RESUMO
Huntington's disease (HD) is associated with expansion of a CAG repeat in a novel gene. We have assessed 21 sporadic cases of HD to investigate sequential events underlying HD. We show the existence of an intermediate allele (IA) in parental alleles of 30-38 CAG repeats in the HD gene which is greater than usually seen in the general population but below the range seen in patients with HD. These IAs are meiotically unstable and in the sporadic cases, expand to the full mutation associated with the phenotype of HD. This expansion has been shown to occur only during transmission through the male germline and is associated with advanced paternal age. These findings suggest that new mutations for HD are more frequent than prior estimates and indicate a previously unrecognized risk of inheriting HD to siblings of sporadic cases of HD and their children.
Assuntos
Alelos , Doença de Huntington/genética , Mutação , Adulto , Idade de Início , Sequência de Bases , Primers do DNA , Feminino , Síndrome do Cromossomo X Frágil/genética , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Distrofia Miotônica/genética , Linhagem , Sequências Repetitivas de Ácido Nucleico , Caracteres SexuaisRESUMO
Huntington disease is associated with an unstable and expanded (CAG) trinucleotide repeat. We have analysed the CAG expansion in different tissues from 12 affected individuals. All tissues examined were found to display some repeat mosaicism, with the greatest levels detected in brain and sperm. Regions within the brain showing most obvious neuropathology, such as the basal ganglia and the cerebral cortex, displayed the greatest mosaicism, whereas the cerebellar cortex, which is seldom involved, displayed the lowest degree of CAG instability. In two cases of childhood onset disease we detected differences of 8 and 13 trinucleotides between the cerebellum and other regions of the brain. Our results provide evidence for tissue specific instability of the CAG repeat, with the largest CAG repeat lengths in affected regions of the brain.
Assuntos
Células Sanguíneas/química , Química Encefálica , DNA/genética , Doença de Huntington/genética , Mosaicismo , Músculos/química , Sequências Repetitivas de Ácido Nucleico , Espermatozoides/química , Vísceras/química , Adulto , Idade de Início , Gânglios da Base/química , Criança , Pré-Escolar , DNA/isolamento & purificação , Feminino , Humanos , Doença de Huntington/epidemiologia , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos , Reação em Cadeia da PolimeraseRESUMO
Huntington's disease (HD) is associated with the expansion of a CAG trinucleotide repeat in a novel gene. We have assessed 360 HD individuals from 259 unrelated families and found a highly significant correlation (r = 0.70, p = 10(-7)) between the age of onset and the repeat length, which accounts for approximately 50% of the variation in the age of onset. Significant associations were also found between repeat length and age of death and onset of other clinical features. Sib pair and parent-child analysis revealed that the CAG repeat demonstrates only mild instability. Affected HD siblings had significant correlations for trinucleotide expansion (r = 0.66, p < 0.001) which was not apparent for affected parent-child pairs.
Assuntos
Doença de Huntington/genética , Sequências Repetitivas de Ácido Nucleico , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Criança , Pré-Escolar , Estudos de Coortes , DNA/sangue , DNA/genética , DNA/isolamento & purificação , Feminino , Genótipo , Humanos , Doença de Huntington/epidemiologia , Doença de Huntington/fisiopatologia , Leucócitos/fisiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Núcleo Familiar , Oligodesoxirribonucleotídeos , FenótipoRESUMO
Extra structurally abnormal chromosomes (ESACs) are small supernumerary chromosomes often associated with developmental abnormalities and malformations. We present 50 probands with ESACs characterized by fluorescence in situ hybridization using centromere-specific probes and chromosome-specific libraries. ESAC-specific libraries were constructed by flow sorting and subsequent amplification by DOP-PCR. Using such ESAC-specific libraries we were able to outline the chromosome regions involved. Twenty-three of the 50 ESACs were inverted duplications of chromosome 15 [inv dup(15)], including patients with normal phenotypes and others with similar clinical symptoms. These 2 groups differed in size and shape of the inv dup(15). Patients with a large inv dup(15), which included the Prader-Willi region, had a high risk of abnormality, whereas patients with a small inv dup(15), not including the Prader-Willi region, were normal. ESACs derived from chromosomes 13 or 21 appeared to have a low risk of abnormality, while one out of 3 patients with an ESAC derived from chromosome 14 had discrete symptoms. One out of 3 patients with an ESAC derived from chromosome 22 had severe anomalies, corresponding to some of the manifestations of the cat eye syndrome. Small extra ring chromosomes of autosomal origin and ESACs identified as i(12p) or i(18p) were all associated with a high risk of abnormality.
Assuntos
Anormalidades Múltiplas/genética , Aberrações Cromossômicas , Adolescente , Adulto , Criança , Pré-Escolar , Cromossomos Humanos 13-15/genética , Cromossomos Humanos 21-22 e Y/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 18/genética , Feminino , Marcadores Genéticos , Variação Genética , Heterozigoto , Humanos , Hibridização in Situ Fluorescente , Lactente , Deficiência Intelectual/genética , Masculino , Síndrome de Prader-Willi/genéticaRESUMO
There now exists a set of tightly linked markers to the gene causing multiple endocrine neoplasia type 2A. In this report we discuss the use of these markers for early and accurate prediction of gene carrier status in three different families. Factors that influence the probability of obtaining useful information with DNA markers are available family size, in particular the number of available affected individuals, and the extent of clinical and biochemical screening in the family. At present, DNA analysis has about a 3% risk of misdiagnosis; this risk is even lower if it is used in conjunction with the pentagastrin stimulation test for C-cell hyperplasia. With the combined tests, an individual at age 20 years may be scored as carrier or not with an estimated accuracy of 99%.
Assuntos
DNA de Neoplasias/análise , Ligação Genética , Marcadores Genéticos/análise , Neoplasia Endócrina Múltipla/genética , Adolescente , Adulto , Idoso , Alelos , Feminino , Triagem de Portadores Genéticos/métodos , Testes Genéticos/métodos , Humanos , Masculino , Neoplasia Endócrina Múltipla/diagnóstico , Valor Preditivo dos TestesAssuntos
Neoplasia Endócrina Múltipla/genética , Adulto , Idoso , Análise Mutacional de DNA , Feminino , Marcadores Genéticos , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Neoplasia Endócrina Múltipla/prevenção & controle , Neoplasia Endócrina Múltipla/terapia , Linhagem , Sistema de Registros , Pesquisa , SuéciaRESUMO
The past year has witnessed outstanding developments in research on Huntington's disease (HD). A gene was identified that contains an expanded CAG trinucleotide repeat on HD chromosomes. Patterns of expression of this gene and the nature of two transcripts were identified. CAG repeat size ranges between 36 and 121 in affected persons, and it is highly sensitive and specific marker for HD. A correlation between CAG repeat size and the age of onset of HD was demonstrated. Identification of this mutation has facilitated direct approaches to predictive testing for HD. The new mutation rate, previously deemed to be exceedingly rare, is now shown to be responsible for up to 3% of affected persons. Although the mechanism by which CAG repeat length induces neuronal death is not known, there is evidence that the pathogenesis involves a gain of function in the HD gene.
Assuntos
Doença de Huntington/genética , Morte Celular/genética , Cromossomos Humanos Par 4 , Marcadores Genéticos/genética , Humanos , Doença de Huntington/diagnóstico , Biologia Molecular , Mutação/genética , Linhagem , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição/genéticaRESUMO
Sorted chromosomes from each of the 20 clusters of the male porcine bivariate flow karyotype were amplified and biotinylated using DOP-PCR. The chromosomes comprising each cluster were identified by fluorescence in situ hybridization (FISH) of the 20 probes to R-banded male pig metaphase spreads. A standard flow karyotype for the pig is presented.
Assuntos
Hibridização in Situ Fluorescente/veterinária , Cariotipagem/veterinária , Suínos/genética , Animais , Sequência de Bases , Biotina , Células Cultivadas , Cromossomos/ultraestrutura , Primers do DNA , Feminino , Citometria de Fluxo/veterinária , Corantes Fluorescentes , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/veterináriaRESUMO
We obtained DNA clones specific to tomato chromosome 2 from a small number of chromosomes collected by flow sorting. Suspensions of metaphase chromosomes were prepared from 3-month-old tomato cell cultures of Lycopersicon pennellii. Isolated chromosomes stained with chromomycin A3 and Hoechst 33258 were analyzed on an EPICS 753 flow cytometer using a UV laser to excite Hoechst fluorescence and a 458 nm laser to excite chromomycin A3 fluorescence. Chromosomes from well-resolved peaks on a bivariate flow karyotype were sorted directly onto membrane filters for spot-blot analysis. The filters were processed and hybridized with chromosome-specific repetitive DNA probes. In this way tomato chromosome 1 and chromosome 2 were assigned to peaks in the bivariate flow karyotypes. One thousand copies of the putative chromosome 2 were flow-sorted directly into microfuge tubes. DNA specific to chromosome 2 was amplified by a polymerase chain reaction (PCR) technique using universal 22mer degenerate oligonucleotide primers (DOP) sequences. DOP-PCR yields a smear of fragments of various sizes from 250 to 1600 bp. Amplified products were cloned into the Bluescript plasmid vector. Approximately 11% of the clones contained sequences with highly repetitive elements, and 85% contained only low-copy-number sequences. Eleven clones containing low-copy-number sequences that detect restriction fragment length polymorphisms were placed on the molecular linkage map of tomato. All showed linkage to chromosome 2.
Assuntos
Cromossomos , Genes de Plantas , Verduras/genética , Sequência de Aminoácidos , Southern Blotting , Clonagem Molecular , Sondas de DNA , Citometria de Fluxo , Biblioteca Genômica , Cariotipagem , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
Vinculin is a cytoskeletal protein component of adherens type cell junctions. The gene had been mapped to 10q11.2-qter. We have used a combination of physical and genetic mapping techniques to refine this localization. Hybridization of the vinculin cDNA probe, HV1, to a human-rodent somatic hybrid panel initially suggested a position of either 10q11.2 or 10q22.1-10q23. Genetic recombination mapping in three-generation families with multiple endocrine neoplasia type 2 (MEN2) indicated a position distal to D10S22 (10q21.1) in 10q22.1-10q23. This was confirmed by hybridization of the vinculin cDNA to flow-sorted translocation derivative chromosomes containing the q21-qter portion of chromosome 10. We conclude that the vinculin locus maps in 10q22.1-q23, distal to D10S22.
Assuntos
Cromossomos Humanos Par 10 , Vinculina/genética , Animais , Bandeamento Cromossômico , Mapeamento Cromossômico , Cricetinae , DNA/genética , Sondas de DNA , Humanos , Células Híbridas , Metáfase , CamundongosRESUMO
Biotinylated chromosome-specific probes were prepared from flow-sorted pig chromosomes 1, 13, 18, X, and Y using the degenerate oligonucleotide-primed polymerase chain reaction. Probes prepared in this way can be used to confirm the identity of chromosomes in the bivariate pig flow karyotype and in pig x mouse somatic cell hybrids.
Assuntos
Citometria de Fluxo , Hibridização In Situ , Cariotipagem/métodos , Sondas de Oligonucleotídeos/genética , Animais , Sequência de Bases , Células Híbridas , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , SuínosRESUMO
Marker chromosome are small supernumerary chromosomes that are sometimes associated with developmental abnormalities. Hence, the genes involved in such cases provide an interesting approach to understanding developmental abnormalities in man. As a first step towards isolating such sequences, marker chromosomes need complete characterization. By combining chromosome isolation by flow sorting and the "degenerate oligonucleotide primed - polymerase chain reaction", we have constructed a DNA library specific for a marker chromosome found in a child with severe developmental abnormalities. We used fluorescent in situ hybridization of the library onto normal metaphase spreads ("reverse chromosome painting") and were thus able to determine that the marker consists of the centromeric part of chromosome 7, the telomeric region of the long arm of chromosome 5 and the telomeric region of the short arm of the X-chromosome. Subsequently, we hybridized normal chromosome-specific libraries of the relevant chromosomes onto metaphases containing the marker chromosome ("forward chromosome painting") and could in this manner establish the precise location of the different chromosome regions on the marker chromosome itself. This is a general approach suitable for outlining marker chromosomes in detail, and will aid the identification of the genes involved.
Assuntos
Marcadores Genéticos , Translocação Genética , Adulto , Sequência de Bases , Linhagem Celular Transformada , Cromossomos Humanos Par 5 , Cromossomos Humanos Par 7 , DNA de Cadeia Simples , Feminino , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Cariotipagem , Masculino , Metáfase , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
A version of the polymerase chain reaction (PCR), termed degenerate oligonucleotide-primed PCR (DOP-PCR), which employs oligonucleotides of partially degenerate sequence, has been developed for genome mapping studies. This degeneracy, together with a PCR protocol utilizing a low initial annealing temperature, ensures priming from multiple (e.g., approximately 10(6) in human) evenly dispersed sites within a given genome. Furthermore, as efficient amplification is achieved from the genomes of all species tested using the same primer, the method appears to be species-independent. Thus, for the general amplification of target DNA, DOP-PCR has advantages over interspersed repetitive sequence PCR (IRS-PCR), which relies on the appropriate positioning of species-specific repeat elements. In conjunction with chromosome flow sorting, DOP-PCR has been applied to the characterization of abnormal chromosomes and also to the cloning of new markers for specific chromosome regions. DOP-PCR therefore represents a rapid, efficient, and species-independent technique for general DNA amplification.
Assuntos
DNA/genética , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Estudos de Avaliação como Assunto , Marcadores Genéticos , Humanos , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genéticaRESUMO
Linkage between seven loci on chromosome 19 and multiple endocrine neoplasia type 2a (MEN2A) was examined in a single large Swedish pedigree. A total of 50 cM was excluded from the male genetic map by pairwise analysis and an estimated 63 cM by multipoint analysis. Using existing data on the likelihood of different marker-marker distances and taking into account current exclusions on other chromosomes, the probability that the gene for MEN2A segregating in this pedigree could still be located on chromosome 19 is approximately 0.28%.
Assuntos
Cromossomos Humanos Par 19 , Ligação Genética , Neoplasia Endócrina Múltipla/genética , Mapeamento Cromossômico , Feminino , Marcadores Genéticos , Humanos , Masculino , Linhagem , Polimorfismo Genético , ProbabilidadeRESUMO
Locus-specific minisatellite probes detect multiple alleles with heterozygosities of greater than 90% when hybridised to HinfI and AluI restriction digests of human DNA. We have hybridised 4 of these probes to a panel of DNAs digested with 6 of the restriction enzymes which commonly reveal di-allelic polymorphisms in the human genome. All 6 enzymes detected multiple resolvable alleles with at least 1 of the 4 minisatellite probes tested, thus providing a rapid and efficient means to check family relationships and paternity on filters already being used for linkage analysis.
Assuntos
DNA Satélite , Marcadores Genéticos , Southern Blotting , Europa (Continente) , Medicina Legal/métodos , Ligação Genética , Humanos , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de RestriçãoRESUMO
Two patients with specific and similar phenotypes were both found to have an unusual marker chromosome present in 70%-80% of their lymphocytes at routine cytogenetic examination. The marker chromosomes were isolated by flow sorting and were amplified by degenerate oligonucleotide-primed PCR. These libraries and a cosmid probe located at 15q26 were used to characterize the marker chromosomes by FISH. Both marker chromosomes were found to consist of duplicated chromosome material from the distal part of chromosome 15q and were identified as inv dup(15) (qter-->q23::q23-->qter) and inv dup(15) (qter-->q24::q24-->qter), respectively. Hence, the markers did not include any known centromere region, and no alpha-satellite DNA could be detected at the site of the primary constriction. Tetrasomy 15q may be a new syndrome, associated with a specific type of marker chromosome. In addition, further analyses of this type of marker chromosome might give new insight into the structure and function of the mammalian centromere.
Assuntos
Anormalidades Múltiplas/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 15 , DNA Satélite/análise , Adulto , Pré-Escolar , Mapeamento Cromossômico , Primers do DNA , Feminino , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Linfócitos/patologia , Masculino , Fenótipo , Reação em Cadeia da Polimerase , TrissomiaRESUMO
A total of 254 affected parent-child pairs with Huntington disease (HD) and 440 parent-child pairs with CAG size in the normal range were assessed to determine the nature and frequency of intergenerational CAG changes in the HD gene. Intergenerational CAG changes are extremely rare (3/440 [0.68%]) on normal chromosomes. In contrast, on HD chromosomes, changes in CAG size occur in approximately 70% of meioses on HD chromosomes, with expansions accounting for 73% of these changes. These intergenerational CAG changes make a significant but minor contribution to changes in age at onset (r2 = .19). The size of the CAG repeat influenced larger intergenerational expansions (> 7 CAG repeats), but the likelihood of smaller expansions or contractions was not influenced by CAG size. Large expansions (> 7 CAG repeats) occur almost exclusively through paternal transmission (0.96%; P < 10(-7)), while offspring of affected mothers are more likely to show no change (P = .01) or contractions in CAG size (P = .002). This study demonstrates that sex of the transmitting parent is the major determinant for CAG intergenerational changes in the HD gene. Similar paternal sex effects are seen in the evolution of new mutations for HD from intermediate alleles and for large expansions on affected chromosomes. Affected mothers almost never transmit a significantly expanded CAG repeat, despite the fact that many have similar large-sized alleles, compared with affected fathers. The sex-dependent effects of major expansion and contractions of the CAG repeat in the HD gene implicate different effects of gametogenesis, in males versus females, on intergenerational CAG repeat stability.
Assuntos
Doença de Huntington/genética , Sequências Repetitivas de Ácido Nucleico , Adolescente , Adulto , Idade de Início , Sequência de Bases , Criança , Pré-Escolar , DNA/análise , Pai , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mães , Fatores SexuaisRESUMO
BACKGROUND: A pilot production facility has been established to isolate mammillian artificial chromosomes at high purity by using flow cytometric techniques. Dicentric chromosomes have been generated by the targeted amplification of pericentric heterochromatic and centromeric DNA by activating the "megareplicator." Breakage of these dicentric chromosomes generates satellite DNA-based artificial chromosomes (SATAC) from 60 to 400 megabases. METHODS: For large-scale production, we have developed cell lines capable of carrying one or two SATACs. A SATAC, because of a high adenine-thymine (AT) composition, is easily identified and sorted by using chromomycin A3 and Hoechst 33258 stains and a dual laser high-speed flow cytometer. A prototype SATAC (60 megabases) has been characterized. The prototype SATAC has been isolated from an original rodent/human hybrid cell line and transferred by using modified microcell fusion into a CHO production cell line. RESULTS: Metaphase chromosomes from this production cell line were isolated in a modified polyamine buffer, stained, and sorted by using a modified sheath buffer that maintains condensed chromosomes. SATACs are routinely sorted at rates greater than 1 million per hour. Sorted SATACs have been transferred to a variety of cells by using microcell fusion technology and were found to be functional. CONCLUSIONS: By developing new SATAC containing cell lines with fewer numbers of chromosomes in conjunction with operating a high speed flow sorter we have effectively generated an efficient production facility geared purely for the isolation of SATACs.
Assuntos
Cromossomos/genética , DNA Satélite/genética , Animais , Células CHO , Linhagem Celular , Cromomicinas/metabolismo , Cricetinae , Fibroblastos/metabolismo , Citometria de Fluxo , Técnicas Genéticas , Humanos , Hibridização in Situ Fluorescente , Infertilidade/diagnóstico , Cariotipagem , Camundongos , Projetos PilotoRESUMO
We describe a phenomenon where a second flow karyotype is superimposed on the normal metaphase flow karyotype of human lymphoblastoid chromosomes. The events of this second flow karyotype contain half the DNA content of corresponding events in the normal flow karyotype and, when sorted, show the morphology of single chromatids. DNA amplified by degenerate oligonucleotide-primed PCR of 300 chromatids sorted from a single peak and hybridized onto normal metaphase spreads painted the entire length of the corresponding chromosome type. In retrospect, we could observe this phenomenon at a low level in 37% of randomly selected, previously analyzed flow karyotypes and in several published flow karyotypes from other laboratories. Experimentally, chromatid frequency was shown to vary with the colcemid blocking conditions as well as with the cell line used. At the extreme, we obtained a 4:10 ratio of chromatids to metaphase chromosomes, whereas this could be reduced tenfold by altering the colcemid blocking conditions in the same cell line. The presence of chromatids in chromosome preparations has important implications for the purity of isolated fractions used for generating libraries and in PCR analysis.
Assuntos
Cromátides , Cromossomos , Citometria de Fluxo , Cariotipagem/métodos , Linhagem Celular , Humanos , Metáfase , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
Juvenile Huntington disease (HD), characterised by onset of symptoms before the age of 20 with rigidity and intellectual decline, is associated with a predominance of affected fathers. In order to investigate the molecular basis for the observed parental effect, we have analysed the CAG trinucleotide repeat within the HD gene in 42 juvenile onset cases from 34 families. A highly significant correlation was found between the repeat length and age of onset (r = -0.86, p < 10(-7) and it was determined that the sex of transmitting parent was the major influence on CAG expansion leading to earlier onset. Neither the size of the parental upper allele, the age of parent at conception of juvenile onset child, nor the grandparental sex conferred a significant effect upon expansion. Affected sib pair analysis of CAG repeat length, however, revealed a high correlation (r = 0.91, p < 10(-7). Furthermore, analysis of nuclear and extended families showed a familial predisposition to juvenile onset disease. This study demonstrates that the sex of transmitting parent is the major influence on trinucleotide expansion and clinical features in juvenile Huntington disease.