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1 HEREDITAS (Beijing) , 33(8): ISSN 研究报告 DOI: /SP.J QF-PCR 在染色体异常男性不育症诊断中的应用齐漫龙, 张媛媛, 刘晓亮, 何蓉, 赵彦艳, 为评估定量荧光 PCR (QF-PCR) 方法在男性不育遗传学诊断中的应用价值, 文章对 78 例非梗阻性男性不育患者, 采用精液常规检测精子情况, 并检测患者性激素水平 ; 采用 QF-PCR 方法对患者性染色体多态性 STR 位点及特异性位点进行检测 ; 采用常规染色体 G 显带方法进行核型分析 ; PCR 检测 AZF 微缺失结果显示 78 例非梗阻性男性不育患者中发现无精子症患者 18 例, 少精子症患者 20 例, 总检出率为 48.72% 采用 QF-PCR 方法检出 3 例 47, XXY 患者, 2 例 46,XX(SRY+) 性反转患者, 1 例 AZFc 区微缺失患者, 与细胞培养染色体分析和 AZF 微缺失 PCR 检测结果相符与传统方法相比, QF-PCR 技术能更迅速直接可靠地检测到男性不育患者的染色体异常区域, 及早发现染色体细微结构异常, 有助于染色体异常造成的男性不育症的鉴别诊断无精子症 ; 遗传学 ; 基因 ; STR; SRY Chromosomal abnormality diagnosis of male infertility by QF-PCR QI Man-Long, ZHANG Yuan-Yuan, LIU Xiao-Liang, HE Rong, ZHAO Yan-Yan Department of Clinical Genetics, Shengjing Hospital, China Medical University, Shenyang , China Abstract: To assess the clinical practice of quantitative fluorescence PCR (QF-PCR) in genetic diagnosis of male infertility patients, 78 nonobstructive male infertility patients were pooled for semen routine screening and sexual hormone determination; QF-PCR was applied to detect the polymorphic short tandem repeat (STR) and specific sequence tagged site (STS) of sex chromosomes; routine chromosome G-band was used for karyotype analysis and PCR was used for the detection of AZF microdeletion. Routine screening of semen found 18 azoospermia and 20 oligospermia patients (48.72%). Three patients with 47, XXY, two with 46,XX(SRY+)and one with AZFc microdeletion were detected using QF-PCR technique which were verified by chromosome G-band and PCR. This study suggests that QF-PCR is a comprehensive, rapid and reliable method for detecting abnormal chromosomal regions and microstructures compared with traditional tests and provides a better candidate for diagnosis of male infertility caused by chromosomal anomalies and gene mutation. Keywords: azoospermia; genetics; gene; STR; SRY 50% [1],,,,,, (Short tandem repeat, STR) STR 收稿日期 : ; 修回日期 : 作者简介 :,, Tel: ; fyuan@sina.com 通讯作者 :,,, yyzhao@sj-hospital.org

2 896 HEREDITAS (Beijing) PCR (Quantitative fluorescence PCR, QF-PCR),,,, QF-PCR , 精液常规与性激素检查, WHO [2] ph 3 3 0, ; < /L BH P9507 (Luteotropic hormone, hlh) (Follicle stimulating hormone, hfsh) (Prolactin, PRL) (Testosterone, Testo) (Chorionic gonadotrophin, TBHCG) (Estradiol, E2), 基因组 DNA 提取,, EDTA 2 ml, DNA ( ), DNA QF-PCR 技术 : X Y STR( AZFc STR DYS448), SRY ( 1) 5 1 X Y (5' 3') (bp) QF-PCR AMEL Xp22.2 CCCTGGGCTCTGTAAAGAATAGTG 106 Yp11.2 ATCAGAGCTTAAACTGGGAAGCTG 112 DXS6803 Xq21.2 GAAATGTGCTTTGACAGGAA CAAAAAGGGACATATGCTACTT X22 Xq28 TCTGTTTAATGAGAGTTGGAAAGAAA Yq(PAR2) ATTGTTGCTACTTGAGACTTGGTG SRY Yp11.31 AGTAAAGGCAACGTCCAGGAT TTCCGACGAGGTCGATACTTA 243 DYS488 AZFc CAAGGATCCAAATAAAGAACAAGA GGTTATTTCTTGATTCCCTGTG AZF PCR sy84 AZFa AGAAGGGTCTGAAAGCAGGT GCCTACTACCTGGAGGCTTC 328 sy86 AZFa GTGACACACAGACTATGCTTC ACACACAGAGGGACAACCCT 320 sy127 AZFb GGCTCACAAACGAAAAGAAA CTGCAGGCAGTAATAAGGGA 271 sy134 AZFb GTCTGCCTCACCATAAAACG ACCACTGCCAAAACTTTCAA 301 sy254 AZFc GGGTGTTACCAGAAGGCA AA GAACCGTATCTACCA AAGCAGC 381 sy255 AZFc GTTACAGGATTCGGCGTGAT CTC GTC ATG TGC AGC CAC 126

3 8 : QF-PCR 897 PCR : DNA 50~100 ng, TransStart Taq (Transgene Biotech ) 25 μl PCR PCR : 95 5 min; s, s, 72 1 min, 30 ; min, 4 PCR : PCR (Axygen ), LIZ500, 95 5 min, 5 min; ABI 3730, GENEMAPPER ( ):, QF-PCR, ( ) 1: 1;, QF-PCR DNA QF-PCR, 染色体核型分析 G, 30, 3~ AZF 微缺失检测 DNA, PCR AZFa AZFb AZFc 2 (Sequence tagged sites, STS), sy84 sy86 sy127 sy134 sy254 sy255( 1) DNA, DNA PCR AZF , 23.08%, 20, 25.64% 2.2 QF-PCR, QF-PCR, 3~5 d, : , XXY 3 例, 检出率为 7.89% ( 1) : X Y AMEL 2:1, X STR DX6803 1:1, X Y STR X22 1:1:1, SRY, AZFc STR DYS448 X, Y ,XX(SRY+) 2, 5.26% 2, X Y 图 1 47, XXY 患者 QF-PCR 检测峰图 X, Y 图 2 46,XX(SRY+) 性反转患者 QF-PCR 检测峰图

4 898 HEREDITAS (Beijing) AMEL, X STR DX6803 1:1, X Y STR X22 1:1, SRY, AZFc STR DYS448 X, SRY 2, 1 Y SRY X,,, 46,X, der(x), t(x;y) (p22; p11) ( 3A); 1 SRY, SRY ( 3B) , XY, AZFc 区微缺失 1 例, 检出率为 2.63% 4, X Y AMEL 1:1, X STR DX6803, X Y STR X22 1:1, SRY, AZFc STR DYS448, 图 3 46,XX(SRY+) 性反转患者染色体核型分析示意图 A: 46,X,der(X),t(X;Y)(p22;p11)(SRY+) ; B: 46,XX(SRY+)

5 8 : QF-PCR 899 图 4 46, XY, AZFc 区微缺失患者 QF-PCR 检测峰图 AZFc AZF AZFa AZFb AZFc 2 STS PCR AZFc sy254 sy255, QF-PCR , XXY, 2 46,XX, QF-PCR QF-PCR,, 2,, 47, XXY 46, XX,,, 47,XXY 46,XX, hlh hfsh 3 47,XXY hlh IU/L IU/L IU/L( 1.24~8.62 IU/L), hfsh IU/L IU/L IU/L( 1.27~19.26 IU/L) 2 46,XX hlh IU/L IU/L, hfsh 48.89IU/L IU/L 3 15%, 50 %, [3],,,,,, QF-PCR,, [4~10], X Y STR,,, QF-PCR,, ;, QF-PCR,,, QF-PCR, 46,XX(SRY+) 46, XX (SRY-) SRY, X STR 1:1, X Y STR 1:1, AZF, 46,XX(SRY+); SRY, X STR 1:1, X Y STR 1:1, AZF, 46, XX(SRY-) 46, XX(SRY+) 46, XX(SRY-), QF-PCR klinefelter 46,XX,, QF-PCR : klinefelter X Y STR n:1(n 1), AZF, 46, XX X Y STR 1:1, AZF

6 900 HEREDITAS (Beijing) , Y Yq11.23 AZF [11] 3 (AZFa AZFb AZFc), Yq11.23 AZFc AZF c STR-DYS448, QF-PCR 1, Y AZFc, PCR AZFc AZF, QF-PCR AZF,, AZFa AZFb AZFc 3 sy84 sy86 sy127 sy134 sy254 sy255, AZF QF-PCR,,, QF-PCR, PCR, : (1), ; (2) PCR,, QF-PCR AZF SRY, 60% ;, QF-PCR STR, QF-PCR QF-PCR,,,, 参考文献 (References): [1] Rowe PJ, Comhaire FH, Hargreave TB, Mahmoud AMA..,,,,,,,,,,,,. :, 2007: 1 2. [2]. - ( 4 ).,,,,,. :, 2001: 45. [3] Dada R, Gupta NP, Kucheria K. Molecular screening for Yq microdeletion in men with idiopathic oligozoospermia and azoospermia. J Biosci, 2003, 28(2): [4] Cirigliano V, Voglino G, Ordoñez E, Marongiu A, Paz Cañadas M, Ejarque M, Rueda L, Lloveras E, Fuster C, Adinolfi M. Rapid prenatal diagnosis of common chromosome aneuploidies by QF-PCR, results of 9 years of clinical experience. Prenat Diagn, 2009, 29(1): [5] Liao C, Yang X, Li FT, Li J, Li DZ. The detection of aneuploidy and maternal contamination by QF-PCR in samples undergoing prenatal diagnosis for thalassemia in Southern China. Eur J Obstet Gynecol Reprod Biol, 2009, 144(2): [6] Onay H, Ugurlu T, Aykut A, Pehlivan S, Inal M, Tinar S, Ozkinay C, Ozkinay F. Rapid prenatal diagnosis of common aneuploidies in amniotic fluid using quantitative fluorescent polymerase chain reaction. Gynecol Obstet Invest, 2008, 66(2): [7] Badenas C, Rodríguez-Revenga L, Morales C, Mediano C, Plaja A, Pérez-Iribarne MM, Soler A, Clusellas N, Borrell A, Sánchez MÁ, Miró E, Sánchez A, Milà M, Jiménez W. Assessment of QF-PCR as the first approach in prenatal diagnosis. J Mol Diagn, 2010, 12(6): [8] Christopoulou S, Christopoulou G, Hatzaki A, Hatzipouliou A, Donoghue J, Karkaletsi M, Kaminopetros P, Sifakis S, Velissariou V. The replacement of cytogenetic analysis by direct chorionic villi sampling preparation with quantitative fluorescence PCR. Gynecol Obstet Invest, 2009, 68(4): [9] Morales C, Sánchez A, Bruguera J, Margarit E, Borrell A, Borobio V, Soler A. Cytogenetic study of spontaneous abortions using semi-direct analysis of chorionic villi samples detects the broadest spectrum of chromosome abnormalities. Am J Med Genet A, 2008, 146A(1): [10],,,,,,,. PCR , 2010, 32(11): [11] Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet, 1976, 34(2):

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