Gailite Linda, Valenzuela-Palomo Alberto, Sanoguera-Miralles Lara, Rots Dmitrijs, Kreile Madara, Velasco Eladio A
Scientific Laboratory of Molecular Genetics, Riga Stradins University, Riga, Latvia.
Splicing and Genetic Susceptibility to Cancer, Instituto de Biología y Genética Molecular (CSIC-UVa), Valladolid, Spain.
Front Genet. 2020 Mar 6;11:169. doi: 10.3389/fgene.2020.00169. eCollection 2020.
A large fraction of DNA variants impairs pre-mRNA splicing in human hereditary disorders. Crigler-Najjar syndrome (CNS) is characterized by a severe unconjugated hyperbilirubinemia caused by variants in the gene. We previously reported one CNS-type II patient with two splice-site variants in trans (c.864+5G>T and c.996+2_996+5del). According to MaxEntScan, both disrupt their corresponding donor sites (c.864+5G>T: 6.99 → 2.28; c.996+2_996+5del: 5.96 → -11.02), so they were selected for subsequent functional tests. Given the unavailability of patient RNA, we constructed an splicing-reporter minigene with exons 1-4 to characterize the underlying splicing anomaly. The variant c.996+2_996+5del generated two aberrant transcripts, Δ(E2) (exon 2 skipping/64%) and ▼(E2q135) (intron retention of 135-nt/36%), which lead to the loss of 18 conserved amino-acids and the gain of 45 new ones of a critical functional domain, respectively. The c.864+5G>T variant mainly produced the aberrant transcript Δ(E1q141) (141-nt deletion/70.4%) and the full-length isoform (29.6%). Δ(E1q141) would provoke the loss of 47 amino-acids of the N-terminal domain that encodes for substrate specificity. Thus, the three anomalous transcripts are likely to inactivate . Moreover, this patient is also homozygous for the promoter variant A(TA)7TAA that decreases expression by 70%, so the full-length transcript produced by c.864+5G>T would be even more reduced (<9%), thus supporting the diagnosis of CNS-type II. Therefore, minigenes represent valuable tools for the functional and clinical classifications of genetic variants.
在人类遗传性疾病中，很大一部分DNA变异会损害前体mRNA剪接。克里格勒 - 纳贾尔综合征（CNS）的特征是由该基因变异导致的严重非结合性高胆红素血症。我们之前报道过一名II型CNS患者，其反式存在两个剪接位点变异（c.864 + 5G>T和c.996 + 2_996 + 5del）。根据MaxEntScan分析，这两个变异均破坏了它们相应的供体位点（c.864 + 5G>T：6.99 → 2.28；c.996 + 2_996 + 5del：5.96 → -11.02），因此被选用于后续功能测试。鉴于无法获取患者RNA，我们构建了一个包含外显子1 - 4的剪接报告小基因，以表征潜在的剪接异常。变异c.996 + 2_996 + 5del产生了两种异常转录本，Δ(E2)（外显子2跳跃/64%）和▼(E2q135)（135个核苷酸的内含子保留/36%），它们分别导致一个关键功能域中18个保守氨基酸的缺失和45个新氨基酸的获得。c.864 + 5G>T变异主要产生异常转录本Δ(E1q141)（141个核苷酸缺失/70.4%）和全长异构体（29.6%）。Δ(E1q141)会导致编码底物特异性的N末端结构域中47个氨基酸的缺失。因此，这三种异常转录本可能会使该基因失活。此外，该患者的启动子变异A(TA)7TAA也是纯合的，这使得该基因的表达降低了70%，所以由c.864 + 5G>T产生的全长转录本会进一步减少（<9%），从而支持II型CNS的诊断。因此，小基因是基因变异功能和临床分类的有价值工具。
