In vivo and in vitro splicing assay of SLC12A1 in an antenatal salt-losing tubulopathy patient with an intronic mutation
In vivo and in vitro splicing assay of SLC12A1 in an antenatal salt-losing tubulopathy patient with an intronic mutation
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DOI：
10.1007/s00439-009-0697-7
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发表时间：
2009-10-01
期刊：
影响因子：
5.3
通讯作者：
Matsuo, Masafumi
中科院分区：
文献类型：
--
作者：
Nozu, Kandai;Iijima, Kazumoto;Matsuo, Masafumi
I型巴特综合征(BS)是一种遗传性缺盐小管病变，由SLC12A1基因突变引起。虽然已经检测到该基因的几个内含子核苷酸变化，但还没有进行转录分析，因为只有当可以获得肾活检标本时才有可能进行mRNA分析，或者偶尔在白细胞中表达mRNA时才有可能进行mRNA分析。这篇报道涉及一例由于SLC12A1基因复合杂合性导致的I型BS患者。基因组DNA测序发现内含子5中存在两个新的杂合突变：内含子5中的c.724+4A>；G和内含子16中的c.2095delG，但前者是否可能影响转录仍有待确定。在这篇报道中，我们利用从先证者尿沉渣中提取的RNA进行了体内RT-PCR分析和微基因构建的体外功能剪接研究，并获得了这种内含子突变导致完整的外显子5跳过的证据。据我们所知，这是第一次将非侵入性方法用于遗传性肾脏疾病的体内检测和体外功能剪接检测。这些分析方法可以适用于所有遗传性肾脏疾病。
Type I Bartter syndrome (BS), an inherited salt-losing tubulopathy, is caused by mutations of the SLC12A1 gene. While several intronic nucleotide changes in this gene have been detected, transcriptional analysis had not been conducted because mRNA analysis is possible only when renal biopsy specimens can be obtained or occasionally when mRNA is expressed in the leukocytes. This report concerns a type I BS patient due to compound heterozygosity for the SLC12A1 gene. Genomic DNA sequencing disclosed the presence of two novel heterozygous mutations of c.724 + 4A > G in intron 5 and c.2095delG in intron 16, but it remains to be determined whether the former would be likely to influence the transcription. In this report, we conducted both in vivo assay of RT-PCR analysis using RNA extracted from the proband's urinary sediments and in vitro functional splicing study by minigene construction, and obtained evidence that this intronic mutation leads to complete exon 5 skipping. To the best of our knowledge, this is the first study to use non-invasive methods for both an in vivo assay and an in vitro functional splicing assay of inherited kidney disease. These analytical assays could be adapted for all inherited kidney diseases.