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Abstract：
OBJECTIVE: Intronic variants of the SCN1A gene are detected in patients with epilepsy with febrile seizures (EFS), which includes a series of phenotypes with different severities. However, the pathogenicity of intronic variants and their genotype-phenotype correlation remain under characterized. The purpose of this study was to determine the changes in mRNA splicing caused by SCN1A intronic variants associated with EFS and their association with phenotypes.
METHODS: Five SCN1A intronic variants detected in patients with focal epilepsy with antecedent febrile seizures plus (FEFS+) and Dravet syndrome (DS) were molecularly cloned. Through an in vitro minigene splicing assay, their influence on mRNA splicing was qualitatively and quantitatively compared and analyzed using reverse-transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative PCR (Q-PCR).
RESULTS: The severe phenotype of DS-associated variants c.602 + 1G > A and c.4853-1G > C, which occurred in canonical splice sites of introns, caused exon skipping and little retention of full-length mRNA, while the milder phenotype of FEFS+-associated variants c.473 + 5G > A, c.473 + 5G > C and c.4853-25T > A, which occurred in potential splice sites or in deep intronic regions, presented partial exon skipping or intronic insertion and significantly higher retention of full-length mRNA at different levels. Full-length mRNA retention was negatively correlated with the location of intronic variants and phenotype severity.
CONCLUSIONS: The different aberrant splicing patterns resulting from SCN1A intronic variants with different positions represent a potential molecular mechanism for phenotypic differences in EFS. This research provides valuable clues for functional studies on the pathogenicity of intronic variants and for the evaluation of genotype-phenotype correlations.
METHODS: Five SCN1A intronic variants detected in patients with focal epilepsy with antecedent febrile seizures plus (FEFS+) and Dravet syndrome (DS) were molecularly cloned. Through an in vitro minigene splicing assay, their influence on mRNA splicing was qualitatively and quantitatively compared and analyzed using reverse-transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative PCR (Q-PCR).
RESULTS: The severe phenotype of DS-associated variants c.602 + 1G > A and c.4853-1G > C, which occurred in canonical splice sites of introns, caused exon skipping and little retention of full-length mRNA, while the milder phenotype of FEFS+-associated variants c.473 + 5G > A, c.473 + 5G > C and c.4853-25T > A, which occurred in potential splice sites or in deep intronic regions, presented partial exon skipping or intronic insertion and significantly higher retention of full-length mRNA at different levels. Full-length mRNA retention was negatively correlated with the location of intronic variants and phenotype severity.
CONCLUSIONS: The different aberrant splicing patterns resulting from SCN1A intronic variants with different positions represent a potential molecular mechanism for phenotypic differences in EFS. This research provides valuable clues for functional studies on the pathogenicity of intronic variants and for the evaluation of genotype-phenotype correlations.
摘要：
目的：在高热惊厥（EFS）的癫痫患者中检测到SCN1A基因的内含子变异，其中包括一系列不同严重程度的表型。然而,内含子变异体的致病性及其基因型-表型相关性仍未得到表征.这项研究的目的是确定与EFS相关的SCN1A内含子变体引起的mRNA剪接变化及其与表型的关联。
方法：分子克隆了在局灶性癫痫患者中检测到的五个SCN1A内含子变异，这些患者伴有先前的高热惊厥（FEFS）和Dravet综合征（DS）。通过体外小基因剪接试验，使用逆转录聚合酶链反应（RT-PCR）和荧光定量PCR（Q-PCR）对mRNA剪接的影响进行了定性和定量比较和分析。
结果：DS相关变体的严重表型c.602+1G>A和c.4853-1G>C，发生在内含子的典型剪接位点，导致外显子跳跃和全长mRNA的少量保留，而FEFS+相关变异的温和表型c.473+5G>A，c.473+5G>C和c.4853-25T>A，发生在潜在的剪接位点或深内含子区域，呈现部分外显子跳跃或内含子插入，并且在不同水平上全长mRNA的保留显着增加。全长mRNA保留与内含子变体的位置和表型严重程度呈负相关。
结论：由具有不同位置的SCN1A内含子变体产生的不同异常剪接模式代表了EFS表型差异的潜在分子机制。这项研究为内含子变异体致病性的功能研究和基因型-表型相关性的评估提供了有价值的线索。
方法：分子克隆了在局灶性癫痫患者中检测到的五个SCN1A内含子变异，这些患者伴有先前的高热惊厥（FEFS）和Dravet综合征（DS）。通过体外小基因剪接试验，使用逆转录聚合酶链反应（RT-PCR）和荧光定量PCR（Q-PCR）对mRNA剪接的影响进行了定性和定量比较和分析。
结果：DS相关变体的严重表型c.602+1G>A和c.4853-1G>C，发生在内含子的典型剪接位点，导致外显子跳跃和全长mRNA的少量保留，而FEFS+相关变异的温和表型c.473+5G>A，c.473+5G>C和c.4853-25T>A，发生在潜在的剪接位点或深内含子区域，呈现部分外显子跳跃或内含子插入，并且在不同水平上全长mRNA的保留显着增加。全长mRNA保留与内含子变体的位置和表型严重程度呈负相关。
结论：由具有不同位置的SCN1A内含子变体产生的不同异常剪接模式代表了EFS表型差异的潜在分子机制。这项研究为内含子变异体致病性的功能研究和基因型-表型相关性的评估提供了有价值的线索。
