Molecular analysis of the androgen receptor gene in 52 patients with complete or partial androgen insensitivity syndrome: a collaborative study.

In patients with androgen insensitivity syndrome (AIS), RFLP study of the androgen receptor gene made it possible to analyze whether deletions or mutations could be responsible for abnormalities in androgen responsiveness. We studied RFLPs of DNA from 25 46,XY patients with partial AIS (PAIS), defined as a concentration of androgen receptor in genital-skin fibroblasts less than 340 fmol/mg DNA, and DNA from 27 46,XY patients with complete AIS (CAIS) with no detectable androgen receptor site. DNA samples were digested with BamHI, EcoRI, HindIII and TaqI restriction enzymes and hybridized with three cDNA probes covering the three domains of the androgen receptor. When we had the maternal and an unaffected brother's DNA, we analyzed the two androgen receptor gene polymorphisms described, the HindIII and the exon 1 CAG repeat polymorphisms, in order to distinguish the two maternal X chromosomes, and to detect carriers of AIS. We did not find any large deletion among the 52 patients. We observed a heterozygous mother in 3 of 14 families studied with the HindIII polymorphism, and in 12 of 25 families using the exon 1 CAG repeat polymorphism. This study suggests that in AIS, abnormalities in androgen receptor response could be related to point mutations or microdeletions rather than to gross structural alterations of the androgen receptor gene. Furthermore, unless the point mutation has been described, exon 1 and HindIII polymorphism studies would enable the identification of carriers in 50% of families, and the prenatal diagnosis of AIS.     

Prenatal prediction of androgen insensitivity syndrome using exon 1 polymorphism of the androgen receptor gene.

Exon 1 polymorphism of the androgen receptor (AR) gene is characterized by a (CAG)n(CAA) repeat at position 172 following the translation start codon. The aim of this study was to determine whether AR gene exon 1 polymorphism could be used to perform prenatal diagnosis in high risk families with complete or partial androgen insensitivity syndrome. After enzymatic amplification of a 1 kilobase exon 1 fragment, each DNA was simultaneously digested by MspI and PstI restriction enzymes. After electrophoresis on a 15% electrophoresis on a 15% acrylamide gel or a 6% Nusieve gel, we measured the size of the obtained fragments and determined the number of CAG repeats since a 282 basepair fragment corresponds to 21 CAG. We previously showed that the number of CAG repeats within the AR gene exon 1 in 23 families with complete or partial androgen insensitivity syndrome was 19 +/- 4. By this method, we detected heterozygosity in 50% of the mothers. We present here 2 exclusion prenatal diagnoses using exon 1 polymorphism of the AR gene. Family A presented a boy with a severe form of partial androgen insensitivity syndrome. The mother had 2 uncles with ambiguous genitalia. In family B, the affected child had a complete androgen insensitivity syndrome. In both families, analysis of the AR gene exon 1 polymorphism of the trophoblastic DNA showed the presence of the normal maternal X chromosome. The parents decided to carry on the gestation. In family A, the newborn had normal male external genitalia. In family B, sonography confirmed the presence of normal male external genitalia. These data suggest that exon 1 polymorphism of the AR gene could be prenatally used to predict androgen insensitivity syndrome.     

A novel primer extension method to detect the number of CAG repeats in the androgen receptor gene in families with X-linked spinal and bulbar muscular atrophy.

X-linked spinal and bulbar muscular atrophy (SBMA), an adult-onset form of motor neuron disease, was recently reported to be caused by amplification of the CAG repeats in the androgen receptor gene. We report here a simple and rapid strategy to detect the precise number of the CAGs. After the DNA fragment containing the CAG repeats is amplified by the polymerase chain reaction, a primer extension is carried out; the extension of the end-labelled reverse primer adjacent to 3' end of CAG repeats stops at the first T after CAG repeats with the incorporation of dideoxy ATP in the reaction mixture. The resultant primer products are analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. This method could be quite useful to detect not only CAG repeats in SBMA but also other polymorphic dinucleotide and trinucleotide repeats.     

Androgeninsensitivität

Mutations of the androgen receptor gene cause a spectrum of androgen insensitivity phenotypes ranging from women with female external genitalia through patients with genital ambiguities to men with male genitalia but infertility. The CAG repeat in the first exon is important for transactivation of target genes of the androgen receptor and is thought to modulate androgen-dependent processes. Expansion of this repeat is the cause of X-linked spinobulbar muscular atrophy.     

The androgen receptor gene and psychological traits: are results consistent in Sweden and Australia?

Studies in Sweden and Australia have examined the relationship between number of CAG repeats in the androgen receptor gene and psychological traits - three Masculinity-Femininity (M-F) measures in Australia, and the Karolinska Scales of Personality (KSP) in Sweden. The present study derived M-F scales from the KSP items, and scales corresponding to several KSP scales from the items of the inventories used in Australia, to permit cross-validation of the Australian results in the Swedish sample, and vice versa. The derivation of scales was facilitated by the fact that items from both inventories had been used with a large twin sample in the US. Correlation of the derived scales with androgen receptor gene CAG-repeat scores for women in the Australian and Swedish samples failed to provide clear evidence of replication of either set of original correlations in the other sample, although there were a few hints of consistency. It was concluded that if the number of CAG repeats on this gene is related to psychological traits at all, the relationship is a weak one.     

CAG repeat polymorphism in the androgen receptor (AR) gene of SBMA patients and a control group

Spinobulbar muscular atrophy (SBMA) is an X-linked form of motor neuron disease characterized by progressive atrophy of the muscles, dysphagia, dysarthria and mild androgen insensitivity. SBMA is caused by CAG repeat expansion in the androgen receptor gene. CAG repeat polymorphism was analysed in a Polish control group (n = 150) and patients suspected of SBMA (n = 60). Normal and abnormal ranges of CAG repeats were established in the control group and in 21 patients whose clinical diagnosis of SBMA was molecularly confirmed. The ranges are similar to those reported for other populations.     

PCR片段测序和基因分型两种方法对Kennedy遗传病的诊断

X连锁脊延髓肌萎缩症（SBMA）或肯尼迪病是一种成年人发病的神经变性疾病,以肌无力与慢性、进行性肌萎缩为特征. 通过PCR片段测序和基因分型法准确检测雄激素受体（AR）基因CAG复制数目,兄弟俩（来自同一个中国家庭）被确诊为隐性遗传性SBMA. 为了得到该中国家庭SMBA家系人员AR基因的CAG复制数目,我们采用了PCR片段测序和基因分型两种方法. 在该SMBA家系中有两个已发病的成年男性、未发病的年轻男性,及女性基因携带者. 两个已发病男性患者AR基因中CAG三核苷酸串重复数目分别是48和45. 以前的研究表明特定三核苷酸串重复数目的扩增可导致人类遗传性神经障碍疾病发病。我们的研究结果完全支持这一观点,SMBA中国家系的三核苷酸CAG拷贝数目检测结果表明,AR基因CAG扩增数目与SMBA发病相关. 关键词雄性激素受体; CAG多重三核苷酸重复; 肯尼迪病; 脊延髓肌萎缩症; X连锁     

High Resolution Melting Analysis Is a More Sensitive and Effective Alternative to Gel-Based Platforms in Analysis of SSR - An Example in Citrus

High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.     

Two polymorphic microsatellites in a coding segment of the canine androgen receptor gene

A 0.6-kb segment of exon 1 of the canine androgen receptor gene contains two polymorphic CAG tandem repeats which encode strings of glutamine homopolymers. The number of CAGs in each tandem repeat was determined by (1) polymerase chain reaction (PCR) amplification of a gene segment containing both repeats, (2) cleavage between repeats with restriction enzyme EcoO109I and (3) fractionation of the restriction fragments containing individual CAG repeats by denaturing polyacrylamide gel electrophoresis (PAGE). Individual genomic DNA samples from 80 unrelated dogs (53 males plus 27 females for a total of 107 X chromosomes) contained 10–12 CAGs in the 5′ repeats and 10–13 CAGs in the 3′ repeats. Thirteen distinct androgen receptor genotypes were identified. Eleven (or 41%) of the 27 unrelated females were heterozygous in one or both repeat regions, whereas all male samples produced single bands as expected for X chromosome markers. A total of seven distinct haplotypes contributed to the 13 genotypes. The ‘polymorphism information content’ or PIC for this seven-allele X chromosome marker was 0.67.     

High-throughput sequencing of core STR loci for forensic genetic investigations using the Roche Genome Sequencer FLX platform

The analysis and profiling of short tandem repeat (STR) loci is routinely used in forensic genetics. Current methods to investigate STR loci, including PCR-based standard fragment analyses and capillary electrophoresis, only provide amplicon lengths that are used to estimate the number of STR repeat units. These methods do not allow for the full resolution of STR base composition that sequencing approaches could provide. Here we present an STR profiling method based on the use of the Roche Genome Sequencer (GS) FLX to simultaneously sequence multiple core STR loci. Using this method in combination with a bioinformatic tool designed specifically to analyze sequence lengths and frequencies, we found that GS FLX STR sequence data are comparable to conventional capillary electrophoresis-based STR typing. Furthermore, we found DNA base substitutions and repeat sequence variations that would not have been identified using conventional STR typing.     

Extent of linkage disequilibrium between the androgen receptor gene CAG and GGC repeats in human populations: implications for prostate cancer risk

While studies have implicated alleles at the CAG and GGC trinucleotide repeats of the androgen receptor gene with high-grade, aggressive prostate cancer disease, little is known about the normal range of variation for these two loci, which are separated by about 1.1 kb. More importantly, few data exist on the extent of linkage disequilibrium (LD) between the two loci in different human populations. Here we present data on CAG and GGC allelic variation and LD in six diverse populations. Alleles at the CAG and GGC repeat loci of the androgen receptor were typed in over 1000 chromosomes from Africa, Asia, and North America. Levels of linkage disequilibrium between the two loci were compared between populations. Haplotype variation and diversity were estimated for each population. Our results reveal that populations of African descent possess significantly shorter alleles for the two loci than non-African populations (P<0.0001). Allelic diversity for both markers was higher among African Americans than any other population, including indigenous Africans from Sierra Leone and Nigeria. Analysis of molecular variance revealed that approx. 20% of CAG and GGC repeat variance could be attributed to differences between the populations. All non-African populations possessed the same common haplotype while the three populations of African descent possessed three divergent common haplotypes. Significant LD was observed in our sample of healthy African Americans. The LD observed in the African American population may be due to several reasons; recent migration of African Americans from diverse rural communities following urbanization, recurrent gene flow from diverse West African populations, and admixture with European Americans. This study represents the largest genotyping effort to be performed on the two androgen receptor trinucleotide repeat loci in diverse human populations.     

Profile--Richard A. Mathies. Interview by Suzanne Berry.

Richard A. Mathies (Fig. 1) is a professor of chemistry at the University of California (UC) at Berkeley. His early work at UC was on the use of resonance Raman and time-resolved optical spectroscopy to elucidate the structure and reaction dynamics of energy and information-transducing photoactive proteins called rhodopsins. His work on the Human Genome Project led to the development of high-throughput platform technologies including capillary array electrophoresis and energy transfer fluorescent dye labels for DNA sequencing and analysis. He has also pioneered the development of microfabricated capillary electrophoresis devices, capillary array electrophoresis microplates and microfabriated integrated sample preparation and detection methods. He is the co-founder of the Center for Analytical Biotechnology at UC Berkeley. Mathies was interviewed at the BIOMEMS and Biomedical Nanotechnology conference in Columbus, Ohio, 21-25 September 2001, where he gave a talk about capillary array electrophoresis-based microprocessors. Such devices could be used as point-of-care clinical and genetic analyzers, in integrated microfluidic sequencing chips and in DNA-based computing.     

The use of capillary electrophoresis for DNA polymorphism analysis

Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.     

Counting CAG repeats in the Huntington's disease gene by restriction endonuclease EcoP15I cleavage

Huntington's disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant inheritance. The disease is caused by a CAG trinucleotide repeat expansion located in the first exon of the HD gene. The CAG repeat is highly polymorphic and varies from 6 to 37 repeats on chromosomes of unaffected individuals and from more than 30 to 180 repeats on chromosomes of HD patients. In this study, we show that the number of CAG repeats in the HD gene can be determined by restriction of the DNA with the endonuclease EcoP15I and subsequent analysis of the restriction fragment pattern by electrophoresis through non-denaturing polyacrylamide gels using the ALFexpress DNA Analysis System. CAG repeat numbers in the normal (30 and 35 repeats) as well as in the pathological range (81 repeats) could be accurately counted using this assay. Our results suggest that this high-resolution method can be used for the exact length determination of CAG repeats in HD genes as well as in genes affected in related CAG repeat disorders.     

Androgen induced cell death in SHSY5Y neuroblastoma cells expressing wild-type and spinal bulbar muscular atrophy mutant androgen receptors

Spinal bulbar muscular atrophy (SBMA) is one of a family of inherited neurodegenerative diseases caused by expansion of CAG encoding polyglutamine repeats; in SBMA the affected gene is the androgen receptor. To understand further the mechanisms that lead to neuronal cell death in SBMA, we generated SHSY5Y neuroblastoma cell lines that stably express identical levels of wild-type (19 polyglutamine repeat) or SBMA (52 polyglutamine repeat) androgen receptor. Parental SHSY5Y cells do not express detectable levels of the androgen receptor. In the absence of androgen, the transfected cell lines have similar phenotypes and growth characteristics to parental SHSY5Y cells. However, upon treatment with androgen, both cell lines undergo a marked dose-dependent loss of viability; this loss was significantly greater in cells expressing the SBMA receptor. Morphological analyses of the androgen treated cells revealed that cell death bore hallmarks of apoptosis involving altered nuclear morphology and cleavage of poly(ADP-ribose) polymerase and of caspase 3 in both wild-type and SBMA cell lines. The caspase inhibitor VAD-fmk was able to decrease loss of viability of both cell lines on exposure to androgen.