Abstract:

Objective    To study the clinical and molecular genetic features of 46，XY ambiguous genitalia and to analyze the relationship between them. Methods    A total of 77 individuals with 46，XY karyotype and sporadic ambiguous genitalia were enrolled for the study in Ruijin Hospital. Genomic DNA were extracted from patients’ peripheral blood leukocytes. To search for DNA point mutations in 15 candidate genes which were potentially associated with sexual development，we amplified the full coding regions and intron-exon boundaries of these genes using PCR which were subsequntly sequenced using next generation sequencing （NGS） by Illumina-Miseq. Results    We were able to identify a likely genetic diagnosis in around 23.4% cases. The study identified nine mutations of SRD5A2 gene including 5 compound heterozygous and 4 homozygous mutations. One of mutations was novel based on this study. Four homozygous mutations of AR gene were detected， in which a novel missense mutation was identified. There were two patients with polymorphisms of MAMLD1 gene. Three patients carried mutations in INSL3 or MID1 or NR5A1 gene， respectively. Conclusion    46，XY ambiguous genitalia patients present a variety of clinical phenotypes and hypospadias is the most common congenital anomaly. Gene mutations that involved in sex development play an important role in the occurence of hypospadias（14/64，21.8% in the study）. SRD5A2 and AR mutations are most frequently but other genes like MAMLD1，INSL3，MID1 and NR5A1 should not be ignored.

Key words: ambiguous genitalia, clinical analysis, candidate genes, NGS

摘要：

目的    探讨46，XY外生殖器畸形的临床及分子遗传学特征，并分析二者相关性。方法    2004年1月至2014年12月因外生殖器畸形就诊于上海交通大学医学院附属瑞金医院儿内科的染色体为46，XY的患儿77例，收集患儿的临床资料， 分析总结其临床特征及实验室检查特点，采用候选基因分析策略， 选择15个候选基因， 利用依托Illumina－Miseq测序平台的二代测序（next generation sequencing， NGS）技术对候选基因的全外显子测序筛查突变。结果    77例46，XY外生殖器畸形患儿临床表型多样。NGS检测到18例患儿存在候选基因有氨基酸改变的突变位点或多态性位点，其中9例患儿存在6种5α-还原酶2型（steroid-5-alpha-reductase， alpha
polypeptide 2 gene，SRD5A2）基因变异， 包括一处以往未见报道突变位点。在4例患儿中检测到4种雄激素受体（androgen receptor ，AR）基因的变异， 包含1处新发现突变。2例患儿检测到MAMLD1基因的1处纯合多态性位点。1例患儿中存在INSL3基因的复合杂合改变。1例患儿检测到MID1基因纯合突变。另有1例患儿检测NR5A1基因多态性纯合改变。结论    46，XY外生殖器畸形临床表现多样， 以严重型尿道下裂为主。外生殖器发育过程中相关基因的突变在尿道下裂患儿中的检出率较高（14/64， 21.8%）。这其中以SRD5A2和AR的突变为最常见， MAMLD1、 INSL3、 MID1和NR5A1作为尿道下裂的候选基因， 尽管阳性检出率不高， 但是亦不可忽视。

关键词: 外生殖器畸形, 临床分析, 候选基因, 二代测序技术