Supplementary Material for: New Insights into the Genetics of Fetal Megacystis: ACTG2 Mutations, Encoding γ-2 Smooth Muscle Actin in Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (Berdon Syndrome)

<b><i>Objective:</i></b> To identify the molecular basis for prenatally suspected cases of megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) (MIM 249210) in 3 independent families with clinical and radiographic evidence of MMIHS. <b><i>Methods:</i></b> Whole-exome sequencing (WES) and Sanger sequencing of the ACTG2 gene. <b><i>Results:</i></b> We identified a novel heterozygous de novo missense variant in <i>ACTG2</i> c.770G>A (p.Arg257His) encoding γ-2 smooth muscle actin (ACTG2) in 2 siblings with MMIHS, suggesting gonadal mosaicism of one of the parents. Two additional de novo missense variants (p.Arg257Cys and p.Arg178His) in ACTG2 were identified in 2 additional MMHIS patients. All of our patients had evidence of fetal megacystis and a normal or slightly increased amniotic fluid volume. Additional findings included bilateral renal hydronephrosis, an enlarged fetal stomach, and transient dilated bowel loops. <i>ACTG2</i> immunostaining of the intestinal tissue showed an altered muscularis propria, a markedly thinned longitudinal muscle layer, and a reduced amount and abnormal distribution of ACTG2. <b><i>Conclusion:</i></b> Our study demonstrates that de novo mutations in <i>ACTG2</i> are a cause of fetal megacystis in MMIHS and that gonadal mosaicism may be present in a subset of cases. These findings have implications for the counseling of families with a diagnosis of fetal megacystis with a preserved amniotic fluid volume and associated gastrointestinal findings.