Testicular development in the complete androgen insensitivity syndrome

The complete androgen insensitivity syndrome (CAIS), caused by mutations in the androgen receptor (AR) gene, is associated with abnormal testicular development and an increased risk of germ cell malignancy. Previous histological studies in CAIS have selected patients purely on the basis of clinical diagnosis and were mostly based on small numbers, many of whom were post-pubertal. Here, we present 44 cases of CAIS, each with molecular pathological confirmation of an AR mutation. The median age at gonadectomy was 5.5 years (5.5; IQR 1-13). We have been able, therefore, to investigate testicular development in infancy, childhood and puberty, and estimate the incidence of premalignant change in this series. In addition, we have investigated whether the presence of epididymides and/or vasa deferentia in CAIS, previously shown to be associated with residual activity of mutant ARs, is related to a particular testicular phenotype. Epididymides/vasa deferentia were present in 36% of cases and these patients showed varying degrees of seminiferous tubule maturation at puberty above those without epididymides/vasa deferentia (p = 0.003). There were no other histological differences between these patient groups. In both groups, features of testicular degeneration and dysgenesis were present and germ cell development was delayed, with prolonged expression of the gonocyte markers, placental-like alkaline phosphatase and activator protein-2gamma. Germ cell numbers rapidly declined after the first year of life (R(2) = 0.42). Only two cases of carcinoma in situ were identified in our study and both patients were postpubertal (17 and 53 years). From these results and the literature, we conclude that the risk of premalignant change in germ cells is low before and during puberty. Patients can be advised, therefore, that gonadectomy can be delayed to allow for a natural puberty, with low risk of malignant transformation. Our study only included one patient over 18 years, so we cannot comment on the risk of malignant transformation in later life.     

Down syndrome in association with features of the androgen insensitivity syndrome.

Three cases of Down syndrome (DS) are reported in association with features of the androgen insensitivity syndrome (AIS). All were 47, XY, +21 and reared as females. One case had a normal female phenotype, and two cases showed minimal clitoromegaly and labial fusion. Minor genital underdevelopment has been reported as common in males with DS; however, AIS has not previously been associated with DS. Androgen binding studies in genital skin fibroblasts were normal in two cases and in the 46,XY brother of the third who has perineal hypospadias. Mutation screening of the androgen receptor (AR) gene by PCR-SSCP was normal in all cases. Normal androgen binding and the absence of an identified mutation in the coding region of the AR gene is very unusual in AIS, particularly in the complete form. This finding suggests the operation of hitherto unrecognised genes on chromosome 21 with a role in androgen response and sex differentiation.     

完全型雄激素不敏感综合征雄激素受体基因突变分析

目的 对完全型雄激素不敏感综合征一家系雄激素受体(androgen receptor,AR)基因进行突变检测;并对发现突变的基因进行分析.方法 应用PCR扩增、DNA序列测定等技术分析所有AR基因外显子及其邻近DNA序列片段;应用核苷酸内切酶诊断方法观察其是否存在于正常人群;应用跨物种比对方法探讨突变所在位置的保守性.结果 3例患者AR基因第4外显子均发生E681D(GAG→GAT)错义突变,患者母亲为此突变杂合子携带者;患者父亲未见异常;正常人群未发现AR基因E681D突变;681位谷氨酸在不同物种间高度保守.结论 AR基因E681D(GAG→GAT)突变可能是导致完全型雄激素不敏感综合征新的突变方式.     

CD99 immunoreactivity in gastrointestinal and pulmonary neuroendocrine tumours

Although considered a specific marker for Ewing’s sarcoma/peripheral neuroectodermal tumour, the MIC2 gene product (CD99) has been immunolocalised in a variety of human tumours. The present study evaluated immunohistochemically the prevalence of CD99 expression in a series of 68 neuroendocrine tumours of different gastrointestinal and pulmonary sites. We now report on membrane and/or granular cytoplasmic immunoreactivity in 25% of these tumours, independent of their anatomical sites. In lung neuroendocrine tumours, CD99 was preferentially confined to typical carcinoids (P=0.009). A statistically significant relationship was observed between the number of CD99 positive cells but not the immunostaining patterns and the presence of local invasion and/or distant metastases (P<0.001). Moreover, there was a tendency for CD99-reactive tumours to show a reduced proliferative activity expressed by a Ki67 index of 2% (P=0.119). The number of CD99 immunoreactive cells or patterns of immunoreactivity did not correlate with the presence of associated clinical syndrome or particular hormonal immunostaining. Although the molecular basis underlying CD99 expression in neuroendocrine tumours is still poorly understood, our data suggest that CD99 may be involved in cell-to-cell adhesion of neuroendocrine tumour cells and in downregulation of their proliferative activity. Received: 10 February 2000 / Accepted: 6 April 2000     

Novel androgen receptor gene mutations in Australian patients with complete androgen insensitivity syndrome

We have identified androgen receptor (AR) gene mutations in eight Australian subjects with complete androgen insensitivity syndrome (AIS). Four individuals, from three families, have novel mutations that introduce premature termination codons. Two siblings have the nonsense mutation Glu681X, and another subject has the nonsense mutation p.Ser884X. The other subject has a CA insertion at codon 829 (c.2847_2848insCA), causing a frameshift mutation that introduces four nonsense amino acids prior to a Stop codon. All the termination codons occur in the ligand binding domain, and cause reduced androgen binding in patient genital skin fibroblasts. Four further patients have missense mutations. One subject has two different mutations, p.Ala645Asp in the hinge region of the receptor, and p.Arg752Gln in the ligand binding domain. Both these mutations have previously been reported in patients with AIS, but the combination of these two mutations in one subject is unique. Another subject has a novel c.2533G>C transversion at the first nucleotide in exon 5, introducing the amino acid change p.Gly724Ala at a highly conserved residue in the ligand binding domain. Androgen binding is normal in fibroblasts from this subject, although other point mutations at this amino acid totally abolish binding. Two other subjects have mutations previously described as causing AIS, namely p.Arg779Trp and p.Val889Met substitutions in the ligand binding domain of the receptor. The p.Arg779Trp mutation is associated with the detection of a truncated AR protein in this patient's fibroblasts, suggesting the mutation renders the receptor susceptible to proteolysis.     

Functional analysis of six androgen receptor mutations identified in patients with partial androgen insensitivity syndrome

Partial androgen insensitivity syndrome (PAIS) is caused by defects in the androgen receptor gene and presents with a wide range of undervirilization phenotypes. We studied the consequences of six androgen receptor ligand-binding domain mutations on receptor function in transfected cells. The mutations, Met742Ile, Met780Ile, Gln798Glu, Arg840Cys, Arg855His and Ile869Met, were identified in PAIS patients with phenotypes representing the full spectrum seen in this condition. In all cases the androgen receptor was found to be defective, suggesting that the mutation is the cause of the clinical phenotype. The Gln798Glu mutation is exceptional in that it did not cause an androgen-binding defect in our system, although the mutant receptor was defective in transactivation assays. This mutation may affect an aspect of binding not tested, or may be part of a functional subdomain of the ligand-binding domain involved in transactivation. Overall we found milder mutations to be associated with milder clinical phenotypes. There is also clear evidence that phenotype is not solely dependent on androgen receptor function. Some of the mutant receptors were able to respond to high doses of androgen in vitro, suggesting that patients carrying these mutations may be the best candidates for androgen therapy. One such mutation is Ile869Met. A patient carrying this mutation has virilized spontaneously at puberty, so in vivo evidence agrees with the experimental result. Thus a more complete understanding of the functional consequences of androgen receptor mutations may provide a more rational basis for gender assignment in PAIS.      

Leu-676-Pro mutation of the androgen receptor causes complete androgen insensitivity syndrome in a large Hutterite kindred

A large Manitoba Hutterite kindred with X-linked receptor negative complete androgen insensitivity syndrome (CAIS) was studied. In attempts to identify all carriers of the syndrome in this kindred, using the androgen receptor (AR) cDNA, we have found a novel diagnostic Mspl polymorphic pattern, which cosegregates with the disease. This polymorphism was not detected in 79 unrelated X-chromosomes of which 22 were from Hutterite controls. We were able to localize the polymorphism to exon 4, which is known to encode part of the androgen receptor hormone binding domain. A single base substitution (T→C) was detected, which creates a new Mspl site. This novel transition mutation replaces Leu-676 with Pro at a site which is conserved in numerous members of the steroid receptor gene family. Sequencing all 8 exons of the AR revealed the Leu-676→Pro mutation as the only change in the primary structure of the receptor. Transfection of COS-l cells with an expression vector of the mutant AR demonstrates that this point mutation of nucleotide 2558 abolishes receptor binding activity. The mutation can easily be detected by MspI digestion of the polymerase chain reaction (PCR) amplified exon 4 product.© 1995 wiley-Liss, Inc.     

Malignant sex cord–stromal tumour in a patient with the androgen insensitivity syndrome

A malignant sex cord–stromal tumour that occurred in a 56-year-old patient with the androgen insensitivity syndrome is reported. Although hamartomas composed of sex cord cells are common in the testes of patients with this syndrome, unequivocal neoplasms of sex cord type are rare. The tumour described herein most closely resembled, but lacked the overall morphology of, a juvenile granulosa cell tumour, and Charcot-Böttcher filaments, indicative of Sertoli cell differentiation, were seen on electron microscopy. The features of the androgen insensitivity syndrome and the various tumours that have been reported in patients with this syndrome are briefly reviewed.     

Carrier detection in the testicular feminisation syndrome: deficient 5 alpha-dihydrotestosterone binding in cultured skin fibroblasts from the mothers of patients with complete androgen insensitivity.

Specific binding of 5 alpha-dihydrotestosterone (androgen receptor activity) could not be detected in cultured genital skin fibroblasts (GSF) from two patients with complete androgen insensitivity (CAI). In GSF from the mother of one patient, androgen receptor activity (8.5 fmol/mg cell protein) was reduced in comparison with controls (34.0 +/- 10.1 (SD) fmol/mg protein n = 15). These results favour X linked inheritance of CAI and X inactivation at the androgen receptor locus. Androgen receptors were not detected in GSF of the second mother. It appears that female carriers of CAI could be detected by decreased 5 alpha-dihydrotestosterone binding in GSF. Androgen receptor activity was also undetectable in non-genital skin fibroblasts (NGSF) from the second mother and two further CAI patients. However, in 1 in 10 control NGSF lines androgen receptor activity was at the lower limit of assay sensitivity (1 to 2 fmol/mg protein) demonstrating that NGSF may not be reliable for family studies of androgen receptor deficiency.     

A novel point mutation (R840S) in the androgen receptor in a Brazilian family with partial androgen insensitivity syndrome

Mutations of the androgen receptor gene causing androgen insensitivity syndrome in 46, XY individuals, result in phenotypes ranging from complete female to ambiguous genitalia to males with minor degrees of undervirilization. We studied two Brazilian brothers with partial androgen insensitivity syndrome. They were born with perineal hypospadias, bifid scrotum, small penis and cryptorchidism, and developed gynecomastia at puberty. Genomic DNA was extracted and denaturinggradient gel electrophoresis of exon 7 of the androgen receptor gene followed by sequence analysis revealed a new mutation, a C A transversion, altering codon 840 from arginine (CGT) to serine (AGT). R840 is located in the androgen binding domain, in a “hot spot” region, important for the formation and function of the hormone receptor‐complex and within the region that is involved in androgen receptor dimerization. Replacement of arginine (basic) by serine (neutral and polar) is a nonconservative substitution. Three mutations in this residue (R840C, R840G nonconservative and R840H, conservative) were previously reported in patients with partial androgen insensitivity syndrome and when expressed “in vitro” lead to a subnormal transactivation of a reporter gene. We conclude that the novel R840 mutation in the androgen receptor is the cause of partial androgen insensitivity syndrome in this Brazilian family. Hum Mutat 14:353, 1999. © 1999 Wiley‐Liss, Inc.     

DHT-receptor in cultured human fibroblasts: binding study in a family with androgen insensitivity (complete testicular feminisation).

3H-DHT binding was examined in cultured skin fibroblasts from a patient with complete testicular feminisation (CTF), from his heterozygote mother, and his clinically normal sister, who menstruated normally. Binding parameters were: Bmax less than 1 fmol/mg protein and KD unmeasurable in CTF; Bmax = 24 fmol/mg protein and KD = 3.63 X 10(-9) mol/l in the mother; and Bmax = 46 fmol/mg protein and KD = 3.7 X 10(-9) mol/l in the sister. Five cultures obtained from genital and non-genital skin of normal male and female subjects were used as controls, in which Bmax ranged from 37 to 62 fmol/mg and KD from 2.0 to 3.6 X 10(-9) mol/l. The considerable reduction of Bmax in the obligate heterozygote and the normal binding capacity in the sister, a probable heterozygote, suggests that it may be possible to use the DHT-receptor assay to identify carriers in families with androgen resistance.     

Androgen receptor gene mutations identified by SSCP in fourteen subjects with androgen insensitivity syndrome.

The androgen insensitivity syndrome (AIS) is a disorder of male sexual development resulting in a wide range of clinical phenotypes. AIS is classified into two phenotypic forms: complete (CAIS) and partial (PAIS). To determine the molecular basis of the phenotypic diversity in AIS, we have studied 27 subjects (13 CAIS, 14 PAIS), spanning the full range of AIS phenotypes. We report the results of a mutation screen of the androgen receptor gene. The coding regions of the gene were amplified by the polymerase chain reaction and screened for single strand conformation polymorphisms to identify mutations. This was followed by DNA sequencing of putative mutant segments. Androgen receptor gene mutations were identified in nine CAIS and five PAIS subjects. Two of the CAIS mutations in exon A resulted in frameshifts. A third CAIS mutation resulted in the deletion of a single amino acid from the ligand binding domain of the receptor. All other mutations caused single amino acid substitutions in the ligand binding domain. These results suggest that mutations affecting the ligand binding domain of the androgen receptor are the most frequent cause of AIS, although some cases of PAIS may be the result of other, as yet undefined, genetic lesions.     

雄激素受体基因新突变引起的完全型雄激素不敏感综合征

目的 对1例完全型雄激素不敏感综合征(complete androgen insensitivity syndrom,CAIS)的雄激素受体(androgen receptor,AR)基因进行分析,寻找致病突变.方法 提取外周血全基因组DNA,扩增位于X染色体上的AR基因所有8个外显子及邻近外显子与内含子之间的剪切位点DNA序列,对扩增片段直接进行DNA序列测定,与基因库中的序列进行比对.结果 该病例AR基因第1外显子的441位密码子发生无义突变(GAA→TAA),由编码谷氨酸(Glu)变为终止密码,导致雄激素受体蛋白翻译至441位时终止,产生没有功能的氨基酸多肽残段.结论 Glu441stop(GAA→TAA)是一种导致CAIS的AR基因新的突变方式,之前在世界范围内均未见报道,丰富了AR基因突变谱,增加对CAIS发病机制的了解.

Abstract：

Objective To identify the mutation of human androgen receptor gene(AR) in a patient with complete androgen insensitivity syndrome (CAIS). Methods DNA sequences of 8 exons and their exon/intron boundaries of the AR gene in the patient were amplified by PCR and directly sequenced. Results DNA sequencing revealed a nonsense mutation in exon 1, resulting in a change of codon 441 GAA (glutamic acid) to a stop codon (TAA). Conclusion A novel mutation Glu441stop (GAA to TAA) of the androgen receptor gene leading to complete androgen insensitivity syndrome was identified in this study in a Chinese patient. It may help us further understanding the pathogenesis of CAIS.     

2例睾丸女性化综合征患者雄激素受体基因中的突变

目的：研究２例完全型睾丸女性化综合征（ＴＦＭ）患者的外阴部皮肤成纤维细胞（ＧＳＦ）中的雄激素受体（ＡＲ）。结果：发现两患者ＡＲ的数量无明显改变，但一患者的ＡＲ亲和力明显降低。用ＰＣＲ－ＳＳＣＰ（单链构象多态性）方法对两患者ＡＲ基因８个外显子中的７个（Ｂ－Ｈ）分别进行分析，发现两患者ＡＲ基因外显子Ｅ均存在着泳动变位，ＤＮＡ序列分析证实两患者外显子Ｅ各有一点突变，分别使得编码第７４３位和第７５２位的氨基酸发生了改变，后一突变还造成了一Ｓａｕ３ＡⅠ酶酶切位点的消失。结论：结合ＡＲ功能改变及临床表型可以认为，两患者的性分化发育异常是由于两位点的突变所致     

CD99 immunoreactivity in atypical fibroxanthoma: a common feature of diagnostic value.

Atypical fibroxanthoma (AFX), a pleomorphic superficial cutaneous tumor of low-grade malignancy, shares many morphologic features with malignant melanoma (MM) and squamous cell carcinoma (SCC). Absence of S-100, keratin, and desmin immunoreactivity is the clue for this diagnosis. In a search for positive markers, we tested 26 cases of AFX with 2 antibodies: O13 (CD99) and protein gene product 9.5 (PGP9.5). We also included 10 cases of poorly differentiated SCC and 10 cases of MM in the study. In AFX, CD99 immunoreactivity was present in 19 cases (73%), whereas focal PGP9.5 immunoreactivity was found in only 9 cases (35%). None of the SCC cases showed CD99 immunostaining. No CD99 immunoreactivity was found in 9 of 10 cases of MM. To our knowledge, this is the first report of CD99 and PGP9.5 immunostaining in AFX. We believe, based on our results, that CD99 may be a helpful "positive" feature in the histopathologic diagnosis of AFX.