RESUMO
Retinoblastoma (RB) is a childhood retinal neoplasm and commonly treated with cytotoxic chemotherapeutic agents. However, these therapeutic approaches often lead to diverse adverse effects. A precise molecular therapy will alleviate these side effects and offer better treatment outcomes. Over the years, kinases have become potential drug targets in cancer therapy. Hence, we aimed to investigate genetic alterations of putative kinase drug targets in RB. Targeted exome sequencing was performed on 35 RB tumors with paired blood samples using a gene panel consisting of 29 FDA-approved kinase genes. Single nucleotide variants were analyzed for pathogenicity using an in-house pipeline and copy number variations (CNVs) were detected by a depth of coverage and CNVPanelizer. The correlation between genetic changes and clinicopathological features was assessed using GraphPad Prism. Three somatic mutations, two in ERBB4 and one in EGFR were identified. Two of these mutations (ERBB4 c.C3836A & EGFR c.A1196T) were not reported earlier. CNV analysis revealed recurrent gains of ALK, MAP2K2, SRC, STK11, and FGFR3 as well as frequent losses of ATM, PI3KCA and ERBB4. Notably, nonresponsive tumors had a higher incidence of amplifications in clinically actionable genes such as ALK. Moreover, ALK gain and ATM loss were strongly correlated with optic nerve head invasion. In conclusion, our study revealed genetic alterations of druggable kinases in RB, providing preliminary insights for the exploration of kinase-targeted therapy in RB.
Assuntos
Variações do Número de Cópias de DNA , Terapia de Alvo Molecular , Mutação , Retinoblastoma , Humanos , Retinoblastoma/genética , Retinoblastoma/tratamento farmacológico , Retinoblastoma/patologia , Masculino , Feminino , Pré-Escolar , Lactente , Criança , Neoplasias da Retina/genética , Neoplasias da Retina/tratamento farmacológico , Neoplasias da Retina/patologia , Receptor ErbB-4/genética , Sequenciamento do Exoma , Inibidores de Proteínas Quinases/uso terapêutico , Receptores ErbBRESUMO
Purpose: To estimate the prevalence of Leber hereditary optic neuropathy (LHON) along with genetic screening at a tertiary eye care center in southern India. Methods: Patients with LHON were identified at the Neuro-Ophthalmology Clinic, Aravind Eye Hospital (AEH; Madurai, India) from 2015 to 2019. Clinical data were collected along with blood samples. Genetic testing was performed for the confirmation of LHON using a multiplex PCR restriction fragment length polymorphism (RFLP) approach to detect the primary mutations 3460A, 11778A, and 14484C in mitochondrial DNA (mtDNA). Results: During the study period, 1,598,441 outpatients attended AEH of whom 40,527 were referred to the Neuro-Ophthalmology Clinic. Among them, 55 patients were diagnosed with LHON. The male to female ratio was 8.2:1.0, and the mean age at onset was 20.95 years (SD 8.940). The estimated prevalence was 1:737 or 13.57 per 10,000 (95% confidence intervals [CI] 10.23-17.66) at the Neuro-Ophthalmology Clinic. The frequency of primary mutations in the patients with LHON was determined as 43.6% (24/55), giving a prevalence of 1:1689 or 5.92 per 10,000 (95% CI 3.78-8.81). Conclusions: The high prevalence of LHON observed at a single hospital highlights the impact of the disease in southern India. As the epidemiology of LHON remains unexplored in this region, these findings will pave the way to evaluate the national prevalence. Further, screening the whole mitochondrial genome may help to increase the detection of mutations to estimate the accurate prevalence of the disease.
Assuntos
Hospitais , Atrofia Óptica Hereditária de Leber/epidemiologia , Atrofia Óptica Hereditária de Leber/genética , Adolescente , Adulto , Idade de Início , Sequência de Bases , Criança , Pré-Escolar , Feminino , Genoma Mitocondrial , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Atrofia Óptica Hereditária de Leber/diagnóstico por imagem , Polimorfismo de Fragmento de Restrição , Prevalência , Estudos Prospectivos , Sexismo , Tomografia de Coerência Óptica , Adulto JovemAssuntos
Deleção Cromossômica , Cromossomos Humanos Par 13/genética , Doença de Hirschsprung/patologia , Retinoblastoma/patologia , Síndrome de Waardenburg/patologia , Feminino , Doença de Hirschsprung/complicações , Doença de Hirschsprung/genética , Humanos , Lactente , Prognóstico , Retinoblastoma/complicações , Retinoblastoma/genética , Síndrome de Waardenburg/complicações , Síndrome de Waardenburg/genéticaRESUMO
India has the highest number of retinoblastoma (RB) patients among the developing countries owing to its increasing population. Of the patients with RB, about 40% have the heritable form of the disease, making genetic analysis of the RB1 gene an integral part of disease management. However, given the large size of the RB1 gene with its widely dispersed exons and no reported hotspots, genetic testing can be cumbersome. To overcome this problem, we have developed a rapid screening strategy by prioritizing the order of exons to be analyzed, based on the frequency of nonsense mutations, deletions and duplications reported in the RB1-Leiden Open Variation Database and published literature on Indian patients. Using this strategy for genetic analysis, mutations were identified in 76% of patients in half the actual time and one third of the cost. This reduction in time and cost will allow for better risk prediction for siblings and offspring, thereby facilitating genetic counseling for families, especially in developing countries.
Assuntos
Genes do Retinoblastoma , Testes Genéticos , Neoplasias da Retina/diagnóstico , Retinoblastoma/diagnóstico , Pré-Escolar , Análise Custo-Benefício , Análise Mutacional de DNA/economia , Análise Mutacional de DNA/métodos , Feminino , Testes Genéticos/economia , Testes Genéticos/métodos , Humanos , Índia , Lactente , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase Multiplex/economia , Neoplasias da Retina/economia , Neoplasias da Retina/genética , Retinoblastoma/economia , Retinoblastoma/genética , Fatores de TempoRESUMO
BACKGROUND: The spectrum of RB1gene mutations in Retinoblastoma (RB) patients and the necessity of multiple traditional methods for complete variant analysis make the molecular diagnosis a cumbersome, labor-intensive and time-consuming process. Here, we have used targeted next generation sequencing (NGS) approach with in-house analysis pipeline to explore its potential for the molecular diagnosis of RB. METHODS: Thirty-three patients with RB and their family members were selected randomly. DNA from patient blood and/or tumor was used for RB1 gene targeted sequencing. The raw reads were obtained from Illumina Miseq. An in-house bioinformatics pipeline was developed to detect both single nucleotide variants (SNVs) and small insertions/deletions (InDels) and to distinguish between somatic and germline mutations. In addition, ExomeCNV and Cn. MOPS were used to detect copy number variations (CNVs). The pathogenic variants were identified with stringent criteria, and were further confirmed by conventional methods and cosegregation in families. RESULTS: Using our approach, an array of pathogenic variants including SNVs, InDels and CNVs were detected in 85% of patients. Among the variants detected, 63% were germline and 37% were somatic. Interestingly, nine novel pathogenic variants (33%) were also detected in our study. CONCLUSIONS: We demonstrated for the first time that targeted NGS is an efficient approach for the identification of wide spectrum of pathogenic variants in RB patients. This study is helpful for the molecular diagnosis of RB in a comprehensive and time-efficient manner.
Assuntos
Variações do Número de Cópias de DNA/genética , Patologia Molecular , Proteína do Retinoblastoma/genética , Retinoblastoma/genética , Criança , Pré-Escolar , Biologia Computacional , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Retinoblastoma/patologiaRESUMO
OBJECTIVES: To analyse the profile and implication of genetic testing in a cohort of retinoblastoma (RB) patients and their families conducted on a single day during World Retinoblastoma Awareness Week 2017. METHODS: Retrospective analysis of blood samples were collected from 411 subjects, including 113 probands at a camp organised for RB awareness and were analysed for RB1 mutations by Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). If germline mutations were detected, the parents and siblings of the proband were tested for the same mutation. RESULTS: Germline RB1 mutations were identified in 61/113(54%) probands with a mutation detection rate of 96% (47/49) and 22% (14/64) for bilateral and unilateral RB, respectively. Ten novel pathogenic mutations were identified. Splice mutation was most common (31%) followed by nonsense mutation (26%). The mean age at RB diagnosis was significantly lower in patients having germline RB1 mutation (mean 10.7 months ±2.5) compared to those without (mean 27.2 months ±6.5) (p = <0.0001). Parental transmission of the mutant allele was detected in 15/61(25%) cases of which 11(18%) parents were unaffected indicating incomplete penetrance. The origin of the variant allele was both paternal (n = 7) and maternal (n = 4) wherein 5 were bilateral and 6 unilateral. CONCLUSIONS: The detection of a germline mutation impacts the proband and family members due to its implications on change in prognosis, frequency of subsequent evaluations, screening for ocular and non-ocular cancers, and surveillance of family and future progeny.
Assuntos
Testes Genéticos , Mutação em Linhagem Germinativa , Neoplasias da Retina , Proteínas de Ligação a Retinoblastoma , Retinoblastoma , Humanos , Retinoblastoma/genética , Retinoblastoma/sangue , Masculino , Feminino , Estudos Retrospectivos , Neoplasias da Retina/genética , Neoplasias da Retina/diagnóstico , Neoplasias da Retina/sangue , Índia/epidemiologia , Lactente , Testes Genéticos/métodos , Proteínas de Ligação a Retinoblastoma/genética , Pré-Escolar , Ubiquitina-Proteína Ligases/genética , Linhagem , Análise Mutacional de DNARESUMO
Retinoblastoma (RB) is a pediatric cancer of the eye that occurs in 1/15000 live births worldwide. Albeit RB is initiated by the inactivation of RB1 gene, the disease progression relies largely on transcriptional alterations. Therefore, evaluating gene expression is vital to unveil the therapeutic targets in RB management. In this study, we employed an RT2 Profiler™ PCR array for a focused analysis of 84 cancer-specific genes in RB. An interaction network was built with gene expression data to identify the dysregulated pathways in RB. The key transcript alterations identified in 13 tumors by RT2 Profiler™ PCR array was further validated in 15 tumors by independent RT-qPCR. Out of 84 cancer-specific genes, 68 were dysregulated in RB tumors. Among the 68 genes, 23 were chosen for further analysis based on statistical significance and abundance across multiple tumors. Pathway analysis of altered genes showed the frequent perturbations of cell cycle, angiogenesis and apoptotic pathways in RB. Notably, upregulation of MCM2, MKI67, PGF, WEE1, CDC20 and downregulation of COX5A were found in all the tumors. Western blot confirmed the dysregulation of identified targets at protein levels as well. These alterations were more prominent in invasive RB, correlating with the disease pathogenesis. Our molecular analysis thus identified the potential therapeutic targets for improving retinoblastoma treatment. We also suggest that PCR array can be used as a tool for rapid and cost-effective gene expression analysis.
Assuntos
Neoplasias da Retina , Retinoblastoma , Retinoblastoma/genética , Retinoblastoma/patologia , Retinoblastoma/metabolismo , Humanos , Neoplasias da Retina/genética , Neoplasias da Retina/patologia , Neoplasias da Retina/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão GênicaRESUMO
OBJECTIVE: To determine the association between the parental age gap and the absolute parental age with the risk of retinoblastoma (RB) development in an offspring. METHODS: RB individuals diagnosed between March 2013 and December 2019 in a single tertiary eye care centre were included. We recorded the demographic data, parental age and RB1 gene mutation status in the patient's tumour, blood and the parental blood. We categorised RB1 mutation inheritance as sporadic RB with somatic mutations (only present in tumour), heritable RB with de novo (present in patient's blood) and familial (present in patient and parents' blood) germline mutations. The statistical significance was confirmed by Fisher's exact/Chi-square test. RESULTS: Out of 259 RB patients, 247 were included in our study. Heritable RB with de novo germline mutations was significantly less common (p value: 0.0387; 95% CI: 0.2676-0.9329) and sporadic RB with somatic mutations was more common (p value: 0.0545; 95% CI: 1.025-3.39), if the parental age gap was <10 years. There were increased odds of a heritable RB with de novo germline mutation with an increase in paternal age and this was more intensified when combined with parental age gap of more than ≥10 years. The heritable RB with de novo germline mutations significantly increased as maternal age progressed, only when it was adjusted to ≥10 years parental age gap (p value: 0.0262; 95% CI: 1.26-17.91). CONCLUSIONS: An increased parental age gap and increased paternal age are independent risk factors for the development of heritable RB with de novo germline mutation.
Assuntos
Neoplasias da Retina , Retinoblastoma , Criança , Demografia , Humanos , Mutação , Pais , Neoplasias da Retina/diagnóstico , Neoplasias da Retina/epidemiologia , Neoplasias da Retina/genética , Retinoblastoma/diagnóstico , Retinoblastoma/epidemiologia , Retinoblastoma/genética , Estudos RetrospectivosRESUMO
BACKGROUND: The diagnosis of retinal dystrophies can be challenging due to the spectrum of protean phenotypic manifestations. This study employed trio-whole-exome sequencing (trio-WES) to unveil the genetic cause of an inherited retinal disorder in a south Indian family. MATERIALS AND METHODS: Proband's initial ophthalmic examinations was performed in the year 2016. WES was performed on a proband-parent trio to identify causative mutation followed by Sanger validation, segregation analysis, sequence and structure-based computational analysis to assess its pathogenicity. Based on the genetic findings, detailed clinical reassessments were performed in year 2020 for the proband and available family members. RESULTS: WES revealed a novel homozygous BEST1 mutation c.G310A (p.D104N) in the proband and heterozygous for the parents, indicating autosomal recessive inheritance. Segregation analysis showed heterozygous mutation in maternal grandfather and normal genotype for younger brother and maternal grandmother. Moreover, the structure-based analysis revealed the mutation p.D104N in the cytoplasmic domain, causing structural hindrance by altering hydrogen bonds and destabilizing the BEST1 protein structure. Proband's clinical assessments were consistent with autosomal recessive bestrophinopathy (ARB) phenotype. Additionally, characteristic absent light rise and decreased light peak-to-dark trough ratio (LP:DT) was observed bilaterally in EOG. CONCLUSIONS: Our study demonstrates the utility of WES and clinical re-evaluations in establishing the precise diagnosis of autosomal recessive bestrophinopathy associated with a novel mutation, thus expanding the BEST1-related mutation spectrum.
Assuntos
Anormalidades do Olho , Distrofias Retinianas , Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina , Bestrofinas/genética , Canais de Cloreto/genética , Eletrorretinografia , Oftalmopatias Hereditárias , Proteínas do Olho/genética , Humanos , Masculino , Mutação , Linhagem , Fenótipo , Doenças Retinianas , Sequenciamento do ExomaRESUMO
The tumor microenvironment (TME) constitutes multiple cell types including cancerous and non-cancerous cells. The intercellular communication between these cells through TME derived exosomes may either enhance or suppress the tumorigenic processes. The tumor-derived exosomes could convert an anti-tumor environment into a pro-tumor environment by inducing the differentiation of stromal cells into tumor-associated cells. The exosomes from tumor-associated stromal cells reciprocally trigger epithelial-to-mesenchymal transition (EMT) in tumor cells, which impose therapeutic resistance and metastasis. It is well known that these exosomes contain the signals of EMT, but how these signals execute chemoresistance and metastasis in tumors remains elusive. Understanding the significance and molecular signatures of exosomes transmitting EMT signals would aid in developing appropriate methods of inhibiting them. In this review, we focus on molecular signatures of exosomes that shuttle between cancer cells and their stromal populations in TME to explicate their impact on therapeutic resistance and metastasis through EMT. Especially Wnt signaling is found to be involved in multiple ways of exosomal transport and hence we decipher the biomolecules of Wnt signaling trafficked through exosomes and their potential in serving as therapeutic targets.
Assuntos
Transição Epitelial-Mesenquimal , Exossomos/patologia , Neoplasias/patologia , Microambiente Tumoral , Animais , Antineoplásicos/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Comunicação Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Humanos , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Purpose: This study is aimed to investigate the presence of Human papillomavirus (HPV) DNA in tumors obtained from sporadic retinoblastoma patients. Methods: One hundred six tumor tissues obtained from sporadic RB patients were analyzed for HPV infection by use of both seminested PCR and real-time quantitative PCR. Results: Of 106 RB patients, 55 were male and 51 were female. The mean age at diagnosis was 26.77 ± 15.36 (mean ± Std. dev) months. Almost all patients presented with leukocoria. Molecular investigation by different methods revealed no HPV positivity in any tumor genome. Conclusion: Our study demonstrates no association between HPV and RB, postulating HPV may not be a major risk factor in the etiology of RB.
Assuntos
Alphapapillomavirus , Infecções por Papillomavirus , Neoplasias da Retina , Retinoblastoma , Feminino , Humanos , Índia/epidemiologia , Masculino , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias da Retina/diagnóstico , Neoplasias da Retina/epidemiologia , Retinoblastoma/diagnóstico , Retinoblastoma/epidemiologiaRESUMO
BACKGROUND: Leber congenital amaurosis (LCA), primarily characterized by retinal degeneration is the most severe form of inherited retinal dystrophy (IRD) responsible for congenital blindness. The presence of phenotypic heterogeneity makes the diagnosis of LCA challenging, especially in the absence of pronounced disease pathognomonic, yet it can be well comprehended by employing molecular diagnosis. Therefore, the present study aimed to reveal the causative mutations in ten LCA patients with variable phenotypes using clinical exome sequencing (CES). METHODS: CES was performed in ten unrelated LCA patients. Ophthalmic information and family history of all patients were obtained to make a meaningful interpretation. The clinical exome data was analyzed and prioritized using a bioinformatics pipeline to identify mutations, which was further validated by Sanger sequencing. Segregation analysis was also performed on available family members. RESULTS: CES led to the identification of causative mutations in nine LCA patients. Seven patients harbored a mutation in six LCA candidate genes, including RPE65, LCA5 (n = 2), CRX, PRPH2, CEP290, and ALMS1, while two patients possess a mutation in IFT80 and RP1, known to cause other diseases. Three novel mutations in LCA5 (c.1823del), CRX (c.848del) and CEP290 (c.2483G > T) were identified. The current study reports for the first time, a mutation in PRPH2, CEP290, and ALMS1 from the Indian population. Additionally, we observed a novel association of LCA phenotype with IFT80 known to cause Jeune syndrome. Based on the genetic finding, the patient AS09, who harbored a mutation in the RP1 gene, was re-diagnosed with early-onset retinitis pigmentosa. CONCLUSION: In conclusion, the results underline the importance of CES in clinically diagnosed LCA patients with variable phenotypes. The correlation between mutations in candidate genes and clinical phenotypes, helps to refine the clinical diagnosis. However, molecular evaluation with a larger cohort of LCA patients is needed for better understanding of the mutational spectrum in southern India.
RESUMO
Background: Gyrate Atrophy (GA) is a rare autosomal recessive disorder characterized by progressive chorioretinal degeneration. It is caused due to mutations in OAT gene that encodes a defective ornithine-δ-aminotransferase enzyme. We aim to identify the molecular cause of the disease and correlate it with the phenotype.Materials and Methods: Clinical, biochemical and genetic analyses were performed in siblings with GA.Case Description: A 10-year-old girl presented with impaired vision was clinically diagnosed to have peripheral chorioretinal degeneration in both eyes due to GA with vitreous hemorrhage in the right eye. Similar chorioretinal degeneration was observed in the patient's sibling, while parents were normal. Biochemical analysis of plasma by LC-MS/MS showed an elevated ornithine level of 892.8 µmol/L in the patient and 572.3 µmol/L in the sibling. Familial genetic screening by Sanger sequencing revealed a nonsense mutation in exon 11 of the OAT gene (c.1192C>T; p.Arg398Ter) in all the family members with a homozygous mutation in the patient and sibling, and heterozygous mutation in the parents. The patient was under follow-up with an arginine-restricted diet. At the last follow-up, the vitreous hemorrhage of right eye had resolved with an improvement in visual acuity and left eye remained stable with 6/12.Conclusion: Our patient is a rare case of gyrate atrophy presented with vitreous hemorrhage and nonsense OAT gene mutation, inherited in the autosomal recessive pattern. This report highlights the phenotypic variability among the siblings with the same mutation in OAT gene for the first time.
Assuntos
Códon sem Sentido/genética , Atrofia Girata/genética , Ornitina-Oxo-Ácido Transaminase/genética , Adolescente , Criança , Cromatografia Líquida , Feminino , Angiofluoresceinografia , Atrofia Girata/diagnóstico , Atrofia Girata/dietoterapia , Humanos , Ornitina-Oxo-Ácido Transaminase/sangue , Linhagem , Fenótipo , Irmãos , Espectrometria de Massas em Tandem , Acuidade Visual/fisiologia , Hemorragia Vítrea/diagnóstico , Hemorragia Vítrea/fisiopatologiaRESUMO
Pseudogenes are duplicated or retrotransposed DNA sequences of native functional genes. Amplification of pseudogenes along with gene of interest often produces false positive results, which is an innate problem in Reverse Transcription-quantitative PCR (RT-qPCR). Selecting a reference gene without any interference from pseudogene amplification is therefore a challenge to overcome. Among the common reference genes used for normalization (ACTB, GAPDH, HPRT1, TUBB, RNA18SN1 and B2M), B2M was found to have no pseudogenes in silico, which has also been confirmed by PCR and RT-qPCR. We also assessed the effect of pseudogenes on the determination of the stability of reference genes through data mining. The phylogenetic analysis of pseudogenes and functional genes revealed high sequence similarity among mammals. In addition, we demonstrated the deduction of pseudogene amplification signal using ValidPrime Assay (VPA) under conditions where genomic DNA contamination could not be avoided. Hence, we recommend the use of pseudo-free reference gene with consistent expression in the samples of interest or use VPA normalization method, where genomic DNA or pseudogene amplification is inevitable.
Assuntos
Perfilação da Expressão Gênica/normas , Pseudogenes , Reação em Cadeia da Polimerase em Tempo Real/normas , Microglobulina beta-2/genética , Animais , Sequência Conservada , Evolução Molecular , Humanos , Padrões de ReferênciaRESUMO
BACKGROUND: Retinoblastoma is a sight and life-threatening embryonal tumor in children. Though chemotherapy is the main mode of therapy, evolving resistance remains a major obstacle in treatment success. The presence of cancer stem cells (CSC) is frequently reported to be responsible for chemoresistance in multiple tumors. OBJECTIVE: Our study aims to identify the molecular factors that facilitate the chemoresistance through cancer stem cells in retinoblastoma. METHODS: We developed etoposide and carboplatin resistant retinoblastoma (Y79) cell lines by stepwise drug increment treatment, validated with MTT and TUNEL assays. Colony forming and invasive ability were studied by soft-agar colony forming and transwell assays, respectively. Similar analysis in non-responsive retinoblastoma tumors were carried out by histopathology. Finally, expression of CSC/neuronal markers and ABC transporters were examined by quantitative PCR and protein expression of neuronal stem cell markers was confirmed by Western blot. RESULTS: Larger colony size of resistant cells in soft-agar assay provided evidence for increased selfrenewability. Histopathology in non-responsive tumors showed poorly differentiated cells predominantly. Besides, both resistant cell lines and non-responsive tumors showed increased invasion with higher expression of neuronal stem cell markers - SOX2, NANOG, OCT4 and ABC transporters - ABCB1 and ABCC3. Increased self-renewal ability and invasion along with overexpression of stemness markers in resistant cells and tumors provide evidence for stemness driving chemoresistance and invasion in retinoblastoma. CONCLUSION: We have demonstrated Neuronal stem cell/CSC markers that facilitate the maintenance of cancer stem cells. Developing therapies targeting these factors will help in overcoming resistance and improving retinoblastoma treatment.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Carboplatina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/uso terapêutico , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Neoplasias da Retina/tratamento farmacológico , Neoplasias da Retina/metabolismo , Retinoblastoma/tratamento farmacológico , Retinoblastoma/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Carboplatina/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Etoposídeo/farmacologia , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteína Homeobox Nanog/metabolismo , Invasividade Neoplásica , Fator 3 de Transcrição de Octâmero , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Fatores de Transcrição SOXB1/metabolismo , Transdução de SinaisRESUMO
Retinoblastoma has an increased inheritance risk of germline RB1 mutations in offspring and siblings, especially twins. Three families, each having one retinoblastoma-affected twin, were selected for genetic analysis and DNA profiling. Germline RB1 mutations were found in all probands. DNA profiling carried on similar-looking twins of families I and II, proved them to be fraternal. This study demonstrates the importance of genetic analysis of RB1 gene for risk prediction in retinoblastoma families. It also emphasizes that DNA profiling is a mandate for genetic screening of families with twins, thus adding a new dimension in counseling of retinoblastoma.
Assuntos
Doenças em Gêmeos , Testes Genéticos/métodos , Neoplasias da Retina/diagnóstico , Retinoblastoma/diagnóstico , DNA/genética , Feminino , Genes do Retinoblastoma/genética , Mutação em Linhagem Germinativa , Humanos , Lactente , Microscopia Acústica , Linhagem , Neoplasias da Retina/genética , Retinoblastoma/genéticaRESUMO
Idiopathic dilated cardiomyopathy (DCM) is a structural heart disease with strong genetic background. The aim of this study was to assess the role of mitochondrial DNA (mtDNA) variations and haplogroups in Indian DCM patients. Whole mtDNA analysis of 221 DCM patients revealed 48 novel, 42 disease-associated and 97 private variations. The frequency of reported variations associated with hearing impairment, DEAF, SNHL and LHON are significantly high in DCM patients than controls. Haplogroups H and HV were over represented in DCM than controls. Functional analysis of two private variations (m.8812A>G & m.10320G>A) showed decrease in mitochondrial functions, suggesting the role of mtDNA variations in DCM.
Assuntos
Cardiomiopatia Dilatada/genética , Variação Genética/genética , Genoma Mitocondrial/genética , Mitocôndrias/genética , Adolescente , Adulto , Idoso , Povo Asiático/genética , Criança , DNA Mitocondrial/genética , Feminino , Perda Auditiva/genética , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
PURPOSE: Aldose reductase (ALR), the first and rate-limiting enzyme involved in polyol pathway plays a central role in diabetes and its related complications, including diabetic retinopathy (DR). Inhibition of ALR may also be an ideal target for reducing the deleterious effects of DR. Therefore, the purpose of the present study was to investigate the protective effect of epalrestat (EPL), ALR inhibitor on glucose-induced toxicity in ARPE-19 cells. METHODS: ARPE-19 cells were challenged with normal glucose (NG, 5 mM) and high glucose (HG1, 25 mM and HG2, 50 mM) in the presence or absence of EPL. ALR and VEGF165 expression in retinal pigment epithelial (RPE) cells under experimental conditions were quantified by real-time polymerase chain reaction using SYBR Green chemistry. Vascular endothelial growth factor (VEGF) secretion in the cell supernatant was measured by Sandwich ELISA. Cytotoxicity of EPL was assessed by MTT assay. ALR inhibitory activity, apoptosis, and sorbitol accumulation were also investigated. RESULTS: EPL at studied concentration did not show any toxicity to RPE cells and showed as maximum as 65% ALR inhibition under high glucose condition (HG1). The presence of EPL significantly reduced ALR expression and VEGF levels as induced by high glucose in ARPE-19 cells. CONCLUSION: Inhibition of ALR appeared to be beneficial in reducing diabetes-related complications in RPE cells under high glucose condition.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glucose/antagonistas & inibidores , Epitélio Pigmentado da Retina/efeitos dos fármacos , Rodanina/análogos & derivados , Tiazolidinas/farmacologia , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Apoptose/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Glucose/toxicidade , Humanos , Epitélio Pigmentado da Retina/patologia , Rodanina/administração & dosagem , Rodanina/farmacologia , Sorbitol/antagonistas & inibidores , Sorbitol/metabolismo , Relação Estrutura-Atividade , Tiazolidinas/administração & dosagem , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Purpose: Leber's hereditary optic neuropathy (LHON; OMIM 535000) is one of the most common maternally inherited mitochondrial disorders. Three mitochondrial DNA point mutations-m.3460G>A (MT-ND1), m.11778G>A (MT-ND4), and m.14484T>C (MT-ND6)-account for the majority of reported LHON cases. Only approximately 50% of males and approximately 10% of females carrying these mutations develop optic neuropathy and blindness. Additional factors, such as mtDNA/nuclear genetic background and environmental modifiers, are likely to contribute toward the observed incomplete penetrance and gender bias. We aimed to investigate whether mtDNA haplogroup influences LHON clinical expression in Indian patients harboring the m.11778G>A mutation. Methods: Detailed clinical assessment and complete mitochondrial genome sequencing was undertaken in 64 LHON families harboring the m.11778G>A mutation. Mitochondrial haplogroup was assigned based on evolutionarily conserved mtDNA variations. Results: A total of 543 individuals (295 male, 248 female) from 64 unrelated families harboring the m.11778G>A mutation were recruited to the study. The overall disease penetrance was 27.07% (146 of 543) and higher in males (37.9%; 112 of 295) than females (13.7%; 34 of 248). The mtDNA haplogroup analysis revealed that all affected probands belonged to different mtDNA haplogroups. No association between the m.11778G>A mutation and the background mtDNA haplogroup was detected. Conclusions: The first detailed study of Indian LHON patients confirm that the m.11778G>A-related LHON in India coexists with multiple different mtDNA haplogroups, unlike the preferential association of west Eurasian haplogroup J and the reported increased clinical penetrance with the J2 subhaplogroup. However, we observed variable penetrance of LHON in different Indian mtDNA haplogroup backgrounds, indicating their possible influence on clinical expression. These data suggest that a similar heterogeneity, resulting from the mtDNA haplogroup, might also exist in other mitochondrial diseases among Indian populations.