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1.
Trends Biochem Sci ; 49(5): 391-400, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38490833

RESUMO

One of the two chromosomal breakage events in recurring translocations in B cell neoplasms is often due to the recombination-activating gene complex (RAG complex) releasing DNA ends before end joining. The other break occurs in a fragile zone of 20-600 bp in a non-antigen receptor gene locus, with a more complex and intriguing set of mechanistic factors underlying such narrow fragile zones. These factors include activation-induced deaminase (AID), which acts only at regions of single-stranded DNA (ssDNA). Recent work leads to a model involving the tethering of AID to the nascent RNA as it emerges from the RNA polymerase. This mechanism may have relevance in class switch recombination (CSR) and somatic hypermutation (SHM), as well as broader relevance for other DNA enzymes.


Assuntos
RNA , Translocação Genética , Humanos , RNA/metabolismo , RNA/genética , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Sítios Frágeis do Cromossomo
2.
Cell ; 135(6): 1130-42, 2008 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-19070581

RESUMO

We have assembled, annotated, and analyzed a database of over 1700 breakpoints from the most common chromosomal rearrangements in human leukemias and lymphomas. Using this database, we show that although the CpG dinucleotide constitutes only 1% of the human genome, it accounts for 40%-70% of breakpoints at pro-B/pre-B stage translocation regions-specifically, those near the bcl-2, bcl-1, and E2A genes. We do not observe CpG hotspots in rearrangements involving lymphoid-myeloid progenitors, mature B cells, or T cells. The stage specificity, lineage specificity, CpG targeting, and unique breakpoint distributions at these cluster regions may be explained by a lesion-specific double-strand breakage mechanism involving the RAG complex acting at AID-deaminated methyl-CpGs.


Assuntos
Linfócitos B/metabolismo , Ilhas de CpG , Leucemia Linfoide/genética , Translocação Genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Quebra Cromossômica , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Genes bcl-1 , Genes bcl-2 , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Linfoide/metabolismo
3.
Cell ; 135(7): 1299-310, 2008 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-19109898

RESUMO

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.


Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Quimera , Epigênese Genética , Feminino , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Quinases da Glicogênio Sintase/antagonistas & inibidores , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Ratos , Ratos Endogâmicos , Transdução de Sinais
5.
Am J Physiol Lung Cell Mol Physiol ; 318(4): L671-L683, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32073882

RESUMO

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.


Assuntos
Furões/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/fisiologia , Feminino , Fibroblastos/citologia , Humanos , Masculino
6.
Stat Appl Genet Mol Biol ; 18(3)2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30956231

RESUMO

Genome-wide sequencing enables evaluation of associations between traits and combinations of variants in genes and pathways. But such evaluation requires multi-locus association tests with good power, regardless of the variant and trait characteristics. And since analyzing families may yield more power than analyzing unrelated individuals, we need multi-locus tests applicable to both related and unrelated individuals. Here we describe such tests, and we introduce SKAT-X, a new test statistic that uses genome-wide data obtained from related or unrelated subjects to optimize power for the specific data at hand. Simulations show that: a) SKAT-X performs well regardless of variant and trait characteristics; and b) for binary traits, analyzing affected relatives brings more power than analyzing unrelated individuals, consistent with previous findings for single-locus tests. We illustrate the methods by application to rare unclassified missense variants in the tumor suppressor gene BRCA2, as applied to combined data from prostate cancer families and unrelated prostate cancer cases and controls in the Multi-ethnic Cohort (MEC). The methods can be implemented using open-source code for public use as the R-package GATARS (Genetic Association Tests for Arbitrarily Related Subjects) .


Assuntos
Estudos de Associação Genética/estatística & dados numéricos , Testes Genéticos , Software , Simulação por Computador , Variação Genética/genética , Estudo de Associação Genômica Ampla/estatística & dados numéricos , Humanos , Modelos Genéticos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética
7.
Am J Hum Genet ; 99(4): 877-885, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27666373

RESUMO

The vast majority of coding variants are rare, and assessment of the contribution of rare variants to complex traits is hampered by low statistical power and limited functional data. Improved methods for predicting the pathogenicity of rare coding variants are needed to facilitate the discovery of disease variants from exome sequencing studies. We developed REVEL (rare exome variant ensemble learner), an ensemble method for predicting the pathogenicity of missense variants on the basis of individual tools: MutPred, FATHMM, VEST, PolyPhen, SIFT, PROVEAN, MutationAssessor, MutationTaster, LRT, GERP, SiPhy, phyloP, and phastCons. REVEL was trained with recently discovered pathogenic and rare neutral missense variants, excluding those previously used to train its constituent tools. When applied to two independent test sets, REVEL had the best overall performance (p < 10-12) as compared to any individual tool and seven ensemble methods: MetaSVM, MetaLR, KGGSeq, Condel, CADD, DANN, and Eigen. Importantly, REVEL also had the best performance for distinguishing pathogenic from rare neutral variants with allele frequencies <0.5%. The area under the receiver operating characteristic curve (AUC) for REVEL was 0.046-0.182 higher in an independent test set of 935 recent SwissVar disease variants and 123,935 putatively neutral exome sequencing variants and 0.027-0.143 higher in an independent test set of 1,953 pathogenic and 2,406 benign variants recently reported in ClinVar than the AUCs for other ensemble methods. We provide pre-computed REVEL scores for all possible human missense variants to facilitate the identification of pathogenic variants in the sea of rare variants discovered as sequencing studies expand in scale.


Assuntos
Doença/genética , Mutação de Sentido Incorreto/genética , Software , Área Sob a Curva , Análise Mutacional de DNA , Exoma/genética , Frequência do Gene , Humanos , Curva ROC
8.
Genet Epidemiol ; 41(4): 297-308, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28211093

RESUMO

Next-generation sequencing technologies have afforded unprecedented characterization of low-frequency and rare genetic variation. Due to low power for single-variant testing, aggregative methods are commonly used to combine observed rare variation within a single gene. Causal variation may also aggregate across multiple genes within relevant biomolecular pathways. Kernel-machine regression and adaptive testing methods for aggregative rare-variant association testing have been demonstrated to be powerful approaches for pathway-level analysis, although these methods tend to be computationally intensive at high-variant dimensionality and require access to complete data. An additional analytical issue in scans of large pathway definition sets is multiple testing correction. Gene set definitions may exhibit substantial genic overlap, and the impact of the resultant correlation in test statistics on Type I error rate control for large agnostic gene set scans has not been fully explored. Herein, we first outline a statistical strategy for aggregative rare-variant analysis using component gene-level linear kernel score test summary statistics as well as derive simple estimators of the effective number of tests for family-wise error rate control. We then conduct extensive simulation studies to characterize the behavior of our approach relative to direct application of kernel and adaptive methods under a variety of conditions. We also apply our method to two case-control studies, respectively, evaluating rare variation in hereditary prostate cancer and schizophrenia. Finally, we provide open-source R code for public use to facilitate easy application of our methods to existing rare-variant analysis results.


Assuntos
Algoritmos , Estudos de Associação Genética/métodos , Variação Genética , Simulação por Computador , Humanos , Modelos Genéticos , Tamanho da Amostra , Estatísticas não Paramétricas
9.
Genet Epidemiol ; 40(6): 461-9, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27312771

RESUMO

Rare variants (RVs) have been shown to be significant contributors to complex disease risk. By definition, these variants have very low minor allele frequencies and traditional single-marker methods for statistical analysis are underpowered for typical sequencing study sample sizes. Multimarker burden-type approaches attempt to identify aggregation of RVs across case-control status by analyzing relatively small partitions of the genome, such as genes. However, it is generally the case that the aggregative measure would be a mixture of causal and neutral variants, and these omnibus tests do not directly provide any indication of which RVs may be driving a given association. Recently, Bayesian variable selection approaches have been proposed to identify RV associations from a large set of RVs under consideration. Although these approaches have been shown to be powerful at detecting associations at the RV level, there are often computational limitations on the total quantity of RVs under consideration and compromises are necessary for large-scale application. Here, we propose a computationally efficient alternative formulation of this method using a probit regression approach specifically capable of simultaneously analyzing hundreds to thousands of RVs. We evaluate our approach to detect causal variation on simulated data and examine sensitivity and specificity in instances of high RV dimensionality as well as apply it to pathway-level RV analysis results from a prostate cancer (PC) risk case-control sequencing study. Finally, we discuss potential extensions and future directions of this work.


Assuntos
Modelos Genéticos , Teorema de Bayes , Estudos de Casos e Controles , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA
10.
Nucleic Acids Res ; 42(21): 13186-93, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25378327

RESUMO

The boundaries of R-loops are well-documented at immunoglobulin heavy chain loci in mammalian B cells. Within primary B cells or B cell lines, the upstream boundaries of R-loops typically begin early in the repetitive portion of the switch regions. Most R-loops terminate within the switch repetitive zone, but the remainder can extend a few hundred base pairs further, where G-density on the non-template DNA strand gradually drops to the genome average. Whether the G-density determines how far the R-loops extend is an important question. We previously studied the role of G-clusters in initiating R-loop formation, but we did not examine the role of G-density in permitting the elongation of the R-loop, after it had initiated. Here, we vary the G-density of different portions of the switch region in a murine B cell line. We find that both class switch recombination (CSR) and R-loop formation decrease significantly when the overall G-density is reduced from 46% to 29%. Short 50 bp insertions with low G-density within switch regions do not appear to affect either CSR or R-loop elongation, whereas a longer (150 bp) insertion impairs both. These results demonstrate that G-density is an important determinant of the length over which mammalian genomic R-loops extend.


Assuntos
Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Animais , Linhagem Celular , DNA/química , Camundongos , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico
11.
Hum Genet ; 133(3): 347-56, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24162621

RESUMO

Previous GWAS studies have reported significant associations between various common SNPs and prostate cancer risk using cases unselected for family history. How these variants influence risk in familial prostate cancer is not well studied. Here, we analyzed 25 previously reported SNPs across 14 loci from prior prostate cancer GWAS. The International Consortium for Prostate Cancer Genetics (ICPCG) previously validated some of these using a family-based association method (FBAT). However, this approach suffered reduced power due to the conditional statistics implemented in FBAT. Here, we use a case-control design with an empirical analysis strategy to analyze the ICPCG resource for association between these 25 SNPs and familial prostate cancer risk. Fourteen sites contributed 12,506 samples (9,560 prostate cancer cases, 3,368 with aggressive disease, and 2,946 controls from 2,283 pedigrees). We performed association analysis with Genie software which accounts for relationships. We analyzed all familial prostate cancer cases and the subset of aggressive cases. For the familial prostate cancer phenotype, 20 of the 25 SNPs were at least nominally associated with prostate cancer and 16 remained significant after multiple testing correction (p ≤ 1E (-3)) occurring on chromosomal bands 6q25, 7p15, 8q24, 10q11, 11q13, 17q12, 17q24, and Xp11. For aggressive disease, 16 of the SNPs had at least nominal evidence and 8 were statistically significant including 2p15. The results indicate that the majority of common, low-risk alleles identified in GWAS studies for all prostate cancer also contribute risk for familial prostate cancer, and that some may contribute risk to aggressive disease.


Assuntos
Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/genética , Alelos , Estudos de Casos e Controles , Seguimentos , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Metanálise como Assunto , Linhagem , Fenótipo , Fatores de Risco
12.
Hum Genet ; 132(1): 5-14, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23064873

RESUMO

Prostate cancer has a strong familial component but uncovering the molecular basis for inherited susceptibility for this disease has been challenging. Recently, a rare, recurrent mutation (G84E) in HOXB13 was reported to be associated with prostate cancer risk. Confirmation and characterization of this finding is necessary to potentially translate this information to the clinic. To examine this finding in a large international sample of prostate cancer families, we genotyped this mutation and 14 other SNPs in or flanking HOXB13 in 2,443 prostate cancer families recruited by the International Consortium for Prostate Cancer Genetics (ICPCG). At least one mutation carrier was found in 112 prostate cancer families (4.6 %), all of European descent. Within carrier families, the G84E mutation was more common in men with a diagnosis of prostate cancer (194 of 382, 51 %) than those without (42 of 137, 30 %), P = 9.9 × 10(-8) [odds ratio 4.42 (95 % confidence interval 2.56-7.64)]. A family-based association test found G84E to be significantly over-transmitted from parents to affected offspring (P = 6.5 × 10(-6)). Analysis of markers flanking the G84E mutation indicates that it resides in the same haplotype in 95 % of carriers, consistent with a founder effect. Clinical characteristics of cancers in mutation carriers included features of high-risk disease. These findings demonstrate that the HOXB13 G84E mutation is present in ~5 % of prostate cancer families, predominantly of European descent, and confirm its association with prostate cancer risk. While future studies are needed to more fully define the clinical utility of this observation, this allele and others like it could form the basis for early, targeted screening of men at elevated risk for this common, clinically heterogeneous cancer.


Assuntos
Proteínas de Homeodomínio/genética , Neoplasias da Próstata/genética , Substituição de Aminoácidos , Estudos de Coortes , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Heterozigoto , Humanos , Agências Internacionais , Masculino , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , População Branca/genética
13.
BMC Res Notes ; 16(1): 66, 2023 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-37106434

RESUMO

OBJECTIVE: Comprehensive and reliable genome-wide variant analysis of a small number of cells has been challenging due to genome coverage bias, PCR over-cycling, and the requirement of expensive technologies. To comprehensively identify genome alterations in single colon crypts that reflect genome heterogeneity of stem cells, we developed a method to construct whole-genome sequencing libraries from single colon crypts without DNA extraction, whole-genome amplification, or increased PCR enrichment cycles. RESULTS: We present post-alignment statistics of 81 single-crypts (each contains four- to eight-fold less DNA than the requirement of conventional methods) and 16 bulk-tissue libraries to demonstrate the consistent success in obtaining reliable coverage, both in depth (≥ 30X) and breadth (≥ 92% of the genome covered at ≥ 10X depth), of the human genome. These single-crypt libraries are of comparable quality as libraries generated with the conventional method using high quality and quantities of purified DNA. Conceivably, our method can be applied to small biopsy samples from many tissues and can be combined with single cell targeted sequencing to comprehensively profile cancer genomes and their evolution. The broad potential application of this method offers expanded possibilities in cost-effectively examining genome heterogeneity in small numbers of cells at high resolution.


Assuntos
DNA , Técnicas de Amplificação de Ácido Nucleico , Humanos , Sequenciamento Completo do Genoma , Análise de Sequência de DNA/métodos , Reação em Cadeia da Polimerase , Sequenciamento de Nucleotídeos em Larga Escala/métodos
14.
Front Cell Dev Biol ; 11: 1134121, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37082620

RESUMO

As effective therapies for relapse and refractory B-cell acute lymphoblastic leukemia (B-ALL) remain problematic, novel therapeutic strategies are needed. Artemis is a key endonuclease in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Inhibition of Artemis would cause chromosome breaks during maturation of RAG-expressing T- and B-cells. Though this would block generation of new B- and T-cells temporarily, it could be oncologically beneficial for reducing the proliferation of B-ALL and T-ALL cells by causing chromosome breaks in these RAG-expressing tumor cells. Currently, pharmacological inhibition is not available for Artemis. According to gene expression analyses from 207 children with high-risk pre-B acute lymphoblastic leukemias high Artemis expression is correlated with poor outcome. Therefore, we evaluated four compounds (827171, 827032, 826941, and 825226), previously generated from a large Artemis targeted drug screen. A biochemical assay using a purified Artemis:DNA-PKcs complex shows that the Artemis inhibitors 827171, 827032, 826941, 825226 have nanomolar IC50 values for Artemis inhibition. We compared these 4 compounds to a DNA-PK inhibitor (AZD7648) in three patient-derived B-ALL cell lines (LAX56, BLQ5 and LAX7R) and in two mature B-cell lines (3301015 and 5680001) as controls. We found that pharmacological Artemis inhibition substantially decreases proliferation of B-ALL cell lines while normal mature B-cell lines are not markedly affected. Inhibition of DNA-PKcs (which regulates Artemis) using the DNA-PK inhibitor AZD7648 had minor effects on these same primary patient-derived ALL lines, indicating that inhibition of V(D)J hairpin opening requires direct inhibition of Artemis, rather than indirect suppression of the kinase that regulates Artemis. Our data provides a basis for further evaluation of pharmacological Artemis inhibition of proliferation of B- and T-ALL.

15.
Prostate ; 72(4): 410-26, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21748754

RESUMO

BACKGROUND: In spite of intensive efforts, understanding of the genetic aspects of familial prostate cancer (PC) remains largely incomplete. In a previous microsatellite-based linkage scan of 1,233 PC families, we identified suggestive evidence for linkage (i.e., LOD ≥ 1.86) at 5q12, 15q11, 17q21, 22q12, and two loci on 8p, with additional regions implicated in subsets of families defined by age at diagnosis, disease aggressiveness, or number of affected members. METHODS: In an attempt to replicate these findings and increase linkage resolution, we used the Illumina 6000 SNP linkage panel to perform a genome-wide linkage scan of an independent set of 762 multiplex PC families, collected by 11 International Consortium for Prostate Cancer Genetics (ICPCG) groups. RESULTS: Of the regions identified previously, modest evidence of replication was observed only on the short arm of chromosome 8, where HLOD scores of 1.63 and 3.60 were observed in the complete set of families and families with young average age at diagnosis, respectively. The most significant linkage signals found in the complete set of families were observed across a broad, 37 cM interval on 4q13-25, with LOD scores ranging from 2.02 to 2.62, increasing to 4.50 in families with older average age at diagnosis. In families with multiple cases presenting with more aggressive disease, LOD scores over 3.0 were observed at 8q24 in the vicinity of previously identified common PC risk variants, as well as MYC, an important gene in PC biology. CONCLUSIONS: These results will be useful in prioritizing future susceptibility gene discovery efforts in this common cancer.


Assuntos
Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 8/genética , Estudo de Associação Genômica Ampla , Cooperação Internacional , Polimorfismo de Nucleotídeo Único/genética , Neoplasias da Próstata/genética , Idoso , Interpretação Estatística de Dados , Ligação Genética/genética , Predisposição Genética para Doença/genética , Genótipo , Humanos , Escore Lod , Masculino , Linhagem
16.
Hum Genet ; 131(7): 1095-103, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22198737

RESUMO

Multiple prostate cancer (PCa) risk-related loci have been discovered by genome-wide association studies (GWAS) based on case-control designs. However, GWAS findings may be confounded by population stratification if cases and controls are inadvertently drawn from different genetic backgrounds. In addition, since these loci were identified in cases with predominantly sporadic disease, little is known about their relationships with hereditary prostate cancer (HPC). The association between seventeen reported PCa susceptibility loci was evaluated with a family-based association test using 1,979 hereditary PCa families of European descent collected by members of the International Consortium for Prostate Cancer Genetics, with a total of 5,730 affected men. The risk alleles for 8 of the 17 loci were significantly over-transmitted from parents to affected offspring, including SNPs residing in 8q24 (regions 1, 2 and 3), 10q11, 11q13, 17q12 (region 1), 17q24 and Xp11. In subgroup analyses, three loci, at 8q24 (regions 1 and 2) plus 17q12, were significantly over-transmitted in hereditary PCa families with five or more affected members, while loci at 3p12, 8q24 (region 2), 11q13, 17q12 (region 1), 17q24 and Xp11 were significantly over-transmitted in HPC families with an average age of diagnosis at 65 years or less. Our results indicate that at least a subset of PCa risk-related loci identified by case-control GWAS are also associated with disease risk in HPC families.


Assuntos
Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/genética , População Branca/genética , Alelos , Estudos de Casos e Controles , Frequência do Gene , Variação Genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Neoplasias da Próstata/etnologia , Fatores de Risco
17.
BMC Med Genet ; 13: 46, 2012 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-22712434

RESUMO

BACKGROUND: Genetic variants are likely to contribute to a portion of prostate cancer risk. Full elucidation of the genetic etiology of prostate cancer is difficult because of incomplete penetrance and genetic and phenotypic heterogeneity. Current evidence suggests that genetic linkage to prostate cancer has been found on several chromosomes including the X; however, identification of causative genes has been elusive. METHODS: Parametric and non-parametric linkage analyses were performed using 26 microsatellite markers in each of 11 groups of multiple-case prostate cancer families from the International Consortium for Prostate Cancer Genetics (ICPCG). Meta-analyses of the resultant family-specific linkage statistics across the entire 1,323 families and in several predefined subsets were then performed. RESULTS: Meta-analyses of linkage statistics resulted in a maximum parametric heterogeneity lod score (HLOD) of 1.28, and an allele-sharing lod score (LOD) of 2.0 in favor of linkage to Xq27-q28 at 138 cM. In subset analyses, families with average age at onset less than 65 years exhibited a maximum HLOD of 1.8 (at 138 cM) versus a maximum regional HLOD of only 0.32 in families with average age at onset of 65 years or older. Surprisingly, the subset of families with only 2-3 affected men and some evidence of male-to-male transmission of prostate cancer gave the strongest evidence of linkage to the region (HLOD = 3.24, 134 cM). For this subset, the HLOD was slightly increased (HLOD = 3.47 at 134 cM) when families used in the original published report of linkage to Xq27-28 were excluded. CONCLUSIONS: Although there was not strong support for linkage to the Xq27-28 region in the complete set of families, the subset of families with earlier age at onset exhibited more evidence of linkage than families with later onset of disease. A subset of families with 2-3 affected individuals and with some evidence of male to male disease transmission showed stronger linkage signals. Our results suggest that the genetic basis for prostate cancer in our families is much more complex than a single susceptibility locus on the X chromosome, and that future explorations of the Xq27-28 region should focus on the subset of families identified here with the strongest evidence of linkage to this region.


Assuntos
Cromossomos Humanos X , Neoplasias da Próstata/genética , Alelos , Ligação Genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Repetições de Microssatélites
18.
DNA Repair (Amst) ; 110: 103271, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34990960

RESUMO

Activation-induced deaminase (AID) only deaminates cytosine within single-stranded DNA. Transcription is known to increase AID deamination on duplex DNA substrates during transcription. Using a purified T7 RNA polymerase transcription system, we recently found that AID deamination of a duplex DNA substrate is reduced if RNase A is added during transcription. This finding prompted us to consider that the mRNA tail may contribute to AID action at the nearby transcribed strand (TS) or non-transcribed strand (NTS) of DNA, which are transiently single-stranded in the wake of RNA polymerase movement. Here, we used a purified system to test whether a single-stranded oligonucleotide (oligo) consisting of RNA in the 5' portion and DNA in the 3' portion (i.e., 5'RNA-DNA3', also termed an RNA-DNA fusion substrate) could be deaminated equally efficiently as the same sequence when it is entirely DNA. We found that AID acts on the RNA-DNA fusion substrate and the DNA-only substrate with similar efficiency. Based on this finding and our recent observation on the importance of the mRNA tail, we propose a model in which the proximity and length of the mRNA tail provide a critical site for AID loading to permit a high local collision frequency with the NTS and TS in the transient wake of the RNA polymerase. When the mRNA tail is not present, we know that AID action drops to levels equivalent to when there is no transcription at all. This mRNA tether model explains several local and global features of Ig somatic hypermutation and Ig class switch recombination, while integrating structural and functional features of AID.


Assuntos
Citidina Desaminase , Hipermutação Somática de Imunoglobulina , Citidina Desaminase/química , Citidina Desaminase/genética , DNA/genética , Switching de Imunoglobulina , RNA , RNA Mensageiro/genética
19.
Cell Rep ; 36(2): 109387, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260910

RESUMO

Analysis of translocation breakpoints in human B cell malignancies reveals that DNA double-strand breaks at oncogenes most frequently occur at CpG sites located within 20-600 bp fragile zones and depend on activation-induced deaminase (AID). AID requires single-stranded DNA (ssDNA) to act, but it has been unclear why or how this region transiently acquires a ssDNA state. Here, we demonstrate the ssDNA state in the 23 bp E2A fragile zone using several methods, including native bisulfite DNA structural analysis in live human pre-B cells. AID deamination within the E2A fragile zone does not require but is increased upon transcription. High C-string density, nascent RNA tails, and direct DNA sequence repeats prolong the ssDNA state of the E2A fragile zone and increase AID deamination at overlapping AID hotspots that contain the CpG sites at which breaks occur in patients. These features provide key insights into lymphoid fragile zones generally.


Assuntos
Linfócitos B/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cromossomos Humanos/genética , Neoplasias/genética , Neoplasias/imunologia , Translocação Genética , Pareamento de Bases/genética , Sequência de Bases , Quebra Cromossômica , Ilhas de CpG/genética , Citidina Desaminase , Desaminação , Humanos , Íntrons/genética , Linfócitos/metabolismo , Ribonuclease Pancreático/metabolismo , Especificidade por Substrato
20.
Front Oncol ; 11: 766888, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34926269

RESUMO

The PI3K/Akt pathway-and in particular PI3Kδ-is known for its role in drug resistant B-cell acute lymphoblastic leukemia (B-ALL) and it is often upregulated in refractory or relapsed B-ALL. Myc proteins are transcription factors responsible for transcribing pro-proliferative genes and c-Myc is often overexpressed in cancers. The chromatin regulator BRD4 is required for expression of c-Myc in hematologic malignancies including B-ALL. Previously, combination of BRD4 and PI3K inhibition with SF2523 was shown to successfully decrease Myc expression. However, the underlying mechanism and effect of dual inhibition of PI3Kδ/BRD4 in B-ALL remains unknown. To study this, we utilized SF2535, a novel small molecule dual inhibitor which can specifically target the PI3Kδ isoform and BRD4. We treated primary B-ALL cells with various concentrations of SF2535 and studied its effect on specific pharmacological on-target mechanisms such as apoptosis, cell cycle, cell proliferation, and adhesion molecules expression usingin vitro and in vivo models. SF2535 significantly downregulates both c-Myc mRNA and protein expression through inhibition of BRD4 at the c-Myc promoter site and decreases p-AKT expression through inhibition of the PI3Kδ/AKT pathway. SF2535 induced apoptosis in B-ALL by downregulation of BCL-2 and increased cleavage of caspase-3, caspase-7, and PARP. Moreover, SF2535 induced cell cycle arrest and decreased cell counts in B-ALL. Interestingly, SF2535 decreased the mean fluorescence intensity (MFI) of integrin α4, α5, α6, and ß1 while increasing MFI of CXCR4, indicating that SF2535 may work through inside-out signaling of integrins. Taken together, our data provide a rationale for the clinical evaluation of targeting PI3Kδ/BRD4 in refractory or relapsed B-ALL using SF2535.

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