Abstract：
(1) Background: Autosomal recessive polycystic kidney disease (ARPKD) is a rare ciliopathy characterized by progressively enlarged kidneys with fusiform dilatation of the collecting ducts. Loss-of-function mutations in the PKHD1 gene, which encodes fibrocystin/polyductin, cause ARPKD; however, an efficient treatment method and drug for ARPKD have yet to be found. Antisense oligonucleotides (ASOs) are short special oligonucleotides which function to regulate gene expression and alter mRNA splicing. Several ASOs have been approved by the FDA for the treatment of genetic disorders, and many are progressing at present. We designed ASOs to verify whether ASOs mediate the correction of splicing further to treat ARPKD arising from splicing defects and explored them as a potential treatment option. (2) Methods: We screened 38 children with polycystic kidney disease for gene detection using whole-exome sequencing (WES) and targeted next-generation sequencing. Their clinical information was investigated and followed up. The PKHD1 variants were summarized and analyzed, and association analysis was carried out to analyze the relationship between genotype and phenotype. Various bioinformatics tools were used to predict pathogenicity. Hybrid minigene analysis was performed as part of the functional splicing analysis. Moreover, the de novo protein synthesis inhibitor cycloheximide was selected to verify the degraded pathway of abnormal pre-mRNAs. ASOs were designed to rescue aberrant splicing, and this was verified. (3) Results: Of the 11 patients with PKHD1 variants, all of them exhibited variable levels of complications of the liver and kidneys. We found that patients with truncating variants and variants in certain regions had a more severe phenotype. Two splicing variants of the PKHD1 genotypes were studied via the hybrid minigene assay: variants c.2141-3T>C and c.11174+5G>A. These cause aberrant splicing, and their strong pathogenicity was confirmed. We demonstrated that the abnormal pre-mRNAs produced from the variants escaped from the NMD pathway with the use of the de novo protein synthesis inhibitor cycloheximide. Moreover, we found that the splicing defects were rescued by using ASOs, which efficiently induced the exclusion of pseudoexons. (4) Conclusion: Patients with truncating variants and variants in certain regions had a more severe phenotype. ASOs are a potential drug for treating ARPKD patients harboring splicing mutations of the PKHD1 gene by correcting the splicing defects and increasing the expression of the normal PKHD1 gene.
摘要：
（1）背景：常染色体隐性遗传性多囊肾病（ARPKD）是一种罕见的纤毛病，其特征是肾脏逐渐增大，集合管梭形扩张。PKHD1基因的功能缺失突变,编码纤维囊素/聚果胶，引起ARPKD；然而，目前尚未找到有效的ARPKD治疗方法和药物。反义寡核苷酸（ASO）是短的特殊寡核苷酸，其功能是调节基因表达和改变mRNA剪接。一些ASO已被FDA批准用于治疗遗传性疾病，许多人目前正在进步。我们设计了ASO来验证ASO是否介导剪接的校正以进一步治疗由剪接缺陷引起的ARPKD，并探索它们作为潜在的治疗选择。(2)方法:采用全外显子组测序(WES)和靶向下一代测序法筛选38例多囊肾病患儿进行基因检测。对他们的临床信息进行了调查和随访。对PKHD1变体进行了总结和分析，并进行关联分析,分析基因型与表型的关系。使用各种生物信息学工具来预测致病性。进行杂合小基因分析作为功能剪接分析的一部分。此外，选择从头蛋白合成抑制剂环己酰亚胺来验证异常前mRNA的降解途径。ASO被设计来拯救异常剪接，这被证实了。(3)结果:11例PKHD1变异患者中,所有患者均表现出不同程度的肝脏和肾脏并发症。我们发现在某些区域具有截短变体和变体的患者具有更严重的表型。通过杂交小基因测定研究了PKHD1基因型的两种剪接变体：变体c.2141-3T>C和c.111745G>A。这些导致异常剪接，并证实了它们的强致病性。我们证明，使用从头蛋白合成抑制剂环己酰亚胺，从变体产生的异常pre-mRNA从NMD途径逃脱。此外，我们发现拼接缺陷是通过使用ASO来拯救的，这有效地诱导了假外显子的排斥。(4)结论:具有截短变异体和某些区域变异体的患者具有更严重的表型。ASO是通过纠正剪接缺陷和增加正常PKHD1基因的表达来治疗具有PKHD1基因剪接突变的ARPKD患者的潜在药物。
