scholarly journals Revertant Mosaicism in Epidermolysis Bullosa

Biomedicines ◽  
2022 ◽  
Vol 10 (1) ◽  
pp. 114
Author(s):  
Cameron Meyer-Mueller ◽  
Mark J. Osborn ◽  
Jakub Tolar ◽  
Christina Boull ◽  
Christen L. Ebens

Epidermolysis bullosa (EB) is a group of genetic blistering diseases characterized by mechanically fragile skin and mucocutaneous involvement. Historically, disease management has focused on supportive care. The development of new genetic, cellular, and recombinant protein therapies has shown promise, and this review summarizes a unique gene and cell therapy phenomenon termed revertant mosaicism (RM). RM is the spontaneous correction of a disease-causing mutation. It has been reported in most EB subtypes, some with relatively high frequency, and has been observed in both keratinocytes and fibroblasts. RM manifests as identifiable patches of unaffected, blister-resistant skin and can occur through a variety of molecular mechanisms, including true back mutation, intragenic crossover, mitotic gene conversion, and second-site mutation. RM cells represent a powerful autologous platform for therapy, and leveraging RM cells as a therapeutic substrate may avoid the inherent mutational risks of gene therapy/editing. However, further examination of the genomic integrity and long-term functionality of RM-derived cells, as well in vivo testing of systemic therapies with RM cells, is required to realize the full therapeutic promise of naturally occurring RM in EB.

2021 ◽  
Vol 22 (13) ◽  
pp. 6663
Author(s):  
Maurycy Jankowski ◽  
Mariusz Kaczmarek ◽  
Grzegorz Wąsiatycz ◽  
Claudia Dompe ◽  
Paul Mozdziak ◽  
...  

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-42-SCI-42
Author(s):  
Marjolijn C. Jongmans

Abstract Many genetic syndromes are characterized by a wide spectrum of clinical severity. Even within one family clinical presentations can show extreme variation. Mosaic tissue distribution caused by spontaneous correction of a germline pathogenic allele is one of multiple explanations for variety in phenotypic expression of an inherited mutation. This phenomenon, called somatic reversion, is infrequently observed and can be easily overlooked. Reversion needs to be considered if a person presents with a milder than expected clinical course or with a mixture of phenotypically normal and abnormal cells.1 Mechanisms that may explain reversion include mitotic gene conversion, back mutation, intragenic mitotic recombination and the occurrence of compensatory mutations. A mosaic pattern of somatic reversion only becomes apparent if several criteria are met. The non-mutant cells need to have a selective growth advantage over surrounding mutant cells. Furthermore, to facilitate expansion of the revertant clone the affected genes need to be expressed in regenerating organ systems like skin and blood.1 The chance of spontaneous correction of a pathogenic allele is likely increased in diseases with an underlying mechanism resulting in genomic instability or high mutation rates, like Bloom syndrome and Fanconi anemia, which are both caused by gene defects in DNA repair pathways.2,3 In skin disorders an evolving mosaic revertant pattern is easily visible. Ichthyosis with confetti, caused by mutations in KRT10, is an example of a skin disorder displaying multiple events of reversion.4 In this condition, normal skin spots appear early in life and increase in number and size over time. Each normal spot results from a separate event of loss of heterozygosity on chromosome 17q, which harbors KRT10, via mitotic recombination. Also in the genetic skin fragility disorder epidermolysis bullosa revertant mosaicism has been described repeatedly.5,6 We have observed reversion, caused by mitotic recombination of mutant TE RC (telomerase RNA component) alleles in a family affected by dyskeratosis congenita (DC).7 DC is a multisystem disorder characterized amongst others by bone marrow failure and lung fibrosis. The observation of mosaic stretches of uniparental disomy (UPD) of chromosome 3q as an indication of revertant mosaicism encouraged us to develop a highly sensitive method for detecting genomic regions with low mosaic UPD in SNP array data. Indeed this tool supported us in identifying additional cases of DC and a mosaic reversion pattern in blood cells. Revertant mosaicism being a recurrent event in DC related conditions was recently confirmed by others.8 Awareness of revertant mosaicism is important for improving diagnostic testing. In DC for instance it is common practice that analysis of the DC genes is performed on DNA isolated from peripheral blood cells. In case no pathogenic mutation is found, an obvious conclusion can be that the phenotype in the family is caused by an aberration in an as yet to be identified DC gene. Based on our findings, we recommend sequence analysis on DNA extracted from other cells, such as skin fibroblasts, particularly in individuals without bone marrow failure. The observation of reversion in hematological conditions is also of importance for the development of future therapies: Isolation of autologous reverted stem cells can probably circumvent more toxic and harmful therapies, like allogeneic stem cell transplantation, in a subset of individuals. 1. Hirschhorn R. In vivo reversion to normal of inherited mutations in humans. J Med Genet 2003;40 (10):721-728. 2 Ellis NA, Ciocci S, German J. Back mutation can produce phenotype reversion in Bloom syndrome somatic cells. Hum Genet 2001;108 (2):167-173. 3 Waisfisz Q, Morgan NV, Savino M, et al. Spontaneous functional correction of homozygous fanconi anaemia alleles reveals novel mechanistic basis for reverse mosaicism. Nat Genet 1999;22 (4):379-383. 4 Choate KA, Lu Y, Zhou J, et al. Mitotic recombination in patients with ichthyosis causes reversion of dominant mutations in KRT10. Science 2010;330 (6000):94-97. 5 Jonkman MF, Scheffer H, Stulp R, et al. Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion. Cell 1997;88 (4):543-51. 6 Kiritsi D, Garcia M, Brander R, et al. Mechanisms of natural gene therapy in dystrophic epidermolysis bullosa. J Invest Dermatol 2014;134 (8):2097-104. 7 Jongmans MC, Verwiel ET, Heijdra Y, et al. Revertant somatic mosaicism by mitotic recombination in dyskeratosis congenita. Am J Hum Genet 2012;90 (3):426-33. 8 Alder JK, Stanley SE, Wagner CL, et al. Exome Sequencing Identifies Mutant TINF2 in a Family With Pulmonary Fibrosis. Chest 2015;147 (5):1361-8. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1979-1979
Author(s):  
Myléne Gerritsen ◽  
Esther Tijchon ◽  
Amit Mandoli ◽  
Joost H.A. Martens ◽  
Jan Jacob Schuringa ◽  
...  

Abstract RUNX1 (AML1) is a transcription factor critically involved in normal haematopoiesis. Inactivating RUNX1 mutations have been frequently described in a variety of myeloid neoplasms, including high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Here, we aimed to functionally and molecularly define the actions of a dominant negative mutant by in vitro and in vivo experiments and RNA- and ChIP-sequencing approaches. Overexpression of the RUNX1 mutant S291fs300X in cord blood (CB) CD34+ cells caused a decline in erythroid colony formation (p= 0.01) while the CFU-GM colonies showed enhanced replating capacity compared to control (>3 times). It appeared that the replating potential was restricted to CD14-/CD15- progenitor cells. Long-term suspension cultures with myeloid growth factors (IL-3, SCF) of RUNX1 S291fs300X CB CD34+ cells provided a rather homogenous cell population after 10 weeks of culture. These cells are growth factor dependent and are phenotypically defined by CD34+/CD38+/CD33+/IL1-RAP+/CD45RA+/CD123+ resembling a GMP phenotype which can be propagated for approximately 20 weeks in suspension. Comparable results were obtained with normal bone marrow CD34+cells transduced with the RUNX1 S291fs300X. Karyotype analyses demonstrated no abnormalities while integration site analysis showed a variety of different integration sites and differences between individual samples, suggesting that the myeloid differentiation block is related to the RUNX1 S291fs300X mutation.Long-term MS5 stromal co-cultures of transduced RUNX1 S291fs300X CB CD34+ cells showed after 8-10 weeks a rather homogenous cell population with limited potential to expand and localized under the stromal layer. This cell population is phenotypically defined by CD34+/CD38-. The interactions with the stroma appear to prevent proliferation but retain quiescence, indicating that sufficient niche-cell interactions might be crucial for transformation. NSG mice experiments are performed to test the reproducibility of these findings in vivo. Q-PCR studies demonstrated reduced expression of C/EBPα in RUNX1 S291fs300X CB CD34+ cells, one of the key targets in myeloid differentiation. Therefore, week 10 RUNX1 S291fs300X CB CD34+ cells were transduced with a retroviral C/EBPα overexpression vector. The re-expression of C/EBPα resulted in a reduction in cell proliferation, decline of undifferentiated blasts and an increase in CD15 expression. RNA- and ChIP-sequencing data revealed a decreased expression of crucial RUNX1 target genes including C/EBPα and Cited2 and also a retained binding of mutant RUNX1 on these loci in conjunction with a decrease of H3K27ac. Further research into the molecular mechanisms by which this RUNX1 S291fs300Xderegulates gene-expression is in progress. Our results implicate that overexpression of RUNX1 S291fs300X mutant leads to impaired erythroid differentiation and a strong differentiation block of the myeloid lineage resulting in the expansion and maintenance of a GMP-like cell population. Disclosures No relevant conflicts of interest to declare.


2007 ◽  
Vol 7 ◽  
pp. 421-430 ◽  
Author(s):  
Matthew C. Catley

Glucocorticoids (GCs) are some of the most important drugs in clinical use today. They are mainly used to suppress disease-related inflammation and are widely used for the treatment of many inflammatory diseases including asthma and arthritis. However, GCs are also associated with debilitating side effects that place limitations on the long-term use of these drugs. The development of a GC with reduced side effects would allow more effective treatments for patients who require long-term suppression of inflammation. GCs exert their effects by binding and activating the GC receptor (GR). The activated receptor then binds GC response elements (GREs) in the promoter of genes, and activates transcription (transactivation) or interferes with the activation of transcription by inhibiting the transactivating function of other transcription factors, such as AP-1 and NF-ĸB (transrepression). Transrepression is believed to be responsible for the majority of the beneficial anti-inflammatory effects of GCs, whereas transactivation is believed to play a bigger role in the unwanted side effects of GCs. Compounds that can dissociate the transactivation function of GCs from the transrepression function may, therefore, have an improved therapeutic index. A number of these dissociated corticosteroids have been developed.In vitroassays using these compounds appear to show good dissociation. However,in vivo, the dissociation appears to be lost and these compounds still produce many of the side effects associated with conventional GCs. A better understanding of the molecular mechanisms behind GC-induced effects would allow the design of novel selective GR modulators with an improved therapeutic index.


Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 560-567 ◽  
Author(s):  
David G. Kent ◽  
Brad J. Dykstra ◽  
Jay Cheyne ◽  
Elaine Ma ◽  
Connie J. Eaves

Abstract Hematopoietic stem cells (HSCs) regenerated in vivo display sustained differences in their self-renewal and differentiation activities. Variations in Steel factor (SF) signaling are known to affect these functions in vitro, but the cellular and molecular mechanisms involved are not understood. To address these issues, we evaluated highly purified HSCs maintained in single-cell serum-free cultures containing 20 ng/mL IL-11 plus 1, 10, or 300 ng/mL SF. Under all conditions, more than 99% of the cells traversed a first cell cycle with similar kinetics. After 8 hours in the 10 or 300 ng/mL SF conditions, the frequency of HSCs remained unchanged. However, in the next 8 hours (ie, 6 hours before any cell divided), HSC integrity was sustained only in the 300 ng/mL SF cultures. The cells in these cultures also contained significantly higher levels of Bmi1, Lnk, and Ezh2 transcripts but not of several other regulators. Assessment of 21 first division progeny pairs further showed that only those generated in 300 ng/mL SF cultures contained HSCs and pairs of progeny with similar differentiation programs were not observed. Thus, SF signaling intensity can directly and coordinately alter the transcription factor profile and long-term repopulating ability of quiescent HSCs before their first division.


2011 ◽  
Vol 14 (4) ◽  
pp. 371-374 ◽  
Author(s):  
Toshihide Mizuno ◽  
Yashushi Nemoto ◽  
Tomonori Tsukiya ◽  
Yoshiaki Takewa ◽  
Yoshiyuki Taenaka ◽  
...  

2012 ◽  
Vol 24 (1) ◽  
pp. 80 ◽  
Author(s):  
O. R. Vaughan ◽  
A. N. Sferruzzi-Perri ◽  
P. M. Coan ◽  
A. L. Fowden

Environmental conditions during pregnancy determine birthweight, neonatal viability and adult phenotype in human and other animals. In part, these effects may be mediated by the placenta, the principal source of nutrients for fetal development. However, little is known about the environmental regulation of placental phenotype. Generally, placental weight is reduced during suboptimal conditions like maternal malnutrition or hypoxaemia but compensatory adaptations can occur in placental nutrient transport capacity to help maintain fetal growth. In vivo studies show that transplacental glucose and amino acid transfer adapt to the prevailing conditions induced by manipulating maternal calorie intake, dietary composition and hormone exposure. These adaptations are due to changes in placental morphology, metabolism and/or abundance of specific nutrient transporters. This review examines environmental programming of placental phenotype with particular emphasis on placental nutrient transport capacity and its implications for fetal growth, mainly in rodents. It also considers the systemic, cellular and molecular mechanisms involved in signalling environmental cues to the placenta. Ultimately, the ability of the placenta to balance the competing interests of mother and fetus in resource allocation may determine not only the success of pregnancy in producing viable neonates but also the long-term health of the offspring.


Author(s):  
Xinhui Guo ◽  
Wei Xue ◽  
Yujiao Wang ◽  
Jiangang Zhang ◽  
Qinghong Meng ◽  
...  

Resveratrol (RSV) as a naturally occurring small molecule has been reported to benefit the cardiovascular system through a dietary supplementation. However, the relevant genetic regulation by RSV has been uncovered. This work is solely to investigate how RSV increases a fecal excretion of total sterols of HF fed C57BL/6J mice to block lipid accumulation in vivo through regulation of PPRAa, HMG-CoA-R, CYP7A1, LDL-R and ABCG5 genes. The results showed that RSV significantly improved the excretion of total sterols through up regulation of mRNA expressions of PPARa, CYP7A1, LDL-R, ABCG5 and down regulation of HMG-CoA-R. These data indicate that beneficial effects from RSV were associated with the favorably altered expression of hepatic and large intestine genes in HF fed mice.


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