Splicing analysis of SLC40A1 missense variations and contribution to hemochromatosis type 4 phenotypes
Splicing analysis of SLC40A1 missense variations and contribution to hemochromatosis type 4 phenotypes
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DOI：
10.1016/j.bcmd.2020.102527
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发表时间：
2021-03-01
影响因子：
2.3
通讯作者：
Le Gac, Gerald
中科院分区：
文献类型：
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作者：
Le Tertre, Marlene;Ka, Chandran;Le Gac, Gerald
血色素沉着症4型，或铁色素沉着病，被认为是继HFE相关性血色素沉着症之后导致原发铁超载的第二大原因。这种疾病主要与SLC40A1基因的错义变异有关，具有广泛的临床异质性。我们测试了一些已报道的错义突变，尽管它们位于外显子内，但可能会导致剪接缺陷。根据两个标准从文献中选择了58个遗传变异：核苷酸变化的准确描述和铁超载的个体证据。选择的变异体通过不同的电子预测工具进行研究，并优先用于微基因剪接分析。在体外测试的15个变异中，只有两个与剪接变化有关。我们证实，c.1402G>；A转换(p.Gly468Ser)破坏了外显子7供体位点，导致了外显子隐蔽剪接位点的使用和截断阅读框的产生。我们首次观察到P.Gly468Ser取代对铁蛋白铁的输出功能没有影响。我们证明了外显子5在不同的细胞系中的选择性剪接，并表明c.430A>；G(p.Asn144Asp)变体促进了外显子5的包含。这可能是一种功能收益机制的一部分。我们得出结论，剪接突变很少影响4型血色素沉着症的表型。对外显子5辅助剪接序列的深入研究有助于阐明剪接调控蛋白调节全长SLC40A1转录本产生的机制，并阐明其生理意义。
Hemochromatosis type 4, or ferroportin disease, is considered as the second leading cause of primary iron overload after HFE-related hemochromatosis. The disease, which is predominantly associated with missense variations in the SLC40A1 gene, is characterized by wide clinical heterogeneity. We tested the possibility that some of the reported missense mutations, despite their positions within exons, cause splicing defects. Fifty-eight genetic variants were selected from the literature based on two criteria: a precise description of the nucleotide change and individual evidence of iron overload. The selected variants were investigated by different in silico prediction tools and prioritized for midigene splicing assays. Of the 15 variations tested in vitro, only two were associated with splicing changes. We confirm that the c.1402G>A transition (p.Gly468Ser) disrupts the exon 7 donor site, leading to the use of an exonic cryptic splicing site and the generation of a truncated reading frame. We observed, for the first time, that the p.Gly468Ser substitution has no effect on the ferroportin iron export function. We demonstrate alternative splicing of exon 5 in different cell lines and show that the c.430A>G (p. Asn144Asp) variant promotes exon 5 inclusion. This could be part of a gain-of-function mechanism. We conclude that splicing mutations rarely contribute to hemochromatosis type 4 phenotypes. An in-depth investigation of exon 5 auxiliary splicing sequences may help to elucidate the mechanism by which splicing regulatory proteins regulate the production of the full length SLC40A1 transcript and to clarify its physiological importance.