RESUMO
BACKGROUND: Non-syndromic autosomal recessive Retinitis Pigmentosa (arRP) is a highly heterogeneous genetic visual disorder with a large number of causative genes. We aimed to determine the power of Whole Exome Sequencing (WES) in the identification of the genes responsible for non-syndromic arRP among Iranian patients. METHODS: We used WES, followed by the Sanger sequencing to identify the underlying gene mutations causing non-syndromic arRP. RESULTS: Our study revealed disease-causing mutations in known arRP genes for 10 of the 13 families studied (76.9%). These mutations included two-frameshift insertion/deletion in CRB1 and ABCA4, one splicing mutation in PDE6B, four missense mutations in RP1, CRB1, PANK2 and IFT140, as well as three stop codon mutations in RDH12, PRCD, and C2orf71. Three remaining families harbored no mutation in previously known RP genes. Of the 10 diseases causing mutations identified among the investigated Iranian patients with non-syndromic arRP, eight variants had not been reported previously. We confirmed segregation of all 10 mutations with disease phenotypes in our studied population. CONCLUSION: This study supports the genetic heterogeneity of non-syndromic arRP in Iranian patients, and provides an opportunity to show the effectiveness of WES in the identification of pathogenic mutations among patients with non-syndromic arRP born to consanguineous parents.
Assuntos
Análise Mutacional de DNA/métodos , Exoma/genética , Retinose Pigmentar/classificação , Retinose Pigmentar/genética , Transportadores de Cassetes de Ligação de ATP/genética , Adolescente , Adulto , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Proteínas do Olho/genética , Feminino , Homozigoto , Humanos , Irã (Geográfico) , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Linhagem , Fenótipo , Adulto JovemRESUMO
PURPOSE: To quantitatively assess the superoxide dismutase 1 (SOD1), transforming growth factor, beta 1 (TGF-ß1), and dual-specificity phosphatase 1 (DUSP1) messenger ribonucleic acid (mRNA) expression levels as the main intracellular reactive oxygen species neutralizers, wound healing mediators, and immunomodulators (respectively) in keratoconic (KCN) and non-KCN corneas. METHODS: Total RNA was extracted from normal and keratoconic cultured corneal stromal fibroblasts. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure the relative expression levels of mRNAs of the SOD1, TGF-ß1, and DUSP1 genes. RESULTS: The mRNA expression of TGF-ß1 and DUSP1 was augmented in the KCN corneas (three- and fivefold, respectively; both p<0.05). The KCN and non-KCN samples showed no difference in comparative SOD1 mRNA levels. CONCLUSIONS: This study demonstrated a higher level of DUSP1 and TGF-ß1 expression as known molecules in the inflammatory process. These results may provide new insight into the complex molecular pathways underlying KCN for investigating other inflammatory molecules.
Assuntos
Fosfatase 1 de Especificidade Dupla/genética , Fibroblastos/metabolismo , Ceratocone/genética , RNA Mensageiro/genética , Fator de Crescimento Transformador beta1/genética , Células Cultivadas , Córnea/imunologia , Córnea/patologia , Córnea/cirurgia , Fosfatase 1 de Especificidade Dupla/imunologia , Feminino , Fibroblastos/imunologia , Fibroblastos/patologia , Expressão Gênica , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Inflamação/cirurgia , Ceratocone/imunologia , Ceratocone/patologia , Ceratocone/cirurgia , Ceratoplastia Penetrante , Masculino , RNA Mensageiro/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/genética , Superóxido Dismutase/imunologia , Superóxido Dismutase-1 , Fator de Crescimento Transformador beta1/imunologiaRESUMO
Glioblastoma is the most common and the most lethal primary brain cancer. This malignancy is highly locally invasive, rarely metastatic and resistant to current therapies. Little is known about the distinct molecular biology of glioblastoma multiforme (GBM) in terms of initiation and progression. So far, several molecular mechanisms have been suggested to implicate in GBM development. Homeodomain (HD) transcription factors play central roles in the expression of genomic information in all known eukaryotes. The TGIFX homeobox gene was originally discovered in human adult testes. Our previous study showed implications of TGIFLX in prostate cancer and azoospermia, although the molecular mechanism by which TGIFLX acts is unknown. Moreover, studies reported that HD proteins are involved in normal and abnormal brain developments. We examined the expression pattern of TGIFLX in different human brain tumor cell lines including U87MG, A172, Daoy and 1321N1. Interestingly, real time RT-PCR and western blot analysis revealed a high level of TGIFLX expression in A172 cells but not in the other cell lines. We subsequently cloned the entire coding sequence of TGIFLX gene into the pEGFP-N1 vector, eukaryotic expression vector encoding eGFP, and transfected into the U-87 MG cell line. The TGIFLX-GFP expression was confirmed by real time RT-PCR and UV-microscopic analysis. Upon transfection into U87 cells, fusion protein TGIFLX-GFP was found to locate mainly in the nucleus. This is the first report to determine the nuclear localization of TGIFLX and evaluation of its expression level between different brain tumor cell lines. Our data also suggest that TGIFLX gene dysregulation could be involved in the pathogenesis of some human brain tumors.
Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Proteínas de Homeodomínio/genética , Sequência de Bases , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Primers do DNA , Progressão da Doença , Feminino , Glioblastoma/patologia , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The mitochondrial DNA (mtDNA) mutations in mitochondrial coding and non coding regions seem to be important in carcinogenesis. The aim of this investigation was to evaluate coding region (mt-tRNA(Phe) and tRNA(Pro)) and non-coding sequence, mitochondrial displacement loop (mtDNA D-loop), in the cancerous and non-cancerous lesions of Iranian patients with breast cancer (BC). Genomic DNA was extracted from 50 breast tumors and surrounding normal tissue pairs as well as from 50 unrelated normal breast tissues from Iranian Kurdish population. Subsequently, PCR amplification was performed using specific primers, and then PCR products were subjected to direct sequencing. 41 genetic variants were identified in mtDNA D-loop among tumoral and non-tumoral tissues but not in tRNA(Phe) and tRNA(Pro) sequences. Our findings indicated that C182T, 194insT, 285insA and 16342delT were just found in BC tumors whereas 302insC, C309T and C16069T found in both tumors and surrounding normal tissues. Although our findings showed that the observed genetic variations were not restricted to breast cancer tissues, some genetic changes were found only in BC tumors. Our results, in agreement with the evidence from earlier studies, confirm that the mtDNA genetic alterations might be implicated in tumor initiation, progression and development.
Assuntos
Neoplasias da Mama/genética , DNA Mitocondrial/genética , Mutação , Adulto , Sequência de Bases , Primers do DNA , Feminino , Humanos , Irã (Geográfico) , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Glaucoma is a major cause of blindness worldwide. A single nucleotide polymorphism of the MTHFR gene (C677T) has been associated with susceptibility to this disease, although this is controversial in the last decade. In this study, the possible association between the MTHFR C677T polymorphism and the risk of developing primary open angle (POAG) and pseudoexfoliation glaucoma (PEXG) was investigated. For this, a prospective study consisting of 73 POAG, 85 PEXG and 90 matched controls was undertaken in an Iranian population. Genomic DNA was extracted from whole blood. Genotyping of all individuals for the MTHFR C677T polymorphism was conducted using the PCR-RFLP technique. Our findings revealed no significant association between the MTHFR C677T polymorphism in POAG and PEXG compared with controls. Consistent with several other studies, our analysis suggests that the MTHFR C677T polymorphism is unlikely to be a factor contributing to the risk of developing specific forms of glaucoma.
Assuntos
Síndrome de Exfoliação/genética , Glaucoma de Ângulo Aberto/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo de Nucleotídeo Único , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Síndrome de Exfoliação/enzimologia , Síndrome de Exfoliação/epidemiologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Glaucoma de Ângulo Aberto/enzimologia , Glaucoma de Ângulo Aberto/epidemiologia , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase , Estudos Prospectivos , Medição de Risco , Fatores de RiscoRESUMO
PURPOSE: To evaluate mutations in the visual system homeobox gene 1 (VSX1) and superoxide dismutase 1 (SOD1) genes with keratoconus (KTCN), direct sequencing was performed in an Iranian population. METHODS: One hundred and twelve autosomal dominant KTCN patients and fifty-two unaffected individuals from twenty-six Iranian families, as well as one hundred healthy people as controls were enrolled. Genomic DNA was extracted from whole blood sample. Then to study the possible linkage between KTCN and six known loci linkage analysis was performed using 12 short tandem repeat (STR) markers. Also, the entire coding region and intron-exon boundaries of VSX1 and SOD1 were amplified by the PCR technique in each proband. Subsequently, PCR products were subjected to direct sequencing. Co-segregation analysis of the identified mutation was conducted in the family members. An Amplification Refractory Mutation System PCR (ARMS-PCR) was additionally employed for detection of the identified mutation in healthy controls. RESULTS: Linkage analysis of aforementioned loci did not detect evidence for linkage to KTCN. Direct PCR sequencing revealed two single nucleotide polymorphisms (SNPs; g.1502T>G and g.9683C>T), as well as two missense mutations that have been previously reported (R166W and H244R) in VSX1. We also found three undescribed SNPs (g.4886G>A, g.4990C>G, and g.9061T>A) in SOD1. The R166W and H244R mutations were co-segregated in affected family members but not in those that were unaffected. Moreover, the ARMS-PCR strategy did not detect the identified mutations in controls. CONCLUSIONS: Our data suggest a significant association between KTCN patients and VSX1 genetic alterations (p.R166W and p.H244R). Although our findings support VSX1 as a plausible candidate gene responsible for keratoconus, other chromosomal loci and genes could be involved in KTCN development. Taken together, our results suggest that p.R166W and p.H244R could have possible pathogenic influences on KTCN.