MAMLD1、SF-1基因和尿道下裂关系研究
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摘要
以尿道褶不完全融合致尿道沿腹侧异常开口为特征,尿道下裂(Hypospadias)是男孩最为常见的泌尿生殖器畸形之一。估计亚洲、欧洲和北美的新生男婴患病率介于0.3%到0.8%,其发病率在全世界范围内呈上升趋势。
尿道下裂是一种复杂的疾病,大部分病例尤其是轻症患者病因不明。环境和内分泌因素参与致病过程,更多调查表明其强烈的遗传背景。部分患者有明显的家族聚集性,多种涉及雄激素信号通路的酶参与尿道发育,编码、调控这些酶的相应基因的功能学变化影响病情发展,到目前为止,如5α-还原酶(5a-reductases, SRD5A2)、雄激素受体(AR)、LH-受体、ATF3和FGF8／10等多种基因被发现为尿道下裂候选基因。
在胚胎睾丸分泌的雄激素影响下,正常的男性外生殖器发育介于第8至第16孕周,生殖结节自近端延长而成阴茎干和阴茎头。由于尿道下裂是胚胎性分化过程中发育停滞造成的,因此可被视为连续过程,融合过程越早被打断,其表现型越严重或复杂。
男性外生殖器形成需要一种特异雄激素：睾酮(testosterone, T)。胎盘人绒毛膜促性腺激素(human chorionic gonadotrophin, hCG)刺激胚胎睾丸内Leydig细胞产生睾酮,在Leydig细胞内,17p-羟基固醇脱氢酶(17beta-hydroxysteroid dehydrogenase 3, HSD17B3)催化雄烯二酮(androsternedione)生成睾酮,睾酮可被类固醇激素5α-还原酶转化成活性更强的双氢睾酮(dihydrotestosterone, DHT),两种酶均可结合至生殖靶组织内的雄激素受体。睾酮诱导吴非氏管(Wolffian duct)或称中肾管(mesonephric duct)发育成附睾等,结合至同一受体的双氢睾酮调节阴茎和阴囊等的分化。
因此,任何有可能影响生殖结节延长或尿生殖褶融合的因素,都有可能造成尿道下裂；任何影响雄激素通路及其受体、5α-还原酶的关键基因突变,造成胚胎性别分化过程发育停滞,都有可能是尿道下裂的易感基因。
X染色体Xq28位点缺失最初发现于肌管性肌病病人：生殖发育在肌管素1(myotubularin, MTM1)基因内突变的患者正常,而在MTM1基因位点缺失的患者则出现了不同程度的异常。后续试验表明,所有出现46,XY性发育异常(Disorder of sexual development, DSD)的肌管性肌病患者,除了Mastermind-like domain containing 1 (MAMLD1)基因,在共同缺失区域未发现其他候选基因。这些发现表明MAMLD1是46,XYDSD极佳的候选基因,特别是尿道下裂。
进一步的实验表明MAMLD1基因在性发育的关键时段表达于胚胎Sertoli和Leydig细胞,小鼠Leydig肿瘤细胞一过性敲除MAMLD1 mRNA表达会导致睾酮表达显著降低。MAMLD1和性发育中调节转录的类固醇生成因子(steroidogenic factor-1, SF-1)共同表达,并在MAMLD1基因发现了SF-1结合位点。据此可认为MAMLD1在性发育过程中调节睾酮产生。相关研究已经证实在尿道下裂病例中发现了多个导致MAMLD1功能减弱的点突变。
在性腺分化为睾丸后,SF-1持续表达于睾丸早期体细胞,在和Y染色体性别决定区(sex determining region Y, SRY)共同维持SOX9表达方面可能起重要作用。在支持细胞,大约从第7孕周开始,SF-1激活苗勒管抑制物(mullerian-inhibiting substance, MIS;也称抗中肾旁管激素,anti-Mullerian hormone (AMH))的表达,诱导发展中的男性Mullerian结构退化。在Leydig细胞,从第8孕周开始,SF-1激活类固醇生成酶系统使外生殖器男性化。
通过下丘脑-垂体-类固醇轴,SF-1转录调控一系列参与肾上腺和性腺发展的基因、性别分化、类固醇合成以及生殖过程,因此在关键时段SF-1相对小的活性改变也可引起内分泌系统和临床的“级联反应”。
然而到目前为止,对如何调节SF-1知之甚少,尽管诸如WT1等因子启动子和增强子,影响SF-1表达的许多重要机制仍需进一步阐明。在此领域的进一步研究对明确SF-1明确在人类疾病中的作用有重要意义。
本论文目的是研究尿道下裂候选基因及其作用机制,由两部分组成：第一部分通过对散发性尿道下裂患者MAMLD1基因直接测序等相关研究和功能预测；第二部分通过测序和基因分型研究SF-1基因在尿道下裂中的作用,尤其是p.G146A (rs1110061)和尿道下裂的关系。
第一部分MAMLD1基因在尿道下裂中作用机制研究目的：在研究性染色体异常在尿道下裂中的发病率以后,在大样本散发性病例中对MAMLD1基因扫描以进一步阐明其突变谱及其在尿道下裂中的作用机制。材料：本实验选择从瑞典收集的500个进行手术的、不同严重程度的的尿道下裂患者样本,从卡罗林斯卡大学附属医院收集的760个健康志愿者作为对照。
(1)从外周血或手术获得的阴茎皮肤组织中提取DNA；
(2)利用Primer3软件针对外显子设计引物,利用PCR扩增出相应片段；
(3)纯化PCR产物,用BigDye(?) Terminator v3.1试剂盒分别利用正向和反向引物对相应片段进行测序,然后在ABI3730测序仪上进行扫描,结果用Seqscape2.5软件进行分析；
(4)基因分型和统计分析：选择rs41313406,rs61740566,rs2073043进行基因分型：
(5)在线软件NetGene 2.0 NNSplice and HSF 2.4进行剪切位点预测；ClustalW2分析同源氨基酸序列；Phyre检测基因突变对蛋白质三级结构和功能的影响。
结果：
(1)非整倍性X染色体筛选
从样本中去除非整倍性X染色体或嵌合子
(2)对99个散发性尿道下裂患者和95个男性健康对照进行直接测序。
实验组中分别发现1个p.V432A,p.Q529K(,c.2065+8a>t和p.D686D,且均未在对照组95个健康人中发现；p.P286S和p.N589S实验组中分别发现11例,对照组未发现；实验组中CAG10>CAG13在3个病例中发现,同时在对照组发现1例。非同义突变p.Q529K、同义突变p.D686D和非编码区突变c.2065+8a>t为新的发现。
(3)在370例患者和418名男性对照中,p.P589S显示统计学意义(p<0.05),而p.P286S和p.V432A无统计学意义。
单元型[p.286S,p.589S]在实验组中轻度增加,和对照组相比p值位于临界值0.05附近。
(4)同源性分析显示p.P286S和p.Q529K分别位于脯氨酸(P)和谷氨酰胺(Q)重复区域；p.53lins3Q位于和p.Q529K谷氨酰胺(Q)同一区域,预测显示可改变蛋白质结构。
结论：我们的发现表明小部分尿道下裂可能由MAMLD1基因突变引起,单元型[p.286S,p.589S]可能是尿道下裂的一个危险因素。
第二部分类固醇生成因子-1(SF-1)基因在尿道下裂中作用机制研究
目的：类固醇生成因子-1(SF-1,NR5A1,Ad4BP)下丘脑-垂体-类固醇轴中起重要作用,在46 XY性发育异常(46 XY DSD) SF-1基因中发现了越来越多的突变,我们试图研究其和散发性尿道下裂的关系。
材料和方法：选择95个不同严重程度的的散发性尿道下裂患者样本,对其编码外显子和侧翼区域直接测序。对单核苷酸多态性rs1110061：G>C进行基因分型：380例尿道下裂患者作为实验组,766个健康志愿者作为对照。
结果：通过直接测序,95例患者中发现2个单核苷酸多态性,p.P125P(rs1110062)和p.G146A(rs1110061)：其中3例为[p.P125P,p.G146A]型,均为严重尿道下裂；2例为纯合子p.G146A,3例为杂合子p.G146A。
选择单核苷酸多态性rs1110061作基因分型：实验组中368名患者发现30例G／C基因型(21例杂合子)或C／C基因(9例纯杂合子),为8.15%；对照组中,760病例发现21例杂合子(2.76%),未见纯合子。显示出明显统计学意义((p<0.0001)。
结论：SF-1 rs1110061：G>C在尿道下裂患者中增加明显,[p.P125P,p.G146A]基因型和散发性尿道下裂的关系尚需进一步实验论证。SF-1应被认为是尿道下裂的易感基因。
Characterized by incomplete fusion of the urethral folds resulting in an abnormal opening of urethra and different degrees of curvature of the penis, hypospadias is one of the most prevalent urogenital malformations in boys. The estimations of frequency are placed between 0.3%and 0.8%of male births in Asian, Europe and North America, and the international trends of the incidence is increasing.
Hypospadias is considered as a complex disorder, the etiology is just beginning to be elucidated. Environmental and endocrinal agents were discovered involved in, more investigations demonstrating strong genetic background. Earlier molecular analyses have revealed mutations of gene responsible for the enzyme activity of 5-alpha-reductase, leading to disturbance of the production of dihydrotestosterone which is necessary for development of the malegenito-urinary tract, are contribute to the etiology of hypospadias. So far, lots of genes such as AR (androgen receptor),5 a-reductases, LH-receptor, ATF3, FGF8/10 were unveiled to be candidate gene for hypospadias.
The normal development of external genitalia in male occurs during the 8th to 16th gestational week under the influence of androgens that secreted by the fetal testes. The genital tubercle elongates to form the shaft and glands of the penis. Since hypospadias are caused by arrested development of the urethra during the period of embryological sexual differentiation, the severity of hypospadias can be seen as a continuum. The earlier the fusion process is interrupted, the more severe or complex it will be.
The formation of male genitalia needs the action of a specific androgen: testosterone (T). Placenta human chorionic gonadotrophin (hCG), stimulate Leydig cells in fetal testis to produce testosterone, In Leydig cells,17beta-hydroxysteroid dehydrogenase (HSD17B3) catalyzes the conversion of androsternedione to testosterone. Testosterone is converted to the more active dihydrotesterone (DHT) by the enzyme steroid 5a-reductase (SRD5A2). Both hormones (T and DHT) bind to the androgen receptor (AR) in the genital target tissue. T induces the differentiation of Wolffian duct into epididymis, and so on. DHT bound to the same androgen receptor, modulates the differentiation of the penis and scrotum.
Thus, any factor that could affect the elongation of the urogenital tubercle, or interfere with the fusion of urogenital folds, could be a susceptibility of hypospadias. Mutations in the crucial genes in the androgen pathway, the androgen receptor and the 5-alpha-reductase genes, could cause arrested development of the urethra during the period of embryological sexual differentiation could account for a subset of hypospadias.
Deletions at the Xq28 locus were first found in patients with myotubular myopathy:genital development is normal in patients with intragenic MTM1 mutations, and invariably abnormal in patients with microdeletions involving MTM1. Subsequent studies have shown loss of Mastermind-like domain containing 1 (MAMLD1) gene MAMLD1 in all patients with myotubular myopathy and 46, XY DSD, and no other candidate gene for 46, XY DSD has been identified within the commonly deleted region. These findings imply that MAMLD1 is an excellent candidate gene for 46, XY DSD, especially hypospadias.
Further studies of MAMLD1 have shown that the gene is expressed in fetal Sertoli and Leydig cells around the critical period for sex development and that transient knockdown of MAMLD1 mRNA expression result in significantly reduced testosterone production in mouse Leydig tumor cells. MAMLD1 is further co-expressed with steroidogenic factor (SF-1), which regulates the transcription of genes involved in sex development, and a SF-1 target site is found within the MAMLD1 gene.
SF-1 expression is consistently maintained in the somatic cells of the early testis after testis determination, where it may play a crucial role together with SRY in supporting SOX9 expression. In Sertoli cells, SF-1 activates expression of Mullerian inhibiting substance (MIS, anti-Mullerian hormone (AMH) from around 7 weeks gestation, which leads to the regression of Mullerian structures in the developing male fetus. In Leydig cells, SF-1 activates the expression of steroidogenic enzyme systems from 8 weeks gestation, which results in androgenization of the external genitalia.
SF-1 transcriptionally regulates a vast array of genes involved in adrenal and gonadal development, sex differentiation, steroidgenesis and reproduction by the hypothalamic-pituitary-steroidogenic axis. So relatively small changes in SF-1 activity may cause'cascade reaction'of endocrine systems and clinically effects in the critical period.
However, much less is known about the regulation of SF-1 expression itself. Despite reports of SF-1 promoter or enhancer regulation by factors such as WT1, many of the key mechanisms influencing SF-1 expression remain to be elucidated. Further research in this area could also have important implications for understanding the role of SF-1 in human disease.
The aim of this thesis is to study candidate genes in hypospadias and composed by two parts:the first segment was focused on the MAMLD1 gene in sporadic cases by associated studies and function prediction; the second part is to study the SF-1 gene on hypospadias by sequencing and Genotyping.
Part One:Study the mechanism of MAMLD1 gene in sporadic hypospadias
Objective:We have analyzed the occurrence of sex chromosome aberrations in hypospadias, further we have screened the MAMLD1 gene for mutations in a large number of sporadic hypospadias cases to further elucidate the mutation spectrum in the pathogenesis of hypospadias.
Material:500 boys surgically treated for different severities of hypospadias in Sweden were recruited for the study. The control group consists of healthy voluntary anonymous blood donors at the Karolinska University Hospital.
Methods:
(1) DNA was extracted from peripheral blood or penile skin tissue.
(2) Exon-flanking primers were designed by Primer 3 program.
(3) After ExoSap-IT enzyme treatment, PCR fragments were sequenced on both directions using BigDye(?) Terminator v3.1 kit and analyzed in ABI Prism 3730 Sequencer. Sequence analysis was preformed with the program SeqScape v2.5.
(4) Genotyping and statistical analyses:Three SNPs in the MAMLD1 gene (rs41313406, rs61740566, rs2073043) were selected for association study.
(5) Splice site prediction:NetGene 2.0 NNSplice and HSF 2.4. HomoloGene: ClustalW2. Three-dimensional structure protein structure:Protein Homology/analogy Recognition Engine (Phyre).
Results:
(1) Screening for sex chromosomal aneuploidy
Patients with sex chromosomal aneuploidy or mosaicism were removed from further analysis.
(2) Direct sequencing 99 cases and 95 male controls.
The p.V432A, p.Q529K, c.2065+8a>t and p.D686D variants were separately detected in one case each and none of the controls. The p.P286S and p.N589S variants were each detected in 11 cases and not in any of the controls while the CAG repeat polymorphism was detected in three cases and one control. The nonsynonymous mutation p.Q529K as well as the synonymous p.D686D and the non-coding c.2065+8a>t mutations are new mutations.
(3) In 370 patients and 418 male controls, the p.P589S showed a weak association with hypospadias (p<0.05) while the p.P286S didn't make it.
The [p.286S, p.589S] haplotype was slightly over represented in controls while the p-value is around the border of significance (p=0.05).
No significance in the p.V432A (rs61740566).
(4) Homology analysis showed that p.P286S and p.Q529K are located in regions of proline (P) and glutamine (Q) repeats respectively. The p.531ins3Q is located in the same stretch of glutamine (Q) residues as the p.Q529K mutation and structure prediction suggests it can change the protein structure.
Conclusion:our findings suggest that a few hypospadias cases are caused by mutations in the MAMLD1 gene and that the haplotype [p.286S, p.589S] possibly is a risk factor for hypospadias.
Part Two:The association between steroidogenic factor-1 and hypospadias
Objective:Steroidogenic factor-1 (SF-1, NR5A1, Ad4BP) plays key role in the hypothalamic-pituitary-steroidogenic organ axis. More and more mutations of SF-1 were discovered in 46 XY DSD (disorders of sex development).We tried to identify its association with hypospadias.
Material and methods:95 patients with different degrees of severities of sporadic hypospadias (recruited by the Swedish Malformation Registry) were selected for direct sequencing of human SF-1 gene in coding exons and flanking region.380 sporadic and familial cases,760 healthy voluntary blood samples were chosen for genotyping:SF-1 polymorphism SNP (rs1110061):G>C.
Results:We revealed 2 SNPs in 95 sporadic patients by direct sequencing:p.P125P (rs1110062) and p.G146A (rs1110061):3 cases are [p.P125P, p.G146A] haplotype, all of them are severe hypospadias; 2 cases are homozygous of p.G146A, and 3 more patients are simply heterozygous of p.G146A.
The polymorphism rs1110061 was chosen for Taqman analysis:30 of 368 patients (8.15%) had G/C genotype (heterozygous,21 cases) or C/C (homozygous,9 cases). In the control group,21 of 753 contained allele C (heterozygous, no homozygous be discovered), a frequency of 2.76%:making significant difference (p<0.0001) between the cases and controls on C allele frequency.
Conclusion:The allele C in Gly146Ala polymorphism of SF-1 increased significantly in hypospadias, the compound heterozygous carrying the p.G146A polymorphism in combination with other mutated alleles needed to further identified. SF-1 should be proposed as a susceptibility factor for hypospadias.
尿道下裂是一种复杂的疾病,大部分病例尤其是轻症患者病因不明。环境和内分泌因素参与致病过程,更多调查表明其强烈的遗传背景。部分患者有明显的家族聚集性,多种涉及雄激素信号通路的酶参与尿道发育,编码、调控这些酶的相应基因的功能学变化影响病情发展,到目前为止,如5α-还原酶(5a-reductases, SRD5A2)、雄激素受体(AR)、LH-受体、ATF3和FGF8／10等多种基因被发现为尿道下裂候选基因。
在胚胎睾丸分泌的雄激素影响下,正常的男性外生殖器发育介于第8至第16孕周,生殖结节自近端延长而成阴茎干和阴茎头。由于尿道下裂是胚胎性分化过程中发育停滞造成的,因此可被视为连续过程,融合过程越早被打断,其表现型越严重或复杂。
男性外生殖器形成需要一种特异雄激素：睾酮(testosterone, T)。胎盘人绒毛膜促性腺激素(human chorionic gonadotrophin, hCG)刺激胚胎睾丸内Leydig细胞产生睾酮,在Leydig细胞内,17p-羟基固醇脱氢酶(17beta-hydroxysteroid dehydrogenase 3, HSD17B3)催化雄烯二酮(androsternedione)生成睾酮,睾酮可被类固醇激素5α-还原酶转化成活性更强的双氢睾酮(dihydrotestosterone, DHT),两种酶均可结合至生殖靶组织内的雄激素受体。睾酮诱导吴非氏管(Wolffian duct)或称中肾管(mesonephric duct)发育成附睾等,结合至同一受体的双氢睾酮调节阴茎和阴囊等的分化。
因此,任何有可能影响生殖结节延长或尿生殖褶融合的因素,都有可能造成尿道下裂；任何影响雄激素通路及其受体、5α-还原酶的关键基因突变,造成胚胎性别分化过程发育停滞,都有可能是尿道下裂的易感基因。
X染色体Xq28位点缺失最初发现于肌管性肌病病人：生殖发育在肌管素1(myotubularin, MTM1)基因内突变的患者正常,而在MTM1基因位点缺失的患者则出现了不同程度的异常。后续试验表明,所有出现46,XY性发育异常(Disorder of sexual development, DSD)的肌管性肌病患者,除了Mastermind-like domain containing 1 (MAMLD1)基因,在共同缺失区域未发现其他候选基因。这些发现表明MAMLD1是46,XYDSD极佳的候选基因,特别是尿道下裂。
进一步的实验表明MAMLD1基因在性发育的关键时段表达于胚胎Sertoli和Leydig细胞,小鼠Leydig肿瘤细胞一过性敲除MAMLD1 mRNA表达会导致睾酮表达显著降低。MAMLD1和性发育中调节转录的类固醇生成因子(steroidogenic factor-1, SF-1)共同表达,并在MAMLD1基因发现了SF-1结合位点。据此可认为MAMLD1在性发育过程中调节睾酮产生。相关研究已经证实在尿道下裂病例中发现了多个导致MAMLD1功能减弱的点突变。
在性腺分化为睾丸后,SF-1持续表达于睾丸早期体细胞,在和Y染色体性别决定区(sex determining region Y, SRY)共同维持SOX9表达方面可能起重要作用。在支持细胞,大约从第7孕周开始,SF-1激活苗勒管抑制物(mullerian-inhibiting substance, MIS;也称抗中肾旁管激素,anti-Mullerian hormone (AMH))的表达,诱导发展中的男性Mullerian结构退化。在Leydig细胞,从第8孕周开始,SF-1激活类固醇生成酶系统使外生殖器男性化。
通过下丘脑-垂体-类固醇轴,SF-1转录调控一系列参与肾上腺和性腺发展的基因、性别分化、类固醇合成以及生殖过程,因此在关键时段SF-1相对小的活性改变也可引起内分泌系统和临床的“级联反应”。
然而到目前为止,对如何调节SF-1知之甚少,尽管诸如WT1等因子启动子和增强子,影响SF-1表达的许多重要机制仍需进一步阐明。在此领域的进一步研究对明确SF-1明确在人类疾病中的作用有重要意义。
本论文目的是研究尿道下裂候选基因及其作用机制,由两部分组成：第一部分通过对散发性尿道下裂患者MAMLD1基因直接测序等相关研究和功能预测；第二部分通过测序和基因分型研究SF-1基因在尿道下裂中的作用,尤其是p.G146A (rs1110061)和尿道下裂的关系。
第一部分MAMLD1基因在尿道下裂中作用机制研究目的：在研究性染色体异常在尿道下裂中的发病率以后,在大样本散发性病例中对MAMLD1基因扫描以进一步阐明其突变谱及其在尿道下裂中的作用机制。材料：本实验选择从瑞典收集的500个进行手术的、不同严重程度的的尿道下裂患者样本,从卡罗林斯卡大学附属医院收集的760个健康志愿者作为对照。
(1)从外周血或手术获得的阴茎皮肤组织中提取DNA；
(2)利用Primer3软件针对外显子设计引物,利用PCR扩增出相应片段；
(3)纯化PCR产物,用BigDye(?) Terminator v3.1试剂盒分别利用正向和反向引物对相应片段进行测序,然后在ABI3730测序仪上进行扫描,结果用Seqscape2.5软件进行分析；
(4)基因分型和统计分析：选择rs41313406,rs61740566,rs2073043进行基因分型：
(5)在线软件NetGene 2.0 NNSplice and HSF 2.4进行剪切位点预测；ClustalW2分析同源氨基酸序列；Phyre检测基因突变对蛋白质三级结构和功能的影响。
结果：
(1)非整倍性X染色体筛选
从样本中去除非整倍性X染色体或嵌合子
(2)对99个散发性尿道下裂患者和95个男性健康对照进行直接测序。
实验组中分别发现1个p.V432A,p.Q529K(,c.2065+8a>t和p.D686D,且均未在对照组95个健康人中发现；p.P286S和p.N589S实验组中分别发现11例,对照组未发现；实验组中CAG10>CAG13在3个病例中发现,同时在对照组发现1例。非同义突变p.Q529K、同义突变p.D686D和非编码区突变c.2065+8a>t为新的发现。
(3)在370例患者和418名男性对照中,p.P589S显示统计学意义(p<0.05),而p.P286S和p.V432A无统计学意义。
单元型[p.286S,p.589S]在实验组中轻度增加,和对照组相比p值位于临界值0.05附近。
(4)同源性分析显示p.P286S和p.Q529K分别位于脯氨酸(P)和谷氨酰胺(Q)重复区域；p.53lins3Q位于和p.Q529K谷氨酰胺(Q)同一区域,预测显示可改变蛋白质结构。
结论：我们的发现表明小部分尿道下裂可能由MAMLD1基因突变引起,单元型[p.286S,p.589S]可能是尿道下裂的一个危险因素。
第二部分类固醇生成因子-1(SF-1)基因在尿道下裂中作用机制研究
目的：类固醇生成因子-1(SF-1,NR5A1,Ad4BP)下丘脑-垂体-类固醇轴中起重要作用,在46 XY性发育异常(46 XY DSD) SF-1基因中发现了越来越多的突变,我们试图研究其和散发性尿道下裂的关系。
材料和方法：选择95个不同严重程度的的散发性尿道下裂患者样本,对其编码外显子和侧翼区域直接测序。对单核苷酸多态性rs1110061：G>C进行基因分型：380例尿道下裂患者作为实验组,766个健康志愿者作为对照。
结果：通过直接测序,95例患者中发现2个单核苷酸多态性,p.P125P(rs1110062)和p.G146A(rs1110061)：其中3例为[p.P125P,p.G146A]型,均为严重尿道下裂；2例为纯合子p.G146A,3例为杂合子p.G146A。
选择单核苷酸多态性rs1110061作基因分型：实验组中368名患者发现30例G／C基因型(21例杂合子)或C／C基因(9例纯杂合子),为8.15%；对照组中,760病例发现21例杂合子(2.76%),未见纯合子。显示出明显统计学意义((p<0.0001)。
结论：SF-1 rs1110061：G>C在尿道下裂患者中增加明显,[p.P125P,p.G146A]基因型和散发性尿道下裂的关系尚需进一步实验论证。SF-1应被认为是尿道下裂的易感基因。
Characterized by incomplete fusion of the urethral folds resulting in an abnormal opening of urethra and different degrees of curvature of the penis, hypospadias is one of the most prevalent urogenital malformations in boys. The estimations of frequency are placed between 0.3%and 0.8%of male births in Asian, Europe and North America, and the international trends of the incidence is increasing.
Hypospadias is considered as a complex disorder, the etiology is just beginning to be elucidated. Environmental and endocrinal agents were discovered involved in, more investigations demonstrating strong genetic background. Earlier molecular analyses have revealed mutations of gene responsible for the enzyme activity of 5-alpha-reductase, leading to disturbance of the production of dihydrotestosterone which is necessary for development of the malegenito-urinary tract, are contribute to the etiology of hypospadias. So far, lots of genes such as AR (androgen receptor),5 a-reductases, LH-receptor, ATF3, FGF8/10 were unveiled to be candidate gene for hypospadias.
The normal development of external genitalia in male occurs during the 8th to 16th gestational week under the influence of androgens that secreted by the fetal testes. The genital tubercle elongates to form the shaft and glands of the penis. Since hypospadias are caused by arrested development of the urethra during the period of embryological sexual differentiation, the severity of hypospadias can be seen as a continuum. The earlier the fusion process is interrupted, the more severe or complex it will be.
The formation of male genitalia needs the action of a specific androgen: testosterone (T). Placenta human chorionic gonadotrophin (hCG), stimulate Leydig cells in fetal testis to produce testosterone, In Leydig cells,17beta-hydroxysteroid dehydrogenase (HSD17B3) catalyzes the conversion of androsternedione to testosterone. Testosterone is converted to the more active dihydrotesterone (DHT) by the enzyme steroid 5a-reductase (SRD5A2). Both hormones (T and DHT) bind to the androgen receptor (AR) in the genital target tissue. T induces the differentiation of Wolffian duct into epididymis, and so on. DHT bound to the same androgen receptor, modulates the differentiation of the penis and scrotum.
Thus, any factor that could affect the elongation of the urogenital tubercle, or interfere with the fusion of urogenital folds, could be a susceptibility of hypospadias. Mutations in the crucial genes in the androgen pathway, the androgen receptor and the 5-alpha-reductase genes, could cause arrested development of the urethra during the period of embryological sexual differentiation could account for a subset of hypospadias.
Deletions at the Xq28 locus were first found in patients with myotubular myopathy:genital development is normal in patients with intragenic MTM1 mutations, and invariably abnormal in patients with microdeletions involving MTM1. Subsequent studies have shown loss of Mastermind-like domain containing 1 (MAMLD1) gene MAMLD1 in all patients with myotubular myopathy and 46, XY DSD, and no other candidate gene for 46, XY DSD has been identified within the commonly deleted region. These findings imply that MAMLD1 is an excellent candidate gene for 46, XY DSD, especially hypospadias.
Further studies of MAMLD1 have shown that the gene is expressed in fetal Sertoli and Leydig cells around the critical period for sex development and that transient knockdown of MAMLD1 mRNA expression result in significantly reduced testosterone production in mouse Leydig tumor cells. MAMLD1 is further co-expressed with steroidogenic factor (SF-1), which regulates the transcription of genes involved in sex development, and a SF-1 target site is found within the MAMLD1 gene.
SF-1 expression is consistently maintained in the somatic cells of the early testis after testis determination, where it may play a crucial role together with SRY in supporting SOX9 expression. In Sertoli cells, SF-1 activates expression of Mullerian inhibiting substance (MIS, anti-Mullerian hormone (AMH) from around 7 weeks gestation, which leads to the regression of Mullerian structures in the developing male fetus. In Leydig cells, SF-1 activates the expression of steroidogenic enzyme systems from 8 weeks gestation, which results in androgenization of the external genitalia.
SF-1 transcriptionally regulates a vast array of genes involved in adrenal and gonadal development, sex differentiation, steroidgenesis and reproduction by the hypothalamic-pituitary-steroidogenic axis. So relatively small changes in SF-1 activity may cause'cascade reaction'of endocrine systems and clinically effects in the critical period.
However, much less is known about the regulation of SF-1 expression itself. Despite reports of SF-1 promoter or enhancer regulation by factors such as WT1, many of the key mechanisms influencing SF-1 expression remain to be elucidated. Further research in this area could also have important implications for understanding the role of SF-1 in human disease.
The aim of this thesis is to study candidate genes in hypospadias and composed by two parts:the first segment was focused on the MAMLD1 gene in sporadic cases by associated studies and function prediction; the second part is to study the SF-1 gene on hypospadias by sequencing and Genotyping.
Part One:Study the mechanism of MAMLD1 gene in sporadic hypospadias
Objective:We have analyzed the occurrence of sex chromosome aberrations in hypospadias, further we have screened the MAMLD1 gene for mutations in a large number of sporadic hypospadias cases to further elucidate the mutation spectrum in the pathogenesis of hypospadias.
Material:500 boys surgically treated for different severities of hypospadias in Sweden were recruited for the study. The control group consists of healthy voluntary anonymous blood donors at the Karolinska University Hospital.
Methods:
(1) DNA was extracted from peripheral blood or penile skin tissue.
(2) Exon-flanking primers were designed by Primer 3 program.
(3) After ExoSap-IT enzyme treatment, PCR fragments were sequenced on both directions using BigDye(?) Terminator v3.1 kit and analyzed in ABI Prism 3730 Sequencer. Sequence analysis was preformed with the program SeqScape v2.5.
(4) Genotyping and statistical analyses:Three SNPs in the MAMLD1 gene (rs41313406, rs61740566, rs2073043) were selected for association study.
(5) Splice site prediction:NetGene 2.0 NNSplice and HSF 2.4. HomoloGene: ClustalW2. Three-dimensional structure protein structure:Protein Homology/analogy Recognition Engine (Phyre).
Results:
(1) Screening for sex chromosomal aneuploidy
Patients with sex chromosomal aneuploidy or mosaicism were removed from further analysis.
(2) Direct sequencing 99 cases and 95 male controls.
The p.V432A, p.Q529K, c.2065+8a>t and p.D686D variants were separately detected in one case each and none of the controls. The p.P286S and p.N589S variants were each detected in 11 cases and not in any of the controls while the CAG repeat polymorphism was detected in three cases and one control. The nonsynonymous mutation p.Q529K as well as the synonymous p.D686D and the non-coding c.2065+8a>t mutations are new mutations.
(3) In 370 patients and 418 male controls, the p.P589S showed a weak association with hypospadias (p<0.05) while the p.P286S didn't make it.
The [p.286S, p.589S] haplotype was slightly over represented in controls while the p-value is around the border of significance (p=0.05).
No significance in the p.V432A (rs61740566).
(4) Homology analysis showed that p.P286S and p.Q529K are located in regions of proline (P) and glutamine (Q) repeats respectively. The p.531ins3Q is located in the same stretch of glutamine (Q) residues as the p.Q529K mutation and structure prediction suggests it can change the protein structure.
Conclusion:our findings suggest that a few hypospadias cases are caused by mutations in the MAMLD1 gene and that the haplotype [p.286S, p.589S] possibly is a risk factor for hypospadias.
Part Two:The association between steroidogenic factor-1 and hypospadias
Objective:Steroidogenic factor-1 (SF-1, NR5A1, Ad4BP) plays key role in the hypothalamic-pituitary-steroidogenic organ axis. More and more mutations of SF-1 were discovered in 46 XY DSD (disorders of sex development).We tried to identify its association with hypospadias.
Material and methods:95 patients with different degrees of severities of sporadic hypospadias (recruited by the Swedish Malformation Registry) were selected for direct sequencing of human SF-1 gene in coding exons and flanking region.380 sporadic and familial cases,760 healthy voluntary blood samples were chosen for genotyping:SF-1 polymorphism SNP (rs1110061):G>C.
Results:We revealed 2 SNPs in 95 sporadic patients by direct sequencing:p.P125P (rs1110062) and p.G146A (rs1110061):3 cases are [p.P125P, p.G146A] haplotype, all of them are severe hypospadias; 2 cases are homozygous of p.G146A, and 3 more patients are simply heterozygous of p.G146A.
The polymorphism rs1110061 was chosen for Taqman analysis:30 of 368 patients (8.15%) had G/C genotype (heterozygous,21 cases) or C/C (homozygous,9 cases). In the control group,21 of 753 contained allele C (heterozygous, no homozygous be discovered), a frequency of 2.76%:making significant difference (p<0.0001) between the cases and controls on C allele frequency.
Conclusion:The allele C in Gly146Ala polymorphism of SF-1 increased significantly in hypospadias, the compound heterozygous carrying the p.G146A polymorphism in combination with other mutated alleles needed to further identified. SF-1 should be proposed as a susceptibility factor for hypospadias.
引文
1.Paulozzi LJ, Erickson JD, Jackson RJ. Hypospadias trends in two US surveillance systems. Pediatrics.1997; 100:831-4.
2.Mondaini N, Ponchietti R, Bonafe M, Biscioni S, Di Loro F, Agostini P, Salvestrini F, Rizzo M. Hypospadias:incidence and effects on psychosexual development as evaluated with the Minnesota Multiphasic Personality Inventory test in a sample of 11,649 young Italian men. Urol Int.2002; 68:81-5.
3.Gallentine ML, Morey AF, Thompson IM Jr. Hypospadias:a contemporary epidemiologic assessment. Urology.2001; 57:788-90.
4.Chong JH, Wee CK, Ho SK, Chan DK. Factors associated with hypospadias in Asian newborn babies. J Perinat Med.2006; 34:497-500.
5.Kurahashi N, Murakumo M, Kakizaki H, Nonomura K, Koyanagi T, Kasai S, Sata F, Kishi R. The estimated prevalence of hypospadias in Hokkaido, Japan. J Epidemiol.2004; 14:73-7.
6.Paulozzi LJ. International trends in rates of hypospadias and cryptorchidism. Environ Health Perspect.1999; 107:297-302.
7.Sun G, Tang D, Liang J, Wu M. Increasing prevalence of hypospadias associated with various perinatal risk factors in Chinese newborns. Urology.2009; 73:1241-5.
8.Khuri FJ, Hardy BE, Churchill BM. Urologic anomalies associated with hypospadias. Urol Clin North Am.1981; 8:565-71.
9.Kenneth CH, Leung AK. Hypospadias:a review. J Singapore.Paediatr Soc.1987; 29:54-6.
10.Soomro NA, Neal DE. Treatment of hypospadias:an update of current practice. Hosp Med.1998; 59:553-6.
11.Leung AK, Robson WL. Current status of cryptorchidism.Adv Pediatr.2004; 51: 351-77.
12.Fukami M, Wada Y, Miyabayashi K, Nishino I, Hasegawa T, Nordenskjold A, Camerino G, Kretz C, Buj-Bello A, Laporte J, Yamada G, Morohashi K, Ogata T. CXorf6 is a causative gene for hypospadias. Nat Genet.2006; 38:1369-71.
13.Bartsch O, Kress W, Wagner A, Seemanova E. The novel contiguous gene syndrome of myotubular myopathy (MTM1), male hypogenitalism and deletion in Xq28:report of the first familial case. Cytogenet Cell Genet.1999; 85: 310-314.
14.Biancalana V, Caron O, Gallati S, Baas F, Kress W, Novelli G, D'Apice MR, Lagier-Tourenne C, Buj-Bello A, Romero NB, Mandel JL:Characterisation of mutations in 77 patients with X-linked myotubular myopathy, including a family with a very mild phenotype. Hum Genet.2003; 112:135-142.
15. Hu LJ, Laporte J, Kress W, Kioschis P, Siebenhaar R, Poustka A, Fardeau M, Metzenberg A, Janssen EA, Thomas N, Mandel JL, Dahl N:Deletions in Xq28 in two boys with myotubular myopathy and abnormal genital development define a new contiguous gene syndrome in a 430 kb region. Hum Mol Genet.1996; 5: 139-143.
16. Laporte J, Guiraud-Chaumeil C, Vincent MC, Mandel JL, Tanner SM, Liechti-Gallati S, Wallgren-Pettersson C, Dahl N, Kress W, Bolhuis PA, Fardeau M, Samson F, Bertini E:Mutations in the MTM1 gene implicated in X-linked myotubular myopathy. Hum Mol Genet.1997; 6:1505-1511.
17. Fukami M, Wada Y, Okada M, Kato F, Katsumata N, Baba T, Morohashi K, Laporte J, Kitagawa M, Ogata T. Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF-1 target sequence. J Biol Chem.2008; 283: 5525-32.
18. Sadovsky Y, Dorn C.Function of steroidogenic factor 1 during development and differentiation of the reproductive system. Rev Reprod.2000; 5:136-42.
19. Kalfa N, Liu B, Klein O, Audran F, Wang MH, Mei C, Sultan C, Baskin LS. Mutations of CXorf6 are associated with a range of severities of hypospadias. Eur J Endocrinol.2008; 150:453-8.
20. Luo X, Ikeda Y, Parker KL. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell.1994; 77:481-490.
21. Sadovsky Y, Crawford PA, Woodson KG, Polish JA, Clements MA, et al. Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids. Proc Natl Acad Sci USA. 1995; 92:10939-10943.
22. Shinoda K, Lei H, Yoshii H, Nomura M, Nagano M, Shiba H, Sasaki H, Osawa Y, Ninomiya Y, Niwa O, Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice. Dev Dyn.1995; 204:22-9.
23. Morohashi K, Honda S, Inomata Y, Handa H, Omura T. A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s. J Biol Chem.1992; 267:17913-9.
24. Kurrasch DM, Cheung CC, Lee FY, Tran PV, Hata K, Ingraham HA. The neonatal ventromedial hypothalamus transcriptome reveals novel markers with spatially distinct patterning. J Neurosci.2007; 27:13624-13634.
25. Ikeda Y, Shen WH, Ingraham HA, Parker KL. Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol.1994; 8:654-662.
26. Nef S, Schaad O, Stallings NR, Cederroth CR, Pitetti JL, et al. Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development. Dev Biol.2005; 287:361-377.
27. Val P, Martinez-Barbera JP, Swain A. Adrenal development is initiated by Cited2 and Wtl through modulation of Sf-1 dosage. Development.2007; 134: 2349-2358.
28. Ramayya MS, Zhou J, Kino T, Segars JH, Bondy CA, Chrousos GP. Steroidogenic factor 1 messenger ribonucleic acid expression in steroidogenic
and nonsteroidogenic human tissues:Northern blot and in situ hybridization studies. J Clin Endocrinol Metab.1997; 82:1799-1806.
29. Hanley NA, Ball SG, Clement-Jones M, Hagan DM, Strachan T, et al. Expression of steroidogenic factor 1 and Wilms'tumour 1 during early human gonadal development and sex determination. Mech Dev.1999; 87:175-180.
30. Yu RN, Ito M, Jameson JL. The murine Dax-1 promoter is stimulated by SF-1 (steroidogenic factor-1) and inhibited by COUP-TF (chicken ovalbumin upstream promoter-transcription factor) via a composite nuclear receptor-regulatory element. Mol Endocrinol.1998; 12:1010-1022.
31. Sekido R, Lovell-Badge R. Sex determination involves synergistic action of SRY and SF-1 on a specific Sox9 enhancer. Nature.2008; 453:930-934.
32. Wilhelm D, Englert C. The Wilms tumor suppressor WT1 regulates early gonad development by activation of SF-1. Genes Dev.2002; 16:1839-1851.
33. Xue Q, Lin Z, Yin P, Milad MP, Cheng YH, Confino E, Reierstad S, Bulun SE. Transcriptional activation of steroidogenic factor-1 by hypomethylation of the 5'CpG island in endometriosis. J Clin Endocrinol Metab.2007; 92: 3261-3267.
34. Shima Y, Zubair M, Komatsu T, Oka S, Yokoyama C, Tachibana T, Hjalt TA, Drouin J, Morohashi K. Pituitary homeobox 2 regulates adrenal4 binding protein/steroidogenic factor-1 gene transcription in the pituitary gonadotrope through interaction with the intronic enhancer. Mol Endocrinol.2008; 22:1633-46.
35. Watanabe M, Yoshida R, Ueoka K, Aoki K, Sasagawa I, Hasegawa T, Sueoka K,Kamatani N, Yoshimura Y, Ogata T. Haplotype analysis of the estrogen receptor 1 gene in male genital and reproductive abnormalities. Hum Reprod. 2007; 22:1279-84.
36. Kallen B, Winberg J. An epidemiological study of hypospadias in Sweden. Acta Paediatr Scand Suppl.1982; 293:1-21.
37.Calzolari E, Contiero MR, Roncarati E, Mattiuz PL, Volpato S. Aetiological factors in hypospadias. J Med Genet.1986; 23:333-7.
38.Weidner IS, Moller H, Jensen TK, Skakkebaek NE. Risk factors for cryptorchidism and hypospadias. J Urol.1999; 161:1606-9.
39.Fredell L, Kockum I, Hansson E, Holmner S, Lundquist L, Lackgren G, Pedersen J, Stenberg A, Westbacke G, Nordenskjold A. Heredity of hypospadias and the significance of low birth weight. J Urol.2002; 167:1423-7.
40.Fredell L, Lichtenstein P, Pedersen NL, Svensson J, Nordenskjold A. Hypospadias is related to birth weight in discordant monozygotic twins. J Urol. 1998; 160:2197-9.
41.Gatti JM, Kirsch AJ, Troyer WA, Perez-Brayfield MR, Smith EA, Scherz HC. Increased incidence of hypospadias in small-for-gestational age infants in a neonatal intensive-care unit. BJU Int.2001; 87:548-50.
42.Sweet RA, Schrott HG, Kurland R, Culp OS. Study of the incidence of hypospadias in Rochester, Minnesota,1940-1970, and a case-control comparison of possible etiologic factors. Mayo Clin Proc.1974; 49:52-8.
43.Czeizel A. Increasing trends in congenital malformations of male external genitalia. Lancet.1985; 1:462-3.
44.Brouwers MM, Feitz WF, Roelofs LA, Kiemeney LA, de Gier RP, Roeleveld N. Hypospadias:a transgenerational effect of diethylstilbestrol? Hum Reprod.2006; 21:666-9.
45.Fritz G, Czeizel AE. Abnormal sperm morphology and function in the fathers of hypospadiacs. J Reprod Fertil.1996; 106:63-6.
46.Silver RI, Rodriguez R, Chang TS, Gearhart JP. In vitro fertilization is associated with an increased risk of hypospadias. J Urol.1999; 161:1954-7.
47.Vilela ML, Willingham E, Buckley J, Liu BC, Agras K, Shiroyanagi Y, Baskin LS. Endocrine disruptors and hypospadias:role of genistein and the fungicide vinclozolin. Urology.2007; 70:618-21.
48. Kurzrock EA, Jegatheesan P, Cunha GR, Baskin LS. Urethral development in the fetal rabbit and induction of hypospadias:a model for human development. J Urol.2000; 164:1786-92.
49. Kojima Y, Hayashi Y, Mizuno K, Mogami M, Sasaki S, Kohri K. Spermatogenesis, fertility and sexual behavior in a hypospadiac mouse model. J Urol.2002; 167:1532-7.
50. Klip H, Verloop J, van Gool JD, Koster ME, Burger CW, van Leeuwen FE; OMEGA Project Group. Hypospadias in sons of women exposed to diethylstilbestrol in utero:a cohort study. Lancet.2002; 359:1102-7.
51. North K, Golding J. A maternal vegetarian diet in pregnancy is associated with hypospadias. The ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood. BJU Int.2000; 85:107-13.
52. Stoll C, Alembik Y, Roth MP, Dott B. Genetic and environmental factors in hypospadias. J Med Genet.1990; 27:559-63.
53. Bauer SB, Bull MJ, Retik AB. Hypospadias:a familial study. J Urol.1979; 121: 474-7.
54. Stokowski LA. Hypospadias in the neonate. Adv Neonatal Care.2004; 4: 206-15.
55. Frisen L, Lagerstedt K, Tapper-Persson M, Kockum I, Nordenskjold A. A novel duplication in the HOXA13 gene in a family with atypical hand-foot-genital syndrome. J Med Genet.2003; 40:e49.
56. Schnack TH, Zdravkovic S, Myrup C, Westergaard T, Christensen K, Wohlfahrt J, Melbye M. Familial aggregation of hypospadias:a cohort study. Am J Epidemiol.2008; 167:251-6.
57. Schafer AJ, Goodfellow PN. Sex determination in humans. Bioessays.1996; 18: 955-63.
58. Zhou L, Mei H, Liu T, Guang W. Identification of mutations of SRD5A2 gene and SRY gene in patients with hypospadias. Zhonghua Yi Xue Yi Chuan Xue Za Zhi.1999; 16:311-4.
59. Lopez M, Torres L, Mendez JP, Cervantes A, Alfaro G, Perez-Palacios G, Erickson RP, Kofman-Alfaro S. SRY alone can induce normal male sexual differentiation. Am J Med Genet.1995; 55:356-8.
60. Yamaguchi TP, Bradley A, McMahon AP, Jones S. A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development.1999; 126:1211-23.
61. Haraguchi R, Suzuki K, Murakami R, Sakai M, Kamikawa M, Kengaku M, Sekine K, Kawano H, Kato S, Ueno N, Yamada G. Molecular analysis of external genitalia formation:the role of fibroblast growth factor (Fgf) genes during genital tubercle formation. Development.2000; 127:2471-9.
62. Haraguchi R, Mo R, Hui C, Motoyama J, Makino S, Shiroishi T, Gaffield W, Yamada G. Unique functions of Sonic hedgehog signaling during external genitalia development. Development.2001; 128:4241-50.
63. Perriton CL, Powles N, Chiang C, Maconochie MK, Cohn MJ. Sonic hedgehog signaling from the urethral epithelium controls external genital development. Dev Biol.2002; 247:26-46.
64. Suzuki K, Bachiller D, Chen YP, Kamikawa M, Ogi H, Haraguchi R, Ogino Y, Minami Y, Mishina Y, Ahn K, Crenshaw EB 3rd, Yamada G. Regulation of outgrowth and apoptosis for the terminal appendage:external genitalia development by concerted actions of BMP signaling. Development.2003; 130: 6209-20.
65. Yamada G, Suzuki K, Haraguchi R, Miyagawa S, Satoh Y, Kamimura M, Nakagata N, Kataoka H, Kuroiwa A, Chen Y. Molecular genetic cascades for external genitalia formation:an emerging organogenesis program. Dev Dyn. 2006; 235:1738-52.
66. Wilhelm D, Koopman P. The makings of maleness:towards an integrated view of male sexual development. Nat Rev Genet.2006; 7:620-31.
67. Lin C, Yin Y, Long F, Ma L. Tissue-specific requirements of beta-catenin in external genitalia development. Development.2008; 135:2815-25.
68. Morgan EA, Nguyen SB, Scott V, Stadler HS. Loss of Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia. Development.2003; 130:3095-109.
69. Kojima Y, Mizuno K, Nakane A, Kato T, Kohri K, Hayashi Y. Long-term physical, hormonal, and sexual outcome of males with disorders of sex development. J Pediatr Surg.2009; 44:1491-6.
70. Thigpen AE, Davis DL, Milatovich A, Mendonca BB, Imperato-McGinley J, Griffin JE, Francke U, Wilson JD, Russell DW. Molecular genetics of steroid 5 alpha-reductase 2 deficiency. J Clin Invest.1992; 90:799-809.
71. Kojima Y, Hayashi Y, Mizuno K, Mogami M, Sasaki S, Kohri K. Spermatogenesis, fertility and sexual behavior in a hypospadiac mouse model. J Urol.2002; 167:1532-7.
72. Uda A, Kojima Y, Hayashi Y, Mizuno K, Asai N, Kohri K. Morphological features of external genitalia in hypospadiac rat model:3-dimensional analysis. J Urol.2004; 171:1362-6.
73. Nordenskjold A, Friedman E, Tapper-Persson M, Soderhall C, Leviav A, Svensson J, Anvret M. Screening for mutations in candidate genes for hypospadias. Urol Res.1999; 27:49-55.
74. Muroya K, Sasagawa I, Suzuki Y, Nakada T, Ishii T, Ogata T. Hypospadias and the androgen receptor gene:mutation screening and CAG repeat length analysis. Mol Hum Reprod.2001; 7:409-13.
75. Tria A, Hiort O, Sinnecker GH. Steroid 5alpha-reductase 1 polymorphisms and testosterone/dihydrotestosterone ratio in male patients with hypospadias. Horm Res.2004; 61:180-3.
76. Wang Y, Li Q, Xu J, Liu Q, Wang W, Lin Y, Ma F, Chen T, Li S, Shen Y. Mutation analysis of five candidate genes in Chinese patients with hypospadias. Eur J Hum Genet.2004; 12:706-12.
77. Ogata T, Muroya K, Ishii T, Suzuki Y, Nakada T, Sasagawa I. Undermasculinized genitalia in a boy with an abnormally expanded CAG repeat length in the androgen receptor gene. Clin Endocrinol (Oxf).2001; 54:835-8.
78.Lim HN, Nixon RM, Chen H, Hughes IA, Hawkins JR. Evidence that longer androgen receptor polyglutamine repeats are a causal factor for genital abnormalities. J Clin Endocrinol Metab.2001; 86:3207-10.
79.Silver RI, Russell DW.5alpha-reductase type 2 mutations are present in some boys with isolated hypospadias. J Urol.1999; 162:1142-5.
80.Bowman CJ, Barlow NJ, Turner KJ, Wallace DG, Foster PM. Effects of in utero exposure to finasteride on androgen-dependent reproductive development in the male rat. Toxicol Sci.2003; 74:393-406.
81.Lee YS, Kirk JM, Stanhope RG, Johnston DI, Harland S, Auchus RJ, Andersson S, Hughes IA. Phenotypic variability in 17beta-hydroxysteroid dehydrogenase-3 deficiency and diagnostic pitfalls. Clin Endocrinol (Oxf).2007; 67:20-8.
82.Sherbet DP, Tiosano D, Kwist KM, Hochberg Z, Auchus RJ. CYP17 mutation E305G causes isolated 17,20-lyase deficiency by selectively altering substrate binding. J Biol Chem.2003; 278:48563-9.
83.Wu L, Sun T, Kobayashi K, Gao P, Griffin JD. Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors. Mol Cell Biol.2002; 22:7688-700.
84.Kojima Y, Sasaki S, Hayashi Y, Umemoto Y, Morohashi K, Kohri K. Role of transcription factors Ad4bp/SF-1 and DAX-1 in steroidogenesis and spermatogenesis in human testicular development and idiopathic azoospermia. Int J Urol.2006; 13:785-93.
85.Dravis C, Yokoyama N, Chumley MJ, Cowan CA, Silvany RE, Shay J, Baker LA, Henkemeyer M. Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development. Dev Biol.2004; 271:272-90.
86.Yucel S, Dravis C, Garcia N, Henkemeyer M, Baker LA. Hypospadias and anorectal malformations mediated by Eph/ephrin signaling. J Pediatr Urol.2007; 3:354-363.
87.Nishida H, Miyagawa S, Matsumaru D, Wada Y, Satoh Y, Ogino Y, Fukuda S, Iguchi T, Yamada G. Gene expression analyses on embryonic external genitalia: identification of regulatory genes possibly involved in masculinization processes. Congenit Anom (Kyoto).2008; 48:63-7.
88.Staib P, Kau N, Romalo G, Schweikert HU. Oestrogen formation in genital and non-genital skin fibroblasts cultured from patients with hypospadias. Clin Endocrinol (Oxf).1994; 41:237-43.
89.Hajek RA, Robertson AD, Johnston DA, Van NT, Tcholakian RK, Wagner LA, Conti CJ, Meistrich ML, Contreras N, Edwards CL, Jones LA. During development,17alpha-estradiol is a potent estrogen and carcinogen. Environ Health Perspect.1997; 105:577-81.
90.Beleza-Meireles A, Kockum I, Lundberg F, Soderhall C, Nordenskjold A. Risk factors for hypospadias in the estrogen receptor 2 gene. J Clin Endocrinol Metab. 2007; 92:3712-8.
91.Ban S, Sata F, Kurahashi N, Kasai S, Moriya K, Kakizaki H, Nonomura K, Kishi R.Genetic polymorphisms of ESR1 and ESR2 that may influence estrogen activity and the risk of hypospadias. Hum Reprod.2008; 23:1466-71.
92.Liu B, Wang Z, Lin G, Agras K, Ebbers M, Willingham E, Baskin LS. Activating transcription factor 3 is up-regulated in patients with hypospadias. PediatrRes.2005; 58:1280-3
93.Kalfa N, Liu B, Klein O, Wang MH, Cao M, Baskin LS. Genomic variants of ATF3 in patients with hypospadias. J Urol.2008; 180:2183-8.
94.Moreno-Garcia M, Miranda EB. Chromosomal anomalies in cryptorchidism and hypospadias. J Urol.2002; 168:2170-2.
95.Cox MJ, Coplen DE, Austin PF.The incidence of disorders of sexual differentiation and chromosomal abnormalities of cryptorchidism and hypospadias stratified by meatal location. J Urol.2008; 180:2649-52.
96.Yong PJ, Barrett IJ, Kalousek DK, Robinson WP. Clinical aspects, prenatal diagnosis, and pathogenesis of trisomy 16 mosaicism. J Med Genet.2003; 40: 175-82.
97.Quigley DI, McDonald MT, Krishnamuthy V, Kishnani PS, Lee MM, Haqq AM, Goodman BK. Triploid mosaicism in a 45,X/69,XXY infant. Am J Med Genet A.2005; 138:171-4.
98.Paliwal P, Sharma A, Sahoo J, Ammini AC.An unusual association of hypospadias with partial deletion of chromosome 1q. Fertil Steril.2010; doi:10.1016/j.fertnstert.2009.12.003.
99.Robin NH, Feldman GJ, Aronson AL, Mitchell HF, Weksberg R, Leonard CO, Burton BK, Josephson KD, Laxova R, Aleck KA, Allanson JE, Guion-Almeida ML, Martin RA, Leichtman LG, Price RA, Opitz JM, Muenke M. Opitz syndrome is genetically heterogeneous, with one locus on Xp22, and a second locus on 22q11.2. Nat Genet.1995; 11:459-61.
100.Andrieux J, Dubourg C, Rio M, Attie-Bitach T, Delaby E, Mathieu M, Journel H, Copin H, Blondeel E, Doco-Fenzy M, Landais E, Delobel B, Odent S, Manouvrier-Hanu S, Holder-Espinasse M. Genotype-phenotype correlation in four 15q24 deleted patients identified by array-CGH. Am J Med Genet A.2009; 149A:2813-9.
[1]O. Bartsch, et al., The novel contiguous gene syndrome of myotubular myopathy (MTM1), male hypogenitalism and deletion in Xq28:report of the first familial case, Cytogenet Cell Genet.85 (1999) 310-314.
[2]A. Beleza-Meireles, et al., Activating transcription factor 3:a hormone responsive gene in the etiology of hypospadias, Eur J Endocrinol.158 (2008) 729-739.
[3]L. Cartegni, S.L. Chew, and A.R. Krainer, Listening to silence and understanding nonsense:exonic mutations that affect splicing, Nat Rev Genet.3 (2002) 285-298.
[4]F.O. Desmet, et al., Human Splicing Finder:an online bioinformatics tool to predict splicing signals, Nucleic Acids Res.37 (2009) e67.
[5]W.G.Fairbrother, et al., Predictive identification of exonic splicing enhancers in human genes, Science.297 (2002) 1007-1013.
[6]M. Fukami, et al., CXorf6 is a causative gene for hypospadias, Nat Genet.38 (2006) 1369-1371.
[7]M. Fukami, et al., Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF-1 target sequence, J Biol Chem.283 (2008) 5525-5532.
[8]S.M. Hebsgaard, et al., Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information, Nucleic Acids Res.24 (1996) 3439-3452.
[9]M.W. Hentze and A.E. Kulozik, A perfect message:RNA surveillance and nonsense-mediated decay, Cell.96 (1999) 307-310.
[10]L.J. Hu, et al., Deletions in Xq28 in two boys with myotubular myopathy and abnormal genital development define a new contiguous gene syndrome in a 430 kb region, Hum Mol Genet.5 (1996) 139-143.
[11]N. Kalfa, et al., Mutations of CXorf6 are associated with a range of severities of hypospadias, Eur J Endocrinol.159 (2008) 453-458.
[12]B. Kallen and J. Winberg, An epidemiological study of hypospadias in Sweden, Acta Paediatr Scand Suppl.293 (1982) 1-21.
[13]L.A. Kelley and M.J. Sternberg, Protein structure prediction on the Web:a case study using the Phyre server, Nat Protoc.4 (2009) 363-371.
[14]J. Laporte, et al., Cloning and characterization of an alternatively spliced gene in proximal Xq28 deleted in two patients with intersexual genitalia and myotubular myopathy, Genomics.41 (1997) 458-462.
[15]T. Ogata, J. Laporte, and M. Fukami, MAMLD1 (CXorf6):a new gene involved in hypospadias, Horm Res.71 (2009) 245-252.
[16]C.M. Ogilvie, et al., Rapid prenatal diagnosis of aneuploidy using quantitative fluorescence-PCR (QF-PCR), J Histochem Cytochem.53 (2005) 285-288.
[17]L.J. Paulozzi, International trends in rates of hypospadias and cryptorchidism, Environ Health Perspect.107 (1999) 297-302.
[18]M.G.Reese, et al., Improved splice site detection in Genie, J Comput Biol.4 (1997)311-323.
[19]Y. Sadovsky and C. Dorn, Function of steroidogenic factor 1 during development and differentiation of the reproductive system, Rev Reprod.5 (2000) 136-142.
[20]C. Sultan, et al., Disorders of androgen action, Semin Reprod Med.20 (2002) 217-228.
[21]C. Sultan, et al., Disorders linked to insufficient androgen action in male children, Hum Reprod Update.7 (2001) 314-322.
[22]A.E. Thigpen, et al., Molecular genetics of steroid 5 alpha-reductase 2 deficiency, J Clin Invest.90 (1992) 799-809.
[23]G.L. Warne and S. Kanumakala, Molecular endocrinology of sex differentiation, Semin Reprod Med.20 (2002) 169-180.
[24]J.D. Wilson, J.E. Griffin, and D.W. Russell, Steroid 5 alpha-reductase 2 deficiency, Endocr Rev.14 (1993) 577-593.
[1]B Kallen, J Winberg, An epidemiological study of hypospadias in Sweden, Acta Paediatr Scand.293 (1982) 1-21.
[2]A Czeizel, J Toth, E Czvenits, Increased birth prevalence of isolated hypospadias in Hungary, Acta Paediatr Hung.27 (1986) 329-337.
[3]LJ Paulozzi, JD Erickson, RJ Jackson,Hypospadias trends in two US surveillance systems, Pediatrics.100 (1997) 831-834.
[4]LJ Paulozzi, International trends in rates of hypospadias and cryptorchidism, Environ Health Perspect.107 (1999) 297-302.
[5]FH Pierik, A Burdorf, JM Nijman, et al., A high hypospadias rate in the Netherlands, Hum Reprod.17 (2002) 1112-1115.
[6]CP Nelson, JM Park, J Wan, et al., The increasing incidence of congenital penile anomalies in the United States, J Urol.174 (2005) 1573-1576.
[7]M Wong, MS Ramayya, GP Chrousos, et al., Cloning and sequence analysis of the human gene encoding steroidogenic factor 1, J Mol Endocrinol.17 (1996) 139-147.
[8]K.L Parker., et al., A unified nomenclature system for the nuclear receptor superfamily, Cell.97 (1999) 161-163.
[9]Y Ikeda, WH Shen, HA Ingraham, et al., Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases, Mol Endocrinol.8 (1994) 654-662.
[10]K Morohashi, S Honda, Y Inomata, et al., A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s, J Biol Chem.267 (1992)17913-17919.
[11]RV Correa, S Domenice, NC Bingham, et al., A microdeletion in the ligand binding domain of human steroidogenic factor 1 causes XY sex reversal without adrenal insufficiency, J Clin Endocrinol Metab.89 (2004) 1767-1772.
[12]B Kohler, L Lin, I Mazen, et al., The spectrum of phenotypes associated with mutations in steroidogenic factor 1 (SF-1, NR5A1, Ad4BP) includes severe penoscrotal hypospadias in 46,XY males without adrenal insufficiency, Eur J Endocrinol.161(2009)237-42.
[13]X Luo, Y Ikeda, KL Parker, A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation, Cell.77 (1994) 481—490.
[14]Y Sadovsky, PA Crawford, KG Woodson, et al., Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids, Proc Natl Acad Sci USA.92 (1995) 10939-10943.
[15]K Shinoda, H Lei, H Yoshii, et al., Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice, Dev Dyn.204(1995)22-29.
[16]JC Achermann, M Ito, M Ito, et al., A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans, Nat Genet.22 (1999) 125-126.
[17]JC Achermann, G Ozisik, M Ito, et al., Gonadal determination and adrenal development are regulated by the orphan nuclear receptor steroidogenic factor-1, in a dose-dependent manner, J Clin Endocrinol Metab.87 (2002) 1829-1833.
[18]A Biason-Lauber, EJ Schoenle, Apparently normal ovarian differentiation in a prepubertal girl with transcriptionally inactive steroidogenic factor 1 (NR5A1/SF-1) and adrenocortical insufficiency, Am J Hum Genet.67 (2000) 1563-1568.
[19]R Coutant, D Mallet, N Lahlou, et al., Heterozygous mutation of steroidogenic factor-1 in 46,XY subjects may mimic partial androgen insensitivity syndrome, J Clin Endocrinol Metab.92 (2007) 2868-2873.
[20]L Lin, P Philibert, B Ferraz-de-Souza, et al., Heterozygous mis-sense mutations in steroidogenic factor 1 (SF-1/Ad4BP, NR5A1) are associated with 46,XY disorders of sex development with normal adrenal function, J Clin Endocrinol Metab. 92(2007)991-999.
[21]AL Reuter, K Goji, NC Bingham, et al., A novel mutation in the accessory DNA-binding domain of human steroidogenic factor 1 causes XY gonadal dysgenesis without adrenal insufficiency, Eur J Endocrinol.157 (2007) 233-238.
[22]B Kohler, L Lin, B Ferraz-de-Souza, et al., Five novel mutations in steroidogenic factor 1 (SF-1, NR5A1) in 46, XY patients with severe underandrogenization but without adrenal insufficiency, Hum Mutat.29 (2008) 59-64.
[23]F WuQiang, T Yanase, L Wei, et al., Functional characterization of a new human Ad4BP/SF-1 variation, G146A, Biochem Biophys Res Commun.311 (2003) 987-994.
[24]Y Wada, M Okada, T Hasegawa, et al., Association of severe micropenis with Gly146Ala polymorphism in the gene for steroidogenic factor-1, Endocr J.52 (2005) 445-448.
[25]Y Wada, M Okada, M Fukami, et al., Association of cryptorchidism with Gly146Ala polymorphism in the gene for steroidogenic factor-1, Fertil Steril.85 (2006)787-790.
[26]W Liu, M Liu, W Fan, et al., The Gly146Ala variation in human SF-1 gene:its association with insulin resistance and type 2 diabetes in Chinese, Diabetes Research and Clinical Practice.73 (2006) 322-328.
2.Mondaini N, Ponchietti R, Bonafe M, Biscioni S, Di Loro F, Agostini P, Salvestrini F, Rizzo M. Hypospadias:incidence and effects on psychosexual development as evaluated with the Minnesota Multiphasic Personality Inventory test in a sample of 11,649 young Italian men. Urol Int.2002; 68:81-5.
3.Gallentine ML, Morey AF, Thompson IM Jr. Hypospadias:a contemporary epidemiologic assessment. Urology.2001; 57:788-90.
4.Chong JH, Wee CK, Ho SK, Chan DK. Factors associated with hypospadias in Asian newborn babies. J Perinat Med.2006; 34:497-500.
5.Kurahashi N, Murakumo M, Kakizaki H, Nonomura K, Koyanagi T, Kasai S, Sata F, Kishi R. The estimated prevalence of hypospadias in Hokkaido, Japan. J Epidemiol.2004; 14:73-7.
6.Paulozzi LJ. International trends in rates of hypospadias and cryptorchidism. Environ Health Perspect.1999; 107:297-302.
7.Sun G, Tang D, Liang J, Wu M. Increasing prevalence of hypospadias associated with various perinatal risk factors in Chinese newborns. Urology.2009; 73:1241-5.
8.Khuri FJ, Hardy BE, Churchill BM. Urologic anomalies associated with hypospadias. Urol Clin North Am.1981; 8:565-71.
9.Kenneth CH, Leung AK. Hypospadias:a review. J Singapore.Paediatr Soc.1987; 29:54-6.
10.Soomro NA, Neal DE. Treatment of hypospadias:an update of current practice. Hosp Med.1998; 59:553-6.
11.Leung AK, Robson WL. Current status of cryptorchidism.Adv Pediatr.2004; 51: 351-77.
12.Fukami M, Wada Y, Miyabayashi K, Nishino I, Hasegawa T, Nordenskjold A, Camerino G, Kretz C, Buj-Bello A, Laporte J, Yamada G, Morohashi K, Ogata T. CXorf6 is a causative gene for hypospadias. Nat Genet.2006; 38:1369-71.
13.Bartsch O, Kress W, Wagner A, Seemanova E. The novel contiguous gene syndrome of myotubular myopathy (MTM1), male hypogenitalism and deletion in Xq28:report of the first familial case. Cytogenet Cell Genet.1999; 85: 310-314.
14.Biancalana V, Caron O, Gallati S, Baas F, Kress W, Novelli G, D'Apice MR, Lagier-Tourenne C, Buj-Bello A, Romero NB, Mandel JL:Characterisation of mutations in 77 patients with X-linked myotubular myopathy, including a family with a very mild phenotype. Hum Genet.2003; 112:135-142.
15. Hu LJ, Laporte J, Kress W, Kioschis P, Siebenhaar R, Poustka A, Fardeau M, Metzenberg A, Janssen EA, Thomas N, Mandel JL, Dahl N:Deletions in Xq28 in two boys with myotubular myopathy and abnormal genital development define a new contiguous gene syndrome in a 430 kb region. Hum Mol Genet.1996; 5: 139-143.
16. Laporte J, Guiraud-Chaumeil C, Vincent MC, Mandel JL, Tanner SM, Liechti-Gallati S, Wallgren-Pettersson C, Dahl N, Kress W, Bolhuis PA, Fardeau M, Samson F, Bertini E:Mutations in the MTM1 gene implicated in X-linked myotubular myopathy. Hum Mol Genet.1997; 6:1505-1511.
17. Fukami M, Wada Y, Okada M, Kato F, Katsumata N, Baba T, Morohashi K, Laporte J, Kitagawa M, Ogata T. Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF-1 target sequence. J Biol Chem.2008; 283: 5525-32.
18. Sadovsky Y, Dorn C.Function of steroidogenic factor 1 during development and differentiation of the reproductive system. Rev Reprod.2000; 5:136-42.
19. Kalfa N, Liu B, Klein O, Audran F, Wang MH, Mei C, Sultan C, Baskin LS. Mutations of CXorf6 are associated with a range of severities of hypospadias. Eur J Endocrinol.2008; 150:453-8.
20. Luo X, Ikeda Y, Parker KL. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell.1994; 77:481-490.
21. Sadovsky Y, Crawford PA, Woodson KG, Polish JA, Clements MA, et al. Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids. Proc Natl Acad Sci USA. 1995; 92:10939-10943.
22. Shinoda K, Lei H, Yoshii H, Nomura M, Nagano M, Shiba H, Sasaki H, Osawa Y, Ninomiya Y, Niwa O, Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice. Dev Dyn.1995; 204:22-9.
23. Morohashi K, Honda S, Inomata Y, Handa H, Omura T. A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s. J Biol Chem.1992; 267:17913-9.
24. Kurrasch DM, Cheung CC, Lee FY, Tran PV, Hata K, Ingraham HA. The neonatal ventromedial hypothalamus transcriptome reveals novel markers with spatially distinct patterning. J Neurosci.2007; 27:13624-13634.
25. Ikeda Y, Shen WH, Ingraham HA, Parker KL. Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol.1994; 8:654-662.
26. Nef S, Schaad O, Stallings NR, Cederroth CR, Pitetti JL, et al. Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development. Dev Biol.2005; 287:361-377.
27. Val P, Martinez-Barbera JP, Swain A. Adrenal development is initiated by Cited2 and Wtl through modulation of Sf-1 dosage. Development.2007; 134: 2349-2358.
28. Ramayya MS, Zhou J, Kino T, Segars JH, Bondy CA, Chrousos GP. Steroidogenic factor 1 messenger ribonucleic acid expression in steroidogenic
and nonsteroidogenic human tissues:Northern blot and in situ hybridization studies. J Clin Endocrinol Metab.1997; 82:1799-1806.
29. Hanley NA, Ball SG, Clement-Jones M, Hagan DM, Strachan T, et al. Expression of steroidogenic factor 1 and Wilms'tumour 1 during early human gonadal development and sex determination. Mech Dev.1999; 87:175-180.
30. Yu RN, Ito M, Jameson JL. The murine Dax-1 promoter is stimulated by SF-1 (steroidogenic factor-1) and inhibited by COUP-TF (chicken ovalbumin upstream promoter-transcription factor) via a composite nuclear receptor-regulatory element. Mol Endocrinol.1998; 12:1010-1022.
31. Sekido R, Lovell-Badge R. Sex determination involves synergistic action of SRY and SF-1 on a specific Sox9 enhancer. Nature.2008; 453:930-934.
32. Wilhelm D, Englert C. The Wilms tumor suppressor WT1 regulates early gonad development by activation of SF-1. Genes Dev.2002; 16:1839-1851.
33. Xue Q, Lin Z, Yin P, Milad MP, Cheng YH, Confino E, Reierstad S, Bulun SE. Transcriptional activation of steroidogenic factor-1 by hypomethylation of the 5'CpG island in endometriosis. J Clin Endocrinol Metab.2007; 92: 3261-3267.
34. Shima Y, Zubair M, Komatsu T, Oka S, Yokoyama C, Tachibana T, Hjalt TA, Drouin J, Morohashi K. Pituitary homeobox 2 regulates adrenal4 binding protein/steroidogenic factor-1 gene transcription in the pituitary gonadotrope through interaction with the intronic enhancer. Mol Endocrinol.2008; 22:1633-46.
35. Watanabe M, Yoshida R, Ueoka K, Aoki K, Sasagawa I, Hasegawa T, Sueoka K,Kamatani N, Yoshimura Y, Ogata T. Haplotype analysis of the estrogen receptor 1 gene in male genital and reproductive abnormalities. Hum Reprod. 2007; 22:1279-84.
36. Kallen B, Winberg J. An epidemiological study of hypospadias in Sweden. Acta Paediatr Scand Suppl.1982; 293:1-21.
37.Calzolari E, Contiero MR, Roncarati E, Mattiuz PL, Volpato S. Aetiological factors in hypospadias. J Med Genet.1986; 23:333-7.
38.Weidner IS, Moller H, Jensen TK, Skakkebaek NE. Risk factors for cryptorchidism and hypospadias. J Urol.1999; 161:1606-9.
39.Fredell L, Kockum I, Hansson E, Holmner S, Lundquist L, Lackgren G, Pedersen J, Stenberg A, Westbacke G, Nordenskjold A. Heredity of hypospadias and the significance of low birth weight. J Urol.2002; 167:1423-7.
40.Fredell L, Lichtenstein P, Pedersen NL, Svensson J, Nordenskjold A. Hypospadias is related to birth weight in discordant monozygotic twins. J Urol. 1998; 160:2197-9.
41.Gatti JM, Kirsch AJ, Troyer WA, Perez-Brayfield MR, Smith EA, Scherz HC. Increased incidence of hypospadias in small-for-gestational age infants in a neonatal intensive-care unit. BJU Int.2001; 87:548-50.
42.Sweet RA, Schrott HG, Kurland R, Culp OS. Study of the incidence of hypospadias in Rochester, Minnesota,1940-1970, and a case-control comparison of possible etiologic factors. Mayo Clin Proc.1974; 49:52-8.
43.Czeizel A. Increasing trends in congenital malformations of male external genitalia. Lancet.1985; 1:462-3.
44.Brouwers MM, Feitz WF, Roelofs LA, Kiemeney LA, de Gier RP, Roeleveld N. Hypospadias:a transgenerational effect of diethylstilbestrol? Hum Reprod.2006; 21:666-9.
45.Fritz G, Czeizel AE. Abnormal sperm morphology and function in the fathers of hypospadiacs. J Reprod Fertil.1996; 106:63-6.
46.Silver RI, Rodriguez R, Chang TS, Gearhart JP. In vitro fertilization is associated with an increased risk of hypospadias. J Urol.1999; 161:1954-7.
47.Vilela ML, Willingham E, Buckley J, Liu BC, Agras K, Shiroyanagi Y, Baskin LS. Endocrine disruptors and hypospadias:role of genistein and the fungicide vinclozolin. Urology.2007; 70:618-21.
48. Kurzrock EA, Jegatheesan P, Cunha GR, Baskin LS. Urethral development in the fetal rabbit and induction of hypospadias:a model for human development. J Urol.2000; 164:1786-92.
49. Kojima Y, Hayashi Y, Mizuno K, Mogami M, Sasaki S, Kohri K. Spermatogenesis, fertility and sexual behavior in a hypospadiac mouse model. J Urol.2002; 167:1532-7.
50. Klip H, Verloop J, van Gool JD, Koster ME, Burger CW, van Leeuwen FE; OMEGA Project Group. Hypospadias in sons of women exposed to diethylstilbestrol in utero:a cohort study. Lancet.2002; 359:1102-7.
51. North K, Golding J. A maternal vegetarian diet in pregnancy is associated with hypospadias. The ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood. BJU Int.2000; 85:107-13.
52. Stoll C, Alembik Y, Roth MP, Dott B. Genetic and environmental factors in hypospadias. J Med Genet.1990; 27:559-63.
53. Bauer SB, Bull MJ, Retik AB. Hypospadias:a familial study. J Urol.1979; 121: 474-7.
54. Stokowski LA. Hypospadias in the neonate. Adv Neonatal Care.2004; 4: 206-15.
55. Frisen L, Lagerstedt K, Tapper-Persson M, Kockum I, Nordenskjold A. A novel duplication in the HOXA13 gene in a family with atypical hand-foot-genital syndrome. J Med Genet.2003; 40:e49.
56. Schnack TH, Zdravkovic S, Myrup C, Westergaard T, Christensen K, Wohlfahrt J, Melbye M. Familial aggregation of hypospadias:a cohort study. Am J Epidemiol.2008; 167:251-6.
57. Schafer AJ, Goodfellow PN. Sex determination in humans. Bioessays.1996; 18: 955-63.
58. Zhou L, Mei H, Liu T, Guang W. Identification of mutations of SRD5A2 gene and SRY gene in patients with hypospadias. Zhonghua Yi Xue Yi Chuan Xue Za Zhi.1999; 16:311-4.
59. Lopez M, Torres L, Mendez JP, Cervantes A, Alfaro G, Perez-Palacios G, Erickson RP, Kofman-Alfaro S. SRY alone can induce normal male sexual differentiation. Am J Med Genet.1995; 55:356-8.
60. Yamaguchi TP, Bradley A, McMahon AP, Jones S. A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development.1999; 126:1211-23.
61. Haraguchi R, Suzuki K, Murakami R, Sakai M, Kamikawa M, Kengaku M, Sekine K, Kawano H, Kato S, Ueno N, Yamada G. Molecular analysis of external genitalia formation:the role of fibroblast growth factor (Fgf) genes during genital tubercle formation. Development.2000; 127:2471-9.
62. Haraguchi R, Mo R, Hui C, Motoyama J, Makino S, Shiroishi T, Gaffield W, Yamada G. Unique functions of Sonic hedgehog signaling during external genitalia development. Development.2001; 128:4241-50.
63. Perriton CL, Powles N, Chiang C, Maconochie MK, Cohn MJ. Sonic hedgehog signaling from the urethral epithelium controls external genital development. Dev Biol.2002; 247:26-46.
64. Suzuki K, Bachiller D, Chen YP, Kamikawa M, Ogi H, Haraguchi R, Ogino Y, Minami Y, Mishina Y, Ahn K, Crenshaw EB 3rd, Yamada G. Regulation of outgrowth and apoptosis for the terminal appendage:external genitalia development by concerted actions of BMP signaling. Development.2003; 130: 6209-20.
65. Yamada G, Suzuki K, Haraguchi R, Miyagawa S, Satoh Y, Kamimura M, Nakagata N, Kataoka H, Kuroiwa A, Chen Y. Molecular genetic cascades for external genitalia formation:an emerging organogenesis program. Dev Dyn. 2006; 235:1738-52.
66. Wilhelm D, Koopman P. The makings of maleness:towards an integrated view of male sexual development. Nat Rev Genet.2006; 7:620-31.
67. Lin C, Yin Y, Long F, Ma L. Tissue-specific requirements of beta-catenin in external genitalia development. Development.2008; 135:2815-25.
68. Morgan EA, Nguyen SB, Scott V, Stadler HS. Loss of Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia. Development.2003; 130:3095-109.
69. Kojima Y, Mizuno K, Nakane A, Kato T, Kohri K, Hayashi Y. Long-term physical, hormonal, and sexual outcome of males with disorders of sex development. J Pediatr Surg.2009; 44:1491-6.
70. Thigpen AE, Davis DL, Milatovich A, Mendonca BB, Imperato-McGinley J, Griffin JE, Francke U, Wilson JD, Russell DW. Molecular genetics of steroid 5 alpha-reductase 2 deficiency. J Clin Invest.1992; 90:799-809.
71. Kojima Y, Hayashi Y, Mizuno K, Mogami M, Sasaki S, Kohri K. Spermatogenesis, fertility and sexual behavior in a hypospadiac mouse model. J Urol.2002; 167:1532-7.
72. Uda A, Kojima Y, Hayashi Y, Mizuno K, Asai N, Kohri K. Morphological features of external genitalia in hypospadiac rat model:3-dimensional analysis. J Urol.2004; 171:1362-6.
73. Nordenskjold A, Friedman E, Tapper-Persson M, Soderhall C, Leviav A, Svensson J, Anvret M. Screening for mutations in candidate genes for hypospadias. Urol Res.1999; 27:49-55.
74. Muroya K, Sasagawa I, Suzuki Y, Nakada T, Ishii T, Ogata T. Hypospadias and the androgen receptor gene:mutation screening and CAG repeat length analysis. Mol Hum Reprod.2001; 7:409-13.
75. Tria A, Hiort O, Sinnecker GH. Steroid 5alpha-reductase 1 polymorphisms and testosterone/dihydrotestosterone ratio in male patients with hypospadias. Horm Res.2004; 61:180-3.
76. Wang Y, Li Q, Xu J, Liu Q, Wang W, Lin Y, Ma F, Chen T, Li S, Shen Y. Mutation analysis of five candidate genes in Chinese patients with hypospadias. Eur J Hum Genet.2004; 12:706-12.
77. Ogata T, Muroya K, Ishii T, Suzuki Y, Nakada T, Sasagawa I. Undermasculinized genitalia in a boy with an abnormally expanded CAG repeat length in the androgen receptor gene. Clin Endocrinol (Oxf).2001; 54:835-8.
78.Lim HN, Nixon RM, Chen H, Hughes IA, Hawkins JR. Evidence that longer androgen receptor polyglutamine repeats are a causal factor for genital abnormalities. J Clin Endocrinol Metab.2001; 86:3207-10.
79.Silver RI, Russell DW.5alpha-reductase type 2 mutations are present in some boys with isolated hypospadias. J Urol.1999; 162:1142-5.
80.Bowman CJ, Barlow NJ, Turner KJ, Wallace DG, Foster PM. Effects of in utero exposure to finasteride on androgen-dependent reproductive development in the male rat. Toxicol Sci.2003; 74:393-406.
81.Lee YS, Kirk JM, Stanhope RG, Johnston DI, Harland S, Auchus RJ, Andersson S, Hughes IA. Phenotypic variability in 17beta-hydroxysteroid dehydrogenase-3 deficiency and diagnostic pitfalls. Clin Endocrinol (Oxf).2007; 67:20-8.
82.Sherbet DP, Tiosano D, Kwist KM, Hochberg Z, Auchus RJ. CYP17 mutation E305G causes isolated 17,20-lyase deficiency by selectively altering substrate binding. J Biol Chem.2003; 278:48563-9.
83.Wu L, Sun T, Kobayashi K, Gao P, Griffin JD. Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors. Mol Cell Biol.2002; 22:7688-700.
84.Kojima Y, Sasaki S, Hayashi Y, Umemoto Y, Morohashi K, Kohri K. Role of transcription factors Ad4bp/SF-1 and DAX-1 in steroidogenesis and spermatogenesis in human testicular development and idiopathic azoospermia. Int J Urol.2006; 13:785-93.
85.Dravis C, Yokoyama N, Chumley MJ, Cowan CA, Silvany RE, Shay J, Baker LA, Henkemeyer M. Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development. Dev Biol.2004; 271:272-90.
86.Yucel S, Dravis C, Garcia N, Henkemeyer M, Baker LA. Hypospadias and anorectal malformations mediated by Eph/ephrin signaling. J Pediatr Urol.2007; 3:354-363.
87.Nishida H, Miyagawa S, Matsumaru D, Wada Y, Satoh Y, Ogino Y, Fukuda S, Iguchi T, Yamada G. Gene expression analyses on embryonic external genitalia: identification of regulatory genes possibly involved in masculinization processes. Congenit Anom (Kyoto).2008; 48:63-7.
88.Staib P, Kau N, Romalo G, Schweikert HU. Oestrogen formation in genital and non-genital skin fibroblasts cultured from patients with hypospadias. Clin Endocrinol (Oxf).1994; 41:237-43.
89.Hajek RA, Robertson AD, Johnston DA, Van NT, Tcholakian RK, Wagner LA, Conti CJ, Meistrich ML, Contreras N, Edwards CL, Jones LA. During development,17alpha-estradiol is a potent estrogen and carcinogen. Environ Health Perspect.1997; 105:577-81.
90.Beleza-Meireles A, Kockum I, Lundberg F, Soderhall C, Nordenskjold A. Risk factors for hypospadias in the estrogen receptor 2 gene. J Clin Endocrinol Metab. 2007; 92:3712-8.
91.Ban S, Sata F, Kurahashi N, Kasai S, Moriya K, Kakizaki H, Nonomura K, Kishi R.Genetic polymorphisms of ESR1 and ESR2 that may influence estrogen activity and the risk of hypospadias. Hum Reprod.2008; 23:1466-71.
92.Liu B, Wang Z, Lin G, Agras K, Ebbers M, Willingham E, Baskin LS. Activating transcription factor 3 is up-regulated in patients with hypospadias. PediatrRes.2005; 58:1280-3
93.Kalfa N, Liu B, Klein O, Wang MH, Cao M, Baskin LS. Genomic variants of ATF3 in patients with hypospadias. J Urol.2008; 180:2183-8.
94.Moreno-Garcia M, Miranda EB. Chromosomal anomalies in cryptorchidism and hypospadias. J Urol.2002; 168:2170-2.
95.Cox MJ, Coplen DE, Austin PF.The incidence of disorders of sexual differentiation and chromosomal abnormalities of cryptorchidism and hypospadias stratified by meatal location. J Urol.2008; 180:2649-52.
96.Yong PJ, Barrett IJ, Kalousek DK, Robinson WP. Clinical aspects, prenatal diagnosis, and pathogenesis of trisomy 16 mosaicism. J Med Genet.2003; 40: 175-82.
97.Quigley DI, McDonald MT, Krishnamuthy V, Kishnani PS, Lee MM, Haqq AM, Goodman BK. Triploid mosaicism in a 45,X/69,XXY infant. Am J Med Genet A.2005; 138:171-4.
98.Paliwal P, Sharma A, Sahoo J, Ammini AC.An unusual association of hypospadias with partial deletion of chromosome 1q. Fertil Steril.2010; doi:10.1016/j.fertnstert.2009.12.003.
99.Robin NH, Feldman GJ, Aronson AL, Mitchell HF, Weksberg R, Leonard CO, Burton BK, Josephson KD, Laxova R, Aleck KA, Allanson JE, Guion-Almeida ML, Martin RA, Leichtman LG, Price RA, Opitz JM, Muenke M. Opitz syndrome is genetically heterogeneous, with one locus on Xp22, and a second locus on 22q11.2. Nat Genet.1995; 11:459-61.
100.Andrieux J, Dubourg C, Rio M, Attie-Bitach T, Delaby E, Mathieu M, Journel H, Copin H, Blondeel E, Doco-Fenzy M, Landais E, Delobel B, Odent S, Manouvrier-Hanu S, Holder-Espinasse M. Genotype-phenotype correlation in four 15q24 deleted patients identified by array-CGH. Am J Med Genet A.2009; 149A:2813-9.
[1]O. Bartsch, et al., The novel contiguous gene syndrome of myotubular myopathy (MTM1), male hypogenitalism and deletion in Xq28:report of the first familial case, Cytogenet Cell Genet.85 (1999) 310-314.
[2]A. Beleza-Meireles, et al., Activating transcription factor 3:a hormone responsive gene in the etiology of hypospadias, Eur J Endocrinol.158 (2008) 729-739.
[3]L. Cartegni, S.L. Chew, and A.R. Krainer, Listening to silence and understanding nonsense:exonic mutations that affect splicing, Nat Rev Genet.3 (2002) 285-298.
[4]F.O. Desmet, et al., Human Splicing Finder:an online bioinformatics tool to predict splicing signals, Nucleic Acids Res.37 (2009) e67.
[5]W.G.Fairbrother, et al., Predictive identification of exonic splicing enhancers in human genes, Science.297 (2002) 1007-1013.
[6]M. Fukami, et al., CXorf6 is a causative gene for hypospadias, Nat Genet.38 (2006) 1369-1371.
[7]M. Fukami, et al., Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF-1 target sequence, J Biol Chem.283 (2008) 5525-5532.
[8]S.M. Hebsgaard, et al., Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information, Nucleic Acids Res.24 (1996) 3439-3452.
[9]M.W. Hentze and A.E. Kulozik, A perfect message:RNA surveillance and nonsense-mediated decay, Cell.96 (1999) 307-310.
[10]L.J. Hu, et al., Deletions in Xq28 in two boys with myotubular myopathy and abnormal genital development define a new contiguous gene syndrome in a 430 kb region, Hum Mol Genet.5 (1996) 139-143.
[11]N. Kalfa, et al., Mutations of CXorf6 are associated with a range of severities of hypospadias, Eur J Endocrinol.159 (2008) 453-458.
[12]B. Kallen and J. Winberg, An epidemiological study of hypospadias in Sweden, Acta Paediatr Scand Suppl.293 (1982) 1-21.
[13]L.A. Kelley and M.J. Sternberg, Protein structure prediction on the Web:a case study using the Phyre server, Nat Protoc.4 (2009) 363-371.
[14]J. Laporte, et al., Cloning and characterization of an alternatively spliced gene in proximal Xq28 deleted in two patients with intersexual genitalia and myotubular myopathy, Genomics.41 (1997) 458-462.
[15]T. Ogata, J. Laporte, and M. Fukami, MAMLD1 (CXorf6):a new gene involved in hypospadias, Horm Res.71 (2009) 245-252.
[16]C.M. Ogilvie, et al., Rapid prenatal diagnosis of aneuploidy using quantitative fluorescence-PCR (QF-PCR), J Histochem Cytochem.53 (2005) 285-288.
[17]L.J. Paulozzi, International trends in rates of hypospadias and cryptorchidism, Environ Health Perspect.107 (1999) 297-302.
[18]M.G.Reese, et al., Improved splice site detection in Genie, J Comput Biol.4 (1997)311-323.
[19]Y. Sadovsky and C. Dorn, Function of steroidogenic factor 1 during development and differentiation of the reproductive system, Rev Reprod.5 (2000) 136-142.
[20]C. Sultan, et al., Disorders of androgen action, Semin Reprod Med.20 (2002) 217-228.
[21]C. Sultan, et al., Disorders linked to insufficient androgen action in male children, Hum Reprod Update.7 (2001) 314-322.
[22]A.E. Thigpen, et al., Molecular genetics of steroid 5 alpha-reductase 2 deficiency, J Clin Invest.90 (1992) 799-809.
[23]G.L. Warne and S. Kanumakala, Molecular endocrinology of sex differentiation, Semin Reprod Med.20 (2002) 169-180.
[24]J.D. Wilson, J.E. Griffin, and D.W. Russell, Steroid 5 alpha-reductase 2 deficiency, Endocr Rev.14 (1993) 577-593.
[1]B Kallen, J Winberg, An epidemiological study of hypospadias in Sweden, Acta Paediatr Scand.293 (1982) 1-21.
[2]A Czeizel, J Toth, E Czvenits, Increased birth prevalence of isolated hypospadias in Hungary, Acta Paediatr Hung.27 (1986) 329-337.
[3]LJ Paulozzi, JD Erickson, RJ Jackson,Hypospadias trends in two US surveillance systems, Pediatrics.100 (1997) 831-834.
[4]LJ Paulozzi, International trends in rates of hypospadias and cryptorchidism, Environ Health Perspect.107 (1999) 297-302.
[5]FH Pierik, A Burdorf, JM Nijman, et al., A high hypospadias rate in the Netherlands, Hum Reprod.17 (2002) 1112-1115.
[6]CP Nelson, JM Park, J Wan, et al., The increasing incidence of congenital penile anomalies in the United States, J Urol.174 (2005) 1573-1576.
[7]M Wong, MS Ramayya, GP Chrousos, et al., Cloning and sequence analysis of the human gene encoding steroidogenic factor 1, J Mol Endocrinol.17 (1996) 139-147.
[8]K.L Parker., et al., A unified nomenclature system for the nuclear receptor superfamily, Cell.97 (1999) 161-163.
[9]Y Ikeda, WH Shen, HA Ingraham, et al., Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases, Mol Endocrinol.8 (1994) 654-662.
[10]K Morohashi, S Honda, Y Inomata, et al., A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s, J Biol Chem.267 (1992)17913-17919.
[11]RV Correa, S Domenice, NC Bingham, et al., A microdeletion in the ligand binding domain of human steroidogenic factor 1 causes XY sex reversal without adrenal insufficiency, J Clin Endocrinol Metab.89 (2004) 1767-1772.
[12]B Kohler, L Lin, I Mazen, et al., The spectrum of phenotypes associated with mutations in steroidogenic factor 1 (SF-1, NR5A1, Ad4BP) includes severe penoscrotal hypospadias in 46,XY males without adrenal insufficiency, Eur J Endocrinol.161(2009)237-42.
[13]X Luo, Y Ikeda, KL Parker, A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation, Cell.77 (1994) 481—490.
[14]Y Sadovsky, PA Crawford, KG Woodson, et al., Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids, Proc Natl Acad Sci USA.92 (1995) 10939-10943.
[15]K Shinoda, H Lei, H Yoshii, et al., Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice, Dev Dyn.204(1995)22-29.
[16]JC Achermann, M Ito, M Ito, et al., A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans, Nat Genet.22 (1999) 125-126.
[17]JC Achermann, G Ozisik, M Ito, et al., Gonadal determination and adrenal development are regulated by the orphan nuclear receptor steroidogenic factor-1, in a dose-dependent manner, J Clin Endocrinol Metab.87 (2002) 1829-1833.
[18]A Biason-Lauber, EJ Schoenle, Apparently normal ovarian differentiation in a prepubertal girl with transcriptionally inactive steroidogenic factor 1 (NR5A1/SF-1) and adrenocortical insufficiency, Am J Hum Genet.67 (2000) 1563-1568.
[19]R Coutant, D Mallet, N Lahlou, et al., Heterozygous mutation of steroidogenic factor-1 in 46,XY subjects may mimic partial androgen insensitivity syndrome, J Clin Endocrinol Metab.92 (2007) 2868-2873.
[20]L Lin, P Philibert, B Ferraz-de-Souza, et al., Heterozygous mis-sense mutations in steroidogenic factor 1 (SF-1/Ad4BP, NR5A1) are associated with 46,XY disorders of sex development with normal adrenal function, J Clin Endocrinol Metab. 92(2007)991-999.
[21]AL Reuter, K Goji, NC Bingham, et al., A novel mutation in the accessory DNA-binding domain of human steroidogenic factor 1 causes XY gonadal dysgenesis without adrenal insufficiency, Eur J Endocrinol.157 (2007) 233-238.
[22]B Kohler, L Lin, B Ferraz-de-Souza, et al., Five novel mutations in steroidogenic factor 1 (SF-1, NR5A1) in 46, XY patients with severe underandrogenization but without adrenal insufficiency, Hum Mutat.29 (2008) 59-64.
[23]F WuQiang, T Yanase, L Wei, et al., Functional characterization of a new human Ad4BP/SF-1 variation, G146A, Biochem Biophys Res Commun.311 (2003) 987-994.
[24]Y Wada, M Okada, T Hasegawa, et al., Association of severe micropenis with Gly146Ala polymorphism in the gene for steroidogenic factor-1, Endocr J.52 (2005) 445-448.
[25]Y Wada, M Okada, M Fukami, et al., Association of cryptorchidism with Gly146Ala polymorphism in the gene for steroidogenic factor-1, Fertil Steril.85 (2006)787-790.
[26]W Liu, M Liu, W Fan, et al., The Gly146Ala variation in human SF-1 gene:its association with insulin resistance and type 2 diabetes in Chinese, Diabetes Research and Clinical Practice.73 (2006) 322-328.
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