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BRIEF REPORT |
Department of Clinical Sciences (K.B.L., A.G.), Fertility Centre, and Department of Clinical Sciences (K.B.L., Y.L.G.), Division of Urological Research, Malmö University Hospital, Lund University, SE-205 02 Malmö, Sweden; and Department of Molecular Medicine (A.N.), Clinical Genetics Unit, Karolinska Hospital, SE-171 76 Stockholm, Sweden
Address all correspondence and requests for reprints to: Yvonne L. Giwercman, Clinical Research Centre, Lund University, Building 91, Plan 10, Malmö University Hospital, Entrance 72, SE-205 02 Malmö, Sweden. E-mail: yvonne.giwercman{at}med.lu.se.
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Abstract |
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Objective: Because of this phenotypic variation, we screened a cohort of men from the general population (n = 293) as well as men with the following disorders of the genital tract for the mutation: men with prostate cancer (n = 89), testicular cancer (n = 87), and infertility (n = 103). We also investigated the influence of the polymorphic CAG and GGN repeats on the phenotypic outcome.
Results: The A645D variant was found in three men from the general population (1.0%). These men did not differ regarding testosterone or LH concentrations, compared with the rest of this population. In addition, two men with prostate cancer (2.3%) and one infertile man (1.0%) presented with the mutation. No statistical differences in frequency were noted between the study groups, and none of these individuals had any genital malformations. All men who presented with the mutation carried an extraordinarily short GGN repeat of 10 base triplets in combination with long CAG repeats of 26–28 (average 27.3). In contrast, men with GGN=10, but CAG less than 26 did not have the A645D mutation. A single-nucleotide polymorphism analysis revealed that the A645D variant has emerged from the most common haplogroup in our population.
Conclusions: We conclude that the A645D mutation, which is present in 1% of the general Swedish population, is linked to GGN10 and long CAG repeats. Its effect on androgen receptor function is currently unknown.
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Introduction |
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The A645D substitution was first described in a child with partial AIS (2) and later in a phenotypically normal boy (3). The mutation is located in the hinge region of the AR, between the DNA and the hormone binding domain of the receptor. Although more than 300 germ line AR mutations have been found, only six have been reported in the hinge region to date (www.androgendb.mcgill.ca). The relatively low numbers of mutations in this part of the AR indicates that either this region escapes mutations or mutations occur with the same frequency as in other regions of the molecule but are of activating character or of minor importance for AR function and for that reason not discovered.
Although the A645D mutation was absent in 108 normal chromosomes (3), it could be a false-negative result due to too few subjects included (type II error). Because AR mutations in the hinge region are rare and the A645D variant was present in an intersex patient as well as a boy with normal genital development, we wished to investigate whether this mutation was a normal polymorphism and whether the polymorphic CAG and GGN stretches in the AR could contribute to the discrepancies in reported phenotypes. We screened a group of more than 300 men from the general Swedish population as well as different patient categories of androgen-dependent disorders (prostate cancer, testicular cancer, and infertility) for the mutation. Furthermore, to investigate whether the A645D followed a specific pattern of inheritance, a single-nucleotide polymorphism (SNP) analysis was performed.
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Subjects and Methods |
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Phenotypically normal boy. A 15-yr-old phenotypically normal boy was found to carry the A645D mutation (3). He had a history of Wilms’ tumor but without undervirilization and a family history without intersex conditions. The boy was a chance finding, used as a normal control in a study of subjects with AIS.
Military conscripts. Three hundred five men who were born between 1979 and 1982 (mean age 18.1 yr), who were under medical examination for military service, and who represent the general Swedish population were included in the study (4). All participants underwent a physical examination, including examination of the genital organs, and delivered a semen sample, which was investigated according to the World Health Organization guidelines (5). None of the men had any genital malformations.
Infertile men. The cohort of infertile men consisted of 103 consecutive potential assisted reproduction candidates referred to the Scanian Andrology Centre at Malmö University Hospital (Malmö, Sweden). All patients had a minimum of 1 yr of infertility and sperm counts 5.0 x 106/ml or less as determined by at least two consecutive semen analyses. Patients were excluded a priori if they had the following: endocrine disturbances due to hypogonadotropic hypogonadism or abuse of anabolic steroids, obstructive syndromes of the urogenital tract, or treatment with chemotherapeutic agents.
Testicular germ cell cancer patients. During the period 2001–2002, all consecutive patients with testicular germ cell cancer passing through the outpatient clinic of the Department of Oncology, Lund University Hospital, were asked to participate in a study of reproductive function (75% participation rate). The analysis of the CAG and the GGN repeat length in DNA extracted from blood leukocytes was performed in the first 87 consecutive men entering the study.
Men with prostate cancer. The study population consisted of 134 Swedish men undergoing ultrasound-guided biopsies of the prostate on suspicion of prostate cancer due to serum prostate-specific antigen concentration 4 ng/ml or more. Prostate cancer was confirmed in 89 patients, whereas 45 had benign prostate hyperplasia (BPH).
All men participated after giving written informed consent according to protocols approved by the Ethical Review Board of Lund University.
Allele-specific PCR
Allele-specific PCR to detect the A645D variant was performed. Two reactions for each subject were used, each containing one mutant and one wild-type specific primer, together with an upstream and a downstream primer. PCR conditions were established to generate a short, allele-specific band in the presence of the variant and only a long control fragment in its absence. To verify the results, sequencing of samples representing each genotype was performed using the Big Dye primer cycle sequencing ready reaction kit and the ABI Prism 310 DNA sequencer (PerkinElmer Corp., Foster City, CA).
Exon 1 polymorphisms
The CAG and the GGN repeat lengths were determined by direct sequencing (6).
SNP analysis
The men from the general population as well as the normal boy were genotyped by a SNP analysis. Eight SNPs (962458, 2070757, 6152, 2207040, 2223823, 2870448, 1204039, and 1204038) were chosen from the National Center for Biotechnology Information SNP database (www.ncbi.nlm.nih.gov), spanning the entire AR gene, including flanking regions (40 kb) and surrounding the CAG and GGN repeats in exon 1. The SNP analysis was performed using an ABI 3730 (PerkinElmer).
Hormone analysis
Testosterone and LH concentration in blood was measured on an automated fluorescence detection system (Autodelfia; Wallac Oy, Turku, Finland) at the routine clinical chemistry laboratory, Uppsala University Hospital. Intra- and total assay variation was below the level of 4.0 and 7.5%, respectively.
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Results |
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Screening for the A645D mutation revealed three men from the general population carrying the mutation [1.03%; 95% confidence interval (CI) 0.21–3.0%]. Of the infertile men, one presented with the A645D variant (0.97%; 95% CI 0.020–5.3%), whereas the mutation was present in two men diagnosed with prostate cancer (2.3%; 95% CI 0.27–7.9%) and absent among men with testicular cancer (0.0%; 95% CI 0.0–3.4%) and men with BPH (0.0%; 95% CI 0.0–6.4%). The overall frequency of the A645D genotype was 0.98% (95% CI 0.36–2.1%).
Exon 1 polymorphisms
When analyzed by direct sequencing, the phenotypically normal boy displayed CAG=26 and GGN=10.
All men from the general population, who in addition were carriers of the A645D mutation, also presented with GGN=10. Their CAG tracts were 26, 27, and 28, respectively. Of these men, one stated responsibility for a pregnancy. Two additional subjects, without the mutation, but with GGN=10 presented with 16 and 28 CAG repeats, respectively.
The infertile man with the A645D variant also had GGN=10 and his CAG length was 28. GGN=10 was found in an additional man with infertility but without the mutation. His CAG repeat length was 16.
Only one of the men with testicular cancer included in the study carried GGN=10. He did not have the mutation and his CAG repeat consisted of 20 base triplets.
The two prostate cancer patients with theA645D mutation presented with GGN=10 and CAG=28. The third allele with GGN=10, but without the exon 4 variant, contained CAG=16. The men with BPH were carriers of neither the A645D variant nor GGN=10.
In all populations taken together, 1.8% (95% CI) had the GGN=10 allele. Of these, 63.6% harbored the A645D mutation, and in this group, the CAG repeat length was on average 27.3, which is in the upper normal range among Caucasians and much longer than the average of 22.1 in the general Swedish population (6).
SNP analysis
In 12 cases SNP analysis failed. Thus, all results are based on 293 individuals. In the general population, four different haplotypes emerged (HPT1–4) (Fig. 1
). HPT2 was the most common haplogroup, comprising 82% of the population, whereas HPT1 comprised 3%; HPT3, 6%; and HPT4, 8%. Mean CAG repeat length decreased, whereas GGN repeat length increased, with increasing haplogroup number. The A645D containing haplogroup has developed from HPT2 (Fig. 1).
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The number of conscripts with the A645D genotype was not sufficient to perform any statistical analysis. However, semen volume, sperm concentration, sperm counts, and testosterone as well as LH concentrations were within the range of the remaining conscripts (Table 1).
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Discussion |
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In the current work, in total 615 individuals of different patient categories were genotyped. The A645D mutation was found in six of these subjects (0.98%), indicating that the A645D variant mutation is so frequent that it might be regarded as a normal polymorphism. Notably, the A645D variant was not found in a higher proportion in any male disorder tested. However, the numbers of patients included were too small to allow any firm conclusion in relation to their disease.
The mutation was exclusively present in combination with the rare GGN=10 and long CAG repeats (
26). According to the SNP analysis performed, this haplogroup has emerged from the most common, and probably original, main group (HPT2), most likely due to a founder effect. The coinheritance of the mutation with GGN=10 and long CAG repeats is intriguing. One could imagine that a low activity due to the length of one of the repeats would be compensated for by the other. An inverse relationship between CAG number and AR function has previously been demonstrated (8), but regarding the GGN repeat so far, only two in vitro studies have been performed, in which a complete deletion of the GGN segment showed a 30% reduction in receptor activity (9), whereas in the second work, ARs expressing 19–23 GGN triplets were tested (10). No significant difference in AR transcriptional activity was noted between ARs with different GGN lengths. However, only one concentration of R1881 was used, and all constructs contained a CAG=24, which is above average length in all populations genotyped to date.
In vivo data have supported the hypothesis of an inversed relationship between the CAG number and AR function (11, 12, 13). Much less is known regarding the GGN repeat, but available information indicates that in terms of receptor function GGN=23 is superior to other GGN numbers (14, 15, 16). Thus, it appears unlikely that A645D implies a reduction in AR function, which would be compensated by more favorable GGN and/or CAG repeat lengths. Semen parameters and hormone values within the normal range in conscripts carrying the A645D variant also support that this genotype per se is not associated with severe AR dysfunction. Thus, finding of this genotype in an intersex patient might be by chance. Another possibility is that the A645D variant containing haplogroup could be predisposing to insufficient androgen response, which under specific circumstances during gestation, e.g. low testosterone production, could be manifest as genital abnormalities. Functional studies are warranted for further insight of the impact of this variant on AR activity.
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Acknowledgments |
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Footnotes |
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Disclosure statement: None of the authors have financial and/or personal relationships with people or organizations that could inappropriately influence (bias) their work. The corresponding author had full access to all data in the study and had the final responsibility for the decision to submit the manuscript for publication.
First Published Online May 16, 2006
Abbreviations: AIS, Androgen insensitivity syndrome; AR, androgen receptor; BPH, benign prostate hyperplasia; CI, confidence interval; SNP, single-nucleotide polymorphism.
Received February 21, 2006.
Accepted May 8, 2006.
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