RESUMO
Blindness caused by advanced stages of inherited retinal diseases and age-related macular degeneration are characterized by photoreceptor loss. Cell therapy involving replacement with functional photoreceptor-like cells generated from human pluripotent stem cells holds great promise. Here, we generated a human recombinant retina-specific laminin isoform, LN523, and demonstrated the role in promoting the differentiation of human embryonic stem cells into photoreceptor progenitors. This chemically defined and xenogen-free method enables reproducible production of photoreceptor progenitors within 32 days. We observed that the transplantation into rd10 mice were able to protect the host photoreceptor outer nuclear layer (ONL) up to 2 weeks post transplantation as measured by full-field electroretinogram. At 4 weeks post transplantation, the engrafted cells were found to survive, mature, and associate with the host's rod bipolar cells. Visual behavioral assessment using the water maze swimming test demonstrated visual improvement in the cell-transplanted rodents. At 20 weeks post transplantation, the maturing engrafted cells were able to replace the loss of host ONL by extensive association with host bipolar cells and synapses. Post-transplanted rabbit model also provided congruent evidence for synaptic connectivity with the degenerated host retina. The results may pave the way for the development of stem cell-based therapeutics for retina degeneration.
Assuntos
Células-Tronco Pluripotentes , Degeneração Retiniana , Humanos , Camundongos , Animais , Coelhos , Laminina/genética , Retina , Células Fotorreceptoras , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Diferenciação CelularRESUMO
PURPOSE: To examine the retinal and choroidal foveal and parafoveal vasculature in patients with bilateral geographic atrophy (GA) secondary to age-related macular degeneration using optical coherence tomography angiography (OCTA). METHODS: Fourteen eyes from 7 patients with and without fovea-sparing bilateral GA at St. Erik Eye Hospital. All patients were examined by optical coherence tomography angiography, en face OCT and fundus autofluorescence (FAF). Segmented optical coherence tomography angiography flow scans were obtained from the superficial retinal vascular layer (SL) and the choriocapillaris (CC) and correlated with areas of retinal pigment epithelial (RPE) loss on fundus autofluorescence. The foveal avascular zone (FAZ) was measured on superficial retinal vascular layer scans and compared to the GA area of each patient. RESULTS: No significant correlation (r = -0.17, P = 0.58) was found between superficial retinal vascular layer foveal avascular zone (0.49 mm ± 0.23 mm) and GA area (7.36 mm ± 4.36 mm). Absent or severely impaired CC flow was observed inside all GA lesions and to varied extent outside the GA margins including areas of fovea sparing. A high level of symmetry was observed in CC flow between fellow eyes. CONCLUSION: In this cross-sectional study, no relation was found between superficial retinal vascular layer foveal avascular zone and GA area. CC flow inside the GA was severely impaired, whereas CC flow outside the GA correlated poorly with both RPE integrity and visual acuity. Fellow eye symmetry suggests that CC monitoring may be a relevant clinical end point in interventional GA studies.
Assuntos
Angiofluoresceinografia/métodos , Atrofia Geográfica/patologia , Tomografia de Coerência Óptica/métodos , Idoso , Idoso de 80 Anos ou mais , Corioide/diagnóstico por imagem , Corioide/patologia , Estudos Transversais , Feminino , Fóvea Central/diagnóstico por imagem , Fóvea Central/patologia , Atrofia Geográfica/diagnóstico por imagem , Humanos , Degeneração Macular/complicações , Degeneração Macular/patologia , Vasos Retinianos/diagnóstico por imagem , Vasos Retinianos/patologiaRESUMO
Spindle cell lipomas (SCL) are circumscribed, usually s.c. tumors that typically occur on the posterior neck, shoulder, and back of middle aged men. Cytogenetically, almost all SCL are characterized by deletions of chromosome arm 13q, often in combination with loss of 16q. Deletions of 13q are seen also in approximately 15% of conventional lipomas. Through single nucleotide polymorphism (SNP) array analyses, we identified two minimal deleted regions (MDR) in 13q14 in SCL. In MDR1, four genes were located, including the tumor suppressor gene RB1. MDR1 in SCL overlapped with the MDR detected in conventional lipomas with 13q14 deletion. In MDR2 in SCL there were 34 genes and the two microRNA (miRNA) genes miR-15a and miR-16-1. Global gene expression analysis was used to study the impact of the deletions on genes mapping to the two SCL-associated MDR. Five genes (C13orf1, DHRS12, ATP7B, ALG11, and VPS36) in SCL and one gene (C13orf1) in conventional lipomas with 13q-deletions were found to be significantly underexpressed compared with control tissues. Quantitative real-time PCR showed that miR-16-1 was expressed at lower levels in SCL than in the control samples. No mutations were found at sequencing of RB1, miR-15a, and miR-16-1. Our findings further delineate the target region for the 13q deletion in SCL and conventional lipomas and show that the deletions are associated with down-regulated expression of several genes, notably C13orf1, which was the only gene to be significantly down-regulated in both tumor types.
Assuntos
Transtornos Cromossômicos/genética , Lipoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Deleção Cromossômica , Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 13/genética , Expressão Gênica , Genes Supressores de Tumor , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Proteína do Retinoblastoma/genética , Deleção de Sequência/genéticaRESUMO
Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here, we conduct single-cell transcriptomic profiling of an hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, whereby cells rapidly acquired a rostral embryo patterning signature before converging toward the RPE lineage. At intermediate steps, we identified and examined the potency of an NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.
Assuntos
Células-Tronco Embrionárias Humanas , Degeneração Macular , Animais , Diferenciação Celular/genética , Humanos , Degeneração Macular/genética , Degeneração Macular/terapia , Epitélio Pigmentado da Retina , Pigmentos da RetinaRESUMO
As pluripotent stem cell (PSC)-based reparative cell therapies are reaching the bedside, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical trials using these promising strategies are in development, and treatment for age-related macular degeneration is one of the first that has reached patients. We have previously established a xeno-free and defined differentiation protocol to generate functional human embryonic stem cells (hESCs)-derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole-genome sequencing (WGS) to assess genome stability, single-cell RNA sequencing to ensure cell purity, and biodistribution and tumorigenicity analysis to rule out potential migratory or tumorigenic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro culture or differentiation, and enforces the importance to examine the genome integrity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC-based therapies and the data support safety of the hESC-RPE cells generated through our in vitro differentiation methodology.
Assuntos
Células-Tronco Embrionárias Humanas/metabolismo , Degeneração Macular/terapia , Células-Tronco Pluripotentes/metabolismo , Idoso , Diferenciação Celular , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration. Although the inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging. To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. We show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Finally, these surface markers aided to develop a robust, direct and scalable monolayer differentiation protocol on human recombinant laminin-111 and -521 without the need for manual isolation.
Assuntos
Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Neurônios/metabolismo , Pigmentos da Retina/metabolismo , Animais , Antígeno CD56 , Células-Tronco Embrionárias , Humanos , Laminina/genética , Degeneração Macular/metabolismo , Coelhos , Epitélio Pigmentado da Retina/metabolismoRESUMO
Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.
Assuntos
Células Epiteliais/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Epitélio Pigmentado da Retina/citologia , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Citotoxicidade Imunológica , Xenoenxertos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Imunomodulação , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Linfócitos T/metabolismo , Transativadores/metabolismo , Microglobulina beta-2/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
BACKGROUND: The HMGA2 gene encodes a protein that alters chromatin structure. Deregulation, typically through chromosomal rearrangements, of HMGA2 has an important role in the development of several mesenchymal neoplasms. These rearrangements result in the expression of a truncated protein lacking the acidic C-terminus, a fusion protein consisting of the AT-hook domains encoded by exons 1-3 and parts from another gene, or a full-length protein; loss of binding sites for regulatory microRNA molecules from the 3' untranslated region (UTR) of HMGA2 has been suggested to be a common denominator. METHODS: Seventy adipocytic tumors, representing different morphologic and cytogenetic subgroups, were analyzed by qRT-PCR to study the expression status of HMGA2; 18 of these tumors were further examined by PCR to search for mutations or deletions in the 3'UTR. RESULTS: Type (full-length or truncated) and level of expression varied with morphology and karyotype, with the highest levels in atypical lipomatous tumors and lipomas with rearrangements of 12q13-15 and the lowest in lipomas with 6p- or 13q-rearrangements, hibernomas, spindle cell lipomas and myxoid liposarcomas. All 18 examined tumors showed reduced or absent expression of the entire, or parts of, the 3'UTR, which was not due to mutations at the DNA level. CONCLUSION: In adipocytic tumors with deregulated HMGA2 expression, the 3'UTR is consistently lost, either due to physical disruption of HMGA2 or a shift to production of shorter 3'UTR.
Assuntos
Perfilação da Expressão Gênica/métodos , Rearranjo Gênico , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Lipoma/genética , Regiões 3' não Traduzidas/genética , Análise Citogenética , Humanos , Hibridização in Situ Fluorescente , Lipoma/classificação , Lipoma/metabolismo , Lipoma/patologia , Metáfase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.
Assuntos
Atrofia Geográfica/diagnóstico , Células-Tronco Embrionárias Humanas/transplante , Degeneração Macular/terapia , Retina/transplante , Epitélio Pigmentado da Retina/transplante , Animais , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Atrofia Geográfica/patologia , Humanos , Coelhos , Retina/citologia , Epitélio Pigmentado da Retina/citologia , Transplante HeterólogoRESUMO
Purpose: Subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have the capacity to form functional monolayers in naive eyes. We explore hESC-RPE integration when transplanted in suspension to a large-eyed model of geographic atrophy (GA). Methods: Derivation of hESC-RPE was performed in a xeno-free and defined manner. Subretinal bleb injection of PBS or sodium iodate (NaIO3) was used to induce a GA-like phenotype. Suspensions of hESC-RPE were transplanted to the subretinal space of naive or PBS-/NaIO3-treated rabbits using a transvitreal pars plana technique. Integration of hESC-RPE was monitored by multimodal real-time imaging and by immunohistochemistry. Results: Subretinal blebs of PBS or NaIO3 caused different degrees of outer neuroretinal degeneration, RPE hyperautofluorescence, focal RPE loss, and choroidal atrophy; that is, hallmark characteristics of GA. In nonpretreated naive eyes, hESC-RPE integrated as subretinal monolayers with preserved overlying photoreceptors, yet not in areas with outer neuroretinal degeneration and native RPE loss. When transplanted to eyes with PBS-/NaIO3-induced degeneration, hESC-RPE failed to integrate. Conclusions: In a large-eyed preclinical model, subretinal suspension transplants of hESC-RPE did not integrate in areas with GA-like degeneration.
Assuntos
Células Epiteliais/transplante , Atrofia Geográfica/terapia , Células-Tronco Embrionárias Humanas/citologia , Epitélio Pigmentado da Retina/citologia , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Humanos , Injeções Intraoculares , CoelhosRESUMO
Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.
Assuntos
Diferenciação Celular/fisiologia , Atrofia Geográfica/fisiopatologia , Células-Tronco Embrionárias Humanas/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultura/química , Meios de Cultura/farmacologia , Modelos Animais de Doenças , Atrofia Geográfica/terapia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/transplante , Humanos , Laminina/metabolismo , Microscopia Confocal , Coelhos , Epitélio Pigmentado da Retina/citologia , Transplante de Células-Tronco/métodos , Imagem com Lapso de Tempo , Transplante Heterólogo , Xenobióticos/química , Xenobióticos/farmacologiaRESUMO
PURPOSE: To analyze the morphologic effects of subretinal blebs in rabbits using real-time imaging by spectral-domain optical coherence tomography (SD-OCT), infrared-confocal scanning laser ophthalmoscopy (IR-cSLO), and blue-light fundus autofluorescence (BAF). METHODS: Subretinal blebs of PBS or balanced salt solution (BSS) were induced in albino or pigmented rabbits using a transvitreal pars plana technique. Spectral-domain optical coherence tomography, IR-cSLO, and BAF were done at multiple intervals for up to 12 weeks after subretinal bleb injection. The morphologic effects were compared with histologic analysis on hematoxylin-eosin-stained sections of the neurosensory retina and on flat-mounts of phalloidin-labeled RPE. RESULTS: Scans of SD-OCT of the normal rabbit posterior segment revealed 11 bands including six layers of the photoreceptors. Subretinal blebs of PBS or BSS caused acute swelling of the neurosensory retina followed by gradual atrophy. Outer retinal thickness was significantly reduced with pronounced degeneration of all the photoreceptor OCT layers. En face IR-cSLO showed a hyperreflective area corresponding to the progressive photoreceptor degeneration, whereas BAF revealed both hyper- and hypofluorescent changes in the RPE layer. The in vivo results were confirmed by histology and on subretinal flatmounts demonstrating extensive photoreceptor loss and disruption of the RPE mosaic. CONCLUSIONS: Subretinal blebs induce pronounced photoreceptor degeneration and RPE changes in the rabbit as demonstrated by in vivo imaging using SD-OCT, IR-cSLO, and BAF.
Assuntos
Diagnóstico por Imagem , Angiofluoresceinografia/métodos , Oftalmoscopia/métodos , Células Fotorreceptoras de Vertebrados/patologia , Segmento Posterior do Olho/patologia , Degeneração Retiniana/diagnóstico , Tomografia de Coerência Óptica/métodos , Animais , Modelos Animais de Doenças , Fundo de Olho , Coelhos , Reprodutibilidade dos Testes , Degeneração Retiniana/etiologiaRESUMO
Ordinary lipomas are cytogenetically characterized primarily by simple balanced chromosome aberrations with stable morphologies, most of which affect chromosome segment, 12q13-15, where the HMGA2 gene plays a key pathogenetic role. Atypical lipomatous tumors (ALTs) display supernumerary ring or giant marker chromosomes with amplification of several genes including HMGA2 and MDM2. A study of HMGA2 expression in a variety of adipocytic tumors showed aberrant expression in lipomas with 12q13-15 aberrations and ring chromosomes as well as in ALTs and well-differentiated liposarcomas (WDLSs), and frequent differential expression of HMGA2 exons 1-2 versus that of exons 4-5. A minor subset of adipocytic tumors harbors unbalanced karyotypes with extra copies of 12q sequences in structures that are not giant marker or ring chromosomes. Out of a series of ten such tumors, three lipomas and four ALTs with more than two copies of 12q13-15 and breakpoints in 12q13-15 could be analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) to find out whether HMGA2 and MDM2 expression was more similar to the levels seen in lipomas with cytogenetically balanced aberrations of 12q13-15, or to ALTs with giant ring or marker chromosomes. One of two ALTs with more complex, hyperdiploid karyotypes had expression levels closer to those seen in ALT, whereas the remaining six cases were similar to lipomas with 12q13-15 changes and ring chromosomes. Differential expression was seen in two ALTs and all three lipomas. Two cases showed MDM2 expression levels similar to those found among WDLSs, two cases showed levels similar to those found among lipomas, whereas the remaining three cases displayed intermediate expression levels. The studied cases represent intermediates between lipoma and ALT, insofar as they shared 12q13-15 rearrangements and karyotypic stability with lipomas and gain of 12q sequences with ALTs. Neither of these characteristics can be used to discriminate between lipoma and ALT.
Assuntos
Cromossomos Humanos Par 12 , Proteína HMGA2/biossíntese , Lipoma/metabolismo , Neoplasias Lipomatosas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Citogenética , Feminino , Proteína HMGA2/genética , Humanos , Hibridização in Situ Fluorescente , Lipoma/genética , Lipoma/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Lipomatosas/genética , Neoplasias Lipomatosas/patologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Low-grade fibromyxoid sarcoma (LGFMS) is a rare, low-grade malignant soft tissue tumor that is often mistaken for either benign or more malignant tumor types. Commonly, this tumor affects young adults and typically arises in the deep proximal extremities or trunk with frequent recurrences and can metastasize to the lungs many years later. Most cases have a recurrent balanced translocation involving chromosomes 7 and 16, t(7;16)(q32-34;p11), which leads to the fusion of the FUS and CREB3L2 genes. However, supernumerary ring chromosomes have been identified in a subset of FUS/CREB3L2-positive LGFMS, but it has not yet been formally demonstrated that such ring chromosomes harbor the FUS/CREB3L2 fusion gene. Here, we report the genetic findings of a supernumerary ring chromosome from an LGFMS from a 77-year-old man. Chromosome banding analysis revealed a supernumerary ring chromosome, and further studies with fluorescence in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the ring contained material from chromosomes 7 and 16, that the FUS gene was present in two rearranged copies, and that it expressed the FUS/CREB3L2 fusion gene. Moreover, an assessment of previously reported cases showed that tumors with ring chromosomes relapsed more often than tumors with a balanced t(7;16), suggesting that ring formation in LGFMS is correlated with tumor progression.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Fibrossarcoma/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Proteína FUS de Ligação a RNA/genética , Cromossomos em Anel , Idoso , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 7/genética , Fibrossarcoma/patologia , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Neoplasias Pulmonares/patologia , Masculino , Translocação Genética/genéticaRESUMO
Lipoblastoma is a rare benign tumor that arises from embryonic adipose tissue and usually occurs in young children. Here, we present a review of available cytogenetic data and the karyotypes of 10 new cases of lipoblastoma, of which 7 could be studied further by fluorescence in situ hybridization (FISH) with regard to the involvement of the PLAG1 gene. All seven tumors with clonal aberrations harbored breakpoints in 8q11 approximately q13, in agreement with literature data. Including previously published cases, 33/40 (82%) lipoblastomas had rearrangement of the 8q11 approximately q13 region. These rearrangements target the PLAG1 gene, which becomes upregulated through promoter swapping. FISH revealed that five of seven cases in our series had a rearrangement of the PLAG1 gene. Occasionally, there can be difficulties in distinguishing a lipoblastoma from a conventional lipoma or a myxoid liposarcoma. As 8q11 approximately q13 rearrangements have been reported in only 3% of conventional lipomas and never in myxoid liposarcoma, cytogenetic analysis or FISH for the PLAG1 gene can provide useful differential diagnostic information.
Assuntos
Aberrações Cromossômicas , Lipoma/genética , Técnicas de Diagnóstico Molecular , Parede Abdominal/patologia , Nádegas/patologia , Pré-Escolar , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 5 , Cromossomos Humanos Par 8 , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Lactente , Cariotipagem , Masculino , Coxa da Perna/patologia , Parede Torácica/patologiaRESUMO
Conventional lipomas harbor karyotypic changes that could be subdivided into four, usually mutually exclusive, categories: rearrangement, in particular through translocations, of chromosome bands 12q13-15, resulting in deregulation of the HMGA2 gene, loss of material from or rearrangement of chromosome 13, supernumerary ring or giant marker chromosomes, and aberrations of chromosome band 6p21. In the present study, 272 conventional lipomas, two-thirds of them deep-seated, with acquired clonal chromosome changes were assessed with regard to karyotypic and clinical features. A nonrandom distribution of breakpoints and imbalances could be confirmed, with 83% of the cases harboring one or more of the previously known cytogenetic hallmarks. Correlation with clinical features revealed that lipomas with rings/giant markers were larger, occurred in older patients, were more often deep-seated, and seemed to have an increased tendency to recur locally, compared with tumors with other chromosome aberrations. The possible involvement of the HMGA2 gene in cases that did not show any of the characteristic cytogenetic changes was further evaluated by locus-specific metaphase fluorescence in situ hybridization (FISH) and RT-PCR, revealing infrequent cryptic disruption of the gene but abundant expression of full length or truncated transcripts. By FISH, we could also show that breakpoints in bands 10q22-23 do not affect the MYST4 gene, whereas breakpoints in 6p21 or 8q11-12 occasionally target the HMGA1 or PLAG1 genes, respectively, also in conventional lipomas.