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1.  Associations between androgen receptor CAG & GGN repeat polymorphism & recurrent spontaneous abortions in Chinese women 
Background & objectives:
Recurrent spontaneous abortion (RSA) is a reproductive problem that occurs in women in reproductive age with a frequency of 1-3 per cent. Previous studies have reported high levels of serum androgens to be associated with RSAs. At the molecular level, the effect of androgens is mediated through the activation of the androgen receptor (AR). The CAG and GGN repeat polymorphisms of the AR gene are associated with the AR activity. We hypothesize that the AR CAG/GGN repeat polymorphism may be associated with levels of serum androgens. Thus, this study as undertaken to evaluate the relationship between CAG/GGN repeats in exon 1 of the AR gene in women with RSAs.
Methods:
This case-control study was performed in Ningxia, PR China, including 149 women with RSAs and 210 controls. The CAG and GGN repeats of the AR gene were genotyped using a PCR-based assay and were analyzed using Peak Scanner Software v1.0 to determine the CAG/GGN repeat length.
Results:
CAG repeats ranged from 15 to 29 in the RSA patients, compared to 14 to 35 in the control group. The median value of CAG repeats was 22 for the RSA group and 24 for control group. The total AR CAG alleles (≤22 repeats), shorter AR CAG alleles (≤22 repeats), and biallelic means (≤22.5 repeats) were significantly different in the RSA group in comparison to the control group (P<0.001, P<0.01). The median value of the GGN repeats was 23 for the cases and 22 for controls. The total number of AR GGN alleles (≤23 repeats) was significantly different in the RSA group compared to the control group (P<0.5). There was no difference between the RSA group and the control groups in regards to shorter alleles, longer alleles, and biallelic means.
Interpretation & conclusions:
Our observation suggests that the CAG and GGN repeat length is shorter in women with RSAs as compared with controls and that shorter CAG and GGN repeats may be pathogenic for RSAs in Chinese women. Further studies need to be done in different ethnic populations.
PMCID: PMC4140038  PMID: 25027083
Androgen; androgen receptor gene; CAG repeats; GGN repeats; recurrent spontaneous abortion
2.  Androgen Receptor CAG Repeat Polymorphism and Epigenetic Influence among the South Indian Women with Polycystic Ovary Syndrome 
PLoS ONE  2010;5(8):e12401.
The present study was carried out to assess the role of androgen receptor CAG repeat polymorphism and X chromosome inactivation (XCI) pattern among Indian PCOS women and controls which has not been hitherto explored and also to test the hypothesis that shorter CAG alleles would be preferentially activated in PCOS. CAG repeat polymorphism and X chromosome methylation patterns were compared between PCOS and non-PCOS women. 250 PCOS women and 299 controls were included for this study. Androgen receptor CAG repeat sizes, XCI percentages, and clinical and biochemical parameters were measured. The mean CAG repeat number is similar between the cases (18.74±0.13) and controls (18.73±0.12). The obese PCOS women were significantly more frequent in the <18 and >20 CAG repeat category than the lean PCOS women, yielding a highly significant odds (p = 0.001). Among the women with non-random X-inactivation, alleles with <19 repeats were more frequently activated among cases than controls (p = 0.33). CAG repeat polymorphism by itself cannot be considered as a useful marker for discriminating PCOS. We observed a trend of preferential activation of the shorter allele among the PCOS cases with non random XCI pattern. In the obese PCOS women, this microsatellite variation may account for the hyperandrogenicity to a larger extent than the lean PCOS women.
doi:10.1371/journal.pone.0012401
PMCID: PMC2928732  PMID: 20865044
3.  The role of X-chromosome inactivation in female predisposition to autoimmunity 
Arthritis Research  2000;2(5):399-406.
We propose that the phenomenon of X-chromosome inactivation in females may constitute a risk factor for loss of T-cell tolerance; specifically that skewed X-chromosome inactivation in the thymus may lead to inadequate thymic deletion. Using a DNA methylation assay, we have examined the X-chromosome inactivation patterns in peripheral blood from normal females (n = 30), female patients with a variety of autoimmune diseases (n = 167). No differences between patients and controls were observed. However, locally skewed X-chromsome inactivation may exist in the thymus, and therefore the underlying hypothesis remains to be disproved.
Introduction:
A reduction in the sex ratio (male : female) is characteristic of most autoimmune disorders. The increased prevalence in females ranges from a modest 2:1 for multiple sclerosis [1], to approximately 10:1 for systemic lupus erythematosus [2]. This tendency toward autoimmunity in females is often ascribed to hormonal differences, because in a number of experimental disease models estrogens exacerbated disease, and androgens can inhibit disease activity [3,4]. However, human studies have failed to demonstrate a clear-cut influence of hormonal environment on disease susceptibility to lupus or other autoimmune disorders. In addition, many childhood forms of autoimmunity, such as juvenile rheumatoid arthritis, exhibit female predominance [5]. Interestingly, juvenile (type 1) diabetes is an exception to this general trend, with a sex ratio close to 1 in most studies [6]. Therefore, it is reasonable to consider alternative explanations for the increased prevalence of autoimmune diseases in human females.
A unifying feature of autoimmune disorders appears to be the loss of immunologic tolerance to self-antigens, and in many of these diseases there is evidence that T-cell tolerance has been broken. The most profound form of T-cell tolerance involves deletion of potentially self-reactive T cells during thymic selection. Thus, lack of exposure to a self-antigen in the thymus may lead to the presence of autoreactive T cells and may increase the risk of autoimmunity. An elegant example of this has recently been reported [7].
The existence of X-chromosome inactivation in females offers a potential mechanism whereby X-linked self-antigens may escape presentation in the thymus or in other peripheral sites that are involved in tolerance induction. Early in female development, one of the two X chromosomes in each cell undergoes an ordered process of inactivation, with subsequent silencing of most genes on the inactive X chromosome [8]. This phenomenon occurs at a very early embryonic stage [9], and thus all females are mosaic and may occasionally exhibit extreme skewing towards one or the other parental X chromosome. In theory, this may result in a situation in which polymorphic self-antigens on one X chromosome may fail to be expressed at sufficiently high levels in a tolerizing compartment, such as the thymus, and yet may be expressed at a considerable frequency in the peripheral soma. Thus, females may be predisposed to a situation in which they can occasionally express X-linked autoantigens in the periphery to which they have been inefficiently tolerized. Stewart [10] has recently speculated that such a mechanism may play a role in the predisposition to systemic lupus.
This hypothesis predicts that females with autoimmunity may be particularly prone to this mechanism of `inadequate tolerization' by virtue of extremely skewed X-chromosome inactivation. We therefore performed a comprehensive analysis of X-chromosome inactivation patterns in populations of females with multiple sclerosis, systemic lupus erythematosus, juvenile rheumatoid arthritis, and type 1 (insulin-dependent) diabetes mellitus, and in female control individuals. The results do not provide support for a major role for skewed X-chromosome inactivation in female predisposition to autoimmunity; however, neither is the underlying hypothesis disproved by the present data.
Materials and method:
DNA was obtained from female patients from the following sources: 45 persons with juvenile diabetes seen at the Virginia Mason Research Center in Seattle, Washington; 58 multiple sclerosis patients seen at the New York Hospital Multiple Sclerosis Center; 46 patients with systemic lupus erythematosus seen at the Hospital for Special Surgery (New York); 18 patients with juvenile rheumatoid arthritis seen at the Children's Hospital Medical Center in Cleveland. In addition, 30 healthy age-matched females were studied as normal controls.
Employing a modification of previously described methods [11], we utilized a fluorescent Hpa II/PCR assay of the androgen receptor (AR) locus to assess X-chromosome inactivation patterns. The AR gene contains a polymorphic CAG repeat, which is flanked by Hpa II sites. These Hpa II sites are methylated on the inactive X chromosome, and are unmethylated on the active X chromosome. By performing PCR amplification across this region after cutting with the methylation-sensitive enzyme Hpa II, the relative amounts of the methylated AR alleles can be quantitatively determined with a high degree of accuracy; variance on repeated assays is approximately 4% [12].
Skewing of X-chromosome inactivation is expressed as percentage deviation from equal (50:50) inactivation of the upper and lower AR alleles. Therefore, the maximal possible deviation is 50%, in which case all of the X chromosomes bearing one of the AR alleles are inactivated.
Results:
We examined X-chromosome inactivation patterns in several different populations. The results are summarized in Fig. 1. A wide range of X-inactivation skewing was observed in all five groups. Approximately 5% (nine out of 197) of individuals exhibited extreme skewing (greater than 40% deviation from a 50:50 distribution). However, there was no difference between the groups, either in the overall mean skewing, or in the fraction of individuals with extreme skewing (>40%).
Although the present study was not initiated in order to examine allelic variation in the AR gene per se, the data provide an opportunity to address this question. Excessively long CAG repeats in the AR are a rare cause of spinal-bulbar muscular atrophy [13], and AR repeat length appears to have an influence on the biology of certain tumors [14,15]. In this context, it has been shown that transcription of AR correlates inversely with repeat length [16]. We therefore compared AR repeat length in control individuals and patients with autoimmunity. No differences were observed for mean repeat length, or for maximum and minimum repeat length, among the five groups.
Discussion:
The reason for the female predominance in most autoimmune diseases remains obscure. The present study was initiated in order to address the hypothesis that a nonhormonal mechanism related to X inactivation might be involved. The hypothesis rests on the idea that skewing of X inactivation might lead to a deficiency of tolerance induction in the thymus, particularly with respect to polymorphic X-linked autoantigens. The hypothesis predicts that skewed X inactivation would be more prevalent in females with autoimmune diseases than in female control individuals. This was not observed.
Nevertheless, these negative data do not rule out a role for X inactivation in female predisposition to loss of tolerance. A general model for how this mechanism might operate is shown in Fig. 2. Thymocytes undergo selection in the thymic parenchyma and, in the case of negative selection, the selecting elements appear to be derived from the bone marrow and consist mainly of thymic dendritic cells. If the thymic dendritic cell population exhibits random X inactivation, it is highly likely that differentiating thymocytes will contact dendritic cells that express self-antigens on both X chromosomes. This situation is outlined schematically on the left side of Fig. 2. However, if there is extremely skewed X inactivation in the thymic dendritic cell population, a particular thymocyte might not come into contact with dendritic cells that express one of the two X chormosomes. This would lead to a situation where T cells may undergo thymic maturation without having been negatively selected for antigens that are expressed on the predominantly inactive X chromosome. This situation is shown on the right side of Fig. 2.
In order for this mechanism to be physiologically relevant, some assumptions must be made. First, defective tolerance from skewed X inactivation should only be directed at X-linked antigens that are polymorphic, and for which the individual is heterozygous. Thus, this mechanism would not be expected to lead to lack of tolerance commonly, unless there are at least several highly polymorphic X-linked autoantigens in the population that are involved in thymic deletion events. Second, if this actually leads to autoimmunity, it also predicts that the initial break in tolerance that leads to disease should involve an X-linked autoantigen that is expressed in a peripheral nontolerizing site or circumstance.
A recent report [7] has elegantly demonstrated the importance of thymic deletion events in predisposition to autoimmune disease. The proteolipid protein (PLP) autoantigen is expressed in alternatively spliced forms, which exhibit tissue specific expression. A nonspliced variant is expressed in peripheral neural tissue. However, in the thymus a splice variant results in the lack of thymic expression of an immunodominant peptide. This results in loss of tolerace of T cells to this peptide, presumably on the basis of lack of thymic deletion of thymocytes that are reactive with this antigen. Interestingly, PLP is encoded on the X chromsome. However, there is no evidence that genetic polymorphisms control the level splicing of PLP within the thymus. Nevertheless, these data illustrate the potential importance of deficiencies in thymic deletion for autoimmune T-cell reactivity.
The present results suggest that if skewed X inactivation is relevant to thymic tolerance induction, then the effect does not depend on global skewing of X-chromosome inactivation, at least in the hematopoietic compartment. In this study we examined X-inactivation patterns in peripheral blood mononuclear cells, and the results should reflect the state of X inactivation in all mesenchymal tissues, including dendritic cells. X inactivation occurs at a very early time point in development, and thus the results in one tissue should reflect the general situation in the rest of the body. However, there may be exceptions to this. We have occasionally observed differences in X-inactivation patterns between buccal mucosa (an ectodermally derived tissue) and peripheral blood in the same individiual (unpublished observations). This could be a chance event, or it may result from selection for certain X-linked alleles during embryonic development, as has been described in carriers of X-linked immunodeficiencies [17].
Another consideration is that certain tissue microenvironments may be derived from very small numbers of founder cells, and thus may exhibit skewed utilization of one or the other X chromosome, even if the tissue as a whole is not skewed. This situation could vary over time. Thus, there may be time points at which certain thymic microenvironments are populated by dendritic cells that, for stochastic reasons, all utilize the same X chromosome. This would create a `window of opportunity' in which a given thymocyte, in a given selecting location, could escape negative selection by antigens on the inactive X chromosome. The likelihood of this happening would obviously depend on the number of dendritic cells that are usually contacted by a thymocyte during thymic selection. There is limited information on this point, although Stewart [10] has theorized that this number may be as low as 15. If this is the case, then escape from thymic deletion may still occur in females who are heterozygous for a relevant X-linked antigen, even if the hematopoietic cells in general do not exhibit extreme skewing.
In conclusion, we suggest that X-chromosome inactivation needs to be considered as a potential factor in the predominance of females in most autoimmune diseases. Our inability to show an increase in X-chromosome skewing in females with autoimmunity does not eliminate this as an etiologic contributor to loss of immunologic tolerance. Future experiments must be directed at a detailed analysis of tissue patterns of X inactivation, as well as at a search for potential X-linked autoantigens.
PMCID: PMC17816  PMID: 11056674
autoimmunity; gender; immune tolerance; X chromosome
4.  CAG repeat length in the androgen receptor gene is related to age at diagnosis of prostate cancer and response to endocrine therapy, but not to prostate cancer risk 
British Journal of Cancer  1999;81(4):672-676.
The length of the polymorphic CAG repeat in the N-terminal of the androgen receptor (AR) gene is inversely correlated with the transactivation function of the AR. Some studies have indicated that short CAG repeats are related to higher risk of prostate cancer. We performed a case–control study to investigate relations between CAG repeat length and prostate cancer risk, tumour grade, tumour stage, age at diagnosis and response to endocrine therapy. The study included 190 AR alleles from prostate cancer patients and 186 AR alleles from female control subjects. All were whites from southern Sweden. The frequency distribution of CAG repeat length was strikingly similar for cases and controls, and no significant correlation between CAG repeat length and prostate cancer risk was detected. However, for men with non-hereditary prostate cancer (n = 160), shorter CAG repeats correlated with younger age at diagnosis (P = 0.03). There were also trends toward associations between short CAG repeats and high grade (P = 0.07) and high stage (P = 0.07) disease. Furthermore, we found that patients with long CAG repeats responded better to endocrine therapy, even after adjusting for pretreatment level of prostate-specific antigen and tumour grade and stage (P = 0.05). We conclude that short CAG repeats in the AR gene correlate with young age at diagnosis of prostate cancer, but not with higher risk of the disease. Selection of patients with early onset prostate cancer in case–control studies could therefore lead to an over-estimation of the risk of prostate cancer for men with short CAG repeats. An association between long CAG repeats and good response to endocrine therapy was also found, but the mechanism and clinical relevance are unclear. © 1999 Cancer Research Campaign
doi:10.1038/sj.bjc.6690746
PMCID: PMC2362888  PMID: 10574254
prostatic neoplasms; cancer risk; androgen receptor; genetics; epidemiology
5.  Preliminary evidence of a noncausal association between the X-chromosome inactivation pattern and thyroid autoimmunity: a twin study 
An increased frequency of skewed X-chromosome inactivation (XCI) is found in clinically overt autoimmune thyroid disease (AITD) compared with controls. Whether skewed XCI is involved in the pathogenesis of autoantibodies to thyroid peroxidase (TPOAb) in euthyroid subjects is unknown. To examine the impact of XCI on the serum concentration of TPOAb, we studied whether within-cohort and within-twin-pair differences in XCI are associated with differences in serum concentrations of TPOAb. A total of 318 euthyroid female twin individuals distributed in 159 pairs were investigated. XCI was determined by PCR analysis of a polymorphic CAG repeat in the first exon of the androgen receptor gene. TPOAb concentrations were measured using a solid-phase time-resolved fluoroimmunometric assay. Overall (within cohort), there was a significant association between XCI and serum concentrations of TPOAb; regression coefficient (β)=1.45 (95% confidence interval, 0.52–2.38), P=0.003. The association remained significant in the within-pair analysis; β=1.74 (0.79–2.69), P<0.001. The relationship was nonsignificant within the 82 monozygotic pairs (β=0.57 (−0.78–1.92), P=0.405), whereas the association was significant in the 77 dizygotic pairs (β=2.17 (0.81–3.53), P=0.002). This preliminary finding of a significant association between TPOAb concentrations and XCI within cohort and within dizygotic but not within monozygotic twin pairs may indicate that XCI per se does not have a major role in the pathogenesis of TPOAb. More likely, XCI and TPOAb are influenced by shared genetic determinants.
doi:10.1038/ejhg.2009.156
PMCID: PMC2987197  PMID: 19789576
X-chromosome inactivation; thyroid peroxidase antibodies; thyroid autoantibodies; thyroid autoimmunity; twins; epigenetics
6.  Analysis of skewed X-chromosome inactivation in females with rheumatoid arthritis and autoimmune thyroid diseases 
Arthritis Research & Therapy  2009;11(4):R106.
Introduction
The majority of autoimmune diseases such as rheumatoid arthritis (RA) and autoimmune thyroid diseases (AITDs) are characterized by a striking female predominance superimposed on a predisposing genetic background. The role of extremely skewed X-chromosome inactivation (XCI) has been questioned in the pathogenesis of several autoimmune diseases.
Methods
We examined XCI profiles of females affected with RA (n = 106), AITDs (n = 145) and age-matched healthy women (n = 257). XCI analysis was performed by enzymatic digestion of DNA with a methylation sensitive enzyme (HpaII) followed by PCR of a polymorphic CAG repeat in the androgen receptor (AR) gene. The XCI pattern was classified as skewed when 80% or more of the cells preferentially inactivated the same X-chromosome.
Results
Skewed XCI was observed in 26 of the 76 informative RA patients (34.2%), 26 of the 100 informative AITDs patients (26%), and 19 of the 170 informative controls (11.2%) (P < 0.0001; P = 0.0015, respectively). More importantly, extremely skewed XCI, defined as > 90% inactivation of one allele, was present in 17 RA patients (22.4%), 14 AITDs patients (14.0%), and in only seven controls (4.1%, P < 0.0001; P = 0.0034, respectively). Stratifying RA patients according to laboratory profiles (rheumatoid factor and anti-citrullinated protein antibodies), clinical manifestations (erosive disease and nodules) and the presence of others autoimmune diseases did not reveal any statistical significance (P > 0.05).
Conclusions
These results suggest a possible role for XCI mosaicism in the pathogenesis of RA and AITDs and may in part explain the female preponderance of these diseases.
doi:10.1186/ar2759
PMCID: PMC2745787  PMID: 19589151
7.  Involvement of different mechanisms for the association of CAG repeat length polymorphism in androgen receptor gene with prostate cancer 
While androgen and androgen receptor (AR) activity have been strongly implicated in prostate cancer development and therapy, the influence of the CAG repeat, which is found within the first exon of the AR gene, on prostate carcinogenesis is still unclear. We investigated the differences in the length of the CAG repeat between prostate cancer patients and controls in the Chinese population as well as between TMPRSS2:ERG fusion positive and negative samples. A general association between prostate cancer and either longer or shorter AR CAG repeat length was not observed in the Chinese population. However, our data suggest that certain CAG repeat lengths may increase or decrease prostate cancer risk. Shorter CAG repeat length was also not shown to be associated with a higher induction rate of TMPRSS2 and ERG proximity, an essential step for TMPRSS2:ERG fusion formation. However, samples with a CAG repeat of 17 were found more frequently in the TMPRSS2:ERG fusion positive than negative prostate cancer cases and mediated a higher rate of androgen-induced TMPRSS2 and ERG co-localisation than AR with longer (24) and shorter (15) CAG repeats. This suggests that 17 CAG repeats may be associated with TMPRSS2:ERG fusion positive prostate cancer, but may have a preventive role for prostate cancer in the Chinese population, which has a low TMPRSS2:ERG fusion frequency. This study suggests that different mechanisms for the association of CAG repeat length polymorphism and prostate cancer exist in different ethnic populations.
PMCID: PMC4266720  PMID: 25520876
CAG repeat; length polymorphism; androgen receptor gene; prostate cancer
8.  CAG Repeat Number in the Androgen Receptor Gene and Prostate Cancer 
Prostate cancer (PC) is the second leading cause of cancer deaths in men. The effects of androgens on prostatic tissue are mediated by the androgen receptor (AR) gene. The 5′ end of exon 1 of the AR gene includes a polymorphic CAG triplet repeat that numbers between 10 to 36 in the normal population. The length of the CAG repeats is inversely related to the transactivation function of the AR gene. There is controversy over association between short CAG repeat numbers in the AR gene and PC. This retrospective case-control study evaluates the possible effect of short CAG repeats on the AR gene in prostate cancer risk in Macedonian males. A total of 392 male subjects, 134 PC patients, 106 patients with benign prostatic hyperplasia (BPH) and 152 males from the general Macedonian population were enrolled in this study. The CAG repeat length was determined by fluorescent polymerase chain reaction (PCR) amplification of exon1 of the AR gene followed by capillary electrophoresis (CE) on a genetic analyzer. The mean repeat length in PC patients was 21.5 ± 2.65, in controls 22.28 ± 2.86 (p = 0.009) and in BPH patients 22.1 ± 2.52 (p = 0.038). Short CAG repeats (<19) were found in 21.64% of PC patients vs. 9.43% in BPH patients (p = 0.0154). We also found an association of low Gleason score (<7) with short CAG repeat (<19) in PC patients (p = 0.0306), and no association between the age at diagnosis of PC and BPH and CAG repeat length. These results suggest that reduced CAG repeat length may be associated with increased prostate cancer risk in Macedonian men.
doi:10.2478/v10034-012-0005-z
PMCID: PMC3776652  PMID: 24052720
Prostrate cancer (PC); Androgen receptor (AR) gene; CAG repeat; Benign prostatic hyperplasia (BPH)
9.  Androgen Receptor Cytosine, Adenine, Guanine Repeats, and Haplotypes in Relation to Ovarian Cancer Risk 
Cancer research  2005;65(13):5974-5981.
Biological and epidemiologic evidence suggest that androgen or its receptor may play a role in ovarian cancer pathogenesis. The most notable genetic factor influencing androgen receptor (AR) activity is the functional cytosine, adenine, guanine (CAG) repeat in which length is inversely proportional to its transactivational activity. Additional genetic variation due to single nucleotide polymorphisms in the AR gene may be captured through haplotypes. We genotyped the CAG microsatellite and six haplotype-tagging single nucleotide polymorphisms (rs962458, rs6152, rs1204038, rs2361634, rs1337080, rs1337082) of the androgen receptor gene in 987 ovarian cancer cases and 1,034 controls from a study conducted in New Hampshire and eastern Massachusetts between May 1992 and July 2003. We estimated haplotype frequencies and calculated odds ratios with 95% confidence intervals to evaluate the association between the haplotypes and the AR CAG microsatellite with ovarian cancer risk. We observed that carriage of two alleles with ≥22 CAG repeats was associated with an increased risk of ovarian cancer compared with carriage of two alleles with <22 CAG repeats (covariate-adjusted odds ratios, 1.31; 95% confidence intervals, 1.01-1.69). Five common haplotypes in the AR gene were identified, but no association between these and ovarian cancer risk was observed. Our results suggest that possession of two long AR alleles (≥22 CAG repeats) may be associated with increased risk of ovarian cancer compared with women with two short AR alleles (<22 CAG repeats).
doi:10.1158/0008-5472.CAN-04-3885
PMCID: PMC1364476  PMID: 15994977
10.  Androgen Receptor Trinucleotide Polymorphism in Leiomyoma 
Objective: Androgen receptor (AR) was detected in leiomyoma. AR gene has a polymorphic microsatellite encoding cytosine, adenine, and guanine (CAG) repeats. We aimed to investigate the association between the AR gene CAG repeats and leiomyoma.
Methods: Women were divided into two groups: (1) leiomyoma (n=159); (2) non- leiomyoma groups (n=129). Their CAG repeats were detected by polymerase chain reaction. The CAG repeats ranged in length from 168 bp (9 CAG repeats, genotype A) to 234 bp (31 CAG repeats, genotype W). Distributions of CAG repeats in both groups were compared.
Results: Genotype proportions of different CAG repeats for AR gene in both groups were significantly different. The genotype S (27 CAG repeats) is associated with higher susceptibility of leiomyoma. Distribution of CAG repeats in both groups appeared mono-peak distributions. Percentages of genotypes K-S (19–27 CAG repeats) in leiomyoma and non-leiomyoma groups were: (1) 5, 11, 19.5, 10.4, 12.9, 8.8, 7.5, 5.7, 4.4%; (2) 5.4, 14.3, 16.7, 12.8, 12.4, 5.8, 9.3, 7, 1.2%.
Conclusions: AR trinucleotide polymorphism is associated with leiomyoma susceptibility. The 27 CAG repeats is related with higher risk of leiomyoma.
doi:10.1007/s10815-004-8762-6
PMCID: PMC3455613  PMID: 15704521
Androgen receptor; leiomyoma; multiallele polymorphism; trinucleotide repeat polymorphism
11.  Androgen Receptor CAG Repeats Length Polymorphism and the Risk of Polycystic Ovarian Syndrome (PCOS) 
PLoS ONE  2013;8(10):e75709.
Objective
Polycystic ovarian syndrome (PCOS) refers to an inheritable androgen excess disorder characterized by multiple small follicles located at the ovarian periphery. Hyperandrogenism in PCOS, and inverse correlation between androgen receptor (AR) CAG numbers and AR function, led us to hypothesize that CAG length variations may affect PCOS risk.
Methods
CAG repeat region of 169 patients recruited following strictly defined Rotterdam (2003) inclusion criteria and that of 175 ethnically similar control samples, were analyzed. We also conducted a meta-analysis on the data taken from published studies, to generate a pooled estimate on 2194 cases and 2242 controls.
Results
CAG bi-allelic mean length was between 8.5 and 24.5 (mean = 17.43, SD = 2.43) repeats in the controls and between 11 and 24 (mean = 17.39, SD = 2.29) repeats in the cases, without any significant difference between the two groups. Further, comparison of bi-allelic mean and its frequency distribution in three categories (short, moderate and long alleles) did not show any significant difference between controls and various case subgroups. Frequency distribution of bi-allelic mean in two categories (extreme and moderate alleles) showed over-representation of extreme sized alleles in the cases with marginally significant value (50.3% vs. 61.5%, χ2 = 4.41; P = 0.036), which turned insignificant upon applying Bonferroni correction for multiple comparisons. X-chromosome inactivation analysis showed no significant difference in the inactivation pattern of CAG alleles or in the comparison of weighed bi-allelic mean between cases and controls. Meta-analysis also showed no significant correlation between CAG length and PCOS risk, except a minor over-representation of short CAG alleles in the cases.
Conclusion
CAG bi-allelic mean length did not differ between controls and cases/case sub-groups nor did the allele distribution. Over-representation of short/extreme-sized alleles in the cases may be a chance finding without any true association with PCOS risk.
doi:10.1371/journal.pone.0075709
PMCID: PMC3792992  PMID: 24116069
12.  A preliminary case study of androgen receptor gene polymorphism association with impulsivity in women with alcoholism 
Objective
The androgen receptor (AR) gene, located on the X chromosome, contains a common polymorphism involving cytosine–adenine–guanine (CAG) repeats, which impacts disease and could contribute to the unequal sex ratio in alcoholism. CAG repeats in the AR gene are known to correlate with impulsivity in males. We report the first preliminary study examining the association between the number of CAG repeats and measures of impulsivity in females with chronic alcoholism.
Methods
A total of 35 women and 85 men with chronic alcoholism were previously recruited for a nutritional clinical trial, and 26 well-characterized females (19 African–American and seven Caucasian) with alcoholism agreed to participate for genetic testing. Genomic deoxyribonucleic acid (DNA) was isolated from peripheral blood and CAG repeats determined by analyzing polymerase chain reaction (PCR)-amplified products, using the polymorphic AR gene assay. CAG repeat length was correlated with raw scores from the Barratt Impulsivity Scale, version 11 and the Alcoholism Severity Scale.
Results
CAG repeat lengths were significantly longer in Caucasian alcoholic women compared with African–Americans, and the average number of CAG repeats were significantly, positively correlated (P<0.05) with impulsivity scores. Women with average CAG repeat length (CAGave) ≥18, representing the upper quartile of the repeat range, showed significantly greater mean raw impulsivity scores. CAG repeat length appeared to have less effect in African–American compared with Caucasian women, possibly due to a shorter average repeat length.
Conclusion
We found an association between the number of CAG repeats and impulsivity in females with chronic alcoholism, specifically in women with CAGave ≥18, seen more commonly in Caucasian compared with African–American women.
doi:10.2147/AGG.S57771
PMCID: PMC4067054  PMID: 24966714
AR gene; CAG repeat; African-American; Caucasian; behavior
13.  Androgen Receptor CAG Repeat Length and Association with Prostate Cancer Risk: Results from the Prostate Cancer Prevention Trial 
The Journal of urology  2010;184(6):2297-2302.
PURPOSE
We investigated the association between the length of the polymorphic trinucleotide CAG microsatellite repeats in exon 1 of the androgen receptor (AR) gene and the risk of prostate cancer.
METHODS
This is a nested case-control study of 1159 cases and 1353 controls drawn from the Prostate Cancer Prevention Trial, a randomized, placebo-controlled trial testing whether finasteride, a 5α-reductase inhibitor, could reduce the 7-year period prevalence of prostate cancer. During the course of the PCPT, men underwent annual DRE and PSA measures and a prostate biopsy was recommended for all men with an abnormal DRE or a finasteride-adjusted PSA of > 4.0 ng/mL. Cases were drawn from men with biopsy-determined prostate cancer identified either by a for-cause or end-of-study biopsy and controls were selected from men who completed the end-of-study biopsy procedure.
RESULTS
CAG repeat mean lengths were not different between cases and controls. The frequency distribution for cases and controls for the AR CAG repeat length is similar. There were no significant associations of CAG repeat length with prostate cancer risk, either when stratified by treatment arm (finasteride or placebo) or when combined together. There was also no significant association between CAG repeat length and the risk of low- or high-grade prostate cancer.
CONCLUSIONS
There were no associations of the AR CAG repeat length and prostate cancer risk. Knowledge of AR CAG repeat length provides no clinical useful information for predicting prostate cancer risk.
doi:10.1016/j.juro.2010.08.005
PMCID: PMC3930175  PMID: 20952028
androgen receptor; CAG repeat length; prostate cancer
14.  Role of Progesterone Receptor Polymorphisms in the Recurrent Spontaneous Abortions: Indian Case 
PLoS ONE  2010;5(1):e8712.
Background
We attempt to ascertain if the 3 linked single nucleotide polymorphisms (SNPs) of the Progesterone Receptor (PR) gene (exon 1: G 1031 C; S344T, exon 4: G 1978 T; L660V and exon 5: C 2310 T; H770H) and the PROGINS insertion in the intron G, between exons 7 and 8, are associated with Recurrent Spontaneous Abortion (RSA) in the Indian population.
Methodology/Principal Findings
A total of 143 women with RSA and 150 controls were sequenced for all the 8 exons looking for the above 3 linked SNPs of the PR gene earlier implicated in the RSA, as well as for any new SNPs that may be possibly found in the Indian population. PROGINS insertion was screened by electrophoresis. We did not find any new mutations, not observed earlier, in our population. Further, we did not find significant role of the *2 allele (representing the mutant allele at the three SNP loci) or the T2 allele (PROGINS insertion) in the manifestation of RSA. We also did not find an LD pattern between each of the 3 SNPs and the PROGINS insertion.
Conclusions/Significance
The results suggest that the PR gene mutations may not play any exclusive role in the manifestation of RSA, and instead, given significantly higher frequency of the *2 allele among the normal women, we surmise if it does not really confer a protective role among the Indian populations, albeit further studies are required in the heterogeneous populations of this region before making any conclusive statement.
doi:10.1371/journal.pone.0008712
PMCID: PMC2806831  PMID: 20090851
15.  StuI polymorphism on the androgen receptor gene is associated with recurrent spontaneous abortion 
Purpose
This is a case- control study to determine whether G1733A polymorphism of androgen receptor gene is associated with an increased risk for recurrent spontaneous abortion (RSA).
Method
A total of 85 women with at least two recurrent spontaneous abortion before 20th week of gestation composed the study group. Subjects were genotyped by the polymerase chain reaction restriction fragment length polymorphism method.
Results
The observed frequencies of GG, GA and AA genotypes of the G1733A polymorphism were 5.89 %, 82.35 % and 11.76 %, respectively, for the patient group and 71.76 %, 23.51 % and 4.71 %, respectively, for the control group. Allele frequencies of the G1733A polymorphism among patients and controls were 0.47 and 0.84, respectively, for the dominant allele (G) (wild type) and 0.53 and 0.16, respectively, for the A allele (mutant type).
Conclusions
These results indicated that the androgen receptor G1733A polymorphism is strongly associated with increased risk for RSA.
doi:10.1007/s10815-013-9949-5
PMCID: PMC3607689  PMID: 23430227
Recurrent spontaneous abortion; Androgen receptor; Polymorphism; RFLP PCR
16.  X-chromosome Inactivation Patterns in Korean Women with Idiopathic Recurrent Spontaneous Abortion 
Journal of Korean Medical Science  2004;19(2):258-262.
Recurrent spontaneous abortion (RSA) defines as two or more consecutive losses at ≤20 weeks of gestation and affects an estimated 1 of every 100 couples wishing to have children. However, it remains a poorly understood phenomenon. Recent reports observed a significant association between highly skewed X chromosome and RSA, supporting that X chromosome inactivation might be an important and previously unknown cause of RSA. X-inactivation pattern, using polymeric X-linked women with idiopathic RSA and 80 control subjects with a single successful pregnancy and no history of spontaneous abortion. The ratio of heterozygotes was 68.2% (45/66) in women with RSA and 67.5% (54/80) in control group. Among 45 informative RSA cases, only 1 (2.2%) woman showed extreme skewed X inactivation (≥90%) and 4 (8.9%) had mild skewed inactivation (≥85%). In 54 heterozygous control subjects, 5 (9.3%) women showed extreme skewed X inactivation and 7 (13.0%) had mild one. The frequency of skewed X inactivation between RSA patients and control group was not significantly different (p>0.05). This finding suggests that skewed X chromosome be not associated with unexplained RSA patients.
doi:10.3346/jkms.2004.19.2.258
PMCID: PMC2822308  PMID: 15082900
Abortion, Spontaneous; X chromosome; Receptors, Androgen
17.  Long CAG Repeat Sequence and Protein Expression of Androgen Receptor Considered as Prognostic Indicators in Male Breast Carcinoma 
PLoS ONE  2012;7(12):e52271.
Background
The androgen receptor (AR) expression and the CAG repeat length within the AR gene appear to be involved in the carcinogenesis of male breast carcinoma (MBC). Although phenotypic differences have been observed between MBC and normal control group in AR gene, there is lack of correlation analysis between AR expression and CAG repeat length in MBC. The purpose of the study was to investigate the prognostic value of CAG repeat lengths and AR protein expression.
Methods
81 tumor tissues were used for immunostaining for AR expression and CAG repeat length determination and 80 normal controls were analyzed with CAG repeat length in AR gene. The CAG repeat length and AR expression were analyzed in relation to clinicopathological factors and prognostic indicators.
Results
AR gene in many MBCs has long CAG repeat sequence compared with that in control group (P = 0.001) and controls are more likely to exhibit short CAG repeat sequence than MBCs. There was statistically significant difference in long CAG repeat sequence between AR status for MBC patients (P = 0.004). The presence of long CAG repeat sequence and AR-positive expression were associated with shorter survival of MBC patients (CAG repeat: P = 0.050 for 5y-OS; P = 0.035 for 5y-DFS AR status: P = 0.048 for 5y-OS; P = 0.029 for 5y-DFS, respectively).
Conclusion
The CAG repeat length within the AR gene might be one useful molecular biomarker to identify males at increased risk of breast cancer development. The presence of long CAG repeat sequence and AR protein expression were in relation to survival of MBC patients. The CAG repeat length and AR expression were two independent prognostic indicators in MBC patients.
doi:10.1371/journal.pone.0052271
PMCID: PMC3522691  PMID: 23272232
18.  CAG and GGC repeat polymorphisms in the androgen receptor gene and breast cancer susceptibility in BRCA1/2 carriers and non-carriers 
British Journal of Cancer  2001;85(1):36-40.
Variation in the penetrance estimates for BRCA1 and BRCA2 mutations carriers suggests that other genetic polymorphisms may modify the cancer risk in carriers. A previous study has suggested that BRCA1 carriers with longer lengths of the CAG repeat in the androgen receptor (AR) gene are at increased risk of breast cancer (BC). We genotyped 188 BRCA1/2 carriers (122 affected and 66 unaffected with breast cancer), 158 of them of Ashkenazi origin, 166 BC cases without BRCA1/2 mutations and 156 Ashkenazi control individuals aged over 56 for the AR CAG and GGC repeats. In carriers, risk analyses were conducted using a variant of the log-rank test, assuming two sets of risk estimates in carriers: penetrance estimates based on the Breast Cancer Linkage Consortium (BCLC) studies of multiple case families, and lower estimates as suggested by population-based studies. We found no association of the CAG and GGC repeats with BC risk in either BRCA1/2 carriers or in the general population. Assuming BRCA1/2 penetrance estimates appropriate to the Ashkenazi population, the estimated RR per repeat adjusted for ethnic group (Ashkenazi and non-Ashkenazi) was 1.05 (95%CI 0.97–1.17) for BC and 1.00 (95%CI 0.83–1.20) for ovarian cancer (OC) for CAG repeats and 0.96 (95%CI 0.80–1.15) and 0.90 (95%CI 0.60–1.22) respectively for GGC repeats. The corresponding RR estimates for the unselected case–control series were 1.00 (95%CI 0.91–1.10) for the CAG and 1.05 (95%CI 0.90–1.22) for the GGC repeats. The estimated relative risk of BC in carriers associated with ≥28 CAG repeats was 1.08 (95%CI 0.45–2.61). Furthermore, no significant association was found if attention was restricted to the Ashkenazi carriers, or only to BRCA1 or BRCA2 carriers. We conclude that, in contrast to previous observations, if there is any effect of the AR repeat length on BRCA1 penetrance, it is likely to be weak. © 2001 Cancer Research Campaign http://www.bjcancer.com
doi:10.1054/bjoc.2001.1777
PMCID: PMC2363908  PMID: 11437399
BRCA1/2; androgen receptor; polymorphisms; breast cancer risk
19.  Salivary Testosterone and a Trinucleotide (CAG) Length Polymorphism in the Androgen Receptor Gene Predict Amygdala Reactivity in Men 
Psychoneuroendocrinology  2010;35(1):94-104.
SUMMARY
In studies employing functional magnetic resonance imaging (fMRI), reactivity of the amygdala to threat-related sensory cues (viz., facial displays of negative emotion) has been found to correlate positively with interindividual variability in testosterone levels of women and young men and to increase on acute administration of exogenous testosterone. Many of the biological actions of testosterone are mediated by intracellular androgen receptors (ARs), which exert transcriptional control of androgen-dependent genes and are expressed in various regions of the brain, including the amygdala. Transactivation potential of the AR decreases (yielding relative androgen insensitivity) with expansion a polyglutamine stretch in the N-terminal domain of the AR protein, as encoded by a trinucleotide (CAG) repeat polymorphism in exon 1 of the X-chromosome AR gene. Here we examined whether amygdala reactivity to threat-related facial expressions (fear, anger) differs as a function of AR CAG length variation and endogenous (salivary) testosterone in a mid-life sample of 41 healthy men (mean age = 45.6 yr, range: 34–54 yr; CAG repeats, range: 19–29). Testosterone correlated inversely with participant age (r = −0.39, p = 0.012) and positively with number of CAG repeats (r = 0.45, p = 0.003). In partial correlations adjusted for testosterone level, reactivity in the ventral amygdala was lowest among men with largest number of CAG repeats. This inverse association was seen in both the right (rp = −0.34, p<0.05) and left (rp = −0.32, p<0.05) hemisphere. Activation of dorsal amygdala, correlated positively with individual differences in salivary testosterone, also in right (r = 0.40, p<0.02) and left (r = 0.32, p<0.05) hemisphere, but was not affected by number of CAG repeats. Hence, androgenic influences on threat-related reactivity in the ventral amygdala may be moderated partially by CAG length variation in the AR gene. Because individual differences in salivary testosterone also predicted dorsal amygdala reactivity and did so independently of CAG repeats, it is suggested that androgenic influences within this anatomically distinct region may be mediated, in part, by non-genomic or AR-independent mechanisms.
doi:10.1016/j.psyneuen.2009.04.013
PMCID: PMC2825741  PMID: 19493626
testosterone; androgen receptor; CAG repeat polymorphism; fMRI; amygdala; facial expressions of emotion
20.  5meCpG Epigenetic Marks Neighboring a Primate-Conserved Core Promoter Short Tandem Repeat Indicate X-Chromosome Inactivation 
PLoS ONE  2014;9(7):e103714.
X-chromosome inactivation (XCI) is the epigenetic transcriptional silencing of an X-chromosome during the early stages of embryonic development in female eutherian mammals. XCI assures monoallelic expression in each cell and compensation for dosage-sensitive X-linked genes between females (XX) and males (XY). DNA methylation at the carbon-5 position of the cytosine pyrimidine ring in the context of a CpG dinucleotide sequence (5meCpG) in promoter regions is a key epigenetic marker for transcriptional gene silencing. Using computational analysis, we revealed an extragenic tandem GAAA repeat 230-bp from the landmark CpG island of the human X-linked retinitis pigmentosa 2 RP2 promoter whose 5meCpG status correlates with XCI. We used this RP2 onshore tandem GAAA repeat to develop an allele-specific 5meCpG-based PCR assay that is highly concordant with the human androgen receptor (AR) exonic tandem CAG repeat-based standard HUMARA assay in discriminating active (Xa) from inactive (Xi) X-chromosomes. The RP2 onshore tandem GAAA repeat contains neutral features that are lacking in the AR disease-linked tandem CAG repeat, is highly polymorphic (heterozygosity rates approximately 0.8) and shows minimal variation in the Xa/Xi ratio. The combined informativeness of RP2/AR is approximately 0.97, and this assay excels at determining the 5meCpG status of alleles at the Xp (RP2) and Xq (AR) chromosome arms in a single reaction. These findings are relevant and directly translatable to nonhuman primate models of XCI in which the AR CAG-repeat is monomorphic. We conducted the RP2 onshore tandem GAAA repeat assay in the naturally occurring chimeric New World monkey marmoset (Callitrichidae) and found it to be informative. The RP2 onshore tandem GAAA repeat will facilitate studies on the variable phenotypic expression of dominant and recessive X-linked diseases, epigenetic changes in twins, the physiology of aging hematopoiesis, the pathogenesis of age-related hematopoietic malignancies and the clonality of cancers in human and nonhuman primates.
doi:10.1371/journal.pone.0103714
PMCID: PMC4117532  PMID: 25078280
21.  Handedness and the X chromosome: The role of androgen receptor CAG-repeat length 
Scientific Reports  2015;5:8325.
Prenatal androgen exposure has been suggested to be one of the factors influencing handedness, making the androgen receptor gene (AR) a likely candidate gene for individual differences in handedness. Here, we examined the relationship between the length of the CAG-repeat in AR and different handedness phenotypes in a sample of healthy adults of both sexes (n = 1057). Since AR is located on the X chromosome, statistical analyses in women heterozygous for CAG-repeat lengths are complicated by X chromosome inactivation. We thus analyzed a sample of women that were homozygous for the CAG-repeat length (n = 77). Mixed-handedness in men was significantly associated with longer CAG-repeat blocks and women homozygous for longer CAG-repeats showed a tendency for stronger left-handedness. These results suggest that handedness in both sexes is associated with the AR CAG-repeat length, with longer repeats being related to a higher incidence of non-right-handedness. Since longer CAG-repeat blocks have been linked to less efficient AR function, these results implicate that differences in AR signaling in the developing brain might be one of the factors that determine individual differences in brain lateralization.
doi:10.1038/srep08325
PMCID: PMC4321186  PMID: 25659367
22.  The relationship between anogenital distance and the androgen receptor CAG repeat length 
Asian Journal of Andrology  2013;15(2):286-289.
Anogenital distance (AGD) is used to define degree of virilization of genital development, with shorter length being associated with feminization and male infertility. The first exon of the androgen receptor (AR) consists of a polymorphic sequence of cytosine–adenine–guanine (CAG) repeats, with longer CAG repeat lengths being associated with decreased receptor function. We sought to determine if there is an association between AGD and AR CAG repeat length. A cross-sectional, prospective cohort of men evaluated at a urology clinic at a single institution was recruited. AGD (the distance from the posterior scrotum to the anal verge) and penile length (PL) were measured. Sanger DNA sequence analysis was used to define CAG repeat length. AGD and CAG repeat lengths in 195 men were determined. On unadjusted analysis, there was no linear relationship between CAG repeat length and PL (P=0.17) or AGD (P=0.31). However, on sub-population analyses, those men with longer CAG repeat lengths (>26) had significantly shorter AGDs compared to men with shorter CAG repeat lengths. For example, the mean AGD was 41.9 vs. 32.4 mm with a CAG repeat length ≤26 vs. >26 (P=0.01). In addition, when stratifying the cohort based on AGD, those with AGD less than the median (i.e. 40 mm) had a longer CAG repeat length compared to men with an AGD >40 mm (P=0.02). In summary, no linear relationship was found between AGD and AR CAG repeat length overall.
doi:10.1038/aja.2012.126
PMCID: PMC3739145  PMID: 23334200
androgen receptor (AR); anogenital distance (AGD); genitalia; penile length (PL); perineum
23.  Androgen receptor gene polymorphism and sex hormones in elderly men: the Tromsø study 
Asian Journal of Andrology  2009;11(2):222-228.
The aim of this study was to examine whether CAG/GGN repeats are significant modulators of serum concentrations of total and free testosterone (T) as well as of luteinizing hormone (LH) in elderly men. Sixty-nine 60- to 80-year-old men with subnormal T levels (≤ 11.0 nmol L−1) and 104 men with normal T levels taking part in a nested case-control study were used for these analyses. Sex hormones were measured and free T was calculated. The CAG and GGN polymorphisms in the androgen receptor gene were determined by polymerase chain reaction and subsequent direct sequencing. There were no differences in the CAG and GGN repeat lengths between the groups. In cross-sectional analyses of the whole cohort, total and free T were positively associated with CAG length (all P < 0.05) before, but not after, waist circumference or body mass index was added to the model. CAG repeat lengths were weakly, but not independently, associated with total and free T. These findings indicate that when clinically evaluating T and LH levels in elderly men, the CAG and GGN repeat lengths do not need to be taken into consideration.
doi:10.1038/aja.2008.7
PMCID: PMC3735021  PMID: 19137002
androgen receptor gene polymorphism; luteinizing hormone; testosterone
24.  Polymorphic repeat in AIB1 does not alter breast cancer risk 
Breast Cancer Research : BCR  2000;2(5):378-385.
We assessed the association between a glutamine repeat polymorphism in AIB1 and breast cancer risk in a case-control study (464 cases, 624 controls) nested within the Nurses' Health Study cohort. We observed no association between AIB1 genotype and breast cancer incidence, or specific tumor characteristics. These findings suggest that AIB1 repeat genotype does not influence postmenopausal breast cancer risk among Caucasian women in the general population.
Introduction:
A causal association between endogenous and exogenous estrogens and breast cancer has been established. Steroid hormones regulate the expression of proteins that are involved in breast cell proliferation and development after binding to their respective steroid hormone receptors. Coactivator and corepressor proteins have recently been identified that interact with steroid hormone receptors and modulate transcriptional activation [1]. AIB1 (amplified in breast 1) is a member of the steroid receptor coactivator (SRC) family that interacts with estrogen receptor (ER)α in a ligand-dependent manner, and increases estrogen-dependent transcription [2]. Amplification and overexpression of AIB1 has been observed in breast and ovarian cancer cell lines and in breast tumors [2,3]. A polymorphic stretch of glutamine amino acids, with unknown biologic function, has recently been described in the carboxyl-terminal region of AIB1 [4]. Among women with germline BRCA1 mutations, significant positive associations were observed between AIB1 alleles with 26 or fewer glutamine repeats and breast cancer risk [5]
Aim:
To establish whether AIB1 repeat alleles are associated with breast cancer risk and specific tumor characteristics among Caucasian women.
Patients and methods:
We evaluated associations prospectively between AIB1 alleles and breast cancer risk in the Nurses' Health Study using a nested case-control design. The Nurses' Health Study was initiated in 1976, when 121 700 US-registered nurses between the ages of 30 and 55 years returned an initial questionnaire reporting medical histories and baseline health-related exposures. Between 1989 and 1990 blood samples were collected from 32 826 women. Eligible cases in this study consisted of women with pathologically confirmed incident breast cancer from the subcohort who gave a blood specimen. Cases with a diagnosis anytime after blood collection up to June 1, 1994, with no previously diagnosed cancer except for nonmelanoma skin cancer were included. Controls were randomly selected participants who gave a blood sample and were free of diagnosed cancer (except nonmelanoma skin cancer) up to and including the interval in which the cases were diagnosed, and were matched to cases on year of birth, menopausal status, postmenopausal hormone use, and time of day, month and fasting status at blood sampling. The nested case-control study consisted of 464 incident breast cancer cases and 624 matched controls. The protocol was approved by the Committee on Human Subjects, Brigham and Womens' Hospital, Boston, Massachusetts USA. Information regarding breast cancer risk factors was obtained from the 1976 baseline questionnaire, subsequent biennial questionnaires, and a questionnaire that was completed at the time of blood sampling. Histopathologic characteristics, such as stage, tumor size and ER and progesterone receptor (PR) status, were ascertained from medical records when available and used in case subgroup analyses.
AIB1 repeat alleles were determined by automated fluorescence-based fragment detection from polymerase chain reaction (PCR)-amplified DNA extracted from peripheral blood lymphocytes. Fluorescent 5' -labeled primers were utilized for PCR amplification, and glutamine repeat number discrimination was performed using the ABI Prism 377 DNA Sequencer (Perkin-Elmer, Foster City, CA, USA). Genotyping was performed by laboratory personnel who were blinded to case-control status, and blinded quality control samples were inserted to validate genotyping identification procedures (n = 110); concordance for the blinded samples was 100%. Methods regarding plasma hormone assays have previously been reported [6]. Conditional and unconditional logistic regression models, including terms for the matching variables and other potential confounders, were used to assess the association of AIB1 alleles and breast cancer characterized by histologic subtype, stage of disease, and ER and PR status. We also evaluated whether breast cancer risk associated with AIB1 genotype differed within strata of established breast cancer risk factors, and whether repeat length in AIB1 indirectly influenced plasma hormone levels.
Results:
The case-control comparisons of established breast cancer risk factors among these women have previously been reported [7], and are generally consistent with expectation. The mean age of the women was 58.3 (standard deviation [SD] 7.1) years, ranging from 43 to 69 years at blood sampling. There were 188 premenopausal and 810 postmenopausal women, with mean ages of 48.1 (SD 2.8) years and 61.4 (SD 5.0) years, respectively, at blood sampling. Women in this study were primarily white; Asians, African-Americans and Hispanics comprised less than 1% of cases or controls.
The distribution of AIB1 glutamine repeat alleles and AIB1 genotypes for cases and controls are presented in Table 1. Women with AIB1 alleles of 26 glutamine repeats or fewer were not at increased risk for breast cancer (odds ratio [OR] 1.01, 95% confidence interval [CI] 0.75-1.36; Table 2). Results were also similar by menopausal status and in analyses additionally adjusting for established breast cancer risk factors. Among premenopausal women, the OR for women with at least one allele with 26 glutamine repeats or fewer was 0.82 (95% Cl 0.37-1.81), and among postmenopausal women the OR was 1.09 (95% Cl 0.78-1.52; Table 2). We did not observe evidence of a positive association between shorter repeat length and advanced breast cancer, defined as women with breast cancer having one or more involved nodes (OR 1.07, 95% Cl 0.64-1.78), or with cancers with a hormone-dependent phenotype (ER-positive: OR 1.16, 95% Cl 0.81-1.65; Table 3). No associations were observed among women who had one or more alleles with 26 glutamine repeats or fewer, with or without a family history of breast cancer (family history: OR 1.09; 95% Cl 0.46-2.58; no family history: OR 0.94; 95% Cl 0.68-1.31; test for interaction P = 0.65). We also did not observe associations with breast cancer risk to be modified by other established breast cancer risk factors. Among postmenopausal controls not using postmenopausal hormones, geometric least-squared mean plasma levels of estrone sulfate and estrone were similar among carriers and noncarriers of AIB1 alleles with 26 glutamine repeats or fewer (both differences: ≤ +3.5%; P >0.50). Mean levels of estradiol were slightly, but nonsignificantly elevated among carriers of alleles with 26 glutamine repeats or fewer (+11.6%; P = 0.08).
Discussion:
In this population-based nested case-control study, women with at most 26 repeating glutamine codons (CAG/CAA) within the carboxyl terminus of AIB1 were not at increased risk for breast cancer. We did not observe shorter repeat alleles to be positively associated with breast cancer grouped by histologic subtype, stage of disease, or by ER and PR status. These data suggest that AIB1 repeat length is not a strong independent risk factor for postmenopausal breast cancer, and does not modify the clinical presentation of the tumor among Caucasian women in the general population.
PMCID: PMC13920  PMID: 11056690
AIB1 polymorphism; breast cancer; genetic susceptibility; molecular epidemiology
25.  Relationships among androgen receptor CAG repeat polymorphism, sex hormones and penile length in Han adult men from China: a cross-sectional study 
Asian Journal of Andrology  2014;16(3):478-481.
This study aimed to investigate the correlations among androgen receptor (AR) CAG repeat polymorphism, sex hormones and penile length in healthy Chinese young adult men. Two hundred and fifty-three healthy men (aged 22.8 ± 3.1 years) were enrolled. The individuals were grouped as CAG short (CAGS) if they harbored repeat length of ≤20 or as CAG long (CAGL) if their CAG repeat length was >20. Body height/weight, penile length and other parameters were examined and recorded by the specified physicians; CAG repeat polymorphism was determined by the polymerase chain reaction (PCR) method; and the serum levels of the sex hormones were detected by radioimmunoassay. Student's t-test or linear regression analysis was used to assess the associations among AR CAG repeat polymorphism, sex hormones and penile length. This investigation showed that the serum total testosterone (T) level was positively associated with the AR CAG repeat length (P = 0.01); whereas, no significant correlation of T or AR CAG repeat polymorphism with the penile length was found (P = 0.593). Interestingly, an inverse association was observed between serum prolactin (PRL) levels and penile length by linear regression analyses (β= −0.024, P = 0.039, 95% confidence interval (CI): −0.047, 0). Collectively, this study provides the first evidence that serum PRL, but not T or AR CAG repeat polymorphism, is correlated with penile length in the Han adult population from northwestern China.
doi:10.4103/1008-682X.124560
PMCID: PMC4023382  PMID: 24589466
androgen receptor; CAG repeat; testosterone; prolactin; penile length

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