Supplementary Material for: Novel Heterozygous Mutations of <b><i>NR5A1</i></b> and Their Functional Characteristics in Patients with 46,XY Disorders of Sex Development without Adrenal Insufficiency
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<b><i>Background/Aims:</i></b> Heterozygous mutations of <i>NR5A1</i>, which encodes steroidogenic factor 1 (SF1), were identified in patients with 46,XY disorders of sex development (DSD) with normal adrenal function. This study was aimed to identify and functionally characterize mutations of <i>NR5A1</i> in patients with 46,XY DSD. <b><i>Methods:</i></b> This study included 51 patients from 49 unrelated families with 46,XY DSD. Genomic DNA was extracted from peripheral blood leukocytes, and direct sequencing of all coding exons and their flanking introns of <i>NR5A1</i> was performed. Transient transfections and dual-luciferase® reporter assays were performed to evaluate the effect of <i>NR5A1</i> variants on transcriptional activity. <b><i>Results:</i></b> Four of 49 patients (8.2%) harbored a novel heterozygous sequence variant of <i>NR5A1</i>: c.80G>C (p.G26A), c.847T>C (p.C283R), c.1151del (p.L384Rfs*7), and c.1333G>T (p.E445*). They presented with female external genitalia with clitoromegaly in infancy or childhood, or primary amenorrhea in adolescence. In vitro functional studies of SF1 activity determined that each variant, except p.E445*, led to a reduced expression of downstream target genes and disturbed the regulation of gonadal development. <b><i>Conclusions:</i></b> Loss-of-function mutations of <i>NR5A1</i> are a relatively common cause of 46,XY DSD. Therefore, genetic defects of <i>NR5A1</i> should be considered as an etiology in subjects with 46,XY DSD without adrenal insufficiency.
**背景与目的**：编码类固醇生成因子1（steroidogenic factor 1, SF1）的NR5A1基因杂合突变，此前已在伴肾上腺功能正常的46,XY性发育障碍（disorders of sex development, DSD）患者中被鉴定出。本研究旨在鉴定并功能表征46,XY DSD患者体内的NR5A1基因突变。**方法**：本研究纳入了来自49个无关家系的51例46,XY DSD患者。从外周血白细胞中提取基因组DNA，对NR5A1的全部编码外显子及其侧翼内含子进行直接测序。通过瞬时转染与双荧光素酶（dual-luciferase）报告基因实验，评估NR5A1变异体的转录活性。**结果**：49例患者中共4例（占比8.2%）携带NR5A1的新型杂合序列变异：c.80G>C（p.G26A）、c.847T>C（p.C283R）、c.1151del（p.L384Rfs*7）以及c.1333G>T（p.E445*）。此类患者在婴幼儿或儿童时期表现为伴阴蒂肥大的女性外生殖器表型，或在青春期出现原发性闭经。体外SF1活性功能实验结果显示，除p.E445*变异体外，其余所有变异体均导致下游靶基因表达水平降低，并扰乱性腺发育调控通路。**结论**：NR5A1基因的功能丧失型突变是引发46,XY DSD的一类相对常见的病因。因此，对于无肾上腺功能不全的46,XY DSD患者，应将NR5A1基因缺陷纳入其潜在病因的考量范围。
