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1.
SAR QSAR Environ Res ; 32(7): 595-614, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34148451

RESUMO

The quantitative structure-property relationship (QSPR) method is commonly used to predict different physicochemical characteristics of interest of chemical compounds with an objective to accelerate the process of design and development of novel chemical compounds in the biotechnology and healthcare industries. In the present report, we have employed a QSPR approach to predict the different properties of the aminoglycoside-derived polymers (i.e. polymer DNA binding and aminoglycoside-derived polymers mediated transgene expression). The final QSPR models were obtained using the partial least squares (PLS) regression approach using only specific categories of two-dimensional descriptors and subsequently evaluated considering different internationally accepted validation metrics. The proposed models are robust and non-random, demonstrating excellent predictive ability using test set compounds. We have also developed different kinds of consensus models using several validated individual models to improve the prediction quality for external set compounds. The present findings provide new insight for exploring the design of an aminoglycoside-derived polymer library based on different identified physicochemical properties as well as predict their property before their synthesis.


Assuntos
Aminoglicosídeos/química , DNA/metabolismo , Polímeros/química , Polímeros/farmacologia , Relação Quantitativa Estrutura-Atividade , Expressão Gênica/efeitos dos fármacos , Análise dos Mínimos Quadrados , Polímeros/metabolismo , Transgenes
2.
SAR QSAR Environ Res ; 30(5): 363-382, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31112078

RESUMO

In the current study, we have developed predictive quantitative structure-activity relationship (QSAR) models for cellular response (foetal rate lung fibroblast proliferation) and protein adsorption (fibrinogen adsorption (FA)) on the surface of tyrosine-derived biodegradable polymers designed for tissue engineering purpose using a dataset of 66 and 40 biodegradable polymers, respectively, employing two-dimensional molecular descriptors. Best four individual models have been selected for each of the endpoints. These models are developed using partial least squares regression with a unique combination of six and four descriptors for cellular response and protein adsorption, respectively. The generated models were strictly validated using internal and external metrics to determine the predictive ability and robustness of proposed models. Subsequently, the validated individual models for each response endpoints were used for the generation of 'intelligent' consensus models ( http://teqip.jdvu.ac.in/QSAR_Tools/DTCLab/ ) to improve the quality of predictions for the external data set. These models may help in prediction of virtual polymer libraries for rational design/optimization for properties relevant to biomedical applications prior to their synthesis.


Assuntos
Materiais Biocompatíveis/química , Fibrinogênio/química , Modelos Moleculares , Polímeros/química , Adsorção/efeitos dos fármacos , Materiais Biocompatíveis/farmacologia , Fenômenos Fisiológicos Celulares/efeitos dos fármacos , Análise dos Mínimos Quadrados , Estrutura Molecular , Polímeros/farmacologia , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes
3.
SAR QSAR Environ Res ; 29(12): 935-956, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30392386

RESUMO

The glass transition temperature is a vital property of polymers with a direct impact on their stability. In the present study, we built quantitative structure-property relationship models for the prediction of the glass transition temperatures of polymers using a data set of 206 diverse polymers. Various 2D molecular descriptors were computed from the single repeating units of polymers. We derived five models from different combinations of six descriptors in each case by employing the double cross-validation technique followed by partial least squares regression. The selected models were subsequently validated by methods such as cross-validation, external validation using test set compounds, the Y-randomization (Y-scrambling) test and an applicability domain study of the developed models. All of the models have statistically significant metric values such as r2 ranging from 0.713-0.759, Q2 ranging from 0.662-0.724 and [Formula: see text] ranging 0.702-0.805. Finally, a comparison was made with recently published models, though the previous models were based on a much smaller data set with limited diversity. We also used a true external set to demonstrate the performance of our developed models, which may be used for the prediction and design of novel polymers prior to their synthesis.


Assuntos
Modelos Químicos , Polímeros/química , Temperatura de Transição , Resinas Acrílicas/química , Vidro , Estrutura Molecular , Poliestirenos/química , Polivinil/química , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes
4.
Chem Pharm Bull (Tokyo) ; 48(11): 1771-3, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11086911

RESUMO

A novel beta-glucuronidase inhibiting triterpene (1) has been isolated from the chloroform fraction of Paeonia emodi beta-glucuronidase inhibit established ase chloroform fraction of Pa 11,beta,5alpha,23,24-pentahydroxy-30-norolean-12,20(29)-d ien-28-oic acid on the basis of spectroscopic analysis, including high resolution mass spectroscopy, one and two-dimensional NMR techniques. Oleanolic acid, betulinic acid, ethyl gallate, methyl grevillate and 1,5-dihydroxy-3-methylanthraquinone are also reported for the first time from Paeonia emodi.


Assuntos
Inibidores Enzimáticos/isolamento & purificação , Glucuronidase/antagonistas & inibidores , Plantas Medicinais/química , Triterpenos/síntese química , Triterpenos/isolamento & purificação , Acetilação , Ásia , Inibidores Enzimáticos/química , Metilação , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectrofotometria Infravermelho , Triterpenos/química , Triterpenos/farmacologia
5.
Blood ; 94(9): 2955-62, 1999 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-10556177

RESUMO

We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, predicting a Pro172-->Ser replacement. Analysis of G6PD RNA from the patient's leukocytes and fibroblasts showed only transcripts with the 514C-->T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.


Assuntos
Anemia Hemolítica/genética , Glucosefosfato Desidrogenase/genética , Granulócitos/fisiologia , Adulto , Anemia Hemolítica/complicações , Anemia Hemolítica/enzimologia , Anemia Hemolítica/fisiopatologia , Doença Crônica , Doenças Transmissíveis/etiologia , Doenças Transmissíveis/genética , Ativação Enzimática , Feminino , Predisposição Genética para Doença , Glucosefosfato Desidrogenase/metabolismo , Humanos , Mutação , Linhagem , Reação em Cadeia da Polimerase
6.
Genes Dev ; 13(10): 1309-21, 1999 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-10346819

RESUMO

The adenomatous polyposis coli (APC) gene is considered as the true gatekeeper of colonic epithelial proliferation: It is mutated in the majority of colorectal tumors, and mutations occur at early stages of tumor development in mouse and man. These mutant proteins lack most of the seven 20-amino-acid repeats and all SAMP motifs that have been associated with down-regulation of intracellular beta-catenin levels. In addition, they lack the carboxy-terminal domains that bind to DLG, EB1, and microtubulin. APC also appears to be essential in development because homozygosity for mouse Apc mutations invariably results in early embryonic lethality. Here, we describe the generation of a mouse model carrying a targeted mutation at codon 1638 of the mouse Apc gene, Apc1638T, resulting in a truncated Apc protein encompassing three of the seven 20 amino acid repeats and one SAMP motif, but missing all of the carboxy-terminal domains thought to be associated with tumorigenesis. Surprisingly, homozygosity for the Apc1638T mutation is compatible with postnatal life. However, homozygous mutant animals are characterized by growth retardation, a reduced postnatal viability on the B6 genetic background, the absence of preputial glands, and the formation of nipple-associated cysts. Most importantly, Apc1638T/1638T animals that survive to adulthood are tumor free. Although the full complement of Apc1638T is sufficient for proper beta-catenin signaling, dosage reductions of the truncated protein result in increasingly severe defects in beta-catenin regulation. The SAMP motif retained in Apc1638T also appears to be important for this function as shown by analysis of the Apc1572T protein in which its targeted deletion results in a further reduction in the ability of properly controlling beta-catenin/Tcf signaling. These results indicate that the association with DLG, EB1, and microtubulin is less critical for the maintenance of homeostasis by APC than has been suggested previously, and that proper beta-catenin regulation by APC appears to be required for normal embryonic development and tumor suppression.


Assuntos
Proteínas do Citoesqueleto/fisiologia , Proteínas de Drosophila , Neoplasias Experimentais/etiologia , Transativadores , Proteínas Supressoras de Tumor , Proteína da Polipose Adenomatosa do Colo , Fatores Etários , Animais , Peso Corporal , Cistos/genética , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/química , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Genitália Masculina/anatomia & histologia , Genitália Masculina/embriologia , Proteínas de Insetos/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Mutagênese Insercional , Fenótipo , Glândulas Sebáceas/fisiologia , Dermatopatias/genética , Células-Tronco/metabolismo , Tubulina (Proteína)/análise , beta Catenina
7.
J Med Genet ; 36(1): 65-7, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9950370

RESUMO

Familial adenomatous polyposis (FAP) is characterised by hundreds of colorectal adenomas. Endocrine neoplasms have occasionally been reported, as have gastric polyps, which are usually hamartomatous in the fundus of the stomach and adenomatous in the antrum. A 57 year old man with colorectal, gastric, and periampullary adenomatous polyposis, in association with three bilateral adrenocortical adenomas, is presented. Mutation screening showed a 5960delA germline mutation in the adenomatous polyposis coli (APC) gene predicted to lead to a premature stop codon. This mutation was found in three of the four children of the patient. Western blot analysis of a lymphoblastoid cell line derived from the patient failed to detect any truncated APC polypeptide. This rare 3' mutation is responsible for an unusually complex and late onset phenotype of FAP.


Assuntos
Polipose Adenomatosa do Colo/genética , Adenoma Adrenocortical/genética , Genes APC , Mutação em Linhagem Germinativa , Polipose Adenomatosa do Colo/patologia , Adenoma Adrenocortical/patologia , Análise Heteroduplex , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Análise de Sequência de DNA
9.
N Engl J Med ; 339(8): 511-8, 1998 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-9709044

RESUMO

BACKGROUND: Germ-line mutations in DNA mismatch-repair genes (MSH2, MLH1, PMS1, PMS2, and MSH6) cause susceptibility to hereditary nonpolyposis colorectal cancer. We assessed the prevalence of MSH2 and MLH1 mutations in families suspected of having hereditary nonpolyposis colorectal cancer and evaluated whether clinical findings can predict the outcome of genetic testing. METHODS: We used denaturing gradient gel electrophoresis to identify MSH2 and MLH1 mutations in 184 kindreds with familial clustering of colorectal cancer or other cancers associated with hereditary nonpolyposis colorectal cancer. Information on the site of cancer, the age at diagnosis, and the number of affected family members was obtained from all families. RESULTS: Mutations of MSH2 or MLH1 were found in 47 of the 184 kindreds (26 percent). Clinical factors associated with these mutations were early age at diagnosis of colorectal cancer, the occurrence in the kindred of endometrial cancer or tumors of the small intestine, a higher number of family members with colorectal or endometrial cancer, the presence of multiple colorectal cancers or both colorectal and endometrial cancers in a single family member, and fulfillment of the Amsterdam criteria for the diagnosis of hereditary nonpolyposis colorectal cancer (at least three family members in two or more successive generations must have colorectal cancer, one of whom is a first-degree relative of the other two; cancer must be diagnosed before the age of 50 in at least one family member; and familial adenomatous polyposis must be ruled out). Multivariate analysis showed that a younger age at diagnosis of colorectal cancer, fulfillment of the Amsterdam criteria, and the presence of endometrial cancer in the kindred were independent predictors of germ-line mutations of MSH2 or MLH1. These results were used to devise a logistic model for estimating the likelihood of a mutation in MSH2 and MLH1. CONCLUSIONS: Assessment of clinical findings can improve the rate of detection of mutations of DNA mismatch-repair genes in families suspected of having hereditary nonpolyposis colorectal cancer.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA , Mutação , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Fatores Etários , Idoso , Proteínas de Transporte , Análise por Conglomerados , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , Análise Mutacional de DNA , Eletroforese em Gel de Campo Pulsado , Neoplasias do Endométrio/epidemiologia , Neoplasias do Endométrio/genética , Feminino , Testes Genéticos , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Neoplasias Primárias Múltiplas/epidemiologia , Neoplasias Primárias Múltiplas/genética , Proteínas Nucleares
10.
Recent Results Cancer Res ; 146: 20-31, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9670246

RESUMO

About 15% of patients with colorectal cancer report a family history of this disease. An estimated 1%-5% of patients have hereditary non-polyposis colorectal cancer (HNPCC). Recently, DNA mismatch repair genes associated with this syndrome were identified. For about 50% of families in which HNPCC occurs, DNA-based diagnosis and presymptomatic DNA testing are now feasible. Diagnosis of a hereditary tumour syndrome is relevant for both the patient with cancer and his or her close relatives. The complexities of family studies warrant the forming of a multidisciplinary team which may choose to work within a specialized cancer family clinic.


Assuntos
Pólipos Adenomatosos/cirurgia , Neoplasias Colorretais Hereditárias sem Polipose/cirurgia , Pólipos Adenomatosos/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , DNA de Neoplasias/análise , Feminino , Humanos , Masculino , Linhagem
11.
Gastroenterology ; 114(2): 275-83, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9453487

RESUMO

BACKGROUND & AIMS: Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to the formation of multiple colorectal adenomas. Moreover, patients with FAP are at high risk of developing several extracolonic manifestations, including desmoids, cutaneous cysts, and tumors of the upper gastrointestinal tract. Although by definition desmoids are nonmalignant, because of their aggressive invasion of local structures, they represent one of the major causes of morbidity and mortality among patients with FAP. METHODS: This study describes the histopathologic and molecular characterization of Apc1638N, a mouse model for the broad spectrum of extracolonic manifestations characteristic of FAP. RESULTS: Heterozygous Apc+/Apc1638N animals develop fully penetrant and multifocal cutaneous follicular cysts and desmoid tumors in addition to attenuated polyposis of the upper gastrointestinal tract. Moreover, breeding of Apc+/Apc1638N mice in a p53-deficient background results in a dramatic seven-fold increase of the desmoid multiplicity. CONCLUSIONS: Because of the attenuated nature of their intestinal phenotype, these mice survive longer than other murine models for Apc-driven tumorigenesis. Therefore, Apc1638N represents an ideal laboratory tool to test various therapeutic intervention strategies for the management of intestinal as well as extraintestinal tumors.


Assuntos
Polipose Adenomatosa do Colo/complicações , Cistos/etiologia , Modelos Animais de Doenças , Fibromatose Agressiva/etiologia , Dermatopatias/etiologia , Polipose Adenomatosa do Colo/genética , Idade de Início , Animais , Cistos/patologia , Feminino , Fibromatose Agressiva/patologia , Genes APC/genética , Genes p53/genética , Perda de Heterozigosidade , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mutação/fisiologia , Neoplasias Primárias Múltiplas/complicações , Fenótipo , Distribuição por Sexo , Dermatopatias/patologia
12.
Carcinogenesis ; 18(11): 2197-203, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9395221

RESUMO

Seven-week-old Apc1638N mice were exposed to a single dose of 5 Gy total-body X-irradiation resulting in a 8-fold increase in the number of intestinal tumors and a reduction of the lifespan to an average of 6 months. The distribution of tumors along the intestinal tract as well as the adenoma/carcinoma ratio, were similar between non-irradiated and irradiated animals. Semi-quantitative PCR analysis of intestinal-tumor DNA revealed that 10 out of 14 tumors had lost the wild-type Apc allele. However, in contrast to spontaneous Apc1638N intestinal tumors in which the LOH event at the Apc locus involves the entire chromosome 18 (1), in 6 out of 10 tumors derived from X-irradiated animals the Apc loss is associated with only a partial intrachromosomal deletion. The remaining tumors have lost all chromosome 18 markers tested. In addition to the intestinal tumors, female Apc1638N mice are susceptible to the development of mammary tumors. Upon X-irradiation, Apc1638N mice show a striking 15-fold increase in mammary tumors. Moreover, Apc1638N mice spontaneously develop other extra-intestinal neoplasia, such as desmoid-like lesions similar to those associated with familial adenomatous polyposis (FAP), the human syndrome caused by germline mutations in the APC gene. Spontaneous desmoid growth is sex-dependent, as male Apc1638N mice develop 3-fold more desmoids than female mice. Interestingly, X-irradiation seemed to increase the number of desmoids per animal nearly twofold only in female Apc1638N mice. Five out of 9 desmoids found in Apc1638N mice exposed to X-ray displayed loss of the wild-type Apc allele.


Assuntos
Mutação da Fase de Leitura , Genes APC , Neoplasias Intestinais/etiologia , Neoplasias Experimentais/etiologia , Neoplasias Induzidas por Radiação/etiologia , Alelos , Animais , Feminino , Neoplasias Intestinais/genética , Perda de Heterozigosidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/genética , Neoplasias Induzidas por Radiação/genética , Irradiação Corporal Total , Raios X
14.
Am J Hum Genet ; 61(2): 329-35, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9311737

RESUMO

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer-susceptibility condition characterized by early onset colorectal cancer. Germ-line mutations in one of four DNA mismatch repair (MMR) genes, hMSH2, hMLH1, hPMS1, or hPMS2, are known to cause HNPCC. Although many mutations in these genes have been found in HNPCC kindreds complying with the so-called Amsterdam criteria, little is known about the involvement of these genes in families not satisfying these criteria but showing clear-cut familial clustering of colorectal cancer and other cancers. Here, we applied denaturing gradient-gel electrophoresis to screen for hMSH2 and hMLH1 mutations in two sets of HNPCC families, one set comprising families strictly complying with the Amsterdam criteria and another set in which at least one of the criteria was not satisfied. Interestingly, hMSH2 and hMLH1 mutations were found in 49% of the kindreds fully complying with the Amsterdam criteria, whereas a disease-causing mutation could be identified in only 8% of the families in which the criteria were not satisfied fully. In correspondence with these findings, 4 of 6 colorectal tumors from patients belonging to kindreds meeting the criteria showed microsatellite instability, whereas only 3 of 11 tumors from the other set of families demonstrated this instability. Although the number of tumors included in the study admittedly is small, the frequencies of mutations in the MMR genes show obvious differences between the two clinical sets of families. These results also emphasize the practical importance of the Amsterdam criteria, which provide a valid clinical subdivision between families, on the basis of their chance of carrying an hMSH2 or an hMLH1 mutation, and which bear important consequences for genetic testing and counseling and for the management of colorectal cancer families.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA , Mutação em Linhagem Germinativa , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Estudos de Casos e Controles , Neoplasias Colorretais Hereditárias sem Polipose/enzimologia , Neoplasias Colorretais Hereditárias sem Polipose/etnologia , República Tcheca , Dinamarca , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Itália , Repetições de Microssatélites , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Países Baixos , Proteínas Nucleares , Ácidos Nucleicos Heteroduplexes , Desnaturação Proteica , Padrões de Referência
15.
J Exp Zool ; 277(3): 245-54, 1997 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-9062998

RESUMO

In an effort to generate a good mouse model for human colorectal cancer, we generated mice which carry a mutation in the adenomatous polyposis coli (Apc) gene. Mice which are heterozygous for the mutation, designated Apc1638, develop colonic polyps and tumors of the small intestine. Neoplasms were found in 96% of animals studied, and they included adenomas, adenocarcinomas, and polypoid hyperplasias. The mice developed an average of 3.3 tumors, with the highest number in duodenum, followed by jejunum, stomach, ileum, and colon. Focal areas of dysplasias were observed in the colonic mucosa in 50% of mice which were 10 months old or older. These results suggest that mice carrying the Apc1638 mutation can serve as a good model to study the initiation, progression, and inhibition of gastrointestinal tumors.


Assuntos
Polipose Adenomatosa do Colo , Modelos Animais de Doenças , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Animais , Carcinoma/patologia , Feminino , Mucosa Gástrica/patologia , Neoplasias Gastrointestinais/patologia , Genes APC/genética , Heterozigoto , Humanos , Hiperplasia , Mucosa Intestinal/patologia , Masculino , Camundongos , Mutação , Lesões Pré-Cancerosas/patologia
16.
Hum Mutat ; 9(1): 7-16, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-8990002

RESUMO

Germline mutations of the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to colorectal cancer. We screened the entire coding region of the APC gene for mutations in an unselected series of 105 Dutch FAP kindreds. For the analysis of exons 1-14, we employed the GC-clamped denaturing gradient gel electrophoresis (DGGE), while the large exon 15 was examined using the protein truncation test. Using this approach, we identified 65 pathogenic mutations in the above 105 apparently unrelated FAP families. The mutations were predominantly either frameshifts (39/65) or single base substitutions (18/65), resulting in premature stop codons. Mutations that would predict abnormal RNA splicing were identified in seven cases. In one of the families, a nonconservative amino acid change was found to segregate with the disease. In spite of the large number of APC mutations reported to date, we identified 27 novel germline mutations in our patients, which reiterates the great heterogeneity of the mutation spectrum in FAP. In addition to the point mutations identified in our patients, structural rearrangements of APC were found in two pedigrees, by Southern blot analysis. The present study indicates that the combined use of DGGE, protein truncation test, and Southern blot analysis offers an efficient strategy for the presymptomatic diagnosis of FAP by direct mutation detection. We found that the combined use of the currently available molecular approaches still fails to identify the underlying genetic defect in a significant subset of the FAP families. The possible causes for this limitation are discussed.


Assuntos
Polipose Adenomatosa do Colo/genética , Genes APC , Mutação em Linhagem Germinativa , Southern Blotting , Eletroforese em Gel de Poliacrilamida , Éxons , Feminino , Rearranjo Gênico , Humanos , Masculino , Países Baixos , Linhagem
17.
Hum Mutat ; 9(2): 157-63, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9067756

RESUMO

Three germline mutations in the TP53 tumor-suppressor gene are reported, two of which are not reported previously. A missense mutation at codon 265 of TP53 was found in three patients of a family that complied with the definition of the Li-Fraumeni syndrome. A nonsense mutation in codon 306 was found in a woman who had had a rhabdomyosarcoma at age 4 and a subsequent breast cancer at age 22. She was part of a Li-Fraumeni-like family, but the parental origin of the mutation could not be traced. Finally, while screening for somatic alterations in TP53 in a series of 141 sporadic breast tumors, we detected a constitutional missense mutation in codon 235 in a woman diagnosed with breast cancer at age 26 and a recurrence 4 years later. The recurrence, but not the primary tumor, showed an additional missense mutation at codon 245 as well as loss of the wild-type allele. This suggests that the 245 mutation was particularly important for tumor progression and that there might exist heterogeneity in terms of cancer predisposition potential among the various germline TP53 mutations.


Assuntos
Neoplasias da Mama/genética , Genes p53/genética , Mutação em Linhagem Germinativa , Síndrome de Li-Fraumeni/genética , Rabdomiossarcoma/genética , Neoplasias da Mama/patologia , Separação Celular , DNA/análise , Análise Mutacional de DNA , DNA de Neoplasias/análise , Feminino , Citometria de Fluxo , Mutação em Linhagem Germinativa/genética , Heterozigoto , Humanos , Imuno-Histoquímica , Síndrome de Li-Fraumeni/patologia , Masculino , Linhagem , Polimorfismo Genético , Rabdomiossarcoma/patologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo
18.
Am J Hum Genet ; 59(4): 818-24, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8808596

RESUMO

Hereditary nonpolyposis colorectal cancer (HNPCC) is a cancer syndrome inherited in an autosomal dominant fashion. Four susceptibility genes are known, which code for DNA mismatch repair enzymes. The purpose of this study was to identify the HNPCC gene defects in a cohort of Australian HNPCC families and to evaluate the use of RNA-based screening methods. Six mutations were identified, four in the hMLH1 gene and two in hMSH2, by using a combination of DNA-based and RNA-based methods. One of the hMLH1 defects was a missense mutation, and the other five mutations would be expected to result in a shortened protein. These included a rare type of mRNA splicing mutation in hMLH1 exon 17. By use of reverse-transcriptase (RT) PCR, defective transcripts were detectable for three of the hMLH1 mutations but not for the fourth one, which was predicted to cause skipping of exon 15. Furthermore, many more alternative transcripts for the hMLH1 gene were found than previously described, and these were more abundant in the RNA samples prepared from whole blood than from lymphoblastoid cell lines. This confounded RNA-based screening for HNPCC mutations, because it was difficult to determine which aberrant RT-PCR fragment was the real hereditary defect. One of the splice-site mutations reported here causes skipping of exons 9 and 10, which also occurs as an alternative transcript. When the protein-truncation test was used, the results were indistinguishable between the patients in this family and controls. Other aberrant transcripts were also observed that varied in size between individuals but were unrelated to the hereditary defects. This study has important implications for the design of reliable diagnostic tests for HNPCC gene defects.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteínas de Ligação a DNA , Mutação , RNA/genética , Linhagem Celular , Estudos de Coortes , Testes Genéticos , Humanos , Linfócitos/química , Proteína 2 Homóloga a MutS , Reação em Cadeia da Polimerase , Proteínas/genética , Proteínas Proto-Oncogênicas/genética
19.
Gastroenterology ; 110(4): 1020-7, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8612988

RESUMO

BACKGROUND & AIMS: Hereditary nonpolyposis colorectal cancer is characterized by early-onset colorectal cancer and the occurrence of various other cancers. The recent isolation of four mismatch repair genes responsible for hereditary nonpolyposis colorectal cancer allows for the identification of carriers within affected families. The purpose of this study was to assess the age-specific cancer risk in a large series of gene carriers. METHODS: Thirty-four families were studied by mutation analysis. In 19 of these families, pathogenic mutations were found at hMSH2 or hMLH1. Of 382 relatives, 124 had a mutation in hMLH1 and 86 in hMSH2. RESULTS: The lifetime risk of colorectal cancer was the same in both groups of gene carriers (80%). The risk of endometrial cancer was greater in hMSH2 gene carriers compared with hMLH1 gene carriers (61% vs. 42%), but the difference was not statistically significant. A very high relative risk of cancer of the small bowel (relative risk of >100) was observed in carriers of either gene. Only the carriers of hMSH2 mutations had a significantly increased relative risk of cancer of the urinary tract (kidney and ureter) (relative risk of 75.3), stomach (relative risk of 19.3), and ovaries (relative risk of 8.0). CONCLUSIONS: This study provides estimates of cancer risk that may contribute to the appropriate management of gene carriers within families with hereditary nonpolyposis colorectal cancer.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Saúde da Família , Mutação , Adolescente , Adulto , Idoso , Análise Mutacional de DNA , Reparo do DNA/genética , Feminino , Genes , Heterozigoto , Humanos , Neoplasias Intestinais/genética , Intestino Delgado , Masculino , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Risco , Neoplasias Gástricas/genética , Neoplasias Urológicas/genética
20.
Am J Hum Genet ; 58(2): 300-7, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8571956

RESUMO

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer susceptibility condition. Inherited mutations in at least four DNA mismatch repair genes, hMSH2, hMLH1, hPMS1, and hPMS2, are known to cause HNPCC. In this study we used denaturing gradient gel electrophoresis (DGGE) to screen for hMLH1 mutations in 34 unrelated HNPCC families (30 Dutch, 3 Italian, and 1 Danish). Ten novel pathogenic germ-line mutations (seven affecting splice sites, two frameshifts, and one in-frame deletion of a single amino acid) have been identified in 12 (35%) of these families. In a previous study, hMSH2 mutations were found in 21% of the same families. While the spectrum of mutations at the hMSH2 gene among HNPCC patients appears heterogeneous, a cluster of hMLH1 mutations has been found in the region encompassing exons 15 and 16, which accounts for 50% of all the independent hMLH1 mutations described to date and for > 20% of the unrelated HNPCC kindreds here analyzed. This unexpected finding has a great practical value in the clinical scenario of genetic services.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteínas de Ligação a DNA , Proteínas de Neoplasias/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Proteínas de Transporte , Primers do DNA/química , Reparo do DNA/genética , Eletroforese em Gel de Poliacrilamida , Europa (Continente) , Éxons/genética , Feminino , Genes Dominantes , Mutação em Linhagem Germinativa , Humanos , Masculino , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Mutação , Proteínas Nucleares , Linhagem , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/genética
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