Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder 
characterized by the absence of ganglion cells along variable lengths of the 
distal gastrointestinal tract, which results in tonic contraction of the 
aganglionic colon segment and functional intestinal obstruction. The RET 
proto-oncogene is the major gene associated to HSCR with differential 
contributions of its rare and common, coding and noncoding mutations to the 
multifactorial nature of this pathology. In addition, many other genes have been 
described to be associated with this pathology, including the semaphorins class 
III genes SEMA3A (7p12.1) and SEMA3D (7q21.11) through SNP array analyses and by 
next-generation sequencing technologies. Semaphorins are guidance cues for 
developing neurons implicated in the axonal projections and in the determination 
of the migratory pathway for neural-crest derived neural precursors during 
enteric nervous system development. In addition, it has been described that 
increased SEMA3A expression may be a risk factor for HSCR through the 
upregulation of the gene in the aganglionic smooth muscle layer of the colon in 
HSCR patients. Here we present the results of a comprehensive analysis of SEMA3A 
and SEMA3D in a series of 200 Spanish HSCR patients by the mutational screening 
of its coding sequence, which has led to find a number of potentially 
deleterious variants. RET mutations have been also detected in some of those 
patients carrying SEMAs variants. We have evaluated the A131T-SEMA3A, 
S598G-SEMA3A and E198K-SEMA3D mutations using colon tissue sections of these 
patients by immunohistochemistry. All mutants presented increased protein 
expression in smooth muscle layer of ganglionic segments. Moreover, A131T-SEMA3A 
also maintained higher protein levels in the aganglionic muscle layers. These 
findings strongly suggest that these mutants have a pathogenic effect on the 
disease. Furthermore, because of their coexistence with RET mutations, our data 
substantiate the additive genetic model proposed for this rare disorder and 
further support the association of SEMAs genes with HSCR.