Academic literature on the topic 'Cellular mosaicism'

Intercellular karyotypic variability has been a focus of genetic research for more than 50 years. It has been repeatedly shown that chromosome heterogeneity manifesting as chromosomal mosaicism is associated with a variety of human diseases. Due to the ability of changing dynamically throughout the ontogeny, chromosomal mosaicism may mediate genome/chromosome instability and intercellular diversity in health and disease in a bottleneck fashion. However, the ubiquity of negligibly small populations of cells with abnormal karyotypes results in difficulties of the interpretation and detection, which may be nonetheless solved by post-genomic cytogenomic technologies. In the post-genomic era, it has become possible to uncover molecular and cellular pathways to genome/chromosome instability (chromosomal mosaicism or heterogeneity) using advanced whole-genome scanning technologies and bioinformatic tools. Furthermore, the opportunities to determine the effect of chromosomal abnormalities on the cellular phenotype seem to be useful for uncovering the intrinsic consequences of chromosomal mosaicism. Accordingly, a post-genomic review of chromosomal mosaicism in the ontogenetic and pathogenetic contexts appears to be required. Here, we review chromosomal mosaicism in its widest sense and discuss further directions of cyto(post)genomic research dedicated to chromosomal heterogeneity.

Abstract Context Molecular mechanisms causing the broad phenotypic diversity of external masculinization in individuals with 45,X/46,XY mosaicism are poorly understood. Objective Analysis of androgen receptor (AR) expression and function as a putative influencing factor for the genital phenotype in patients with 45,X/46,XY mosaicism. Design Measurement of AR mRNA expression levels, AR activity [DHT-mediated APOD (apolipoprotein D) induction] and cellular 45,X/46,XY ratios in genital skin fibroblasts from individuals with 45,X/46,XY mosaicism and male reference individuals, and determination of the external virilization scale from individuals with 45,X/46,XY mosaicism. Setting University hospital endocrine research laboratory. Patients or Other Participants: 30 genital skin fibroblast cultures (GFs) from male reference individuals and 15 GFs from individuals with 45,X/46,XY mosaicism. Intervention None Main Outcome Measures Determination of AR mRNA expression and AR activity in male reference GFs and 45,X/46,XY GFs and correlation of the obtained data with the cellular 45,X/46,XY ratios and the patients’ external virilization scale. Results In 6 of 15 45,X/46,XY GFs, AR mRNA expression and AR activity were significantly lower compared with those in the 46,XY reference GFs. In this subgroup of reduced AR mRNA expression, a positive trend was seen between AR mRNA expression and the percentage of XY-positive cells. Furthermore, we found a positive correlation between AR activity and the external virilization scale in the 15 45,X/46,XY GF samples (P = 0.03). Conclusion Our results suggest that AR expression and AR activity might influence the phenotypic variability seen in patients with 45,X/46,XY mosaicism.

Clonal mosaicism (a detectable post-zygotic mutational event in cellular subpopulations) is common in cancer patients. Detected segments of clonal mosaicism are usually bundled into large-locus regions for statistical analysis. However, low-frequency genes are overlooked and are not sufficient to elucidate qualitative differences between cancer patients and non-patients. Therefore, it is of interest to develop and describe a tool named Sub-GOFA for Sub-Gene Ontology function analysis in clonal mosaicism using semantic similarity. Sub-GOFA measures the semantic (logical) similarity among patients using the sub-GO network structures of various sizes segmented from the gene ontology (GO) for clustering analysis. The sub-GO’s root-terms with significant differences are extracted as disease-associated genetic functions. Sub-GOFA selected a high ratio of cancer-associated genes under validation with acceptable threshold.

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.

Abstract Abstract 4401 Proinflammatory cytokines, TNF-α and IFN-γ, are potent inhibitors of hematopoiesis, and may be relevant in the pathogenesis of bone marrow failure in inherited bone marrow failure syndromes (IBMFS). Increased levels of these cytokines in sera and in bone marrow CD3+ cells have been reported in Fanconi anemia (FA) patients. However, our study did not find increased TNF-α or IFN-γ in sera, or supernatants from phytohemagglutinin-stimulated peripheral blood mononuclear cells from IBMFS patients. To assess whether production of these cytokines is dysregulated in BM of these patients, we examined intracellular expression of TNF-α and IFN-γ in BM mononuclear cells from 16 FA, 20 dyskeratosis congenita (DC), 21 Diamond-Blackfan anemia (DBA) and 7 Shwachman-Diamond syndrome (SDS) patients by flow cytometry; 14 healthy adults were studied as controls. To detect intracellular TNF-α and IFN-γ, BM lymphocytes and monocytes were stimulated with phorbol 12-myristate 13-acetate plus ionomycin (P+I), or lipopolysaccharide (LPS), respectively. Separately, unstimulated cells were stained with antibodies to CD45, CD3, CD19, CD14, and CD34 to determine the proportion of cellular subsets. Percentages of T cells in patients with IBMFS were comparable to the controls, while DC patients had lower proportion of B cells (p=0.02). The percentages of monocytes were lower in FA (p=0.04), DC (p=0.009), and DBA (p<0.001) patients. The proportions of CD34+ cells were also lower in IBMFS patients (≤0.02 for all) except for those with DBA, who had similar proportions as the controls. When we compared the effect of cytopenia (counts below normal for age), only the proportion of CD34+ cells in DC patients was significantly affected. DC patients with cytopenia (n=15) had lower numbers of CD34+ cells (p=0.007) compared with those without (n=5). We also analyzed the effect of somatic mosaicism in FA because it may correct the hematopoietic defect in these patients. FA patients without mosaicism (n=11) had lower proportions of CD19+, CD14+, and CD34+ cells than those with mosaicism (n=5), while the CD3+ cell numbers were unaffected. We detected both intracellular TNF-α and IFN-γ in T cells, but only TNF-α in B cells in response to P+I, while LPS stimulation led to TNF-α production only in monocytes. Percentages of cytokine-producing T and B cells were significantly lower for patients with DBA when compared with healthy adult controls (p<0.006 for T cells and p=0.001 for B cells). There were no significant differences in the other syndromes. Comparison of intracellular cytokines between cytopenic and non-cytopenic patients showed that TNF-α-producing T cells were affected in FA (p=0.03), where the cytopenic patients had a higher proportion of TNF-α-positive T cells. For the LPS-stimulated monocytes, FA (p=0.01) and DBA (p=0.05) patients had significantly lower proportions of TNF-α-producing cells than the controls, and this was independent of cytopenia. There was no effect of mosaicism on cytokine production. Contrary to previous reports, we did not find an increase in intracellular TNF-α or IFN-γ in T cells from FA patients. However, the number of TNF-α-producing monocytes in FA was lower than that in healthy adult controls. This is consistent with reported dysregulation of monocytes in FA patients. We also identified reduced cytokine expression in lymphocytes and monocytes from DBA patients, but not from DC or SDS. As expected, we found reduced proportions of CD34+ cells in FA, DC and SDS, syndromes associated with multilineage cytopenia, and not in DBA which is associated with pure red cell aplasia. And, we ascertained that FA patients with somatic mosaicism had significantly higher percentages of cells including CD34+, suggesting that the corrected stem cell pool in FA mosaics is able to maintain hematopoiesis in contrast to non-mosaic FA patients who develop progressive cytopenia over time. Overall, the effect of cytopenia on cytokine production was mild; however, this may be related to the small sample size. In conclusion, our results suggest that mechanisms other than an excess of inflammatory cytokines may be responsible for bone marrow failure in IBMFS, and this area of research deserves a further attention in larger studies. Disclosures: No relevant conflicts of interest to declare.

It is known that age-related changes impacting multiple organ systems occur earlier in people with Down syndrome (Ds), but the biological basis underlying this trisomy 21-associated propensity for premature aging is poorly understood. Given that the trisomic/normal cells from people with mosaic Ds (mDs) are identical with regards to environmental exposures and genes (except for chromosome 21 copy number), comparisons of these isogenic trisomic/disomic cells allow one to “unmask” the cellular consequences of trisomy 21 by removing extraneous factors. The primary aim of this study was to determine if trisomy 21 results in an increase in the acquisition of age-related somatic chromosomal changes. To meet this aim, chromosome-specific telomere lengths, senescence-associated distension of satellites (SADS), and chromosomal instability frequencies were compared between the isogenic trisomic/disomic cells of people with mDs ranging from 1 to 44 years of age. Chromosome-specific telomere lengths were quantified using a Q-FISH (pantelomeric probe) method. The average trisomic cell telomere length (3.609 mean, +/- 0.082 SE) was significantly less than the average disomic cell telomere length (3.888 +/- 0.083) (n=28; p

La technique de micro-injection des spermatozoides humains sous la pellucide (suzi) des ovocytes de hamster est developpee afin d'etudier le pouvoir fecondant des spermatozoides humains et de proceder a l'analyse de leur caryotype. L'analyse de l'interaction gametique apres micro-injection de plusieurs milliers d'ovocytes dans differentes conditions environnementales a mis en evidence la relation existant entre la dynamique de la reaction acrosomiale et la capacite des spermatozoides a fusionner avec l'ovocyte. D'autre part la selection de sous populations spermatiques presentant differents etats acrosomiques et pouvoirs fecondants a ete obtenue par centrifugation sur gradient de percoll. Une technique originale d'induction de la reaction acrosomiale par l'ionophore calcique a23187 est proposee pour apprecier le pouvoir fusiogene des spermatozoides humains apres micro-injection. Le pouvoir fusiogene des spermatozoides humains micro-injectes sous la pellucide a ete compare dans les modeles heterospecifique et homospecifique. La valeur predictive de la suzi-hamster pour la suzi humaine est discutee. La maitrise des parametres de la fecondation apres suzi-hamster a permis de realiser l'analyse des spermatozoides micro-injectes. Chez deux sujets temoins, 72 metaphases interpretables de spermatozoides ont ete obtenus. Cependant la suzi se revele etre une technique moins performante pour l'analyse cytogenetique des spermatozoides humains que la technique conventionnelle d'insemination des ovocytes depellucides. Elle semble donc difficilement applicable aux sujets dont la spermatogenese est deficiente. De facon concomitante, l'analyse cytogenetique des spermatozoides d'un patient porteur d'un syndrome de klinefelter en mosaique (46,xy/47,xxy) a ete realisee par la technique conventionnelle d'insemination in vitro des ovocytes depellucides de hamster. 543 caryotypes spermatiques ont ete obtenus. Un taux significativement augmente de spermatozoides 24,xy a ete mis en evidence, suggerant que les cellules germinales de la lignee 47,xxy seraient aptes a franchir toutes les etapes de la meiose

Girls Clustering Epilepsy is the second most common developmental and epileptic encephalopathy. GCE is due to variants in the X chromosome gene PCDH19 and is underpinned by cellular mosaicism due to X-chromosome inactivation (XCI) in females or somatic variant in males. The hallmark feature is that seizures occur in clusters and mainly affect females. At the time of thesis submission, GCE was re-named “X-linked clustering epilepsy” (XCE) to accommodate the growing number of affected male cases. Seizures typically present as generalized tonic-clonic and/or focal, which may evolve to bilateral, tonic-clonic. The clinical profile includes variable cognitive impairment and psychiatric features. The prevalence of these comorbidities and cause of the variable clinical expressivity is unknown. We performed a systematic review and meta-analysis to identify the comorbidities associated with PCDH19 variants and examine phenotype- and genotype-phenotype associations. Data from 38 peer-reviewed original articles were used and included 271 individual cases. We found that seizure onset ≤ 12 months was significantly associated (p = 4.127 x 10-7) with more severe intellectual disability compared with onset > 12 months. We identified two recurrent variants p.Asn340Ser and p.Tyr366Leufs*10, occurring in 25 (20 unrelated) and 30 (11 unrelated) cases, respectively. PCDH19 variants were associated with psychiatric comorbidities in approximately 60% females, 80% affected mosaic males, and reported in nine hemizygous males. Executive dysfunction, and hyperactive, autistic, and obsessive-compulsive features were most frequently associated with PCDH19 variants. We developed a PCDH19 survey to systematically examine the comorbidities identified in our review using standardized neuropsychiatric assessments. The survey was completed by 122/186 (66%) participants diagnosed with GCE or with a confirmed likely pathogenic PCDH19 variant. Executive functions were measured using the Behavior Rating Inventory of Executive Function. Psychiatric comorbidities were assessed via the Social Responsiveness Scale or Social Communication Questionnaire, the Strengths and Difficulties Questionnaire, and the Dimensional Obsessive-Compulsive Scale. Genetic, seizure, and developmental information were also collected. Of the 112 evaluated participants (15 males), there were 70 unique variants. Thirty-five variants were novel and included a newly identified recurrent variant Ile781Asnfs*3. There were no phenotypic differences between published and unpublished cases. Seizures occurred in clusters in 94% individuals, with seizures resolving in 28% at an average age of 17.5 years. Developmental delay prior to seizure onset occurred in 18% of our cohort. Executive dysfunction and autism spectrum disorder (ASD) occurred in approximately 60% of individuals. The ASD profile included features of attention-deficit hyperactivity disorder. Obsessive-compulsive symptomology was observed in 21% individuals. There were no phenotypic differences between heterozygous females and mosaic males. We describe a mosaic male and two hemizygous males with atypical clinical profiles. Earlier seizure onset age and increased number of seizures within a cluster were associated with more severe clinical outcomes. No clinical profile was observed for transmitting males. The penetrance of GCE is incomplete; estimated to be around 80-90%, and might be explained by cellular interference. Cellular interference postulates that the coexistence of PCDH19 wild-type and variant cells would be pathogenic, whereas a homogenous cell population would be tolerated; an idea supported by the presence of asymptomatic PCDH19-negative hemizygous and symptomatic PCDH19-mosaic males. The cellular interference hypothesis was tested through analyses of GCE penetrant and non-penetrant female fibroblast cell lines using assays to determine XCI patterns and relative PCDH19 cDNA expression. Specifically, we hypothesized that XCI and PCDH19 cDNA expression will be skewed towards complete wild-type or variant expression in non-penetrant females. We have shown that XCI patterns do not correlate with relative PCDH19 cDNA expression in fibroblasts, thus invalidating use of this assay to infer PCDH19 expression. No clear association was observed between penetrance in XCE and the degree of variant and wild-type PCDH19 mRNA expression in skin fibroblasts. Although we were able to identify three non-penetrant females with 100% wild-type PCDH19 expression, we were unable to provide support for the mechanism of cellular interference through our finding clinical phenotypes in individuals with markedly skewed XCI. Neuropsychiatric disorders can be very responsive to early intervention; therefore, a better understanding of these comorbidities may help to inform treatment and ultimately lead to better developmental outcomes for individuals affected by GCE. We show that both seizure onset age and activity are associated with clinical outcomes. Clinicians can use this information to inform prognosis and provide targeted intervention and guidance for patients and their families.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2020

Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina
A doença de Machado-Joseph (DMJ), também conhecida como ataxia espinocerebelosa do tipo 3 é uma doença neurodegenerativa caracterizada por uma expansão anormal do tripleto CAG na região codificante do gene MJD1/ATXN3, o que se traduz numa expansão de uma cadeia de poliglutaminas na proteína ataxina-3. Esta expansão de CAGs anormalmente longa confere toxicidade à proteína ataxina-3 produzida que através de múltiplos mecanismos patogénicos culminando em neurodegenerescência em diversas regiões do cérebro.As abordagens terapêuticas atuais consistem principalmente em sessões de fisioterapia e no alívio farmacológico sintomático de sintomas específicos, constituindo a força motriz para o desenvolvimento de novas abordagens terapêuticas.Os diversos modelos existentes de DMJ têm sido ferramentas indispensáveis para a identificação dos mecanismos intrínsecos da doença, bem como para a validação de novas terapias. No entanto, modelos transgénicos de murganho são altamente dispendiosos, necessitam de longos períodos para o desenvolvimento de fenótipo, não recapitulando algumas das características desta doença. O nosso grupo foi pioneiro no desenvolvimento de um modelo roedor da DMJ com base em vetores lentivirais. Apesar das inúmeras vantagens deste modelo, este envolve intervenção cirúrgica (craniotomia), injeção localizada no parênquima cerebral, estando a patologia confinada ao local de injeção. Estas evidências demonstram a urgência no desenvolvimento de novos modelos que expressem ataxina-3 mutante de forma ubíqua no Sistema Nervoso Central (SNC), providenciando novas perspetivas relacionadas com os mecanismos da doença, permitindo ainda a avaliação do potencial de novas terapias.O rápido desenvolvimento de ferramentas baseadas em Vírus Adeno-Associados (AAV), tornou-se um dos mais promissores sistemas de entrega de genes a uma grande variedade de tipos celulares, através de diferentes vias de administração.O objetivo do presente trabalho foi o desenvolvimento de novas estratégias baseadas no uso de vetores mosaico de AAV para gerar modelos in vitro e in vivo de DMJ. Para tal, foram desenvolvidos vetores mosaico AAV1/2 e AAV2/9 codificando para a ataxina-3 humana mutada direcionados para o SNC. Esta abordagem providenciou o desenvolvimento de modelos de DMJ com elevada relevância fisiológica em tempo-útil e com uma boa relação custo-benefício, ultrapassando algumas das limitações mencionadas anteriormente.Resumidamente, este estudo fornece forte evidências que os vetores AAVs gerados são capazes de transduzir o SNC após injeção intracraniana (AAV1/2) ou intravenosa (AAV2/9), sobre expressando a ataxina-3 mutante completa não só em modelos in vitro como também in vivo, recapitulando algumas das principais características da DMJ.
Machado-Joseph Disease (MJD) or Spinocerebellar Ataxia type 3 (SCA3) is a neurodegenerative disorder characterized by an abnormal expansion of the CAG triplet in the coding region of MJD-1/ATXN3 gene, translating into an expanded polyglutamine tract within the ataxin-3 protein. This abnormally long CAG expansion, confers a toxic gain of function to the ataxin-3 protein that through multiple pathogenic mechanisms leads to neurodegeneration in several brain regions. Current therapeutic approaches consist mainly in the use of physiotherapy and in the pharmacological alleviation of specific symptoms, thus encouraging further investigation towards possible therapeutic approaches.The several existing models of MJD have been useful tools that largely contributed to the identification of intrinsic pathways affecting the disease as well as the validation of new therapies. However, transgenic mouse models are expensive, take long periods of time to develop a phenotype, or do not recapitulate some of the hallmarks of this disease. Our group was pioneer in developing cost-effective lentiviral-based rodent models of MJD. Despite the numerous advantages of this model, it involves craniotomy, in situ injection in the brain parenchyma and the pathology is only confined to the local of injection. In light of these evidences, there is an urgent need for new mouse models that widely express mutant ataxin-3 throughout the Central Nervous System (CNS), potentially providing new insights into the disease mechanisms and allowing screening of novel therapies.The rapidly expanding Adeno-Associated Virus (AAV) vector toolkit has become one of the most promising viral vectors delivering genetic cargo to a wide range of cell types through different routes of administration. In the present work, we aimed to develop new strategies based on the use of mosaic rAAV vectors, to generate in vitro and in vivo models of MJD. For that purpose, mosaic vectors AAV1/2, and AAV2/9 encoding the mutant human ataxin-3 have been developed to efficiently target and transduce the CNS. This approach provided physiologically relevant, time-effective, and cost-effective models for MJD, circumventing some of the limitations of above-mentioned models.In summary, this study provides compelling evidence that the generated mosaic rAAVs are able to efficiently transduce the CNS upon intracranial (AAV1/2) or intravenous injection (AAV2/9), and overexpress full-length mutant ataxin-3 both in vitro and in vivo, recapitulating some of the hallmarks of MJD.
FCT
Outro - American Portuguese Biomedical Research Fund (APBRF)
Outro - BrainHealth2020 (CENTRO-01-0145-FEDER-000008)
Outro - CortaCAGs (POCI-01-0145-FEDER-016719)
Outro - H2020 da União Europeia, GA No. 643417
Outro - “National Ataxia Foundation” (USA)
Outro - POCI-01-0145-FEDER-007440
Outro - Richard Chin and Lily Lock Machado Joseph Disease Research Fund
Outro - ViraVector (CENTRO-01-0145-FEDER- 022095)