Whole-genome sequencing identified ALK as a novel susceptible gene of Hirschsprung disease.

Background And Study Aims: Hirschsprung disease (HD) is a complex developmental disease that resulted from impaired proliferation and migration of neural crest cells. Despite the genetic causation of enteric nervous system have been found to be responsible for part of HD cases, the genetic aetiology of most HD patients still needs to be explored.

Patients And Methods: Whole-genome sequencing and subsequent Sanger sequencing validation analysis were performed in 13 HD children and their unaffected parents. Autophagy assays were performed to validate the functions of the identified locus.

Results: After the initial quality checking (SNP quality score ≥ 40, coverage ≥ 10X) of the raw data, we identified 3182 single nucleotide variants (SNVs). We subsequently compared these SNVs against the public databases and got a total of 15 suspicious genes shared among these patients. Subsequent Sanger sequencing and bioinformatics analyses revealed that amino acid-altering de novo mutation c.1648C > T(p.L550F) in ALK was responsible for HD. For validation, we sequenced all 29 exons ofALKin 76 additional sporadic HD cases and 200 healthy children and identified this variant in five of the HD cases. We further illustrated that ALK mutation c.1648C > T interrupted the interactions between ALK and its ligand midkine (Mdk), and induced autophagic cell death through AKT/mTORC1 axis.

Conclusion: These finding implied that the abnormal variant of ALK c.1648C > T may account for the pathogenesis of HD.