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1.
Ann Oncol ; 33(11): 1186-1199, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35988656

RESUMO

BACKGROUND: Germline variant evaluation in precision oncology opens new paths toward the identification of patients with genetic tumor risk syndromes and the exploration of therapeutic relevance. Here, we present the results of germline variant analysis and their clinical implications in a precision oncology study for patients with predominantly rare cancers. PATIENTS AND METHODS: Matched tumor and control genome/exome and RNA sequencing was carried out for 1485 patients with rare cancers (79%) and/or young adults (77% younger than 51 years) in the National Center for Tumor Diseases/German Cancer Consortium (NCT/DKTK) Molecularly Aided Stratification for Tumor Eradication Research (MASTER) trial, a German multicenter, prospective, observational precision oncology study. Clinical and therapeutic relevance of prospective pathogenic germline variant (PGV) evaluation was analyzed and compared to other precision oncology studies. RESULTS: Ten percent of patients (n = 157) harbored PGVs in 35 genes associated with autosomal dominant cancer predisposition, whereof up to 75% were unknown before study participation. Another 5% of patients (n = 75) were heterozygous carriers for recessive genetic tumor risk syndromes. Particularly, high PGV yields were found in patients with gastrointestinal stromal tumors (GISTs) (28%, n = 11/40), and more specifically in wild-type GISTs (50%, n = 10/20), leiomyosarcomas (21%, n = 19/89), and hepatopancreaticobiliary cancers (16%, n = 16/97). Forty-five percent of PGVs (n = 100/221) supported treatment recommendations, and its implementation led to a clinical benefit in 40% of patients (n = 10/25). A comparison of different precision oncology studies revealed variable PGV yields and considerable differences in germline variant analysis workflows. We therefore propose a detailed workflow for germline variant evaluation. CONCLUSIONS: Genetic germline testing in patients with rare cancers can identify the very first patient in a hereditary cancer family and can lead to clinical benefit in a broad range of entities. Its routine implementation in precision oncology accompanied by the harmonization of germline variant evaluation workflows will increase clinical benefit and boost research.


Assuntos
Neoplasias , Adulto Jovem , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Mutação em Linhagem Germinativa , Predisposição Genética para Doença , Estudos Prospectivos , Síndrome , Medicina de Precisão/métodos
2.
ESMO Open ; 6(6): 100310, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34808524

RESUMO

BACKGROUND: Approvals of cancer therapeutics are primarily disease entity specific. Current molecular diagnostic approaches frequently identify actionable alterations in rare cancers or rare subtypes of common cancers for which the corresponding treatments are not approved and unavailable within clinical trials due to entity-related eligibility criteria. Access may be negotiated with health insurances. However, approval rates vary, and critical information required for a scientific evaluation of treatment-associated risks and benefits is not systematically collected. Thus clinical trials with optimized patient selection and comprehensive molecular characterization are essential for translating experimental treatments into standard care. PATIENTS AND METHODS: Continuous ReAssessment with Flexible ExTension in Rare Malignancies (CRAFT) is an open-label phase II trial for adults with pretreated, locally advanced, or metastatic solid tumors. Based on the evaluation by a molecular tumor board, patients are assigned to combinations of six molecularly targeted agents and a programmed death-ligand 1 (PD-L1) antagonist within seven study arms focusing on (i) BRAF V600 mutations; (ii) ERBB2 amplification and/or overexpression, activating ERBB2 mutations; (iii) ALK rearrangements, activating ALK mutations; (iv and v) activating PIK3CA and AKT mutations, other aberrations predicting increased PI3K-AKT pathway activity; (vi) aberrations predicting increased RAF-MEK-ERK pathway activity; (vii) high tumor mutational burden and other alterations predicting sensitivity to PD-L1 inhibition. The primary endpoint is the disease control rate (DCR) at week 16; secondary and exploratory endpoints include the progression-free survival ratio, overall survival, and patient-reported outcomes. Using Simon's optimal two-stage design, 14 patients are accrued for each study arm. If three or fewer patients achieve disease control, the study arm is stopped. Otherwise, 11 additional patients are accrued. If the DCR exceeds 7 of 25 patients, the null hypothesis is rejected for the respective study arm. CONCLUSIONS: CRAFT was activated in October 2021 and will recruit at 10 centers in Germany. TRIAL REGISTRATION NUMBERS: EudraCT: 2019-003192-18; ClinicalTrials.gov: NCT04551521.


Assuntos
Antineoplásicos , Neoplasias , Adulto , Antineoplásicos/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Humanos , Estudos Multicêntricos como Assunto , Mutação , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/uso terapêutico , Intervalo Livre de Progressão
3.
Psychopharmacology (Berl) ; 235(6): 1821-1833, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29700576

RESUMO

RATIONALE: The corticotropin-releasing hormone (CRH) system is a key mediator of stress-induced responses in alcohol-seeking behavior. Recent research has identified the central nucleus of the amygdala (CeA), a brain region involved in the regulation of fear and stress-induced responses that is especially rich in CRH-positive neurons, as a key player in mediating excessive alcohol seeking. However, detailed characterization of the specific influences that local neuronal populations exert in mediating alcohol responses is hampered by current limitations in pharmacological and immunohistochemical tools for targeting CRH receptor subtype 1 (CRHR1). OBJECTIVE: In this study, we investigated the effect of cell- and region-specific overexpression of CRHR1 in the CeA using a novel transgenic tool. METHODS: Co-expression of CRHR1 in calcium-calmodulin-dependent kinase II (αCaMKII) neurons of the amygdala was demonstrated by double immunohistochemistry using a Crhr1-GFP reporter mouse line. A Cre-inducible Crhr1-expressing adeno-associated virus (AAV) was site-specifically injected into the CeA of αCaMKII-CreERT2 transgenic rats to analyze the role of CRHR1 in αCaMKII neurons on alcohol self-administration and reinstatement behavior. RESULTS: Forty-eight percent of CRHR1-containing cells showed co-expression of αCaMKII in the CeA. AAV-mediated gene transfer in αCaMKII neurons induced a 24-fold increase of Crhr1 mRNA in the CeA which had no effect on locomotor activity, alcohol self-administration, or cue-induced reinstatement. However, rats overexpressing Crhr1 in the CeA increased responding in the stress-induced reinstatement task with yohimbine serving as a pharmacological stressor. CONCLUSION: We demonstrate that CRHR1 overexpression in CeA-αCaMKII neurons is sufficient to mediate increased vulnerability to stress-triggered relapse into alcohol seeking.


Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Núcleo Central da Amígdala/metabolismo , Comportamento de Procura de Droga/fisiologia , Etanol/administração & dosagem , Neurônios/metabolismo , Receptores de Hormônio Liberador da Corticotropina/biossíntese , Consumo de Bebidas Alcoólicas/genética , Animais , Núcleo Central da Amígdala/efeitos dos fármacos , Comportamento de Procura de Droga/efeitos dos fármacos , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Receptores de Hormônio Liberador da Corticotropina/genética , Autoadministração
4.
Clin Genet ; 84(6): 531-8, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23425300

RESUMO

Acrodysostosis is characterized by a peripheral dysostosis that is accompanied by short stature, midface hypoplasia, and developmental delay. Recently, it was shown that heterozygous point mutations in the PRKAR1A gene cause acrodysostosis with hormone resistance. By mutational analysis of the PRKAR1A gene we detected four different mutations (p.Arg368Stop, p.Ala213Thr, p.Tyr373Cys, and p.Arg335Cys) in four of seven affected patients with acrodysostosis. The combination of clinical results, endocrinological parameters and in silico mutation analysis gives evidence to suppose a pathogenic effect of each mutation. This assumption is supported by the de novo origin of these mutations. Apart from typical radiological abnormalities of the hand bones, elevated thyroid stimulating hormone and parathyroid hormone values as well as short stature are the most common findings. Less frequent features are characteristic facial dysmorphisms, sensorineural hearing loss and mild intellectual disability. These results lead to the conclusion that mutations of PKRAR1A are the major molecular cause for acrodysostosis with endocrinological abnormalities. In addition, in our cohort of 44 patients affected with brachydactyly type E (BDE) we detected only one sequence variant of PRKAR1A (p.Asp227Asn) with an unclear effect on protein function. Thus, we conclude that PRKAR1A mutations may play no major role in the pathogenesis of BDE.


Assuntos
Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Disostoses/genética , Deficiência Intelectual/genética , Mutação , Osteocondrodisplasias/genética , Adolescente , Adulto , Alelos , Criança , Análise Mutacional de DNA , Disostoses/diagnóstico , Disostoses/metabolismo , Feminino , Ossos da Mão/diagnóstico por imagem , Ossos da Mão/patologia , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/metabolismo , Masculino , Osteocondrodisplasias/diagnóstico , Osteocondrodisplasias/metabolismo , Fenótipo , Radiografia , Adulto Jovem
5.
Klin Padiatr ; 224(6): 386-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23143765

RESUMO

Two related boys who died from fulminant infectious mononucleosis were diagnosed with X-linked lymphoproliferative disease type 1 (XLP-1). Family screening (n=17) identified 6 female mutation carriers and 2 more XLP-1 patients in whom, despite recurrent infections, agammaglobulinemia, and Hodgkin's Disease, the genetic basis had been unknown; demonstrating that awareness and early genetic testing are crucial to reveal underlying primary immunodeficiencies and improve outcome. Furthermore, XLP should be included routinely in the differential diagnosis of severe hypogammaglobulinemia and/or lymphoma in males.


Assuntos
Mononucleose Infecciosa/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfo-Histiocitose Hemofagocítica/genética , Transtornos Linfoproliferativos/genética , Adolescente , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/genética , Pré-Escolar , Análise Mutacional de DNA , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/genética , Éxons/genética , Evolução Fatal , Triagem de Portadores Genéticos , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Testes Genéticos , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/genética , Humanos , Lactente , Mononucleose Infecciosa/diagnóstico , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/mortalidade , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/mortalidade , Masculino , Meningoencefalite/complicações , Meningoencefalite/diagnóstico , Meningoencefalite/genética , Mutação de Sentido Incorreto , Linhagem , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Adulto Jovem
6.
Gene Ther ; 19(2): 210-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21654820

RESUMO

Cell surface targeting of recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to modify AAV's natural tropism. As modification of the capsid surface is likely to affect the mechanism of vector internalization and consequently the vector's intracellular fate, we investigated early steps in cell transduction of rAAV capsid insertion mutants. Mutants displaying peptides with neutral overall charge at position 587 transduced cells independently of AAV2's primary receptor heparan sulfate proteoglycan (HSPG), whereas mutants carrying positively charged insertions were capable of HSPG binding with affinities correlating with their net positive charge. Whereas rAAV2 is internalized via an HSPG- and clathrin-dependent pathway, HSPG-binding mutants used a clathrin- and caveolin-independent mechanism. Surprisingly, although this pathway was as efficient in mediating vector entry as the one used by rAAV2, successful cell transduction was hampered at a post-entry step, presumably caused by inefficient endosomal escape. In contrast, HSPG-independent, clathrin-dependent internalization used by non-HSPG-binding mutants correlated with efficient nuclear delivery of vector genomes and robust transgene expression. These findings indicate that cell surface targeting strategies should direct uptake of rAAV targeting vectors to clathrin-mediated endocytosis, the naturally evolved entry route of AAV, to promote successful intracellular processing and re-targeting of rAAV's tropism.


Assuntos
Clatrina/genética , Dependovirus/genética , Endocitose/genética , Terapia Genética , Proteoglicanas de Heparan Sulfato/metabolismo , Proteínas do Capsídeo/genética , Clatrina/metabolismo , Vetores Genéticos/administração & dosagem , Células HEK293 , Células HeLa , Células Hep G2 , Proteoglicanas de Heparan Sulfato/genética , Humanos , Mutagênese Insercional/genética , Mutação , Transdução Genética , Internalização do Vírus
7.
Clin Genet ; 73(1): 62-70, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18042262

RESUMO

Cardio-facio-cutaneous (CFC) and Costello syndrome (CS) are congenital disorders with a significant clinical overlap. The recent discovery of heterozygous mutations in genes encoding components of the RAS-RAF-MAPK pathway in both CFC and CS suggested a similar underlying pathogenesis of these two disorders. While CFC is heterogeneous with mutations in BRAF, MAP2K1, MAP2K2 and KRAS, HRAS alterations are almost exclusively associated with CS. We carried out a comprehensive mutation analysis in 51 CFC-affected patients and 31 individuals with CS. Twelve different BRAF alterations were found in twenty-four patients with CFC (47.0%), two MAP2K1 mutations in five (9.8%) and two MAP2K2 sequence variations in three CFC-affected individuals (5.9%), whereas three patients had a KRAS alteration (5.9%). We identified four different missense mutations of HRAS in twenty-eight cases with CS (90.3%), while KRAS mutations were detected in two infants with a phenotype meeting criteria for CS (6.5%). In 14 informative families, we traced the parental origin of HRAS alterations and demonstrated inheritance of the mutated allele exclusively from the father, further confirming a paternal bias in the parental origin of HRAS mutations in CS. Careful clinical evaluation of patients with BRAF and MAP2K1/2 alterations revealed the presence of slight phenotypic differences regarding craniofacial features in MAP2K1- and MAP2K2-mutation positive individuals, suggesting possible genotype-phenotype correlations.


Assuntos
Anormalidades Múltiplas/genética , Fácies , Cardiopatias Congênitas/genética , Mutação , Anormalidades da Pele/genética , Adulto , Criança , Análise Mutacional de DNA , Deficiências do Desenvolvimento , Humanos , Deficiência Intelectual , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Fenótipo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Síndrome , Proteínas ras/genética
8.
Artigo em Alemão | MEDLINE | ID: mdl-14605482

RESUMO

BACKGROUND: Glaucoma is one of the most frequent causes of blindness worldwide. The main risk factor for this optic nerve disease is high individual intraocular pressure. OBJECTIVE: The aim of this observational study was to investigate the effect of acupuncture therapy on individual internal eye pressure in glaucoma and ocular hypertension patients. MATERIAL AND METHODS: 18 patients (11 female; age 38-75 years) with a diagnosis of glaucoma (n = 3) or ocular hypertension (n = 15) were included in this study. Patients received one session of a standardized acupuncture treatment (acupuncture points Liv 3, Li 4, Gb 37). Target parameters were the change in intraocular pressure and the compatibility of treatment. Measurements of intraocular pressure were carried out 15 min before, 15 min after, and 24 h after acupuncture. RESULTS: Compared with the mean intraocular pressure in both eyes before treatment (21.94 +/- 2.4 mm Hg), patients showed a significant decrease 15 min after treatment (-2.67 +/- 1.34 mm Hg) as well as 24 h after treatment (-2.5 +/- 2.13 mm Hg). There was no significant difference between the mean intraocular pressure at 15 min after treatment and at 24 h after treatment. No adverse events were observed. CONCLUSION: The results of this study indicate that acupuncture therapy may be a valid treatment option for glaucoma and ocular hypertension patients. Additional and, if possible, randomized studies investigating the effectiveness of acupuncture in the treatment of the above-mentioned conditions still need to be conducted.


Assuntos
Terapia por Acupuntura , Glaucoma/terapia , Hipertensão Ocular/terapia , Adulto , Idoso , Feminino , Humanos , Pressão Intraocular , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento
9.
J Med Genet ; 39(10): 734-40, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12362030

RESUMO

As a result of the increasing use of genome wide telomere screening, it has become evident that a significant proportion of people with idiopathic mental retardation have subtle abnormalities involving the telomeres of human chromosomes. However, during the course of these studies, there have also been telomeric imbalances identified in normal people that are not associated with any apparent phenotype. We have begun to scrutinize cases from both of these groups by determining the extent of the duplication or deletion associated with the imbalance. Five cases were examined where the telomere rearrangement resulted in trisomy for the 16p telomere. The size of the trisomic segment ranged from approximately 4-7 Mb and the phenotype included mental and growth retardation, brain malformations, heart defects, cleft palate, pancreatic insufficiency, genitourinary abnormalities, and dysmorphic features. Three cases with telomeric deletions without apparent phenotypic effects were also examined, one from 10q and two from 17p. All three deletions were inherited from a phenotypically normal parent carrying the same deletion, thus without apparent phenotypic effect. The largest deletion among these cases was approximately 600 kb on 17p. Similar studies are necessary for all telomeric regions to differentiate between those telomeric rearrangements that are pathogenic and those that are benign variants. Towards this goal, we are developing "molecular rulers" that incorporate multiple clones at each telomere that span the most distal 5 Mb region. While telomere screening has enabled the identification of telomere rearrangements, the use of molecular rulers will allow better phenotype prediction and prognosis related to these findings.


Assuntos
Telômero/genética , Calibragem , Criança , Deleção Cromossômica , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 17/genética , Evolução Fatal , Feminino , Amplificação de Genes/genética , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Fenótipo , Diagnóstico Pré-Natal , Trissomia/diagnóstico , Trissomia/genética
10.
Ther Apher ; 5(4): 252-9, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11724509

RESUMO

Hypercoagulability is a key contributor to acute cardiovascular syndromes and to various microcirculatory disorders. The use of heparin-mediated extracorporeal low-density lipoprotein/fibrinogen precipitation (HELP) apheresis makes a controlled, immediately effective reduction of clotting factors possible, and induces subsequent positive effects on plasma viscosity, erythrocyte aggregation, and microcirculation. Oxygen supply to an ischemic artery can thus be increased without hemodilution, qualifying the HELP system as a possible therapeutic tool in the treatment of acute cardiovascular syndromes and microcirculatory disorders.


Assuntos
Transtornos da Coagulação Sanguínea/terapia , Fatores de Coagulação Sanguínea/análise , Doenças Cardiovasculares/terapia , Hipercolesterolemia/terapia , Plasmaferese/métodos , Idoso , Coagulação Sanguínea/fisiologia , Doença da Artéria Coronariana/prevenção & controle , Trombose Coronária/prevenção & controle , Feminino , Hemorreologia , Humanos , Masculino , Pessoa de Meia-Idade
11.
Cytometry ; 44(1): 7-15, 2001 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-11309803

RESUMO

BACKGROUND: Routine application of multicolor fluorescence in situ hybridization (M-FISH) technology for molecular cytogenetic diagnostics has been hampered by several technical limitations. First, when using chromosome-specific painting probes, there is a limit in cytogenetic resolution of approximately 2-3 Mb, which can mask hidden structural abnormalities that have a significant clinical effect. Second, using whole chromosome painting probes, intrachromosomal rearrangements cannot be detected and the exact localization of breakpoints is often not possible. METHODS: We suggest the use of multiplex-labeled region or locus- specific probes in combination with an optimal probe design to improve the sensitivity and resolution of the M-FISH technology. To allow the application of this assay in routine diagnostics, we developed a multipurpose image analysis system. RESULTS: goldFISH was applied to the study of cryptic translocations in mental retardation patients and to the study of high-resolution breakpoint mapping in non-small cell lung cancer patients. For an individual with mental retardation, who had an apparently normal karyotype by G-banding, we detected an unbalanced translocation involving chromosomes 2 and 7. CONCLUSIONS: In combination with optimally designed probe kits, goldFISH overcomes most of the present limitations of the M-FISH technology and results in virtually 100% reliability for detecting interchromosomal and intrachromosomal rearrangements.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Hibridização in Situ Fluorescente/métodos , Deficiência Intelectual/genética , Neoplasias Pulmonares/genética , Automação , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Humanos , Deficiência Intelectual/diagnóstico , Neoplasias Pulmonares/diagnóstico , Metáfase , Sensibilidade e Especificidade , Translocação Genética
13.
Lab Invest ; 80(7): 1031-41, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10908148

RESUMO

Lung cancer has a considerable impact on morbidity and mortality throughout the world. Despite extensive effort, no lung cancer-specific cytogenetic changes, such as lineage-specific translocations or inversions, have been described to date. In this study we used multiplex fluorescence in situ hybridization (M-FISH), comparative genomic hybridization, and multicolor bar coding to analyze eight cell lines derived from non-small cell lung cancers. M-FISH did not identify any balanced translocations, which are the dominating feature in leukemias and lymphomas. Instead, M-FISH unraveled an enormous number of numerical and structural aberrations, with each tumor having its own "private" pattern of chromosomal changes. In contrast, comparative genomic hybridization demonstrated similarities between tumors, because each cell line shared some chromosomal segments that were commonly gained or lost. One of these involved chromosome 12. Chromosome 12 specific bar code probe sets were constructed and used to demonstrate that breaks on chromosome 12 occur preferentially within specific bands. With the progressive use of higher resolution approaches, more information can be gained about the chromosomal alterations in cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Cromossomos/genética , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Cromossomos Humanos Par 12/genética , Cor , Processamento Eletrônico de Dados , Humanos , Hibridização in Situ Fluorescente/métodos , Cariotipagem , Hibridização de Ácido Nucleico , Células Tumorais Cultivadas
14.
Am J Hum Genet ; 66(1): 16-25, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10631133

RESUMO

In the search for genetic causes of mental retardation, we have studied a five-generation family that includes 10 individuals in generations IV and V who are affected with mild-to-moderate mental retardation and mild, nonspecific dysmorphic features. The disease is inherited in a seemingly autosomal dominant fashion with reduced penetrance. The pedigree is unusual because of (1) its size and (2) the fact that individuals with the disease appear only in the last two generations, which is suggestive of anticipation. Standard clinical and laboratory screening protocols and extended cytogenetic analysis, including the use of high-resolution karyotyping and multiplex FISH (M-FISH), could not reveal the cause of the mental retardation. Therefore, a whole-genome scan was performed, by linkage analysis, with microsatellite markers. The phenotype was linked to chromosome 16p13.3, and, unexpectedly, a deletion of a part of 16pter was demonstrated in patients, similar to the deletion observed in patients with ATR-16 syndrome. Subsequent FISH analysis demonstrated that patients inherited a duplication of terminal 3q in addition to the deletion of 16p. FISH analysis of obligate carriers revealed that a balanced translocation between the terminal parts of 16p and 3q segregated in this family. This case reinforces the role of cryptic (cytogenetically invisible) subtelomeric translocations in mental retardation, which is estimated by others to be implicated in 5%-10% of cases.


Assuntos
Anormalidades Múltiplas/genética , Deleção Cromossômica , Deficiência Intelectual/genética , Translocação Genética/genética , Adolescente , Adulto , Criança , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 3/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Escore Lod , Masculino , Pessoa de Meia-Idade , Linhagem , Síndrome , Telômero
16.
Am J Hum Genet ; 65(2): 448-62, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10417288

RESUMO

For >3 decades, Giemsa banding of metaphase chromosomes has been the standard karyotypic analysis for pre- and postnatal diagnostic applications. However, marker chromosomes or structural abnormalities are often encountered that cannot be deciphered by G-banding alone. Here we describe the use of multiplex-FISH (M-FISH), which allows the visualization of the 22 human autosomes and the 2 sex chromosomes, in 24 different colors. By M-FISH, the euchromatin in marker chromosomes could be readily identified. In cases of structural abnormalities, M-FISH identified translocations and insertions or demonstrated that the rearranged chromosome did not contain DNA material from another chromosome. In these cases, deleted or duplicated regions were discerned either by chromosome-specific multicolor bar codes or by comparative genomic hybridization. In addition, M-FISH was able to identify cryptic abnormalities in patients with a normal G-karyotype. In summary, M-FISH is a reliable tool for diagnostic applications, and results can be obtained in

Assuntos
Testes Genéticos/métodos , Hibridização in Situ Fluorescente , Diagnóstico Pré-Natal/métodos , Cromatina/genética , Aberrações Cromossômicas/genética , Bandeamento Cromossômico , Coloração Cromossômica , Cromossomos Humanos/genética , Sondas de DNA , Feminino , Marcadores Genéticos/genética , Humanos , Deficiência Intelectual/genética , Cariotipagem , Masculino , Fenótipo , Recombinação Genética/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Cytogenet Cell Genet ; 82(3-4): 160-71, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9858809

RESUMO

Multiplex-FISH (M-FISH) is a recently developed technique by which each of the two dozen human chromosomes-the 22 autosomes and the X and Y sex chromosomes-can be stained or "painted" with uniquely distinctive colors. Using a combinatorial labeling technique and a specially designed filter set, each DNA probe can be identified by its unique spectral signature. Here we present several significant optimizations of the M-FISH technology. First, a new strategy for labeling the probes is described which allows for easy and fast production of the complex M-FISH probe mix. Second, a newly developed, completely motorized microscope equipped with an eight-position filter wheel and a new generation of filter sets is presented that allows fully automatic imaging of a complete metaphase spread within seconds. Third, to determine the characteristic spectral signatures for all different combinations of fluorochromes, we developed a novel multichannel image analysis method. The spectral analysis is solely guided by the image information itself and does not require any user interaction. A complete analysis of a metaphase spread can be accomplished in less than 3 min. Sophisticated built-in quality controls were developed, and the value of visual inspection of M-FISH images as a simple means of controlling the computer-generated chromosome classification are illustrated. In addition, we discuss advantages of adding new fluorochromes to the traditionally used five fluorochromes.


Assuntos
Aberrações Cromossômicas , DNA de Neoplasias/análise , Rearranjo Gênico , Hibridização in Situ Fluorescente/métodos , Bandeamento Cromossômico/métodos , Corantes Fluorescentes , Humanos , Processamento de Imagem Assistida por Computador/métodos , Indóis , Microscopia de Fluorescência/métodos
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