Variants in the ACTG2 gene, encoding a protein crucial for correct enteric muscle contraction, have been found in patients affected with chronic intestinal pseudo-obstruction, either congenital or late-onset visceral myopathy, and megacystis-microcolon-intestinal hypoperistalsis syndrome. Here we report about ten pediatric and one adult patients, from nine families, carrying ACTG2 variants: four show novel still unpublished missense variants, including one that is apparently transmitted according to a recessive mode of inheritance. Four of the remaining five probands carry variants affecting arginine residues, that have already been associated with a severe phenotype. A de novo occurrence of the variants could be confirmed in six of these families. Since a genotype-phenotype correlation is affected by extrinsic factors, such as, diagnosis delay, quality of clinical management, and intra-familial variability, we have undertaken 3D molecular modeling to get further insights into the effects of the variants here described. The present findings and further ACTG2 testing of patients presenting with intestinal pseudo-obstruction, will improve our understanding of visceral myopathies, including implications in the prognosis and genetic counseling of this set of severe disorders.

BACKGROUND: Hirschsprung disease is a functional obstruction of the gastrointestinal tract due to the congenital absence of ganglion cells in the intermyenteric plexuses of the distal bowel. Gastrointestinal motility requires intact muscular layers as well as neural network connection to function properly. The Actin G2 gene is the main gene encoding actin gamma 2; a smooth muscle actin found in enteric tissues.
OBJECTIVE: This study of the Actin G2 gene in patients with Hirschsprung disease explores a possible molecular basis abnormal muscle function and post-surgical pseudo-obstruction in a group of patients. As far as the authors are aware, this is the first report confirming structural muscle deficits in Hirschsprung disease.
METHODS: Ethical permission and informed consent were obtained. DNA was extracted from whole blood samples in 10 patients with histologically proven HSCR patients. PCR amplification of the ACTG2 gene, were subjected to semi-automated bi-directional sequencing analysis. Sequencing results were analyzed using FinchTV Sequence Alignment Software (http:/en.biosoft.net) to read chromatogram files. Further predicting bioinformatic investigation was obtained by PolyPhen 2 software to evaluate the significance of the observed amino acid changes.
RESULTS: Ten new patients with similar HSCR phenotypes were prospectively investigated for variation in the Actin G2 gamma gene (ACTG2) variations. The results of ACTG2 gene analysis showing variation in exons 5, 8 and 10 of the ACTG2 gene in 7 of them (64%). The c.109C > G S345 L was the most frequent occurring in 6 of the 10 patients (54%), the c.171 A > A K119E in 2 and the significant c.108 T > G W357G variation in exon 10 (1 patient) Four patients had a combination of different variants in different exons which were less significant. Allele frequency on a control sample of the South African population showed no comparable pathology link scores (http://gnomad.broadinstitute.org/). Bioinformatic in silico modeling showed that the residue replacements in both variants (Lys to Glu and Trp to Gly) are highly non-conservative and variation can alter interactions within the protein conformation.
CONCLUSIONS: The Actin smooth muscle gene showed variation in 64% of samples, indicating a reason for abnormal functioning muscle in many HSCR patients. Hirschsprung disease is part of a complex spectrum which also includes smooth muscle.
METHODS: VI.