Molecular studies on Hirschsprung disease and "Ondine's curse"

Sammanfattning: This thesis is based on molecular studies of two diseases that arise from dysfunction of, defective migration or inappropriate apoptosis of neural crest derived cells: Hirschsprung disease (HSCR) and "Ondine's curse" (congenital central hypoventilation syndrome, CCHS). The neural crest (NC) is a condensation of pluripotent cells of ectodermal origin. Neural crest cells migrate during fetal life to form, among other tissues and cell types, the sympathetic part of the autonomic nervous system. Sympathetic neuroblasts invade the gastrointestinal canal in a cranial to caudal direction, cluster and differentiate to become the neuronal intestinal ganglia of Meissner and Auerbach. NC cells also form the cholinergic pathways in the ventral medulla. Patients with HSCR lack neuronal ganglia in the hindgut. This results in poor co-ordination of propulsion and a contracted distal segment with subsequent constipation or intestinal obstruction. The disease affects one in 5000 live births and males are more often affected than females. Familial occurrence and an increased risk for siblings indicate a genetic cause of HSCR. The studies on HSCR (Papers I-III) were based on 69 unrelated HSCR patients comprising 7 familial and 62 sporadic cases, accrued from the Stockholm region. Being a population-based study, there were no bias of ascertainment towards familial or long segment aganglionosis. Mutational screening was performed with single stranded conformation polymorphism analysis (SSCP) in candidate genes for NC development: the RET, endothelin receptor-B (EDNRB) and endothelin-3 (EDN-3) genes. Five germline mutations were detected in the RET gene: two frameshift and one missense mutation (C620R) typical for multiple endocrine neoplasia type 2A (MEN 2A) in familial cases; two missense mutations in sporadic cases. A girl with HSCR and MEN2A mutation developed medullary thyroid carcinoma at the age of 13 years (paper I). Six sporadic HSCR cases had one out of three different missense mutations in the EDNRB gene. Five of the mutations were inherited, one de novo. In all families with an inherited mutation, there were multiple cases of constipation in the family branch segregating the EDNRB mutation. These could constitute cases of subclinical Hirschsprung disease (paper II). An additional non-consanguineous family segregated two identical EDNRB mutations (G57S), as well as a RET mutation (R982C). The male proband, with short segment HSCR carries the RET and is heterozygous for the EDNRB mutation. The mother and sister both carry the RET mutation. The mother is heterozygous, the sister homozygous for the EDNRB mutation. The female carriers of both mutations were clinically unaffected. This suggests a possible additional sex-dependent modifying locus in this family (paper III). The original population-based cohort of 69 cases was expanded to 75 for the EDN-3 study described in paper IV. Among these 75 cases, a male patient with non-syndromic HSCR was found to carry a heterozygous frameshift mutation, predicted to result in haploinsufficiency. The mutation is inherited from the mother, who has a history of moderate constipation (paper IV). "Ondine's curse" is a disease characterised by the absence of autonomous breathing due to dysfunction of neural crest derived cells in the ventral medulla. An association with HSCR is reported in 15-50% of "Ondine's curse" patients. 25 cases, four with associated HSCR, where nonselectively ascertained and screened for mutations in the RET, glial cell line-derived neurotrophic factor (GDNF), EDNRB, EDN-3 and EDN-l genes. A missense mutation was detected in the RET gene in a patient with late onset "Ondine's curse" and hyperphagia. This mutation was inherited from the clinically unaffected father. A mutation was also detected in the EDN-l gene 3'UTR domain, in a patient with classical isolated "Ondine's curse" (paper V).