PCR-SSCP银染色法检测尿道下裂患儿雄激素受体基因突变

研究尿道下裂的发生与雄激素受体基因突变间的关系。方法运用盐析法从２７例不同类型尿道下裂患儿外周血中提取基因组ＤＮＡ,然后通过聚合酶反应－单链构象多态性分析银染色法检测雄激素受体基因第２和第８外显子基因突变情况。结论激素受体基因突变可能与尿道下裂的发生有关,并可能与严重尿道下裂的发生关系更为密切。     

Objective use of testosterone reveals androgen insensitivity in patients with proximal hypospadias

ObjectiveWe report preoperative testosterone stimulation based on glans width measurements in patients with midshaft and proximal hypospadias, revealing androgen resistance in those with proximal hypospadias.MethodsPatients had maximum glans width measured preoperatively. Those <14 mm initially received 2 mg/kg testosterone cypionate intramuscularly for two to three doses, with the aim of increasing glans width ≥15 mm. Not all patients achieved targeted growth, and some were subsequently treated with escalating doses of testosterone.Results5/15 midshaft patients had two to three doses of 2 mg/kg testosterone, with all increasing glans width to ≥15 mm. 29/47 proximal patients had testosterone, with 13 (57%) not reaching desired glans width. Six of these and another six patients had escalating doses from 4 to 32 mg/kg testosterone, with 11 then achieving targeted glans width. Relative androgen resistance was found in 19/29 (66%) proximal cases, including all treated patients with perineal hypospadias.Conclusions39/62 (63%) patients met objective criteria for preoperative testosterone stimulation based on glans width <14 mm, which is less than the average normal newborn glans diameter. Evidence of relative androgen resistance was found in 19 (49%), all with proximal hypospadias.     

中国汉族人群AR基因多态性与男性尿道下裂的相关性研究

目的：研究中国汉族人群中雄激素受体基因（AR）第一外显子上的（CAG）n多态性与男性尿道下裂发病风险的相关性。方法研究对象为342例男性尿道下裂患儿以及351例健康人。从外周血淋巴细胞中提取全基因组DNA,对AR基因上的第一外显子（CAG）n进行PCR扩增,扩增产物以Sanger测序方法对其测序,计算各样本的（CAG）n分布情况。采用卡方检验（Chi-square test）比较病例组与对照组之间的（CAG）n分布,并分析（CAG）n对男性尿道下裂发病风险的影响。结果病例组（CAG）n为15～44,平均长度23．65±3．36;对照组为19～31,平均长度23．14±1．87,两组CAG重复序列有明显统计学差异（P＝0.013）。当n≥23,24,25,26时,尿道下裂发病风险分别为1．47（CI：1．081～2．001）,1．356（CI：1．004～1．830）,1．484（CI：1．063～2．071）,1．565（CI：1．034～2．369）,各组均有明显统计学意义（P＜0.05）;而当n≤19时,尿道下裂患病风险为2．111（CI：0．935～4．767）,P＝0.05。结论 AR基因第一外显子上的（CAG）n与男性尿道下裂发病有明显的相关性,CAG重复序列越长,尿道下裂的发病风险越高。     

Functional assessment and clinical classification of androgen sensitivity in patients with mutations of the androgen receptor gene

In the genetic male, mutations of the androgen receptor (AR) gene cause phenotypes ranging from female to subfertile male. Binding assays on genital skin fibroblasts and DNA analysis alone provide incomplete information about receptor function. We used the sex hormone-binding globulin (SHBG) response to stanozolol as a measure of AR function and correlated the results with phenotypes which were classified according to the degree of defective masculinization. Of the 34 patients investigated, 9 had complete, and 14 had partial androgen insensitivity syndrome (AIS) with predominantly female, ambiguous, or predominantly male phenotype. Eleven subjects served as controls. Mutations were characterized using polymerase chain reaction-single strand conformation polymorphism analysis and direct DNA sequencing. DNA analysis revealed two major deletions, two minor defects leading to premature stop codons in exon 1, and 19 point mutations in the DNA- and hormone-binding domains of the AR gene. After stanozolol, SHBG remained unchanged in patients with complete AIS (102.0 ± 3.8 [SE]%; range 92.4%–129% of the initial value). The SHBG decrease was diminished in partial AIS with predominantly female (83.8% ± 1.7%; range 81.3%–87.0%), ambiguous (80.4% ± 4.4%, range 68.4%–89.1%), and predominantly male (mean 65.9% ± 4.9%, range 48.6%– 80.8%) phenotypes, and normal in controls (51.4% ± 2.1%, range 35.6%–62.1%). Differences between controls and each AIS group were statistically significant (P< < 0.05 – < 0.0001). A close correlation was found between the degree of undermasculinization (AIS phenotype) and the SHBG response. Conclusions The SHBG test provides functional information about the severity of the receptor defect in vivo and hence adds to the structural information provided by DNA analysis. It detects receptor defects due to mutations within the entire gene, including the DNA-binding domain, and is a rapid, simple, and cost effective procedure. It may provide useful information for the diagnosis and management of affected children. Received: 13 February 1996 / Accepted: 4 June 1996     

Ex-premature infant boys with hypospadias are similar in size to age-matched, ex-premature infant boys without hypospadias

Objective

Studies have postulated that hypospadias, prematurity, and low birth weight are linked by defects in androgen signaling. To determine whether premature, hypospadiac boys are small and remain so, we compared their size at birth and at hypospadias repair to premature boys who underwent post-neonatal circumcision.

Methods

We identified premature boys admitted to Texas Children’s Hospital who underwent either hypospadias repair or circumcision after 4 months of age. Age, weight, and height at birth and surgery were recorded.

Results

Fifty-four boys had hypospadias and 34 did not. For hypospadiac boys, the mean birth weight and age, height, and weight at surgery were lower than for boys without hypospadias. More importantly, length-for-age and weight-for-age percentiles were also lower for hypospadiac boys. When subset analysis was performed on boys younger than 2 years at surgery, however, there were no significant differences in height or weight between hypospadiac and non-hypospadiac boys.

Conclusion

Our series suggests that premature, hypospadiac boys are born smaller than age-matched, non-hypospadiac controls. However, there were no age-corrected size differences between hypospadiac and non-hypospadiac boys at surgery. This implies that hypospadiac boys exhibit post-neonatal ‘rebound’ growth. Global growth deficits, if any, do not persist in hypospadiac boys.     

Study on the relationship among testicular dysgenesis syndrome, androgen receptor gene polymorphism and associated perinatal factors

目的 探讨睾丸发育不全综合症(testicular dysgenesis syndrome,TDS)的幼年期发病(尿道下裂、隐睾)与雄激素受体(Androgen receptor,AR)基因CAG重复长度多态性改变及相关围产因素的关系.方法 收集2009年5月至2010年12月共60例尿道下裂、隐睾32例(单侧29例,双侧3例,其中尿道下裂合并单侧隐睾6例)及正常对照组70例,采血提取基因组DNA,检测AR基因CAG重复序列次数;收集围产资料,包括小儿出生时体重及胎龄、母亲分娩时年龄、母孕早期保胎史(黄体酮使用)、是否人工辅助生育、母亲职业、居住地、孕期患妊娠高血压情况,并进行统计分析.结果 尿道下裂组和隐睾组CAG重复次数均明显大于对照组(P分别为0.008和0.028);相关围产因素中,尿道下裂的发生与低出生体重(P=0.003)、母亲低分娩年龄(P=0.007)及孕早期黄体酮使用(P =0.000)有关.隐睾与母亲低分娩年龄(P=0.003)及从事农业生产(P=0.017)有关.结论 TDS 的发生与多因素相关,当AR基因CAG重复次数增加,合并其他相关危险因素,如低出生体重、年轻的母亲,尤其是孕期暴露于雌激素或抗雄激素物质,如黄体酮、杀虫剂等,可能使发生TDS的风险累加而致病.     

先天性尿道下裂阴茎皮肤细胞雌雄激素受体表达及胞核内的聚集

目的：探索先天性尿道下裂与阴茎皮肤细胞雄激素受体（ＡＲ）、雌激素受体（ＥＲ）的表达及细胞核内的聚集的关系。方法：通过受体的放射配基结合分析，对２８例不同程度的先天性尿道下裂患儿外生殖器皮肤细胞内的ＡＲ、ＥＲ的表达及细胞内的分布进行了测定。结果：实验显示尿道下裂患儿外生殖器皮肤细胞内及胞核内ＡＲ量较正常对照减少（Ｐ＜０．０１），尿道下裂的严重程度与细胞内及细胞核内的ＡＲ量无关（Ｐ＞０．０５）。细胞内总ＥＲ与正常无显著性差异（Ｐ＞０．０５），胞核内ＥＲ量较正常对照减少（Ｐ＜０．０５）。结论：ＡＲ异常是先天性尿道下裂的原因之一，先天性尿道下裂也属于雄激素不敏感综合征的范畴，而核内ＥＲ的减少可能为继发性改变     

先天性尿道下裂阴茎雄激素受体的研究

通过对２４例不同临床类型和不同阴茎发育状态的尿道下裂患儿的包皮、龟头及纤维索带组织上雄激素受体（ＡＲ）的研究发现：前两种组织上存在有相似数量的ＡＲ，说明尿道下裂临床表现上的差异可能是由于受体质量或受体亚单位的异常所致，与受体总体数量关系不大。其进一步的研究可能不仅有助于尿道下裂病因的探讨而且还有助于临床内分泌治疗方案的选择。纤维索带组织中没有发现ＡＲ阳性的细胞，因此，它对雄激素无反应，是引起和加重阴茎下屈的重要原因，在重建尿道时必须彻底清除该组织。     

Expression of two functionally different androgen receptors in a patient with androgen insensitivity

Recently, we demonstrated a previously unknown high rate of de novo mutations of the androgen receptor (AR) gene in androgen insensitivity syndrome (AIS) with some resulting in somatic mosaicism of mutant and wild type AR alleles. However, data on the genotype-phenotype relationship in the latter patients are sparse. We present here a 46,XY newborn with ambiguous genitalia carrying a mosaic of an 866 GTG (Val) → ATG (Met) mutation with the wild type AR gene. This mutation has usually been associated with complete AIS. Accordingly, we found markedly impaired transactivation due to the mutant Met⁸⁶⁶ AR. Essential information arose from Scatchard analysis of methyltrienolone binding on cultured genital skin fibroblasts. We demonstrated for the first time the expression of two functionally different ARs (K_d1: 5.58 nM = mutant, K_d2: 0.06 nM = wild type) in one AIS individual. This finding not only represents an important confirmation for the presence of the somatic mosaicism in the patient, it also indicates the most likely molecular mechanism responsible for the unexpectedly strong virilization of the patient: Androgen action through the wild type AR expressed by part of the somatic cells. Conclusions The present case clearly demonstrates the molecular mechanism by which somatic mosaicism of the androgen receptor gene can modulate in vivo androgen action. It underlines the importance of particular notice on somatic mosaicism in all androgen insensitivity syndrome patients carrying de novo mutations of the androgen receptor gene. Received: 24 August 1998 / Accepted in revised form: 5 January 1999     

先天性原发性甲状腺功能减低症50例TSHR基因研究

目的探讨促甲状腺激素受体（TSHR）基因在先天性原发性甲状腺功能减低症（甲低）病因学中的地位。方法对50例先天性甲状腺功能减低症患者,从外周血白细胞中提取基因组DNA,采用PCR-SSCP方法对TSHR基因外显子10、6、1进行分析。结果在TSHR基因外显子10、6、1中未发现突变。结论已发现的TSHR基因编码区的结构改变,在中国人甲低的病因学中可能不具有重要地位。     

尿道下裂患儿包皮组织中表皮生长因子

目的　探讨表皮生长因子（EGF）及其表皮生长因子受体（EGFR）在尿道下裂发生中的作用。方法　应用免疫组织化学染色方法对15例尿道下裂患儿包皮组织和尿道板中EGF、EGFR表达进行检测。结果　15例尿道下裂患儿包皮组织中EGF、EGFR阳性细胞指数分别为195±4、153±8,均低于正常儿童为212±4，225±6（P<0.01），尿道板中EGF、EGFR阳性细胞指数分别为153±8、118±7,亦明显少于其包皮组织（P<0.01）。结论　EGF、EGFR在包皮组织中的不足表达和不均衡分布可能与尿道下裂的发生有关。     

FGFR2单核苷酸多态性rs2981582位点(C/T)与尿道下裂的相关性研究

目的 寻找尿道下裂遗传易感性的基因,特别是成纤维细胞生长因子受体2(FGFR2)基因的多态性与尿道下裂发病的相关性,探讨FGFR2基因单核苷酸多态位点rs2981582与尿道下裂的关系.方法 收集2007年至2009年小儿外科确诊后实施手术并且无其他畸形的尿道下裂188例,作为实验病例组.无任何先天畸形的健康儿童118例做为正常对照组,收集血液样本,提取基因组DNA.设计针对FGFR2基因rs2981582位点的PCR引物、进行聚合酶链反应--限制性片段长度多态性方法(PCR-RFLP)分析尿道下裂患儿及正常人群中的基因分型.结果 我国北方儿童FG-FR2基因rs2981582位点的基因型以C/C为主,占78.81%,C/T基因型占21.19%,本组未检测到T/T基因型.两种基因型频率在北方儿童尿道下裂中分别为C/C基因型150例,占87.77%,C/T基因型23例,占12.23%;正常对照组中C/C基因型93例,占78.81%,C/T基因型25例,占21.19%.两组之间的基因型频率分布比较有差异,χ2=4.396,P=0.028.结论 在所研究的人群中,FGFR2单核苷酸多态性位点rs2981582与尿道下裂的发病相关,FGFR2基因rs2981582位点的基因型多态性可能是尿道下裂的致病因素之一.

Abstract：

Objective To investigate the association between FGFR2 gene rs2981582 single nucleotide polymorphism and hypospadias in Chinese patients. Methods We collected 188 patients with isolated hypopadias and 118 healthy individuals' DNA and designed the PCR primers. Genotyping of FGFR2 gene rs2515733 was carried in 188 patients and 118 healthy individuals by PCR-RFLP assay respectively. Results The genotypes in this series consisted of C/C (78. 81%) and C/T (21.19%).T/T genotype was not detected in both groups. The rs2981582 genotypes in the hypospadias patients consisted of homozygote 150 (87. 77%) C/C and 23(12. 23%) C/T, while the genotypes in the controls were composed of 93(78. 81%)C/C and 25(21.19%)C/T. Significant difference was found in the distribution of rs2981582 genotypes between the two groups (χ2 = 4. 396, P = 0. 028). Conclusions There may be association between the FGFR2 gene rs2981582 SNP and hypospadias in northern Chinese population.     

先天性甲状腺功能减低症汉族儿童的促甲状腺素受体基因失活突变

目的探讨汉族儿童促甲状腺素受体（TSHR）基因失活突变与先天性甲状腺功能减低症（CH）的相关性。方法（1）选择79例CH汉族儿童（亚临床甲减14例,年龄1～5.5岁,男8例,女6例;甲减65例,年龄1.5～6岁,男27例,女38例）为研究对象;100名正常儿童（男40例,女60例,年龄1～8岁）作为对照组。（2）采用PCR和DNA测序技术检测TSHR基因失活突变。结果（1）79例CH患儿中有1例发生复合杂合子突变,其突变位点为（Pro52Thr／Val689Gly）。1例发生杂合子突变,其突变位点为（Gly245Ser）。30例患儿在第10外显子2181位核苷酸处发生C-G转换（GAC→GAG）,使727位密码子天冬氨酸被谷氨酸代替（Asp727Glu）。47例患儿在第7外显子561位核苷酸处发生T-c转换（AAT→AAc）,相应的187位氨基酸（Asn）不发生改变。（2）33例正常对照儿童在第10外显子2181位核苷酸处发生C-G转换;50例正常对照儿童在第7外显子561位核苷酸处发生T-c转换（AAT→AAc）。结论浙江汉族CH患儿TSHR基因有3个杂合子突变位点：（Pr052Thr）、（Gly245Ser）、（Val689Gly）,第10外显子2181位核苷酸处（GAc→GAG）及第7外显子561位核苷酸处（AAT→AAC）存在TSHR基因多态性。