RESUMEN
Comprehensive genetic profiling is increasingly important for the clinical workup of hematologic tumors, as specific alterations are now linked to diagnostic characterization, prognostic stratification and therapy selection. To characterize relevant genetic and genomic alterations in myeloid malignancies maximally, we utilized a comprehensive strategy spanning fluorescence in situ hybridization (FISH), classical karyotyping, Chromosomal Microarray (CMA) for detection of copy number variants (CNVs) and Next generation Sequencing (NGS) analysis. In our cohort of 569 patients spanning the myeloid spectrum, NGS and CMA testing frequently identified mutations and copy number changes in the majority of genes with important clinical associations, such as TP53, TET2, RUNX1, SRSF2, APC and ATM. Most importantly, NGS and CMA uncovered medically actionable aberrations in 75.6% of cases normal by FISH/cytogenetics testing. NGS identified mutations in 65.5% of samples normal by CMA, cytogenetics and FISH, whereas CNVs were detected in 10.1% cases that were normal by all other methodologies. Finally, FISH or cytogenetics, or both, were abnormal in 14.1% of cases where NGS or CMA failed to detect any changes. Multiple mutations and CNVs were found to coexist, with potential implications for patient stratification. Thus, high throughput genomic tumor profiling through targeted DNA sequencing and CNV analysis complements conventional methods and leads to more frequent detection of actionable alterations.
Asunto(s)
Cromosomas Humanos/genética , Citogenética/métodos , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Hibridación Fluorescente in Situ/métodos , Trastornos Mieloproliferativos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Estudios de Cohortes , Variaciones en el Número de Copia de ADN/genética , Humanos , Mutación/genética , Trastornos Mieloproliferativos/diagnóstico , Carga TumoralRESUMEN
Genome-wide association (GWA) analyses have generally been used to detect individual loci contributing to the phenotypic diversity in a population by the effects of these loci on the trait mean. More rarely, loci have also been detected based on variance differences between genotypes. Several hypotheses have been proposed to explain the possible genetic mechanisms leading to such variance signals. However, little is known about what causes these signals, or whether this genetic variance-heterogeneity reflects mechanisms of importance in natural populations. Previously, we identified a variance-heterogeneity GWA (vGWA) signal for leaf molybdenum concentrations in Arabidopsis thaliana. Here, fine-mapping of this association reveals that the vGWA emerges from the effects of three independent genetic polymorphisms that all are in strong LD with the markers displaying the genetic variance-heterogeneity. By revealing the genetic architecture underlying this vGWA signal, we uncovered the molecular source of a significant amount of hidden additive genetic variation or "missing heritability". Two of the three polymorphisms underlying the genetic variance-heterogeneity are promoter variants for Molybdate transporter 1 (MOT1), and the third a variant located ~25 kb downstream of this gene. A fourth independent association was also detected ~600 kb upstream of MOT1. Use of a T-DNA knockout allele highlights Copper Transporter 6; COPT6 (AT2G26975) as a strong candidate gene for this association. Our results show that an extended LD across a complex locus including multiple functional alleles can lead to a variance-heterogeneity between genotypes in natural populations. Further, they provide novel insights into the genetic regulation of ion homeostasis in A. thaliana, and empirically confirm that variance-heterogeneity based GWA methods are a valuable tool to detect novel associations of biological importance in natural populations.
Asunto(s)
Proteínas de Transporte de Anión/genética , Proteínas de Arabidopsis/genética , Estudio de Asociación del Genoma Completo , Proteínas de Transporte de Membrana/genética , Sitios de Carácter Cuantitativo/genética , Alelos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Heterogeneidad Genética , Genoma de Planta , Genotipo , Proteínas de Transporte de Membrana/metabolismo , Molibdeno/química , Molibdeno/metabolismo , Hojas de la Planta/genética , Polimorfismo de Nucleótido Simple , Proteínas SLC31RESUMEN
BACKGROUND & AIMS: A new gene expression profile test may distinguish eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD), but the optimal tissue preparation and biopsy location are unknown. We aimed to determine if formalin-fixed paraffin-embedded (FFPE) and RNA-later (RNAL) preserved specimens from newly diagnosed EoE patients have equivalent gene expression scores and whether scores vary by esophageal biopsy location. METHODS: We analyzed prospectively collected and banked esophageal biopsies from EoE patients and GERD controls. Paired FFPE and RNAL samples from the distal, mid, and proximal esophagus were used. RNA was extracted, and gene expression for a previously constructed 96 gene panel was quantified with a summary expression score. Scores were compared between EoE and GERD patients, between FFPE and RNAL samples, and between the different esophageal locations. RESULTS: A total of 72 samples, representing paired FFPE and RNAL specimens from 9 EoE cases and 3 GERD controls, were analyzed. Overall median gene expression scores were similar between FFPE and RNAL (238 vs 227; p=0.64), correlation was excellent between FFPE and RNAL (Spearman's rho=0.90; p<0.001), and there were no differences by biopsy level. Median gene scores distinguished EoE from controls (134 vs 402; p=0.02), and overall agreement between preservation methods and EoE case status was perfect (kappa=1.0; p<0.001). CONCLUSIONS: Gene expression scores were equivalent in FFPE and RNAL, and were also similar across three esophageal locations. This implies that a single biopsy in either FFPE or RNAL from anywhere in the esophagus may have the potential for genetic diagnosis of EoE.
Asunto(s)
Esofagitis Eosinofílica/genética , Esófago/química , Perfilación de la Expresión Génica/métodos , Adulto , Anciano , Biopsia , Estudios de Casos y Controles , Esofagitis Eosinofílica/diagnóstico , Esófago/patología , Femenino , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Valor Predictivo de las Pruebas , Estudios Prospectivos , Adulto JovenRESUMEN
Protein-coding sequences can arise either from duplication and divergence of existing sequences, or de novo from noncoding DNA. Unfortunately, recently evolved de novo genes can be hard to distinguish from false positives, making their study difficult. Here, we study a more tractable version of the process of conversion of noncoding sequence into coding: the co-option of short segments of noncoding sequence into the C-termini of existing proteins via the loss of a stop codon. Because we study recent additions to potentially old genes, we are able to apply a variety of stringent quality filters to our annotations of what is a true protein-coding gene, discarding the putative proteins of unknown function that are typical of recent fully de novo genes. We identify 54 examples of C-terminal extensions in Saccharomyces and 28 in Drosophila, all of them recent enough to still be polymorphic. We find one putative gene fusion that turns out, on close inspection, to be the product of replicated assembly errors, further highlighting the issue of false positives in the study of rare events. Four of the Saccharomyces C-terminal extensions (to ADH1, ARP8, TPM2, and PIS1) that survived our quality filters are predicted to lead to significant modification of a protein domain structure.
Asunto(s)
Codón de Terminación , Evolución Molecular , Polimorfismo Genético , Proteínas/genética , Regiones no Traducidas 3' , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Fúngicas/genética , Conformación Proteica , Saccharomyces/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
As sessile organisms, plants have to cope with diverse environmental constraints that may vary through time and space, eventually leading to changes in the phenotype of populations through fixation of adaptive genetic variation. To fully comprehend the mechanisms of evolution and make sense of the extensive genotypic diversity currently revealed by new sequencing technologies, we are challenged with identifying the molecular basis of such adaptive variation. Here, we have identified a new variant of a molybdenum (Mo) transporter, MOT1, which is causal for fitness changes under artificial conditions of both Mo-deficiency and Mo-toxicity and in which allelic variation among West-Asian populations is strictly correlated with the concentration of available Mo in native soils. In addition, this association is accompanied at different scales with patterns of polymorphisms that are not consistent with neutral evolution and show signs of diversifying selection. Resolving such a case of allelic heterogeneity helps explain species-wide phenotypic variation for Mo homeostasis and potentially reveals trade-off effects, a finding still rarely linked to fitness.