9例46，XY完全性性腺发育不全患者分子遗传学分析

doi:10.3969/j.issn.1674-8115.2022.01.009

上海交通大学学报(医学版) ›› 2022, Vol. 42 ›› Issue (1): 63-69.doi: 10.3969/j.issn.1674-8115.2022.01.009

9例46，XY完全性性腺发育不全患者分子遗传学分析

王海城¹(), 刘宇¹, 叶荟¹, 倪琳¹, 曹英¹, 孙云龙¹, 肖冰¹, 马彩霞², 唐利芳²()

^1.上海交通大学医学院附属新华医院上海市儿科医学研究所小儿内分泌与遗传代谢病研究室，上海 200092
^2.复旦大学附属中山医院青浦分院儿科，上海 201700

收稿日期:2021-07-29 出版日期:2022-01-28 发布日期:2022-02-18
通讯作者: 唐利芳 E-mail:seacity@yeah.net;tanglifang1119@126.com
作者简介:王海城（1988—），男，初级技师，学士；电子信箱：seacity@yeah.net。
基金资助:
国家自然科学基金(81401193);上海市自然科学基金(19ZR1442100)

Molecular genetic diagnosis of 9 cases with 46,XY complete gonadal dysgenesis

Haicheng WANG¹(), Yu LIU¹, Hui YE¹, Lin NI¹, Ying CAO¹, Yunlong SUN¹, Bing XIAO¹, Caixia MA², Lifang TANG²()

^1.Department of Pediatric Endocrinology and Genetic Metabolism, Shanghai Institute for Pediatric Research, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
^2.Department of Pediatrics, Qingpu Branch of Zhongshan Hospital Affiliated to Fudan University, Shanghai 201700, China

Received:2021-07-29 Online:2022-01-28 Published:2022-02-18
Contact: Lifang TANG E-mail:seacity@yeah.net;tanglifang1119@126.com
Supported by:
National Natural Science Foundation of China(81401193);Municipal Natural Science Foundation of Shanghai(19ZR1442100)

摘要/Abstract

摘要：

目的·分析9例46，XY完全性性腺发育不全（complete gonadal dysgenesis，CGD）患者的遗传学病因。方法·应用SRY基因测序、二代测序（next generation sequencing，NGS）和染色体微阵列（chromosome microarray，CMA）等分子诊断技术综合分析9例46，XY CGD患者的基因变异情况。结果·9例46，XY CGD先证者中检出4例携带SRY基因突变，其中2例携带未见报道的SRY基因致病/疑似致病突变，分别为SRY缺失突变和c.208T>C（p.Trp70Arg）错义突变；2例携带已报道的SRY基因致病突变，即c.169C>T（p.Gln57X）和c.264dup （p.Glu89fs*15）。NGS检测出1例MAP3K1基因杂合突变c.1016G>A（p.Arg339Gln），该突变为疑似致病突变。有2例散发病例经NGS检测、CMA分析未发现致病性拷贝数变异（copy number variations，CNVs）。有2例为家族病例，通过对父母为近亲结婚的两姐妹中的妹妹进行全外显子组测序，在筛选纯合区域的纯合变异后未发现明确的致病突变或可能与性发育相关的变异；CNVs分析发现两姐妹DMRT1基因内含子2中有约14 kb纯合缺失（Chr9：866，388-880，086，hg19），扩增覆盖断点的PCR测序显示健康双亲为杂合缺失携带者；而后采用PhastCons软件对内含子的缺失序列进行保守片段分析，发现2个保守的非编码元件（conserved non-coding elements，CNEs）。结论·SRY基因突变是46，XY CGD较常见的遗传学病因；发现2个新的SRY基因致病/疑似致病突变。DMRT1内含子2中14 kb纯合缺失可能是该研究中两姐妹的候选致病突变。采用不同序贯的遗传学方法逐步分析46，XY CGD患者的遗传病因，有助于全面了解46，XY CGD患者的分子病因。

关键词: 46，XY完全性性腺发育不全, 性发育不全, SRY基因, MAP3K1基因, 二代测序

Abstract:

Objective·To identify the genetic causes of 9 patients with 46,XY complete gonadal dysgenesis (CGD) .

Methods·The genetic variations of 9 patients with 46,XY CGD were analyzed by combining SRY mutation screening, next generation sequencing (NGS) and chromosome microarray (CMA).

Results·SRY mutations were identified in 4 of nine 46,XY CGD probands, including 2 patients with novel SRY gene pathogenic/likely pathogenic mutations, namely SRY deletion and c.208T>C (p.Trp70Arg) missense variation, and 2 patients with reported SRY pathogenic mutation, namely c.169C>T(p.Gln57X) and c.264dup(p.Glu89fs*15). One patient was identified with a heterozygous mutation in MAP3K1 gene c.1016G>A (p.Arg339Gln) by NGS, which was likely pathogenic mutation. In addition, CMA analysis and NGS found no pathogenic copy number variations (CNVs) in other 2 sporadic patients. Whole exome sequencing (WES) was performed on the younger of two sisters, whose parents were consanguineous marriage, filtered homozygous variants in the homozygous regions, no specific deleterious variants or likely variants associated with sexual development were found. CNVs analysis found an approximate 14 kb homozygous deletion in intron 2 of DMRT1 gene in both sister cases (Chr9: 866, 388-880, 086, hg19), and PCR sequencing with amplified spanned the junction revealed that healthy parents were heterozygous deletion carriers. Then PhastCons software was used to analyze the conserved fragments of the deletion intron sequences and two conserved non-coding elements (CNEs) were found.

Conclusion·SRY mutation was the frequent cause accounted for 46,XY CGD, and 2 novel pathogenic mutations in SRY gene were found. The 14 kb homozygous deletion in intron 2 of DMRT1 might be candidate pathogenic mutation for the sister patients. Stepwise analysis of genetic causes of 46,XY CGD patients might help to fully learn about the molecular changes in these patients.

Key words: 46,XY complete gonadal dysgenesis, disorders of sex development, SRY gene, MAP3K1 gene, next generation sequencing

中图分类号:

R394

王海城, 刘宇, 叶荟, 倪琳, 曹英, 孙云龙, 肖冰, 马彩霞, 唐利芳. 9例46，XY完全性性腺发育不全患者分子遗传学分析[J]. 上海交通大学学报(医学版), 2022, 42(1): 63-69.

Haicheng WANG, Yu LIU, Hui YE, Lin NI, Ying CAO, Yunlong SUN, Bing XIAO, Caixia MA, Lifang TANG. Molecular genetic diagnosis of 9 cases with 46,XY complete gonadal dysgenesis[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(1): 63-69.

图/表 6

参考文献 23

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Proband	Age/year	Clinical manifestation	Gynecologic ultrasonography	Sex hormone					SRY Sanger sequencing/TS/WES/CNVs
Proband	Age/year	Clinical manifestation	Gynecologic ultrasonography	FSH/ （mIU· mL^-1）	LH/ （mIU· mL^-1）	PRL/ （ng· mL^-1）	E₂/ （pg· mL^-1）	T/ （ng· mL^-1）	SRY Sanger sequencing/TS/WES/CNVs
P1	11	No breast development， a height of 150 cm	Left ovariectomy for gondal tumor	127.00	21.30	/	32.50	/	SRY deletion
P2	11	Short stature， vulva young female， clitoris slightly larger	Small uterus， none gonad	79.28	5.84	/	43.00	0	TS （‒）
P3	16	Primary amenorrhea， absent secondary sex characteristics	Small uterus， bilateral streak gonad	81.03	17.89	12.46	14.00	0.55	NM_003140.1：c.264dup （p.Glu89fs*15）
P4	18	Primary amenorrhea， absent secondary sex characteristics	Small uterus， streak gonad on left side， no gonad on right	77.59	22.96	5.47	12.75	0.21	NM_003140.1：c.169C>T （p.Gln57X）
P5	24	Primary amenorrhea， absent secondary sex characteristics	Small uterus， none gonad	35.60	80.45	0.81	73.40	0.77	NM_003140.1：c.208T>C （p.Trp70Arg）
P6	26	Primary amenorrhea， absent secondary sex characteristics， prehypertension	Small uterus， none gonad	97.34	24.28	14.14	1.00	0.45	TS： HSD17B3， NM_000197.1：c.590T>C （p.Met197Thr）
P7	20	Primary amenorrhea， absent secondary sex characteristics， a height of 162 cm	Small uterus， bilateral streak gonad	116.80	26.16	16.31	28.00	0.26	WES（‒）， CNVs： Chr9：856， 388-870， 086 del； homo
P8	19	Primary amenorrhea， absent secondary sex characteristics	Small uterus， bilateral streak gonad	/	/	/	/	/	WES（‒）， CNVs： Chr9：856，388-870，086 del； homo
P9	14	Primary amenorrhea， absent secondary sex characteristics	No uterus and gonads	83.69	32.61	/	61.68	1.27	TS： MAP3K1， NM_005921.1：c.1016G>A （p.Arg339Gln）

Proband	Age/year	Clinical manifestation	Gynecologic ultrasonography	Sex hormone					SRY Sanger sequencing/TS/WES/CNVs
Proband	Age/year	Clinical manifestation	Gynecologic ultrasonography	FSH/ （mIU· mL^-1）	LH/ （mIU· mL^-1）	PRL/ （ng· mL^-1）	E₂/ （pg· mL^-1）	T/ （ng· mL^-1）	SRY Sanger sequencing/TS/WES/CNVs
P1	11	No breast development， a height of 150 cm	Left ovariectomy for gondal tumor	127.00	21.30	/	32.50	/	SRY deletion
P2	11	Short stature， vulva young female， clitoris slightly larger	Small uterus， none gonad	79.28	5.84	/	43.00	0	TS （‒）
P3	16	Primary amenorrhea， absent secondary sex characteristics	Small uterus， bilateral streak gonad	81.03	17.89	12.46	14.00	0.55	NM_003140.1：c.264dup （p.Glu89fs*15）
P4	18	Primary amenorrhea， absent secondary sex characteristics	Small uterus， streak gonad on left side， no gonad on right	77.59	22.96	5.47	12.75	0.21	NM_003140.1：c.169C>T （p.Gln57X）
P5	24	Primary amenorrhea， absent secondary sex characteristics	Small uterus， none gonad	35.60	80.45	0.81	73.40	0.77	NM_003140.1：c.208T>C （p.Trp70Arg）
P6	26	Primary amenorrhea， absent secondary sex characteristics， prehypertension	Small uterus， none gonad	97.34	24.28	14.14	1.00	0.45	TS： HSD17B3， NM_000197.1：c.590T>C （p.Met197Thr）
P7	20	Primary amenorrhea， absent secondary sex characteristics， a height of 162 cm	Small uterus， bilateral streak gonad	116.80	26.16	16.31	28.00	0.26	WES（‒）， CNVs： Chr9：856， 388-870， 086 del； homo
P8	19	Primary amenorrhea， absent secondary sex characteristics	Small uterus， bilateral streak gonad	/	/	/	/	/	WES（‒）， CNVs： Chr9：856，388-870，086 del； homo
P9	14	Primary amenorrhea， absent secondary sex characteristics	No uterus and gonads	83.69	32.61	/	61.68	1.27	TS： MAP3K1， NM_005921.1：c.1016G>A （p.Arg339Gln）

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9例46，XY完全性性腺发育不全患者分子遗传学分析

Molecular genetic diagnosis of 9 cases with 46,XY complete gonadal dysgenesis

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