次世代定序是一項新興技術，主要用於DNA和RNA定序以及與生物資訊學結合使用的變異/突變檢測。基於次世代定序技術的全外顯子定序可以對大部分編碼基因組進行定序，並徹底革新基因分析。性發育異常/差異(DSD)是一群人類先天性狀況，其特徵是染色體、性腺和/或解剖性別發育不典型。臨床表現差異很大，而有些患者可能需要在生命早期進行性別指派的醫療和手術介入。使用全外顯子定序合併生物資訊計算，可以對與 DSD發病機制相關的候選基因變異進行優先排序。我們建立了根據全外顯子定序的可行診斷流程，於25名DSD患者中辨識出15名的遺傳病因：7名單獨性DSD患者被發現存在AR、MAP3K1 和FLNA 基因的致病變異，另外8名患者除男性化不足的生殖器外還表現出其他表型，可符合CHARGE症候群(CHD7)、Robinow症候群(DVL1)、低促性腺激素性性腺功能低下症(FGFR1、LHX4 和SEMA3A)、以及先天性腎臟和泌尿道症候群(PBX1)。而為了進一步驗證PBX1 c.710G>C (p.R237T) 的功能影響，我們產生穩定的誘導型Flp-In T-REx™ HEK293 細胞株分別由攜帶野生型(WT)或突變體PBX1的質體轉染。功能分析顯示，存在於核定位序列內的這種變異降低蛋白質穩定性並阻礙核轉位。透過誘導表現Flp-In PBX1 p.R237T所產生的增殖抑制作用不如Flp-In PBX1 WT明顯，但細胞黏附作用沒有差異。RNA定序分析發現Flp-In HEK293 PBX1 WT和p.R237T細胞之間存在多個差異表現基因，其中2個DSD相關基因上升(MAP3K4和EMX2)，2個基因下降(KISS1R和HOXA13)。我們的結果強調全外顯子定序在DSD基因診斷中的臨床實用性。功能研究證明了PBX1 c.710G>C (p.R237T)的有害後果，使雙向分化潛能的性腺傾向於卵巢分化。

Next-generation sequencing (NGS) is an emerging technology that is principally used for DNA and RNA sequencing and variant/mutation detection if combined with the use of bioinformatics. Whole-exome sequencing (WES) based on NGS techniques allows for most of the coding genome to be sequenced and has revolutionized genetic analysis. Disorders/Differences of sex development (DSDs) are a group of human congenital conditions characterized by atypical development of the chromosomal, gonadal and/or anatomic sex. The clinical presentations vary considerably, while some patients may require medical and surgical interventions for gender assignment in early life. Using WES combined with bioinformatics computation, the candidate genetic variants can be prioritized in association with DSD pathogenesis. We established a feasible diagnostic pipeline based on WES to identify genetic etiologies for 15 out of 25 recruited patients with DSDs: 7 patients with isolated DSD were found to have causative variants in AR, MAP3K1, and FLNA genes, and the other 8 patients presenting additional phenotypes beyond undervirilized genitalia were compatible with CHARGE syndrome (CHD7), Robinow syndrome (DVL1), hypogonadotropic hypogonadism (FGFR1, LHX4, and SEMA3A), and congenital anomalies of kidney and urinary tract syndrome (PBX1). To further validate the functional impacts of PBX1 c.710G>C (p.R237T), We generated stable, inducible Flp-In T-REx™ HEK293 cell lines transfected with plasmids carrying either wild-type (WT) or mutant PBX1. Functional analysis showed that this variant residing within the nuclear localization sequence reduced protein stability and hampered nuclear translocation. The proliferation suppression through the induced expression of Flp-In PBX1 p.R237T was less prominent than that of Flp-In PBX1 WT, while the cell adhesion was not different. RNA sequencing analysis found several differentially expressed genes between Flp-In HEK293 PBX1 WT and p.R237T cells, with three DSD-related genes upregulated (MAP3K4 and EMX2) and two genes downregulated (KISS1R and HOXA13). Our results highlighted the clinical utility of WES in the genetic diagnosis of DSDs. The functional investigations demonstrated the deleterious consequences of PBX1 c.710G>C (p.R237T), tilting biopotential gonads toward pro-ovarian differentiation.

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