ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.

Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the absence of enteric nervous system (ENS) in the distal region of the intestine. Down Syndrome (DS) patients have a >50-fold higher risk of developing HSCR than the general population, suggesting that overexpression of human chromosome 21 (Hsa21) genes contribute to HSCR etiology. However, identification of responsible genes remains challenging. Here, we describe a genetic screening of potential candidate genes located on Hsa21, using the zebrafish. Candidate genes were located in the DS-HSCR susceptibility region, expressed in the human intestine, were known potential biomarkers for DS prenatal diagnosis, and were present in the zebrafish genome. With this approach, four genes were selected: RCAN1, ITSN1, ATP5PO and SUMO3. However, only overexpression of ATP5PO, coding for a component of the mitochondrial ATPase, led to significant reduction of ENS cells. Paradoxically, in vitro studies showed that overexpression of ATP5PO led to a reduction of ATP5PO protein levels. Impaired neuronal differentiation and reduced mitochondrial ATP production, were also detected in vitro, after overexpression of ATP5PO in a neuroblastoma cell line. Finally, epistasis was observed between ATP5PO and ret, the most important HSCR gene. Taken together, our results identify ATP5PO as the gene responsible for the increased risk of HSCR in DS patients in particular if RET variants are also present, and show that a balanced expression of ATP5PO is required for normal ENS development.

Diagnostic accuracy of calretinin and acetylcholinesterase staining of rectal suction biopsies in Hirschsprung disease examined by unexperienced pathologists.

Rectal suction biopsy (RSB) is a gold standard for diagnosing Hirschsprung disease (HD). Calretinin staining of RSB is increasingly used by experienced pathologists due to non-complex examination and comparable diagnostic accuracy with acetylcholinesterase (AChE). However, the diagnostic accuracy of calretinin examined by unexperienced pathologists remains to be elucidated. Therefore, we aim to compare diagnostic accuracy of calretinin with AChE on RSB for diagnosing HD when examined by unexperienced pathologists. We prospectively analyzed sections from RSB stained with AChE + HE and calretinin. Blinded examination was done by five unexperienced pathologists (pathology residents) and three experienced pathologists (senior pediatric gastro-enterology pathologists) assessing for the presence of HD. Cases for the study included ones proven to be HD on resection specimens and cases without HD. Diagnostic accuracy was determined calculating area under the curve (AUC), sensitivity, specificity, likelihood ratio, and posttest probability. Fleiss' kappa analysis was performed to assess interobserver agreement between reviewers. Eleven of 18 included patients (61%) were diagnosed with HD. Comparing the diagnostic accuracy of unexperienced pathologists, calretinin versus AChE + HE showed sensitivity of 80.0% versus 74.5% and specificity of 100% versus 65.4%, AUC of 0.87 (0.78-0.96) versus 0.59 (0.45-0.72). Unexperienced pathologists showed substantial agreement with calretinin (kappa 0.72 [0.61-0.84]) and fair agreement with AChE + HE (kappa 0.34 [0.23-0.44]). We found calretinin having higher diagnostic accuracy in diagnosing HD compared to AChE + HE when examined by unexperienced pathologists. Therefore, we recommend to use calretinin as the standard technique for staining RSB in diagnosing HD.