RESUMEN
OBJECTIVE: Epigenetic mechanisms, including DNA methylation (DNAm), have been proposed to play a key role in Crohn's disease (CD) pathogenesis. However, the specific cell types and pathways affected as well as their potential impact on disease phenotype and outcome remain unknown. We set out to investigate the role of intestinal epithelial DNAm in CD pathogenesis. DESIGN: We generated 312 intestinal epithelial organoids (IEOs) from mucosal biopsies of 168 patients with CD (n=72), UC (n=23) and healthy controls (n=73). We performed genome-wide molecular profiling including DNAm, bulk as well as single-cell RNA sequencing. Organoids were subjected to gene editing and the functional consequences of DNAm changes evaluated using an organoid-lymphocyte coculture and a nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 (NLRC5) dextran sulphate sodium (DSS) colitis knock-out mouse model. RESULTS: We identified highly stable, CD-associated loss of DNAm at major histocompatibility complex (MHC) class 1 loci including NLRC5 and cognate gene upregulation. Single-cell RNA sequencing of primary mucosal tissue and IEOs confirmed the role of NLRC5 as transcriptional transactivator in the intestinal epithelium. Increased mucosal MHC-I and NLRC5 expression in adult and paediatric patients with CD was validated in additional cohorts and the functional role of MHC-I highlighted by demonstrating a relative protection from DSS-mediated mucosal inflammation in NLRC5-deficient mice. MHC-I DNAm in IEOs showed a significant correlation with CD disease phenotype and outcomes. Application of machine learning approaches enabled the development of a disease prognostic epigenetic molecular signature. CONCLUSIONS: Our study has identified epigenetically regulated intestinal epithelial MHC-I as a novel mechanism in CD pathogenesis.
Asunto(s)
Enfermedad de Crohn , Metilación de ADN , Epigénesis Genética , Mucosa Intestinal , Organoides , Humanos , Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Enfermedad de Crohn/metabolismo , Organoides/metabolismo , Organoides/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Animales , Femenino , Masculino , Ratones Noqueados , Bancos de Muestras Biológicas , Adulto , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Modelos Animales de Enfermedad , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
Clinical whole-genome sequencing (WGS) has been shown to deliver potential benefits to children with cancer and to alter treatment in high-risk patient groups. It remains unknown whether offering WGS to every child with suspected cancer can change patient management. We collected WGS variant calls and clinical and diagnostic information from 281 children (282 tumors) across two English units (n = 152 from a hematology center, n = 130 from a solid tumor center) where WGS had become a routine test. Our key finding was that variants uniquely attributable to WGS changed the management in ~7% (20 out of 282) of cases while providing additional disease-relevant findings, beyond standard-of-care molecular tests, in 108 instances for 83 (29%) cases. Furthermore, WGS faithfully reproduced every standard-of-care molecular test (n = 738) and revealed several previously unknown genomic features of childhood tumors. We show that WGS can be delivered as part of routine clinical care to children with suspected cancer and can change clinical management by delivering unexpected genomic insights. Our experience portrays WGS as a clinically impactful assay for routine practice, providing opportunities for assay consolidation and for delivery of molecularly informed patient care.