Neurodevelopmental disorders (NDDs) are a heterogeneous class of brain diseases, with a complex genetic basis estimated to account for up to 50% of cases. Nevertheless, genetic diagnostic yield is about 20%. Array-comparative genomic hybridization (array-CGH) is an established first-level diagnostic test able to detect pathogenic copy number variants (CNVs), however, most identified variants remain of uncertain significance (VUS). Failure of interpretation of VUSs may depend on various factors, including complexity of clinical phenotypes and inconsistency of genotype-phenotype correlations. Indeed, although most NDD-associated CNVs are de novo, transmission from unaffected parents to affected children of CNVs with high risk for NDDs has been observed. Moreover, variability of genetic components overlapped by CNVs, such as long non-coding genes, genomic regions with long-range effects, and additive effects of multiple CNVs can make CNV interpretation challenging. We report on 12 patients with complex phenotypes possibly explained by complex genetic mechanisms, including involvement of antisense genes and boundaries of topologically associating domains. Eight among the 12 patients carried two CNVs, either de novo or inherited, respectively, by each of their healthy parents, that could additively contribute to the patients’ phenotype. CNVs overlapped either known NDD-associated or novel candidate genes (PTPRD, BUD13, GLRA3, MIR4465, ABHD4, and WSCD2). Bioinformatic enrichment analyses showed that genes overlapped by the co-occurring CNVs have synergistic roles in biological processes fundamental in neurodevelopment. Double CNVs could concur in producing deleterious effects, according to a two-hit model, thus explaining the patients’ phenotypes and the incomplete penetrance, and variable expressivity, associated with the single variants. Overall, our findings could contribute to the knowledge on clinical and genetic diagnosis of complex forms of NDD.

神经发育障碍 (NDD) 是一类异质的脑部疾病，据估计，多达 50% 的病例具有复杂的遗传基础。尽管如此，基因诊断的产率约为 20%。阵列比较基因组杂交 (array-CGH) 是一种既定的一级诊断测试，能够检测致病性拷贝数变异 (CNV)，但是，大多数已鉴定的变异仍然具有不确定的意义 (VUS)。VUS 解释失败可能取决于多种因素，包括临床表型的复杂性和基因型-表型相关性的不一致。事实上，虽然大多数 NDD 相关的 CNV 是从头开始，已经观察到从未受影响的父母向受影响的儿童传播具有 NDD 高风险的 CNV。此外，CNV 重叠的遗传成分的变异性，例如长非编码基因、具有远距离效应的基因组区域以及多个 CNV 的累加效应，可能使 CNV 解释具有挑战性。我们报告了 12 名可能由复杂遗传机制解释的复杂表型的患者，包括反义基因的参与和拓扑相关域的边界。12 名患者中有 8 名携带两个 CNV，要么从头开始或分别由他们的每个健康父母遗传，这可能会增加患者的表型。CNV 与已知的 NDD 相关基因或新的候选基因重叠（PTPRD, BUD13, GLRA3、MIR4465、ABHD4、 和 WSCD2）。生物信息学富集分析表明，由共同发生的 CNV 重叠的基因在神经发育的基本生物过程中具有协同作用。根据两次打击模型，双 CNV 可能同时产生有害影响，从而解释了患者的表型和不完整的外显率，以及与单一变异相关的可变表达。总体而言，我们的发现可能有助于了解复杂形式的 NDD 的临床和基因诊断。