SASH1基因杂合变异c.1574C>G（p.T525R）可促进黑色素合成增加

doi:10.11958/20221703

天津医药 ›› 2023, Vol. 51 ›› Issue (7): 756-761.doi: 10.11958/20221703

SASH1基因杂合变异c.1574C>G（p.T525R）可促进黑色素合成增加

陈红宇¹^,²(), 张景², 曾珍¹^,², 杨娉娉¹^,², 熊宇¹^,², 张淼³, 周定安²^,^△()

1 贵州医科大学医学检验学院（邮编550004）
2 贵州医科大学附属医院临床研究中心
3 贵州医科大学附属医院内分泌代谢科

收稿日期:2022-10-27 修回日期:2023-02-16 出版日期:2023-07-15 发布日期:2023-07-18
通讯作者: ^△周定安 E-mail:460318918@qq.com
作者简介:陈红宇（1995），女，硕士在读，主要从事单基因遗传病的致病基因筛选和功能学方面研究。E-mail：806677383@qq.com
基金资助:
国家自然科学基金地区项目(31860319);贵州省科技计划基础研究（科学技术基金）项目(黔科合基础-ZK[2021]重点031)

The heterozygous variant c. 1574C > G (p.T525R) of SASH1 gene can promote the increase of melanin synthesis

CHEN Hongyu¹^,²(), ZHANG Jing², ZENG Zhen¹^,², YANG Pingping¹^,², XIONG Yu¹^,², ZHANG Miao³, ZHOU Ding’an²^,^△()

1 School of Medical Laboratory Science, Guizhou Medical University, Guiyang 550004, China
2 Clinical Research Center, the Affiliated Hospital of Guizhou Medical University
3 Department of Endocrinology, the Affiliated Hospital of Guizhou Medical University

Received:2022-10-27 Revised:2023-02-16 Published:2023-07-15 Online:2023-07-18
Contact: ^△ZHOU Ding’an E-mail:460318918@qq.com

陈红宇, 张景, 曾珍, 杨娉娉, 熊宇, 张淼, 周定安. SASH1基因杂合变异c.1574C>G（p.T525R）可促进黑色素合成增加[J]. 天津医药, 2023, 51(7): 756-761.

CHEN Hongyu, ZHANG Jing, ZENG Zhen, YANG Pingping, XIONG Yu, ZHANG Miao, ZHOU Ding’an. The heterozygous variant c. 1574C > G (p.T525R) of SASH1 gene can promote the increase of melanin synthesis[J]. Tianjin Medical Journal, 2023, 51(7): 756-761.

摘要/Abstract

摘要：

目的报告1例由SASH1基因c.1574C>G（p.T525R）变异位点导致的遗传性泛发性色素异常症（DUH）患者，并通过体外功能实验探讨该变异对黑色素表达的影响。方法采集先证者及其父母外周血，提取DNA，利用全外显子组测序、Sanger测序技术明确致病性基因；生物信息学分析基因变异位点有害性；小鼠黑色素瘤细胞B16为细胞模型，Western blot分析SASH1基因c.1574C>G（p.T525R）变异对小眼相关转录因子（MITF）、酪氨酸酶（Tyrosinase）蛋白表达量的影响；体外黑色素定量实验分析该变异位点对人皮肤黑色素瘤细胞SK-MEL-1和B16细胞黑色素合成的影响。结果基因测序结果显示，先证者检出SASH1基因c.1574C>G（p.T525R）变异；生物信息学分析该变异对蛋白质结构和功能造成影响的概率较大；Western blot结果显示该变异上调了黑色素合成过程中最关键的MITF及Tyrosinase的表达量；体外黑色素定量分析该变异可促使B16和SK-MEL-1细胞合成黑色素增加。结论 SASH1基因c.1574C>G（p.T525R）杂合变异与DUH发生相关，且该变异可促进黑色素瘤细胞合成黑色素增加。

Abstract:

Objective To report a case of dyschromatosis universalis hereditaria (DUH) caused by the variant site of c.1574C>G (p.T525R) of SASH1 gene and discuss the effect of mutation on melanin synthesis by functional experiments in vitro. Methods DNA was extracted from the peripheral blood of the proband and his parents, and pathogenic genes were identified by whole-exome sequencing and Sanger sequencing. Bioinformatics was used to analyze the harmful gene variant site. Mouse melanoma cell B16 was used as the cell model, and Western blot assay was used to analyze the effect of SASH1 gene c.1574C>G (p.T525R) variation on the expression of microphthalmia-associated transcription factor (MITF) and Tyrosinase proteins. In vitro melanin quantification experiment was used to analyze the effect of this variant site on melanin synthesis in human melanoma cells SK-MEL-1 and B16 cells. Results Gene sequencing results showed that the proband was detected SASH1 gene c.1574C>G (p.T525R) mutation. Bioinformatics analysis showed that the variant had a high probability affecting protein structure and function. Western blot results showed that this variant site up-regulated the expression levels of MITF and Tyrosinase, which were the most critical in melanin synthesis. This variation analyzed by in vitro melanin quantification can increase the melanin synthesis in B16 and SK-MEL-1 cells. Conclusion The heterozygous variant c.1574C>G (p.T525R) of SASH1 gene is associated with DUH, and this mutation can promote the increase of melanin synthesis in melanoma cells.

Key words: melanins, microphthalmia-associated transcription factor, dyschromatosis universalis hereditaria, SASH1 gene

中图分类号:

R394.3

图/表 8

图1 先证者临床表现 A：先证者腿部分布大小不一、形状不规则色素沉着斑；B：先证者左右手背分布色素沉着斑和色素减退斑。

Fig.1 Clinical manifestations of the proband

图2 先证者及其父母SASH1 c.1574C>G变异位点的Sanger测序

Fig.2 Sanger sequencing of SASH1 c.1574C>G of the proband and his parents

图3 PolyPhen-2预测T525R-SASH1变异位点对该蛋白结构和功能的影响

Fig. 3 PolyPhen-2 predicted the effect of T525R-SASH1 mutation on the structure and function of the protein

图4 MEGA7预测Thr525位点在不同物种之间的保守性

Fig. 4 MEGA7 predicted that the conserved nature of Thr525 locus in different species

图5 共聚焦显微镜显示SASH1在PIG1细胞中的定位（免疫荧光染色×200）

Fig.5 Confocal microscopy showed the localization of SASH1 in PIG1 cells (Immunofluorescence staining ×200)

图6 T525R-SASH1上调B16细胞MITF和Tyrosinase蛋白表达

Fig.6 T525R-SASH1 overexpression increased MITF and Tyrosinase expression in B16 cells

图7 过表达T525R-SASH1促进黑色素瘤细胞产生黑色素 A、B：对照腺病毒组、野生型SASH1腺病毒组、T525R-SASH1腺病毒组过表达于B16细胞、SK-MEL-1细胞；C、D：各组B16细胞、SK-MEL-1细胞的黑色素含量检测；a与对照腺病毒组，b与过表达野生型SASH1腺病毒组比较，P<0.05。

Fig.7 T525R-SASH1 over expression increased melanin synthesis in melanoma cells

表1 3组MITF、Tyrosinase蛋白表达水平比较

Tab.1 Comparison of expression levels of MITF and Tyrosinase proteins between the three groups

组别	MITF	Tyrosinase
空白对照组	0.97±0.02	0.16±0.02
WT-SASH1组	1.22±0.08^a	0.48±0.03^a
T525R-SASH1组	1.76±0.04^ab	1.12±0.1^ab
F	107.884^＊＊	179.575^＊＊

参考文献 15

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SASH1基因杂合变异c.1574C>G（p.T525R）可促进黑色素合成增加

The heterozygous variant c. 1574C > G (p.T525R) of SASH1 gene can promote the increase of melanin synthesis

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