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     

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.     

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     

Molecular characterization of the androgen receptor gene in boys with hypospadias

Development of male external genitalia is dependent on androgens, and karyotypic males lacking appropriate levels of androgens or functionally normal receptors may show abnormal virilization. Mutations in the androgen receptor gene cause abnormal receptor function and diverser mutations may be associated with heterogenous clinical signs of androgen insensitivity. In this study, we have searched for the existence of androgen receptor gene mutations carried by some patients with hypospadias. Genomic DNA samples from peripheral blood leucocytes from 21 patients with different degrees of hypospadias were studied. Analysis of the androgen receptor gene was performed by exon-specific amplification using polymerase chain reaction, single strand conformation polymorphism analysis, and direct genomic sequencing. Although a silent polymorphism was identified in exon 1 of the androgen receptor gene, the majority of patients studied (20/21) did not carry androgen receptor gene mutations. One patient with severe hypospadias and bilateral cryptorchidism was found to carry a point mutation in exon 8. We conclude that mutations in the androgen receptor gene may be carried by subset of patients with genital ambiguity presenting primarily with hypospadias, but this is not the underlying cause in the majority of cases. Characterization of this genetic defect may be important for classification and subsequent conservative therapeutic approaches for these patients.     

Robinow or “fetal face syndrome” in a male infant with ambiguous genitalia and androgen receptor deficiency

Typical features of the fetal face or Robinow syndrome are reported in a male infant who presented with ambiguous genitalia and persistence of the Mullerian ducts. Histology of the testes was normal whereas endocrinological studies showed partial deficiency of androgen receptors.Dedicated to Professor Dr. K. Stehr on the occasion of his 60th birthday     

Novel mutations, no detectable mRNA and familial genetic analysis of the Wiskott-Aldrich syndrome protein gene in six Japanese patients with Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is a primary X-linked immunodeficiency disease caused by mutations of the Wiskott-Aldrich syndrome protein (WASP) gene. The present molecular studies of six Japanese WAS patients identified five different mutations of WASP, including two novel mutations (45delG, 395insGGAGAT), the latter appearing to have occurred de novo. Familial carriers were detected by polymerase chain reaction-single strand conformational polymorphism analysis, restriction enzyme digestion and direct sequencing of PCR products. Neither mRNA nor the protein product were detectable in any of the patients, while various amounts of WASP protein were expressed in carriers, normal controls, haematopoietic cell lines of all lineages and in one patient after receiving allogeneic bone marrow transplantation. Conclusion Genetic and protein analysis is useful in the definite diagnosis and follow up of Wiskott-Aldrich syndrome patients and in carrier detection, especially of atypical or sporadic patients. Received: 14 January 1999 / Accepted: 8 April 1999     

一个Laron 综合征家系患者临床特点和生长激素受体基因突变分析

Laron 综合征是一种常染色体隐性遗传病,生长激素受体（GHR）基因缺陷是导致Laron 综合征的主要病因。Laron 综合征主要临床特征为生后严重的生长落后伴特殊面容,血生化特点为高生长激素(GH)、低胰岛素样生长因子- (IGF-I) 和低胰岛素样生长因子结合蛋白- (IGFBP-3)。该研究报道一家系2例Laron 综合征患者的临床特点及GHR基因突变。这两个病人为同胞姐弟。弟弟8岁,身高80.0 cm (-8.2 SDS),姐姐11岁,身高96.6 cm (-6.8 SDS)。他们出生体重和身长无特殊,自生后出现生长落后,身高明显落后于同龄正常儿童,并均呈现了典型Laron 综合征外貌特征：身材矮、肥胖、前额突出、大眼睛、塌鼻梁、头发稀软。这两个病人空腹血清GH值均明显高于正常儿童,空腹血清IGF-I明显低于同年龄同性别正常儿童,血浆IGFBP-3和生长激素结合蛋白（GHBP）低于检测线。其中1例（8岁男孩）胰岛素和可乐定刺激后GH峰值大于350 ng/mL,给予重组人生长激素治疗1年,身高由治疗前的80.0 cm 增加至83.3 cm。GHR基因序列测定结果显示2例患者均存在外显子4上第65位氨基酸的纯合突变S65H(TCA → CCA),为新发现的突变。Laron 综合征患者存在特殊的面貌特征,结合血GH、IGF-I、IGFBP-3和GHBP测定可以明确诊断。GHR基因外显子4上S65H突变可能是这两位Laron 综合征患者的致病原因。［中国当代儿科杂志,2007,9（4）：335-338］     

Laron综合征家系患者生长激素受体基因突变分析

目的研究Laron综合征患者的临床特点及生长激素受体（GHR）的基因突变。方法对一家系中2例表现为身材明显落后的患儿进行研究，分别进行体格测量和GH-IGF—I轴功能测定。提取外周血白细胞基因组DNA，运用聚合酶链式反应（PCR）扩增GHR基因第2～9外显子及其两侧的侧翼序列，直接进行测序，将所测结果与正常人GHR基因序列进行对比，确定突变位点和突变类型。对突变基因均经2次测定证实。结果2例患儿自出生后身长明显落后于同龄儿童，呈现Laron综合征的独特面貌，空腹血清GH值均明显高于正常儿童。空腹血清IGF-I明显低于同年龄同性别正常儿童。血浆IGFBP-3和GHBP低于检测线。GHR基因序列测定结果显示2例患儿均存在外显子4上第65为氨基酸的纯合突变S65H，为新发现突变。结论Laron综合征患者存在典型的面貌特征，结合血GH、IGF-I、IGFBP-3和GHBP测定可以明确诊断。GHR基因外显子4上S65H突变可能是该Laron综合征患者的致病原因。     

半乳糖血症患儿半乳糖-1-磷酸尿苷酰转移酶基因突变的鉴定与分析

目的 从分子水平研究半乳糖血症患儿半乳糖-1-磷酸尿苷酰转移酶基因(GALT基因)突变情况.方法 选取2例确诊为半乳糖血症的患儿,分离其外周血淋巴细胞,提取总RNA,RT-PCR扩增GALT基因全长cDNA;将PCR产物亚克隆至T-easy载体,筛选阳性克隆并测序;同时,采用限制性片段多态性的方法将PCR产物进行酶切鉴定.结果 2例患儿均出现了未报道过的突变.其中1例患儿GALT基因1006位A突变为G,导致M336V氨基酸突变;另1例患儿GALT基因779位A突变为T,导致H260L突变.这2例突变均为碱基替换形成的错义突变,且均为杂合型突变.结论 基因诊断可提高半乳糖血症诊断的准确性,并为该病的产前筛查、造血干细胞移植甚至基因治疗提供有价值的参考.     

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.     

Use of human androgen receptor gene analysis to aid the diagnosis of JMML in female noonan syndrome patients

Noonan syndrome (NS) patients are at increased risk for developing juvenile myelomonocytic leukemia (JMML), an aggressive clonal disorder of aberrant cell proliferation. Many NS patients exhibit spontaneously remitting monocytosis and transient myeloproliferation. The distinction between bone marrow hyperproliferation due to germline mutation and leukemia resulting from clonal transformation can be difficult in NS patients. The GM-CSF hypersensitivity assay, diagnostic of sporadic JMML, can be positive in NS patients at baseline. In this report, we demonstrate the utility of determining the clonal status of the monocyte population by the HUMARA assay in distinguishing JMML and benign myeloproliferation in female NS patients.     

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.     

Six novel mutations of the fibrillin-1 gene in Korean patients with Marfan syndrome

BACKGROUND: Mutations in the FBN1 gene, encoding fibrillin-1, result in Marfan syndrome (MFS). According to previous reports, the mutations in FBN1 share certain characteristics in each family with variable penetrance and overlapping symptoms, even in the same genotype. In the present study, we report six novel mutations and evaluate the clinical significance of these nucleotide changes. METHODS: To screen for nucleotide changes in all 65 exons of the FBN1 gene in 38 unrelated Korean patients, we performed polymerase chain reaction, single-strand conformational polymorphism (SSCP) and sequencing for the shift of the band in SSCP. RESULTS: We identified six mutations: a 2253 del 7 b.p., N1043S, C1254S, L1421F, C1895R and S2662P. CONCLUSIONS: These results suggest that many different mutations are responsible for MFS in the Korean population.     

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目的了解穿孔素基因(PRF1)突变和序列变异在中国儿童噬血细胞综合征(HLH)中的发生情况,探讨基因突变型与临床表现之间可能的关系。方法应用聚合酶链反应(PCR)结合直接测序方法,对2006年1月至2008年5月在首都医科大学附属北京儿童医院治疗的临床诊断为HLH的30例患儿(HLH组)及50名新生儿(对照组)PRF1基因外显子编码区进行突变筛查。结果在3例HLH患儿的PRF1基因外显子编码区发现3个杂合错义突变,这3个突变均导致氨基酸改变(C102F、S108N和T450M),而在对照组中却未发现。1例患儿为复合杂合错义突变(S108N和T450M),从遗传学上可明确诊断为家族性HLH亚型2(FHL2);1个同义序列变异(Q540Q)在1例患儿中发现,而在对照组中未发现;在HLH组和对照组的PRF1基因编码区发现2个单核苷酸多态位点(SNP)(A274A、H300H),但这2个SNP的基因型频率在HLH组和对照组之间的分布差异无统计学意义(P均>0.05)。结论我国HLH患儿中存在PRF1基因突变,而突变位点(C102F和S108N)目前仅在中国患儿中发现。显示了我国HLH患儿PRF1基因突变具有自身的特点...     

Prevalence of germline mutations in the TSH receptor gene as a cause of juvenile thyrotoxicosis

Aim: To ascertain the prevalence of germline mutations in the TSH receptor gene as a cause of juvenile thyrotoxicosis (JT) in non-autoimmune patients. TSH receptor gene mutations are not seen in autoimmune-active patients. Methods: In a nationwide study on JT, 123 patients were re-examined 10 y (range 4 to 21 y) after diagnosis. Two patients with toxic adenoma were excluded. In 25 patients, no TPO, TG or TSH-R antibodies were found. In 17 patients, DNA material was available for TSH receptor gene analysis. The entire TSH receptor gene was sequenced in five patients. TSH receptor “hot spots” for mutations in exon 9 and 10 were sequenced in the remaining 12 patients. Results: A TSH receptor gene germline mutation was identified in only one patient of a total number of 121 patients with JT, of which 17 patients were presumed to have non-autoimmune JT by the lack of thyroid autoantibodies.

Conclusion: In Denmark the prevalence of germline mutations in the TSH receptor gene is one in 121 patients with JT (0.8%; 95% CI: 0.02-4.6%) and one in 17 patients with presumed non-autoimmune JT (6%; 95% CI: 5.88% (0.15-28.69)).