RESUMO
Hirschsprung disease (HSCR) or colonic aganglionosis is a congenital disorder characterized by an absence of intramural ganglia along variable lengths of the colon resulting in intestinal obstruction. The incidence of HSCR is 1 in 5,000 live births. Mutations in the RET gene, which codes for a receptor tyrosine kinase, and in EDNRB which codes for the endothelin-B receptor, have been shown to be associated with HSCR in humans. The lethal-spotted mouse which has pigment abnormalities, but also colonic aganglionosis, carries a mutation in the gene coding for endothelin 3 (Edn3), the ligand for the receptor protein encoded by EDNRB. Here, we describe a mutation of the human gene for endothelin 3 (EDN3), homozygously present in a patient with a combined Waardenburg syndrome type 2 (WS2) and HSCR phenotype (Shah-Waardenburg syndrome). The mutation, Cys159Phe, in exon 3 in the ET-3 like domain of EDN3, presumably affects the proteolytic processing of the preproendothelin to the mature peptide EDN3. The patient's parents were first cousins. A previous child in this family had been diagnosed with a similar combination of HSCR, depigmentation and deafness. Depigmentation and deafness were present in other relatives. Moreover, we present a further indication for the involvement of EDNRB in HSCR by reporting a novel mutation detected in one of 40 unselected HSCR patients.
Assuntos
Endotelinas/genética , Doença de Hirschsprung/complicações , Doença de Hirschsprung/genética , Mutação , Síndrome de Waardenburg/complicações , Síndrome de Waardenburg/genética , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , Primers do DNA/genética , Feminino , Homozigoto , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , Síndrome de Waardenburg/classificaçãoRESUMO
We investigated a possible role of the mismatch-repair gene MLH3 in hereditary nonpolyposis colorectal cancer by scanning for mutations in 39 HNPCC families and in 288 patients suspected of having HNPCC. We identified ten different germline MLH3 variants, one frameshift and nine missense mutations, in 12 patients suspected of HNPCC. Three of the 12 also carried a mutation in MSH6.
Assuntos
Proteínas de Transporte/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Sequência de Bases , DNA , Reparo do DNA/genética , Marcadores Genéticos , Humanos , Proteínas MutL , MutaçãoRESUMO
Denaturing gradient gel electrophoresis (DGGE) has proven to be a powerful pre-screening method for the detection of DNA variants. If such variants occur, however, in DNA fragments that are very rich in G and C, they may escape detection. To overcome this limitation, we tested a novel gel system which combines DGGE and constant denaturant gel electrophoresis (CDGE), as it might have the advantages of both methods. Indeed, this combination had the advantages of both methods, good separation of hetero-duplex molecules and prevention of total strand dissociation, and it proved successful in the detection of DNA variants in several GC-rich fragments.
Assuntos
Proteínas de Ligação a DNA , DNA/genética , Proteínas de Drosophila , Eletroforese em Gel de Poliacrilamida/métodos , Mutação , Proteínas de Saccharomyces cerevisiae , Sequência de Bases , Citosina , DNA/química , Éxons , Proteínas Fúngicas/genética , Guanina , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Denaturing gradient gel electrophoresis (DGGE) is believed to be the most powerful pre-screening method for mutation detection currently available, being used mostly on an exon-by-exon basis. Broad-range DGGE for the analysis of multiple fragments or an entire gene is rarely applied. We and others have already shown that one or two DGGE conditions are usually sufficient to analyse an entire gene. Conditions, however, have never been profoundly tested and compared with alternative methods suggested in the literature. Trying to do so in this study, we found significant differences between the various gel systems. The optimal conditions we found for broad-range DGGE include 9% polyacrylamide for the gel, a denaturing gradient with a difference of 30-50% between the lowest and the highest concentration of denaturant, and electrophoresis in 0.5x TAE buffer at a voltage >100 V and <200 V.
Assuntos
Análise Mutacional de DNA/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Resinas Acrílicas/química , DNA de Neoplasias/química , DNA de Neoplasias/genética , Eletroquímica , Géis , Desnaturação de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
A malignant stromal tumor of the testis with an osteosarcoma component and five of its metastases mainly containing osteosarcoma have been analyzed for RB1 and TP53 abnormalities. Whereas in the primary tumor and in some of the metastases loss of heterozygosity could not be detected for RB1 or for the 17p13 region in which TP53 is located, other metastases showed such losses of heterozygosity. By polymerase chain reaction analysis an 18-base pair deletion from exon 5 of the TP53 gene was found in a small proportion of primary tumor cells and in one of the metastases, but not in the other metastases. Therefore, in this case neither RB1 nor TP53 seems to play an essential role in the initiation of osteosarcoma.
Assuntos
DNA de Neoplasias/química , Genes do Retinoblastoma/genética , Genes p53/genética , Osteossarcoma/genética , Neoplasias Testiculares/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Análise Mutacional de DNA , Humanos , Neoplasias Pulmonares/secundário , Masculino , Dados de Sequência Molecular , Osteossarcoma/patologia , Reação em Cadeia da Polimerase , Neoplasias Testiculares/patologiaRESUMO
Mutation of one p53 allele and loss of the normal p53 allele [loss of heterozygosity (LOH)] occur in many tumors including lung cancers. These alterations apparently contribute to development of cancer by interfering with the tumor suppressor activity of p53. We directly sequenced amplified DNA in the mutational hot spots (exons 4-8) of p53 in DNA samples from 40 lung cancers. Most (31 of 40) samples were preselected for LOH in the region of p53. We detected 23 p53 mutations within these exons in 22 lung cancers; no p53 mutations were found in normal tissue of the patients. One-half of the mutations were G to T transversions on the nontranscribed strand, consistent with mutagenesis by tobacco smoke. Mutations of C to A on the nontranscribed strand, which would result from G to T mutations on the transcribed strand, were detected only in one sample. Three of 23 mutations were nonsense mutations; to date, nonsense mutations of p53 have not been reported in lung cancer. Mutation of this p53-coding region was detected in 20 of 27 small cell lung cancer samples, representing a 70% occurrence. Mutation of the p53 gene is apparently very frequent in small cell lung cancers. When LOH in the p53 region could be determined, complete concordance occurred between a sample having both a p53 mutation and LOH in the region of p53 (18 of 18 samples). Twelve samples of lung cancer had LOH in the region of p53, but the samples had no detectable p53 mutations, suggesting either alterations outside the known mutational hot spots of p53 or alterations of another unidentified tumor suppressor gene in the region of p53.
Assuntos
Genes p53 , Neoplasias Pulmonares/genética , Mutação , Proteína Supressora de Tumor p53/genética , Sequência de Aminoácidos , Sequência de Bases , Carcinoma de Células Pequenas/genética , Códon , Éxons , Humanos , Dados de Sequência Molecular , OligodesoxirribonucleotídeosRESUMO
Germ line mutations in one allele of the RET proto-oncogene predispose to the multiple endocrine neoplasia type 2 (MEN 2) syndromes. To investigate whether these inherited mutations alone can cause the development of tumors in vivo (oncogene model) or whether somatic mutations in the homologous RET allele are required for tumorigenesis (tumor suppressor gene model), we analyzed the entire coding region of both alleles of the RET gene in two MEN 2A and two MEN 2B tumors by reverse transcription-PCR and direct sequencing. No tumor-specific mutations could be detected in either allele of the RET gene in these tumors. Unlike the molecular mechanism in other hereditary tumor syndromes, somatic mutations in the homologous allele are apparently not required in MEN 2 tumorigenesis. Thus, RET genes with MEN 2-specific germ line mutations act as dominantly transforming oncogenes in vivo.
Assuntos
Proteínas de Drosophila , Heterozigoto , Neoplasia Endócrina Múltipla Tipo 2a/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , DNA Complementar/química , Humanos , Masculino , Polimorfismo Genético , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-retRESUMO
Three new, well growing cell lines (GLC-1, GLC-2, and GLC-3) have been established from small cell lung carcinoma (SCLC) and characterized. A subclone (GLC-1-M13) markedly different from its parent line GLC-1 was also isolated and characterized. Cytogenetic analysis of the cell lines revealed deletions in the short arm of chromosome 3 as a most consistent chromosomal aberration. The deleted region was not identical in all metaphases, 3p(21-23) being the shortest region of overlap. Despite their SCLC origin GLC-1, GLC-2, and GLC-3 do not show pronounced SCLC differentiation features. Neurosecretory granula were very rare (GLC-1) or completely absent (GLC-2 and GLC-3), whereas the SCLC-related enzyme and hormone markers L-3,4-dihydroxyphenylalanine decarboxylase, neuron-specific enolase, creatine kinase BB, and bombesin-like immunoreactivity were variably expressed. Although the subclone GLC-1-M13 was derived from the poorly differentiated GLC-1, it behaved according to the above criteria as a differentiated "classic" SCLC cell line. When assessed with specific monoclonal antibodies the different cell lines appeared to express different subsets of intermediate filament proteins, indicative for different stages and directions of differentiation: "undifferentiated" (GLC-1 and GLC-2); "neural tissue related" (GLC-2); "simple epithelium" related (GLC-1-M13); and a combination of simple and squamous epithelium related (GLC-3). We conclude that GLC-1, GLC-2, and GLC-3 represent dedifferentiated forms of SCLC, related to the recently described "variant" type of SCLC, whereas the clonal derivate GLC-1-M13 behaves like a differentiated "classic" SCLC cell line.
Assuntos
Carcinoma de Células Pequenas/patologia , Neoplasias Pulmonares/patologia , Carcinoma de Células Pequenas/metabolismo , Linhagem Celular , Hormônios/metabolismo , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Cariotipagem , Neoplasias Pulmonares/metabolismo , Microscopia EletrônicaRESUMO
Three classic-type, small cell lung cancer cell lines (GLC-14, GLC-16, and GLC-19) have been established from one patient during longitudinal follow-up. During this period the tumor changed from sensitive to completely resistant to (chemo)therapy. A phenotypical and functional characterization of the different cell lines is given in combination with the matching clinical data. (a) The cell lines have been compared with the biopsies from which they were derived. There was a good match between the morphological, biochemical, and immunohistological findings in the cell lines as compared to those obtained in the biopsies. When the biopsy and cell line (GLC-14) obtained before the start of therapy were compared to the biopsies and cell lines (GLC-16 and GLC-19) acquired after the first and second reinduction therapy, respectively, no major changes could be observed. The only clear alteration was the loss of a neuroendocrine antigen (defined by monoclonal antibody MOC-51) in the posttherapy specimens. (b) The doxorubicin, melphalan, and etoposide sensitivity in vitro reflected the clinically observed development of resistance to treatment. The cell line (GLC-14) established before the start of therapy was more sensitive than the lines (GLC-16 and GLC-19) obtained after treatment. It is concluded that the cell lines described in this paper represent a well-characterized in vitro model in which the development of drug resistance in small cell lung cancer can be studied.
Assuntos
Carcinoma de Células Pequenas/patologia , Neoplasias Pulmonares/patologia , Antígenos de Diferenciação/análise , Antígenos de Neoplasias/análise , Antineoplásicos/farmacologia , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/imunologia , Linhagem Celular , Aberrações Cromossômicas , Resistência a Medicamentos , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Estudos Longitudinais , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Pessoa de Meia-Idade , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), a M(r) 38,000 transmembrane antigen also known as 17-1A or Ep-CAM, is commonly used for targeted immunotherapy of carcinomas because it is strongly expressed by most carcinomas. EGP-2 is, however, also expressed in most normal epithelia. To evaluate anti-EGP-2-directed treatment-associated effects on tumors and on EGP-2-positive normal tissue, we generated EGP-2-expressing transgenic mice. A 55-kb DNA fragment consisting of the 14-kb genomic coding sequence of the human EGP-2 gene with approximately 10-kb-upstream and approximately 31-kb-downstream sequences was isolated and used to direct EGP-2 expression in an epithelium-specific manner. In the EGP-2 transgenic mice, EGP-2 appeared to be specifically expressed in all of those epithelial tissues that also express EGP-2 in humans, whereas all of the other tissues were negative. The specific in vivo localization of the i.v. administered anti-EGP-2 monoclonal antibody MOC31 was studied in EGP-2-positive and -negative tumors induced s.c. in this EGP-2 transgenic mouse model. Immunohistochemical analysis showed specific localization of MOC31 in the EGP-2-positive tumors but not in the EGP-2-negative tumors. No anti-EGP-2 monoclonal antibody localization was observed in any of the EGP-2-positive normal mouse tissues, which indicated a limited in vivo accessibility. In conclusion, an EGP-2 transgenic mouse model has been generated that expresses the EGP-2 antigen as in humans and, therefore, can serve as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities in both tumors and normal tissue.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Imunoterapia/métodos , Melanoma Experimental/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos de Neoplasias/biossíntese , Moléculas de Adesão Celular/biossíntese , Modelos Animais de Doenças , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Masculino , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras GenéticasRESUMO
The MEN2A oncogene encodes for a constitutive active member of the RET receptor tyrosine kinase family. Here, we report that MEN2A-RET activates Signal Transducer and Activator of Transcription 3 (STAT3) via two YxxV/Q STAT3 docking sites, Tyr752 and Tyr928. MEN2A-RET induces both Tyr705 and Ser727 phosphorylation of STAT3, and STAT3 serine phosphorylation is required for its maximal transcriptional activity. Stable NIH3T3 cell lines expressing both MEN2A-RET and STAT3alpha but not STAT3beta, are characterized by enhanced proliferation and cyclin-D1 promoter activity, and enhanced growth in soft agar. These data indicate that malignant cell growth induced by MEN2A-RET involves its activation of STAT3.
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transativadores/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Células COS , Divisão Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Genes Reporter , Humanos , Camundongos , Oncogenes/genética , Fosforilação , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ret , Fator de Transcrição STAT3 , Serina/química , Fatores de Tempo , Ativação Transcricional , Transfecção , Tirosina/química , Regulação para CimaRESUMO
The RET proto-oncogene, a transmembrane tyrosine kinase receptor, is involved in the development of at least five different disease phenotypes. RET is activated through somatic rearrangements in a number of cases of papillary thyroid carcinoma while germ-line point mutations are associated with three inherited cancer syndromes MEN 2A, MEN 2B and FMTC. Moreover, point mutations or heterozygous deletions of RET are found in the dominant form of Hirschsprung disease or congenital colonic aganglionosis. We cloned the entire RET genomic sequence in a contig of cosmids encompassing 150 kb, from the CA repeat sTCL-2 to the region upstream the RET promoter, and established the position of the 20 exons of the RET gene with respect to a detailed restriction map based on eight endonucleases. A new highly polymorphic CA repeat sequence was identified within intron 5 of RET (RET-INT5). Finally the orientation of RET on chromosome 10q11.2 made it possible to orientate three other genes rearranged with RET in papillary thyroid carcinomas, namely H4/D10S170 on 10q21, R1 alpha on 17q23 and RFG2/Ele1 on 10q11.2.
Assuntos
Cromossomos Humanos Par 10 , Proteínas de Drosophila , Rearranjo Gênico , Hominidae/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Animais , Sequência de Bases , Carcinoma Medular/genética , Carcinoma Papilar/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 17 , Cosmídeos , Primers do DNA , Éxons , Doença de Hirschsprung/genética , Humanos , Íntrons , Dados de Sequência Molecular , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2b/genética , Mutação Puntual , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Neoplasias da Glândula Tireoide/genéticaRESUMO
High frequencies of allelic loss on the short arm of chromosome 3 in small cell lung cancer (SCLC) and a number of other tumors suggest the existence of a tumor suppressor gene(s) within the deleted regions. Two small cell lung cancer lines, NCI H740 and GLC20, have been described which have homozygous deletions in the region 3p21.3. The deleted region overlaps with a 2 Mb fragment of human DNA present in the interspecies hybrid HA(3)BB9F, that suppresses tumor formation by mouse A9 fibrosarcoma cells. Human sequences from this cell hybrid were isolated using inter Alu PCR. From this starting point, a P1 contig was developed for the region of 450 Kb that is common to the homozygous deletions seen in the SCLC lines NCI H740 and GLC20 and is also present in HA(3)BB9F, the suppressed A9 hybrid. Individual P1 clones were assayed for their ability to suppress the tumorigenicity of the mouse fibrosarcoma cell line A9 as assayed by injection of transfected A9 cells into athymic nude mice. The introduction of one of the P1 clones into A9 cells resulted in suppression of tumor growth whereas two other P1 clones from the contig failed to suppress tumor formation in athymic nude mice. These data functionally delimit a tumor suppressor locus to a region of 80 kb within a P1 clone at 3p21.3.
Assuntos
Carcinoma de Células Pequenas/genética , Cromossomos Humanos Par 3/genética , Genes Supressores de Tumor/genética , Neoplasias Pulmonares/genética , Deleção de Sequência , Animais , Carcinoma de Células Pequenas/patologia , Fibrossarcoma/genética , Marcadores Genéticos , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Sondas de Oligonucleotídeos/genética , Análise de Sequência de DNA/métodos , Células Tumorais CultivadasRESUMO
The concept that cells can become malignant upon the elimination of parts of chromosomes inhibiting cell division dates back to Boveri in 1914. Deletions occurring in tumor cells are therefore considered a first indication of possible locations of tumor suppressor gene. Approaches used to localize and identify the paradigm of tumor suppressors, RB1, have also been applied to localize tumor suppressor genes on 3p, the short arm of chromosome 3. This review discusses the methodological advantages and limitations of the various approaches. From a review of the literature on losses of 3p in different types of solid tumors it appears that some tumor types show involvement of the same region, while between others the regions involved clearly differ. Also discussed are results of functional assays of tumor suppression by transfer of part of chromosome 3 into tumor cell lines. The likelihood that a common region of deletions would contain a tumor suppressor is strongly enhanced by coincidence of that region with a chromosome fragment suppressing tumorigenicity upon introduction in tumor cells. Such a situation exists for a region in 3p21.3 as well as for one or more in 3p12-p14. The former region is considered the location of a lung cancer suppressor. The same gene or a different one in the same region may also play a role in the development of other cancers including renal cell cancer. In the latter cancer, there may be additional roles of the VHL region and/or a 3p12-p14 region. The breakpoint region of a t(3;8) originally found to be constitutively present in a family with hereditary renal cell cancer now seems to be excluded from such a role. Specific genes on 3p have been suggested to act as suppressor genes based on either their location in a common deletion region, a markedly reduced expression or presence of aberrant transcripts, their capacity to suppress tumorigenicity upon transfection in to tumor cells, the presumed function of the gene product, or a combination of several of these criteria. A number of genes are evaluated for their possible role as a tumor suppressor according to these criteria. General agreement on such a role seems to exist only for VHL. Though hMLH1 plays an obvious role in the development of specific mismatch repair-deficient cancers, it cannot revert the tumor phenotype and therefore cannot be considered a proper tumor suppressor. The involvement of VHL and MLH1 also in some specific hereditary cancers allowed to successfully apply linkage analysis for their localization. TGFBR2 might well have a tumor suppressor function. It does reduce tumorigenicity upon transfection. Other 3p genes coding for receptor proteins THRB and RARB, are unlikely candidates for tumor suppression. Present observations on a possible association of FHIT with tumor development leave a number of questions unanswered, so that provisionally it cannot be considered a tumor suppressor. Regions that have been identified as crucial in solid tumor development appear to be at the edge of synteny blocks that have been rearranged through the chromosome evolution which led to the formation of human chromosome 3. Although this may merely represent a chance occurrence, it might also reflect areas of genomic instability.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 3 , Genes Supressores de Tumor , Neoplasias/genética , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Evolução Biológica , Proteínas de Transporte , Fusão Celular , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Heterozigoto , Humanos , Cariotipagem , Proteína 1 Homóloga a MutL , Proteínas de Neoplasias/genética , Proteínas Nucleares , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Transfecção , Doença de von Hippel-Lindau/genéticaRESUMO
The clearance from the blood and the conformation of serum albumin modified by nitroguanidination and labeled with 125-I have been studied. Like formaldehyde-denatured albumin, but in contrast to native albumin, the nitroguanidinated derivative is rapidly cleared from the blood and taken up in lysosomes of liver sinusoidal cells. Although 94% of the free amino groups were blocked by nitroguanidination, we could not detect significant conformational changes using gel filtration, determination of reducible disulfide groups, and titration of tyrosine residues. It is concluded that extensive denaturation is no prerequisite for the uptake of albumin derivatives in liver sinusoidal cells. It is suggested that the nitroguanidinated protein, in contrast to native albumin, is bound on membrane receptors of sinusoidal cells. The nitroguanidino groups themselves might be bound on these receptors, but it seems equally possible that the blocking of positive charges of the albumin molecule or minor, local conformational changes of the protein are sufficient for the binding on the receptors.
Assuntos
Fígado/metabolismo , Soroalbumina Bovina/metabolismo , Fosfatase Ácida/metabolismo , Animais , Transporte Biológico , Catepsinas/metabolismo , Cromatografia em Gel , Guanidinas , Fígado/citologia , Nitrocompostos , Ligação Proteica , Conformação Proteica , Ratos , Soroalbumina Radioiodada/metabolismo , Fatores de TempoRESUMO
We identified a novel mutation (867delA) in the glucose-6-phosphatase gene of two siblings with glycogen storage disease type Ia. Although both siblings share the same mutations, their phenotype regarding adult height and hepatomegaly differs. In glycogen storage disease type Ia, substantial heterogeneity in phenotype is observed. So far, no evidence for a clear genotype-phenotype correlation has been found. Hum Mutat 15:381, 2000.
Assuntos
Mutação da Fase de Leitura , Glucose-6-Fosfatase/genética , Doença de Depósito de Glicogênio/enzimologia , Doença de Depósito de Glicogênio/genética , Adulto , Éxons/genética , Feminino , Genótipo , Humanos , Masculino , Núcleo Familiar , Fenótipo , Polimorfismo Conformacional de Fita SimplesRESUMO
In patients with the major forms of epidermolysis bullosa simplex, either of the keratin genes KRT5 or KRT14 is mutated. This causes a disturbance of the filament network resulting in skin fragility and blistering. For KRT5, a genomic mutation detection system has been described previously. Mutation detection of KRT14 on a DNA level is, however, hampered by the presence of a highly homologous but nontranscribed KRT14 pseudogene. Consequently, mutation detection in epidermolysis bullosa simplex has mostly been carried out on cDNA synthesized from KRT5 and KRT14 transcripts in mRNA isolated from skin biopsies. Here we present a genomic mutation detection system for exons 1, 4, and 6 of KRT14 that encode the 1A, L1-2, and 2B domains of the keratin 14 protein containing the mutation hotspots. After cutting the KRT14 pseudogene genomic sequences with restriction enzymes while leaving the homologous genomic sequences of the functional gene intact, only the mutation hotspot-containing exons of the functional KRT14 gene are amplified. This is followed by direct sequencing of the polymerase chain reaction products. In this way, three novel mutations could be identified, Y415H, L419Q, and E422K, all located in the helix termination motif of the keratin 14 rod domain 2B, resulting in moderate, severe, and mild epidermolysis bullosa simplex phenotype, respectively. By obviating the need of KRT14 cDNA synthesis from RNA isolated from skin biopsies, this approach substantially facilitates the detection of KRT14 hotspot mutations.
Assuntos
Análise Mutacional de DNA , Queratinas/genética , Pseudogenes/genética , Epidermólise Bolhosa Simples , Mutação da Fase de Leitura , Amplificação de Genes , Humanos , Queratina-14 , Mutação PuntualRESUMO
In several families, multiple endocrine neoplasia type 2A (MEN 2A) has been found in association with cutaneous lichen amyloidosis. It has been debated, however, whether the skin amyloidosis found in MEN 2A families, localized exclusively in the interscapular area, represents the same anomaly as that found in autosomal dominant familial cutaneous lichen amyloidosis, which is more generalized. We screened two MEN 2A families with associated skin amyloidosis for germline mutations in the RET gene responsible for the MEN 2A cancer syndrome, and found the same mutation characteristic of MEN 2A in both families. We also screened probands from three pedigrees with familial cutaneous lichen amyloidosis for RET mutations. In none of the RET coding and flanking intronic sequences was a mutation detected. This most probably indicates that skin amyloidosis found in some MEN 2A families and familial cutaneous lichen amyloidosis are different conditions. Consequently, patients with apparent familial cutaneous lichen amyloidosis do not appear to be at risk for MEN 2A.
Assuntos
Amiloidose/genética , Testes Genéticos , Erupções Liquenoides/complicações , Erupções Liquenoides/genética , Neoplasia Endócrina Múltipla Tipo 2a/complicações , Mutação , Dermatopatias/genética , Sequência de Bases , Haplótipos , Humanos , Sondas Moleculares/genética , Dados de Sequência Molecular , Neoplasia Endócrina Múltipla Tipo 2a/genética , Linhagem , Polimorfismo GenéticoRESUMO
In adults, the TP53 tumor suppressor gene is frequently mutated in astrocytic brain tumors which is supposed to represent an early event in their development. In juvenile pilocytic and low-grade astrocytomas, however, TP53 mutations have until now been reported as rare, which has led to the suggestion that these tumors may follow a different molecular pathogenesis with an involvement of genes other than TP53. Our analysis of 20 pilocytic and two low-grade astrocytomas of childhood, based on a comprehensive denaturing gradient gel electrophoresis (DGGE) mutation detection assay of the entire coding region, including all splice site junctions of TP53, showed mutations considered as causative in 7 of the 20 (35%) pilocytic astrocytomas and in one of the two low-grade astrocytomas. Our finding is significantly different from the mutation frequency of 1.3% (2/155) previously reported for these tumor types. This may be attributed to the mutation detection system used, which also detects mutations occurring outside the evolutionary conserved region of TP53. Our results suggest that, contrary to the present notion, TP53 mutations may well play a role in the development of juvenile astrocytomas. Furthermore, no mutations were found in tumors of patients with progression of residual tumor after postoperative follow-up. This suggests that TP53 mutations may be associated with less aggressive forms of juvenile astrocytomas, analogous to the situation in adult astrocytomas.
Assuntos
Astrocitoma/genética , Proteína Supressora de Tumor p53/genética , Adolescente , Substituição de Aminoácidos , Criança , Pré-Escolar , Análise Mutacional de DNA , Eletroforese em Gel de Poliacrilamida , Éxons , Homozigoto , Humanos , Lactente , Recém-Nascido , Íntrons , Mutação , Neoplasia Residual/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Specific mutations in the ret protooncogene have been found associated with multiple endocrine neoplasia type 2A (MEN 2A) and type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). Mutations in one of five cysteine residues in the extracellular domain have been found in over 95% of families with MEN 2A and 88% of families with FMTC. In MEN 2B patients, a specific mutation at codon 918, substituting a threonine for a methionine, has been found in 95% of cases. In FMTC, in addition to the mutations of the extracellular cysteines, three intracellular base pair changes have been reported at codons 768 and 804. Here we describe a novel intracellular mutation in exon 15 of the ret gene that leads to the substitution of an alanine for a serine at codon 891 in a family with medullary thyroid carcinoma. This amino acid change may be important in determining substrate specificity or, alternatively, may play a role in ATP binding.