Estudo de candidatos a biomarcadores moleculares de prognóstico em carcinoma renal de células claras / Study of molecular biomarker candidates for prognosis in clear cell renal carcinoma

O carcinoma de células renais (CCR) é o tumor mais agressivo que afeta o rim de pessoas adultas. O CCR é uma doença heterogênea, com diferentes alterações moleculares e variados patrões histológicos e clínicos que apresentam evolução diferente. Atualmente apenas variáveis anatomopatológicas clássicas são utilizadas para determinar o prognóstico dos pacientes. Utilizando uma plataforma de microarranjos de DNA, nosso grupo identificou em um trabalho anterior um conjunto de genes que se encontram diferencialmente expressos em tumores de rim. Neste estudo, nove candidatos foram selecionados para avaliação como marcadores de prognóstico no CCR. Foi confirmada a alteração na expressão dos genes ARNTL, ACTN4 e EPAS1 (p < 0,05) em amostras tumorais de CCR através de PCR em tempo real. Adicionalmente, foi observada a alteração da expressão dos genes ARNTL, EPAS1 e CASP7 em linhagens celulares imortalizadas derivadas de tumores renais, recapitulando por tanto, as alterações observadas nos tumores obtidos de pacientes. Posteriormente investigamos o padrão de expressão proteica destes candidatos por imunohistoquímica utilizando microarranjos de tecidos. Foi detectada a diminuição significativa (p < 0,05) da expressão das proteínas ACTN4, ARNTL, CASP7 e EPAS1 em tumores de pacientes com CCR relativamente ao tecido renal não tumoral. Além disso, foi possível determinar valores de imunomarcação capazes de estratificar pacientes com CCR em diferentes grupos de risco quanto à sobrevida câncer-específica, que adicionalmente apresentaram associação significativa com parâmetros anatomopatológicos utilizados na clínica. As imunomarcações de ACTN4, ARNTL, e EPAS1 se mostraram parâmetros independentes de prognóstico de sobrevida dos pacientes. A imunomarcação de CASP7 foi capaz de identificar subgrupos de pacientes com pior prognóstico dentro de um conjunto de pacientes de baixo risco em função do estadio clinico, além de identificar pacientes com menor risco de morte pelo câncer entre aqueles apresentaram recorrência em até 5 após a cirurgia. O conjunto de resultados obtidos aponta para um novo conjunto de biomarcadores moleculares com potencial relevância para auxiliar no prognóstico de pacientes com carcinoma de células renais

The renal cell carcinoma (RCC) is the most aggressive tumor that affects the kidney in adult people. The RCC is a heterogeneous disease, with many different molecular alterations and varied histological and clinical patterns with different outcome. Currently, only classic anatomopathological variables are used to determine patients' prognosis. Using a DNA microarray platform, our group identified in a previous work a set of genes differentially expressed in renal tumors. In this study, nine candidates were selected for evaluation as prognostic biomarkers in RCC. Alteration of the gene expression in RCC tumor samples was confirmed for ARNTL, ACTN4 and EPAS1 (p < 0.05) by real time PCR. Additionally, gene expression changes of ARNTL, EPAS1 and CASP7 were also observed in immortalized cell lines derived from renal tumors, recapitulating the expression changes detected in the patients' tumors. Next, we used tissue microarrays to investigate the protein expression of the selected candidates by immunohistochemistry. Expression of the proteins ACTN4, ARNTL, CASP7 and EPAS1 was detected as significantly downregulated (p < 0.05) in patients´ tumors relative to non-tumor renal tissue. Furthermore, immunostaining patterns of the selected candidates were able to stratify patients with RCC in different risk groups according to cancer-specific survival, which also showed significant associations with anatomopathological parameters used in the clinics. ACTN4, ARNTL and EPAS1 immunostaining resulted as independent prognostic parameters of patient survival. CASP7 immunostaining was able to identify subgroups of patients with worse prognosis in a set of low risk patients as determined by their clinical stage, and also identified patients with lower risk of death from cancer amongst patients that relapsed within 5 years after surgery. Overall, these results point to a new set of molecular biomarkers with potential relevance to help in the prognosis of patients with renal cell carcinoma

Mutations of ACTN4 and SYNPO genes promoter in primary focal segmental glomerulosclerosis / 中华肾脏病杂志

Objective To investigate the mutations ACTN4 and SYNPO genes promoter in sporadic primary focal segmental glomerulosclerosis (FSGS) and to analyze the role of mutations in FSGS. Methods The study consisted of 82 Chinese primary FSGS, including 39 females and 43 males, ranged from 12 to 76 years old. Seventy volunteers were selected as healthy control group. Genomie DNA was extracted from peripheral blood cells of FSGS patients and hair of patients' parents by polymerase chain reaction (PCR) and direct sequencing to analyze ACTN4 and SYNPO gene promoter mutations. Mutations were matched with GenBank and TRANSFAC software database (www.ncbi.nlm.nih.gov; www.genometix.de; www.gene-regulation, corn). Dual luciferase assay system was used to analyze the promoter region mutations, based on PGL3-Basie vector, pRL-SV40 and PCI2 cell line. Hair DNA of novel mutation patients' parents was sequenced. Expression of alpha-actinin-4 and synaptopodin in patients' kidney tissue was examined by immunofluorescence. Results Three patients with 1-34C>T, 1-590delA and (1-1044delT)+ (I-797T >C) +(1-769A >G) heterozygous mutations were found in ACTN4 gene promoter respectively, and two patients with 1-24G>A and 1-851C>T heterozygous mutations in SYNPO gene promoter respectively. The same mutations were not found in the control group of 70 healthy people. Except one patient accepting her parents' 1-1044delT and 1-797T>C mutated chromosome respectively, no same mutations were found in patients' parents. Protein expression of alpha-actinin-4 and synaptopodin was reduced in mutated patients' kidneys. Except 1-1044delT group, luciferase activity in mutated groups decreased. (1-1044delT)+(1-797T>C)+(1-769A>G) mutation was associated with poor outcome and patient with these mutations progressed to end-stage renal failure. Conclusion Mutations of ACTN4 and SYNPO gene promoters affect gene transcription and protein translation, which may contribute to the onset of sporadic primary FSGS.

Mutations of podocyte molecules in patients with late onset familial focal segmental glomerular sclerosis / 中华肾脏病杂志

Objective To investigate the mutations of pedocyte molecules in patients with late onset familial focal segmental glomerular sclerosis (FSGS). Methods Thirty-one pedigrees of late onset familial FSGS in Department of Nephrology, Shanghai Ruijin Hospital from Sep 1997 to Oct 2007 were enrolled in this study. The diagnosis standard of familial FSGS was as follows:(1) the age of presentation was more than 12 years old. (2) in one pedigree, two or more individuals were proven as FSGS by renal biopsy, or at least one was proven to be FSGS by renal biopsy, the others presented renal insufficiency or pmteinuria without precise causes. One hundred unrelated healthy people were screened as control group. Genomic DNA extracted from peripheral blood cells were amplified by PCR and then sequenced for mutations of NPHS2, ACTN4 and TRPC6. Results A novel missense heterozygotic mutation L316P of ACTN4 was identified inone pedigree. The mean onset age of the affected members of this pedigree was (38.7±7.4) years old and their kidney injury progress was slow. Proteinuria of the proband's brother was not improved by immunosuppressor. All 3 affected members of this family had such heterozygotic mutation. A novel missense heterozygotic mutation Q889K of TRPC6 was found in another pedigree. The mean onset age of the affected members in this pedigree was (38.0±4.2) years old. Three members presenting renal disease in this family all had such heterozygotic mutation but with different clinical manifestations. A quiescent mutation G467G of TRPC6 was also identified. Above variants were not found in healthy controls. No NPHS2 mutation was found to cause familial FSGS in these pedigrees. Conclusions A novel mutation L316P of ACTN4 and a new mutation Q889K of TRPC6 are identified in Chinese patients of late onset familial FSGS. No NPHS2 mutation is found to induce FSGS in these pedigrees.