Genetic and functional analysis of SHROOM1-4 in a Chinese neural tube defect cohort

Neural tube defects (NTDs), which include spina bifida and anencephaly, are the second most common form of human structural congenital malformations. While it is well established that SHROOM3 plays a pivotal role in the complex morphogenetic processes involved in neural tube closure (NTC), the underlying genetic contributions of SHROOM gene family members in the etiology of human NTDs remain poorly understood. Herein, we systematically investigated the mutation patterns of SHROOM1-4 in a Chinese population composed of 343 NTD cases and 206 controls, using targeted next-generation sequencing. Functional variants were further confirmed by western blot and the mammalian two-hybrid assays. Loss of function (LoF) variants were identified in SHROOM3. We observed 1.56 times as many rare [minor allele frequency (MAF) < 0.01] coding variants (p = 2.9 x 10(-3)) in SHROOM genes, and 4.5 times as many rare D-Mis (deleterious missense) variants in SHROOM2 genes in the NTD cases compared with the controls. D-Mis variants of SHROOM2 (p.A1331S; p.R1557H) were confirmed by Sanger sequencing, and these variants were determined to have profound effects on gene function that disrupted their binding with ROCK1 in vitro. These findings provide genetic and molecular insights into the effects of rare damaging variants in SHROOM2, indicating that such variants of SHROOM2 might contribute to the risk of human NTDs. This research enhances our understanding of the genetic contribution of the SHROOM gene family to the etiology of human NTDs.