Supplementary Material for: An Intron 9 CYP19 Gene Variant (IVS9+5G>A), Present in an Aromatase-Deficient Girl, Affects Normal Splicing and Is Also Present in Normal Human Steroidogenic Tissues

<strong><em>Background/Aims:</em></strong> Splicing CYP19 gene variants causing aromatase deficiency in 46,XX disorder of sexual development (DSD) patients have been reported in a few cases. A misbalance between normal and aberrant splicing variants was proposed to explain spontaneous pubertal breast development but an incomplete sex maturation progress. The aim of this study was to functionally characterize a novel <i>CYP19A1</i> intronic homozygote mutation (IVS9+5G&gt;A) in a 46,XX DSD girl presenting spontaneous breast development and primary amenorrhea, and to evaluate similar splicing variant expression in normal steroidogenic tissues. <b><i>Methods:</i></b> Genomic DNA analysis, splicing prediction programs, splicing assays, and in vitro protein expression and enzyme activity analyses were carried out. <i>CYP19A1 </i>mRNA expression in human steroidogenic tissues was also studied. <b><i>Results:</i></b> A novel IVS9+5G&gt;A homozygote mutation was found. In silico analysis predicts the disappearance of the splicing donor site in intron 9, confirmed by patient peripheral leukocyte cP450arom and in vitro studies. Protein analysis showed a shorter and inactive protein. The intron 9 transcript variant was also found in human steroidogenic tissues. <b><i>Conclusions:</i></b> The mutation IVS9+5G&gt;A generates a splicing variant that includes intron 9 which is also present in normal human steroidogenic tissues, suggesting that a misbalance between normal and aberrant splicing variants might occur in target tissues, explaining the clinical phenotype in the affected patient.

背景与研究目的：目前仅见少数病例报道，在46,XX型性发育异常（disorder of sexual development, DSD）患者中存在可导致芳香化酶缺乏的CYP19基因剪接变异体。有研究提出，正常剪接变异体与异常剪接变异体之间的失衡，可解释患者出现自发性青春期乳腺发育，但性成熟进程不完全的表型。本研究旨在对1例表现为自发性乳腺发育及原发性闭经的46,XX型性发育异常（DSD）女童体内发现的新型CYP19A1内含子纯合突变（IVS9+5G>A）进行功能鉴定，并检测正常类固醇生成组织中是否存在相似的剪接变异体表达。
研究方法：本研究采用基因组DNA分析、剪接预测程序、剪接实验以及体外蛋白质表达与酶活性分析等方法，并对人类类固醇生成组织中的CYP19A1 mRNA表达水平进行了检测。
研究结果：本研究发现了新型IVS9+5G>A纯合突变。计算机模拟分析预测，第9内含子的剪接供体位点会消失，这一结果经患者外周血白细胞的细胞色素P450芳香化酶检测及体外实验得到证实。蛋白质分析显示，该突变产生的蛋白质长度更短且无活性。此外，研究还在人类类固醇生成组织中发现了包含第9内含子的转录变异体。
研究结论：IVS9+5G>A突变可产生包含第9内含子的剪接变异体，该变异体同样存在于正常人类类固醇生成组织中，这提示靶组织中正常剪接变异体与异常剪接变异体的失衡可能是导致该患者出现临床表型的原因。