Alterations in neuronal physiology, development, and function associated with a common duplication of chromosome 15 involving CHRNA7.

Copy number variants at chromosome 15q13.3 contribute to liability for multiple intellectual and developmental disabilities (IDDs) including Autism Spectrum Disorder (ASD). Individuals with duplications of this interval, which includes the gene CHRNA7, have IDDs with variable penetrance. However, the basis of such differential affectation remains uncharacterized.Methods: Induced pluripotent stem cell (iPSC) models were generated from two first degree relatives with the same 15q13.3 duplication, a boy with distinct features of autism and emotional dysregulation (the affected proband, AP) and his clinically unaffected mother (the UM). These models were compared to unrelated control subjects lacking this duplication (UC, male and female). iPSC-derived neural progenitors and cortical neuroids consisting of cortical excitatory and inhibitory neurons were used to model potential contributors to neuropsychiatric impairment. Results: The AP-derived model uniquely exhibited disruptions of cellular physiology and neurodevelopment not observed in either the UM or in unrelated male and female controls. These included enhanced neural progenitor proliferation but impaired neuronal differentiation, maturation, and migration, and increased endoplasmic reticulum (ER) stress in neural progenitors. Both the AP’s neuronal migration deficit and elevated ER stress could be selectively rescued by different pharmacologic agents. Neuronal gene expression was also specifically dysregulated in the AP, including altered expression of genes related to behavior, psychological disorders, neuritogenesis, neuronal migration, and WNT signaling. By contrast with these AP-specific phenotypes, both the AP- and UM-derived neurons exhibited shared alterations of neuronal function, including elevated cholinergic activity consistent with increased homomeric CHRNA7 channel activity.Conclusion: Together, these data define both affectation-specific phenotypes seen only in the AP, as well as abnormalities observed in both individuals with CHRNA7 duplication, the AP and UM, but not in UC-derived neurons. This is, to our knowledge, the first study to use a human stem cell-based platform to study the basis of variable affectation in cases of 15q13.3 duplication at the cellular, molecular, and functional levels. This work suggests potential approaches for suppressing abnormal neurodevelopment or physiology that may contribute to severity of affectation in this proband. Some of these AP-specific neurodevelopmental anomalies, or the functional anomalies observed in both 15q13.3 duplication carriers (the AP and UM), could also contribute to the differential phenotypic penetrance seen in other individuals with 15q13.3 duplication. iPSC's were generated from patients with 15q13.3 duplication involving two first degree relatives (Affected Proband (AP), Unaffected Mother (UM)), with and without affectation. The Neural Progenitor Cells were generated from the iPSCs, and they are further matured for 15 days as a cortical neuroids. This study also includes two unrelated male (UC-M) and female (UC-F) control samples. Four independent biological experiments were performed and RNA was isolated from cortical neuroids after 15 days of differentiation. A batch correction was used to identify genes that were reproducibly differentially expressed genes (DEGs) across comparisons of paired mutant versus control samples.15q13.3染色体区域的拷贝数变异可增加包括孤独症谱系障碍（Autism Spectrum Disorder, ASD）在内的多种智力与发育障碍（intellectual and developmental disabilities, IDDs）的患病易感性。携带该区域（含CHRNA7基因）拷贝数重复的个体，可出现外显率不均一的智力与发育障碍。然而，此类表型差异的分子基础仍未明确。 方法：本研究从携带相同15q13.3拷贝数重复的一对一级亲属中构建诱导多能干细胞（induced pluripotent stem cell, iPSC）模型：一名表现出典型孤独症与情绪调节异常特征的男性先证者（affected proband, AP），以及其表型正常的母亲（unaffected mother, UM）。将上述模型与不携带该重复变异的无关对照个体（UC，涵盖男、女性）进行比对。本研究借助iPSC诱导分化的神经前体细胞，以及由皮层兴奋性与抑制性神经元构成的皮层类器官，构建了用于研究神经精神损伤潜在致病机制的体外模型。 结果：先证者来源的模型特异性出现了细胞生理与神经发育紊乱，此类异常在母亲来源的模型及无关男女对照中均未观测到。此类异常包括：神经前体细胞增殖亢进，但神经元分化、成熟与迁移受损，且神经前体细胞的内质网应激（endoplasmic reticulum, ER stress）水平升高。其中，先证者的神经元迁移缺陷与升高的内质网应激，均可通过不同的药理学试剂实现选择性挽救。先证者的神经元基因表达亦出现特异性失调，涵盖与行为、精神疾病、神经突发生、神经元迁移及WNT信号通路相关的基因表达异常。与上述先证者特异性表型不同，先证者与母亲来源的神经元均出现共同的神经元功能异常，包括胆碱能活性升高，该表型与同源CHRNA7通道活性增强的效应一致。 结论：综上，本研究明确了仅在先证者中出现的患病特异性表型，以及同时在两名CHRNA7拷贝数重复携带者（先证者与母亲）中出现、但未在对照来源神经元中观测到的异常。据我们所知，本研究首次借助人类干细胞体外平台，在细胞、分子与功能层面解析了15q13.3拷贝数重复病例中表型不均一外显的分子基础。本研究提出了可用于抑制异常神经发育或生理紊乱的潜在干预策略，此类紊乱可能加重先证者的病情严重程度。部分先证者特异性神经发育异常，或两名15q13.3拷贝数重复携带者共有的功能异常，亦可解释其他15q13.3拷贝数重复个体中观测到的表型不均一外显现象。本研究从携带15q13.3拷贝数重复的一对一级亲属——患病先证者（AP）与表型正常的母亲（UM）——中诱导构建了iPSC模型，并设置了两名无关男性（UC-M）与女性（UC-F）对照样本。研究人员从iPSC中诱导分化获得神经前体细胞，并将其进一步诱导成熟为皮层类器官，培养周期为15天。本研究共开展4次独立生物学重复，并在细胞分化15天后从皮层类器官中提取总RNA。通过批次校正分析，本研究在配对突变组与对照组样本的比较中，筛选出可重复的差异表达基因（differentially expressed genes, DEGs）。