The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G; TRAPPC4: NM_016146.5; c.454+3A>G). Single nucleotide polymorphism array analysis revealed there was no haplotype shared between the tested Turkish and Caucasian families suggestive of a variant hotspot region rather than a founder effect. In silico analysis predicted the variant to cause aberrant splicing. Consistent with this, experimental evidence showed both a reduction in full-length transcript levels and an increase in levels of a shorter transcript missing exon 3, suggestive of an incompletely penetrant splice defect. TRAPPC4 protein levels were significantly reduced whilst levels of other TRAPP complex subunits remained unaffected. Native polyacrylamide gel electrophoresis and size exclusion chromatography demonstrated a defect in TRAPP complex assembly and/or stability. Intracellular trafficking through the Golgi using the marker protein VSVG-GFP-ts045 demonstrated significantly delayed entry into and exit from the Golgi in fibroblasts derived from one of the affected subjects. Lentiviral expression of wild-type TRAPPC4 in these fibroblasts restored trafficking, suggesting that the trafficking defect was due to reduced TRAPPC4 levels. Consistent with the recent association of the TRAPP complex with autophagy, we found that the fibroblasts had a basal autophagy defect and a delay in autophagic flux, possibly due to unsealed autophagosomes. These results were validated using a yeast trs23 temperature sensitive variant that exhibits constitutive and stress-induced autophagic defects at permissive temperature and a secretory defect at restrictive temperature. In summary we provide strong evidence for pathogenicity of this variant in a member of the core TRAPP subunit, TRAPPC4 that associates with vesicular trafficking and autophagy defects. This is the first report of a TRAPPC4 variant, and our findings add to the growing number of TRAPP-associated neurological disorders.

中文翻译：

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TRAPPC4 介导的囊泡运输缺陷与严重的综合征性智力障碍有关。

保守的转运蛋白颗粒 (TRAPP) 复合物调节关键的转运事件，并且是自噬所必需的。TRAPPC4 与其酵母 Trs23 直系同源物一样，是 TRAPP 复合物的核心成分，也是 Rab1 GTPase 鸟嘌呤核苷酸交换因子活性的重要亚基之一。特定 TRAPP 亚基的致病变异与神经系统疾病相关。我们对三个不相关的白种人、土耳其人和法裔加拿大人家庭的七名受影响儿童进行了外显子组测序，这些儿童表现出早发性癫痫、发育迟缓、小头畸形、感音神经性耳聋、痉挛性四肢瘫痪以及进行性皮质和小脑萎缩的特征确定神经发育障碍的遗传病因。所有七名受影响的受试者在 TRAPPC4 内的非规范且高度保守的剪接位点上具有相同的罕见、纯合、潜在致病性变异（hg19:chr11:g.118890966A>G；TRAAPPC4: NM_016146.5；c.454+3A >G）。单核苷酸多态性阵列分析显示，测试的土耳其人和白人家族之间没有共享单倍型，这表明存在变异热点区域而不是创始人效应。计算机分析预测该变体会导致异常剪接。与此一致的是，实验证据表明全长转录本水平降低，而缺少外显子 3 的较短转录本水平增加，表明存在不完全渗透剪接缺陷。TRAPPC4 蛋白水平显着降低，而其他 TRAPP 复合体亚基的水平不受影响。天然聚丙烯酰胺凝胶电泳和尺寸排阻色谱法证明 TRAPP 复合物组装和/或稳定性存在缺陷。使用标记蛋白 VSVG-GFP-ts045 通过高尔基体的细胞内运输表明，来自受影响受试者之一的成纤维细胞进入和离开高尔基体的时间显着延迟。这些成纤维细胞中野生型 TRAPPC4 的慢病毒表达恢复了运输，表明运输缺陷是由于 TRAPPC4 水平降低所致。与最近 TRAPP 复合物与自噬的关联一致，我们发现成纤维细胞具有基础自噬缺陷和自噬流延迟，可能是由于自噬体未封闭。这些结果使用酵母 trs23 温度敏感变体进行了验证，该变体在允许温度下表现出组成型和应激诱导的自噬缺陷，在限制温度下表现出分泌缺陷。总之，我们为核心 TRAPP 亚基成员 TRAPPC4 中该变异的致病性提供了强有力的证据，该变异与囊泡运输和自噬缺陷相关。这是 TRAPPC4 变异的第一份报告，我们的发现增加了 TRAPP 相关神经系统疾病数量的增加。