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1.
Hum Mutat ; 43(1): 74-84, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34747535

RESUMO

Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in ∼86% of familial and ∼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification.


Assuntos
Neurilemoma , Neurofibromatoses , Cromossomos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neurilemoma/genética , Neurilemoma/patologia , Neurofibromatoses/genética , Proteína SMARCB1/genética , Fatores de Transcrição/genética
2.
Neurogenetics ; 18(3): 169-174, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28285357

RESUMO

Multiplex ligation-dependent probe amplification (MLPA) has been widely used to identify copy-number variations (CNVs), but MLPA's sensitivity and specificity in mosaic CNV detection are largely unknown. Here, we present two mosaic deletions identified by MLPA as NF1 deletion of exons 17-21 and NF2 deletion of exons 9-10. Through cDNA analysis, genomic breakpoint-spanning PCR and Sanger sequencing, we found however both NF1 and NF2 deletions are each composed of two consecutive deletions, which cannot be differentiated by MLPA. Importantly, these consecutive deletions are most likely originating from a single genomic rearrangement and have been preserved independently in different populations of cells.


Assuntos
Variações do Número de Cópias de DNA/genética , Éxons/genética , Neurofibromatose 1/genética , Neurofibromatose 2/genética , Deleção de Genes , Genoma Humano , Genômica , Humanos , Mutação/genética , Reação em Cadeia da Polimerase/métodos
3.
Mol Microbiol ; 92(5): 903-9, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24865634

RESUMO

On 19 January 2014 Rolf ('Roffe') Bernander passed away unexpectedly. Rolf was a dedicated scientist; his research aimed at unravelling the cell biology of the archaeal domain of life, especially cell cycle-related questions, but he also made important contributions in other areas of microbiology. Rolf had a professor position in the Molecular Evolution programme at Uppsala University, Sweden for about 8 years, and in January 2013 he became chair professor at the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University in Sweden. Rolf was an exceptional colleague and will be deeply missed by his family and friends, and the colleagues and co-workers that he leaves behind in the scientific community. He will be remembered for his endless enthusiasm for science, his analytical mind, and his quirky sense of humour.


Assuntos
Archaea/citologia , Ciclo Celular/fisiologia , História do Século XX , História do Século XXI , Suécia
4.
Nat Genet ; 46(2): 182-7, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24362817

RESUMO

Constitutional SMARCB1 mutations at 22q11.23 have been found in ∼50% of familial and <10% of sporadic schwannomatosis cases. We sequenced highly conserved regions along 22q from eight individuals with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ∼80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1.


Assuntos
Cromossomos Humanos Par 22/genética , Predisposição Genética para Doença/genética , Mutação em Linhagem Germinativa/genética , Modelos Moleculares , Neurilemoma/genética , Conformação Proteica , Fatores de Transcrição/genética , Sequência de Bases , Proteínas Cromossômicas não Histona/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Componentes do Gene , Genes Dominantes/genética , Humanos , Perda de Heterozigosidade , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Neurofibromatose 2/genética , Linhagem , Proteína SMARCB1 , Análise de Sequência de DNA , Fatores de Transcrição/química
5.
Eur J Hum Genet ; 18(5): 560-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20051991

RESUMO

Breast cancer is a major cause of morbidity and mortality in women and its metastatic spread is the principal reason behind the fatal outcome. Metastasis-related research of breast cancer is however underdeveloped when compared with the abundant literature on primary tumors. We applied an unexplored approach comparing at high resolution the genomic profiles of primary tumors and synchronous axillary lymph node metastases from 13 patients with breast cancer. Overall, primary tumors displayed 20% higher number of aberrations than metastases. In all but two patients, we detected in total 157 statistically significant differences between primary lesions and matched metastases. We further observed differences that can be linked to metastatic disease and there was also an overlapping pattern of changes between different patients. Many of the differences described here have been previously linked to poor patient survival, suggesting that this is a viable approach toward finding biomarkers for disease progression and definition of new targets useful for development of anticancer drugs. Frequent genetic differences between primary tumors and metastases in breast cancer also question, at least to some extent, the role of primary tumors as a surrogate subject of study for the systemic disease.


Assuntos
Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Progressão da Doença , Metástase Linfática/genética , Adulto , Idoso , Cromossomos Humanos Par 11/genética , Variações do Número de Cópias de DNA/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos
6.
Hum Mutat ; 29(9): 1118-24, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18570184

RESUMO

Two major types of genetic variation are known: single nucleotide polymorphisms (SNPs), and a more recently discovered structural variation, involving changes in copy number (CNVs) of kilobase- to megabase-sized chromosomal segments. It is unknown whether CNVs arise in somatic cells, but it is, however, generally assumed that normal cells are genetically identical. We tested 34 tissue samples from three subjects and, having analyzed for each tissue < or =10(-6) of all cells expected in an adult human, we observed at least six CNVs, affecting a single organ or one or more tissues of the same subject. The CNVs ranged from 82 to 176 kb, often encompassing known genes, potentially affecting gene function. Our results indicate that humans are commonly affected by somatic mosaicism for stochastic CNVs, which occur in a substantial fraction of cells. The majority of described CNVs were previously shown to be polymorphic between unrelated subjects, suggesting that some CNVs previously reported as germline might represent somatic events, since in most studies of this kind, only one tissue is typically examined and analysis of parents for the studied subjects is not routinely performed. A considerable number of human phenotypes are a consequence of a somatic process. Thus, our conclusions will be important for the delineation of genetic factors behind these phenotypes. Consequently, biobanks should consider sampling multiple tissues to better address mosaicism in the studies of somatic disorders.


Assuntos
Dosagem de Genes , Mosaicismo , Polimorfismo Genético , Adulto , Cromossomos Humanos , Predisposição Genética para Doença , Genômica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Distribuição Tecidual
7.
Am J Hum Genet ; 82(3): 763-71, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18304490

RESUMO

The exploration of copy-number variation (CNV), notably of somatic cells, is an understudied aspect of genome biology. Any differences in the genetic makeup between twins derived from the same zygote represent an irrefutable example of somatic mosaicism. We studied 19 pairs of monozygotic twins with either concordant or discordant phenotype by using two platforms for genome-wide CNV analyses and showed that CNVs exist within pairs in both groups. These findings have an impact on our views of genotypic and phenotypic diversity in monozygotic twins and suggest that CNV analysis in phenotypically discordant monozygotic twins may provide a powerful tool for identifying disease-predisposition loci. Our results also imply that caution should be exercised when interpreting disease causality of de novo CNVs found in patients based on analysis of a single tissue in routine disease-related DNA diagnostics.


Assuntos
Cromossomos Humanos/genética , Variação Genética , Doenças Neurodegenerativas/genética , Gêmeos Monozigóticos/genética , DNA/química , DNA/genética , Feminino , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo
8.
Hum Mutat ; 29(3): 398-408, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18058796

RESUMO

To further explore the extent of structural large-scale variation in the human genome, we assessed copy number variations (CNVs) in a series of 71 healthy subjects from three ethnic groups. CNVs were analyzed using comparative genomic hybridization (CGH) to a BAC array covering the human genome, using DNA extracted from peripheral blood, thus avoiding any culture-induced rearrangements. By applying a newly developed computational algorithm based on Hidden Markov modeling, we identified 1,078 autosomal CNVs, including at least two neighboring/overlapping BACs, which represent 315 distinct regions. The average size of the sequence polymorphisms was approximately 350 kb and involved in total approximately 117 Mb or approximately 3.5% of the genome. Gains were about four times more common than deletions, and segmental duplications (SDs) were overrepresented, especially in larger deletion variants. This strengthens the notion that SDs often define hotspots of chromosomal rearrangements. Over 60% of the identified autosomal rearrangements match previously reported CNVs, recognized with various platforms. However, results from chromosome X do not agree well with the previously annotated CNVs. Furthermore, data from single BACs deviating in copy number suggest that our above estimate of total variation is conservative. This report contributes to the establishment of the common baseline for CNV, which is an important resource in human genetics.


Assuntos
Dosagem de Genes , Variação Genética , Grupos Raciais/genética , Algoritmos , Povo Asiático/genética , População Negra/genética , Cromossomos Artificiais Bacterianos/genética , Cromossomos Humanos X/genética , Feminino , Duplicação Gênica , Rearranjo Gênico , Genoma Humano , Humanos , Masculino , Cadeias de Markov , Análise de Sequência com Séries de Oligonucleotídeos , População Branca/genética
9.
J Microbiol Methods ; 69(1): 161-73, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17289189

RESUMO

The aim of this study was to analyze a total euryarchaeal community at DNA and RNA levels in a Swedish barley field with relation to soil depth (0-10 and 20-30 cm layers), soil fraction (bulk soil and rhizosphere) and time (August and November sample collection). Amplification of 16S rRNA gene using the archaeal universal A2F and Euryarchaea specific EK510R/(EURY498) primer pair, combined with denaturing gradient gel electrophoresis (DGGE), revealed distinct differences between rDNA and rRNA DGGE profiles. The soil depth, time, or rhizosphere effects did not significantly influence Archaeal community structure. Surprisingly, sequence analysis of DGGE-derived amplicons revealed the presence of Euryarchaea as well as uncultured soil Crenarchaea affiliated with group 1. In agreement, sequence comparison analyses showed that the majority of uncultured Crenarchaea group 1 had almost 100% sequence complementarity to the 3' end of the EK510R/(EURY498) primer. Therefore, we propose that EK510R/(EURY498R) is a universal archaeal primer rather than a Euryarchaea specific SSUrRNA primer. Hence, considerable care should be taken during application of this primer in studies of euryarchaeal biodiversity in soil environments.


Assuntos
Archaea/isolamento & purificação , Primers do DNA/química , DNA Arqueal/química , Hordeum , RNA Arqueal/genética , RNA Ribossômico 16S/genética , Solo , Archaea/classificação , Archaea/genética , Biodiversidade , DNA Ribossômico/química , Ecossistema , Eletroforese em Gel de Poliacrilamida , Filogenia
10.
J Biol Chem ; 280(17): 16571-8, 2005 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-15722359

RESUMO

The opportunistic human pathogen Pseudomonas aeruginosa is one of a few microorganisms that code for three different classes (I, II, and III) of the enzyme ribonucleotide reductase (RNR). Class II RNR of P. aeruginosa differs from all hitherto known class II enzymes by being encoded by two consecutive open reading frames denoted nrdJa and nrdJb and separated by 16 bp. Split nrdJ genes were also found in the few other gamma-proteobacteria that code for a class II RNR. Interestingly, the two genes encoding the split nrdJ in P. aeruginosa were co-transcribed, and both proteins were expressed. Exponentially growing aerobic cultures were predominantly expressing the class I RNR (encoded by the nrdAB operon) compared with the class II RNR (encoded by the nrdJab operon). Upon entry to stationary phase, the relative amount of nrdJa transcript increased about 6-7-fold concomitant with a 6-fold decrease in the relative amount of nrdA transcript. Hydroxyurea treatment known to knock out the activity of class I RNR caused strict growth inhibition of P. aeruginosa unless 5'-deoxyadenosylcobalamin, a cofactor specifically required for activity of class II RNRs, was added to the rich medium. Rescue of the hydroxyurea-treated cells in the presence of the vitamin B12 cofactor strongly implies that P. aeruginosa produces a functionally active NrdJ protein. Biochemical studies showed for the first time that presence of both NrdJa and NrdJb subunits were absolutely essential for enzyme activity. Based on combined genetic and biochemical results, we suggest that the two-component class II RNR in P. aeruginosa is primarily used for DNA repair and/or possibly DNA replication at low oxygen tension.


Assuntos
Pseudomonas aeruginosa/enzimologia , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/fisiologia , Transcrição Gênica , Animais , Proteínas de Bactérias , Sequência de Bases , Cobamidas/química , Cobamidas/farmacologia , Meios de Cultura/farmacologia , DNA/metabolismo , Reparo do DNA , Relação Dose-Resposta a Droga , Hidroxiureia/farmacologia , Modelos Genéticos , Dados de Sequência Molecular , Oligonucleotídeos/química , Fases de Leitura Aberta , Oxigênio/metabolismo , Filogenia , Plasmídeos/metabolismo , Proteobactérias/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Fatores de Tempo
11.
EMBO Rep ; 3(8): 792-7, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12151340

RESUMO

Mini-chromosome maintenance (MCM) proteins form a conserved family found in all eukaryotes and are essential for DNA replication. They exist as heteromultimeric complexes containing as many as six different proteins. These complexes are believed to be the replicative helicases, functioning as hexameric rings at replication forks. In most archaea a single MCM protein exists. The protein from Methanobacterium thermoautotrophicum (mtMCM) has been reported to assemble into a large complex consistent with a dodecamer. We show that mtMCM can assemble into a heptameric ring. This ring contains a C-terminal helicase domain that can be fit with crystal structures of ring helicases and an N-terminal domain of unknown function. While the structure of the ring is very similar to that of hexameric replicative helicases such as bacteriophage T7 gp4, our results show that such ring structures may not be constrained to have only six subunits.


Assuntos
Proteínas Arqueais/química , DNA Helicases/química , Methanobacterium/metabolismo , Motivos de Aminoácidos , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , DNA/biossíntese , DNA Helicases/metabolismo , Escherichia coli/metabolismo , Methanobacterium/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Estrutura Terciária de Proteína
12.
Microbiology (Reading) ; 146 ( Pt 3): 749-757, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10746779

RESUMO

As a basis for studing the essential cellular processes of hyperthermophilic archaea, thermosensitive mutants of Sulfolobus acidocaldarius were isolated and characterized. Exponential-phase liquid cultures were shifted to the nonpermissive temperature and growth, viability, and distributions of cell mass and DNA content were measured as a function of time after the shift. The observed phenotypes demonstrate that chromosome replication, nucleoid organization, nucleoid partition and cell division, which normally are tightly co-ordinated during cellular growth, can be inhibited or uncoupled by mutation in this hyperthermophilic archaeon.


Assuntos
Mutação , Sulfolobus acidocaldarius/crescimento & desenvolvimento , Sulfolobus acidocaldarius/genética , Ciclo Celular , Meios de Cultura , DNA Arqueal/metabolismo , Citometria de Fluxo , Fenótipo , Sulfolobus acidocaldarius/ultraestrutura , Temperatura
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