RESUMO
BACKGROUND: Cadherin switch in melanoma, with loss of E-cadherin and upregulation of N-cadherin, is believed to underlie melanoma cell detachment from the epidermis and promotion of dermal and vascular melanoma invasion. The tumour suppressor phosphatase and tensin homolog (PTEN) has been suggested as a potential regulator of this cadherin switch. OBJECTIVES: To study the biological and clinical implications of cadherin switch and PTEN expression in melanoma progression. METHODS: We constructed tissue microarrays from primary tumour samples from 394 formalin-fixed paraffin-embedded melanomas diagnosed between 2001 and 2006. Median follow-up was 10 years. Tissue microarray sections were stained by immunohistochemistry for E-cadherin, N-cadherin and PTEN, and expression was analysed semiquantitatively. RESULTS: Breslow thickness correlated strongly with reduced/absent PTEN expression (P < 0·0001), low E-cadherin expression (P < 0·0001), high N-cadherin expression (P < 0·0001) and the combination of low E-cadherin and high N-cadherin expression (cadherin switch profile; P = 0·001). There was a significant association between reduced/absent PTEN and the presence of the cadherin switch profile (P = 0·03). In univariate analyses, low E-cadherin expression significantly predicted an adverse overall relapse-free (P = 0·04), melanoma-specific (P = 0·03) and distant-metastasis-free (P = 0·01) survival; reduced/absent PTEN predicted an adverse overall relapse-free survival (P = 0·006), and the cadherin switch profile predicted adverse melanoma-specific (P = 0·005) and distant-metastasis-free (P = 0·01) survival. In multivariate analysis, the cadherin switch profile was an independent prognostic marker of melanoma-specific (P = 0·04) and distant-metastasis-free survival (P = 0·02). CONCLUSIONS: Cadherin switch and reduced/absent PTEN expression are associated in melanoma, and both factors may play important roles in the progression of melanoma.
Assuntos
Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Melanoma/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias Cutâneas/metabolismo , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Melanoma/mortalidade , Pessoa de Meia-Idade , Variações Dependentes do Observador , Neoplasias Cutâneas/mortalidade , Análise Serial de Tecidos , Regulação para CimaRESUMO
We have mapped the autosomal sex reversal locus, SRA1, associated with campomelic dysplasia (CMPD1) to 17q24.3-q25.1 by three independent apparently balanced de novo reciprocal translocations. Chromosome painting indicates that the translocated segment of 17q involves about 15% of chromosome 17 in all three translocations, corresponding to a breakpoint at the interphase between 17q24-q25. All three 17q breakpoints were localized distal to the growth hormone locus (GH), and proximal to thymidine kinase (TK1). Due to the distal location of the breakpoints, previously mentioned candidate genes, HOX2 and COL1A1, can be excluded as being involved in CMPD1/SRA1. The mouse mutant tail-short (Ts) which maps to the homologous syntenic region on mouse chromosome 11, displays some of the features of CMPD1.
Assuntos
Anormalidades Múltiplas/genética , Doenças do Desenvolvimento Ósseo/genética , Aberrações Cromossômicas/genética , Cromossomos Humanos Par 13/ultraestrutura , Cromossomos Humanos Par 17/ultraestrutura , Cromossomos Humanos Par 1/ultraestrutura , Cromossomos Humanos Par 7/ultraestrutura , Transtornos do Desenvolvimento Sexual , Disgenesia Gonadal/genética , Análise para Determinação do Sexo , Translocação Genética , Adulto , Sequência de Bases , Transtornos Cromossômicos , Mapeamento Cromossômico , Feminino , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Masculino , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
Malignant melanoma is an aggressive, heterogeneous disease where new biomarkers for diagnosis and clinical outcome are needed. We searched for chromosomal aberrations that characterize its pathogenesis using 47 different melanoma cell lines and tiling-resolution bacterial artificial chromosome-arrays for comparative genomic hybridization. Major melanoma genes, including BRAF, NRAS, CDKN2A, TP53, CTNNB1, CDK4 and PTEN, were examined for mutations. Distinct copy number alterations were detected, including loss or gain of whole chromosomes but also minute amplifications and homozygous deletions. Most common overlapping regions with losses were mapped to 9p24.3-q13, 10 and 11q14.1-qter, whereas copy number gains were most frequent on chromosomes 1q, 7, 17q and 20q. Amplifications were delineated to oncogenes such as MITF (3p14), CCND1 (11q13), MDM2 (12q15), CCNE1 (19q12) and NOTCH2 (1p12). Frequent findings of homozygous deletions on 9p21 and 10q23 confirmed the importance of CDKN2A and PTEN. Pair-wise comparisons revealed distinct sets of alterations, for example, mutually exclusive mutations in BRAF and NRAS, mutual mutations in BRAF and PTEN, concomitant chromosome 7 gain and 10 loss and concomitant chromosome 15q22.2-q26.3 gain and 20 gain. Moreover, alterations of the various melanoma genes were associated with distinct chromosomal imbalances suggestive of specific genomic programs in melanoma development.
Assuntos
Aberrações Cromossômicas , Genes Neoplásicos/genética , Melanoma/genética , Neoplasias Cutâneas/genética , Linhagem Celular Tumoral , Análise Mutacional de DNA , Amplificação de Genes , Dosagem de Genes , Genômica , Humanos , Mutação , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The mts1/S100A4 gene encodes a small acidic calcium-binding protein that is expressed in a cell-specific manner in development, tumorigenesis and certain tissues of adult mice. A composite enhancer that is active in murine mammary adenocarcinoma cells was previously identified in the first intron of the mts1/S100A4 gene. Here we present a detailed analysis of the structure and function of this enhancer in the Mts1/S100A4-expressing CSML100 and non-expressing CSML0 mouse adenocarcinoma cell lines. In CSML100 cells the enhancer activity is composed of at least six cis-elements interacting with Sp1 and AP-1 family members and CBF/AML/PEBP2 and KRC transcription factors. In addition, a minisatellite-like DNA sequence significantly contributes to the enhancer activity via interaction with abundant proteins, which likely have been described previously under the name minisatellite-binding proteins. Extensive mutational analysis of the mts1/S100A4 enhancer revealed a cooperative function of KRC and the factors binding minisatellite DNA. This is the first example of an enhancer where two nuclear factors earlier implicated in different recombination processes cooperate to activate transcription. In Mts1/S100A4-negative CSML0 cells the strength of the enhancer was 7- to 12.5-fold lower compared to that in CSML100 cells, when referred to the activities of three viral promoters. In CSML0 cells the enhancer could be activated by exogenous AP-1 and CBF transcription factors.
Assuntos
Elementos Facilitadores Genéticos/genética , Genes p16/genética , Íntrons/genética , Repetições Minissatélites/genética , Metástase Neoplásica/genética , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Sítio Alostérico , Animais , Sequência de Bases , Proteína de Ligação a CREB , DNA/genética , DNA/metabolismo , Pegada de DNA , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Virais/genética , Camundongos , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Fator de Transcrição Sp1/metabolismo , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Células Tumorais CultivadasRESUMO
Silencing of the cyclin-dependent kinase inhibitor gene p15INK4B by cytosine methylation of the promoter region has been associated with some types of hematological malignancy. To study in detail the patterns of p15INK4B methylation in patients with acute myeloid leukemia, we adopted a novel approach based on PCR amplification of bisulfite-treated DNA followed by resolution of differentially methylated sequences by denaturing gradient gel electrophoresis. This method visually displays the degree and heterogeneity of DNA methylation and enables the isolation and characterization of distinct clonotypic epigenotypes. A surprisingly high degree of intra- and interindividual heterogeneity of p15INK4B methylation was observed in the 65 acute myeloid leukemia patients examined. Methylation was detected in 46 (71%) of the patients and was observed more frequently in the French-American-British subtypes M1/M2 than in M4/M5 (P < 0.025). Examination of the same panel of samples using a highly sensitive methylation-specific PCR method showed methylated p15INK4B alleles in 61 (94%) of the samples. We present evidence that the higher frequency of p15INK4B methylation determined by methylation-specific PCR may, at least in part, be due to the presence of a small fraction of p15INK4B-methylated lymphocytes in normal blood.
Assuntos
Proteínas de Ciclo Celular , Metilação de DNA , Leucemia Mieloide Aguda/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor , Alelos , Sequência de Bases , Inibidor de Quinase Dependente de Ciclina p15 , Humanos , Leucócitos Mononucleares/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
The MMAC1/PTEN gene, located at 10q23.3, is a candidate tumor suppressor commonly mutated in glioma. We have studied the pattern of deletion, mutation, and expression of MMAC1/PTEN in 35 unrelated melanoma cell lines. Nine (26%) of the cell lines showed partial or complete homozygous deletion of the MMAC1/PTEN gene, and another six (17%) harbored a mutation in combination with loss of the second allele. Mutations could also be demonstrated in uncultured tumor specimens from which the cell lines had been established, and cell lines derived from two different metastases from one individual carried the same missense mutation. Collectively, these findings suggest that disruption of MMAC1/PTEN by allelic loss or mutation may contribute to the pathogenesis or neoplastic evolution in a large proportion of malignant melanomas.
Assuntos
Cromossomos Humanos Par 10/genética , Deleção de Genes , Genes Supressores de Tumor/genética , Melanoma/genética , Mutação , Monoéster Fosfórico Hidrolases , Proteínas Tirosina Fosfatases/genética , Neoplasias Cutâneas/genética , Proteínas Supressoras de Tumor , Sequência de Bases , Éxons/genética , Humanos , Dados de Sequência Molecular , PTEN Fosfo-Hidrolase , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
The p16Ink4/CDKN2, D-type cyclins, their partners Cdk4/Cdk6, and pRb constitute a G1 regulatory pathway commonly targeted in tumorigenesis. Genetic, immunochemical, and functional cell cycle analyses showed abnormalities of this pathway in each of 22 human melanoma cell lines examined. Normal melanocytes and all melanoma lines expressed Cdk4, Cdk6, and cyclins D1 and D3. The tumor suppressors p16Ink4/CDKN2 and pRb were lost in 17 and 4 cases, respectively, due to various genetic mechanisms, including transcriptional block of p16 and nonsense mutations of RB1. Ectopic expression of p16 prevented S-phase entry of Rb+/p16- but not Rb-deficient melanoma lines. The SK29-MEL-1 cell line harboring an R24C mutation in Cdk4 expressed wild-type pRb and overabundant p16, the latter preventing endogenous Cdk6 but not Cdk4 from associating with cyclin D1. Microinjection of cyclin D1-neutralizing antibody arrested the SK29-MEL-1 cells in G1, whereas pl6 did not, indicating that the cyclin D1/Cdk4-R24C complex is required for G1 progression, and the resistance of the complex to p16 in vivo. These data strongly support the candidacy of Cdk4 as a novel proto-oncogene, provide further evidence for the p16-cyclin D/Cdk-pRb pathway as a functional unit, and suggest that deregulation of this checkpoint may represent a common step in the multistep progression of sporadic malignant melanomas.
Assuntos
Proteínas de Transporte/fisiologia , Transformação Celular Neoplásica/genética , Quinases Ciclina-Dependentes/fisiologia , Ciclinas/fisiologia , Fase G1/fisiologia , Melanoma/etiologia , Proteínas Proto-Oncogênicas , Proteína do Retinoblastoma/fisiologia , Transdução de Sinais/fisiologia , Sequência de Bases , Proteínas de Transporte/genética , Ciclina D , Quinase 4 Dependente de Ciclina , Inibidor p16 de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Progressão da Doença , Deleção de Genes , Genes do Retinoblastoma , Humanos , Melanoma/genética , Melanoma/fisiopatologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proto-Oncogene Mas , Proto-Oncogenes , Células Tumorais CultivadasRESUMO
p27Kip1 is a regulator of the mammalian cell cycle and a putative tumor suppressor. Distinct altered patterns of p27Kip1 protein expression are found in a variety of human carcinomas, and p27Kip1 expression levels usually correlate directly with disease-free survival. The mechanism(s) by which p27Kip1 expression is reduced or lost during tumorigenesis remains unclear. Specific alterations of the p27Kip1 gene, including mutations and homozygous deletions, are exceedingly rare in human cancers. We have used methylation-specific PCR and bisulfite genomic sequencing to examine the methylation status of p27Kip1 in 61 primary and metastatic tumors and 35 cell lines from patients with malignant melanoma. Dense methylation of a CpG island in the promoter region of p27Kip1 was detected in four of 45 metastatic tumors (9%) and three of the cell lines (9%), including two cell lines established from two different metastases from the same patient. Examination of a naturally occurring, allele-specific sequence variant demonstrated that p27Kip1 methylation is associated with transcriptional silencing in situ. Cell lines with p27Kip1 methylation showed retention of the wild-type allele and detectable p27Kip1 protein whose abundance was reduced compared with normal melanocytes. Collectively, our data suggest that DNA methylation may be a cause of monoallelic p27Kip1 silencing in malignant melanoma, which would support a role for p27Kip1 haploinsufficiency in human cancer.
Assuntos
Proteínas de Ciclo Celular , Metilação de DNA , Melanoma/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Supressoras de Tumor , Substituição de Aminoácidos , Ilhas de CpG/fisiologia , Inibidor de Quinase Dependente de Ciclina p27 , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Humanos , Melanócitos/metabolismo , Melanoma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ativação TranscricionalRESUMO
Mutations in LKB1/STK11, a gene mapping to chromosome 19p13.3 and encoding a widely expressed serine/threonine kinase, were recently identified as the cause of Peutz-Jeghers syndrome. Despite the hamartomatous polyps and increased cancer risk associated with this syndrome, somatic alterations in LKB1/STK11 have not been identified in human tumours. Prompted by another feature of the syndrome, lentigines of the lips and oral mucosa, we evaluated the status of LKB1/STK11 expression, deletion, and mutation in cell lines and tumour samples from 35 patients with sporadic malignant melanoma. Two somatic mutations were identified, a nonsense mutation (Glu170Stop) causing exon skipping and intron retention, and a missense mutation (Asp194Tyr) affecting an invariant residue in the catalytic subunit of LKB1/STK11. Our data suggest that LKB1/STK11 may contribute to tumorigenesis in a small fraction of malignant melanomas.
Assuntos
Mutação , Síndrome de Peutz-Jeghers/enzimologia , Síndrome de Peutz-Jeghers/genética , Proteínas Serina-Treonina Quinases/genética , Quinases Proteína-Quinases Ativadas por AMP , Humanos , Melanoma , Células Tumorais CultivadasRESUMO
The INK4a/ARF locus at chromosome 9p21 encodes two structurally and functionally distinct molecules with tumor-suppressive properties. p16INK4a controls cell cycle progression by inhibiting phosphorylation of the retinoblastoma protein (Rb), while ARF prevents MDM2-mediated degradation of p53. By using a panel of PCR-based methods, we have examined the status of the p16INK4a, ARF and p53 genes in 123 cases of non-Hodgkin's lymphoma (NHL) at diagnosis. Alterations of one or more of these genes were detected in seven of 36 (19%) cases with low- to intermediate-grade histology, and in 35 of 87 (40%) cases with aggressive histology. For the aggressive lymphomas, the Kaplan-Meier estimate of overall survival for cases with disruption of either p16INK4a or the ARF-p53 pathway was not different from cases with retention of both pathways (5 year survival 45% vs 35%; P= 0.85), suggesting that selective inactivation of one of the pathways does not significantly influence overall survival. By contrast, the 5-year survival was only 7% for cases with concurrent disruption of p16INK4a and the ARF-p53 pathway vs 38% for cases with retention of one or both pathways (P = 0.005). Similar results were obtained when the analysis was confined to diffuse large B cell lymphomas (P= 0.019). On stepwise multivariate regression analysis including factors from the international prognostic index, concurrent disruption of p16INK4a and the ARF-p53 pathway was an independent negative prognostic factor in NHL with aggressive histology (P = 0.006). Our results suggest that the compound status of the p16INK4a and ARF-p53 pathways is a major determinant of outcome in NHL.
Assuntos
Fatores de Ribosilação do ADP/genética , Genes p16 , Genes p53 , Linfoma não Hodgkin/genética , Metilação de DNA , Deleção de Genes , Humanos , Linfoma não Hodgkin/patologia , Mutação , Prognóstico , Regiões Promotoras Genéticas , Análise de SobrevidaRESUMO
Mantle cell lymphomas (MCL) are morphologically and immunophenotypically distinctive lymphoid neoplasms characterised by overexpression of cyclin D1. Recent studies have suggested that co-operating aberrations of cell cycle associated genes may provide a growth advantage to a tumour. To address this issue further, we investigated five typical and three aggressive (blastoid) MCL for alterations in the cell cycle regulating genes p15, p16, CDK4, Rb and p53. In 3/3 aggressive cases with cyclin D1 overexpression we found aberration of at least one additional gene. One case showed diminished expression of the retinoblastoma protein (pRb); one case harboured deletion of both p15 and p16; and one case exhibited both deletion of p16 and point mutation of p53. However, we also identified two typical cases which in addition to cyclin D1 overexpression exhibited diminished pRb expression and p15 and p16 hypermethylation, respectively. Our findings confirm and extend other recent investigations and indicate that co-operating genetic alterations of cell cycle-associated genes may contribute to the pathogenesis of MCL.
Assuntos
Proteínas de Ciclo Celular , Genes cdc , Linfoma não Hodgkin/genética , Proteínas Proto-Oncogênicas , Proteínas Supressoras de Tumor , Adulto , Idoso , Idoso de 80 Anos ou mais , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
TP53 mutation is a strong independent marker for survival in breast cancer with some heterogeneity in the clinical phenotype of various types of mutations. Based on 315 patients with breast carcinoma, we suggest a new model for the differentiation of TP53 mutations. Although TP53 mutation in general was associated with aggressive tumor/patient characteristics, missense mutations outside any conserved or structural domain did not affect the clinical outcome (risk of disseminated disease and death). In contrast, patients with missense mutations affecting amino acids directly involved in DNA or zinc binding displayed a very aggressive clinical phenotype. Null mutations (including missense mutations disrupting the tetramerization domain) and the remaining missense mutations displayed an intermediate aggressive clinical phenotype. When patients with primary early breast cancer were divided into three groups (wild-type together with missense mutations outside structural/conserved domains, null mutations and missense mutations with intermediate clinical phenotype, and very aggressive missense mutations), disease-specific survival rates were 89%, 58%, and 35% (5-year actuarial values, P < 0.0001), respectively. In a Cox proportional hazards analysis, separation of TP53 mutations according to these criteria eliminated the prognostic importance of all investigated classical factors except nodal status.
Assuntos
Neoplasias da Mama/genética , Genes p53/genética , Mutação , Alelos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Códon sem Sentido , Análise Mutacional de DNA , Intervalo Livre de Doença , Eletroforese , Éxons , Feminino , Mutação da Fase de Leitura , Deleção de Genes , Humanos , Modelos Estatísticos , Mutação de Sentido Incorreto , Metástase Neoplásica , Segunda Neoplasia Primária/genética , Fenótipo , Prognóstico , Estrutura Terciária de Proteína , Fatores de Tempo , Proteína Supressora de Tumor p53/químicaRESUMO
The PTEN/MMAC1 gene on chromosome 10q23 encodes a lipid phosphatase with tumor-suppressive properties. Germline PTEN/MMAC1 mutations have been implicated as the predisposing factor in Cowden disease and other hamartoma syndromes, and somatic mutations and deletions have been identified in a wide range of human cancers, including 30-40% of metastatic melanoma cell lines. To study further the possible role of PTEN/MMAC1 in the pathogenesis and progression of malignant melanoma, we examined uncultured specimens from 16 primary and 61 metastatic tumors from 67 patients. Denaturing gradient gel electrophoresis was used to analyze systematically the coding region of PTEN/MMAC1 and revealed mutations in four of the metastatic samples (7%). Sequence analysis of the mutants identified a 1 bp frameshift insertion, a 2 bp frameshift deletion, an 11 bp frameshift deletion, and a single base substitution resulting in the generation of a premature stop codon. Analysis of two intragenic polymorphisms showed allelic loss in three of eight informative primary tumors (38%) and in 18 of 31 metastatic tumors (58%). One of the mutant cases showed allelic loss, suggesting that both PTEN/MMAC1 alleles were inactivated in this tumor. Altogether, these results suggest that mutation and deletion of PTEN/MMAC1 may contribute to the development and progression of malignant melanoma.
Assuntos
Melanoma/patologia , Melanoma/secundário , Monoéster Fosfórico Hidrolases/genética , Proteínas Supressoras de Tumor , Biópsia , Genes Supressores de Tumor , Mutação em Linhagem Germinativa , Humanos , Perda de Heterozigosidade , PTEN Fosfo-Hidrolase , Reação em Cadeia da Polimerase , Polimorfismo GenéticoRESUMO
The cloning of genes encoding melanoma antigens has opened new possibilities for the treatment of patients with cancer; however, most tumor rejection antigens recognized by tumor infiltrating lymphocytes are the products of genes that are also expressed by normal melanocytes. Hence, a large set of antigenic determinants of the self have not induced self-tolerance and these peptide determinants furnish target structures for immune responses directed against tumors. The notion that the immunotherapeutic targets involved in cancer regression comprise normal differentiation antigens is stressed by the association between vitiligo-like leukoderma, due to destruction of normal melanocytes, and melanoma regression, due to destruction of cancer cells. Nevertheless, this is the first report to demonstrate by means of a new technique based on reverse transcription polymerase chain reaction and denaturing gradient gel electrophoresis, the presence of clonally expanded T cells with identical BV regions in areas of destruction of both normal and neoplastic cells.
Assuntos
Melanoma/imunologia , Linfócitos T/imunologia , Vitiligo/imunologia , Sequência de Aminoácidos , Antígenos de Neoplasias , Sequência de Bases , Humanos , Linfócitos do Interstício Tumoral/imunologia , Antígeno MART-1 , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Neoplasias/análise , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Iceland was settled during the late 9th and early 10th centuries AD by Vikings who arrived from Norway and the British Isles. Although it is generally acknowledged that the Vikings brought with them Celtic slaves, the relative contribution of these peoples to the modern Icelandic gene pool has been a matter of considerable discussion. Most population genetic studies using classical markers have indicated a large Irish genetic contribution. We have investigated the molecular basis of phenylketonuria (PKU) in 17 Icelandic patients and found 9 different mutations in the phenylalanine hydroxylase gene. One novel mutation, Y377fsdelT, accounts for more than 40% of the mutant chromosomes. Haplotype data support a common ancestral origin of the mutation, and genealogical examination extending back more than 5 generations shows that this mutation has probably arisen in an isolated part of southern Iceland and was enriched by a founder effect. At least 7 PKU mutations have originated outside iceland. The almost exclusively Scandinavian background of these mutations and the complete absence of common Irish PKU mutations strongly support historical and linguistic evidence of a predominant Scandinavian heritage of the Icelandic people.
Assuntos
Efeito Fundador , Fenilcetonúrias/genética , Análise Mutacional de DNA , Mutação da Fase de Leitura/genética , Frequência do Gene , Pool Gênico , Haplótipos/genética , Humanos , Islândia , Mapas como Assunto , Linhagem , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/epidemiologia , Polimorfismo Genético/genética , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNARESUMO
Using a simple, standardized denaturing gradient gel electrophoresis (DGGE) based mutation screening technique, a novel G-to-A mutation in the last base of the intron 12 splice acceptor site of the LDL receptor gene was found in 2 Danish families with familial hypercholesterolemia (FH). The mutation is shown to result in 2 mRNA splice variants, both leading to truncated LDLR proteins, containing only the first 594 of the normal 839 amino acids. In one of the FH-families harbouring the mutation, a striking difference in the clinical picture amongst biochemically diagnosed FH patients was clarified when genetic analysis showed that 2 hypercholesterolemic family members, who despite advanced age had no atherosclerotic disease, had not inherited the family LDLR mutation. DGGE analyses of the LDLR exons, LDLR promoter, and apolipoprotein B codon 3456-3553 as well as Southern blotting of the LDLR gene were without signs of other mutations in the non-atherosclerotic hypercholesterolemics of the family. Availability of the clinically applicable mutation screening assay for FH may thus aid in defining reasons for phenotypic differences in FH families and potentially supply information allowing a more differentiated therapeutic approach to individual members of FH families.
Assuntos
Hiperlipoproteinemia Tipo II/genética , Íntrons/genética , Mutação Puntual , Splicing de RNA/genética , RNA Mensageiro/genética , Receptores de LDL/genética , Adolescente , Adulto , Idoso , Northern Blotting , Eletroforese , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNARESUMO
We performed a screening of exon 9 of the low density lipoprotein receptor (LDLR) gene in 14 Danish families with familial hypercholesterolemia (FH) using the denaturing gradient gel electrophoresis (DGGE) technique. In one of the probands from these families an abnormal band pattern in the gradient gel was detected. Subsequent DGGE analysis of the family of this index patient revealed that the DGGE pattern cosegregated with the disease in this family. Sequencing of the exon showed a deletion of a C in codon 424 of the LDLR gene resulting in a frame shift with the introduction of a stop codon 5 codons further downstream. The mutation is referred to as FH-Odense. The predicted truncated receptor protein consists of the 428 amino terminal amino acids. Consequently, the cytosolic and membrane spanning parts of the mature LDL receptor, which normally secure the receptor in the plasma membrane, are missing. The FH-Odense mutation results in severe premature coronary atherosclerosis as shown by the clinical expression in 5 generations of the affected family.
Assuntos
Hiperlipoproteinemia Tipo II/genética , Mutação , Receptores de LDL/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Criança , Pré-Escolar , Códon , Dinamarca , Eletroforese em Gel Bidimensional , Feminino , Humanos , Hiperlipoproteinemia Tipo II/mortalidade , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Isquemia Miocárdica/genética , Isquemia Miocárdica/mortalidade , Linhagem , Reação em Cadeia da Polimerase , SobreviventesRESUMO
Accumulation of T cells carrying identical T-cell receptors (TCR) is associated with a number of immunological and non-immunological diseases. Therefore, it is of interest to be able to analyze complex T-cell populations for the presence of clonally expanded subpopulations. Here, we describe a simple method combining reverse transcription (RT)-PCR and denaturing gradient gel electrophoresis (DGGE) for rapid detection and characterization of T-cell clonality. The detection of clonally expanded T cells by DGGE relies on the fact that clonal transcripts have no junctional diversity and therefore resolve at a fixed position in the gel, which is determined by their melting properties. For polyclonal populations with a high degree of junctional diversity, the different DNA molecules will resolve at different positions in the gel and together will be revealed as a smear. For each of the TCR beta-variable gene (BV) 1-24 families, cloned transcripts were amplified and shown to resolve as distinct bands in the denaturing gradient gel, whereas the analysis of polyclonal T-cell populations resulted in a smear in the gel. The present method might prove useful to test for clonotypic T-cells in a variety of pathological and physiological conditions and for monitoring T-cell responses in diagnostic and therapeutic settings.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Desnaturação de Ácido Nucleico , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/metabolismo , Células Cultivadas , Células Clonais , Primers do DNA , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
OBJECTIVE: To examine the relationship of phenylalanine hydroxylase (PAH) genotypes to biochemical phenotype and cognitive development in maternal phenylketonuria (PKU). METHODOLOGY: PAH gene mutations were examined in 222 hyperphenylalaninemic females enrolled in the Maternal PKU Collaborative Study (MPKUCS). A total of 84 different mutations were detected, and complete genotype was obtained in 199 individuals. Based on previous knowledge about mutation-phenotype associations, 78 of the mutations could be assigned to one of four classes of severity (severe PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia [MHP]). Then, 189 MPKUCS subjects were grouped according to the various combinations of mutation classifications. The sample sizes were large enough for statistical testing in four groups with at least one mutation that completely abolishes enzyme activity. These patients are considered functionally hemizygous. RESULTS: The biochemical phenotype predicted from the genotype in functionally hemizygous patients was related significantly to the assigned phenylalanine level. Cognitive performance (IQ) was also significantly related to genotype. The IQ of PAH-deficient mothers with a severe PKU mutation in combination with a MHP mutation or a mild PKU mutation was 99 and 96, respectively, whereas the IQ of PKU mothers with two severe PKU mutations or with one severe and one moderate PKU mutation was 83 and 84, respectively. Of the patients with PKU, 92% had been treated during childhood. Those who were untreated or treated late had lower than average IQ scores for their group of mutation combinations. Females with moderate or mild PKU who were treated early and treated for >6 years showed IQ scores 10 points above average for their group. CONCLUSIONS: The reproductive outcome in maternal phenylketonuria is dependent on prenatal metabolic control and postnatal environmental circumstances. Both factors depend on the intellectual resources of the mother with PKU. The significant relationship among genotype, biochemical phenotype, and cognitive performance observed in the present study is of importance for the development of an optimal strategy for future treatment of females with PKU who plan pregnancy.
Assuntos
Inteligência , Fenilalanina Hidroxilase/deficiência , Fenilcetonúrias , Feminino , Genótipo , Humanos , Mutação , Fenótipo , Fenilcetonúrias/genética , Fenilcetonúrias/metabolismoRESUMO
During recent years it has become increasingly evident that L&H cells in nodular lymphocytic predominance (LP) Hodgkin's disease (HD) and Hodgkin and Reed-Sternberg (H-RS) cells in approximately half the cases of classical HD originate from B-lymphocytes, and that H-RS cells in most of the remaining cases of classical HD express a null phenotype. The pathogenesis of HD is unknown. An association with Epstein-Barr virus (EBV) has been suggested and there are also indications that genes involved in programmed cell death (apoptosis) may be implicated. In this study, the expression of four apoptosis-related proteins (bcl-2, bcl-x, bax and p53) in 53 cases of HD was examined and the data were correlated with the genotype, the EBV status and the phenotype (B, T or null) of the neoplastic cells. The H-RS cells expressed a B-cell phenotype in 3/3 cases of nodular LP and in 19/ 50 (38%) cases of classical HD. The remaining cases showed a null-cell phenotype in 29/50 (58%) and a T-cell phenotype in 2/50 (4%). EBV was more often positive in B (14/19, 74%) than in null (7/29, 24%) type HD. The H-RS cells were bcl-2-positive in 19/53 (36%), bcl-x-positive in 17/53 (32%), bax-positive in 1/53, and p53-positive in 41/53 (77%) cases. No relationship was found between bcl-2 expression and EBV status, or between bcl-2 and bcl-x expression. A t(14;18) translocation was seen in 2 of 34 cases. P53 point mutations were not detected. Our findings indicate that nodular LP and classical HD originate from B-cells in a high proportion of cases. They also suggest a role for bcl-2, bcl-x and p53 in tumorigenesis. The pathogenesis is not known at this stage.