Department of Rehabilitation, Hunan Children's Hospital, Changsha, China.
Mol Genet Genomic Med. 2023 Jul;11(7):e2169. doi: 10.1002/mgg3.2169. Epub 2023 Mar 19.
Neurodevelopmental disorders associated with periventricular nodular heterotopia (PVNH) are characterized by phenotypic and genetic heterogeneity. NEDD4L mutation can lead to PVNH7. However, at present, only eight NEDD4L pathogenic variants have been identified across 15 cases of PVNH7 worldwide. Given this dearth of evidence, the precise correlations between genetic pathogenesis and phenotypes remain to be determined.
This report discusses the case of a 19-month-old male child with cleft palate, seizures, psychomotor retardation, and hypotonia, for whom we verified the genetic etiology using Trio-whole-exome and Sanger sequencing to analyze the potential pathogenicity of the mutant protein structure. Mutant plasmids were constructed for in vitro analyses. After transfection into human 293 T cells, the mutant transcription process was analyzed using real-time PCR (RT-PCR), and levels of mutant protein expression were examined using western blotting (WB) and immunofluorescence (IF) experiments.
Genetic analyses revealed a novel missense mutation Gln900Arg, located in the homologous to E6-APC terminal (HECT) domain of NEDD4L and that the parents were wild-type, suggestive of a de novo mutation. The variant was predicted to be pathogenic by bioinformatics software, which also suggested alterations in the structural stability of the mutant protein. RT-PCR results indicated that the mutation did not affect mRNA expression, whereas WB and IF results indicated that the level of mutant protein was significantly reduced by 41.07%.
Functional experiments demonstrated that Gln900Arg probably did not lead to transcriptional abnormalities in this patient, instead leading to increased ubiquitination activity owing to the constitutive activation of the HECT domain, thereby promoting protein degradation. Extensive clinical reports should be generated for patients presenting with PVNH and/or polymicrogyria, developmental delay, syndactyly, and hypotonia to increase the pool of evidence related to NEDD4L.
与脑室周围结节性异位（PVNH）相关的神经发育障碍的特点是表型和遗传异质性。NEDD4L 突变可导致 PVNH7。然而，目前全球仅在 15 例 PVNH7 中发现了 8 种 NEDD4L 致病变异。鉴于证据不足，遗传发病机制与表型之间的确切相关性仍有待确定。
本报告讨论了一名 19 个月大的男性患儿，患有腭裂、癫痫、精神运动发育迟缓伴张力减退，我们使用 Trio-whole-exome 和 Sanger 测序来验证遗传病因，分析潜在突变蛋白结构的致病性。构建突变质粒进行体外分析。转染入人 293T 细胞后，使用实时 PCR（RT-PCR）分析突变转录过程，使用 Western blot（WB）和免疫荧光（IF）实验检测突变蛋白表达水平。
遗传分析显示一种新的错义突变 Gln900Arg，位于 NEDD4L 的同源 E6-APC 末端（HECT）结构域，父母均为野生型，提示为新生突变。生物信息学软件预测该变异具有致病性，同时也提示突变蛋白结构稳定性发生改变。RT-PCR 结果表明该突变不影响 mRNA 表达，而 WB 和 IF 结果表明突变蛋白水平显著降低了 41.07%。
功能实验表明，Gln900Arg 可能不会导致该患者转录异常，而是由于 HECT 结构域的持续激活导致泛素化活性增加，从而促进蛋白降解。应针对出现 PVNH 和/或脑回发育不良、发育迟缓、并指和张力减退的患者进行广泛的临床报告，以增加与 NEDD4L 相关的证据。
