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
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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

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Figure 3

Characterization of CRS patients with digenic impairments in TRPS1 and IL6ST.

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Characterization of CRS patients with digenic impairments in TRPS1 and I...
(A and B) Pedigree and genotype information of the 2 individuals with rare variants in TRPS1 and IL6ST. 3D reconstruction images of skull computed tomography reveal premature fusions of lambdoidal and sagittal sutures (indicated by black arrows) in P093 and P051, respectively. (C and D) Schematic representation of the variants identified in TRPS1 and IL6ST. The positions of all variants are indicated, and they are shown to be located in highly conserved sites across mammalian species. (E) TRPS1 functional activity was measured using the osteoblast-specific cis-acting element (OSE2) reporter assay in HepG2 cells, which minimally express endogenous TRPS1. Cells were cotransfected with plasmids for RUNX2 to activate OSE2 reporter transcriptional activity. Subsequently, TRPS1-mediated transcriptional repression activity was measured using the dual luciferase assay in cells transfected with WT or variant TRPS1 plasmids. The Q181R and R814L variants showed lower TRPS1 activity than WT. EV, empty vector. (F) Immunoblot analyses show comparable protein expression levels among TRPS1 WT and variant proteins. (G) IL6ST functional activity was measured using the IL6 SIE/STAT3 dual luciferase reporter assays with IL-11 stimulation in HEK293 cells. To eliminate the potential effects of endogenous IL6ST, we treated cells with siRNA against the 3′-UTR of IL6ST 6 hours before plasmid transfection. Cells were then transfected with plasmids for dual luciferase assay and plasmids for the expression of IL-11 receptor and IL6STs (WT, N360S, and S580F). The N360S and S580F variants showed lower IL6ST functional activity than WT. (H) Immunoblot analyses show comparable protein expression levels among WT and variant types of IL6ST. n.s., not significant. *P < 0.05, ***P < 0.001.