Auswahl der wissenschaftlichen Literatur zum Thema „Somatic genetic rescue“

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.

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.

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.

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.

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.

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.

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.

Le syndrome de Shwachman Diamond (SDS) est une ribosomopathie génétique rare entraînant une altération de la synthèse protéique associée à de nombreux symptômes, notamment une insuffisance médullaire et une neutropénie pouvant évoluer vers un syndrome de myélodysplasie ou une leucémie myéloïde aiguë. Les mutations bialléliques du gène SBDS sont responsables de plus de 90 % des cas de SDS et nous avons récemment identifié des mutations bialléliques EFL1 comme une nouvelle cause génétique de SDS. SBDS et EFL1 évincent le facteur elF6 de la sous-unité ribosomale pré60S, permettant à cette dernière d'interagir avec la sous-unité 40S pour former le ribosome mature 80S. L'acquisition naturelle d'événements génétiques somatiques au fil du temps participe au développement des maladies liées à l'âge et au développement des cancers. Cependant, dans les maladies mendéliennes, ces événements peuvent, dans de rares cas, contrer l'effet délétère de la mutation germinale et conférer un avantage sélectif aux cellules somatiquement modifiées, un phénomène appelé sauvetage génétique somatique (SGR). Nous avons récemment montré que plusieurs événements génétiques somatiques affectantl'expression ou la fonction d'elF6 sont fréquemment détectés dans les clones sanguins de patients atteints de SDS mais pas chez les individus sains, suggérant un mécanisme de SGR. Alors que la plupart de ces mutations somatiques induisent une déstabilisation de elF6 ou une haploinsuffisance d'EIF6, une mutation récurrente (N106S) n'affecte pas l'expression/stabilité d'elF6 mais réduit sa capacité à interagir avec la sous-unité 60S. Afin d'étudier plus en détail les conséquences fonctionnelles de l'haploinsuffisance de EIF6 et de la mutation N106S dans un contexte de SDS, j'ai introduit via CRISPR/Cas9 ces mutations dans des lignées fibroblastiques immortalisées de patients SDS et de contrôle. Ces modèles cellulaires originaux ont permis de déterminer l'impact de la mutation N106S sur la la localisation et la fonction d'elF6 mais aussi de préciser les effets de ces mutations sur plusieurs aspects du « fitness » cellulaire, notamment la biogenèse des ribosomes, le taux de traduction et la prolifération cellulaire. Dans l'ensemble, le développement de ce modèle a aidé à caractériser comment la mutation N106S et l'haploinsuffisance somatique de elF6 confèrent un avantage sélectif dans les cellules déficientes en SBDS ou EFL1
Shwachman Diamond syndrome (SDS) is a rare genetic ribosomopathy leading to impaired protein synthesis, which causes numerous symptoms including bone marrow failure and neutropenia that can evolve to myelodysplasia syndrome or acute myeloid leukaemia. Biallelic mutations in the SBDS gene are responsible of above 90% of the SDS cases and we recently identified biallelic EFL1 mutations as a novel cause of SDS. SBDS together with EFL1 remove the anti-association factor elF6 from the pre60S ribosomal subunit, allowing its interaction with the 40S subunit to form the mature ribosome 80S. Natural acquisition of somatic genetic events over time participâtes to age-related diseases and cancer development. However, in Mendelian diseases these events can, in rare case, counteract the deleterious effect of the germline mutation and provide a sélective advantage to the somatically modified cells, a phenomenon dubbed Somatic Genetic Rescue (SGR). We recently showed that several somatic genetic events affecting the expression or function of elF6 are frequently detected in blood clones from SDS patients but not in healthy individuals, suggesting a mechanism of SGR. While most of these somatic mutations induce elF6 destabilization or EIF6 haploinsufficiency, one récurrent mutation (N106S) did not affect the expression of elF6 but rather impact its ability to interact with the 60S subunit. In order to further investigate the functional conséquences of ElF6 haploinsufficiency and N106S mutation in a context of SDS, I introduced via CRISPR/Cas9 these mutations in immortalized fibroblastic cell line from SDS patients and control. These original cellular models hâve made it possible to détermine the impact of the N106S mutation on the localisation and function of elF6 and also to clarify the effects of these mutations on several aspects of cellular fitness, in particular ribosome biogenesis, translation rate and cell prolifération. Overall, the development of these cellular models has helped to characterise how the somatic N106S mutation and elF6 haploinsufficiency confer a sélective advantage in cells déficient in SBDS or EFL1

Papaya (Carica papaya L.) holds significant importance as a fruit crop in tropical and sub-tropical regions. In India, it gives earnings rivaling (higher yield per hectare) next to banana It is a fast growing herbaceous plant and also used as a filler plant in orchards.. And within this chapter, our focus will be directed towards an exploration of diverse methodologies of unconventional and biotechnological approaches in papaya which include micropropagation, organogenesis, embryo rescue, anther culture, somatic embryogenesis, protoplast culture for improvement of papaya. Most important topic we are covering here is genetic engineering. Severe loss causing disease of papaya is papaya ring spot, attributed to the papaya ring spot virus (PRSV), is the subject under consideration and for this virus control several transgenic plant had been developed which is based on coat protein (CP) and replicase mediated resistance. For future suitable method to control PRSV will be post-transcriptional gene silencing (PTGS). Generally farmers use conventional methods rather than nonconventional or biotechnological method because they don’t want to take risk and they don’t have trust on these PSRV transgenic papaya.

Abstract K+ channel See ion channel; action potential; resting membrane potential. Kahlbaum, Karl Ludwig (1828–1899) German psychiatrist; catatonia. Kahlbaum syndrome Nonmalignant catatonia(q.v.). Kahlbaum-Wernicke syndrome Presbyophrenia(q.v.). kayak gene See clock, biological. kaif, kif Pleasure or feeling of contentment and ease, as in a dream or state of ecstasy. The term is used in Morocco to refer to hashish or marijuana (q.v.). kainate An activator of the kainate subtype of EAA (q.v.). See AMPA. Kalinowsky, Lothar B. (1899–1992) German-born neuropsychiatrist; in United States after 1940; electroconvulsive and other somatic treatments. Kallmann, Franz J. (1897–1965) German-born psychoanalyst and geneticist; genetics of human behavior, especially schizophrenia, manic-depressive psychosis. Kalmuk idiocy Obs. (Kalmuk, member of a nomad Tartar tribe) Down syndrome (q.v.). Kandinsky-Clérambault complex See Clérambault-Kandinsky complex. Kanner, Leo (1894–1981) Austrian-born psychiatrist; emigrated to United States in 1924; founded Johns Hopkins Children ‘s Psychiatric Clinic (1930); wrote first text in Child Psychiatry (1935); early infantile autism. Kardiner, Abraham (1891–1981) Cofounder of first psychoanalytic training school in the United States (1930); The Individual and His Society (1939); Psychological Frontiers of Society (1945). Karpman Drama Triangle A conceptualization of traumatic experiences in terms of victim, rescuer, and persecutor. Originally suggested as one way of viewing intrafamilial dynamics of alcoholics and their families, it has since been extended to other disorders, such as dissociative identity disorder (DID).

Abstract As currently practiced, gene therapy can be defined as the transfer of exogenous genes to somatic cells of a patient in order to correct an inherited or acquired gene defect, or to introduce a new function or trait. In the last 15 years, gene therapy has moved from the speculative stage to clinical applications as clinical gene transfer protocols have been applied to inherited genetic defects, malignancies, and infectious diseases. Primary immunodeficiciency diseases (PIDs), especially severe combined immunodeficiency (SCID) disorders, have played a major role in this process as they have provided a proving ground for the first therapeutic applications (Blaese et al., 1995) as well as the first evidence that gene therapy can be curative (CavazzanaCalvo et al., 2000; Hacein-Bey-Abina et al., 2002). Several advantageous characteristics have made PIDs attractive candidate diseases for gene therapy. First, PIDs are often curable by allogeneic hematopoietic stem cell transplantation (HSCT), which translates into the possibility that they should also be treatable with procedures combining ex vivo manipulation and reinfusion of autologous, gene-corrected hematopoietic stem cells. Second, the target cells reside in the bone marrow, a readily accessible tissue, which makes them amenable to harvest for ex vivo gene transfer, the procedure that currently offers the best success. In addition, the regulation of genes responsible for many PIDs (i.e., adenosine deaminase deficiency, chronic granulomatous disease, X-linked severe combined immunodeficiency, leukocyte adhesion deficiency) appears relatively simple and, while currently available technology does not allow reproducing the physiological control mechanisms of gene expression, the available gene transfer vectors are more than adequate to provide expression of these genes at levels required for clinical benefit. Finally, for several PIDs, gene-corrected progenitors and mature cells have a survival advantage over the unmodified, affected cell populations. This selective survival translates into the very advantageous circumstance that even low gene transfer efficiency can have therapeutic effects.