated an eif6 knockout (KO) line which died earlier (~7-10 days post-fertilization) than the sbds KO line (~15 days post-fertilization). To determine the role of a Eif6 dose effect, we generated zebrafish mutants with low Eif6 protein expression (10% of the wildtype). Surprisingly, we observed that these eif6 hypomorph mutants survived to adulthood. Polysome profiling revealed significant reductions in the 80S monosomes and 40S ribosomal subunits in the eif6 KO at 5 dpf. However, the eif6 hypomorph mutants displayed polysome profiles similar to the eif6 wild type. We crossed the eif6 KO or hypomorph mutants with sbds-null fish and analyzed their phenotypic and molecular characteristics. In the sbds-null background, expression of eif6+/- significantly but partially extended their survival at 15 days post fertilization. The eif6 hypomorph mutants also increased larvae survival at 20 days post fertilization with Mendelian ratios. Low Eif6 levels did not rescue neutropenia in Sbds-deficient zebrafish. Notably, the eif6 hypomorph mutants in sbds-null background reduced the expression of Tp53-dependent targets (e.g., cdkn1a, bax, and puma). SDS patient-derived cell lines also showed accumulation of EIF6, TP53, and CDKN1A (p21). Knocking down EIF6 significantly decreased CDKN1A mRNA levels. These observations support the hypothesis that low levels of Eif6 mitigate Tp53 pathway activation and partially alleviate cellular stress in Sbds-deficient cells. These findings highlight the complex relationships between SBDS, EIF6, TP53, and stress responses in the pathogenesis of SDS. EIF6 dosage determines the degree of intracellular stress mediated by TP53, which may explain the somatic genetic rescue in SDS and the decreased risk for malignant transformation. Understanding these mechanisms will aid in developing therapeutic strategies for SDS. In addition, this partial dosage effect suggests additional stress pathways that are unlikely to be TP53-dependent. We are currently identifying TP53-independent mechanisms.

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Loi, Pasqualino, Grazyna Ptak, Barbara Barboni, Josef Fulka, Pietro Cappai e Michael Clinton. "Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells". Nature Biotechnology 19, n. 10 (ottobre 2001): 962–64. http://dx.doi.org/10.1038/nbt1001-962.

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Theodoropoulou, Marily, Martin Reincke, Martin Fassnacht e Masayuki Komada. "Decoding the genetic basis of Cushing's disease: USP8 in the spotlight". European Journal of Endocrinology 173, n. 4 (ottobre 2015): M73—M83. http://dx.doi.org/10.1530/eje-15-0320.

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Abstract (sommario):

Cushing's disease (CD) arises from pituitary-dependent glucocorticoid excess due to an ACTH-secreting corticotroph tumor. Genetic hits in oncogenes and tumor suppressor genes that afflict other pituitary tumor subtypes are not found in corticotrophinomas. Recently, a somatic mutational hotspot was found in up to half of corticotrophinomas in the USP8 gene that encodes a protein that impairs the downregulation of the epidermal growth factor receptor (EGFR) and enables its constitutive signaling. EGF is an important regulator of corticotroph function and its receptor is highly expressed in Cushing's pituitary tumors, where it leads to increased ACTH synthesis in vitro and in vivo. The mutational hotspot found in corticotrophinomas hyper-activates USP8, enabling it to rescue EGFR from lysosomal degradation and ensure its stimulatory signaling. This review presents new developments in the study of the genetics of CD and focuses on the USP8-EGFR system as trigger and target of corticotroph tumorigenesis.

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Ozminkowski, Richard H., e Pablo Jourdan. "Comparing the Resynthesis of Brassica napus L. by Interspecific Somatic and Sexual Hybridization. I. Producing and Identifying Hybrids". Journal of the American Society for Horticultural Science 119, n. 4 (luglio 1994): 808–15. http://dx.doi.org/10.21273/jashs.119.4.808.

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Brassica napus (genome aacc), a natural allotetraploid derived from hybridization between B. oleracea L. (genome cc) and B. rapa L. (genome aa), was synthesized by sexual and somatic interspecific hybridizations from the same parent plants to compare the two methods of combining genomes and assess the genetic consequences of bypassing the gametophytic phase before hybrid formation. Highly heterozygous species parents were first produced by intraspecific hybridization between two subspecies each of B. oleracea and B. rapa. Leaf tissue from young plants of both parental species served as a source of protoplasts for fusion; the same plants were later used for crosses. Seventy-two somatic hybrids were produced using a polyethylene glycol-mediated fusion protocol and 27 sexual hybrids were obtained by embryo rescue. Somatic hybrids were produced between one B. oleracea and two sibling B. rapa plants. Sexual hybrids were successfully produced with only one of the two B. rapa siblings. Hybrids were identified by morphology, isozyme patterns, and total DNA content. Although fertile allotetraploid somatic hybrids were obtained within 7 months after seeding parent lines, >1 year was required to produce fertile sexual hybrids.

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Donaires, Flavia S., Sophie de Tocqueville, Marilia Bazzo Catto, Luiz Fernando Bazzo Catto, Emma M. Groarke, Barbara A. Santana, Settara C. Chandrasekharappa et al. "Novel Germline ZCCHC8 Mutations Causes Dyskeratosis Congenita with Genetic Rescue By Uniparental Disomy of Chromosome 12q". Blood 144, Supplement 1 (5 novembre 2024): 1330. https://doi.org/10.1182/blood-2024-205711.

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Telomere-biology disorders (TBD) are caused by germline defects in telomere maintenance genes, most commonly in TERT, TERC, DKC1, TINF2 and RTEL1. Recently, ZCCHC8 was added to the list of genes that cause an adult-onset TBD as it is required for TERC maturation and telomerase function. The heterozygous ZCCHC8 p.P186L and p.E192K germline mutations are etiologic in familial pulmonary fibrosis with short telomeres. Here we expand the phenotypes associated with ZCCHC8 by describing two unrelated young patients with dyskeratosis congenita (DC) identified with distinct de novo pathogenic ZCCHC8 mutations in the p.G184 codon. The first patient (UPN1) presented as a 4 year old boy with cerebellar hypoplasia, pancytopenia, and the clinical mucocutaneous triad. Bone marrow (BM) revealed signs of dyserythropoiesis and aplasia. Further workup showed he had very short telomere length (TL < 1st percentile of age-matched controls) and normal karyotype. He was identified with a ZCCHC8 p.Gly184Arg mutation. UPN2 presented at age 25; she had typical mucocutaneous findings, mild anemia and thrombocytopenia, a hypocellular BM (<5%) without dysplasia, normal karyotype, and very short TL. Her sequence data revealed a ZCCHC8 p.Gly184Val mutation. Neither patient had signs of pulmonary or liver disease. In both cases, the novel ZCCHC8 variants were in complete heterozygosity in patients' fibroblasts but detected at somatic ranges in peripheral blood (PB) leukocytes by targeted sequencing (p.Gly184Arg/Val variant allele frequency [VAF] of 7.5% and 40-20%, respectively). The unusual VAFs in PB prompted the investigation of genomic imbalances by SNP array. A large copy-neutral loss of heterozygosity (uniparental disomy; UPD) of chromosome 12q was identified in both patients, confirming a somatic genetic rescue (SGR) of the germline p.Gly184Arg/Val mutation in 85% and >40% of PB cells, respectively. Direct SGR of these germline mutations is a proof-of-concept that they are pathogenic and disease-causing. In both cases, the parents and multiple siblings were wild-type. Longitudinal screening of PB from UPN02 showed that the VAF of the p.Gly184Val mutation decreased from 41% to 22% in 7 years of follow-up, a consequence of the UPD(12q) clonal expansion. This coincided with an increased megakaryocyte frequency and preserved trilineage hematopoiesis in the BM. Despite the hypocellular BM and the typical DC phenotype, patients' thrombocytopenia improved (from 33 to 74x103cell/µL) and the absolute neutrophil and monocyte counts slightly increased without any specific treatment. Error-corrected sequencing (ECS) also detected four additional somatic ZCCHC8 mutations at VAFs <0.5-2.5% in her PB, mostly truncations before the p.G184 position. At last follow-up, SGR characterization in BM mononuclear cells from UPN2 by single-cell proteogenomic sequencing (Mission Bio) revealed that the UPD(12q) arose in hematopoietic stem cells and propagated downstream in all megakaryocyte-erythroid, myeloid and lymphoid progenitors cells; 84%, 77% and 64% of these progenitors were wild-type (as a consequence of UPD12q), respectively. Most myeloid and NK cells (>85%) harbored the UPD12q, while only less than 15% and 30% of T (both CD8+ and CD4+) and B cells, respectively, carried this SGR. These findings were validated in sorted populations from peripheral blood by ECS and Sanger sequencing. In conclusion, we report novel de novo ZCCHC8 mutations that cause DC and very short telomeres; the disease onset of UPN2 was later than classically observed (>20 vs. <10 years old, respectively). Blood count recovery of UPN2 coincided with UPD(12q) expansion, suggesting that this natural gene therapy mechanism ameliorated marrow failure. The strong selective pressure for the p.G184 SGR by UPD(12q) in unrelated cases highlights the biological relevance of this protein, specifically this codon, for telomere maintenance. Further study is ongoing to precisely determine the functional impact of the p.G184 mutations on telomerase activity and telomere integrity. The somatic rescue of the germline mutations by UPD(12q) in myeloid and NK cells but not T/B lymphocytes changed the expected germline VAFs of this mutation to somatic ranges in PB. Therefore, SGR mechanisms should be considered when screening TBD patients, particularly those who have undergone extensive sequencing without a clear molecular etiology (about 20%).

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Magioli, Claudia, e Elisabeth Mansur. "Eggplant (Solanum melongena L.): tissue culture, genetic transformation and use as an alternative model plant". Acta Botanica Brasilica 19, n. 1 (marzo 2005): 139–48. http://dx.doi.org/10.1590/s0102-33062005000100013.

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Eggplant is an agronomically important non-tuberous solanaceous crop grown primarily for its large oval fruit. In popular medicine, eggplant is indicated for the treatment of several diseases, including diabetes, arthritis, asthma and bronchitis. Eggplant is susceptible to a number of diseases and pests capable of causing serious crop losses. This problem has been addressed by hybridizing eggplant with wild resistant Solanum species, which present a wide genetic diversity and are source of useful agronomic traits. The application of in vitro methodologies to eggplant has resulted in considerable success. Eggplant tissues present a high morphogenetic potential that is useful for developmental studies as well as for establishing biotechnological approaches to produce improved varieties, such as embryo rescue, in vitro selection, somatic hybridization and genetic transformation. Taken together, these characteristics also make eggplant a complete model for studies on different areas of plant science, including control of gene expression and assessment of genetic stability of somaclones derived from different morphogenetic processes. In the present study, important factors that affect the efficiency of in vitro regeneration through organogenesis and embryogenesis as well as genetic transformation are analyzed. The potential of this species as a model plant for studying various aspects of plant genetics and physiology is also discussed.

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Schratz, Kristen E. "Clonal evolution in inherited marrow failure syndromes predicts disease progression". Hematology 2023, n. 1 (8 dicembre 2023): 125–34. http://dx.doi.org/10.1182/hematology.2023000469.

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Abstract Progression to myelodysplastic syndromes (MDS) and acute myeloid leukemia is one of the most serious complications of the inherited bone marrow failure and MDS-predisposition syndromes. Given the lack of predictive markers, this risk can also be a source of great uncertainty and anxiety to patients and their providers alike. Recent data show that some acquired mutations may provide a window into this risk. While maladaptive mechanisms, such as monosomy 7, are associated with a high risk of leukemogenesis, mutations that offset the inherited defect (known as somatic genetic rescue) may attenuate this risk. Somatic mutations that are shared with age-acquired clonal hematopoiesis mutations also show syndrome-specific patterns that may provide additional data as to disease risk. This review focuses on recent progress in this area with an emphasis on the biological underpinnings and interpretation of these patterns for patient care decisions.

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Chlon, Timothy, Emily Stepanchick, Christy M. Finke, Terra L. Lasho, Talha Badar, Alejandro Ferrer e Mrinal M. Patnaik. "Genetic Complementation Studies Reveal That Many Disease-Associated DDX41 Variants Do Not Cause Loss of Protein Function". Blood 142, Supplement 1 (28 novembre 2023): 4104. http://dx.doi.org/10.1182/blood-2023-181881.

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Germline variants in DDX41 are the most common cause of inherited predisposition to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). DDX41 codes for an RNA helicase with many described functions, and thus the precise role of these variants in disease is uncertain. Approximately 2/3 of disease-associated DDX41 variants cause truncation of the DDX41 protein due to frameshift, loss of a translation start, or splice alterations, while the other 1/3 of DDX41 variants are missense. The effect of these missense variants on DDX41 function is largely unknown and thus they are typically categorized as variants of unknown significance (VUSs). Importantly, 30-50% of MDS/AML patients with either truncating or missense DDX41 variants acquire a somatic mutation in the other allele of DDX41, and this mutation has strong preference for the amino acid substitution R525H. Our previous work demonstrated that the R525H mutation causes loss of DDX41 function. To better understand how germline variants affect DDX41 function, we designed a genetic complementation strategy in immortalized hematopoietic progenitor cells with inducible deletion of DDX41. We screened six of the most common missense VUSs by expressing them from a lentivirus in DDX41-proficient cells and then inducing deletion of endogenous DDX41 to determine the effect of each VUS on proliferation and survival of the cells. Empty vector-transduced DDX41-deleted cells undergo cell-cycle arrest and apoptosis within 3-4 days. In contrast, viral expression of wild-type DDX41 rescues the survival and proliferation of the cells, while expression of the R525H mutant fails to rescue the cells. We screened the Y33H, M155H, G173R, R219H, P258L, and I396T variants of DDX41 in this assay, and all six of the mutants successfully rescued the survival and proliferation of DDX41-deleted cells long-term. This indicates that the mutant proteins coded for by these variants are functional. To test the effect of commonly observed truncating variants in the same assay, we expressed M1I, Q52fs, and D140Gfs variants of DDX41. Surprisingly, we saw complete rescue of cell proliferation and survival by the M1I and Q52fs variants. In contrast, the D140Gfs variant failed to rescue the cells. These data indicate that frameshift at D140 or later causes loss of DDX41 function. It is likely that translation from alternative methionine residues located at M127 or M132 of DDX41 accounts for the rescue of the cells by truncating variants affecting sites upstream of M127. In support of this theory, we targeted DDX41 for CRISPR-mediated knockout in MOLM13 cells and found that guide RNAs targeting exons 1-4, which are upstream of M127, produced homozygous frameshift mutations in proliferating cells from single-cell clones. However, guides targeting exon 6, which is downstream of M127 and M132, did not produce any surviving cells with homozygous frameshift mutations, despite screening 96 clones. This finding indicates that short DDX41 isoforms expressed from alternative start sites are functional and can support cell proliferation when full-length protein is lost. Collectively, these data raise important questions about the precise role of DDX41 variants in hematologic malignancy. Data from recently published patient cohorts indicates that patients with missense DDX41 variants have similar patient characteristics to those with truncating variants and thus these aberrations are thought to cause disease by similar mechanisms. While these cell line models may not fully recapitulate the pathogenic mechanism by which DDX41 variants cause MDS/AML predisposition, the results of this study demonstrate that the germline variants are not complete loss-of-function alleles in all cases and more complex mechanisms may be involved.

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Song, Wenjie, Xin Xia, Yue Fan, Bo Zhang e Xiaowei Chen. "Functional and Genetic Analyses Unveil the Implication of CDC27 in Hemifacial Microsomia". International Journal of Molecular Sciences 25, n. 9 (26 aprile 2024): 4707. http://dx.doi.org/10.3390/ijms25094707.

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Hemifacial microsomia (HFM) is a rare congenital genetic syndrome primarily affecting the first and second pharyngeal arches, leading to defects in the mandible, external ear, and middle ear. The pathogenic genes remain largely unidentified. Whole-exome sequencing (WES) was conducted on 12 HFM probands and their unaffected biological parents. Predictive structural analysis of the target gene was conducted using PSIPRED (v3.3) and SWISS-MODEL, while STRING facilitated protein-to-protein interaction predictions. CRISPR/Cas9 was applied for gene knockout in zebrafish. In situ hybridization (ISH) was employed to examine the spatiotemporal expression of the target gene and neural crest cell (NCC) markers. Immunofluorescence with PH3 and TUNEL assays were used to assess cell proliferation and apoptosis. RNA sequencing was performed on mutant and control embryos, with rescue experiments involving target mRNA injections and specific gene knockouts. CDC27 was identified as a novel candidate gene for HFM, with four nonsynonymous de novo variants detected in three unrelated probands. Structural predictions indicated significant alterations in the secondary and tertiary structures of CDC27. cdc27 knockout in zebrafish resulted in craniofacial malformation, spine deformity, and cardiac edema, mirroring typical HFM phenotypes. Abnormalities in somatic cell apoptosis, reduced NCC proliferation in pharyngeal arches, and chondrocyte differentiation issues were observed in cdc27−/− mutants. cdc27 mRNA injections and cdkn1a or tp53 knockout significantly rescued pharyngeal arch cartilage dysplasia, while sox9a mRNA administration partially restored the defective phenotypes. Our findings suggest a functional link between CDC27 and HFM, primarily through the inhibition of CNCC proliferation and disruption of pharyngeal chondrocyte differentiation.

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B. M, Harish,, Shreedevi Badiger, D. R. Chaudhary e Jasdeep Kaur. "Wild Genetic Resource in Vegetable Improvement: Applications and Strategies". Journal of Advances in Biology & Biotechnology 27, n. 9 (26 agosto 2024): 247–60. http://dx.doi.org/10.9734/jabb/2024/v27i91295.

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Domestication of feral types of the present-day cultivated types was in vogue in earlier days of farming followed by the selection and hybridization that led to many improved varieties. Expression of traits faded due to the development of homozygosity due to repeated interventions in the same set. Relying on wild relatives that are found to be the source of many target genes is found significant. While, this is not so easy, because of the barriers that reside in distant hybridization, consequences like failure in zygote and embryo formation, embryo death, linkage drags, and incompatibility within wild and cultivated groups, etc are need of concern. Strategies like bridge crosses, somatic hybridization, grafting, embryo rescue, etc., techniques are solely or in combination can combat these issues. Vegetables are the group of crops that are mainly cultivated for their high returns and quality aspects for the healthy food routine of the consumer. This review will spotlight the recent advances in vegetable improvement through their wild relatives and available strategies to overcome the barriers to the usage of wild relatives in vegetable improvement, meanwhile the conservation of these resources for the future utilization in vegetable improvement.

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23

Liu, Min, e Zai-Yun Li. "Genome doubling and chromosome elimination with fragment recombination leading to the formation of Brassica rapa–type plants with genomic alterations in crosses with Orychophragmus violaceus". Genome 50, n. 11 (novembre 2007): 985–93. http://dx.doi.org/10.1139/g07-071.

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In distant hybridization of plants, nonclassical hybrids with unexpected chromosome complements, chromosome elimination, and genetic introgression have been well documented. We obtained intergeneric hybrids between Brassica rapa , B. rapa var. chinensis, and another cruciferous species, Orychophragmus violaceus , following embryo rescue. Hybrids mainly displayed phenotypes of B. rapa, although certain O. violaceus or novel characteristics also appeared. Variable numbers of chromosomes were observed in somatic cells in the roots of plantlets on medium and in ovaries and pollen mother cells (PMCs). However, higher numbers were recorded in the roots. GISH revealed that the majority of ovary cells and PMCs contained 20 chromosomes of B. rapa with or without individual O. violaceus chromosomes or fragments added or introgressed. AFLP analysis showed that fragments deleted from the B. rapa genome were much more frequent than novel and O. violaceus fragments. The mechanisms involved genome doubling and successive elimination of O. violaceus chromosomes accompanied by fragment recombination and introgression, producing B. rapa–type plants with modified genetic constitutions and phenotypes.

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Fernando, W. M. U., L. Perera e R. R. A. Peries. "An Overview of Breeding Research in Coconut — the Sri Lankan Experience". Outlook on Agriculture 26, n. 3 (settembre 1997): 191–98. http://dx.doi.org/10.1177/003072709702600310.

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Coconut is so important, not only economically but also socially, that it was described as ‘one of Nature's greatest gifts to man’ by Burkill (1966). Despite the inherent constraints to the breeding of palm species, important progress in coconut improvement has been obtained using standard breeding techniques. A continuous effort to search for new genetic material within Sri Lanka and to conserve valuable material threatened with erosion is in progress. Somatic embryogenesis, which is the only method of vegetative propagation, has been achieved on a limited scale but consistent plant regeneration is yet to be accomplished. Zygotic embryo rescue has been exploited to propagate an indigenous ‘Makapuno ’ type coconut. Development of DNA markers in order to exploit the existing variability in coconut germplasm to its maximum potential is being explored.

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Kury, Patrick, Marita Führer, Sebastian Fuchs, Myriam R. Lorenz, Orlando Bruno Giorgetti, Shahrzad Bakhtiar, Andreas P. Frei et al. "Long-term robustness of a T-cell system emerging from somatic rescue of a genetic block in T-cell development". EBioMedicine 59 (settembre 2020): 102961. http://dx.doi.org/10.1016/j.ebiom.2020.102961.

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26

Echols, Josh, Amna Siddiqui, Yanying Dai, Viktoria Havasi, Richard Sun, Aneta Kaczmarczyk e Kim M. Keeling. "A regulated NMD mouse model supports NMD inhibition as a viable therapeutic option to treat genetic diseases". Disease Models & Mechanisms 13, n. 8 (31 luglio 2020): dmm044891. http://dx.doi.org/10.1242/dmm.044891.

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ABSTRACTNonsense-mediated mRNA decay (NMD) targets mRNAs that contain a premature termination codon (PTC) for degradation, preventing their translation. By altering the expression of PTC-containing mRNAs, NMD modulates the inheritance pattern and severity of genetic diseases. NMD also limits the efficiency of suppressing translation termination at PTCs, an emerging therapeutic approach to treat genetic diseases caused by in-frame PTCs (nonsense mutations). Inhibiting NMD may help rescue partial levels of protein expression. However, it is unclear whether long-term, global NMD attenuation is safe. We hypothesize that a degree of NMD inhibition can be safely tolerated after completion of prenatal development. To test this hypothesis, we generated a novel transgenic mouse that expresses an inducible, dominant-negative form of human UPF1 (dnUPF1) to inhibit NMD in mouse tissues by different degrees, allowing us to examine the effects of global NMD inhibition in vivo. A thorough characterization of these mice indicated that expressing dnUPF1 at levels that promote relatively moderate to strong NMD inhibition in most tissues for a 1-month period produced modest immunological and bone alterations. In contrast, 1 month of dnUPF1 expression to promote more modest NMD inhibition in most tissues did not produce any discernable defects, indicating that moderate global NMD attenuation is generally well tolerated in non-neurological somatic tissues. Importantly, a modest level of NMD inhibition that produced no overt abnormalities was able to significantly enhance in vivo PTC suppression. These results suggest that safe levels of NMD attenuation are likely achievable, and this can help rescue protein deficiencies resulting from PTCs.

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Donadieu, Jean, e Christine Bellanné-Chantelot. "Genetics of severe congenital neutropenia as a gateway to personalized therapy". Hematology 2022, n. 1 (9 dicembre 2022): 658–65. http://dx.doi.org/10.1182/hematology.2022000392.

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Abstract Severe congenital neutropenias (SCNs) are rare diseases, and to date about 30 subtypes have been described according to their genetic causes. Standard care aims to prevent infections and limit the risk of leukemic transformation; however, several subtypes may have additional organ dysfunction(s), requiring specialized care. Granulocyte colony-stimulating factor and hematopoietic stem cell transplantation are now the bedrock of standard care. Better understanding of SCN mechanisms now offers the possibility of adapted therapy for some entities. An inhibitor of sodium glucose cotransporter, an antidiabetic drug, may attenuate glycogen storage disease type Ib and glucose-6-phosphatase catalytic subunit 3 neutropenias by clearing 1,5-anhydroglucitol, the precursor of the phosphate ester responsible for these SCNs. Chemokine receptor CXCR4 inhibitors contribute to reversing the leukocyte defect in warts, hypoglobulinemia, infections, and myelokathexis syndrome. All these new approaches use oral drugs, which notably improve quality of life. Additionally, improved research into clonal evolution has highlighted some ways to potentially prevent leukemia, such as stimulating somatic genetic rescue, a physiological process that might limit the risk of leukemic transformation.

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Nogueira, Iara Pastor Martins, Guilherme Mattos Jardim Costa e Samyra Maria dos Santos Nassif Lacerda. "Avian iPSC Derivation to Recover Threatened Wild Species: A Comprehensive Review in Light of Well-Established Protocols". Animals 14, n. 2 (10 gennaio 2024): 220. http://dx.doi.org/10.3390/ani14020220.

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Induced pluripotent stem cells (iPSCs) were first generated by Yamanaka in 2006, revolutionizing research by overcoming limitations imposed by the use of embryonic stem cells. In terms of the conservation of endangered species, iPSC technology presents itself as a viable alternative for the manipulation of target genetics without compromising specimens. Although iPSCs have been successfully generated for various species, their application in nonmammalian species, particularly avian species, requires further in-depth investigation to cover the diversity of wild species at risk and their different protocol requirements. This study aims to provide an overview of the workflow for iPSC induction, comparing well-established protocols in humans and mice with the limited information available for avian species. Here, we discuss the somatic cell sources to be reprogrammed, genetic factors, delivery methods, enhancers, a brief history of achievements in avian iPSC derivation, the main approaches for iPSC characterization, and the future perspectives and challenges for the field. By examining the current protocols and state-of-the-art techniques employed in iPSC generation, we seek to contribute to the development of efficient and species-specific iPSC methodologies for at-risk avian species. The advancement of iPSC technology holds great promise for achieving in vitro germline competency and, consequently, addressing reproductive challenges in endangered species, providing valuable tools for basic research, bird genetic preservation and rescue, and the establishment of cryobanks for future conservation efforts.

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Visconte, Valeria, Carlos Bravo-Perez, Arda Durmaz, Zachary Brady, Luca Guarnera, Matteo D'Addona, Carmelo Gurnari, Chao-Yie Yang e Jaroslaw Maciejewski. "Genetic and Molecular Dynamics of DDX41-variants: Insights on the Variant Effect Prediction". Blood 144, Supplement 1 (5 novembre 2024): 4096. https://doi.org/10.1182/blood-2024-208366.

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About 50% of germline DDX41 (DDX41GL) occur with biallelic somatic hits (including the recurrent R525H) while the remainder seems to harbor only monoallelic (germline or somatic) hits. DDX41GL are heterozygous and typically occur in a frameshift fashion (including the canonical D140fs) implying a loss of function and defining DDX41 a bona fide TSG. Lack of inactivating biallelic somatic hits and presence of the conserved R525H led to unanswered questions as to the impact of variants on the protein function. We reviewed our tally of 158 DDX41 mutants obtained by screening 1,893 patients with BMF. Overall, 61% of the patients carried sole germline, 25% somatic + germline and 14% carried sole somatic hits. Among biallelic configurations, there were canonical D140fs/R525H (n=4), germline missense/R525H (n=16), D140fs/non R525H (n=1) and missense germline/somatic (n=10). We then undertook an integrated genomic, enzymatic and molecular dynamics analysis of DDX41 variants to clarify their effect including maladaptive RNA unwinding properties of mutant DDX41. Our original study1 postulated that R525H is possibly hypomorphic because R525 is in the ATP binding domain. This location makes R525 a preferential sweet spot forming hydrogen bonds with the α and β phosphates of bound ATP to potentially regulate the dynamical motion of DDX41 required for RNA unwinding. The histidine replacement may alter but retain the basal unwinding activity albeit through a different hydrogen bonding pattern. To clarify the impact of missense hits on RNA unwinding rate and the efficiency of remodeling activity, we applied state-of-the-art computer modeling. The AI based system, AlphaFold2, was deployed to predict 3D structure and generate DDX41/RNA models for molecular dynamics simulation to study the effect of R525H on DDX41/RNA structure. To explore the unwinding impairment, we examined R525 and the close R522 in the ATP binding site. In the wild type DDX41, R525 forms strong salt bridge interactions with 2 oxygens (O2A, O2G) in ATP at shorter distances whereas R522 maintains weaker interactions with another oxygen (O3G) in ATP. In R525H, H525 loses the strong salt bridge interaction with ATP; however, R522 replaces H525 to form a strong salt bridge interaction with ATP based on a distance <4 Å deemed a strong salt bridge interaction. The simulations further clarified that the distances between R525 in DDX41 and O2G in ATP followed a bimodal distribution likely reflecting the on (<4Å) and off (peaked at 5 or 5.5 Å) switch between DEAD and HELICc. Loss of salt bridge interaction between H525 with ATP undermines the role of R525 to control the relative motion between two domains required for efficient progressive RNA unwinding and potentially it reduces/ slows down the unwinding rate further supported by kinetics experiments measuring the actual RNA unwinding. We then studied differences in other missense variants. We first analyzed15 somatic mutants at residueslocated distantly andproximally to R525H and predicted theimpact of each of them on DDX41 function. In 2 missense, the amino acid changes altered the residues binding ATP with lesser impact than R525H, favorably (Q208E) and unfavorably (E345D). 3 variants (D446A, H463L, R471Q) abolished the RNA grab, 4 affected allosteric regulation (P321L, M378I, L548H, L552R), 2 affected conformational changes (A225D, G586R) while in 2 variants the impact could not be predicted (K482R, A488T). Of note is that the variant located in the closest triplet to R525H (G524R) seemed to exhibit different functional effects compared to R525H. A similar in silico analysis was conducted by modeling 5 assumed non pathogenic DDX41GL (M155I, Y151H, K184R, R164Q, I169V) and predicted that such spots might alter the binding with other protein partners possibly expanding the function of DDX41 which to date is restricted to pre mRNA and innate immunity, both failing to explain the smoldering phenotype of cases lacking the biallelic configuration. Our study suggests that we can't paint all mutations with the same brush and their functional impact is likely diverse. Novel insights in mutant DDX41 structure further clarify the impact of single variants which may exhibit functional effects distinct from those of the canonical configuration. In our presentation we will show alternate modes of biallelic somatic vs germline hits including loss of TSG function vs somatic gene rescue contingent on the lesions involved.

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K.C., Mamata, e Anuj Lamichhane. "Advances in Agricultural Biotechnology". Nepal Journal of Biotechnology 9, n. 1 (31 luglio 2021): 85–92. http://dx.doi.org/10.3126/njb.v9i1.38643.

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Agricultural biotechnology is becoming the major sector in crop improvement through the use of scientific techniques for the modification of genes conferring resistance to biotic, abiotic stress and improving the quality of crops. With the evolvement from Mendelian genetics to molecular biotechnology, there have been several developments in the field of crop improvement. Recent biotechnological advances have aimed towards removing the physiological constraints of the crops and increasing crop yield potential. With the use of different tools of agricultural biotechnologies like genetic engineering, tissue culture, embryo rescue, somatic hybridization, molecular marker-assisted selection, genome doubling, and omics technologies, various transgenic crops have been developed over the decades and have been approved for commercialization. This development and adoption of transgenic technology have been shown to increase crop yields, reduce CO2 emission, reduce pesticide and insecticide use and decrease the costs of crop production. Even though the biotechnological approach and transgenic organisms have immense potential to contribute to the world’s food security, several concerns of genetically modified crops being a threat to the environment and human health have developed. This review will address applications and concerns of biotechnology in crop improvement considering health hazards and ecological risks.

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Unlu, Serhan, Christopher Haddad, Tariq Kewan, Olisaemeka Ogbue, Arda Durmaz, Waled Bahaj, Arooj Ahmed et al. "Could Somatic Gene Rescue Explain Frequency of Compound Heterozygous and Concurrent Receptor Tyrosine Kinase Mutations in Hematological Diseases?" Blood 144, Supplement 1 (5 novembre 2024): 3199. https://doi.org/10.1182/blood-2024-205865.

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CSF3R plays a key role in regulation of myelopoiesis. In general, CSF3R mutations may be sub-grouped into two classes by receptor distribution with class I mainly consisting of missense mutations in the transmembrane domain (TMD) and seen in CNL and atypical CML. Class II are, instead mainly nonsense mutations in the cytoplasmic domain (CD), resulting in loss of carboxy-terminal domain, often associated with transformation to AML in the context of severe congenital neutropenia (SCN). Somatic (SM) gain of function (GOF) mutations following a hypomorphic mutation may represent a form of maladaptive somatic genetic rescue (SGR). By analogy to this “lesson of nature” we argue that in adult patients affected by SM CSFR3 mutations, a similar mechanism may be observed. The rescued germline (GL) alteration may be less pathogenic or recessive and thus their penetrance might be weaker with a longer disease anticipation, explaining their presentation later in life and in conjunction with more cryptic clinical presentation. We investigated this theory for GL and SM CSF3R variants and other receptor tyrosine kinase (RTK) variants. We analyzed a total of 7312 cases from multiple institutions, and after exclusion of benign variants we found a total of 54 adult patients with assumed GL suspicious VUS and likely pathogenic (LP) CSF3R variants but confirmed pathogenic GL CSF3R variants were not found. We identified 65 patients with a total of 76 confirmed SM CSF3R variants, of which 36 were in the cytoplasmic domain (47%), 27 in the transmembrane domain (36%), and 13 in the extracellular domain (ECD) (17%). 52% of CD variants were found in AML and 14% in MDS, while 48% of TMD variants were found in MPN or MDS/MPN and 16% in MDS. We investigated whether any of these SM mutations were associated with hypomorphic GL variants typical of SCN and variants of other RTK (FLT3, CSF1R, CSF2RA, CSF2RB, KIT). We found 3 cases of compound heterozygous GL CSF3RT640I with CSF3RR583H, and one case each of GL CSF3RP782Qfs*6 with CSF3RT618I, GL CSF3RH599N with CSF3RT618I, GL CSF2RBV890I with CSF3RT618I, GL CSF2RBP508H with CSF3RQ741*,T618I and GL ELANER81P with CSF3RQ739*. In total, 8/65 (12%) of adult patients with somatic CSF3R mutations had concurrent GL RTK or hypomorphic alterations. Of patients with compound heterozygous GL and SM CSF3R mutations, the most common somatic variants included R583H and T618I (4.5%, 3% of SM variants). Of note is that CSF3RR583H was always in a compound heterozygous configuration. GL CSF3R variants were located in ECD, (n=17), CD (n=26), and TMD (n=11). Variants with high likelihood of effect on receptor function (T640I, L723V, G687S) were more frequently observed in our cohort (12/7312), vs general population databases (gnomAD, 104/251,194, p=0.0001). Combined allelic burden of GL VUS/LP CSF3R variants in our cohort was similar to the general population (0.37% vs 0.44%, p=0.24) suggesting some variants may only affect myelopoiesis marginally. Higher frequency of cytopenia without overt myeloproliferation or dysplasia was observed with SM CSF3R alterations in CD compared to TMD (18% vs 0%, p=0.032), while lower frequency of cytopenia was observed with GL CSF3R alterations in CD compared to TMD (11% vs 46%, p=0.031). As compared to the rest of our cohort, GL CSF3R variants had more somatic mutations in SETBP1, CEBPA, NF1, WT1, while SF3B1 and TP53 mutations were less common (all p<0.05). Overall, cases of GL CSF3R concurrent with SM RTK alterations were enriched for AML/high risk MDS compared to GL CSF3R alterations alone (60% vs 7%, p=0.0017). In total, 13/54 (24%) GL CSF3R variants were found to be associated with SM CSF3R (n=5), CSF2RB (n=3), CSF2RA (n=1), FLT3 (n=4), KIT (n=1) mutations. GL VUS variants of CSF2RB (n=7), CSF2RA (n=5), CSF1R (n=4), KIT (n=1) and FLT3 (n=1) were found, GL CSF2RBP508H was concurrent with SM FLT3V592A. Of note is that, 2 patients with GL CSF3RR698C and CSF2RBL276V acquired -7, none of which acquired any SM variants of RTK. In summary, we present our findings showing that alterations in GL hypomorphic and RTK variants may present in patients who acquired other SM RTK mutations by analogy to SCN whereby the acquisition of concurrent mutations represents a maladaptive SGR. Patients with GL RTK mutations may constitute a suitable genetic milieu for selection of acquired hits that can reverse the underlying hypomorphic phenotype and proceed to develop myeloproliferative or dysplastic disorders.

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Magerus-Chatinet, Aude, Benedicte Neven, Marie-Claude Stolzenberg, Cecile Daussy, Peter Arkwright, Marina Cavazzana-Calvo, Capucine Picard, Alain Fischer e Frederic Rieux-Laucat. "Onset of Autoimmunity In ALPS as a Consequence of Genetic Defects Accumulation". Blood 116, n. 21 (19 novembre 2010): 278. http://dx.doi.org/10.1182/blood.v116.21.278.278.

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Abstract Abstract 278 Autoimmune diseases develop in about 5% of human beings and arise with a delayed stochastic penetrance when lymphocytes directed towards self components, such as chromatin in lupus or insulin-producing cells in diabetes, are generated. This occurs when self-tolerance checkpoints of the immune system are bypassed as a consequence of inherited mutations of key genes involved in activation, survival or death of lymphocytes. The autoimmune lymphoproliferative syndrome (ALPS) is a model of self-tolerance checkpoints defects as autoimmune manifestations occur in 2/3 of ALPS patients. Mutations in the TNFRSF6 gene (encoding FAS, also known as the CD95 receptor) is the most common cause of ALPS. Patients carrying a homozygous TNFRSF6 mutation have an early-onset, severe phenotype, whereas subjects with heterozygous TNFRSF6 mutations present ALPS of variable intensity. T cells from patients with homozygous or heterozygous TNFRSF6 mutations respectively exhibit a complete or partial functional impairment in an in vitro FAS-induced apoptosis assay. Moreover, we recently described somatic heterozygous mutations in TNFRSF6 in patients with a clinical ALPS phenotype in which activated T cells showed normal sensitivity to FAS-induced apoptosis in vitro. The inability to detect an apoptosis defect was due to spontaneous in vitro apoptosis of the mutant T cells. However, more than 80% of these patients' DN T cells were mutated. These patients displayed mosaic expression of a somatic TNFRSF6 mutation, which provided the affected cells a selective advantage and accounted for the observed lymphoproliferation and autoimmunity. Heterozygous germline TNFRSF6 mutations are not always associated with clinical expression (partial clinical penetrance) since some mutation-positive relatives (MPRs) remain asymptomatic despite an in vitro FAS-induced apoptosis impairment on their T cells. This observation led us to postulate that a second event is necessary for disease expression in patients with TNFRSF6 mutations when an incomplete penetrance is observed. We analyzed double-negative (DN) T lymphocytes from seven ALPS patients with low penetrance mutations for FAS expression and defects in the second TNFRSF6 allele and compared with data from healthy, mutation-positive relatives and controls. Missense mutations (in three patients) and loss of heterozygosity (LOH, in four patients) were found in the second TNFRSF6 allele. The LOH observed in 4 of our patients with germline TNFRSF6 ECD mutations resulted from loss of the wild-type allele and concomitant duplication of the mutant one, i.e. telomeric UPD. This type of mechanism is usually observed in genetic disorders of development, where it can be involved in trisomy or monosomy rescue during embryonic development. In 6 patients, alteration or loss of the second TNFRSF6 allele resulted in the absence of FAS cell surface expression on DN T lymphocytes. In these seven patients the disease develop as a consequence of somatic genetic events of TNFRSF6 in a subpopulation of lymphocytes combined with inherited heterozygous mutations of the same gene. This finding is the first demonstration that combined germline and somatic mutations of the same gene is leading to a non-malignant autoimmune disease in humans. Disclosures: No relevant conflicts of interest to declare.

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Brown, Anna L., Jane Churpek, Christopher N. Hahn e Hamish S. Scott. "Clonal Evolution in the Setting of Germline Predisposition". Blood 130, Suppl_1 (7 dicembre 2017): SCI—39—SCI—39. http://dx.doi.org/10.1182/blood.v130.suppl_1.sci-39.sci-39.

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Abstract Germline predisposition to haematological malignancy is an entity with an increasing awareness amongst hematologists in both clinical and research domains, with new predisposition genes being identified at a rapid rate. Recent advances in sequencing technologies, allowing the molecular profiling of hematological malignancies, have revealed clonal evolution of tumours in germline mutation carriers. RUNX1, as the first autosomal dominant hematological malignancy predisposition gene, is the best characterised in this regard. A consensus of findings from several groups indicates that in tumours of germline RUNX1 mutation carriers, somatic alteration of the second RUNX1 allele is the most frequent event observed. This is similarly true for DDX41 germline mutated tumours, where up to 50% of tumours have mutation of the second DDX41 allele, predominantly p.R525H. Studies on germline CEBPA mutated tumours also indicate a high selection for somatic mutation of the second allele, with temporally distinct leukemias arising in patients each carrying different somatic CEBPA mutations. These findings are in contrast to germline GATA2 mutated malignancy where biallelic mutations are rare and instead monosomy 7 and somatic mutation of ASXL1 are most commonly seen. How acquired mutations and epigenetic changes, as well as additional germline modifiers, can affect malignancy phenotype, age of onset and disease penetrance within families is an emerging theme that will also be discussed. Germline predisposition syndromes also offer a unique opportunity to identify acquired changes that occur prior to overt malignancy development in carriers. Although not yet well understood, this is an area of great interest. Recent studies in germline RUNX1 mutation carries without malignancy, has identified acquired mutations in genes associated with clonal hematopoiesis (e.g. DNMT3A). These mutations occur at an earlier age than that observed in the general population for clonal hematopoiesis, raising the possibility that they may be triggers for early malignancy onset. In contrast to clonal evolution that promotes tumour development; somatic alterations may also act to rescue consequences of germline mutations through somatic revertant mosaicism. This is well established in bone marrow failure syndromes such as Fanconi Anemia and Diamond Blackfan Anemia. More recently, SAMD9 and SAMD9L germline mutated syndromes (MIRAGE syndrome and Ataxia Pancytopenia syndrome, respectively) have allowed the examination of both phenomena operating contemporaneously in families. Selective pressure to remove or correct germline gain of function mutations, that cause growth deficiencies in hematopoietic cells, leads to acquisition of mutations and chromosomal abnormalities that act to rescue cytopenias, but can also predispose to subsequent development of myelodysplastic syndrome (e.g. monosomy 7). Finally, treatment related manipulation of the hematopoietic system offers its own risks for clonal evolution leading to tumour development where germline predisposition is concerned. In severe congenital neutropenia, treatment with G-CSF may lead to an increased risk of MDS and AML, associated with somatic CSF3R and RUNX1 mutations. Additionally, where asymptomatic germline carriers are inadvertently used as stem cell donors in genetic predisposition syndromes, stem cell stress experienced from reconstituting the hematopoietic system of the host may trigger development of donor cell leukemia. A better understanding of the contexts in which clonal haematopoiesis in germline predisposition syndromes occur, offers the hope of more sensitive molecular monitoring techniques and development of improved and earlier treatment interventions. Disclosures No relevant conflicts of interest to declare.

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Kent, David, Heather Machado, Nina Obro, Anna Clay, Megan Davies, E. Joanna Baxter, Peter Campbell e Alan Warren. "1020 – HEMATOPOIETIC STEM CELL CLONAL TRACKING BY WHOLE GENOME SEQUENCING IDENTIFIES MULTIPLE INDEPENDENT SOMATIC GENETIC RESCUE MECHANISMS IN BONE MARROW FAILURE DISORDERS." Experimental Hematology 88 (agosto 2020): S24. http://dx.doi.org/10.1016/j.exphem.2020.09.019.

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Wang, Linghua, Sabina Swierczek, Lucie Piterkova, Kimberly Hickman, David A. Wheeler e Josef T. Prchal. "Whole Exome Sequencing of Polycythemia Vera Reveals Novel Recurrent Somatic and Germline Variation". Blood 120, n. 21 (16 novembre 2012): 705. http://dx.doi.org/10.1182/blood.v120.21.705.705.

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Abstract Abstract 705 Polycythemia vera (PV) is a clonally derived hematopoietic malignancy arising from a multipotent hematopoietic stem cell (HSC). Almost all patients have the acquired somatic mutation, JAK2V617F. There is strong evidence suggesting that the JAK2V617F mutation is not a PV-initiating genetic event in its pathogenesis. Thus, “pre- JAK2V617F mutation(s)” account for clonal dominance before the acquisition of the JAK2V617Fmutation. The existence of PV family clustering indicates that germline genetic variations contribute to PV genesis. To explore possible PV-initiating events, we performed deep exome sequencing of 31 JAK2V617F-positive PV patients. DNA from granulocytes (clonal cells) and their matched T-lymphocytes (polyclonal cells with germline DNA) were collected and exonic regions were sequenced to ∼130-fold coverage to identify somatic and germline base substitutions, insertions and deletions (indels), as well as copy number alterations (CNA). DNA from skin was also examined in 6 PV patients and was sequenced to ∼117-fold coverage in order to rescue any germline events that may have been missed in the T-lymphocytes. Our findings confirm the somatic loss of heterozygosity (LOH) and trisomy at 9p. The proportion of JAK2V617 as determined by the fraction of the reads harboring the mutations in the patient's granulocytes varied from almost undetectable to 100% of the reads. As previously reported, the degree of LOH at 9p correlated with the variant fraction of JAK2V617F with stronger LOH associated with higher allelic burden of JAK2V617F. We also observed single examples of LOH on 22q and 18p of which the latter is a novel finding in this study. In addition, we observed focal amplification at the PRSS1 locus in 75% of the PV granulocytes. Nine patients also harbor recurrent somatic mutations in genes with known roles in hematologic malignancies including four patients (13%) carrying IDH2 somatic mutations, 1 previously described and 2 novel. Additional somatic mutations were found in several other genes functioning in the growth signaling and chromatin remodeling processes. Remarkably, four patients exhibited novel germline variation in JAK2 itself, including one canonical JAK2V617F variant and 3 other missense variants. Moreover, nearly one half of the patients harbor novel germline variants in FAT2, a tumor suppressor essential for controlling cell proliferation. Interestingly, we also observed in 71% of patients, a germline common allele of EXO1, rs1047840, which has been linked to multiple cancers by genome-wide association studies (GWAS). Our study, using whole exome sequencing, recapitulated all of the major findings of PV genomic alteration from earlier studies. Prior to this study JAK2V617 or K539L and associated 9p LOH, were the only known somatic variation. We have extended previous observations by demonstrating recurrent germline and somatic variation that potentially could contribute to the initiation of the disease. Both CNA alterations and point mutations in important cancer genes were seen in a significant number of patients and germline variants were seen in almost all patients in the cohort. This study finds strong evidence in support of the hypothesis of a second hit contributing to PV onset and progression as well as germline predisposition of PV. Disclosures: No relevant conflicts of interest to declare.

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Tao, Yong, Jianming Liu, Yunhai Zhang, Meiling Zhang, Junshun Fang, Wei Han, Zhizhong Zhang, Ya Liu, Jianping Ding e Xiaorong Zhang. "Fibroblast cell line establishment, cryopreservation and interspecies embryos reconstruction in red panda (Ailurus fulgens)". Zygote 17, n. 2 (maggio 2009): 117–24. http://dx.doi.org/10.1017/s0967199408004966.

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SummaryIn evolution, the red panda (Ailurus fulgens) plays a pivotal role in the higher level phylogeny of arctoides carnivore mammals. The red panda inhabits certain Asian countries only and its numbers are decreasing. Therefore, the development of feasible ways to preserve this species is necessary. Genetic resource cryopreservation and somatic cell nuclear transfer (SCNT) have been used extensively to rescue this endangered species. The present study describes the establishment, for the first time, of a red panda ear fibroblast cell line, which was then cryopreserved, thawed and cultured. Through micromanipulation, interspecies embryos were reconstructed using the cryopreserved–thawed fibroblasts of the red panda as the donor and rabbit oocytes as recipients. A total of 194 enucleated rabbit oocytes were reconstructed with red panda ear fibroblasts; enucleated oocytes were activated without fusion as the control. The results show that the fibroblast cell line was established successfully by tissue culture and then cryopreserved in liquid nitrogen. Supplementation with 20% fetal bovine serum and 8% dimethyl sulphoxide in basic medium facilitated the cryopreservation. The interspecies embryos were successfully reconstructed. The cleavage, morulae and blastocyst rates after in vitro culture were 71, 47 and 23% (31/194), respectively. This study indicated that a somatic cell line could be established and cryopreserved from red panda and that rabbit cytoplast supports mitotic cleavage of the red panda karyoplasts and is capable of reprogramming the nucleus to achieve blastocysts.

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Li, Xueting, Ce Chen, Mingyue He, Lidong Yu, Renhao Liu, Chunmeng Ma, Yu Zhang, Jianbo Jia, Bingsheng Li e Li Li. "Lead Exposure Causes Spinal Curvature during Embryonic Development in Zebrafish". International Journal of Molecular Sciences 23, n. 17 (24 agosto 2022): 9571. http://dx.doi.org/10.3390/ijms23179571.

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Lead (Pb) is an important raw material for modern industrial production, they enter the aquatic environment in several ways and cause serious harm to aquatic ecosystems. Lead ions (Pb2+) are highly toxic and can accumulate continuously in organisms. In addition to causing biological deaths, it can also cause neurological damage in vertebrates. Our experiment found that Pb2+ caused decreased survival, delayed hatching, decreased frequency of voluntary movements at 24 hpf, increased heart rate at 48 hpf and increased malformation rate in zebrafish embryos. Among them, the morphology of spinal malformations varied, with 0.4 mg/L Pb2+ causing a dorsal bending of the spine of 72 hpf zebrafish and a ventral bending in 120 hpf zebrafish. It was detected that spinal malformations were mainly caused by Pb2+-induced endoplasmic reticulum stress and apoptosis. The genetic changes in somatic segment development which disrupted developmental polarity as well as osteogenesis, resulting in uneven myotomal development. In contrast, calcium ions can rescue the series of responses induced by lead exposure and reduce the occurrence of spinal curvature. This article proposes new findings of lead pollution toxicity in zebrafish.

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Goodings-Harris, Charnise, Sushree Sangita Sahoo, Sara Lewis, Emilia J. Kozyra, Marc Arribas-Layton, Senthilkumar Ramamoorthy, Rohith Jesudas et al. "Germline SAMD9/9L MDS Predisposition Syndromes Are Characterized By Complex Clonal Architecture and Lineage-Specific Escape Mechanisms Including Somatic Genetic Rescue in T and B Lymphocytes". Blood 140, Supplement 1 (15 novembre 2022): 1195–96. http://dx.doi.org/10.1182/blood-2022-163337.

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Nakitandwe, Joy, Shann-Ching Chen, Noel T. Lenny, Christopher B. Miller, Xiaoping Su, Charles G. Mullighan e James R. Downing. "Acute Lymphoblastic Leukemia-Associated PAX5 Mutations Induce Aberrant B Cell Development". Blood 116, n. 21 (19 novembre 2010): 10. http://dx.doi.org/10.1182/blood.v116.21.10.10.

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Abstract Abstract 10 Over 60% of pediatric B progenitor acute lymphoblastic leukemia (ALL) cases contain somatic mutations in genes regulating B cell development, with PAX5 being the most common target of mutation (>32% of cases). The transcription factor PAX5 is required for commitment and maintenance of the B lymphoid lineage. A variety of PAX5 mutations has been identified including, mono-allelic deletions, sequence mutations, internal deletions, frame-shift mutations and translocations. We have previously shown that these PAX5 mutations result in reduced transcriptional activity either as a result of haploinsufficiency or the generation of altered PAX5 isoforms with reduced DNA-binding and/or transcriptional activity. However, the direct effect of the mutations on normal B cell development remains unknown. To address this question, we assessed the ability of a series of PAX5 mutations to rescue normal B cell development in Pax5-/- bone marrow (BM) cells using a murine in vitro culture system. Whole BM or transduced cells were grown in IL-7 producing stromal-supported cultures for two weeks and then assessed for their extent of B cell differentiation using flow cytometry. Under these in vitro conditions, both Pax5+/+ and Pax5+/− BM cells differentiated to a Hardy fraction D pre-B cell stage of differentiation (CD43−/B220+/CD19+/BP1+), with only a slight decrease in the level of expression of BP1 detected in the Pax5+/− cells. By contrast, Pax5-/- cells failed to undergo significant differentiation under these in vitro growth conditions and were arrested at an early pro-B stage of development (CD43+/−/B220+/CD19−/BP1−). To assess the biological activity of the identified PAX5 mutants, we then transduced lineage-depleted BM cells from Pax5+/+, Pax5+/− and Pax5-/- mice with MSCV-based retroviral vectors expressing either wild type (WT) or mutant PAX5 followed by in vitro culture. Three classes of PAX5 mutations were assessed: DNA binding domain mutations (P80R, P34Q, and V26G), an internal deletion mutation (Δe6-8), and translocation-induced PAX5 chimeric genes (PAX5-ETV6, PAX5-FOXP1 and PAX5-ZNF521). As expected, expression of WTPAX5 resulted in full rescue of Pax5-/- cells and induced no significant effects on the ability of Pax5+/+ and +/− cells to differentiate. By contrast, PAX5 DNA-binding domain mutants resulted in only partial rescue of Pax5-/- cells, with P80R inducing B220+/CD19−/BP1−, P34Q producing B220+/CD19+ cells with weak BP1 expression, and V26G yielding CD19+/BP1+ cells with minimally reduced levels of BP1. Similarly, expression of Δe6-8 resulted in partial rescue with the expansion of B220+/CD19+/−/BP1− cells. In stark contrast, expression of the translocation encoded PAX5 fusion proteins failed to induce any evidence of rescue. Moreover, these fusion proteins induced only minimal perturbations in the ability of Pax5+/+ and +/− cells to differentiate, suggesting that these fusion proteins were weak competitive inhibitors of normal Pax5 transcriptional activity under intra-cellular conditions. To further characterize the effects of these PAX5 mutations on B cell differentiation, we next analyzed the gene expression patterns of the resultant cell populations using the Mouse Genome 430 2.0 Arrays (Affymetrix) and compared the profiles to those obtained from purified Hardy fractions from normal murine BM. The expression signatures of the Pax5-/- cells were identical to those for normal Hardy fraction A and shifted to the signature of Hardy fraction C following rescue with WTPAX5. Transduction of Pax5-/- cells with either V26G or P34Q resulted in a near complete rescue with expression signatures similar to those obtained for Hardy fractions B/C. By contrast, transduction with P80R or Δe6-8 yielded a more incomplete rescue with expression profiles that were between Hardy fractions A and B. Interestingly, a number of genes within the B cell receptor signaling pathway were altered in cells rescued by P80R and Δe6-8, including the down regulation of CD19, Btk and Blnk. In summary, our data demonstrate that leukemia-associated PAX5 mutations have a graded effect on the transcriptional network that controls normal B cell development and differentiation. Defining the differential target gene specificity of the various PAX5 mutants should provide valuable insights into the molecular mechanisms through which these genetic lesions contribute to leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Vasic, Verica, Mattson S. O. Jones, Denise Haslinger, Lisa S. Knaus, Michael J. Schmeisser, Gaia Novarino e Andreas G. Chiocchetti. "Translating the Role of mTOR- and RAS-Associated Signalopathies in Autism Spectrum Disorder: Models, Mechanisms and Treatment". Genes 12, n. 11 (30 ottobre 2021): 1746. http://dx.doi.org/10.3390/genes12111746.

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Mutations affecting mTOR or RAS signaling underlie defined syndromes (the so-called mTORopathies and RASopathies) with high risk for Autism Spectrum Disorder (ASD). These syndromes show a broad variety of somatic phenotypes including cancers, skin abnormalities, heart disease and facial dysmorphisms. Less well studied are the neuropsychiatric symptoms such as ASD. Here, we assess the relevance of these signalopathies in ASD reviewing genetic, human cell model, rodent studies and clinical trials. We conclude that signalopathies have an increased liability for ASD and that, in particular, ASD individuals with dysmorphic features and intellectual disability (ID) have a higher chance for disruptive mutations in RAS- and mTOR-related genes. Studies on rodent and human cell models confirm aberrant neuronal development as the underlying pathology. Human studies further suggest that multiple hits are necessary to induce the respective phenotypes. Recent clinical trials do only report improvements for comorbid conditions such as epilepsy or cancer but not for behavioral aspects. Animal models show that treatment during early development can rescue behavioral phenotypes. Taken together, we suggest investigating the differential roles of mTOR and RAS signaling in both human and rodent models, and to test drug treatment both during and after neuronal development in the available model systems.

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Stiernholm, N., B. Kuzniar e NL Berinstein. "Absence of immunoglobulin variable region hypermutation in a large cell lymphoma after in vivo and in vitro propagation". Blood 80, n. 3 (1 agosto 1992): 738–43. http://dx.doi.org/10.1182/blood.v80.3.738.738.

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Abstract Several genetic mechanisms have been shown to diversity the expressed antibody repertoire of committed B lymphocytes. These include somatic hypermutation, V gene replacement, and ongoing gene rearrangement. These mechanisms may be operational at discrete points in the B-cell differentiation pathway and may generate idiotypic diversity in various malignant B-cell tumors. Hypermutation of the Ig variable region has been shown to occur in follicular lymphoma, but not in pre-B cell acute lymphoblastic leukemia, Burkitt's lymphoma, chronic lymphocytic leukemia, or myeloma. To study hypermutation in a large cell lymphoma, we use a polymerase chain reaction-based approach, employing consensus VH and JH primers, to clone and sequence rearranged Ig heavy chain variable regions. Neither tumor cells immortalized in rescue fusions nor idiotypic variants of a tumor-derived cell line generated through ongoing lambda light chain gene rearrangements show any significant number of variable region mutations. Thus, at the in vivo stage of B- cell differentiation from which this large cell lymphoma arose, Ig variable region hypermutation was not occurring, nor did it occur during propagation in vitro of these tumor cells. Thus, the window of hypermutation in malignant B-cell tumors is more precisely defined, which may have clinical implications for diagnostic and therapeutic approaches directed at the Ig variable region.

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Stiernholm, N., B. Kuzniar e NL Berinstein. "Absence of immunoglobulin variable region hypermutation in a large cell lymphoma after in vivo and in vitro propagation". Blood 80, n. 3 (1 agosto 1992): 738–43. http://dx.doi.org/10.1182/blood.v80.3.738.bloodjournal803738.

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Abstract (sommario):

Several genetic mechanisms have been shown to diversity the expressed antibody repertoire of committed B lymphocytes. These include somatic hypermutation, V gene replacement, and ongoing gene rearrangement. These mechanisms may be operational at discrete points in the B-cell differentiation pathway and may generate idiotypic diversity in various malignant B-cell tumors. Hypermutation of the Ig variable region has been shown to occur in follicular lymphoma, but not in pre-B cell acute lymphoblastic leukemia, Burkitt's lymphoma, chronic lymphocytic leukemia, or myeloma. To study hypermutation in a large cell lymphoma, we use a polymerase chain reaction-based approach, employing consensus VH and JH primers, to clone and sequence rearranged Ig heavy chain variable regions. Neither tumor cells immortalized in rescue fusions nor idiotypic variants of a tumor-derived cell line generated through ongoing lambda light chain gene rearrangements show any significant number of variable region mutations. Thus, at the in vivo stage of B- cell differentiation from which this large cell lymphoma arose, Ig variable region hypermutation was not occurring, nor did it occur during propagation in vitro of these tumor cells. Thus, the window of hypermutation in malignant B-cell tumors is more precisely defined, which may have clinical implications for diagnostic and therapeutic approaches directed at the Ig variable region.

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Pérez-Luz, Sara, e Javier Díaz-Nido. "Prospects for the Use of Artificial Chromosomes and Minichromosome-Like Episomes in Gene Therapy". Journal of Biomedicine and Biotechnology 2010 (2010): 1–16. http://dx.doi.org/10.1155/2010/642804.

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Artificial chromosomes and minichromosome-like episomes are large DNA molecules capable of containing whole genomic loci, and be maintained as nonintegrating, replicating molecules in proliferating human somatic cells. Authentic human artificial chromosomes are very difficult to engineer because of the difficulties associated with centromere structure, so they are not widely used for gene-therapy applications. However, OriP/EBNA1-based episomes, which they lack true centromeres, can be maintained stably in dividing cells as they bind to mitotic chromosomes and segregate into daughter cells. These episomes are more easily engineered than true human artificial chromosomes and can carry entire genes along with all their regulatory sequences. Thus, these constructs may facilitate the long-term persistence and physiological regulation of the expression of therapeutic genes, which is crucial for some gene therapy applications. In particular, they are promising vectors for gene therapy in inherited diseases that are caused by recessive mutations, for example haemophilia A and Friedreich's ataxia. Interestingly, the episome carrying the frataxin gene (deficient in Friedreich's ataxia) has been demonstrated to rescue the susceptibility to oxidative stress which is typical of fibroblasts from Friedreich's ataxia patients. This provides evidence of their potential to treat genetic diseases linked to recessive mutations through gene therapy.

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Felipe, M. Y., M. D. Rodríguez, L. D. Ratner, A. De Stéfano, A. M. Valdez e D. F. Salamone. "15 Blastocysts altered CDX2 and SOX2 gene expression and pregnancy failure after embryo transfer in yak heterospecific somatic cell nuclear transfer". Reproduction, Fertility and Development 33, n. 2 (2021): 115. http://dx.doi.org/10.1071/rdv33n2ab15.

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Heterospecific cloning is a tool for the genetic rescue of endangered animals. Our objective was to evaluate the effects of heterospecific yak (Bos grunniens) cloned embryo aggregation on the expression levels of NANOG, OCT4, CDX2, and SOX2 genes, and to compare with IVF, parthenogenetic zona-free (P-ZF), and homospecific bovine cloned embryos (BB1x). Oocytes were recovered from the ovaries of slaughtered cows and invitro matured for 22h. The zona pellucida was removed by protease treatment and then mature oocytes were enucleated by micromanipulation. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with a somatic donor cell followed by electrofusion (with two 30-µs pulses of 1.2 kV/cm, 0.1s apart). Two hours after fusion, reconstructed embryos were activated using ionomycin followed by 6-(dimethylamino)purine (6-DMAP) treatment for 3h and cultured in synthetic oviductal fluid (SOF) medium for 7 days. The experimental groups were IVF, P-ZF, BB1x, heterospecific yak-bovine cloned embryos (1 embryo per microwell, YB1x), and heterospecific yak-bovine cloned embryos aggregated (2 embryos per microwell, YB2x). In all experimental groups, cleavage and blastocyst rates were assessed 7 days after activation. In addition, 5 blastocysts were pooled for each biological replicate, and pluripotency-specific genes (NANOG, SOX2, CDX2, and OCT4) were analysed by quantitative PCR. Data were analysed by the ΔΔCT method using the geometric mean of ACTB (actin) and GAPDH as internal standard followed by one-way ANOVA. Cleavages rates were significantly lower in the YB1x group compared with the other groups. Moreover, blastocyst rates in YB2x (31.34%, n=67) were significantly higher than in YB1x (13.86%, n=101) and BB1x (13.33%, n=45) groups, but there were no significant differences compared with the IVF (43.82%, n=89) and P-ZF (25%, n=68) groups. In contrast, although no significant differences were observed among groups in the expression of NANOG and OCT4 genes, the expression of CDX2 was lower in YB2x and YB1x blastocysts compared with the BB1X, P-ZF, and IVF (control) groups. In addition, a decrease in SOX2 gene expression was observed in the YB2x and YB1x blastocysts compared with the BB1X group. Blastocysts from YB1x (n=5) and YB2x (n=18) groups were transferred to recipient cows (n=23) on Day 7. Forty days after embryo transfer, presence of uterine fluid was detected by ultrasound in 3 recipient cows (from YB2x), suggesting embryo loss. In concordance with our previous reports, yak heterospecific SCNT blastocysts showed underexpression of CDX2 and SOX2 compared with the overexpression observed for these genes in bovine homospecific SCNT blastocysts. Thus, yak heterospecific SCNT blastocysts may have compromised developmental competence associated with altered expression of CDX2 and SOX2 that cannot be rescued by the aggregation of 2 reconstructed embryos.

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Felipe, M. Y., M. D. Rodríguez, L. D. Ratner, A. De Stéfano, A. M. Valdez e D. F. Salamone. "15 Blastocysts altered CDX2 and SOX2 gene expression and pregnancy failure after embryo transfer in yak heterospecific somatic cell nuclear transfer". Reproduction, Fertility and Development 33, n. 2 (2021): 115. http://dx.doi.org/10.1071/rdv33n2ab15.

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Abstract (sommario):

Heterospecific cloning is a tool for the genetic rescue of endangered animals. Our objective was to evaluate the effects of heterospecific yak (Bos grunniens) cloned embryo aggregation on the expression levels of NANOG, OCT4, CDX2, and SOX2 genes, and to compare with IVF, parthenogenetic zona-free (P-ZF), and homospecific bovine cloned embryos (BB1x). Oocytes were recovered from the ovaries of slaughtered cows and invitro matured for 22h. The zona pellucida was removed by protease treatment and then mature oocytes were enucleated by micromanipulation. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with a somatic donor cell followed by electrofusion (with two 30-µs pulses of 1.2 kV/cm, 0.1s apart). Two hours after fusion, reconstructed embryos were activated using ionomycin followed by 6-(dimethylamino)purine (6-DMAP) treatment for 3h and cultured in synthetic oviductal fluid (SOF) medium for 7 days. The experimental groups were IVF, P-ZF, BB1x, heterospecific yak-bovine cloned embryos (1 embryo per microwell, YB1x), and heterospecific yak-bovine cloned embryos aggregated (2 embryos per microwell, YB2x). In all experimental groups, cleavage and blastocyst rates were assessed 7 days after activation. In addition, 5 blastocysts were pooled for each biological replicate, and pluripotency-specific genes (NANOG, SOX2, CDX2, and OCT4) were analysed by quantitative PCR. Data were analysed by the ΔΔCT method using the geometric mean of ACTB (actin) and GAPDH as internal standard followed by one-way ANOVA. Cleavages rates were significantly lower in the YB1x group compared with the other groups. Moreover, blastocyst rates in YB2x (31.34%, n=67) were significantly higher than in YB1x (13.86%, n=101) and BB1x (13.33%, n=45) groups, but there were no significant differences compared with the IVF (43.82%, n=89) and P-ZF (25%, n=68) groups. In contrast, although no significant differences were observed among groups in the expression of NANOG and OCT4 genes, the expression of CDX2 was lower in YB2x and YB1x blastocysts compared with the BB1X, P-ZF, and IVF (control) groups. In addition, a decrease in SOX2 gene expression was observed in the YB2x and YB1x blastocysts compared with the BB1X group. Blastocysts from YB1x (n=5) and YB2x (n=18) groups were transferred to recipient cows (n=23) on Day 7. Forty days after embryo transfer, presence of uterine fluid was detected by ultrasound in 3 recipient cows (from YB2x), suggesting embryo loss. In concordance with our previous reports, yak heterospecific SCNT blastocysts showed underexpression of CDX2 and SOX2 compared with the overexpression observed for these genes in bovine homospecific SCNT blastocysts. Thus, yak heterospecific SCNT blastocysts may have compromised developmental competence associated with altered expression of CDX2 and SOX2 that cannot be rescued by the aggregation of 2 reconstructed embryos.

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46

Villeneuve, A. M., e B. J. Meyer. "The role of sdc-1 in the sex determination and dosage compensation decisions in Caenorhabditis elegans." Genetics 124, n. 1 (1 gennaio 1990): 91–114. http://dx.doi.org/10.1093/genetics/124.1.91.

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Abstract (sommario):

Abstract Our previous work demonstrated that mutations in the X-linked gene sdc-1 disrupt both sex determination and dosage compensation in Caenorhabditis elegans XX animals, suggesting that sdc-1 acts at a step that is shared by the sex determination and dosage compensation pathways prior to their divergence. In this report, we extend our understanding of early events in C. elegans sex determination and dosage compensation and the role played by sdc-1 in these processes. First, our analysis of 14 new sdc-1 alleles suggests that the phenotypes resulting from the lack of sdc-1 function are (1) an incompletely penetrant sexual transformation of XX animals toward the male fate, and (2) increased levels of X-linked gene transcripts in XX animals, correlated with XX-specific morphological defects but not significant XX-specific lethality. Further, all alleles exhibit strong maternal rescue for all phenotypes assayed. Second, temperature-shift experiments suggest that sdc-1 acts during the first half of embryogenesis in determining somatic sexual phenotype, long before sexual differentiation actually takes place, and consistent with our previous proposal that sdc-1 acts at an early step in the regulatory hierarchy controlling the choice of sexual fate. Other temperature-shift experiments suggest that sdc-1 may be involved in establishing but not maintaining the XX mode of dosage compensation. Third, a genetic mosaic analysis of sdc-1 produced an unusual result: the genotypic mosaics failed to display the sdc-1 sexual transformation phenotypes. This result suggests several possible interpretations: (1) sdc-1 is expressed immediately, in the one- or two-celled embryo; (2) sdc-1 acts non-cell-autonomously, such that expression of the gene in either the AB or P1 lineage can supply sdc-1(+) function to cells of the other lineage; (3) the X/A ratio is assessed immediately, in the one- or two-celled embryo; or (4) the X/A signal directs the choice of sexual fate in a non-cell-autonomous fashion. Finally, examination of the classes of sexual phenotypes produced in sdc-1 mutant strains suggests that different cells in the organism may not choose their sexual fates independently.

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47

Orland, Mark, Yasuo Kubota, Danai Dima, Carlos Bravo-Perez, Luca Guarnera, Carmelo Gurnari, Jaroslaw P. Maciejewski e Valeria Visconte. "Del(20q) As a Somatic Gene Rescue in Adult Patients: Can Germline Mutations Other Than SBDS be Found in Affected Patients?" Blood 142, Supplement 1 (28 novembre 2023): 4095. http://dx.doi.org/10.1182/blood-2023-191101.

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Abstract (sommario):

Deletions involving chromosome 20 (20q-) are present in up to 5-8% of myeloid neoplasms (MN) and have been associated with either myelodysplastic (MDS) or myeloproliferative (MPN) features, chiefly myelofibrosis. Theoretically, any chromosomal deletion may result in haploinsufficient expression of tumor suppressor genes (TSGs) or in loss of a protective allele in heterozygous carriers of germline (GL) variants or heterozygous somatic (SM) mutations. However, deletions have also been implicated as a maladaptive somatic gene rescue (SGR) event in carriers of GL bone marrow failure (BMF) genes such as SAMD9 and SBDS. The latter is found in Shwachman-Diamond syndrome whereby the upregulated EIF6 gene, responsible for hematopoietic dysfunction, is clonally removed by SM 20q deletion event. We identified an interesting 35-years-old patient with mild cytopenia and 20q- in which a cryptic homozygous SBDS c.A148T GL mutation was found. This prompted us to search in other adult patients with 20q deletion for: i) other cases with SBDS gene alteration (both hetero and homozygous), ii) other GL variants which could be clonally relieved by compensatory 20q- or, iii) disease-prone GL variants on 20q, whereby either disease-prone suppressive allele is included in the deleted region. Within a cohort of 5,308 cases with various MN, 215 patients with 20q- were identified. WES results were available for 350 patients. We applied a stringent bioanalytic algorithm to identify unique GL alterations (not deposited in SM databases) in genes expressed in hematopoietic tissues and involved in BMF and cancer, and a population allelic frequency of <1% which were classified as pathogenic (P), likely pathogenic (LP), and VUS with particular level of clinical suspicion (sVUS). In 76 of 215 patients with 20q-, 346 GL mutations in genes of potential interest were identified and further analyzed. No P/LP variants were detected on genes within commonly deleted region on chr.20q, and no additional carriers of SBDS variants were found. However, we identified 9 pathogenic GL variants in potentially pertinent genes including GL alterations in DDX41, STAG2 and TP53 which have been described in the context of MN. However, these gene variants were also present in patients with diploid 20q. The remaining 6 variants included heterozygous P/LP GL alterations in 3 different Fanconi anemia genes ( FANCA, FANCM, FANCD2), SUZ12, and TINF2. The latter, TINF2, a telosome gene, known to be mutated in dyskeratosis congenita (DC), carried an LOF c.605-1G>A in a patient with MDS/MPN with a family history of cancer who later developed 20q-. The patient did not show any symptoms of DC-like features, which usually present at an early age. Since FANCA LOF with SGR via overepression of EIF6 has previously been described 1, we investigated and did not identify any cases in our cohort of EIF6 SGR resultant overexpression. While occasional patients with no previously diagnosed SBDS can be found among MDS with 20q-, screening for SBDS genes demonstrates that such a genetic configuration among adult patients with 20q is very rare. In addition, only 9 out of 215 patients with 20q- carried other pathogenic GL variants in non-recurrent genes which appear to be likely coincidental. This suggests that del20q is not a SGR in response to a GL mutation, but rather a SGR due to other aforementioned mechanisms, which are currently being explored in our cohort of patients. References: 1. Gueiderikh A, Maczkowiak-Chartois F, Rouvet G, Souquère-Besse S, Apcher S, Diaz JJ, Rosselli F. Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis. Sci Adv. 2021 Jan 1;7(1):eabb5414. doi: 10.1126/sciadv.abb5414. PMID: 33523834; PMCID: PMC7775781.

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Tsai, J. Y., e L. M. Silver. "Escape from genomic imprinting at the mouse T-associated maternal effect (Tme) locus." Genetics 129, n. 4 (1 dicembre 1991): 1159–66. http://dx.doi.org/10.1093/genetics/129.4.1159.

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Abstract (sommario):

Abstract Genomic imprinting occurs at the paternally inherited allele of the mouse T-associated maternal effect (Tme) locus. As a consequence, maternal transmission of a functional Tme gene is normally required for viability and individuals that receive a Tme-deleted chromosome (Thp or tlub2) from their mother die late in gestation or shortly thereafter. Here we report that a rearranged paternally derived chromosome duplicated for the Tme locus can act to rescue animals that have not received a maternal copy of the Tme locus. Unexpectedly, all rescued animals display an abnormal short/kinky tail phenotype. Somatic transfer of genomic imprinting between homologs by means of a transvection-like process between paired Tme and T loci is proposed as a model to explain the results obtained.

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49

Young, Neal S., Bogdan Dumitriu e Seishi Ogawa. "Acquired Aplastic Anemia: New Genetics, New Genomics". Blood 124, n. 21 (6 dicembre 2014): SCI—21—SCI—21. http://dx.doi.org/10.1182/blood.v124.21.sci-21.sci-21.

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Abstract (sommario):

Clinical and laboratory data have established that aplastic anemia is an immune-mediated disease in which T lymphocytes destroy hematopoietic stem and progenitor cells. Immunosuppressive therapies are effective in the majority of patients. Addition of the thrombopoietin mimetic eltrombopag increases the overall and complete response rates to anti-thymocyte globulin and as a single agent can rescue patients refractory to immunosuppressive therapy (IST). Multi-lineage robust blood count improvements with increased marrow cellularity suggest activity of eltrombopag on the hematopoietic stem cell. Recently, genetic factors have been identified that increase the risk of bone marrow failure: adults may present in the clinic with late manifestations of a pediatric syndrome (dyskeratosis congenita), but more typically there are no physical anomalies, often no family history, and earlier blood counts may be normal. In the telomeropathies, which in later life are almost always due to mutations in TERT (which encodes the telomerase) or TERC (which encodes the RNA template), there may be personal or familial multi-organ involvement, especially of liver and lung; early hair greying is a useful clinical clue. Detection of extremely short telomeres of leukocytes, accompanied by gene sequencing, is used to establish the diagnosis. In the syndrome defined by GATA2 mutations, there may be a history of unusual or persistent mycobacterial and viral infections, and monocytopenia preceding pancytopenia. Diagnosis requires sophisticated interpretation of gene sequencing. Large genomic data sets are now available acquired (somatic) mutations in aplastic anemia. For almost 300 NIH aplastic anemia patients treated with IST, candidate gene sequencing of myeloid cells obtained six months after treatment revealed somatic mutations in about one-third of cases. PIGA was most frequently abnormal, followed by BCOR1, DNMT3A, and ASXL1. Initial variant allele frequency was usually low. Mutations in a subset of genes predicted poor survival and progression to myelodysplastic syndrome and acute myeloid leukemia, while mutations in BCOR and PIGA correlated favorably with outcomes. When we also examined a subset of patients at the time of progression to monosomy 7 with pancytopenia and/or incipient leukemia by whole exome sequencing.DNMT3A and ASXL1 were implicated in only two cases, RUNX1 in another, and there were no novel recurring mutations. Telomere attrition was markedly accelerated in these monosomy 7 cases. A good model of evolution from bone marrow failure to myeloid malignancy centers on haploinsufficiency of specific genes, by mutations or chromosome loss, both of which would be favored in a stressed, regenerative environment. The failed marrow environment may select for defective cells, as, for example, in differentiation capability. Malignant evolution should be predictable in the clinic and amenable to therapeutic intervention. Disclosures Off Label Use: Eltrombopag in bone marrow failure syndromes, research protocols.

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50

de Bono, Mario, e Jonathan Hodgkin. "Evolution of Sex Determination in Caenorhabditis: Unusually High Divergence of tra-1 and Its Functional Consequences". Genetics 144, n. 2 (1 ottobre 1996): 587–95. http://dx.doi.org/10.1093/genetics/144.2.587.

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Abstract (sommario):

Abstract The tra-1 gene is a terminal regulator of somatic sex in Caenorhabditis elegans: high tra-1 activity elicits female development, low tra-1 activity elicits male development. To investigate the function and evolution of tra-1, we examined the tra-1 gene from the closely related nematode C. briggsae. Ce-tra-1 and Cb-tra-1 are unusually divergent. Each gene generates two transcripts, but only one of these is present in both species. This common transcript encodes TRA-1A, which shows only 44% amino acid identity between the species, a figure much lower than that for previously compared genes. A Cb-tra-1 transgene rescues many tissues of tra-1(nul1) mutants of C. elegans but not the somatic gonad or germ line. This transgene also causes nongonadal feminization of XO animals, indicating incorrect sexual regulation. Alignment of Ce-TRA-1A and Cb-TRA-1A defines several conserved regions likely to be important for tra-1 function. The phenotypic differences between Ce-tra-1(null) mutants rescued by Cb-tra-1 transgenes and wild-type C. elegans indicate significant divergence of regulatory regions. These molecular and functional studies suggest that evolution of sex determination in nematodes is rapid and genetically complex.

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