Supplementary Material for: Novel Splice Site Mutation in MAMLD1 in a Patient with Hypospadias

<i>MAMLD1</i> is a causative gene for disorders of sex development. Several <i>MAMLD1</i> mutations have been shown to cause hypospadias by generating dysfunctional proteins and/or unstable mRNAs. Here, we identified an intronic mutation of <i>MAMLD1</i> (g.IVS4−2A&gt;G) in 1 of 180 hypospadias patients. RT-PCR of the patient's skin sample showed normal expression of full-length<i> MAMLD1</i> 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 <i>Hes3</i> promoter, which reflected limited expression of the mutant protein. These results imply that the pathogenic events resulting from <i>MAMLD1</i> mutations include splice errors. Furthermore, this study raises the possibility of translation failure of MAMLD1 mutants, which deserves further investigation.