多囊卵巢综合征表型分析及易感基因基因型—表型关联性研究
|
摘要
第一部分
多囊卵巢综合征患者临床亚型特点分析
背景：
多囊卵巢综合征(PCOS)是一种常见的内分泌代谢性疾病,主要影响育龄期女性,发病率可高达6-8％。它不只是单一的病理生理损害,而是一组症状的集合,因而具有高度的临床异质性。主要症状包括月经异常、多毛、肥胖、高黄体生成素(LH)血症、高胰岛素血症、胰岛素抵抗、高脂血症等,其中具有诊断意义的为月经稀发或闭经(OA)、高雄激素血症和／或多毛(HA)以及卵巢形态学改变(多囊卵巢或卵巢体积增大)(PCO)。2003年欧洲生殖协会和美国人类生殖协会提出的Rotterdam标准对PCOS的诊断进行了明确规定,以上三条中只要满足两条并排除其它疾病即可诊断。基于该标准,PCOS可分为四种亚型：亚型A患者表现为高雄激素和稀发／无排卵,卵巢形态正常(经典型1,OA+HA);亚型B则同时具有三种特征性表型(经典型2,OA+HA+PCO);亚型C患者排卵功能正常,有规律的月经周期,但存在高雄激素和卵巢多囊样改变的证据(高雄PCO型,HA+PCO);亚型D患者表现为稀发／无排卵和卵巢大量小卵泡推积,其血清雄激素在正常范围内,亦无临床高雄激素表现(无排卵PCO型,OA+PCO)。大量研究证实不同亚型患者在代谢损害和生殖障碍方面都有不同程度的表现。无PCO表现者(亚型A)代谢损害可能更严重,而雄激素正常的患者(亚型D)则相对较轻,也有学者认为同时具有无排卵和高雄激素表型的患者(亚型A和亚型B)肥胖、胰岛素抵抗和心血管疾病及糖尿病发生风险均高于其它亚型。这种高度的临床异质性给PCOS基础和临床相关研究均造成很大困难。因此,准确识别各亚型的临床特点,正确评估其内分泌及代谢改变对合理诊断和治疗PCOS,探索疾病的发病机制意义重大。
目的：
本研究旨在通过大样本临床数据分析,明确不同亚型PCOS患者临床特点及意义,为进一步的基础研究和临床试验提供理论基础。
方法：
选取Rotterdam标准诊断的PCOS患者5287例,正常对照女性2016例。按患者临床特点分为四个亚型组：亚型A(OA+HA)、亚型B(OA+HA+PCO)、亚型C(HA+PCO)、亚型D (OA+PCO)、比较各组患者以及正常对照间人体测量学参数、内分泌指标和代谢特点的差异。
结果：
1.基本特点：四种亚型BMI相近,均显著高于对照组(P<0.01)。无排卵PCO型患者(亚型D) m-FG评分低于有高雄激素表现的亚型(亚型A、B、C)(P<0.01),后者各组间无差异。
2.内分泌特点：各亚型患者LH、LH/FSH均高于对照组(P<0.01)。在患者人群内,同时具有无排卵、高雄激素和PCO三种表现的患者(亚型B)LH水平和LH/FSH比值较其它三个亚型组更高(P值均<0.01)。血清T水平在各组受试者间也存在差异,有高雄激素表现的亚型组(亚型A、B、C)血清T最高,无排卵PCO型患者(亚型D)其次,对照组最低。三者呈阶梯状递减,均数差异显著(组间比较P值均<0.01)。
3.血糖代谢特点：亚型B(经典型2)和亚型D(无排卵PCO型)组患者INSO水平高于对照组(P<0.01)。关于HOMA-IR,亚型D(无排卵PCO型)最高,亚型B(经典型2)其次,亚型A(经典型1)、亚型C(高雄PCO型)和对照组最低(P值均<0.01),且后三组间无显著差异。上述结果在较正BMI后仍有统计学意义(P值均<0.01)。
4.血脂代谢特点：各组受试者间CHOL差异不大。四个患者组TG水平均高于对照,而HDL-C水平低于对照(P值均<0.01)。在患者人群中,亚型D(无排卵PCO型)血清LDL-C最高,亚型B(经典型2)其次,亚型C(高雄PCO型)最低(P值均<0.01)。较正BMI后除TG(P=0.721)外,其它结果仍有统计学意义(P值均<0.05)。
结论：
与对照人群相比,四种亚型的PCOS患者均存在一定程度的内分泌紊乱和雄激素过量状态,其中同时具有OA、HA和PCO三个特点的亚型组(亚型B)患者表现更重,而无排卵PCO型患者(亚型D)则相对较轻。但后者在所有患者中所占比重最大,在糖脂代谢方面的异常改变也更明显。可见,各亚型PCOS可能具有不同的病理生理机制。
第二部分
基于PCOS患者的PCOS易感基因基因型-表型关联性分析
背景：
多囊卵巢综合征(PCOS)患者临床表型具有高度异质性,高雄激素血症、高LH血症等内分泌异常以及高血糖、胰岛素抵抗、高脂血症等代谢异常在PCOS女性中发生率均较高。该综合征病因复杂,目前较为公认的观点为遗传因素和环境因素共同作用的结果。既往大量分子遗传学研究已发现多个PCOS(?)目关基因,但作为复杂遗传性疾病,多基因致病以及遗传与环境之间的相互作用致使单基因关联性研究在探索PCOS候选基因方面作用甚微。全基因组关联研究(GWAS)基于基因芯片技术,通过对个体全部或大部分基因的高效检测和比对,为我们打开了一扇研究复杂性疾病的大门。它将患者与对照组全基因组范围内检测出的SNP位点进行比较,确定所有的变异等位基因频率。与候选基因策略需要预先假设致病基因相比,GWAS研究能更系统、更全面的反应疾病的遗传易感性。关于PCOS的前期GWAS研究对上万例患者和等比例对照进行检测和分析,共发现15个PCOS易感SNP(PCOS-SNP),分别位于11个基因座。其中一期GWAS提示了2p16.3、2p21和9q33.3三个易感区域与PCOS发病相关,LHCGR、THADA和DENND1A基因内的5个SNP (rs13405728、rs13429458、rs12478601、rs2479106、rs1018854)被证实为PCOS-SNP.之后二期GWAS在重复了上述结果外又新发现了8个基因座,经两次独立样本的验证证实C9orf3基因rs4385527和rs3802457位点、YAP1基因rs1894116位点、RAB5B基因rs705702位点、HMGA2基因rs2272046位点、TOX3基因rs4784165位点、INSR基因rs2059807位点、SUM01P1基因rs6022786位点以及FSHR基因rs2268361和rs2349415位点等位基因频率在病例和对照之间存在显著差异。在这些候选基因中,LHCGR, FSHR1及(?)NSR由于处于卵泡发育或胰岛素代谢的重要通路,早在其它单基因筛查中得以证实。但其它基因在之前的PCOS相关报道中并未提及。通过复习既往基础研究,推测THADA和HMGA2作为糖尿病易感基因可能在胰岛素代谢方面发挥作用；YAP、HMGA2基因由于与细胞增殖相关,可能参与卵巢形态学改变的机制；LHCGR、RAB5B基因与部分细胞活动如胞吞作用、钙离子转运等有关,可能影响了一些效应细胞的功能。但对于上述基因与PCOS表型的关系,以及其对于不同表型的遗传贡献仍属未知。明确这些疾病易感基因与表型的关联性有助于进一步深入探讨基因的功能和在PCOS发病机制中的作用。
目的：
对纳入GWAS研究的PCOS患者表型信息进行分析,探讨PCOS易感基因的基因型与表型的相关性,以期为进一步探索上述基因在PCOS发病机制中的作用提供依据。
方法：
统计分析前期GWAS研究所纳入的患者的临床资料,分别根据各SNP的基因型频率选择合适的遗传模型对PCOS患者进行分组,比较不同基因型患者间内分泌及代谢表型的差异。
结果：
1. rs13429458(THADA)位点AA基因型患者血清T、LH及LH/FSH比值高于AC+CC基因型(P值分别为0.02、0.03、0.02)。在较正年龄和BMI后,结果均仍有统计学意义(Padjus,分别为<0.01、0.02、<0.01)。
2. rs12478601(THADA)位点CC基因型患者血清LDL较CT+TT组更高(P=0.02)。结果在较正年龄和BMI后仍有统计学意义(Padjust=0.02)。
3. rs2479106(DENND1A)位点GG+AG基因型患者OGTT后2小时血清胰岛素水平显著高于AA组(P=0.02)。结果在较正年龄和BMI后仍有统计学意义(Padjust=0.02)。
4. rs2349415(FSHR)位点CC基因型患者血清T水平较CT+TT基因型者明显增加(P<0.01)。结果在较正年龄和BMI后仍有统计学意义(Padjust<0.01)。
5. rs4385527(C9orf3)位点AG+AA基因型组BMI较GG组高(P=0.01),其LDL-C水平也较高,而HDL-C水平则较低(P值均<0.01)。较正年龄和BMI后结果仍有统计学意义(Padjust<0.01)
6. rs2272046(HMGA2)位点CC+CA基因型组LDL-C水平显著高于从组(P<0.01),较正年龄和BMI对结果无影响(Padjust<0.01)
7. rs4784165(TOX3)位点各基因型组间mFG评分存在显著差异,GG组评分低于另外两组(P<0.01)。
8. rs2059807(INSR)位点AA基因型组OGTT后2小时血糖水平高于GG+AG组(P=0.03),结果在较正年龄和BMI后仍有意义(Padjust=0.042)。
9. rs6022786(SUMO1P1)位点各基因型组间血清T水平存在差异(P<0.01),GG和GA组T浓度显著高于AA组。
结论：
THADA、FSHR、TOX3、SUMO1P1基因与PCOS高雄表型相关,且THADA以及DENND1A、 C9orf3、HMGA2、INSR基因与PCOS糖脂代谢异常也存在相关性。这些基因可能在PCOS病理生理机制中发挥关键作用。
第三部分
基于单一表型人群的PCOS易感基因基因型-表型关联性分析
背景：
多囊卵巢综合征(PCOS)是一种复杂的遗传性疾病,具有明显的种族差异性和临床异质性。前期对中国PCOS患者的全基因组关联性研究(GWAS)共发现位于11个基因座的15个寡核苷酸多态位点(PCOS-SNPs)与PCOS易感性相关。其中2p16.3(rs13405728)、2p21(rs13429458、rs12478601)和9q33.3(rs10818854、 rs2479106)为一期GWAS (GWAS Ⅰ)发现,并在欧洲和美国高加索人群中得到验证。所提示基因包括LHCGR、THADA和DENND1A。但考虑到作为一种影响广泛的复杂性综合征,其致病基因应该不仅限于上述三个。因此随后二期GWAS (GWAS Ⅱ)又进一步扩大了样本量,结果除验证了GWAS Ⅰ外又发现了八个新的PCOS易感位点：9q22.32(rs4385527、rs3802457)、11q22.1(rs1894116)、12q13.2(rs705702)、12q14.3(rs2272046)、16q12.1(rs4784165),19p13.3(rs2059807)、20q13.2(rs6022786)以及2p16.3(rs2268361、rs2349415)。所提示基因包括：FSHR、 C9orf3、YAP1、RAB5B、HMGA2、TOX3、INSR(?)受SUMO1P1。
如前所述,PCOS表型多样。大量家系研究表明PCOS的临床特点多具有家族聚集性,患者一级亲属中胰岛素抵抗、糖耐量异常及糖尿病发生率明显增加。其母亲及姐妹血清雄激素水平也高于一般女性,而LH反应却有所下降。与同年龄的正常对照女性相比,患者的女儿在青春期前血清胰岛素、LH、T及17-羟孕酮水平更高,卵巢体积也更大；并且这种异常在青春期持续存在。由此可见,遗传因素可能参与了PCOS表型的形成。但对于易感基因和表型发生风险之间的直接相关性却尚无确切证据,究竟这些表型受哪一个或哪几个基因的调控一直是PCOS病因学研究的关注点。基于此,本部分研究选取了三组单一表型人群,即分别具有稀发／无排卵(OA)、高雄激素(HA)、卵巢多囊样改变(PCO)三者之一,与无任何上述表型的严格对照人群进行病例对照研究,以期分离各表型,排除三种表型间的相互影响,确定易感基因与表型之间的独立联系,明确这些PCOS易感基因对哪个特征性表型贡献更大。
目的：
本研究旨在比较单一表型人群和严格对照间PCOS-SNP等位基因频率的差异,确定这些位点与特征性表型发生风险之间的相关性,以期为下一步易感基因功能学研究提供理论基础。
方法：
本研究共纳入三组单一表型人群,其中OA组746例,HA组278例,PCO组536例。对照组为1790例无任何阳性表型的健康女性。提取所有受试者全血DNA,通过直接测序的方法检测15个PCOS-SNPs的基因型,并分析其与PCOS表型间的相关性。
结果：
1.LHCGR(rs13405728)、C9orf3(rs4385527)和(?)NR(s2059807)基因与OA存在显著相关性,结果在较正年龄和BMI后仍有统计学意义(Padjust分别为9.47E-03、4.40E-09、3.18E-02)。
2.C9rf3rs4385527)与HA显著相关,结果独立于年龄和BMI (Padjust=1.77E-04)。
3.THADA(rs13429458、rs12478601)、DEAAD1A(rs10818854)以及C9orf3(rs4385527)与PCO显著相关,结果在较正年龄和BMI后同样有统计学意义(Padjust分别为1.47E-03、6.38E-04、1.73E-02和2.20E-07)。
结论：
部分PCOS易感基因与其特征性临床表型有直接相关性。其中LHCGR和INSR与OA表型相关,THADA和DENND1A与PCO表型相关,而C9orf3则与三种表型均相关,它们可能在疾病的发病机制中发挥关键作用。
Part Ⅰ
Subtype Analysis in Polycystic Ovary Syndrome
Background:
Polycystic ovary syndrome (PCOS) is one of the most common endocrinal metabolic syndrome affecting6-8%women of reproductive age. It is not a single pathophysiological disorder, but a group of manifestations, which presents extremely heterogeneous consequently. The main symptoms include irregular menstrual cycle, hirsutism, obesity, hyper-luteinizing hormone, hyperinsulinemia, insulin resistance, dyslipidemia, etc., of which oligo-/anovulation (OA), hyperandrogenism (HA), and polycystic ovary (PCO) are considered as the diagnostic phenotypes. According to the consensus of European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine (ESHRE/ASRM) in Rotterdam conference in2003, the diagnosis will be established when two of the above phenotypes existed after excluding for other known disease causing ovulatory dysfunction and hyperandrogenism. Based on Rotterdam consensus, there are four subtypes of the syndrome:a) subtype A presents both OA and HA, but with normal ovary morphology (classical type Ⅰ); b) subtype B presents all of the three phenotypes (classical type Ⅱ); c) subtype C presents both HA and PCO, the ovulatory cycle of whom is regular; d) subtype D presents OA and PCO, with neither biological nor clinical sign of androgen excess. It has been evidenced that there are differences among subtypes in endocrinal and metabolic characteristics. Metabolic disturbances are more severe in PCOS patients without PCO, and relatively milder in patients with normal androgen. The prevalence of the complications, such as obesity, diabetes and cardiovascular disease are demonstrated to be increased in classical types (subtype A and subtype B). Highly heterogeneity brings a huge obstacle in etiological and clinical researches of the syndrome.
Identifying the clinical characteristics of every subtype exactly and evaluating the reproductive and metabolic changes mean a lot to explore the mechanism of the syndrome and choose more suitable diagnostic criteria and reasonable therapy in clinical work.
Objective:
The aim of the present study is to analyze the clinical data of large-scale patients, identify the clinical characteristics of every subtype, and provide theoretical basis to further fundamental researches and clinical trials.
Methods:
A total of5287PCOS patients diagnosed by Rotterdam criteria and2016normal control subjects were enrolled. The patients were divided into four groups according their subtypes:subtype A (OA+HA), subtype B (OA+HA+PCO), subtype C (HA+PCO), and subtype D (OA+PCO). The parameters of anthropometry, endocrine, and metabolism were compared among four groups of patients and normal controls.
Results:
a) Basic characteristics:BMI was similar among four groups of patients, which was significantly higher than controls (P<0.01). The m-FG score of subtype D is lower than other subtypes with hyperandrogenism (subtype A, B, C. P<0.01). No significance was found among the latter three groups.
b) Endocrinal characteristics:LH and LH/FSH were elevated in every subtype of patients compared with controls (P<0.01). In the cohort of patients, subjects presented all of the three phenotypes (subtype B) have the highest LH level and LH/FSH (P<0.01), no significance were found among the other three subtypes. Testosterone was gradient descent from hyperandrogenic subtypes (subtype A, B, and C) to subtype D and then to controls (P<0.01). No significance was found in PRL (P=0.17).
c) Glucose metabolism:INSO in subtype B and D were higher than controls (P<0.01). The highest HOMA-IR was found in subtype D, and then in subtype B. Subtypes A and D as well as controls had the lowest HOMA-IR (all P<0.01). All above results were independent of BMI.
d) Lipid metabolism:Levels of CHOL among each group were similar. Compared with controls, TG in all patients groups were elevated and HDL-C were decreased (all P<0.01). In the cohort of patients, level of LDL-C was highest in subtype D (OA+PCO), and lowest in subtype C (HA+PCO). Suptype B (OA+HA+PCO) was in the middle (all P<0.01). Nevertheless it should be noticed that the level of LDL-C of subtype D (OA+PCO) was close to controls, which was also higher than subtype C (HA+PCO)(P<0.01). All results remained significance adjusting for BMI except for TG (P=0.721).
Conclusion:
The reproductive and metabolic manifestations were different among the four subtypes of PCOS. Endocrinal disturbance and hyperandorgenism were identified in all subtypes, among which subtype B (OA+HA+PCO) was the most severe, while subtype D (OA+PCO) was the mildest. However, the prevalence of the latter is highest in Chinese patients. And they also manifest worse in metabolism. There might be different mechanism underlying each subtype.
Part Ⅱ
Geno-phenotype correlation analysis of PCOS susceptibility genes in PCOS patients
Background:
Polycystic ovary syndrome (PCOS) is a heterogeneous endocrinopathy characterized by oligo-anovulation (OA), hyperandrogenism (HA) and polycystic ovary (PCO). Both genetic and evironmental factors are well accepted as the main causes of the syndrome. The vast molecular genetic researches have identified many PCOS related genes. However, as a complex hereditary disease, polygenetic pathopoiesis and the interaction between gene and environment make the single gene association study less effective in screening the candidate genes of PCOS. Genome-wide association studies (GWAS) based on gene chip technology, brings an efficient way for detection and comparison of all or most of the individual genes, and therefore opens a door to study complex diseases. It determines all the variant allele frequencies in genome-wide by comparing the SNPs between cases and controls. Compared with candidate gene strategy which needs to presuppose disease-causing genes beforehand, GWAS can identify the genetic susceptibility of the disease in more systematic and comprehensive way. The previous GWAS on PCOS has studied tens of thousands of patients and controls, and found15susceptibility SNPs (PCOS-SNPs) at11loci. In first phase of GWAS,5SNPs of LHCGR (rs13405728), THADA (rs13429458and rsl2478601), and DENND1A (rs2479106and rs10818854) genes at2p16.3,2p21, and9q33.3respectively were proved to be associated to PCOS. Then the second one confirmed the three loci above and identified eight new ones in addition. Allele frequencies were found significantly different between patients and controls at rs4385527and rs3802457in C9orf3gene, rs1894116in YAP1gene, rs705702in RAB5B gene, rs2272046in HMGA2gene, rs4784165in TOX3gene, rs2059807in INSR gene, rs6022786in SUMO1P1gene, as well as rs2268361and rs2349415in FSHR gene. In these candidate genes, LHCGR, FSHR and INSR, as acting in the important pathways of follicular genesis or insulin metabolism, have been evidenced in the previous single gene screening earlier. But other genes were not mentioned in PCOS related reports before. According to the previous basic researches, it can be speculated that diabetes susceptibility genes THADA and HMGA2, may play a role in insulin metabolism; YAP and HMGA2genes were associated with cell proliferation, and may consequently participate in the mechanism of ovarian morphology changes; LHCGR and RAB5B genes were found to participate in some of the cellular activities such as endocytosis and calcium transportation, and may thus affect the function of some effect cells. But there is still little data about the association of these candidate genes to the phenotypes of PCOS, which is important for the exploration of gene function and the etiology of the syndrome. Therefore, more studies are expected on the geno-phenotype correlation.
Obejective:
The aim of this study was to identify additional correlations between the phenotypes and genotypes of the15PCOS-SNPs described in the previous studies.
Methods:
Clinical data of patients enrolled in the previous GWAS were collected. Appropriate genetic models were adapted according to the genotypes frequencies and served as group criteria. Endocrinal and metabolic characteristics were compared between different subgroups.
Results:
a) Rs13429458in THADA was associated with increased LH (P<0.01) and testosterone (T)(P=0.02) levels in subjects with PCOS. The LH/FSH ratio was also higher in the AA group (P<0.01). After adjusting for age and BMI, there remained significance (Padjust<0.01, Padjust=0.02, and Padjust<0.01, respectively)
b) Rs12478601in THADA was associated with increased levels of LDL (P=0.02), which was independent of age and BMI (Padjust=0.02).
c) In the dominant model, rs2479106in DENND1A was associated with elevated serum insulin levels2hours after a glucose load in the patients with PCOS (P=0.02). The comparisons was adjusted for age and BMI(Padjust=0.02).
d) In dominant model of rs2349415(FSHR), patients carrying T allele had lower serum T level (P<0.01). After adjusting for age and BMI, there remained significance (Padjust<0.01).
e) As for rs4385527in C9orf3gene, patients carrying A allele had higher BMI and serum LDL-C, as well as lower HDL-C (P<0.01in all variables). Differences in lipid parameters were all independent of BMI(Padjust<0.01in all variables).
f) Similarly, in rs2272046of HMGA2gene, LDL-C level was also elevated in subjects carrying C allele(P<0.01), which was independent of BMI(Padjust<0.01).
g) Rs4784165of TOX3gene was associated with hirsutism. PCOS women with GG genotype had lowest m-FG score of three subgroups (P<0.01).
h) Serum insulin level2hours after OGTT was found increased in AA subgroup of rs2059807(INSR). There remained statistically significance after adjusting for age and BMI (Padjust=0.042).
i) Independent association with hyperandrogenemia was also found in rs6022786(SUMO1P1). Subjects carrying G allele have higher T (P<0.01).
Conclusion:
The PCOS susceptibility genes THADA, FSHR, TOX3, and SUMO1P1are associated with hyperandorgenism, and THADA as well as DENND1A, C9orf3, HMGA2, and INSR carry risk alleles that are associated with metabolic disturbances in PCOS patients of Han Chinese. These genes might play an important role in the pathophysiological mechanism of the syndrome.
Part Ⅲ
Geno-phenotype correlation analysis of PCOS susceptibility genes in single phenotype subjects
Background:
Polycystic ovary syndrome (PCOS) is a complex genetic disorder with significant ethnic diversity and clinical heterogeneous. Two previous GWAS have found15PCOS susceptibility SNPs (PCOS-SNPs) in11locus.2p16.3(rs13405728),2p21(rsl3429458, rs12478601), and9q33.3(rs10818854, rs2479106) were identified in GWAS I, and confirmed in European and American Caucasians. LHCGR, THADA, and DENND1A genes were the suggested candidate genes. But as PCOS is such a complex syndrome affecting so many systems, the casual genes should not be only that three. Therefore GWAS Ⅱ were performed next and provided eight new locus:9q22.32(rs4385527, rs3802457),11q22.1(rs1894116),12q13.2(rs705702),12q14.3(rs2272046),16q12.1(rs4784165),19p13.3(rs2059807),20q13.2(rs6022786), and2p16.3(rs2268361, rs2349415). FSHR, C9orf3, YAP1, RAB5B, HMGA2, TOX3, INSR, and SUMO1P1were the suggested candidate genes.
As mentioned above, the privous studies have evidenced the familial aggregation of PCOS phenotypes. The prevalence of insulin resistance, impaired glucose tolerance, and diabetes were significantly elevated in the first-degree relatives of the patients. The levels of andorgen in mothers and sisters of PCOS women were also higher than normal controls, while the LH response was lower. It is reported that compared to age-matched normal women, the daughters of the patients had higher serum insulin, LH, T, and17-OHP levels and larger ovary volume before puberty and would exist thoughout the whole puberty. Thus, it can be seen that genetic factor might play an important role in the etiology of PCOS phenotypes. However, few evidences were found about the direct correction between susceptibility genes and phenotype genesis. It is also unclear by which gene the specific phenotype was regulated. Therefore, in the present study, three cohorts of women presented only one of the key features, including aligo-/anovulation (OA), hyperandrogenism (HA), and polycystic ovary (PCO), were enrolled as cases and compared with strict controls without any of the above phenotypes in order to determine the genetic contributions of these PCOS susceptibility genes to its phenotypes.
Methods:
Three independent cohorts of women presenting single characteristic phenotype were recruited as cases (746subjects with OA,278subjects with HA, and536subjects with PCO). And a total of1790healthy women without any above pathological phenotypes were enrolled as controls.
Results:
a) After adjusted for age and body mass index (BMI), variants in LHCGR (rs13405728), C9orf3(rs4385527), and INSR (rs2059807) were strongly associated with OA (Padjust=9.47E-03,4.40E-09, and3.18E-02).
b) Positive association were also observed between C9orf3(rs4385527) variation and HA which is independent of age and BMI (Padjust=1.77E-04).
c) In addition, there was significant evidence for association of variants in THADA (rs13429458and rs12478601), DENND1A (rs10818854), and C9orf3(rs4385527) with PCO. The difference remained significant after adjusting for age and BMI.(Padjust=1.47E-03,6.38E-04,1.73E-02and2.20E-07).
Conclusion:
The SNPs in some candidate genes of PCOS () are directly associated to the main phenotypes (LHCGR and INSR with OA; THADA and DENND1A with PCO; C9orf3with all three phenotypes) and therefore likely to be important in their etiology. The results provide indications for further function.
多囊卵巢综合征患者临床亚型特点分析
背景：
多囊卵巢综合征(PCOS)是一种常见的内分泌代谢性疾病,主要影响育龄期女性,发病率可高达6-8％。它不只是单一的病理生理损害,而是一组症状的集合,因而具有高度的临床异质性。主要症状包括月经异常、多毛、肥胖、高黄体生成素(LH)血症、高胰岛素血症、胰岛素抵抗、高脂血症等,其中具有诊断意义的为月经稀发或闭经(OA)、高雄激素血症和／或多毛(HA)以及卵巢形态学改变(多囊卵巢或卵巢体积增大)(PCO)。2003年欧洲生殖协会和美国人类生殖协会提出的Rotterdam标准对PCOS的诊断进行了明确规定,以上三条中只要满足两条并排除其它疾病即可诊断。基于该标准,PCOS可分为四种亚型：亚型A患者表现为高雄激素和稀发／无排卵,卵巢形态正常(经典型1,OA+HA);亚型B则同时具有三种特征性表型(经典型2,OA+HA+PCO);亚型C患者排卵功能正常,有规律的月经周期,但存在高雄激素和卵巢多囊样改变的证据(高雄PCO型,HA+PCO);亚型D患者表现为稀发／无排卵和卵巢大量小卵泡推积,其血清雄激素在正常范围内,亦无临床高雄激素表现(无排卵PCO型,OA+PCO)。大量研究证实不同亚型患者在代谢损害和生殖障碍方面都有不同程度的表现。无PCO表现者(亚型A)代谢损害可能更严重,而雄激素正常的患者(亚型D)则相对较轻,也有学者认为同时具有无排卵和高雄激素表型的患者(亚型A和亚型B)肥胖、胰岛素抵抗和心血管疾病及糖尿病发生风险均高于其它亚型。这种高度的临床异质性给PCOS基础和临床相关研究均造成很大困难。因此,准确识别各亚型的临床特点,正确评估其内分泌及代谢改变对合理诊断和治疗PCOS,探索疾病的发病机制意义重大。
目的：
本研究旨在通过大样本临床数据分析,明确不同亚型PCOS患者临床特点及意义,为进一步的基础研究和临床试验提供理论基础。
方法：
选取Rotterdam标准诊断的PCOS患者5287例,正常对照女性2016例。按患者临床特点分为四个亚型组：亚型A(OA+HA)、亚型B(OA+HA+PCO)、亚型C(HA+PCO)、亚型D (OA+PCO)、比较各组患者以及正常对照间人体测量学参数、内分泌指标和代谢特点的差异。
结果：
1.基本特点：四种亚型BMI相近,均显著高于对照组(P<0.01)。无排卵PCO型患者(亚型D) m-FG评分低于有高雄激素表现的亚型(亚型A、B、C)(P<0.01),后者各组间无差异。
2.内分泌特点：各亚型患者LH、LH/FSH均高于对照组(P<0.01)。在患者人群内,同时具有无排卵、高雄激素和PCO三种表现的患者(亚型B)LH水平和LH/FSH比值较其它三个亚型组更高(P值均<0.01)。血清T水平在各组受试者间也存在差异,有高雄激素表现的亚型组(亚型A、B、C)血清T最高,无排卵PCO型患者(亚型D)其次,对照组最低。三者呈阶梯状递减,均数差异显著(组间比较P值均<0.01)。
3.血糖代谢特点：亚型B(经典型2)和亚型D(无排卵PCO型)组患者INSO水平高于对照组(P<0.01)。关于HOMA-IR,亚型D(无排卵PCO型)最高,亚型B(经典型2)其次,亚型A(经典型1)、亚型C(高雄PCO型)和对照组最低(P值均<0.01),且后三组间无显著差异。上述结果在较正BMI后仍有统计学意义(P值均<0.01)。
4.血脂代谢特点：各组受试者间CHOL差异不大。四个患者组TG水平均高于对照,而HDL-C水平低于对照(P值均<0.01)。在患者人群中,亚型D(无排卵PCO型)血清LDL-C最高,亚型B(经典型2)其次,亚型C(高雄PCO型)最低(P值均<0.01)。较正BMI后除TG(P=0.721)外,其它结果仍有统计学意义(P值均<0.05)。
结论：
与对照人群相比,四种亚型的PCOS患者均存在一定程度的内分泌紊乱和雄激素过量状态,其中同时具有OA、HA和PCO三个特点的亚型组(亚型B)患者表现更重,而无排卵PCO型患者(亚型D)则相对较轻。但后者在所有患者中所占比重最大,在糖脂代谢方面的异常改变也更明显。可见,各亚型PCOS可能具有不同的病理生理机制。
第二部分
基于PCOS患者的PCOS易感基因基因型-表型关联性分析
背景：
多囊卵巢综合征(PCOS)患者临床表型具有高度异质性,高雄激素血症、高LH血症等内分泌异常以及高血糖、胰岛素抵抗、高脂血症等代谢异常在PCOS女性中发生率均较高。该综合征病因复杂,目前较为公认的观点为遗传因素和环境因素共同作用的结果。既往大量分子遗传学研究已发现多个PCOS(?)目关基因,但作为复杂遗传性疾病,多基因致病以及遗传与环境之间的相互作用致使单基因关联性研究在探索PCOS候选基因方面作用甚微。全基因组关联研究(GWAS)基于基因芯片技术,通过对个体全部或大部分基因的高效检测和比对,为我们打开了一扇研究复杂性疾病的大门。它将患者与对照组全基因组范围内检测出的SNP位点进行比较,确定所有的变异等位基因频率。与候选基因策略需要预先假设致病基因相比,GWAS研究能更系统、更全面的反应疾病的遗传易感性。关于PCOS的前期GWAS研究对上万例患者和等比例对照进行检测和分析,共发现15个PCOS易感SNP(PCOS-SNP),分别位于11个基因座。其中一期GWAS提示了2p16.3、2p21和9q33.3三个易感区域与PCOS发病相关,LHCGR、THADA和DENND1A基因内的5个SNP (rs13405728、rs13429458、rs12478601、rs2479106、rs1018854)被证实为PCOS-SNP.之后二期GWAS在重复了上述结果外又新发现了8个基因座,经两次独立样本的验证证实C9orf3基因rs4385527和rs3802457位点、YAP1基因rs1894116位点、RAB5B基因rs705702位点、HMGA2基因rs2272046位点、TOX3基因rs4784165位点、INSR基因rs2059807位点、SUM01P1基因rs6022786位点以及FSHR基因rs2268361和rs2349415位点等位基因频率在病例和对照之间存在显著差异。在这些候选基因中,LHCGR, FSHR1及(?)NSR由于处于卵泡发育或胰岛素代谢的重要通路,早在其它单基因筛查中得以证实。但其它基因在之前的PCOS相关报道中并未提及。通过复习既往基础研究,推测THADA和HMGA2作为糖尿病易感基因可能在胰岛素代谢方面发挥作用；YAP、HMGA2基因由于与细胞增殖相关,可能参与卵巢形态学改变的机制；LHCGR、RAB5B基因与部分细胞活动如胞吞作用、钙离子转运等有关,可能影响了一些效应细胞的功能。但对于上述基因与PCOS表型的关系,以及其对于不同表型的遗传贡献仍属未知。明确这些疾病易感基因与表型的关联性有助于进一步深入探讨基因的功能和在PCOS发病机制中的作用。
目的：
对纳入GWAS研究的PCOS患者表型信息进行分析,探讨PCOS易感基因的基因型与表型的相关性,以期为进一步探索上述基因在PCOS发病机制中的作用提供依据。
方法：
统计分析前期GWAS研究所纳入的患者的临床资料,分别根据各SNP的基因型频率选择合适的遗传模型对PCOS患者进行分组,比较不同基因型患者间内分泌及代谢表型的差异。
结果：
1. rs13429458(THADA)位点AA基因型患者血清T、LH及LH/FSH比值高于AC+CC基因型(P值分别为0.02、0.03、0.02)。在较正年龄和BMI后,结果均仍有统计学意义(Padjus,分别为<0.01、0.02、<0.01)。
2. rs12478601(THADA)位点CC基因型患者血清LDL较CT+TT组更高(P=0.02)。结果在较正年龄和BMI后仍有统计学意义(Padjust=0.02)。
3. rs2479106(DENND1A)位点GG+AG基因型患者OGTT后2小时血清胰岛素水平显著高于AA组(P=0.02)。结果在较正年龄和BMI后仍有统计学意义(Padjust=0.02)。
4. rs2349415(FSHR)位点CC基因型患者血清T水平较CT+TT基因型者明显增加(P<0.01)。结果在较正年龄和BMI后仍有统计学意义(Padjust<0.01)。
5. rs4385527(C9orf3)位点AG+AA基因型组BMI较GG组高(P=0.01),其LDL-C水平也较高,而HDL-C水平则较低(P值均<0.01)。较正年龄和BMI后结果仍有统计学意义(Padjust<0.01)
6. rs2272046(HMGA2)位点CC+CA基因型组LDL-C水平显著高于从组(P<0.01),较正年龄和BMI对结果无影响(Padjust<0.01)
7. rs4784165(TOX3)位点各基因型组间mFG评分存在显著差异,GG组评分低于另外两组(P<0.01)。
8. rs2059807(INSR)位点AA基因型组OGTT后2小时血糖水平高于GG+AG组(P=0.03),结果在较正年龄和BMI后仍有意义(Padjust=0.042)。
9. rs6022786(SUMO1P1)位点各基因型组间血清T水平存在差异(P<0.01),GG和GA组T浓度显著高于AA组。
结论：
THADA、FSHR、TOX3、SUMO1P1基因与PCOS高雄表型相关,且THADA以及DENND1A、 C9orf3、HMGA2、INSR基因与PCOS糖脂代谢异常也存在相关性。这些基因可能在PCOS病理生理机制中发挥关键作用。
第三部分
基于单一表型人群的PCOS易感基因基因型-表型关联性分析
背景：
多囊卵巢综合征(PCOS)是一种复杂的遗传性疾病,具有明显的种族差异性和临床异质性。前期对中国PCOS患者的全基因组关联性研究(GWAS)共发现位于11个基因座的15个寡核苷酸多态位点(PCOS-SNPs)与PCOS易感性相关。其中2p16.3(rs13405728)、2p21(rs13429458、rs12478601)和9q33.3(rs10818854、 rs2479106)为一期GWAS (GWAS Ⅰ)发现,并在欧洲和美国高加索人群中得到验证。所提示基因包括LHCGR、THADA和DENND1A。但考虑到作为一种影响广泛的复杂性综合征,其致病基因应该不仅限于上述三个。因此随后二期GWAS (GWAS Ⅱ)又进一步扩大了样本量,结果除验证了GWAS Ⅰ外又发现了八个新的PCOS易感位点：9q22.32(rs4385527、rs3802457)、11q22.1(rs1894116)、12q13.2(rs705702)、12q14.3(rs2272046)、16q12.1(rs4784165),19p13.3(rs2059807)、20q13.2(rs6022786)以及2p16.3(rs2268361、rs2349415)。所提示基因包括：FSHR、 C9orf3、YAP1、RAB5B、HMGA2、TOX3、INSR(?)受SUMO1P1。
如前所述,PCOS表型多样。大量家系研究表明PCOS的临床特点多具有家族聚集性,患者一级亲属中胰岛素抵抗、糖耐量异常及糖尿病发生率明显增加。其母亲及姐妹血清雄激素水平也高于一般女性,而LH反应却有所下降。与同年龄的正常对照女性相比,患者的女儿在青春期前血清胰岛素、LH、T及17-羟孕酮水平更高,卵巢体积也更大；并且这种异常在青春期持续存在。由此可见,遗传因素可能参与了PCOS表型的形成。但对于易感基因和表型发生风险之间的直接相关性却尚无确切证据,究竟这些表型受哪一个或哪几个基因的调控一直是PCOS病因学研究的关注点。基于此,本部分研究选取了三组单一表型人群,即分别具有稀发／无排卵(OA)、高雄激素(HA)、卵巢多囊样改变(PCO)三者之一,与无任何上述表型的严格对照人群进行病例对照研究,以期分离各表型,排除三种表型间的相互影响,确定易感基因与表型之间的独立联系,明确这些PCOS易感基因对哪个特征性表型贡献更大。
目的：
本研究旨在比较单一表型人群和严格对照间PCOS-SNP等位基因频率的差异,确定这些位点与特征性表型发生风险之间的相关性,以期为下一步易感基因功能学研究提供理论基础。
方法：
本研究共纳入三组单一表型人群,其中OA组746例,HA组278例,PCO组536例。对照组为1790例无任何阳性表型的健康女性。提取所有受试者全血DNA,通过直接测序的方法检测15个PCOS-SNPs的基因型,并分析其与PCOS表型间的相关性。
结果：
1.LHCGR(rs13405728)、C9orf3(rs4385527)和(?)NR(s2059807)基因与OA存在显著相关性,结果在较正年龄和BMI后仍有统计学意义(Padjust分别为9.47E-03、4.40E-09、3.18E-02)。
2.C9rf3rs4385527)与HA显著相关,结果独立于年龄和BMI (Padjust=1.77E-04)。
3.THADA(rs13429458、rs12478601)、DEAAD1A(rs10818854)以及C9orf3(rs4385527)与PCO显著相关,结果在较正年龄和BMI后同样有统计学意义(Padjust分别为1.47E-03、6.38E-04、1.73E-02和2.20E-07)。
结论：
部分PCOS易感基因与其特征性临床表型有直接相关性。其中LHCGR和INSR与OA表型相关,THADA和DENND1A与PCO表型相关,而C9orf3则与三种表型均相关,它们可能在疾病的发病机制中发挥关键作用。
Part Ⅰ
Subtype Analysis in Polycystic Ovary Syndrome
Background:
Polycystic ovary syndrome (PCOS) is one of the most common endocrinal metabolic syndrome affecting6-8%women of reproductive age. It is not a single pathophysiological disorder, but a group of manifestations, which presents extremely heterogeneous consequently. The main symptoms include irregular menstrual cycle, hirsutism, obesity, hyper-luteinizing hormone, hyperinsulinemia, insulin resistance, dyslipidemia, etc., of which oligo-/anovulation (OA), hyperandrogenism (HA), and polycystic ovary (PCO) are considered as the diagnostic phenotypes. According to the consensus of European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine (ESHRE/ASRM) in Rotterdam conference in2003, the diagnosis will be established when two of the above phenotypes existed after excluding for other known disease causing ovulatory dysfunction and hyperandrogenism. Based on Rotterdam consensus, there are four subtypes of the syndrome:a) subtype A presents both OA and HA, but with normal ovary morphology (classical type Ⅰ); b) subtype B presents all of the three phenotypes (classical type Ⅱ); c) subtype C presents both HA and PCO, the ovulatory cycle of whom is regular; d) subtype D presents OA and PCO, with neither biological nor clinical sign of androgen excess. It has been evidenced that there are differences among subtypes in endocrinal and metabolic characteristics. Metabolic disturbances are more severe in PCOS patients without PCO, and relatively milder in patients with normal androgen. The prevalence of the complications, such as obesity, diabetes and cardiovascular disease are demonstrated to be increased in classical types (subtype A and subtype B). Highly heterogeneity brings a huge obstacle in etiological and clinical researches of the syndrome.
Identifying the clinical characteristics of every subtype exactly and evaluating the reproductive and metabolic changes mean a lot to explore the mechanism of the syndrome and choose more suitable diagnostic criteria and reasonable therapy in clinical work.
Objective:
The aim of the present study is to analyze the clinical data of large-scale patients, identify the clinical characteristics of every subtype, and provide theoretical basis to further fundamental researches and clinical trials.
Methods:
A total of5287PCOS patients diagnosed by Rotterdam criteria and2016normal control subjects were enrolled. The patients were divided into four groups according their subtypes:subtype A (OA+HA), subtype B (OA+HA+PCO), subtype C (HA+PCO), and subtype D (OA+PCO). The parameters of anthropometry, endocrine, and metabolism were compared among four groups of patients and normal controls.
Results:
a) Basic characteristics:BMI was similar among four groups of patients, which was significantly higher than controls (P<0.01). The m-FG score of subtype D is lower than other subtypes with hyperandrogenism (subtype A, B, C. P<0.01). No significance was found among the latter three groups.
b) Endocrinal characteristics:LH and LH/FSH were elevated in every subtype of patients compared with controls (P<0.01). In the cohort of patients, subjects presented all of the three phenotypes (subtype B) have the highest LH level and LH/FSH (P<0.01), no significance were found among the other three subtypes. Testosterone was gradient descent from hyperandrogenic subtypes (subtype A, B, and C) to subtype D and then to controls (P<0.01). No significance was found in PRL (P=0.17).
c) Glucose metabolism:INSO in subtype B and D were higher than controls (P<0.01). The highest HOMA-IR was found in subtype D, and then in subtype B. Subtypes A and D as well as controls had the lowest HOMA-IR (all P<0.01). All above results were independent of BMI.
d) Lipid metabolism:Levels of CHOL among each group were similar. Compared with controls, TG in all patients groups were elevated and HDL-C were decreased (all P<0.01). In the cohort of patients, level of LDL-C was highest in subtype D (OA+PCO), and lowest in subtype C (HA+PCO). Suptype B (OA+HA+PCO) was in the middle (all P<0.01). Nevertheless it should be noticed that the level of LDL-C of subtype D (OA+PCO) was close to controls, which was also higher than subtype C (HA+PCO)(P<0.01). All results remained significance adjusting for BMI except for TG (P=0.721).
Conclusion:
The reproductive and metabolic manifestations were different among the four subtypes of PCOS. Endocrinal disturbance and hyperandorgenism were identified in all subtypes, among which subtype B (OA+HA+PCO) was the most severe, while subtype D (OA+PCO) was the mildest. However, the prevalence of the latter is highest in Chinese patients. And they also manifest worse in metabolism. There might be different mechanism underlying each subtype.
Part Ⅱ
Geno-phenotype correlation analysis of PCOS susceptibility genes in PCOS patients
Background:
Polycystic ovary syndrome (PCOS) is a heterogeneous endocrinopathy characterized by oligo-anovulation (OA), hyperandrogenism (HA) and polycystic ovary (PCO). Both genetic and evironmental factors are well accepted as the main causes of the syndrome. The vast molecular genetic researches have identified many PCOS related genes. However, as a complex hereditary disease, polygenetic pathopoiesis and the interaction between gene and environment make the single gene association study less effective in screening the candidate genes of PCOS. Genome-wide association studies (GWAS) based on gene chip technology, brings an efficient way for detection and comparison of all or most of the individual genes, and therefore opens a door to study complex diseases. It determines all the variant allele frequencies in genome-wide by comparing the SNPs between cases and controls. Compared with candidate gene strategy which needs to presuppose disease-causing genes beforehand, GWAS can identify the genetic susceptibility of the disease in more systematic and comprehensive way. The previous GWAS on PCOS has studied tens of thousands of patients and controls, and found15susceptibility SNPs (PCOS-SNPs) at11loci. In first phase of GWAS,5SNPs of LHCGR (rs13405728), THADA (rs13429458and rsl2478601), and DENND1A (rs2479106and rs10818854) genes at2p16.3,2p21, and9q33.3respectively were proved to be associated to PCOS. Then the second one confirmed the three loci above and identified eight new ones in addition. Allele frequencies were found significantly different between patients and controls at rs4385527and rs3802457in C9orf3gene, rs1894116in YAP1gene, rs705702in RAB5B gene, rs2272046in HMGA2gene, rs4784165in TOX3gene, rs2059807in INSR gene, rs6022786in SUMO1P1gene, as well as rs2268361and rs2349415in FSHR gene. In these candidate genes, LHCGR, FSHR and INSR, as acting in the important pathways of follicular genesis or insulin metabolism, have been evidenced in the previous single gene screening earlier. But other genes were not mentioned in PCOS related reports before. According to the previous basic researches, it can be speculated that diabetes susceptibility genes THADA and HMGA2, may play a role in insulin metabolism; YAP and HMGA2genes were associated with cell proliferation, and may consequently participate in the mechanism of ovarian morphology changes; LHCGR and RAB5B genes were found to participate in some of the cellular activities such as endocytosis and calcium transportation, and may thus affect the function of some effect cells. But there is still little data about the association of these candidate genes to the phenotypes of PCOS, which is important for the exploration of gene function and the etiology of the syndrome. Therefore, more studies are expected on the geno-phenotype correlation.
Obejective:
The aim of this study was to identify additional correlations between the phenotypes and genotypes of the15PCOS-SNPs described in the previous studies.
Methods:
Clinical data of patients enrolled in the previous GWAS were collected. Appropriate genetic models were adapted according to the genotypes frequencies and served as group criteria. Endocrinal and metabolic characteristics were compared between different subgroups.
Results:
a) Rs13429458in THADA was associated with increased LH (P<0.01) and testosterone (T)(P=0.02) levels in subjects with PCOS. The LH/FSH ratio was also higher in the AA group (P<0.01). After adjusting for age and BMI, there remained significance (Padjust<0.01, Padjust=0.02, and Padjust<0.01, respectively)
b) Rs12478601in THADA was associated with increased levels of LDL (P=0.02), which was independent of age and BMI (Padjust=0.02).
c) In the dominant model, rs2479106in DENND1A was associated with elevated serum insulin levels2hours after a glucose load in the patients with PCOS (P=0.02). The comparisons was adjusted for age and BMI(Padjust=0.02).
d) In dominant model of rs2349415(FSHR), patients carrying T allele had lower serum T level (P<0.01). After adjusting for age and BMI, there remained significance (Padjust<0.01).
e) As for rs4385527in C9orf3gene, patients carrying A allele had higher BMI and serum LDL-C, as well as lower HDL-C (P<0.01in all variables). Differences in lipid parameters were all independent of BMI(Padjust<0.01in all variables).
f) Similarly, in rs2272046of HMGA2gene, LDL-C level was also elevated in subjects carrying C allele(P<0.01), which was independent of BMI(Padjust<0.01).
g) Rs4784165of TOX3gene was associated with hirsutism. PCOS women with GG genotype had lowest m-FG score of three subgroups (P<0.01).
h) Serum insulin level2hours after OGTT was found increased in AA subgroup of rs2059807(INSR). There remained statistically significance after adjusting for age and BMI (Padjust=0.042).
i) Independent association with hyperandrogenemia was also found in rs6022786(SUMO1P1). Subjects carrying G allele have higher T (P<0.01).
Conclusion:
The PCOS susceptibility genes THADA, FSHR, TOX3, and SUMO1P1are associated with hyperandorgenism, and THADA as well as DENND1A, C9orf3, HMGA2, and INSR carry risk alleles that are associated with metabolic disturbances in PCOS patients of Han Chinese. These genes might play an important role in the pathophysiological mechanism of the syndrome.
Part Ⅲ
Geno-phenotype correlation analysis of PCOS susceptibility genes in single phenotype subjects
Background:
Polycystic ovary syndrome (PCOS) is a complex genetic disorder with significant ethnic diversity and clinical heterogeneous. Two previous GWAS have found15PCOS susceptibility SNPs (PCOS-SNPs) in11locus.2p16.3(rs13405728),2p21(rsl3429458, rs12478601), and9q33.3(rs10818854, rs2479106) were identified in GWAS I, and confirmed in European and American Caucasians. LHCGR, THADA, and DENND1A genes were the suggested candidate genes. But as PCOS is such a complex syndrome affecting so many systems, the casual genes should not be only that three. Therefore GWAS Ⅱ were performed next and provided eight new locus:9q22.32(rs4385527, rs3802457),11q22.1(rs1894116),12q13.2(rs705702),12q14.3(rs2272046),16q12.1(rs4784165),19p13.3(rs2059807),20q13.2(rs6022786), and2p16.3(rs2268361, rs2349415). FSHR, C9orf3, YAP1, RAB5B, HMGA2, TOX3, INSR, and SUMO1P1were the suggested candidate genes.
As mentioned above, the privous studies have evidenced the familial aggregation of PCOS phenotypes. The prevalence of insulin resistance, impaired glucose tolerance, and diabetes were significantly elevated in the first-degree relatives of the patients. The levels of andorgen in mothers and sisters of PCOS women were also higher than normal controls, while the LH response was lower. It is reported that compared to age-matched normal women, the daughters of the patients had higher serum insulin, LH, T, and17-OHP levels and larger ovary volume before puberty and would exist thoughout the whole puberty. Thus, it can be seen that genetic factor might play an important role in the etiology of PCOS phenotypes. However, few evidences were found about the direct correction between susceptibility genes and phenotype genesis. It is also unclear by which gene the specific phenotype was regulated. Therefore, in the present study, three cohorts of women presented only one of the key features, including aligo-/anovulation (OA), hyperandrogenism (HA), and polycystic ovary (PCO), were enrolled as cases and compared with strict controls without any of the above phenotypes in order to determine the genetic contributions of these PCOS susceptibility genes to its phenotypes.
Methods:
Three independent cohorts of women presenting single characteristic phenotype were recruited as cases (746subjects with OA,278subjects with HA, and536subjects with PCO). And a total of1790healthy women without any above pathological phenotypes were enrolled as controls.
Results:
a) After adjusted for age and body mass index (BMI), variants in LHCGR (rs13405728), C9orf3(rs4385527), and INSR (rs2059807) were strongly associated with OA (Padjust=9.47E-03,4.40E-09, and3.18E-02).
b) Positive association were also observed between C9orf3(rs4385527) variation and HA which is independent of age and BMI (Padjust=1.77E-04).
c) In addition, there was significant evidence for association of variants in THADA (rs13429458and rs12478601), DENND1A (rs10818854), and C9orf3(rs4385527) with PCO. The difference remained significant after adjusting for age and BMI.(Padjust=1.47E-03,6.38E-04,1.73E-02and2.20E-07).
Conclusion:
The SNPs in some candidate genes of PCOS () are directly associated to the main phenotypes (LHCGR and INSR with OA; THADA and DENND1A with PCO; C9orf3with all three phenotypes) and therefore likely to be important in their etiology. The results provide indications for further function.
引文
1. Diamanti-Kandarakis, E., et al., A survey of the polycystic ovary syndrome in the Greek island of Lesbos:hormonal and metabolic profile. J Clin Endocrinol Metab,1999.84(11):p.4006-11.
2. Knochenhauer, E.S., et al., Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States:a prospective study. J Clin Endocrinol Metab,1998.83(9):p.3078-82.
3. Azziz, R., et al., The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab,2004.89(6):p.2745-9.
4. March, W.A., et al., The prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria. Hum Reprod,2010.25(2):p.544-51.
5. 赵君利,et al.,汉族育龄多囊卵巢综合征患者的临床特征及分析，中华妇产科杂志,2006.41(6)：p.5.
6. Ma, Y.M., et al., Characteristics of abnormal menstrual cycle and polycystic ovary syndrome in community and hospital populations. Chin Med J (Engl), 2010.123(16):p.2185-9.
7. Azziz, R., et al., Health care-related economic burden of the polycystic ovary syndrome during the reproductive life span. J Clin Endocrinol Metab,2005. 90(8):p.4650-8.
8. Azziz, R., et al., Androgen excess in women:experience with over 1000 consecutive patients. J Clin Endocrinol Metab,2004.89(2):p.453-62.
9. Carmina, E., et al., Extensive clinical experience:relative prevalence of different androgen excess disorders in 950 women referred because of clinical hyperandrogenism. J Clin Endocrinol Metab,2006.91(1):p.2-6.
10. Chang, W.Y., et al., Phenotypic spectrum of polycystic ovary syndrome: clinical and biochemical characterization of the three major clinical subgroups. Fertil Steril,2005.83(6):p.1717-23.
11. Diamanti-Kandarakis, E. and D. Panidis, Unravelling the phenotypic map of polycystic ovary syndrome (PCOS):a prospective study of 634 women with PCOS. Clin Endocrinol (Oxf),2007.67(5):p.735-42.
12. Elting, M.W., et al., Women with polycystic ovary syndrome gain regular menstrual cycles when ageing. Hum Reprod,2000.15(1):p.24-8.
13. Brown, Z.A., et al., The phenotype of polycystic ovary syndrome ameliorates with aging. Fertil Steril,2011.96(5):p.1259-65.
14. Azziz, R., et al., The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome:the complete task force report. Fertil Steril,2009. 91(2):p.456-88.
15. Ferriman, D. and J.D. Gallwey, Clinical assessment of body hair growth in women. J Clin Endocrinol Metab,1961.21:p.1440-7.
16. DeUgarte, C.M., et al., Degree of facial and body terminal hair growth in unselected black and white women:toward a populational definition of hirsutism. J Clin Endocrinol Metab,2006.91(4):p.1345-50.
17. Wijeyaratne, C.N., et al., Clinical manifestations and insulin resistance (IR) in polycystic ovary syndrome (PCOS) among South Asians and Caucasians:is there a difference? Clin Endocrinol (Oxf),2002.57(3):p.343-50.
18. Jonard, S., et al., Ultrasound examination of polycystic ovaries:is it worth counting the follicles? Hum Reprod,2003.18(3):p.598-603.
19. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril,2004.81(1):p.19-25.
20. Carmina, E., et al., Ovarian size and blood flow in women with polycystic ovary syndrome and their correlations with endocrine parameters. Fertil Steril, 2005.84(2):p.413-9.
21. Keettel, W.C., J.T. Bradbury, and F.J. Stoddard, Observations on the polycystic ovary syndrome. Am J Obstet Gynecol,1957.73(5):p.954-62; discussion, 962-5.
22. Shterev, A.D. and S.I. Dokumov, Serum LH and FSH levels in women with polycystic ovary syndrome before and after ovarian wedge resection. Endocrinologie,1982.20(3):p.203-7.
23. Taylor, A.E., et al., Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. J Clin Endocrinol Metab,1997.82(7):p.2248-56.
24. Morales, A.J., et al., Insulin, somatotropic, and luteinizing hormone axes in lean and obese women with polycystic ovary syndrome:common and distinct features. J Clin Endocrinol Metab,1996.81(8):p.2854-64.
25. Arroyo, A., et al., Inappropriate gonadotropin secretion in polycystic ovary syndrome:influence of adiposity. J Clin Endocrinol Metab,1997.82(11):p. 3728-33.
26. Pagan, Y.L., et al., Inverse relationship between luteinizing hormone and body mass index in polycystic ovarian syndrome:investigation of hypothalamic and pituitary contributions. J Clin Endocrinol Metab,2006.91(4):p.1309-16.
27. Srouji, S.S., et al., Pharmacokinetic factors contribute to the inverse relationship between luteinizing hormone and body mass index in polycystic ovarian syndrome. J Clin Endocrinol Metab,2007.92(4):p.1347-52.
28. Balen, A.H., et al., Polycystic ovary syndrome:the spectrum of the disorder in 1741 patients. Hum Reprod,1995.10(8):p.2107-11.
29. Hahn, S., et al., Clinical and biochemical characterization of women with polycystic ovary syndrome in North Rhine-Westphalia. Horm Metab Res,2005. 37(7):p.438-44.
30. Carmina, E., et al., Phenotypic variation in hyperandrogenic women influences the findings of abnormal metabolic and cardiovascular risk parameters. J Clin Endocrinol Metab,2005.90(5):p.2545-9.
31. Azziz, R., et al., Troglitazone improves ovulation and hirsutism in the polycystic ovary syndrome:a multicenter, double blind, placebo-controlled trial. J Clin Endocrinol Metab,2001.86(4):p.1626-32.
32. Legro, R.S., et al., Insulin resistance in the sisters of women with polycystic ovary syndrome:association with hyperandrogenemia rather than menstrual irregularity. J Clin Endocrinol Metab,2002.87(5):p.2128-33.
33. Dunaif, A., et al., Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes,1989.38(9):p.1165-74.
34. Legro, R.S., V.D. Castracane, and R.P. Kauffman, Detecting insulin resistance in polycystic ovary syndrome:purposes and pitfalls. Obstet Gynecol Surv, 2004.59(2):p.141-54.
35. Apridonidze, T., et al., Prevalence and characteristics of the metabolic syndrome in women with polycystic ovary syndrome. J Clin Endocrinol Metab, 2005.90(4):p.1929-35.
36. Zawadski JK and D. A, Diagnostic criteria for polycystic ovary syndrome: towards a rational approach, in Polycystic Ovary Syndrome, G.J. Dunaif A, Haseltine F, Editor.1992, Blackwell Scientic(?):Boston. p.377-384.
37. Lenarcik, A., et al., Hormonal abnormalities in first-degree relatives of women with polycystic ovary syndrome (PCOS). Endokrynol Pol,2011.62(2):p. 129-33.
38. Sir-Petermann, T., et al., Metabolic and reproductive features before and during puberty in daughters of women with polycystic ovary syndrome. J Clin Endocrinol Metab,2009.94(6):p.1923-30.
39. Unluhizarci, K., et al., Investigation of hypothalamo-pituitary-gonadal axis and glucose intolerance among the first-degree female relatives of women with polycystic ovary syndrome. Fertil Steril,2007.87(6):p.1377-82.
40. Yildiz, B.O., et al., Glucose intolerance, insulin resistance, and hyperandrogenemia in first degree relatives of women with polycystic ovary syndrome. J Clin Endocrinol Metab,2003.88(5):p.2031-6.
41. Jakimiuk, A.J., et al., Luteinizing hormone receptor, steroidogenesis acute regulatory protein, and steroidogenic enzyme messenger ribonucleic acids are overexpressed in thecal and granulosa cells from polycystic ovaries. J Clin Endocrinol Metab,2001.86(3):p.1318-23.
42. Wang, Y., et al., A microsatellite polymorphism (tttta)n in the promoter of the CYP11a gene in Chinese women with polycystic ovary syndrome. Fertil Steril, 2006.86(1):p.223-6.
43. Hanzu, F.A., et al., Association of insulin receptor genetic variants with polycystic ovary syndrome in a population of women from Central Europe. Fertil Steril,2010.94(6):p.2389-92.
44. Yoshihara, K., et al., Association of single nucleotide polymorphisms in adiponectin and its receptor genes with polycystic ovary syndrome. J Reprod Med,2009.54(11-12):p.669-74.
45. Mitjans, M. and B. Arias, The genetics of depression:what information can new methodologic approaches provide? Actas Esp Psiquiatr,2012.40(2):p. 70-83.
46. Sladek, R., et al., A genome-wide association study identifies novel risk loci for type 2 diabetes. Nature,2007.445(7130):p.881-5.
47. Weedon, M.N., et al., Genome-wide association analysis identifies 20 loci that influence adult height. Nat Genet,2008.40(5):p.575-83.
48. Tin, A., et al., Genome-wide association study for serum urate concentrations and gout among African Americans identifies genomic risk loci and a novel URAT1 loss-of-function allele. Hum Mol Genet,2011.20(20):p.4056-68.
49. Chen, Z.J., et al., Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3,2p21 and 9q33.3. Nat Genet,2011.43(1):p.55-9.
50. Zeggini, E., et al., Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes. Nat Genet,2008.40(5):p.638-45.
51. Goodarzi, M.O., et al., Replication of association of DENND1A and THADA variants with polycystic ovary syndrome in European cohorts. J Med Genet, 2012.49(2):p.90-5.
52. Welt, C.K., et al., Variants in DENND1A are associated with polycystic ovary syndrome in women of European ancestry. J Clin Endocrinol Metab,2012. 97(7):p.E1342-7.
53. Mutharasan, P., et al., Evidence for chromosome 2p16.3 polycystic ovary syndrome susceptibility locus in affected women of European ancestry. J Clin Endocrinol Metab,2013.98(1):p. E185-90.
54. Shi, Y., et al., Clinical and metabolic characteristics of polycystic ovary syndrome without polycystic ovary:a pilot study on Chinese women. Fertil Steril,2008.90(4):p.1139-43.
55. Zhang, H.Y., et al., Characteristics of different phenotypes of polycystic ovary syndrome based on the Rotterdam criteria in a large-scale Chinese population. BJOG,2009.116(12):p.1633-9.
56. Moran, L. and H. Teede, Metabolic features of the reproductive phenotypes of polycystic ovary syndrome. Hum Reprod Update,2009.15(4):p.477-88.
57. Iwasa, T., et al., Diagnostic performance of serum total testosterone for Japanese patients with polycystic ovary syndrome. Endocr J,2007.54(2):p. 233-8.
58. Norman, R.J., et al., Metabolic approaches to the subclassification of polycystic ovary syndrome. Fertil Steril,1995.63(2):p.329-35.
59. Talbott, E., et al., Coronary heart disease risk factors in women with polycystic ovary syndrome. Arterioscler Thromb Vasc Biol,1995.15(7):p. 821-6.
60. Alborzi, S., R. Khodaee, and M.E. Parsanejad, Ovarian size and response to laparoscopic ovarian electro-cauterization in polycystic ovarian disease. Int J Gynaecol Obstet,2001.74(3):p.269-74.
61. Williamson, K., et al., The impact of ethnicity on the presentation of polycystic ovarian syndrome. Aust N Z J Obstet Gynaecol,2001.41(2):p.202-6.
62. Hsu, M.I., et al., Diagnostic criteria for polycystic ovary syndrome in Taiwanese Chinese women:comparison between Rotterdam 2003 and NIH 1990. Fertil Steril,2007.88(3):p.727-9.
63. Lujan, M.E., D.R. Chizen, and R.A. Pierson, Diagnostic criteria for polycystic ovary syndrome:pitfalls and controversies. J Obstet Gynaecol Can,2008. 30(8):p.671-9.
64. Blank, S.K., et al., Neuroendocrine effects of androgens in adult polycystic ovary syndrome and female puberty. Semin Reprod Med,2007.25(5):p. 352-9.
65. Catteau-Jonard, S., et al., Anti-Mullerian hormone, its receptor, FSH receptor, and androgen receptor genes are overexpressed by granulosa cells from stimulated follicles in women with polycystic ovary syndrome. J Clin Endocrinol Metab,2008.93(11):p.4456-61.
66. Marshall, J.C. and C.A. Eagleson, Neuroendocrine aspects of polycystic ovary syndrome. Endocrinol Metab Clin North Am,1999.28(2):p.295-324.
67. Xu, X., et al., Endocrine and metabolic characteristics of polycystic ovary syndrome in Chinese women with different phenotypes. Clin Endocrinol (Oxf), 2012.76(3):p.425-30.
68. Legro, R.S., et al., Changes in glucose tolerance over time in women with polycystic ovary syndrome:a controlled study. J Clin Endocrinol Metab,2005. 90(6):p.3236-42.
69. de Groot, P.C., et al., PCOS, coronary heart disease, stroke and the influence of obesity:a systematic review and meta-analysis. Hum Reprod Update,2011. 17(4):p.495-500.
70. Cooper, H.E., et al., Hereditary factors in the Stein-Leventhal syndrome. Am J Obstet Gynecol,1968.100(3):p.371-87.
71. Cohen, P.N., et al., Polycystic ovarian disease, maturation arrest of spermiogenesis, and Klinefelter's syndrome in siblings of a family with familial hirsutism. Fertil Steril,1975.26(12):p.1228-38.
72. Ferriman, D. and A.W. Purdie, The aetiology of oligomenorrhoea and/or hirsuties:a study of 467 patients. Postgrad Med J,1983.59(687):p.17-20.
73. Carey, A.H., et al., Evidence for a single gene effect causing polycystic ovaries and male pattern baldness. Clin Endocrinol (Oxf),1993.38(6):p.653-8.
74. Hague, W.M., et al., Familial polycystic ovaries:a genetic disease? Clin Endocrinol (Oxf),1988.29(6):p.593-605.
75. Battaglia, C., et al., Polycystic ovaries in childhood:a common finding in daughters of PCOS patients. A pilot study. Hum Reprod,2002.17(3):p.771-6.
76. Hirschhorn, J.N. and M.J. Daly, Genome-wide association studies for common diseases and complex traits. Nat Rev Genet,2005.6(2):p.95-108.
77. Lerchbaum, E., et al., Susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3,2p21, and 9q33.3 in a cohort of Caucasian women. Horm Metab Res,2011.43(11):p.743-7.
78. Eriksen, M.B., et al., Association of polycystic ovary syndrome susceptibility single nucleotide polymorphism rs2479106 and PCOS in Caucasian patients with PCOS or hirsutism as referral diagnosis. Eur J Obstet Gynecol Reprod Biol,2012.163(1):p.39-42.
79. Atger, M., et al., Structure of the human luteinizing hormone-choriogonadotropin receptor gene:unusual promoter and 5' non-coding regions. Mol Cell Endocrinol,1995.111(2):p.113-23.
80. Rahman, N.A. and C.V. Rao, Recent progress in luteinizing hormone/human chorionic gonadotrophin hormone research. Mol Hum Reprod,2009.15(11): p.703-11.
81. Chambers, A.E. and S. Banerjee, Natural antisense LHCGR could make sense of hypogonadism, male-limited precocious puberty and pre-eclampsia. Mol Cell Endocrinol,2005.241(1-2):p.1-9.
82. Qiao, J., et al., A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism. Hum Mutat,2009.30(9):p. E855-65.
83. Banerjee, S., et al., A link between high serum levels of human chorionic gonadotrophin and chorionic expression of its mature functional receptor (LHCGR) in Down's syndrome pregnancies. Reprod Biol Endocrinol,2005.3: p.25.
84. Jeha, G.S., et al., Variable presentation of precocious puberty associated with the D564G mutation of the LHCGR gene in children with testotoxicosis. J Pediatr,2006.149(2):p.271-4.
85. Costa, M.H., et al., Expression profiles of the glucose-dependent insulinotropic peptide receptor and LHCGR in sporadic adrenocortical tumors. J Endocrinol, 2009.200(2):p.167-75.
86. Yariz, K.O., et al., Inherited mutation of the luteinizing hormone/choriogonadotropin receptor (LHCGR) in empty follicle syndrome. Fertil Steril,2011.96(2):p. e125-30.
87. Wang, Z., et al., Variants in DENND1A and LHCGR are associated with endometrioid adenocarcinoma. Gynecol Oncol,2012.127(2):p.403-5.
88. Capalbo, A., et al., The 312N variant of the luteinizing hormone/choriogonadotropin receptor gene (LHCGR) confers up to 2.7-fold increased risk of polycystic ovary syndrome in a Sardinian population. Clin Endocrinol (Oxf),2012.77(1):p.113-9.
89. Rippe, V., et al., Identification of a gene rearranged by 2p21 aberrations in thyroid adenomas. Oncogene,2003.22(38):p.6111-4.
90. Marat, A.L. and P.S. McPherson, The connecdenn family, Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery. J Biol Chem,2010.285(14):p.10627-37.
91. Hatch, R., et al., Hirsutism:implications, etiology, and management. Am J Obstet Gynecol,1981.140(7):p.815-30.
92. Shi, Y., et al., Analysis of clinical characteristics in large-scale Chinese women with polycystic ovary syndrome. Neuro Endocrinol Lett,2007.28(6):p. 807-10.
93. Matthews, D.R., et al., Homeostasis model assessment:insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia,1985.28(7):p.412-9.
94. Simonis-Bik, A.M., et al., Gene variants in the novel type 2 diabetes loci CDC123/CAMK1D, THADA, ADAMTS9, BCL11A, and MTNR1B affect different aspects of pancreatic beta-cell function. Diabetes,2010.59(1):p. 293-301.
95. Apter, D., et al., Accelerated 24-hour luteinizing hormone pulsatile activity in adolescent girls with ovarian hyper androgenism:relevance to the developmental phase of polycystic ovarian syndrome. J Clin Endocrinol Metab, 1994.79(1):p.119-25.
96. van Hooff, M.H., et al., Variation of luteinizing hormone and androgens in oligomenorrhoea and its implications for the study of polycystic ovary syndrome. Hum Reprod,1999.14(7):p.1684-9.
97. Gilling-Smith, C., et al., Hyper secretion of androstenedione by isolated thecal cells from polycystic ovaries. J Clin Endocrinol Metab,1994.79(4):p. 1158-65.
98. Nelson, V.L., et al., Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries. Mol Endocrinol, 1999.13(6):p.946-57.
99. Dumesic, D.A. and D.H. Abbott, Implications of polycystic ovary syndrome on oocyte development. Semin Reprod Med,2008.26(1):p.53-61.
100. Gleicher, N., A. Weghofer, and D.H. Barad, The role of androgens in follicle maturation and ovulation induction:friend or foe of infertility treatment? Reprod Biol Endocrinol,2011.9:p.116.
101. Ip, S., et al., Systematic review:association of low-density lipoprotein subfractions with cardiovascular outcomes. Ann Intern Med,2009.150(7):p. 474-84.
102. Moran, L.J., et al., Impaired glucose tolerance, type 2 diabetes and metabolic syndrome in polycystic ovary syndrome:a systematic review and meta-analysis. Hum Reprod Update,2010.16(4):p.347-63.
103. Saad, M.F., et al., A two-step model for development of non-insulin-dependent diabetes. Am J Med,1991.90(2):p.229-35.
104. Dale, P.O., et al., The impact of insulin resistance on the outcome of ovulation induction with low-dose follicle stimulating hormone in women with polycystic ovary syndrome. Hum Reprod,1998.13(3):p.567-70.
105. Mather, K.J., F. Kwan, and B. Corenblum, Hyperinsulinemia in polycystic ovary syndrome correlates with increased cardiovascular risk independent of obesity. Fertil Steril,2000.73(1):p.150-6.
106. Moran, M.D., Arguments for rejecting the sequential Bonferroni in ecological studies. Oikos,2003.100:p.3.
107. Shi, Y., et al., Genome-wide association study identifies eight new risk loci for polycystic ovary syndrome. Nat Genet,2012.44(9):p.1020-5.
108. Heckert, L.L., M.A. Daggett, and J. Chen, Multiple promoter elements contribute to activity of the follicle-stimulating hormone receptor (FSHR) gene in testicular Sertoli cells. Mol Endocrinol,1998.12(10):p.1499-512.
109. Livshyts, G., et al., A distribution of two SNPs in exon 10 of the FSHR gene among the women with a diminished ovarian reserve in Ukraine. J Assist Reprod Genet,2009.26(1):p.29-34.
110. Lussiana, C., et al., Mutations and polymorphisms of the FSH receptor (FSHR) gene:clinical implications in female fecundity and molecular biology of FSHR protein and gene. Obstet Gynecol Surv,2008.63(12):p.785-95.
111. Ben-Josef, E., et al., Hormone-refractory prostate cancer cells express functional follicle-stimulating hormone receptor (FSHR). J Urol,1999.161(3): p.970-6.
112. Luo, W. and M.C. Wiltbank, Distinct regulation by steroids of messenger RNAs for FSHR and CYP19A1 in bovine granulosa cells. Biol Reprod,2006. 75(2):p.217-25.
113. Kim, S., et al., Epistasis between FSHR and CYP19A1 polymorphisms is associated with premature ovarian failure. Fertil Steril,2011.95(8):p.2585-8.
114. Parikh, H., et al., Molecular correlates for maximal oxygen uptake and type 1 fibers. Am J Physiol Endocrinol Metab,2008.294(6):p. E1152-9.
115. Ruderman, N., et al., The metabolically obese, normal-weight individual revisited. Diabetes,1998.47(5):p.699-713.
116. Van Dorpe, J., et al., Translocation of the HMGI-C (HMGA2) gene in a benign mesenchymoma (chondrolipoangioma). Virchows Arch,2002.440(5): p.485-90.
117. Chau, K.Y., et al., Derepression of HMGA2 gene expression in retinoblastoma is associated with cell proliferation. Mol Med,2003.9(5-8):p.154-65.
118. Ferguson, M., P.A. Henry, and R.A. Currie, Histone deacetylase inhibition is associated with transcriptional repression of the Hmga2 gene. Nucleic Acids Res,2003.31(12):p.3123-33.
119. Chieffi, P., et al., HMGA1 and HMGA2 protein expression in mouse spermatogenesis. Oncogene,2002.21(22):p.3644-50.
120. Fedele, M., et al., Overexpression of the HMGA2 gene in transgenic mice leads to the onset of pituitary adenomas. Oncogene,2002.21(20):p.3190-8.
121. Voight, B.F., et al., Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis. Nat Genet,2010.42(7):p.579-89.
122. Ligon, A.H., et al., Constitutional rearrangement of the architectural factor HMGA2:a novel human phenotype including overgrowth and lipomas. Am J Hum Genet,2005.76(2):p.340-8.
123. Yuan, S.H., Z. Qiu, and A. Ghosh, TOX3 regulates calcium-dependent transcription in neurons. Proc Natl Acad Sci U S A,2009.106(8):p.2909-14.
124. Dittmer, S., et al., TOX3 is a neuronal survival factor that induces transcription depending on the presence of CITED1 or phosphorylated CREB in the transcriptionally active complex. J Cell Sci,2011.124(Pt 2):p.252-60.
125. Fernandez-Navarro, P., et al., Association analysis between breast cancer genetic variants and mammographic density in a large population-based study (Determinants of Density in Mammographies in Spain) identifies susceptibility loci in TOX3 gene. Eur J Cancer,2013.49(2):p.474-81.
126. Gudmundsdottir, E.T., et al., The risk allele of SNP rs3803662 and the mRNA level of its closest genes TOX3 and LOC643714 predict adverse outcome for breast cancer patients. BMC Cancer,2012.12:p.621.
127. Ruiz-Narvaez, E.A., et al., Polymorphisms in the TOX3/LOC643714 locus and risk of breast cancer in African-American women. Cancer Epidemiol Biomarkers Prev,2010.19(5):p.1320-7.
128. Aliahmad, P. and J. Kaye, Development of all CD4 T lineages requires nuclear factor TOX. J Exp Med,2008.205(1):p.245-56.
129. Talbot, J.A., et al., Molecular scanning of the insulin receptor gene in women with polycystic ovarian syndrome. J Clin Endocrinol Metab,1996.81(5):p. 1979-83.
130. Chen, Z.J., et al., [Correlation between single nucleotide polymorphism of insulin receptor gene with polycystic ovary syndrome]. Zhonghua Fu Chan Ke Za Zhi,2004.39(9):p.582-5.
131. 李曼,邱红玉,孙永玉,李红发,多囊卵巢综合征患者胰岛素受体基因酪氨酸蛋白激酶域突变的研究生殖与避孕,2003.23(5)：p.4.
132. LeBlanc, M., et al., Genome-wide study identifies PTPRO and WDR72 and FOXQ1-SUMO1P1 interaction associated with neurocognitive function. J Psychiatr Res,2012.46(2):p.271-8.
133. Haoula, Z., M. Salman, and W. Atiomo, Evaluating the association between endometrial cancer and polycystic ovary syndrome. Hum Reprod,2012.27(5): p.1327-31.
134. Franks, S., J. Stark, and K. Hardy, Follicle dynamics and anovulation in polycystic ovary syndrome. Hum Reprod Update,2008.14(4):p.367-78.
135. Vink, J.M., et al., Heritability of polycystic ovary syndrome in a Dutch twin-family study. J Clin Endocrinol Metab,2006.91(6):p.2100-4.
136. Kahsar-Miller, M.D., et al., Prevalence of polycystic ovary syndrome (PCOS) in first-degree relatives of patients with PCOS. Fertil Steril,2001.75(1):p. 53-8.
137. Homburg, R., Androgen circle of polycystic ovary syndrome. Hum Reprod, 2009.24(7):p.1548-55.
138. Pakarainen, T., et al., Knockout of luteinizing hormone receptor abolishes the effects of follicle-stimulating hormone on preovulatory maturation and ovulation of mouse graafian follicles. Mol Endocrinol,2005.19(10):p. 2591-602.
139. Latronico, A.C., et al., Brief report:testicular and ovarian resistance to luteinizing hormone caused by inactivating mutations of the luteinizing hormone-receptor gene. N Engl J Med,1996.334(8):p.507-12.
140. Latronico, A.C., et al., A homozygous microdeletion in helix 7 of the luteinizing hormone receptor associated with familial testicular and ovarian resistance is due to both decreased cell surface expression and impaired effector activation by the cell surface receptor. Mol Endocrinol,1998.12(3):p. 442-50.
141. Bentov, Y., S. Kenigsberg, and R.F. Casper, A novel luteinizing hormone/chorionic gonadotropin receptor mutation associated with amenorrhea, low oocyte yield, and recurrent pregnancy loss. Fertil Steril,2012. 97(5):p.1165-8.
142. Cui, L., et al., Genotype-phenotype correlations of PCOS susceptibility SNPs identified by GWAS in a large cohort of Han Chinese women. Hum Reprod, 2013.28(2):p.538-44.
143. Rodriguez, K.F., et al., Insufficient luteinizing hormone-induced intracellular signaling disrupts ovulation in preovulatory follicles lacking estrogen receptor-{beta}. Endocrinology,2010.151(6):p.2826-34.
144. Moller, D.E. and J.S. Flier, Detection of an alteration in the insulin-receptor gene in a patient with insulin resistance, acanthosis nigricans, and the polycystic ovary syndrome (type A insulin resistance). N Engl J Med,1988. 319(23):p.1526-9.
145. Hashiramoto, M., et al., A nonsense mutation in the Arg345 of the insulin receptor gene in a Japanese type A insulin-resistant patient. Endocr J,2005. 52(4):p.499-504.
146. Cocozza, S., et al., NIDDM associated with mutation in tyrosine kinase domain of insulin receptor gene. Diabetes,1992.41(4):p.521-6.
147. Ibanez, L., et al., Anovulation in eumenorrheic, nonobese adolescent girls born small for gestational age:insulin sensitization induces ovulation, increases lean body mass, and reduces abdominal fat excess, dyslipidemia, and subclinical hyperandrogenism. J Clin Endocrinol Metab,2002.87(12):p. 5702-5.
148. Ibanez, L., et al., Additive effects of insulin-sensitizing and anti-androgen treatment in young, nonobese women with hyperinsulinism, hyperandrogenism, dyslipidemia, and anovulation. J Clin Endocrinol Metab,2002.87(6):p. 2870-4.
149. Kerns, S.L., et al., Genome-wide association study to identify single nucleotide polymorphisms (SNPs) associated with the development of erectile dysfunction in African-American men after radiotherapy for prostate cancer. Int J Radiat Oncol Biol Phys.2010.78(5):p.1292-300.
150. Eriksson, K.F. and F. Lindgarde, Poor physical fitness, and impaired early insulin response but late hyperinsulinaemia, as predictors of NIDDM in middle-aged Swedish men. Diabetologia,1996.39(5):p.573-9.
151. Cara. J.F. and R.L. Rosenfield, Insulin-like growth factor Ⅰ and insulin potentiate luteinizing hormone-induced androgen synthesis by rat ovarian thecal-interstitial cells. Endocrinology,1988.123(2):p.733-9.
152. Del Villar, K. and C.A. Miller, Down-regulation of DENN/MADD, a TNF receptor binding protein, correlates with neuronal cell death in Alzheimer's disease brain and hippocampal neurons. Proc Nat) Acad Sci U S A,2004. 101(12):p.4210-5.
153. Olszanecka-Glinianowicz. M., et al., Is the polycystic ovary syndrome associated with chronic inflammation per se? Eur J Obstet Gynecol Reprod Biol,2007.133(2):p.197-202.
2. Knochenhauer, E.S., et al., Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States:a prospective study. J Clin Endocrinol Metab,1998.83(9):p.3078-82.
3. Azziz, R., et al., The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab,2004.89(6):p.2745-9.
4. March, W.A., et al., The prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria. Hum Reprod,2010.25(2):p.544-51.
5. 赵君利,et al.,汉族育龄多囊卵巢综合征患者的临床特征及分析，中华妇产科杂志,2006.41(6)：p.5.
6. Ma, Y.M., et al., Characteristics of abnormal menstrual cycle and polycystic ovary syndrome in community and hospital populations. Chin Med J (Engl), 2010.123(16):p.2185-9.
7. Azziz, R., et al., Health care-related economic burden of the polycystic ovary syndrome during the reproductive life span. J Clin Endocrinol Metab,2005. 90(8):p.4650-8.
8. Azziz, R., et al., Androgen excess in women:experience with over 1000 consecutive patients. J Clin Endocrinol Metab,2004.89(2):p.453-62.
9. Carmina, E., et al., Extensive clinical experience:relative prevalence of different androgen excess disorders in 950 women referred because of clinical hyperandrogenism. J Clin Endocrinol Metab,2006.91(1):p.2-6.
10. Chang, W.Y., et al., Phenotypic spectrum of polycystic ovary syndrome: clinical and biochemical characterization of the three major clinical subgroups. Fertil Steril,2005.83(6):p.1717-23.
11. Diamanti-Kandarakis, E. and D. Panidis, Unravelling the phenotypic map of polycystic ovary syndrome (PCOS):a prospective study of 634 women with PCOS. Clin Endocrinol (Oxf),2007.67(5):p.735-42.
12. Elting, M.W., et al., Women with polycystic ovary syndrome gain regular menstrual cycles when ageing. Hum Reprod,2000.15(1):p.24-8.
13. Brown, Z.A., et al., The phenotype of polycystic ovary syndrome ameliorates with aging. Fertil Steril,2011.96(5):p.1259-65.
14. Azziz, R., et al., The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome:the complete task force report. Fertil Steril,2009. 91(2):p.456-88.
15. Ferriman, D. and J.D. Gallwey, Clinical assessment of body hair growth in women. J Clin Endocrinol Metab,1961.21:p.1440-7.
16. DeUgarte, C.M., et al., Degree of facial and body terminal hair growth in unselected black and white women:toward a populational definition of hirsutism. J Clin Endocrinol Metab,2006.91(4):p.1345-50.
17. Wijeyaratne, C.N., et al., Clinical manifestations and insulin resistance (IR) in polycystic ovary syndrome (PCOS) among South Asians and Caucasians:is there a difference? Clin Endocrinol (Oxf),2002.57(3):p.343-50.
18. Jonard, S., et al., Ultrasound examination of polycystic ovaries:is it worth counting the follicles? Hum Reprod,2003.18(3):p.598-603.
19. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril,2004.81(1):p.19-25.
20. Carmina, E., et al., Ovarian size and blood flow in women with polycystic ovary syndrome and their correlations with endocrine parameters. Fertil Steril, 2005.84(2):p.413-9.
21. Keettel, W.C., J.T. Bradbury, and F.J. Stoddard, Observations on the polycystic ovary syndrome. Am J Obstet Gynecol,1957.73(5):p.954-62; discussion, 962-5.
22. Shterev, A.D. and S.I. Dokumov, Serum LH and FSH levels in women with polycystic ovary syndrome before and after ovarian wedge resection. Endocrinologie,1982.20(3):p.203-7.
23. Taylor, A.E., et al., Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. J Clin Endocrinol Metab,1997.82(7):p.2248-56.
24. Morales, A.J., et al., Insulin, somatotropic, and luteinizing hormone axes in lean and obese women with polycystic ovary syndrome:common and distinct features. J Clin Endocrinol Metab,1996.81(8):p.2854-64.
25. Arroyo, A., et al., Inappropriate gonadotropin secretion in polycystic ovary syndrome:influence of adiposity. J Clin Endocrinol Metab,1997.82(11):p. 3728-33.
26. Pagan, Y.L., et al., Inverse relationship between luteinizing hormone and body mass index in polycystic ovarian syndrome:investigation of hypothalamic and pituitary contributions. J Clin Endocrinol Metab,2006.91(4):p.1309-16.
27. Srouji, S.S., et al., Pharmacokinetic factors contribute to the inverse relationship between luteinizing hormone and body mass index in polycystic ovarian syndrome. J Clin Endocrinol Metab,2007.92(4):p.1347-52.
28. Balen, A.H., et al., Polycystic ovary syndrome:the spectrum of the disorder in 1741 patients. Hum Reprod,1995.10(8):p.2107-11.
29. Hahn, S., et al., Clinical and biochemical characterization of women with polycystic ovary syndrome in North Rhine-Westphalia. Horm Metab Res,2005. 37(7):p.438-44.
30. Carmina, E., et al., Phenotypic variation in hyperandrogenic women influences the findings of abnormal metabolic and cardiovascular risk parameters. J Clin Endocrinol Metab,2005.90(5):p.2545-9.
31. Azziz, R., et al., Troglitazone improves ovulation and hirsutism in the polycystic ovary syndrome:a multicenter, double blind, placebo-controlled trial. J Clin Endocrinol Metab,2001.86(4):p.1626-32.
32. Legro, R.S., et al., Insulin resistance in the sisters of women with polycystic ovary syndrome:association with hyperandrogenemia rather than menstrual irregularity. J Clin Endocrinol Metab,2002.87(5):p.2128-33.
33. Dunaif, A., et al., Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes,1989.38(9):p.1165-74.
34. Legro, R.S., V.D. Castracane, and R.P. Kauffman, Detecting insulin resistance in polycystic ovary syndrome:purposes and pitfalls. Obstet Gynecol Surv, 2004.59(2):p.141-54.
35. Apridonidze, T., et al., Prevalence and characteristics of the metabolic syndrome in women with polycystic ovary syndrome. J Clin Endocrinol Metab, 2005.90(4):p.1929-35.
36. Zawadski JK and D. A, Diagnostic criteria for polycystic ovary syndrome: towards a rational approach, in Polycystic Ovary Syndrome, G.J. Dunaif A, Haseltine F, Editor.1992, Blackwell Scientic(?):Boston. p.377-384.
37. Lenarcik, A., et al., Hormonal abnormalities in first-degree relatives of women with polycystic ovary syndrome (PCOS). Endokrynol Pol,2011.62(2):p. 129-33.
38. Sir-Petermann, T., et al., Metabolic and reproductive features before and during puberty in daughters of women with polycystic ovary syndrome. J Clin Endocrinol Metab,2009.94(6):p.1923-30.
39. Unluhizarci, K., et al., Investigation of hypothalamo-pituitary-gonadal axis and glucose intolerance among the first-degree female relatives of women with polycystic ovary syndrome. Fertil Steril,2007.87(6):p.1377-82.
40. Yildiz, B.O., et al., Glucose intolerance, insulin resistance, and hyperandrogenemia in first degree relatives of women with polycystic ovary syndrome. J Clin Endocrinol Metab,2003.88(5):p.2031-6.
41. Jakimiuk, A.J., et al., Luteinizing hormone receptor, steroidogenesis acute regulatory protein, and steroidogenic enzyme messenger ribonucleic acids are overexpressed in thecal and granulosa cells from polycystic ovaries. J Clin Endocrinol Metab,2001.86(3):p.1318-23.
42. Wang, Y., et al., A microsatellite polymorphism (tttta)n in the promoter of the CYP11a gene in Chinese women with polycystic ovary syndrome. Fertil Steril, 2006.86(1):p.223-6.
43. Hanzu, F.A., et al., Association of insulin receptor genetic variants with polycystic ovary syndrome in a population of women from Central Europe. Fertil Steril,2010.94(6):p.2389-92.
44. Yoshihara, K., et al., Association of single nucleotide polymorphisms in adiponectin and its receptor genes with polycystic ovary syndrome. J Reprod Med,2009.54(11-12):p.669-74.
45. Mitjans, M. and B. Arias, The genetics of depression:what information can new methodologic approaches provide? Actas Esp Psiquiatr,2012.40(2):p. 70-83.
46. Sladek, R., et al., A genome-wide association study identifies novel risk loci for type 2 diabetes. Nature,2007.445(7130):p.881-5.
47. Weedon, M.N., et al., Genome-wide association analysis identifies 20 loci that influence adult height. Nat Genet,2008.40(5):p.575-83.
48. Tin, A., et al., Genome-wide association study for serum urate concentrations and gout among African Americans identifies genomic risk loci and a novel URAT1 loss-of-function allele. Hum Mol Genet,2011.20(20):p.4056-68.
49. Chen, Z.J., et al., Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3,2p21 and 9q33.3. Nat Genet,2011.43(1):p.55-9.
50. Zeggini, E., et al., Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes. Nat Genet,2008.40(5):p.638-45.
51. Goodarzi, M.O., et al., Replication of association of DENND1A and THADA variants with polycystic ovary syndrome in European cohorts. J Med Genet, 2012.49(2):p.90-5.
52. Welt, C.K., et al., Variants in DENND1A are associated with polycystic ovary syndrome in women of European ancestry. J Clin Endocrinol Metab,2012. 97(7):p.E1342-7.
53. Mutharasan, P., et al., Evidence for chromosome 2p16.3 polycystic ovary syndrome susceptibility locus in affected women of European ancestry. J Clin Endocrinol Metab,2013.98(1):p. E185-90.
54. Shi, Y., et al., Clinical and metabolic characteristics of polycystic ovary syndrome without polycystic ovary:a pilot study on Chinese women. Fertil Steril,2008.90(4):p.1139-43.
55. Zhang, H.Y., et al., Characteristics of different phenotypes of polycystic ovary syndrome based on the Rotterdam criteria in a large-scale Chinese population. BJOG,2009.116(12):p.1633-9.
56. Moran, L. and H. Teede, Metabolic features of the reproductive phenotypes of polycystic ovary syndrome. Hum Reprod Update,2009.15(4):p.477-88.
57. Iwasa, T., et al., Diagnostic performance of serum total testosterone for Japanese patients with polycystic ovary syndrome. Endocr J,2007.54(2):p. 233-8.
58. Norman, R.J., et al., Metabolic approaches to the subclassification of polycystic ovary syndrome. Fertil Steril,1995.63(2):p.329-35.
59. Talbott, E., et al., Coronary heart disease risk factors in women with polycystic ovary syndrome. Arterioscler Thromb Vasc Biol,1995.15(7):p. 821-6.
60. Alborzi, S., R. Khodaee, and M.E. Parsanejad, Ovarian size and response to laparoscopic ovarian electro-cauterization in polycystic ovarian disease. Int J Gynaecol Obstet,2001.74(3):p.269-74.
61. Williamson, K., et al., The impact of ethnicity on the presentation of polycystic ovarian syndrome. Aust N Z J Obstet Gynaecol,2001.41(2):p.202-6.
62. Hsu, M.I., et al., Diagnostic criteria for polycystic ovary syndrome in Taiwanese Chinese women:comparison between Rotterdam 2003 and NIH 1990. Fertil Steril,2007.88(3):p.727-9.
63. Lujan, M.E., D.R. Chizen, and R.A. Pierson, Diagnostic criteria for polycystic ovary syndrome:pitfalls and controversies. J Obstet Gynaecol Can,2008. 30(8):p.671-9.
64. Blank, S.K., et al., Neuroendocrine effects of androgens in adult polycystic ovary syndrome and female puberty. Semin Reprod Med,2007.25(5):p. 352-9.
65. Catteau-Jonard, S., et al., Anti-Mullerian hormone, its receptor, FSH receptor, and androgen receptor genes are overexpressed by granulosa cells from stimulated follicles in women with polycystic ovary syndrome. J Clin Endocrinol Metab,2008.93(11):p.4456-61.
66. Marshall, J.C. and C.A. Eagleson, Neuroendocrine aspects of polycystic ovary syndrome. Endocrinol Metab Clin North Am,1999.28(2):p.295-324.
67. Xu, X., et al., Endocrine and metabolic characteristics of polycystic ovary syndrome in Chinese women with different phenotypes. Clin Endocrinol (Oxf), 2012.76(3):p.425-30.
68. Legro, R.S., et al., Changes in glucose tolerance over time in women with polycystic ovary syndrome:a controlled study. J Clin Endocrinol Metab,2005. 90(6):p.3236-42.
69. de Groot, P.C., et al., PCOS, coronary heart disease, stroke and the influence of obesity:a systematic review and meta-analysis. Hum Reprod Update,2011. 17(4):p.495-500.
70. Cooper, H.E., et al., Hereditary factors in the Stein-Leventhal syndrome. Am J Obstet Gynecol,1968.100(3):p.371-87.
71. Cohen, P.N., et al., Polycystic ovarian disease, maturation arrest of spermiogenesis, and Klinefelter's syndrome in siblings of a family with familial hirsutism. Fertil Steril,1975.26(12):p.1228-38.
72. Ferriman, D. and A.W. Purdie, The aetiology of oligomenorrhoea and/or hirsuties:a study of 467 patients. Postgrad Med J,1983.59(687):p.17-20.
73. Carey, A.H., et al., Evidence for a single gene effect causing polycystic ovaries and male pattern baldness. Clin Endocrinol (Oxf),1993.38(6):p.653-8.
74. Hague, W.M., et al., Familial polycystic ovaries:a genetic disease? Clin Endocrinol (Oxf),1988.29(6):p.593-605.
75. Battaglia, C., et al., Polycystic ovaries in childhood:a common finding in daughters of PCOS patients. A pilot study. Hum Reprod,2002.17(3):p.771-6.
76. Hirschhorn, J.N. and M.J. Daly, Genome-wide association studies for common diseases and complex traits. Nat Rev Genet,2005.6(2):p.95-108.
77. Lerchbaum, E., et al., Susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3,2p21, and 9q33.3 in a cohort of Caucasian women. Horm Metab Res,2011.43(11):p.743-7.
78. Eriksen, M.B., et al., Association of polycystic ovary syndrome susceptibility single nucleotide polymorphism rs2479106 and PCOS in Caucasian patients with PCOS or hirsutism as referral diagnosis. Eur J Obstet Gynecol Reprod Biol,2012.163(1):p.39-42.
79. Atger, M., et al., Structure of the human luteinizing hormone-choriogonadotropin receptor gene:unusual promoter and 5' non-coding regions. Mol Cell Endocrinol,1995.111(2):p.113-23.
80. Rahman, N.A. and C.V. Rao, Recent progress in luteinizing hormone/human chorionic gonadotrophin hormone research. Mol Hum Reprod,2009.15(11): p.703-11.
81. Chambers, A.E. and S. Banerjee, Natural antisense LHCGR could make sense of hypogonadism, male-limited precocious puberty and pre-eclampsia. Mol Cell Endocrinol,2005.241(1-2):p.1-9.
82. Qiao, J., et al., A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism. Hum Mutat,2009.30(9):p. E855-65.
83. Banerjee, S., et al., A link between high serum levels of human chorionic gonadotrophin and chorionic expression of its mature functional receptor (LHCGR) in Down's syndrome pregnancies. Reprod Biol Endocrinol,2005.3: p.25.
84. Jeha, G.S., et al., Variable presentation of precocious puberty associated with the D564G mutation of the LHCGR gene in children with testotoxicosis. J Pediatr,2006.149(2):p.271-4.
85. Costa, M.H., et al., Expression profiles of the glucose-dependent insulinotropic peptide receptor and LHCGR in sporadic adrenocortical tumors. J Endocrinol, 2009.200(2):p.167-75.
86. Yariz, K.O., et al., Inherited mutation of the luteinizing hormone/choriogonadotropin receptor (LHCGR) in empty follicle syndrome. Fertil Steril,2011.96(2):p. e125-30.
87. Wang, Z., et al., Variants in DENND1A and LHCGR are associated with endometrioid adenocarcinoma. Gynecol Oncol,2012.127(2):p.403-5.
88. Capalbo, A., et al., The 312N variant of the luteinizing hormone/choriogonadotropin receptor gene (LHCGR) confers up to 2.7-fold increased risk of polycystic ovary syndrome in a Sardinian population. Clin Endocrinol (Oxf),2012.77(1):p.113-9.
89. Rippe, V., et al., Identification of a gene rearranged by 2p21 aberrations in thyroid adenomas. Oncogene,2003.22(38):p.6111-4.
90. Marat, A.L. and P.S. McPherson, The connecdenn family, Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery. J Biol Chem,2010.285(14):p.10627-37.
91. Hatch, R., et al., Hirsutism:implications, etiology, and management. Am J Obstet Gynecol,1981.140(7):p.815-30.
92. Shi, Y., et al., Analysis of clinical characteristics in large-scale Chinese women with polycystic ovary syndrome. Neuro Endocrinol Lett,2007.28(6):p. 807-10.
93. Matthews, D.R., et al., Homeostasis model assessment:insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia,1985.28(7):p.412-9.
94. Simonis-Bik, A.M., et al., Gene variants in the novel type 2 diabetes loci CDC123/CAMK1D, THADA, ADAMTS9, BCL11A, and MTNR1B affect different aspects of pancreatic beta-cell function. Diabetes,2010.59(1):p. 293-301.
95. Apter, D., et al., Accelerated 24-hour luteinizing hormone pulsatile activity in adolescent girls with ovarian hyper androgenism:relevance to the developmental phase of polycystic ovarian syndrome. J Clin Endocrinol Metab, 1994.79(1):p.119-25.
96. van Hooff, M.H., et al., Variation of luteinizing hormone and androgens in oligomenorrhoea and its implications for the study of polycystic ovary syndrome. Hum Reprod,1999.14(7):p.1684-9.
97. Gilling-Smith, C., et al., Hyper secretion of androstenedione by isolated thecal cells from polycystic ovaries. J Clin Endocrinol Metab,1994.79(4):p. 1158-65.
98. Nelson, V.L., et al., Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries. Mol Endocrinol, 1999.13(6):p.946-57.
99. Dumesic, D.A. and D.H. Abbott, Implications of polycystic ovary syndrome on oocyte development. Semin Reprod Med,2008.26(1):p.53-61.
100. Gleicher, N., A. Weghofer, and D.H. Barad, The role of androgens in follicle maturation and ovulation induction:friend or foe of infertility treatment? Reprod Biol Endocrinol,2011.9:p.116.
101. Ip, S., et al., Systematic review:association of low-density lipoprotein subfractions with cardiovascular outcomes. Ann Intern Med,2009.150(7):p. 474-84.
102. Moran, L.J., et al., Impaired glucose tolerance, type 2 diabetes and metabolic syndrome in polycystic ovary syndrome:a systematic review and meta-analysis. Hum Reprod Update,2010.16(4):p.347-63.
103. Saad, M.F., et al., A two-step model for development of non-insulin-dependent diabetes. Am J Med,1991.90(2):p.229-35.
104. Dale, P.O., et al., The impact of insulin resistance on the outcome of ovulation induction with low-dose follicle stimulating hormone in women with polycystic ovary syndrome. Hum Reprod,1998.13(3):p.567-70.
105. Mather, K.J., F. Kwan, and B. Corenblum, Hyperinsulinemia in polycystic ovary syndrome correlates with increased cardiovascular risk independent of obesity. Fertil Steril,2000.73(1):p.150-6.
106. Moran, M.D., Arguments for rejecting the sequential Bonferroni in ecological studies. Oikos,2003.100:p.3.
107. Shi, Y., et al., Genome-wide association study identifies eight new risk loci for polycystic ovary syndrome. Nat Genet,2012.44(9):p.1020-5.
108. Heckert, L.L., M.A. Daggett, and J. Chen, Multiple promoter elements contribute to activity of the follicle-stimulating hormone receptor (FSHR) gene in testicular Sertoli cells. Mol Endocrinol,1998.12(10):p.1499-512.
109. Livshyts, G., et al., A distribution of two SNPs in exon 10 of the FSHR gene among the women with a diminished ovarian reserve in Ukraine. J Assist Reprod Genet,2009.26(1):p.29-34.
110. Lussiana, C., et al., Mutations and polymorphisms of the FSH receptor (FSHR) gene:clinical implications in female fecundity and molecular biology of FSHR protein and gene. Obstet Gynecol Surv,2008.63(12):p.785-95.
111. Ben-Josef, E., et al., Hormone-refractory prostate cancer cells express functional follicle-stimulating hormone receptor (FSHR). J Urol,1999.161(3): p.970-6.
112. Luo, W. and M.C. Wiltbank, Distinct regulation by steroids of messenger RNAs for FSHR and CYP19A1 in bovine granulosa cells. Biol Reprod,2006. 75(2):p.217-25.
113. Kim, S., et al., Epistasis between FSHR and CYP19A1 polymorphisms is associated with premature ovarian failure. Fertil Steril,2011.95(8):p.2585-8.
114. Parikh, H., et al., Molecular correlates for maximal oxygen uptake and type 1 fibers. Am J Physiol Endocrinol Metab,2008.294(6):p. E1152-9.
115. Ruderman, N., et al., The metabolically obese, normal-weight individual revisited. Diabetes,1998.47(5):p.699-713.
116. Van Dorpe, J., et al., Translocation of the HMGI-C (HMGA2) gene in a benign mesenchymoma (chondrolipoangioma). Virchows Arch,2002.440(5): p.485-90.
117. Chau, K.Y., et al., Derepression of HMGA2 gene expression in retinoblastoma is associated with cell proliferation. Mol Med,2003.9(5-8):p.154-65.
118. Ferguson, M., P.A. Henry, and R.A. Currie, Histone deacetylase inhibition is associated with transcriptional repression of the Hmga2 gene. Nucleic Acids Res,2003.31(12):p.3123-33.
119. Chieffi, P., et al., HMGA1 and HMGA2 protein expression in mouse spermatogenesis. Oncogene,2002.21(22):p.3644-50.
120. Fedele, M., et al., Overexpression of the HMGA2 gene in transgenic mice leads to the onset of pituitary adenomas. Oncogene,2002.21(20):p.3190-8.
121. Voight, B.F., et al., Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis. Nat Genet,2010.42(7):p.579-89.
122. Ligon, A.H., et al., Constitutional rearrangement of the architectural factor HMGA2:a novel human phenotype including overgrowth and lipomas. Am J Hum Genet,2005.76(2):p.340-8.
123. Yuan, S.H., Z. Qiu, and A. Ghosh, TOX3 regulates calcium-dependent transcription in neurons. Proc Natl Acad Sci U S A,2009.106(8):p.2909-14.
124. Dittmer, S., et al., TOX3 is a neuronal survival factor that induces transcription depending on the presence of CITED1 or phosphorylated CREB in the transcriptionally active complex. J Cell Sci,2011.124(Pt 2):p.252-60.
125. Fernandez-Navarro, P., et al., Association analysis between breast cancer genetic variants and mammographic density in a large population-based study (Determinants of Density in Mammographies in Spain) identifies susceptibility loci in TOX3 gene. Eur J Cancer,2013.49(2):p.474-81.
126. Gudmundsdottir, E.T., et al., The risk allele of SNP rs3803662 and the mRNA level of its closest genes TOX3 and LOC643714 predict adverse outcome for breast cancer patients. BMC Cancer,2012.12:p.621.
127. Ruiz-Narvaez, E.A., et al., Polymorphisms in the TOX3/LOC643714 locus and risk of breast cancer in African-American women. Cancer Epidemiol Biomarkers Prev,2010.19(5):p.1320-7.
128. Aliahmad, P. and J. Kaye, Development of all CD4 T lineages requires nuclear factor TOX. J Exp Med,2008.205(1):p.245-56.
129. Talbot, J.A., et al., Molecular scanning of the insulin receptor gene in women with polycystic ovarian syndrome. J Clin Endocrinol Metab,1996.81(5):p. 1979-83.
130. Chen, Z.J., et al., [Correlation between single nucleotide polymorphism of insulin receptor gene with polycystic ovary syndrome]. Zhonghua Fu Chan Ke Za Zhi,2004.39(9):p.582-5.
131. 李曼,邱红玉,孙永玉,李红发,多囊卵巢综合征患者胰岛素受体基因酪氨酸蛋白激酶域突变的研究生殖与避孕,2003.23(5)：p.4.
132. LeBlanc, M., et al., Genome-wide study identifies PTPRO and WDR72 and FOXQ1-SUMO1P1 interaction associated with neurocognitive function. J Psychiatr Res,2012.46(2):p.271-8.
133. Haoula, Z., M. Salman, and W. Atiomo, Evaluating the association between endometrial cancer and polycystic ovary syndrome. Hum Reprod,2012.27(5): p.1327-31.
134. Franks, S., J. Stark, and K. Hardy, Follicle dynamics and anovulation in polycystic ovary syndrome. Hum Reprod Update,2008.14(4):p.367-78.
135. Vink, J.M., et al., Heritability of polycystic ovary syndrome in a Dutch twin-family study. J Clin Endocrinol Metab,2006.91(6):p.2100-4.
136. Kahsar-Miller, M.D., et al., Prevalence of polycystic ovary syndrome (PCOS) in first-degree relatives of patients with PCOS. Fertil Steril,2001.75(1):p. 53-8.
137. Homburg, R., Androgen circle of polycystic ovary syndrome. Hum Reprod, 2009.24(7):p.1548-55.
138. Pakarainen, T., et al., Knockout of luteinizing hormone receptor abolishes the effects of follicle-stimulating hormone on preovulatory maturation and ovulation of mouse graafian follicles. Mol Endocrinol,2005.19(10):p. 2591-602.
139. Latronico, A.C., et al., Brief report:testicular and ovarian resistance to luteinizing hormone caused by inactivating mutations of the luteinizing hormone-receptor gene. N Engl J Med,1996.334(8):p.507-12.
140. Latronico, A.C., et al., A homozygous microdeletion in helix 7 of the luteinizing hormone receptor associated with familial testicular and ovarian resistance is due to both decreased cell surface expression and impaired effector activation by the cell surface receptor. Mol Endocrinol,1998.12(3):p. 442-50.
141. Bentov, Y., S. Kenigsberg, and R.F. Casper, A novel luteinizing hormone/chorionic gonadotropin receptor mutation associated with amenorrhea, low oocyte yield, and recurrent pregnancy loss. Fertil Steril,2012. 97(5):p.1165-8.
142. Cui, L., et al., Genotype-phenotype correlations of PCOS susceptibility SNPs identified by GWAS in a large cohort of Han Chinese women. Hum Reprod, 2013.28(2):p.538-44.
143. Rodriguez, K.F., et al., Insufficient luteinizing hormone-induced intracellular signaling disrupts ovulation in preovulatory follicles lacking estrogen receptor-{beta}. Endocrinology,2010.151(6):p.2826-34.
144. Moller, D.E. and J.S. Flier, Detection of an alteration in the insulin-receptor gene in a patient with insulin resistance, acanthosis nigricans, and the polycystic ovary syndrome (type A insulin resistance). N Engl J Med,1988. 319(23):p.1526-9.
145. Hashiramoto, M., et al., A nonsense mutation in the Arg345 of the insulin receptor gene in a Japanese type A insulin-resistant patient. Endocr J,2005. 52(4):p.499-504.
146. Cocozza, S., et al., NIDDM associated with mutation in tyrosine kinase domain of insulin receptor gene. Diabetes,1992.41(4):p.521-6.
147. Ibanez, L., et al., Anovulation in eumenorrheic, nonobese adolescent girls born small for gestational age:insulin sensitization induces ovulation, increases lean body mass, and reduces abdominal fat excess, dyslipidemia, and subclinical hyperandrogenism. J Clin Endocrinol Metab,2002.87(12):p. 5702-5.
148. Ibanez, L., et al., Additive effects of insulin-sensitizing and anti-androgen treatment in young, nonobese women with hyperinsulinism, hyperandrogenism, dyslipidemia, and anovulation. J Clin Endocrinol Metab,2002.87(6):p. 2870-4.
149. Kerns, S.L., et al., Genome-wide association study to identify single nucleotide polymorphisms (SNPs) associated with the development of erectile dysfunction in African-American men after radiotherapy for prostate cancer. Int J Radiat Oncol Biol Phys.2010.78(5):p.1292-300.
150. Eriksson, K.F. and F. Lindgarde, Poor physical fitness, and impaired early insulin response but late hyperinsulinaemia, as predictors of NIDDM in middle-aged Swedish men. Diabetologia,1996.39(5):p.573-9.
151. Cara. J.F. and R.L. Rosenfield, Insulin-like growth factor Ⅰ and insulin potentiate luteinizing hormone-induced androgen synthesis by rat ovarian thecal-interstitial cells. Endocrinology,1988.123(2):p.733-9.
152. Del Villar, K. and C.A. Miller, Down-regulation of DENN/MADD, a TNF receptor binding protein, correlates with neuronal cell death in Alzheimer's disease brain and hippocampal neurons. Proc Nat) Acad Sci U S A,2004. 101(12):p.4210-5.
153. Olszanecka-Glinianowicz. M., et al., Is the polycystic ovary syndrome associated with chronic inflammation per se? Eur J Obstet Gynecol Reprod Biol,2007.133(2):p.197-202.