Mutation screening and CAG repeat length analysis of the androgen receptor gene in Klinefelter's syndrome patients with and without spermatogenesis

BACKGROUND: Mutations of the androgen receptor (AR) gene give rise to a wide array of phenotypic abnormalities. A systematic analysis of the AR gene in patients with 47,XXY has not previously been performed. METHODS: Mutations of the AR gene and expansion of the CAG repeats in exon 1 of the AR gene were studied in 13 patients with Klinefelter's syndrome either with (n = 1) or without (n = 12) spermatogenesis. RESULTS: No abnormalities in the AR gene were detected by single strand conformational polymorphism analysis. The CAG lengths ranged from 17 to 27 (mean +/- SD 22.8 +/- 3.3, median 23) for Klinefelter patients or from 17 to 28 (mean +/- SD 23.2 +/- 2.6, median 23) for control subjects. X-inactivation analysis for the methylation status of the AR gene was performed in seven patients who were heterozygous for CAG repeats of different length, showing that the longer CAG repeat alleles underwent random but more frequent inactivation in five patients and skewed inactivation in two. CONCLUSIONS: An AR gene abnormality does not constitute an important factor for impaired spermatogenesis in patients with Klinefelter's syndrome.     

CAG repeat length variation in the polymerase gamma (POLG) gene: effect on semen quality

Several case–control studies have investigated the effectof CAG repeat length variation in the POLG gene on male fertilityand semen quality. Some described an association between thehomozygous not10 CAG-repeat genotype and male subfertility and/orreduced semen quality, whereas others did not. The aim of ourstudy was to investigate whether the not10/not10 variant isassociated with spermatogenic failure. By direct sequencingmethods, we determined the CAG repeat length of POLG in a cohortof 700 consecutive included men with variable degrees of spermatogenesisto investigate its effect on semen quality. The frequency ofthe not10/not10 variant in our cohort was 4.7%. There were nodifferences in semen quality between groups with various POLGgenotypes. There was a significant difference in frequency ofthe three CAG-repeat genotypes between ethnic subgroups. Inconclusion, the not10/not10 POLG variant is not associated withclinically significant decreases in semen quality, but its frequencyis dependent on ethnic background.     

中国男性雄激素受体基因(CAG)n重复多态性的初步研究

目的 研究正常中国男性雄激素受体（androgen receptor,AR)基因（CAG）n重复序列的多态性。方法 应用DNA测序基础上的[α-^32P]dCTP掺入不对称PCR-变性聚丙烯酰胺凝胶电泳（Polyacrylamide gel electrophoresis,PAGE)方法,对107名正常男性AR基因的（CAG）n重复数进行测定。结果 AR基因（CAG）n序列在正常男性人群中呈现重复多态性,其重复范围为11-29,集中于20-24,以22最多。结论 AR基因（CAG）n序列在正常男性人群中呈现重复多态性,为今后进一步研究其病理学及遗传学意义打下基础。     

Human sperm express a functional androgen receptor: effects on PI3K/AKT pathway

BACKGROUND: Results from mice lacking the androgen receptor (AR) showed that it is critical for the proper development and function of the testes. The aim of this study was to investigate whether a functional AR is present in human sperm. METHODS: The expression of AR and its effects on sperm were evaluated by RT-PCR, Western Blot, Immunocytochemistry, PI3Kinase and DNA laddering assays. RESULTS: We showed in human sperm that AR is located at the head region. Dihydrotestosterone (DHT), in a dose-dependent manner, leads to the rapid phosphorylation of the AR on tyrosine, serine and threonine residues and this effect was reduced by the AR antagonist hydroxyflutamide (OH-Flut). The effects of AR were evaluated on the phosphoinositide-3 kinase/protein kinase B (PI3K/AKT) pathway. Specifically, 0.1 and 1 nM DHT stimulated PI3K activity, whereas 10 nM DHT decreased PI3K activity and levels of p-AKT S473 and p-AKT T308, p-BCL2, and enhanced phosphatase and tensin homologue (PTEN) phosphorylation. In addition, 10 nM DHT was able to induce the cleavage of caspases 8, 9 and 3 and cause DNA laddering, and these effects were reversed either by casodex or OHFlut. By using wortmannin, a specific PI3K inhibitor, the cleavage of caspase 3 was reproduced, confirming that in sperm the PI3K/AKT pathway is involved in caspase activation. CONCLUSIONS: Human sperm express a functional AR that have the ability to modulate the PI3K/AKT pathway, on the basis of androgen concentration.     

Correlation between magnitude of CAG repeat length alterations and length of the paternal repeat in paternally inherited Huntington's disease

An increasing number of diseases are being found to be due to elongation of specific trinucleotide repeat sequences. Inverse correlation between the age at onset and the length of the repeat has been found in most of these. The elongated CAG repeat causing Huntington's disease is highly unstable when inherited from an affected father. In this study we found an average parent-to-offspring difference of +0.08 repeat units in maternally inherited repeats, significantly less than the average difference of +2.92 repeat units with paternal transmission. Large repeat expansions, of more than 5 repeat units, were seen only in paternally inherited cases. With paternal transmission the magnitude of repeat length alterations was directly correlated to increasing paternal repeat length. Increasing variation in repeat length among siblings was correlated to increasing average repeat length in the sibship in both maternally and paternally inherited HD. Comparison of the magnitude of repeat length alterations to parental age at the time of birth of the offspring showed no correlation.     

雄激素受体基因CAG、GGN重复片段多态性与身体质量指数/腰臀比的关联性

目的探讨雄激素受体基因CAG、GGN重复片段多态性与身体质量指数(BMI)、腰臀比(WHR)之间的关联性。方法研究对象来自宁夏医科大学2011级健康学生共654例,男性280例,女性374例,平均年龄(20.35±1.24)岁。用ABI 3730XL测序仪测定AR基因的(CAG)n及(GGN)n重复数目,直接测量法测得身高、体重、腰围与臀围。结果 BMI与WHR的分布存在性别差异。较长GGN等位基因与女性BMI呈正相关(P0.05),AR基因的(CAG)n与女性BMI无相关性(P0.05);男性AR基因的(CAG)n及(GGN)n与BMI及WHR无相关性(P0.05)。结论 BMI与WHR在不同性别间的分布存在差异性;较长GGN等位基因与女性BMI存在关联性。     

South Indian men with reduced CAG repeat length in the androgen receptor gene have an increased risk of prostate cancer

The androgen receptor (AR) gene possesses polymorphic CAG tandem repeats and the repeat length has been inversely related to the risk of prostate cancer (PCa). The distinct ethnic variation in the CAG repeat length may be correlated to differences in PCa risk in different populations. To evaluate the CAG repeat length in the AR gene and the implications for PCa, we screened 87 PCa patients and 120 control subjects from South India. The mean CAG repeat length in PCa patients was significantly smaller than that of controls (17.0 vs 20.7; P<0.001). Men with 19 CAG repeats had a significantly increased risk of cancer compared to those with >19 CAG repeats (age-adjusted OR=7.01; 95% CI=3.52–13.94; P<0.001). However, no significant association was observed between CAG repeats and age of onset or prostate-specific antigen levels. Although there was a trend towards shorter CAG repeat length in high grades of cancer, it was not significant (P=0.085). Thus, our results suggest an association between short CAG repeats in the AR gene and PCa risk in South Indian men. Further, we propose that CAG repeats could be used as a prognostic marker for PCa diagnosis.     

GGN repeat length and GGN/CAG haplotype variations in the androgen receptor gene and prostate cancer risk in south Indian men

The ethnic variation in the GGN and CAG microsatellites of the androgen receptor (AR) gene suggests their role in the substantial racial difference in prostate cancer risk. Hence, we performed a case-control study to assess whether GGN repeats independently or in combination with CAG repeats were associated with prostate cancer risk in South Indian men. The repeat lengths of the AR gene determined by Gene scan analysis, revealed that men with GGN repeats ≤21 had no significant risk compared to those with >21 repeats (OR 0.91 at 95% CI-0.52–1.58). However, when CAG repeats of our earlier study was combined with the GGN repeat data, the cases exhibited significantly higher frequency of the haplotypes CAG ≤19/GGN ≤21 (OR-5.2 at 95% CI-2.17–12.48, P < 0.001) and CAG ≤19/GGN > 21(OR-6.9 at 95%CI-2.85–17.01, P < 0.001) compared to the controls. No significant association was observed between GGN repeats and prostate-specific antigen levels and the age at diagnosis. Although a trend of short GGN repeats length in high-grade was observed, it was not significant (P = 0.09). Overall, our data reveals that specific GGN/CAG haplotypes (CAG ≤19/GGN ≤21 and CAG ≤19/GGN > 21) of AR gene increase the risk of prostate cancer and thus could serve as susceptibility marker for prostate cancer in South Indian men.     

The relationship between human sperm apoptosis, morphology and the sperm deformity index

BACKGROUND: This study aimed to assess the relationship between apoptosis in human ejaculated spermatozoa, sperm morphology and the novel sperm deformity index (SDI). METHODS: Semen specimens from 50 healthy donors were prepared by density-gradient centrifugation followed by incubating the prepared sperm with paramagnetic annexin V-conjugated microbeads and subjecting this to magnetic cell sorting (MACS). The procedure delivers two sperm fractions: annexin-negative (non-apoptotic) and annexin-positive (apoptotic). Activated caspase-3 levels and the integrity of the sperm mitochondrial membrane potential (MMP) were assessed as markers of apoptosis in the annexin-negative and -positive aliquots following MACS. Sperm morphology and the SDI scores were assessed using the strict criteria. RESULTS: Compared with the apoptotic sperm subpopulations, the non-apoptotic sperm subpopulations had an improved sperm morphology profile as demonstrated by significantly higher proportions of sperm with normal morphology and significantly lower SDI scores and percentages of sperm with acrosomal defects, midpiece defects, cytoplasmic droplet and tail defects. There was a significant correlation between sperm morphology attributes studied and the expressed apoptotic markers - caspase-3 activation and MMP integrity. CONCLUSIONS: Non-apoptotic sperm fractions have morphologically superior quality sperm compared with apoptotic fractions as reflected by significantly lower SDI scores. The study results may support abortive apoptosis, where the apoptotic mechanism of sperm is already triggered prior to ejaculation.     

Testosterone levels in relation to oral contraceptive use and the androgen receptor CAG and GGC length polymorphisms in healthy young women

BACKGROUND: The combined effect from the androgen receptor (AR) CAG and GGC length polymorphisms on testosterone levels has not been studied in young women. METHODS: Testosterone levels were measured in blood drawn on both menstrual cycle days 5-10 and 18-23 in 258 healthy women, aged < or =40 years, from high-risk breast cancer families. CAG and GGC length polymorphisms were analysed by PCR and fragment analyses. All women completed a questionnaire including information on oral contraceptive (OC) use and reproductive factors. RESULTS: OC users had lower median testosterone levels than non-users during cycle days 5-10 and 18-23 (P < or = 0.005 for both). The BRCA mutation status was associated neither with testosterone levels nor with CAG or GGC length polymorphism. The CAG length polymorphism was not associated with testosterone levels. The cumulative number of long GGC alleles (> or =17 repeats) was significantly associated with lower testosterone levels in OC users during cycle days 5-10 (P(trend) =0.014), but not during cycle days 18-23 or in non-users. The interaction between the GGC length polymorphism and OC status was highly significant during cycle days 5-10 (P = 0.002) and near-significant during days 18-23 (P = 0.07). Incident breast cancer was more common in women with two short GGC alleles (log-rank P = 0.003). CONCLUSION: The GGC repeat length was the only significant genetic factor modifying the testosterone levels in current OC users from high-risk families. Homozygosity for the short GGC allele may be linked to the increased risk of early-onset breast cancer after OC exposure in high-risk women.     

Association between CAG repeat length in the PPP2R2B gene and Alzheimer disease in the Japanese population

We analyzed the association between PPP2R2B gene CAG repeat length and Alzheimer disease (AD) susceptibility in the Japanese population. Blood samples were collected from 218 late-onset AD patients and 86 controls. DNA fragments containing the target CAG repeat region were amplified using polymerase chain reaction (PCR). PCR products were sequenced using ABI PRISM 310 genetic analyzer. The mean CAG repeat length did not differ significantly between the control and AD groups. In contrast, the frequency of CAG repeats shorter than 15 was significantly higher in AD group, specifically in the AD with APOE4 subgroup, than in the control group. The results suggest that CAG repeat lengths in the PPP2R2B gene may be potential genetic markers for AD susceptibility in the Japanese population.     

Androgen receptor gene (CAG)n repeat analysis in the differential diagnosis between Kennedy disease and other motoneuron disorders

An increase in the number of (CAG)n repeats in the first coding exon of the androgen receptor (AR) gene has been strongly associated with Kennedy disease (KD) (spinal and bulbar muscular atrophy). This is an X-linked hereditary disorder characterized by motoneuron degeneration occurring in adults together with gynecomastia and hyperestrogenemia. We have performed AR gene molecular analysis in several members of a large family with KD as well as in 25 sporadic patients suffering from heterogeneous motoneuron disease (MND). An increase in the length of the (CAG)n repeats was detected, as expected, in all the affected males and in obligatory carrier females, some of which had minor signs of lower motoneuron involvement. There was only one possible exception, one young male with initial signs of the disease, who had an apparent normal length allele. An increased pathological allele was also found in 3 patients with MND. This indicates that the analysis of (CAG)n repeats of the AR gene plays a role in the differential diagnosis of this heterogeneous group of neurological diseases. © 1995 Wiley-Liss, Inc.     

Factors associated with ATXN2 CAG/CAA repeat intergenerational instability in Spinocerebellar ataxia type 2

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by the unstable expansion of a cytosine‐adenine‐guanine (CAG)/cytosine‐adenine‐adenine (CAA) repeat in the ATXN2 gene, which normally encodes 22 glutamines (Q22). A large study was conducted to characterize the CAG/CAA repeat intergenerational instability in SCA2 families. Large normal alleles (Q24‐31) were significantly more unstable upon maternal transmissions. In contrast, expanded alleles (Q32‐750) were significantly more unstable during paternal transmissions, in correlation with repeat length. Significant correlations were found between the instability and the age at conception in paternal transmissions. In conclusion, intergenerational instability at ATXN2 locus is influenced by the sex, repeat length and age at conception of the transmitting parent. These results have profound implications for genetic counseling services.     

Andrology: Human sperm morphology evaluation pre- and post-Percoll gradient centrifugation

Previous experiments have established a relationship betweenthe morphological characteristics of human spermatozoa and theirfertilizing potential in vitro. To assess further the efficiencyof Percoll gradient centrifugation (PGC) as a method of spermselection, we have examined morphological characteristics ofspermatozoa from 86 teratozoospermic patients attending NottinghamUniversity Research and Treatment Unit in Reproduction (NURTURE).Patients were divided into groups according to percentage normalmorphology in the fresh sample: group A (n = 14), <5% normalmorphology; group B (n = 41), 5–14% normal morphology;and group C (n = 31), >14% normal morphology. Morphologyslides were prepared using Diff Quik staining techniques andevaluated by Kruger strict criteria, under oil immersion, ata magnification of x 1000; specific defects, viz. head, neck,cytoplasmic droplets, tail, immature cells, were assessed individually.Following PGC, a sperm sample with enhanced morphology was recoveredfor groups B (P < 0.01) and C (P < 0.005); however, forgroup A (very severe teratozoospermia) PGC did not select asample with significantly improved morphological quality. Specificsperm defects affected by PGC were head, neck and immature cells.No significant difference was found for tail abnormalities orcytoplasmic fragments.     

Association of repeat polymorphisms in the estrogen receptors alpha, beta, and androgen receptor genes with knee osteoarthritis

Genetic factors have been shown to play an important role in the etiology of osteoarthritis (OA). To elucidate the possible role of genetic variation in the estrogen receptors alpha and beta (ER-alpha, ER-beta) and androgen receptor (AR) genes with knee OA, the -1174(TA)(n), c.1092+3607(CA)(n), and c.172(CAG)(n) repeat polymorphisms of ER-alpha, ER-beta, and AR genes were studied. A case-control cohort of 158 patients with idiopathic knee OA and 193 controls were used. A significant difference was observed in the frequency distribution of -1174(TA)(9-25) and c.1092+3607(CA)(13-27) repeat polymorphisms of the ER-alpha and ER-beta genes between OA patients and controls (p<0.005 and p<0.0001, respectively). A significantly increased odds ratio (OR) for knee OA was observed in individuals having long alleles (LL) genotype for ER-alpha gene and LL and one short and one long allele (SL) genotypes for ER-beta gene compared to individuals with the short alleles (SS) genotype (95% CI 1.03-3.5; p=0.04 and CI 2.4-8.3 and 2.5-7.5; p < 0.001, respectively). When ORs were adjusted for various risk factors, it was observed that women with LL genotypes for ER-beta and AR genes showed significantly increased risk for OA development (p=0.002 and 0.001). An association between c.1092+3607(CA)(13-27) and c.172(CAG)(8-34) repeat polymorphisms of the ER-beta and AR genes and knee OA was found in individuals of Greek descent.     

Semen parameters and sperm morphology in men in unexplained recurrent spontaneous abortion, before and during a 3 year follow-up period

To investigate the role of the ‘male factor’ inthe patho-genesis of recurrent spontaneous abortion (RSA), especiallysperm morphology abnormalities, 120 previously selected coupleswith unexplained RSA were studied for sperm parameters retrospectivelyand prospectively. The patients were subdivided into three subgroups,depending on their reproductive outcome during the 3 years offollow-up study: (i) 48 RSA couples who achieved a successfulpregnancy; (ii) 39 RSA couples who experienced further abortions;and (iii) 33 RSA couples who experienced infertility duringthe follow-up period. A semen analysis was performed twice atthe time of inclusion in the study, and twice again during the3 year follow-up period. No significant differences in semenparameters were observed between the RSA males and fertile controls.Instead, significant differences were observed between the groupof RSA couples who experienced infertility during the follow-upand the other two groups (RSA couples who achieved successfulpregnancy and RSA couples who experienced miscarriages and nolive birth during the follow-up) for sperm concentration (P< 0.01 and P < 0.01 respectively), sperm motility (P <0.01 and P < 0.01 respectively) and sperm morphology abnormalities(P < 0.01 and P < 0.01 respectively). Sperm morphologyabnormalities do not seem to be involved in determining RSA;instead, they are an aetiological factor in determining infertilityin patients, along with the other semen parameters, in the RSAcouple‘s subsequent reproductive life. Semen analysisis an important test in the clinical management of RSA couples.