Digenic impairments of haploinsufficient genes in patients with craniosynostosis
Jung Woo Yu, Jihoon G. Yoon, Chaerim Han, Shin Hye Noh, Dong Min Shin, Yu-Mi Yang, Yong Oock Kim, Kyu-Won Shim, Min Goo Lee
Jung Woo Yu, Jihoon G. Yoon, Chaerim Han, Shin Hye Noh, Dong Min Shin, Yu-Mi Yang, Yong Oock Kim, Kyu-Won Shim, Min Goo Lee
View: Text | PDF
Research Article Bone biology Genetics

Digenic impairments of haploinsufficient genes in patients with craniosynostosis

Abstract

Craniosynostosis (CRS) is characterized by the development of abnormal cranial suture ossification and premature fusion. Despite the identification of several associated genetic disorders, the genetic determinants of CRS remain poorly understood. In this study, we conducted integrative analyses on 225 exomes, comprising 121 CRS probands and 104 parental exomes (52 trios). These analyses encompassed de novo and pathogenic variants, and digenic combinations within haploinsufficient genes harboring rare variants. Our analysis unveils a shared molecular network between genes associated with CRS and those linked to skeletal and neurodevelopmental disorders, with a notable enrichment of deleterious variants within haploinsufficient genes. Additionally, we identified a unique digenic pair (IL6ST and TRPS1) within haploinsufficient genes that was present in 2 patients with nonsyndromic CRS but absent in parents or 1,048 population controls. In vitro experiments provided evidence that the identified missense variants were hypomorphs, and accelerated bone mineralization could result from the additive effects of diminished IL6ST and TRPS1 activities in osteoblasts. Overall, our study underscores the important role of rare variations in haploinsufficient genes and suggests that in a subset of undiagnosed patients, the CRS phenotype may arise from multiple genetic variations.

Authors

Jung Woo Yu, Jihoon G. Yoon, Chaerim Han, Shin Hye Noh, Dong Min Shin, Yu-Mi Yang, Yong Oock Kim, Kyu-Won Shim, Min Goo Lee

×

Figure 4

Additive effects of Trps1 and Il6st double knockdown on bone mineralization in osteoblast.

Options: View larger image (or click on image) Download as PowerPoint
Additive effects of Trps1 and Il6st double knockdown on bone mineralizat...
(A) Schematic representation of the osteoblast differentiation process in mouse calvarial cells (MC3T3-E1). Il6st and Trps1 siRNA treatments were performed 48 hours before induction of differentiation. The osteogenic medium was replenished every 3 days, and differentiation was evaluated on days 5–7 after osteogenic induction. (B) Measurement of Il6st and Trps1 expression levels using qRT-PCR after 48 hours of siRNA transfection (Il6st: 25 nM, Trps1: 25 nM) with Lipofectamine RNAiMAX (n = 3, biologically independent samples). (C) ALP staining was performed on day 5 after the induction of differentiation. The double-siRNA-treated groups exhibited the most prominent staining, as measured by the highest optical-density (OD) ratio. (D) ALP activity was measured using ELISA on day 5 after the induction of differentiation. The double-siRNA-treated groups exhibited the most prominent ALP activity, indicating the highest level of osteoblast differentiation. (E) Measurement of osteogenic markers (Alpl, Bglap, Ibsp, Col1a1, and Runx2 mRNA) by qRT-PCR on day 5 after osteogenic induction (n = 5 or 6, biologically independent samples). (F) Representative Western blot images representing the protein abundance of phosphorylated Akt1/2 (P-Akt1/2) and phosphorylated Erk1/2 (P-Erk1/2) in the MC3T3E1 cell line after the 6-hour differentiation period. Densitometry-based quantification of the Western blot results, presenting relative protein expression levels as mean ± SD (n = 3, biological replicates). Remarkably, double-knockdown groups treated with Il6st and Trps1 siRNA exhibited synergistic effects on phospho-Akt levels, consistent with the observed trends in osteogenic markers. *P < 0.05, **P < 0.01, ***P < 0.001.