RESUMEN
The malignant Hodgkin and Reed Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) are scarce in affected lymph nodes, creating a challenge to detect driver somatic mutations. As an alternative to cell purification techniques, we hypothesized that ultra-deep exome sequencing would allow genomic study of HRS cells, thereby streamlining analysis and avoiding technical pitfalls. To test this, 31 cHL tumor/normal pairs were exome sequenced to approximately 1,000× median depth of coverage. An orthogonal error-corrected sequencing approach verified >95% of the discovered mutations. We identified mutations in genes novel to cHL including: CDH5 and PCDH7, novel stop gain mutations in IL4R, and a novel pattern of recurrent mutations in pathways regulating Hippo signaling. As a further application of our exome sequencing, we attempted to identify expressed somatic single-nucleotide variants (SNV) in single-nuclei RNA sequencing (snRNA-seq) data generated from a patient in our cohort. Our snRNA analysis identified a clear cluster of cells containing a somatic SNV identified in our deep exome data. This cluster has differentially expressed genes that are consistent with genes known to be dysregulated in HRS cells (e.g., PIM1 and PIM3). The cluster also contains cells with an expanded B-cell clonotype further supporting a malignant phenotype. This study provides proof-of-principle that ultra-deep exome sequencing can be utilized to identify recurrent mutations in HRS cells and demonstrates the feasibility of snRNA-seq in the context of cHL. These studies provide the foundation for the further analysis of genomic variants in large cohorts of patients with cHL. SIGNIFICANCE: Our data demonstrate the utility of ultra-deep exome sequencing in uncovering somatic variants in Hodgkin lymphoma, creating new opportunities to define the genes that are recurrently mutated in this disease. We also show for the first time the successful application of snRNA-seq in Hodgkin lymphoma and describe the expression profile of a putative cluster of HRS cells in a single patient.
Asunto(s)
Enfermedad de Hodgkin , Humanos , Enfermedad de Hodgkin/genética , Células de Reed-Sternberg/metabolismo , Mutación/genética , Secuenciación de Nucleótidos de Alto Rendimiento , ARN Nuclear Pequeño/metabolismoAsunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Promielocítica Aguda , Proteínas de Fusión Oncogénica , PPAR gamma , Factores de Escisión y Poliadenilación de ARNm , Adulto , Cromosomas Humanos Par 15/genética , Cromosomas Humanos Par 15/metabolismo , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 17/metabolismo , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Masculino , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genética , PPAR gamma/biosíntesis , PPAR gamma/genética , Translocación Genética , Factores de Escisión y Poliadenilación de ARNm/biosíntesis , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.
Asunto(s)
Evolución Clonal , Leucemia Mieloide Aguda/genética , Mutación , Adulto , Anciano , Análisis Mutacional de ADN , Progresión de la Enfermedad , Femenino , Estudio de Asociación del Genoma Completo , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/fisiopatología , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Recurrencia , Piel/metabolismo , Adulto JovenRESUMEN
Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame.