RESUMEN
BACKGROUND: Sebaceous gland carcinomas represent rare malignancies of the skin and some 60% of them demonstrate high-grade microsatellite instability on the background of a defective mismatch repair system. However, a significant fraction of periocular sebaceous gland carcinomas exhibits microsatellite stability associated with a frequent loss of the candidate tumour suppressor fragile histidine triad (FHIT). OBJECTIVES: We hypothesized that in those sebaceous gland carcinomas with microsatellite stability and loss of FHIT, effector molecules participating in homologous recombination repair (HRR), such as BRCA1/2, could be somatically inactivated. METHODS: A pilot series of 10 paraffin-embedded sebaceous gland carcinoma specimens with a defined FHIT status was studied for loss of heterozygosity (LOH) events in the genes BRCA1, BRCA2, FHIT and WWOX. We sequenced the coding exons 5-8 of the p53 gene. RESULTS: Sebaceous gland carcinomas with FHIT negativity displayed LOH and biallelic deletions of the BRCA1 gene in five of 10 (50%) of the sebaceous gland carcinoma specimens analysed. Tumour-specific genomic losses close to BRCA2 were also uncovered. A homozygous p53 R248W gain-of-function mutation as the result of a CGG to TGG transition was identified in one of seven sebaceous gland carcinomas. It has been demonstrated previously that p53 R248W mutants inactivate ATM-directed HRR. This particular sebaceous gland carcinoma presented with concomitant genomic deletions at the BRCA1 and BRCA2 loci, and also at the constitutively fragile sites FRA3B/FHIT and FRA16D/WWOX. CONCLUSIONS: Our study demonstrates for the first time that microsatellite-stable FHIT-negative sebaceous gland carcinomas accumulate mutations that target central components of the HRR network. This observation will prompt investigations in synthetic lethality of BRCA-deficient sebaceous gland carcinomas by therapeutic poly(ADP-ribose) polymerase inhibitors.
Asunto(s)
Ácido Anhídrido Hidrolasas/genética , Adenocarcinoma Sebáceo/genética , Genes BRCA1 , Genes BRCA2 , Proteínas de Neoplasias/genética , Neoplasias de las Glándulas Sebáceas/genética , Proteína p53 Supresora de Tumor/genética , ADN de Neoplasias/análisis , Eliminación de Gen , Humanos , Pérdida de Heterocigocidad/genética , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Extensive exposure to ultraviolet radiation is associated with genetic alterations in basal cell carcinomas (BCCs), which represent some 75% of skin cancers. OBJECTIVES: As recent data suggested the fragile histidine triad (FHIT) gene product to participate in DNA damage responses we wished to address whether functional deletion of this tumour suppressor participates in the development of BCC. Our study focused on epigenetic inactivation of the FHIT gene. METHODS: Paraffin-embedded specimens from 17 patients with BCC were available for methylation-specific polymerase chain reaction (MSP), combined bisulphite-dependent restriction analysis (COBRA) of the FHIT gene and immunohistochemistry of its product. RESULTS: We report for the first time that 100% of BCCs are negative for FHIT by immunostaining. Aberrant methylation of the FHIT promoter occurred in a significant portion of BCCs. MSP detected hypermethylation of the FHIT/FRA3B locus in nine of nine (100%) periocular BCCs and in six of eight (75%) BCCs from other body regions. COBRA yielded similar results, confirming that some 88% of the 17 BCCs analysed harbour epigenetic silencing of the FHIT gene. Loss of FHIT protein was demonstrated immunohistochemically, confirming that promoter hypermethylation correlated with loss of gene expression. CONCLUSIONS: We have identified epigenetic silencing of the FHIT tumour suppressor gene as a frequent inactivation mechanism which is likely to contribute to functional deficiencies in DNA damage response of BCCs.
Asunto(s)
Ácido Anhídrido Hidrolasas/genética , Carcinoma Basocelular/genética , Silenciador del Gen , Genes Supresores de Tumor , Proteínas de Neoplasias/genética , Neoplasias Cutáneas/genética , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Metilación de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Periocular sebaceous gland carcinomas (SGCs) occur in the eyelids either sporadically or as a phenotypic feature of Muir-Torre syndrome (MTS). In knockout mice mismatch-repair (MMR) defects or inactivation of the fragile histidine triad (FHIT) gene are associated with MTS-like signs, including SGC. To dissect the genetic alterations associated with microsatellite instability (MSI) and inactivation of the FHIT gene, we studied nine periocular SGC specimens from MTS patients. Immunohistochemistry was performed for FHIT, MSH2, MLH1, and MSH6. We assessed MSI as well as loss of heterozygosity (LOH) at the FHIT locus with polymorphic markers and genomic multiplex PCR. Epigenetic silencing was detected by methylation-specific PCR (MSP) and combined bisulfite restriction analysis (COBRA). Our analyses identified two SGCs with FHIT positivity and high-grade MSI, and seven cases with loss of FHIT and microsatellite stability (MSS). MSI correlated with loss of MSH2 and MLH1 immunostaining. Loss-of-function mechanisms affecting the FHIT gene were identified as intragenic deletions eliminating the coding exons 5 and 6 on one hand, and complete biallelic methylation of the FHIT transcription regulatory region on the other hand. Germinal FHIT mutations as a predisposing factor for MTS were excluded in two index patients with cancer in three generations, including an FHIT-negative SGC. Our data suggest that either somatic inactivation of the FHIT gene associated with MSS or inactivation of the MMR system resulting in MSI contribute to the development of periocular SGCs in presumptive MTS.
Asunto(s)
Carcinoma/genética , Neoplasias de los Párpados/genética , Neoplasias de las Glándulas Sebáceas/genética , Ácido Anhídrido Hidrolasas/genética , Adulto , Anciano , Disparidad de Par Base , Metilación de ADN , Epigénesis Genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , SíndromeRESUMEN
AIMS: About 10-30% of primary liver cancers represent intrahepatic cholangiocarcinomas (IHCC). Since chromosomal losses of 3p are detectable in about 40% of cholangiocarcinomas our study aimed at the identification of mechanisms leading to functional deletion of tumor suppressor genes in this region. Our efforts focussed on genomic losses and epigenetic inactivation of two tumor suppressor genes, the fragile histidine triad (FHIT) and the ras association domain family 1 (RASSF1A) genes, both located on the short arm of chromosome 3. METHODS: Methylation-specific PCR (MSP) and combined bisulfite-dependent restriction analysis (COBRA) were applied to detect epigenetic silencing of gene promoters. Genomic duplex PCR was used to identify exon losses of the FHIT gene. Nineteen paraffin-embedded samples of intrahepatic cholangiocarcinomas were studied. RESULTS: Here we report for the first time that in addition to frequent losses of the exons 5 and 6, hypermethylation of the FHIT promoter occured in a significant portion of IHCC. Methylation specific PCR (MSP) detected epigenetic inactivation of the FHIT/FRA3B locus in 8 of 19 (42%) cases. Combined bisulfite restriction analysis (COBRA) revealed that high levels of methylated FHIT promoter sequences were present in 6 of the 8 methylation positive samples. In agreement with previous reports MSP identified hypermethylation of the RASSF1A gene in 13 of 19 (68%) IHCC specimens examined. CONCLUSIONS: Epigenetic silencing of the FHIT tumor suppressor gene is a novel inactivation mechanism to be considered in the development of intrahepatic cholangiocarcinomas. However, a statistically significant inverse correlation between K-Ras activation and RASSF1A inactivation was not found.
Asunto(s)
Ácido Anhídrido Hidrolasas/genética , Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos , Colangiocarcinoma/genética , Metilación de ADN , Epigénesis Genética/genética , Exones/genética , Genes Supresores de Tumor , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , Silenciador del Gen , Genes ras , Humanos , Pérdida de Heterocigocidad , Reacción en Cadena de la Polimerasa , Proteínas Supresoras de Tumor/genéticaRESUMEN
This study was intended to evaluate a diagnostic reverse transcriptase polymerase chain reaction based protein-truncation test for the identification of germline mutations in the serine/threonine protein kinase 11 (STK11, also designated LKB1) gene in Peutz-Jeghers syndrome (PJS). Our data exemplify that the inactivation of STK11 can be due to unusual disturbances in splicing regulation which result in truncations of the protein. However, nonsense mediated mRNA decay must be blocked with puromycin to detect shortened STK11 gene products contained in the leucocytic mRNA pool of PJS patients. Interestingly, two mutations escaped from detection by exon sequencing techniques with usual flanking PCR primers, since alterations were located right in the middle of intronic sequences. We describe a compound heterozygous PJS patient who carried two different mutations in intron 1 on separate alleles. Each of the two mutations was transmitted individually to one of his two children. In the course of our RNA based analyses we detected high level expression of a novel STK11/LKB1 mRNA variant retaining intron 4 (STK11 c.597(insertion mark)598insIVS4) in various tissues. This mRNA isoform was initiated from an alternative transcription regulatory region as revealed by primer extension analyses even in cell lines with complete methylation of the normal promoter. As a consequence of novel mutational mechanisms identified we discuss the impact of RNA based strategies for the detection of germinal STK11 mutations in PJS.
Asunto(s)
Empalme Alternativo/genética , Mutación/genética , Síndrome de Peutz-Jeghers/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/análisis , Quinasas de la Proteína-Quinasa Activada por el AMP , Alelos , Secuencia de Bases , Metilación de ADN , Análisis Mutacional de ADN , Exones/genética , Femenino , Pruebas Genéticas , Humanos , Intrones/genética , Masculino , Datos de Secuencia Molecular , Linaje , Polimorfismo Conformacional Retorcido-Simple , Sitios de Empalme de ARN/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia/genéticaRESUMEN
AIM AND METHODS: Data of our patients with at least three primary malignancies were retrospectively analysed to detect any remarkable patterns which might be of interest for follow-up or early tumour detection and to identify a possible hereditary cancer predisposition. From 1.1.1954 to 1.8.1995 57 patients (0.1%) among a grand total of 52 398 cancer patients had a minimum of three malignancies. RESULTS: The 5-year survival rates after colorectal, bladder, prostate, uterine corpus and gastric cancer were higher than those seen in patients with the corresponding solitary tumours. In both sexes, the mean interval between the individual tumours was greater (4.0 years) between the first and second tumours than between the second and third (2.5 years). In women, the intervals were roughly twice as long (6.8 and 3.7 years) as in men (3.7 and 2.0 years). 40.4% (n=23/57) had a colorectal, 28.1% (n=16) a bladder, and 41.7% (n=15/36 men) had a prostate carcinoma. 66.7% (n=14/21 women) contracted at least one gynaecological tumour. In 24 families HNPCC, in one a Li-Fraumeni Syndrome, and in another Hereditary Diffuse Gastric Cancer was suspected.
Asunto(s)
Neoplasias Colorrectales/epidemiología , Predisposición Genética a la Enfermedad/epidemiología , Neoplasias Primarias Múltiples/epidemiología , Neoplasias de la Próstata/epidemiología , Neoplasias de la Vejiga Urinaria/epidemiología , Adulto , Distribución por Edad , Anciano , Análisis por Conglomerados , Neoplasias Colorrectales/genética , Femenino , Alemania/epidemiología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Neoplasias Primarias Múltiples/genética , Linaje , Neoplasias de la Próstata/genética , Estudios Retrospectivos , Factores de Riesgo , Distribución por Sexo , Análisis de Supervivencia , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
Familial adenomatous polyposis (FAP, Mendelian Inheritance in Man number *175,100 [edited by Victor A. McKusick], accessible on line under http:¿www3.ncbi.nlm.nih.gov/htbin-post/ Omim/dispmim?175100) is a dominantly inherited colorectal cancer predisposition syndrome. The designation Gardner Syndrome is used for phenotypic variants of FAP with additional extracolonic symptoms. After the adenomatous polyposis coli (APC) gene was identified with the help of positional cloning strategies in 1991, it became evident that inactivation of this tumor suppressor is based on loss of carboxyterminal protein-protein interaction domains. Identification of multiple molecular constituents binding to the distal half of the APC protein revealed its crucial involvement in wnt-signaling. Because the spectrum of mutations is predominated by small insertions and deletions, nonsense-, and splice-site mutations, a prescreening procedure is employed for the identification of germinal mutations in FAP patients that relies on in vitro synthesis of APC gene products, an approach also known under the acronym PTT (protein truncation test). Absence of nonsense-mediated mRNA decay of mutated APC transcripts allows the application of a cDNA-based coupled in vitro transcription/translation reaction for exons 1 to 14. Examination of four overlapping fragments from genomic DNA of probands reveals stops in the large APC exon 15, encompassing more than 6500 base pairs. Using this procedure, mutations causing the disease will be identified in about 80% of FAP patients. In the other cases of clinically manifest FAP, evidence exists that reduction of the steady state level of APC protein as a result of transcriptional silencing or large genomic deletions could provide for the clinical phenotype. Although some genotype-phenotype correlations have been described, exceptions from the rule have been reported, that is, for CHRPE. Modifier genes for the development of extracolonic manifestations are currently still enigmatic. Knowledge of such genes would essentially contribute to a better presymptomatic treatment of FAP patients.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Genes APC , Poliposis Adenomatosa del Colon/diagnóstico , Poliposis Adenomatosa del Colon/etiología , Femenino , Ligamiento Genético , Humanos , Masculino , MutaciónRESUMEN
Epidemiologic data suggest that an underlying genetic disposition can be detected in up to 10% of all colorectal cancer patients and autosomal dominantly inherited hereditary non-polyposis colorectal cancer (HNPCC) is the entity most frequently identified. It was described first by A. Warthin in 1895 in "Family G" and is characterized by a predisposition to an early onset of colorectal cancer and other intestinal or genitourinary tumors. We report the case of a 61-year-old woman with five different cancers. Although the strict Amsterdam Criteria were not fulfilled, molecular analysis revealed HNPCC; further genetic testing in the family confirmed that the 36-year-old and so far healthy son had inherited the germline mutation of his affected mother. Genetic testing in clinically suspected HNPCC cases is recommended for patients with colorectal cancer meeting the Amsterdam Criteria. In patients meeting one of Bethesda Criteria 2-7 without meeting the Amsterdam Criteria, germline mutation analysis is recommended only in MSI-positive tumors.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales/genética , Neoplasias Primarias Múltiples/genética , Neoplasias Urogenitales/genética , Adulto , Neoplasias Colorrectales/cirugía , Neoplasias Colorrectales Hereditarias sin Poliposis/cirugía , Femenino , Pruebas Genéticas , Mutación de Línea Germinal , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Primarias Múltiples/cirugía , Reoperación , Neoplasias Urogenitales/cirugíaRESUMEN
Sjögren-Larsson syndrome (SLS) is a rare autosomal recessively inherited disorder characterised by mental retardation, spasticity and ichthyosis. SLS patients have a profound deficiency in fatty aldehyde dehydrogenase (FALDH) activity. The human cDNA of FALDH has been shown to map to the SLS locus on chromosome 17p11.2. Here we describe a method based on reverse transcriptase-polymerase chain reaction (RT-PCR) and protein truncation test to identify mutations in the FALDH gene in nine German SLS families. Using this detection system both disease-causing mutations were found in eight of the nine SLS families examined (17/18 chromosomes). Seven different mutations were identified: an exon 2 skipping due to exon 2 splice donor mutation; two different exon 3 splice donor mutations resulting in combined exon 2 and 3 skipping; a 906delT deletion in exon 6; a genomic deletion of about 6 kb including exon 9; a 1277T > G transversion resulting in a Leu426Ter nonsense mutation; and a 1297delGA deletion. Two of the mutations identified, the genomic exon 9 deletion and the 906delT in exon 6 affected five out of seven SLS patients from a small region of Northern Bavaria. Therefore these two mutations accounted for 71% (10/14 chromosomes) of Bavarian SLS alleles and so far have not been described in SLS families from other countries. Our findings do not support our 'historical' hypothesis, that a possible region clustering in Northern Bavaria could be due to the presence of Swedish soldiers during the 30 Years War (1618-1648), but suggest that two mutations causing SLS syndrome originated in Northern Bavaria.
Asunto(s)
Aldehído Oxidorreductasas/genética , ARN/genética , Síndrome de Sjögren-Larsson/genética , Aldehído Oxidorreductasas/metabolismo , Alelos , Secuencia de Bases , Codón de Terminación , Exones , Salud de la Familia , Femenino , Pruebas Genéticas , Variación Genética , Alemania , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Linaje , Polimorfismo Genético , ARN/metabolismo , Estabilidad del ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de SecuenciaRESUMEN
Hereditary non-polyposis colon cancer (HNPCC) is a heterogeneous group of tumour predisposition syndromes caused by germline mutations in at least four different mismatch repair genes. HNPCC patients are prone to the development of carcinomas of the intestinal tract and other specific sites. Identification of presumptive HNPCC patients is primarily based on a positive family history of colorectal cancer in at least two generations. In the course of mutation screening of the MLH1 and MSH2 genes in patients manifesting a carcinoma of the HNPCC tumour spectrum before the age of 45 years, we identified a germline MSH2 344delA frameshift mutation in a male proband. This index patient, at the age of 25 years, initially developed a large rectal adenoma that was removed by polypectomy. Ten years later he was operated on for an invasive right sided colon carcinoma in the caecum (International Union Against Cancer (UICC) stage III). The mother and father, aged 61 and 66 years, respectively, were healthy and had no family history of colorectal cancer. Subsequent molecular analyses excluded the germinal MSH2 344delA alteration identified in their son and at the same time paternity was confirmed with a set of informative polymorphic markers. Thus, the genetic alteration identified in our patient definitely represented a de novo germline mutation in one of the major HNPCC genes. This case report of a patient with colorectal cancer at a relatively young age with no family history is intended to encourage mutation screening of the MSH2 and MLH1 genes in similar cases to find out whether this group of patients contains an increased proportion of de novo mutations in mismatch repair genes.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteínas de Unión al ADN , Mutación de Línea Germinal , Proteínas Proto-Oncogénicas/genética , Proteínas Adaptadoras Transductoras de Señales , Adulto , Anciano , Proteínas Portadoras , ADN de Neoplasias/análisis , Femenino , Pruebas Genéticas , Humanos , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/genética , Proteínas Nucleares , LinajeRESUMEN
Adenomatous polyposis coli (APC) exon 14-skipped transcripts encode putative APC proteins of low molecular weight. To prove that exon 14-skipped mRNA variants do not simply represent tissue culture artifacts, expression of these APC transcript variants was demonstrated in native colorectal epithelium. Fresh surgical specimens of human colon were processed and epithelial cells were affinity-purified with the dynabead-immobilized monoclonal antibody Ber-EP4. Epithelium derived cDNA was PCR-amplified in the range of linear accumulation. RT-PCR products with and without APC exon 14 were evaluated by densitometry. Analyses of normal mucosa (n = 8) and matching mucosa--tumor samples (n = 4) revealed a consistent 4 to 1 ratio of APC exon 14-positive to exon 14-negative mRNA levels. We conclude, a) that APC exon 14-skipped transcripts are physiologically expressed in native human colon mucosa, and b) that ratios of exon 14-negative to exon 14-positive isoforms were not altered when colorectal tumor cells were compared with matching normal mucosa (p = 0.80).
Asunto(s)
Colon/metabolismo , Neoplasias del Colon/genética , Exones , Genes APC , Humanos , Mucosa Intestinal/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: Adenomatous polyposis coli protein, glycogen synthetase kinase-3-beta, T cell transcription factor/lymphoid enhancer-binding factor, and beta-catenin modulate cell differentiation and proliferation via the expression of effector genes. It has recently been postulated that beta-catenin is a potent oncogene of sporadic colorectal carcinogenesis and a prognostic tumor marker. Our aim was to investigate whether the nuclear overexpression of beta-catenin, possibly caused by mutations in exon 3 of beta-catenin (CTNNB1), is correlated with distant metastatic spread or disease-free survival in rectal carcinoma. METHODS: Immunohistochemical analysis was performed with an anti-beta-catenin-monoclonal antibody on paraffin sections of two groups of patients (n = 2 x 77) with rectal carcinoma curatively treated by surgery alone. The patients selected were all free of local disease, to exclude surgical influence. Patient groups were matched for age, gender, International Union Against Cancer stage, and year of operation (1982 to 1991) and differed only in subsequent metachronous distant metastatic spread. Follow-up was prospective (median, 9.6 years). Three staining patterns were defined: membranous (normal), diffuse cytoplasmic (pathologic), and intense nuclear staining (pathologic). When intense nuclear staining was defined, the specimen was microdissected. Then, DNA was isolated, polymerase chain reaction-amplified, and sequenced to detect mutations in exon 3. RESULTS: Nuclear overexpression of beta-catenin correlated neither with distant metastatic spread (chi-squared, 0.37; P = 0.79) nor with disease-free survival (log-rank with trend, P = 0.62). No mutations were found in the area of the serine/threonine-kinase glycogen synthetase kinase-3-beta-phosphorylation site in exon 3 (CTNNB1) of beta-catenin. CONCLUSION: Although beta-catenin seems to play an important role in early colorectal carcinogenesis, its value as a prognostic marker is questionable. It must be assumed that metastatic ability is determined by other factors than the disturbance of the beta-catenin T cell transcription factor/lymphoid enhancer-binding factor cascade and that other mechanisms might cause the observed nuclear translocation of beta-catenin.
Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/secundario , Cadherinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , ADN de Neoplasias/metabolismo , Expresión Génica , Proteínas Nucleares/metabolismo , Neoplasias del Recto/metabolismo , Neoplasias del Recto/patología , Transactivadores , Adenocarcinoma/mortalidad , Adenocarcinoma/cirugía , Biomarcadores de Tumor , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Neoplasias del Recto/mortalidad , Neoplasias del Recto/cirugía , Análisis de Secuencia de ADN , beta CateninaRESUMEN
Although in vitro protein synthesis is a rapid method to screen for translational stops in the adenomatous polyposis coli (APC) gene, truncating mutations at the 5' most end are at risk of being overseen due to their small size. The authors describe a reverse transcriptase-polymerase chain reaction (RT-PCR)-based protein truncation test specifically designed for detecting truncated polypeptide chains of less than 10 kDa. Using this detection system, three novel germline mutations in familial adenomatous polyposis (FAP) patients were identified, i.e. a Gly101 Ter non-sense mutation in exon 3, an exon 4 splice acceptor mutation and a 555delC deletion in exon 5. Morever, a patient manifesting congenital hypertrophy of the retinal pigmented epithelium (CHPRE) was detected with an Arg232Ter mutation in exon 6. This is, to the authors' knowledge, the fourth exception to the rule that FAP patients manifesting CHRPE harbour genetic alternations downstream from APC exon 9. Hence, an alternative hotspot for non-sense mutations associated with CHRPE appears to encompass the codons 215, 216 and 232. Patients reported in this study, exhibited relatively mild clinical symptoms with respect to the age of onset of malignancy (> 50 years of age) and the number of polyps (70-100 adenomas). However, manifestation of severe duodenal adenomatosis was independent of the attenuated colorectal FAP phenotype.
Asunto(s)
Codón de Terminación , Genes APC , Epitelio Pigmentado Ocular/patología , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Eliminación de Secuencia , Proteína de la Poliposis Adenomatosa del Colon , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , ADN/sangre , Cartilla de ADN , Exones , Humanos , Hipertrofia , Intrones , Leucocitos , Sistemas de Lectura Abierta , Enfermedades de la Retina/congénito , Enfermedades de la Retina/genéticaRESUMEN
Expression of the adenomatous polyposis coli (APC) gene derived exon BS (e.g., brain-specific exon) has been analyzed by RT-PCR. Four novel APC mRNA isoforms derived from alternative splicing of exons 1A, BS and 1 were identified, which were ubiquitously expressed. One novel cDNA was characterized by cloning and DNA sequence analysis, which combined the exon 1A (identical with exon 0.3) 3' end with nucleotide position +101 of intron 1A and continued throughout exon BS. A second cDNA isoform was isolated, which joined the 3' end of exon 1A with nucleotide position +118 of exon BS. Both novel isoforms were found to be expressed together with a third novel APC exon connection, which was specified by exon BS/2 joining. This interesting exon junction resulted in novel deduced amino terminal open reading frames, which are completely in-frame with sequences located further downstream. Systematic exon connection analyses revealed that APC transcripts with exon BS/2 junctions were predominantly detected with a fixed exon composition. RT-PCR analyses did not identify facultative skipping of exons 9, 10A and 14 in this type of mRNA, in contrast to exon 1-containing APC transcripts analyzed from the same cDNA pool under identical conditions. Hence, exon 1 skipping of exon BS-positive mRNA molecules might preferentially encode unique APC polypeptide chains, which are characterized by an alternative amino terminus and extended heptad repeat structures due to combined incorporation of exons 9 and 10A.
Asunto(s)
Encéfalo/metabolismo , Exones , Genes APC , Línea Celular , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisisRESUMEN
Physical interaction between the lymphoid high mobility group (HMG)-box architectural transcription factors TCF/LEF and beta-catenin is associated with translocation of the heteromeric complex to the nucleus and regulation of target gene expression. Since formation of molecular complexes among beta-catenin, E-cadherin, p300apc and TCF/LEF depends on balanced expression of these constituents, we investigated the biosynthesis of TCF-1 in colorectal cancer. Here we report detailed analyses of activation and overexpression of lymphoid transcription factor TCF-1 in human colorectal cancer-derived cell lines. Northern blot analyses revealed considerable steady-state expression levels of TCF-1 mRNA of normal size. Genomic rearrangement of the 5' flanking region of the TCF-1 gene was excluded as a cause of ectopic expression. By contrast, CAT-reporter constructs depending on a 515-bp T-cell-regulated TCF-1 genomic upstream region were significantly activated in epithelial tumor cells. RT-PCR analyses revealed a heterogeneic population of mRNA isoforms due to alternative splicing in the TCF-1 gene. On Western blots of colorectal cancer cells, the TCF-1-specific monoclonal antibody 7H3 detected a similar heterogeneous spectrum of TCF-1 specific polypeptide chains. Interestingly, overexpression of TCF-1-specific splice forms correlated with the metastatic behavior of the analyzed cells and with overproduction of lymphoid tyrosine protein kinase p56(lck). We conclude that ectopic expression of the HMG-box factor TCF-1 is associated with late events in tumor progression.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Proteínas de Unión al ADN/fisiología , Factores de Transcripción/fisiología , Empalme Alternativo , Northern Blotting , Neoplasias Colorrectales/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Células HeLa , Humanos , Factor de Unión 1 al Potenciador Linfoide , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Factor 1 de Transcripción de Linfocitos T , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción Genética , Células Tumorales CultivadasRESUMEN
BACKGROUND & AIMS: Infantile and childhood liver tumors have been found in 0.42% of individuals with a germline mutation in the adenomatous polyposis coli (APC) gene. This study analyzed a hepatocellular adenoma of a 2-year-old child at risk for familial adenomatous polyposis to identify genetic alterations in hepatic tumors initiated by APC germline mutations. METHODS: Mutation screening was performed for the APC gene (protein truncation test and DNA sequence analysis), p53 gene (complementary DNA cloning and sequencing), and members of the Ras gene family (complementary DNA sequence analysis). RESULTS: Both the mother and child had a germinal CGA-->TGA transition at codon 1451 leading to an Arg1451Ter stop mutation in the APC gene. Loss of the wild-type APC allele as a second hit revealed hemizygosity of the inherited mutation in the tumor. Furthermore, a CGC-->CAC transition in the p53 gene of the adenoma resulted in an Arg-->His missense mutation in codon 175. No loss of heterozygosity was detected at the p53 locus. Ras gene mutations were not found. CONCLUSIONS: Biallelic inactivation of APC gene and p53 mutation are early events in hepatocellular tumorigenesis. Additional reports will confirm whether inherited APC gene mutations between codon 1444 and 1578 increase the risk for hepatic tumors.
Asunto(s)
Adenoma de Células Hepáticas/genética , Poliposis Adenomatosa del Colon/genética , Genes APC , Genes p53 , Neoplasias Hepáticas/genética , Mutación , Preescolar , Femenino , HumanosRESUMEN
To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.
Asunto(s)
Proteínas de la Membrana/metabolismo , Secuencia de Bases , Western Blotting , Células Cultivadas , Detergentes/farmacología , Genes de la Neurofibromatosis 2 , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Neurofibromina 2 , Polietilenglicoles/farmacología , Proteínas Recombinantes de Fusión/genética , Piel/citología , Piel/metabolismo , Solubilidad , Fracciones Subcelulares/metabolismoRESUMEN
Adenomatous polyposis coli (APC) gene transcripts skipping exon 14 in combination with the alternatively spliced exons 9 and 10A contribute to the heterogeneity of physiological APC mRNA isoforms. Here we report on a novel genotype-phenotype correlation in familial adenomatous polyposis (FAP) with early onset of disease and malignancy due to an APC exon 14 splice defect. Compared to controls, two affected individuals of a FAP kindred presented with a significantly distorted APC mRNA isoform pattern in B lymphocytes. As a result of an A-->G transition in the canonical AG-splice acceptor dinucleotide of exon 14, expression levels of all APC mRNA isoforms without exon 14 were dramatically increased and those with exon 14 were simultaneously decreased. Skipping of exon 14 is a physiological event also seen in nonmalignant cells, which results in a frameshift to produce low-molecular-weight APC proteins. Western blot analysis of the patients' lymphoblastoid B cells revealed the identification of intracellularly stable APC protein isoforms with an Mr of 55-67 kDa and, thus, the first demonstration of APC proteins encoded by exon 14-skipped transcripts. We postulate that the quantitatively imbalanced expression of these physiological APC light chains represents a novel pathogenetic mechanism associated with predisposition to FAP.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Exones , Genes APC , Proteína de la Poliposis Adenomatosa del Colon , Adolescente , Adulto , Empalme Alternativo , Clonación Molecular , Proteínas del Citoesqueleto/genética , Análisis Mutacional de ADN , Femenino , Humanos , Isomerismo , Masculino , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
Reverse transcription-polymerase chain reaction (RT-PCR)-based analyses of the adenomatous polyposis coli (APC) gene encompassing exons 1-15 revealed a complex pattern of products that were due to alternative splicing of exons 9, 10A and 14. The multiplicity of polypeptide chains obtained from T7-promoter-directed in vitro translation of the RT-PCR product pool was confirmed immunochemically to correspond to the mRNA isoforms, but not to represent products of internal initiation of translation. This observation is of particular relevance for the diagnostic protein truncation test (PTT), since this assay will pick up mRNA variants derived from physiological splice events, e.g., skipping of exons 9, 10A and 14. In vitro-translated proteins of reduced molecular weight were therefore detectable in healthy individuals. We extended this observation to the PTT of cDNA encompassing APC exons 1-14 of familial adenomatous polyposis patients. Knowledge of the normal polypeptide pattern seen in the diagnostic in vitro translation assay allowed us not only to identify translational stop mutations, but even to detect a splice acceptor mutation of exon 14 as a result of quantitative changes of the isoform pattern. Western immuno blot analysis on protein extracts of Epstein-Barr virus-immortalized lymphocytes of the same patients revealed that mutations accessible to the RT-PCR PTT yield intracellularly undetectable APC proteins.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Empalme Alternativo , Autorradiografía , Western Blotting , ADN , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
Although the majority of fragile-X patients demonstrate methylation and a much-expanded CGG repeat region in the 5'-untranslated region of exon 1 of the FMR1 gene, exceptional cases have been reported to be due to deletions. However, fine mapping of the deletion breakpoints is still lacking and so far the underlying mechanism is unknown. We identified a fragile-X patient mosaic for a full mutation and a microdeletion. The microdeletion spans 486 bp, involving 168 bp upstream from the CGG repeat region, the entire CGG repeat region, exon 1, and 138 bp of the first intron of the FMR1 gene. In contrast to previous reports, the 5' breakpoint does not fall into the hotspot region. The proximal breakpoint, 5'-GTGGTT/T-3', and the distal breakpoint, 5'-GTTGTT/GG-3', can be characterized as chi-like elements and are flanked by direct tandem repeats. Mosaicism of a full mutation and the microdeletion in the DNA of the patient's leukocytes indicates the mitotic origin of the microdeletion. Since the microdeletion allele is unmethylated, it can be concluded that it is not derived from the methylated full mutation but from an unmethylated premutational allele.