Epigenetics of Hirschsprung's disease

Hirschsprung’s disease - epigenetic factors in the development of the enteric nervous systemHirschsprung’s disease is a congenital disease, and the most common enteric neuropathy affecting 1 in 5000 newborns. It is defined by lack of ganglion cells in a segment of the distal bowel leading to a tonic contraction of the affected segment, and pain. Our project aims to elucidate the epigenetic mechanisms involved in the pathogenesis.Hirschsprung’s disease is a congenital disease, and the most common enteric neuropathy affecting 1 in 5000 newborns. It is defined by lack of ganglion cells in a segment of the distal bowel leading to a tonic contraction of the affected segment, gastrointestinal obstruction and pain. The symptoms most often presents in the neonatal period. Nuclear cells of the human body contain the same sequence of DNA, yet display very diverse functions. To achieve differentially expressed genes without altering the genome sequence, nature has developed an intricate system of epigenetic regulation. Our hypothesis is that aberrant epigenetic regulation of migration and differentiation of enteric neural stem cells contributes to the pathogenesis of Hirschsprung’s disease. If true, this may open for novel treatment e.g. epigenetic drugs or cell-based therapies. According to REC approval, stem cells will be established from tissue samples obtained from healthy and diseased gut. Investigation of epigenetic mechanisms requires already established methods of molecular biology e.g. bisulfite sequencing, chromatin immunoprecipitation, PCR and miRNA analysis. The proposed project is a joint initiative between basic scientists and pediatric surgeons and engages national and international collaborative partners. This far we have purified enteric neural stem cells from surgical specimens by using laser microdissection. We have purified DNA from these cells and are starting to do DNA methyl analyses. In the meantime we have published a review of todays knowedge about epigenetics and Hirschsprung's disease.

Hirschsprung’s disease - epigenetic factors in the development of the enteric nervous systemVi studerer faktorer som styrer utviklingen av nervestamceller til modent nervesystem i tarmen. Seneste tiden har vi: 1) optimalisert lasermikrodisseksjon av nervestamceller fra human tarm, 2) samlet inn data om presisjonen av dagens diagnostikk, 3) skrevet en oversiktsartikkel som oppsummerer resultatene innen feltet, 4) importert en musemodell.Hirschsprungs sykdom er en medfødt tilstand som skyldes at deler av tarmen mangler nerveceller. Denne delen av tarmen er dermed «lammet». Årlig fødes ca 1 av 5000 barn med denne tilstanden. Alle cellene i kroppen inneholder den samme DNA sekvensen, likevel er det stor forskjell mellom cellene når det gjelder utseende og funksjon. Epigenetikk betegner prosessene som endrer gen-uttrykk uten at DNA sekvensen endres. Vår hypotese er at epigenetisk regulering er involvert i utviklingen av nervesystemet i tarmen, og at forstyrrelser i denne reguleringen er medvirkende til utviklingen av Hirschsprungs sykdom. Man forsøker å etablere en stamcellebasert behandling for tilstanden ved å tilføre nye nerveceller til den ”syke” delen av tarmen, men for å lykkes med dette, kreves det en bedre forståelse av de epigenetiske mekanismene som styrer utviklingen av stamceller til nerveceller i tarmen. Vi bruker vevsprøver hentet fra pasienter med Hirschsprungs sykdom, og så renser vi nervestamceller fra både den innerverte ("normale") delen av tarmen og fra den aganglionære ("syke") delen. Opprinnelig forsøkte vi å dyrke disse nervecellene i kultur, men metoden viste seg ikke å være nevneverdig reproduserbar. Siden vår initiale idé ikke fungerte, gikk vi over til såkalt FACS for å sortere ut de cellene vi er interessert i. Det viste seg imidlertid at dette gav for høy bakgrunn, og materiale vi fikk ut holdt ikke tilstrekkelig kvalitet. Seneste tiden har vi optimalisert lasermikrodisseksjon av de relevante cellene fra frysesnitt. Dette ser ut til å fungere utmerket, og vi har nå renset DNA av høy kvalitet fra nervestamceller fra nevnte tarmsegmenter (1). Parallelt med dette har vi smalet inn kliniske data om pasientene og dems utredning i forkant av diagnose. Pasientene våre diagnostiseres vha vevsprøver fra endetarmen, enten såkalte sugebiopsier eller kirurgiske fullveggsbiopsier. Disse farges deretter med ulike immunohistokjemiske markører. Presisjonen rundt dette har, lokalt, vært ukjent, og vi har nå et svært stort materiale rundt dette som skrives sammen til en publikasjon i disse dager (2). Svært lite er kjent om hvordan epigenetiske mekanismer styrer utviklingen av nerveceller i tarmen, og om defekter i disse mekanismene kan lede til Hirschsprungs sykdom. For å oppsummere det som er kjent om dette, har vi skrevet en oversiktsartikkel som sendes inn i løpet av kort tid (3). Til slutt; Hirschsprungs sykdom er, som sagt før, en medfødt tilstand. Mekanismene vi studerer er derfor gjeldende under fosterutviklingen, og vi kan selvsagt ikke benytte humant embryonalt vev til studiene våre. Så, på tampen av forrige år importerte vi en transgen musemodell fra en av våre samarbeidspartnere. Det er en såkalt SOX10-YFP modell. I denne transgene musen vil alle nervecellestamcellene lyse gult, så de er svært lette å sortere ut under fosterutviklingen. Vi tror at denne modellen vil tilføre nyttig kunnskap om hva som skjer under den normale embryonale utviklingen, kunnskap som er direkte overførbart til de humane forsøkene våre, og vil være av uvurderlig verdi (4).

Epigenetics of Hirschsprung's diseaseHirschsprung’s disease is a congenital condition characterized by lack of neural cells in a segment of the distal bowel leading to obstruction and pain. We have established cultures of neural precursor cells harvested from human gut, and will investigate if their epigenetic signature influences their ability to repopulate aganglionic gut.Hirschsprung’s disease is a congenital disease, and the most common enteric neuropathy affecting 1 in 5000 newborns. It is defined by lack of ganglion cells in a segment of the distal bowel leading to a tonic contraction of the affected segment, gastrointestinal obstruction and pain. Today’s best treatment of the disease is surgical excision of the aganglionic bowel and a coloanal anastomosis. However, even with the best available therapy, there is significant morbidity associated with the condition. Postoperative fecal incontinence or constipation has been reported in about 60% of the patients, and Hirschsprung’s enterocolitis is a potentially life-threatening complication that can occur both before and after surgery. Thus; there is a need for improved and novel therapy. Nuclear cells of the human body contain the same sequence of DNA, yet display very diverse functions. To achieve differentially expressed genes without altering the genome sequence, nature has developed an intricate system of epigenetic regulation. We believe that aberrant epigenetic regulation of migration and differentiation of enteric neural stem cells contributes to the pathogenesis of Hirschsprung’s disease. If true, targeting these regulatory pathways may open for novel treatment e.g. epigenetic drugs or cell-based therapies. Initially our goal is to gain a deeper understanding of the molecular mechanisms underlying the development of the enteric nervous system and elucidate how defects in epigenetic regulation can contribute in the pathogenesis of Hirschsprung’s disease. For these studies a PhD student (fully funded by the South-Eastern Norway Regional Health Authority) and a medical student enrolled in the Medical Student Research Program, have established cultures of neural precursor cells harvested from surgical specimens. We have generated cultures from both patients suffering from Hirschsprung’s disease and from controls undergoing gastrointestinal surgery for other reasons. Also, we have received neural precursor cells from our collaborators at Harvard Medical School and Massachusetts General Hospital in Boston, USA. All these cells have been characterized to confirm that they possess the full neural potential and the ability to eventually repopulate aganglionic segments of the gut. In the year to come, we will investigate the DNA methylation status of these cells. We expect to find abnormal distribution of genome methylation in cells derived from patients suffering from the disease and subsequently aberrant gene expression leading to inappropriate cellular migration and differentiation. Moreover, we aim to map the histone modification pattern around genes involved in neural differentiation and expect to find locally disturbed chromatin architecture further exaggerating the dysfunctional innervation of the distal gut in patients with Hirschsprung’s disease. Ultimately the results from our studies may contribute in the development of a cell-based therapy for Hirschsprung’s disease.