锌和环指 3 (ZNRF3) 是 Wnt/β-catenin 信号传导的负反馈调节因子，在人类大脑发育中发挥着重要作用。尽管在癌症中体细胞经常发生突变，但 ZNRF3 种系变异尚未被确定为神经发育障碍 (NDD) 的病因。我们通过 GeneMatcher/Decipher 鉴定了 12 名具有 ZNRF3 变异和各种表型的个体，并评估了基因型-表型相关性。我们进行了结构建模，并使用体外转录报告基因测定（有或没有 Wnt 配体 Wnt3a 和/或 Wnt 增强剂 R-spondin (RSPO)）评估了代表性的有害变异和对照变异。 8 个人携带新的错义变异并出现 NDD。我们发现与大头型 NDD 相关的错义变异在 RING 连接酶结构域中聚集。结构模型预测泛素连接酶功能的破坏可能会损害 Wnt 受体的周转。因此，功能测定显示这些变体的 Wnt/β-连环蛋白信号传导以显性负向方式增强。相反，患有小头型NDD的个体在RSPO结合域中含有错义变异，预计会破坏与RSPO的结合亲和力，并在相同的测定中显示出减弱的Wnt/β-连环蛋白信号传导。此外，有 4 个人携带从头截短或从头或继承了具有非 NDD 表型的大框内缺失变异，包括心脏、肾上腺或肾病问题。与NDD相关的错义变体相比，截短变体和空载体之间以及良性变体和野生型之间对Wnt/β-连环蛋白信号传导的影响是相当的。总之，我们通过蛋白质结构域特异性有害的 ZNRF3 种系错义变体，为 Wnt/β-连环蛋白信号传导中的不同病理机制引起的镜像脑大小表型提供了证据。版权所有 © 2024 作者。由爱思唯尔公司出版。保留所有权利。

Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/β-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using in vitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/β-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/β-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/β-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/β-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants.Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.