Igarashi Maki, Wada Yuka, Kojima Yoshiyuki, Miyado Mami, Nakamura Michiko, Muroya Koji, Mizuno Kentaro, Hayashi Yutaro, Nonomura Katsuya, Kohri Kenjiro, Ogata Tsutomu, Fukami Maki
Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.
Sex Dev. 2015;9(3):130-5. doi: 10.1159/000380842. Epub 2015 Mar 31.
MAMLD1 is a causative gene for disorders of sex development. Several MAMLD1 mutations have been shown to cause hypospadias by generating dysfunctional proteins and/or unstable mRNAs. Here, we identified an intronic mutation of MAMLD1 (g.IVS4-2A>G) in 1 of 180 hypospadias patients. RT-PCR of the patient's skin sample showed normal expression of full-length MAMLD1 and markedly reduced expression of a known splice variant lacking exon 4. A hitherto unreported splice variant that lacks exon 5 was similarly identified in samples of the patient and control individuals. The full-length transcript of the patient contained mutant mRNA lacking the first 10 nucleotides of exon 5 (c.1822_1831delACTCATGTAG, p.K609fsX1070). In vitro assays using cells expressing the full-length wild-type and mutant proteins revealed reduced expression of the mutant. The expression of the wild-type and mutant MAMLD1 showed parallel changes upon treatment with a proteasome inhibitor and a translation inhibitor. The mutant-expressing cells exerted low transactivation activity for the Hes3 promoter, which reflected limited expression of the mutant protein. These results imply that the pathogenic events resulting from MAMLD1 mutations include splice errors. Furthermore, this study raises the possibility of translation failure of MAMLD1 mutants, which deserves further investigation.
MAMLD1是性发育障碍的致病基因。已证实几种MAMLD1突变通过产生功能失调的蛋白质和/或不稳定的mRNA导致尿道下裂。在此，我们在180例尿道下裂患者中的1例中鉴定出MAMLD1的一个内含子突变（g.IVS4 - 2A>G）。对患者皮肤样本进行逆转录聚合酶链反应（RT-PCR）显示全长MAMLD1表达正常，而一个已知的缺少外显子4的剪接变体表达明显降低。在患者和对照个体的样本中同样鉴定出一种迄今未报道的缺少外显子5的剪接变体。患者的全长转录本包含缺少外显子5前10个核苷酸的突变mRNA（c.1822_1831delACTCATGTAG，p.K609fsX1070）。使用表达全长野生型和突变型蛋白质的细胞进行的体外试验显示突变型的表达降低。用蛋白酶体抑制剂和翻译抑制剂处理后，野生型和突变型MAMLD1的表达呈现平行变化。表达突变型的细胞对Hes3启动子的反式激活活性较低，这反映了突变型蛋白质的表达受限。这些结果表明，MAMLD1突变导致的致病事件包括剪接错误。此外，本研究提出了MAMLD1突变体翻译失败的可能性，这值得进一步研究。
