CYP21A2基因单核苷酸多态性与汉族痤疮的关联性研究
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摘要
研究背景：痤疮是一种常见的毛囊皮脂腺慢性炎症性疾病,临床表现多种多样。目前研究公认的痤疮发病机制主要包括4个方面：毛囊皮脂腺导管的异常角化、皮脂分泌增多、机体的炎症反应和痤疮丙酸杆菌的增殖。其中,皮脂分泌增多被认为是一个重要因素,它的产生受雄激素调节,过多的雄激素可能会使易感人群诱发痤疮。有研究表明,在以结节为主要表现的重度痤疮患者中,血清中雄激素水平升高。
CYP21A2基因位于人类6号染色体断臂上,编码21-羟化酶,可催化17-羟孕酮向11-脱氧皮质醇的转化过程。21-羟化酶一旦活性下降甚至失活,便会导致糖皮质激素的合成减少而雄激素的合成增加,但是目前CYP21A2基因和痤疮的关系仍不明确。
目的：研究雄激素代谢相关基因CYP21A2单核苷酸多态性与汉族痤疮发病的相互关系,进一步阐明遗传因素在痤疮发病机制中的作用。
方法：根据r2大于0.8、MAF大于0.1的标准,从HapMap和NCBI数据库筛选出了5个SNP位点,分别是：rs6464, rs6465, rs6467, rs6472, rs6474。从门诊收集300例痤疮患者,伴有多囊卵巢综合征、脂溢性脱发和其他自身免疫性疾病的患者不纳入本次研究。一次性抽取外周静脉血3-4mL,采用德国Qiagen公司试剂盒从外周血细胞中抽提DNA。设计特异性引物,随后进行聚合酶链式反应扩增基因片段。.统计分析采用卡方检验,分别对基因型和等位基因的分布频率进行组内比较,当大于20%单元格的理论值小于5时,使用Fisher确切检验,同时分析单倍型与痤疮不同临床表现的相关性。
结果：
1.Rs6465和rs6474位点因不符合哈迪-温伯格平衡(P值分别为0.01和0.00)而被剔除。
2.Rs6467因在本次人群中最小等位基因频率为0(MAF=0.00)亦被剔除。
3.以轻度痤疮(Ⅰ°)和中重度痤疮(Ⅱ°+Ⅲ°+Ⅳ°)两组患者作比,rs6464位点的CC基因型的分布频率在隐性遗传模式下有统计学意义(P=0.044,0R=0.308),与中重度痤疮相关,但C等位基因的分布频率却无明显差异(X2=2.914,P=0.088)。
4.以轻度痤疮(1。)和中重度痤疮(Ⅱ°+Ⅲ°+Ⅳ°)患者作比,rs6472位点在两组中的基因型及等位基因的分布频率无统计学意义,与痤疮的严重程度不相关。
5.Rs6464和rs6472位点之间存在较强的连锁不平衡,D’=0.9185、r2=0.416(χ2=134.8,P=O)。单倍型分析示C-G单倍型与中重度痤疮相关(P=0.0447,OR=0.5652)。
结论：
1.本研究得到了汉族痤疮患者CYP21A2基因rs6464和rs6472位点的等位基因、基因型及单倍型的频率分布数据,为今后相关研究提供了一些基础资料。
2.首次研究了CYP21A2基因单核苷酸多态性和汉族痤疮的关系。本次研究结果提示,在隐性遗传模式下,rs6464位点与痤疮易感性相关,可能作为一种遗传标记。在显性遗传模式下,C-G单倍型与中重度痤疮相关,可能携带这种单倍型的人群患有较严重痤疮的危险性增加。
3.分析了CYP21A2基因与不同程度痤疮之间的关系,携带某种基因型或者单倍型的人群可能更倾向于表现为某种临床表型,不同临床表型可能存在遗传异质性。
4.为进一步研究CYP21A2基因在痤疮发病中的作用和寻找其易感基因提供了重要线索。
Background:Acne with different symptoms is one of the most common chronic inflammation diseases of pilosebaceous gland. Current research indicates that the pathogenesis of acne involves4main processes:follicular hyperproliferation, excess sebum production, inflammation, and proliferation of Propionibacterium acnes. Seborrhea is recognized as one pathogenic factor for acne. Sebum production is regulated by androgens, and androgen excess may provoke or aggravate acne in susceptible individuals. Increased serum androgen levels correlated with the presence of severe nodular acne in men and women.
CYP21A2gene is located on chromosome6p21.3, encoding adrenal21-hydroxylase (21-OH) which catalyses the conversion of17-hydroxyprogesterone (17-OHP) to11-deoxycorticosterone. Its inactivation or dysfunction results in reduced glucocorticoid and increased androgen production. But the relationship between CYP21A2and acne isn't established.
Objective:The aim of our study was to investigate the relationship between the single nucleotide polymorphism of androgen related gene CYP21A2and acne susceptibility.
Methods:5tag and function SNPs (rs6464, rs6465, rs6467, rs6472, rs6474) with an r2threshold of0.8were selected from the International HapMap Database Release and NCBI. All of them had a minor allele frequency (MAF) of≥0.1.300acne patients were included in the study. DNA was extracted from peripheral blood of individual patients. Patients with Polycystic Ovary Syndrome (PCOS), Seborrheic Alopecia and other immune system diseases were excluded from the study. A polymerase chain reaction (PCR) sequencing technique was used to amplify CYP21A2gene. A χ2test was used for data analysis and genotype was compared in different severity groups. Fisher's exact test was necessary when more than20%cells' values were smaller than five.
Results:
1. Rs6465and rs6474were excluded because they didn't comply with Hardy-Weinberg equilibrium (HWE) at the P=0.01and P=0.00levels respectively.
2. Rs6467was also excluded because the allele frequency was0.00(MAF=0.00) in our test.
3. For locus rs6464in CYP21A2gene, the frequency distribution of the C/C homozygotes was significantly different between mild (Ⅰ°) group and moderate+sever (Ⅱ°+Ⅲ°+Ⅳ°) group in the recessive genetic model (P=0.044, OR=0.308). But allelic frequencies were not significantly different within groups (χ2=2.914, P=0.088).
4. For locus rs6472in CYP21A2gene, no statistically significant difference was observed in genotype and allele distribution between the two groups.
5. The analysis suggested that the LD between rs6464and rs6472was strong, D'was0.9185and r2was0.416(x2=134.8, P=0). In dominant model, haplotype of C-G was associated with moderate and severe acne (P=0.0447, OR=0.5652).
Conclusions:
1. This study provides the database of rs6464and re6472of CYP21A2alleles, genotypes and haplotypes in Chinese Hans people with acne.
2. We first studied the SNPs of CYP21A2association with acne in Chinese Hans. Our findings suggest that SNP rs6464is associated with susceptibility to acne in recessive model. It may be markers for general susceptibility to acne. The haplotype of C-G was associated with moderate and severe acne in dominant model. It was found as high-risk ones in developing acne in this study.
3. This study showed the CYP21A2correlation with different severity of acne. The different of acne may have different genetic background.
4. The results of the study provide information for researching association of CYP21A2with acne and searching for susceptibility gene further.
CYP21A2基因位于人类6号染色体断臂上,编码21-羟化酶,可催化17-羟孕酮向11-脱氧皮质醇的转化过程。21-羟化酶一旦活性下降甚至失活,便会导致糖皮质激素的合成减少而雄激素的合成增加,但是目前CYP21A2基因和痤疮的关系仍不明确。
目的：研究雄激素代谢相关基因CYP21A2单核苷酸多态性与汉族痤疮发病的相互关系,进一步阐明遗传因素在痤疮发病机制中的作用。
方法：根据r2大于0.8、MAF大于0.1的标准,从HapMap和NCBI数据库筛选出了5个SNP位点,分别是：rs6464, rs6465, rs6467, rs6472, rs6474。从门诊收集300例痤疮患者,伴有多囊卵巢综合征、脂溢性脱发和其他自身免疫性疾病的患者不纳入本次研究。一次性抽取外周静脉血3-4mL,采用德国Qiagen公司试剂盒从外周血细胞中抽提DNA。设计特异性引物,随后进行聚合酶链式反应扩增基因片段。.统计分析采用卡方检验,分别对基因型和等位基因的分布频率进行组内比较,当大于20%单元格的理论值小于5时,使用Fisher确切检验,同时分析单倍型与痤疮不同临床表现的相关性。
结果：
1.Rs6465和rs6474位点因不符合哈迪-温伯格平衡(P值分别为0.01和0.00)而被剔除。
2.Rs6467因在本次人群中最小等位基因频率为0(MAF=0.00)亦被剔除。
3.以轻度痤疮(Ⅰ°)和中重度痤疮(Ⅱ°+Ⅲ°+Ⅳ°)两组患者作比,rs6464位点的CC基因型的分布频率在隐性遗传模式下有统计学意义(P=0.044,0R=0.308),与中重度痤疮相关,但C等位基因的分布频率却无明显差异(X2=2.914,P=0.088)。
4.以轻度痤疮(1。)和中重度痤疮(Ⅱ°+Ⅲ°+Ⅳ°)患者作比,rs6472位点在两组中的基因型及等位基因的分布频率无统计学意义,与痤疮的严重程度不相关。
5.Rs6464和rs6472位点之间存在较强的连锁不平衡,D’=0.9185、r2=0.416(χ2=134.8,P=O)。单倍型分析示C-G单倍型与中重度痤疮相关(P=0.0447,OR=0.5652)。
结论：
1.本研究得到了汉族痤疮患者CYP21A2基因rs6464和rs6472位点的等位基因、基因型及单倍型的频率分布数据,为今后相关研究提供了一些基础资料。
2.首次研究了CYP21A2基因单核苷酸多态性和汉族痤疮的关系。本次研究结果提示,在隐性遗传模式下,rs6464位点与痤疮易感性相关,可能作为一种遗传标记。在显性遗传模式下,C-G单倍型与中重度痤疮相关,可能携带这种单倍型的人群患有较严重痤疮的危险性增加。
3.分析了CYP21A2基因与不同程度痤疮之间的关系,携带某种基因型或者单倍型的人群可能更倾向于表现为某种临床表型,不同临床表型可能存在遗传异质性。
4.为进一步研究CYP21A2基因在痤疮发病中的作用和寻找其易感基因提供了重要线索。
Background:Acne with different symptoms is one of the most common chronic inflammation diseases of pilosebaceous gland. Current research indicates that the pathogenesis of acne involves4main processes:follicular hyperproliferation, excess sebum production, inflammation, and proliferation of Propionibacterium acnes. Seborrhea is recognized as one pathogenic factor for acne. Sebum production is regulated by androgens, and androgen excess may provoke or aggravate acne in susceptible individuals. Increased serum androgen levels correlated with the presence of severe nodular acne in men and women.
CYP21A2gene is located on chromosome6p21.3, encoding adrenal21-hydroxylase (21-OH) which catalyses the conversion of17-hydroxyprogesterone (17-OHP) to11-deoxycorticosterone. Its inactivation or dysfunction results in reduced glucocorticoid and increased androgen production. But the relationship between CYP21A2and acne isn't established.
Objective:The aim of our study was to investigate the relationship between the single nucleotide polymorphism of androgen related gene CYP21A2and acne susceptibility.
Methods:5tag and function SNPs (rs6464, rs6465, rs6467, rs6472, rs6474) with an r2threshold of0.8were selected from the International HapMap Database Release and NCBI. All of them had a minor allele frequency (MAF) of≥0.1.300acne patients were included in the study. DNA was extracted from peripheral blood of individual patients. Patients with Polycystic Ovary Syndrome (PCOS), Seborrheic Alopecia and other immune system diseases were excluded from the study. A polymerase chain reaction (PCR) sequencing technique was used to amplify CYP21A2gene. A χ2test was used for data analysis and genotype was compared in different severity groups. Fisher's exact test was necessary when more than20%cells' values were smaller than five.
Results:
1. Rs6465and rs6474were excluded because they didn't comply with Hardy-Weinberg equilibrium (HWE) at the P=0.01and P=0.00levels respectively.
2. Rs6467was also excluded because the allele frequency was0.00(MAF=0.00) in our test.
3. For locus rs6464in CYP21A2gene, the frequency distribution of the C/C homozygotes was significantly different between mild (Ⅰ°) group and moderate+sever (Ⅱ°+Ⅲ°+Ⅳ°) group in the recessive genetic model (P=0.044, OR=0.308). But allelic frequencies were not significantly different within groups (χ2=2.914, P=0.088).
4. For locus rs6472in CYP21A2gene, no statistically significant difference was observed in genotype and allele distribution between the two groups.
5. The analysis suggested that the LD between rs6464and rs6472was strong, D'was0.9185and r2was0.416(x2=134.8, P=0). In dominant model, haplotype of C-G was associated with moderate and severe acne (P=0.0447, OR=0.5652).
Conclusions:
1. This study provides the database of rs6464and re6472of CYP21A2alleles, genotypes and haplotypes in Chinese Hans people with acne.
2. We first studied the SNPs of CYP21A2association with acne in Chinese Hans. Our findings suggest that SNP rs6464is associated with susceptibility to acne in recessive model. It may be markers for general susceptibility to acne. The haplotype of C-G was associated with moderate and severe acne in dominant model. It was found as high-risk ones in developing acne in this study.
3. This study showed the CYP21A2correlation with different severity of acne. The different of acne may have different genetic background.
4. The results of the study provide information for researching association of CYP21A2with acne and searching for susceptibility gene further.
引文
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[62]Sved JA. Linkage Disequilibrium and Its Expectation in Human Populations [J]. Twin Res Hum Genet,2009,12(1):35-43.
[63]Pritchard JK, Przeworski M. Linkage disequilibrium in humans:models and data [J]. Am J Hum Genet,2001,69(1):1-14.
[64]Nackley AG, Shabalina SA, Tchivileva IE, et al. Human catechol-O-methyltransferase haplotypes modulate protein expression by altering mRNA secondary structure [J]. Science,2006,314(5807):1930-1933.
[65]Tanaka Yuichiro, Hirata Hiroshi, Chen Zhong, et al. Polymorphisms of catechol-O-methyltransferase in men with renal cell cancer [J]. Cancer Epidemiol Biomarkers Prev,2007,16(1):92-97.
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[1]COLLIER CN, HARPER JC, CANTRELL WC, et al. The prevalence of acne in adult 20 years and older. J Am Acad Dennatol,2008,58(1):56—59.
[2]Knaggs HE, Wood EJ, Rizer RL, et al. Post-adolescent acne. Int J Cosmet Sci, 2004,26:129-138.
[3]Zouboulis CC, Eady A, Philpott M, et al. What is the pathogenesis of acne? Exp Dermatol,2005,14(2):143-152.
[4]Xu SX, Wang HL, Fan X, et al. The familial risk of acne vulgaris in Chinese Hans—a case-control study. J Eur Acad Dermatol Venereol,2007,21(5):602-605.
[5]Wei B, Pang Y, Zhu H, et al. The epidemiology of adolescent acne in North East China. J Eur Acad Dermatol Venereol,2010,24(8):953—957.
[6]Ibanez L, Ong KK, Mongan N, et al. Androgen receptor gene CAG repeat polymorphism in the development of ovarian hyperandrogenism. J Clin Endocrinol Metab,2003,88(7):3333-3338.
[7]Y Pang, CD He, Y Liu, et al. Combination of short CAG and GGN repeats in the androgen receptor gene is associated with acne risk in North East China. JEAD,2008, 22(12):1445-1451.
[8]Z Yang, HJ Yu, BW Cheng, et al. Relationship between the CAG repeat polymorphism in the androgen receptor gene and acne in the Han ethnic group. Dermatology,2009,218(4):302—306.
[9]Diamanti KE, Bartzis MI, Bergiele AT, et al. Microsatellite polymorphism (tttta) at -528bp of gene CYP11α influences hyperandrogenemia in Patients with PCOS. Fertil Steril,2000,73(4):735-741.
[10]杨智,唐文如,董永利等.云南汉族痤疮与CYP11α基因微卫星多态性相互关系的研究.Chin J DennVenereol,2008,22 (2):65-67.
[11]Miller WL. Early steps in androgen biosynthesis:from cholesterol to DHEA. Baillieres Clin Endocrinol Metab,1998,12(1):67—81.
[12]Perez MS, Cerrone GE, Benencia H, et al. Polymorphism in CYP11 alpha and CYP17 genes and the etiology of hyperandrogenism in patients with polycystic ovary syndrome. Medicina,2008,68(2):129—134.
[13]L He, Z Yang, HJ Yu, et al. The Relationship between CYP17-34 polymorphism and acne in Chinese subjects revealed by sequencing. Dermatology,2006,212(4): 338-342.
[14]Vowels BR, Yang S, Leyden JJ. Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes:Implications for chronic inflammatory acne. Infect Immun,1995,63(8):3158-3165.
[15]Baz K, Emin Erda M, Yazici AC, et al. Association between tumor necrosis factor-alpha gene promoter polymorphism at position-308 and acne in Turkish patients. Arch Dermatol Res,2008,300(7):371-376.