Functional in vitro characterisation of the androgen receptor GGN polymorphism☆
Introduction
Normal androgen action is required for the male sex differentiation of the karyotypic male foetus, development of secondary sex characteristics at puberty and the maintenance of sperm production thereafter (Quigley et al., 1995). Androgen action in the peripheral target cells is mediated via the androgen receptor (AR), which is a nuclear transcription factor, functioning as the main regulator of androgen signalling in the cell. The hormones interacting with the AR are mainly testosterone (T) and 5α-dihydrotestosterone (DHT), which both bind to the ligand binding domain of the receptor.
The N-terminal domain, with the transactivating capacity, contains two polymorphic repeats of glutamines and glycines, encoded by (CAG)nCAA and (GGT)3GGG(GGT)2GGCn, respectively (Lubahn et al., 1989). The latter repeat is generally designated the GGN repeat.
The CAG repeat length is normally distributed in the population, spanning from approximately 10–30 repeats, with a median number of 22 in Caucasians (Lundin et al., 2003). An abnormal expansion of the segment to more than 40 repeats has been linked to spinal and bulbar muscular atrophy, also known as Kennedy's disease (La Spada et al., 1991). The CAG repeat has also been suggested to play a role in the pathogenesis of prostate cancer, testicular cancer and male infertility (Giwercman et al., 2004;Irvine et al., 1995;Tut et al., 1997). Experiments in vitro have confirmed the in vivo findings by showing an inverse relationship between the number of CAG repeats and the transactivating capacity of the receptor, with longer CAG stretches resulting in lower AR transactvating capacity than shorter ones.
Much less is known regarding the functional role of the GGN repeat. In the general Swedish population the repeat number spans from 10–27 (Lundin et al., 2003). One predominant allele of 23 (GGN23) has evolved among Caucasians and is carried by 53% of the Swedish subjects. GGN24, which is the second most common allele, was reported in approximately 32%. However, among patients with hypospadias or cryptorchidism the opposite pattern was found, GGN24 or longer being the most frequent genotype, followed by GGN23 (Aschim et al., 2004). In a recent publication on early onset androgenetic alopecia—male pattern baldness, which is characterized by an androgen dependent defined pattern of hair loss from the scalp, the authors found that GGN23 was associated with the disorder (Hillmer et al., 2005). GGN24 on the other hand, seemed to have a protective effect. These in vivo results indicated that GGN lengths above 23 were associated with lower androgenicity as compared to the most common genotype, GGN23. With respect to GGN lengths shorter than 23, it was recently shown that GGN < 23 was associated with lower semen volume, as compared to GGN = 23 and GGN > 23 (Lundin et al., 2006).
Hence, in vivo data have indicated that GGN23 would have a functional advantage (Table 1) and in vitro studies could shed light upon this matter, but to date, there are only two such studies on the GGN repeat. The first study showed that a complete deletion of the GGN polymorphism resulted in a 30% reduction of the transcriptional activity of the receptor (Gao et al., 1996). In the second, more recent study, ARs expressing 19–23 GGN triplets were tested in response to the synthetic testosterone R1881 (Ding et al., 2005). No significant difference in AR transcriptional activity was noted. However, only one concentration of R1881 (10 nM) was tested, and no comparisons were made with the second most common GGN length of 24 repeats, or with extremely short or long GGN repeats. Furthermore, all constructs contained a CAG repeat length of 24, which is above the average length.
The aim of our study was, therefore, to investigate the transcriptional activity of the androgen receptor carrying the two most common lengths of the GGN repeat, together comprising approximately 85% of the population, as well as very short (GGN = 10) and very long stretches (GGN = 27) in combination with the median CAG repeat length.
Section snippets
Molecular cloning
The CAG repeat was amplified in a 50 μl PCR reaction containing 0.4 μM of each of the primers: AR 4 and AR 1B3′ (5′-AGCCTAGCAGGGCAGATCTT-3′ and 5′-CTGCCTTACACAACTCCTTGGC-3′), 0.25 μM of each dNTP, 1 × Pyrobest II PCR Buffer (Takara Bio Inc., Shiga, Japan), 1.25 U Pyrobest DNA Polymerase (Takara) and approximately 10 ng human leukocyte DNA. Thirty-five cycles of 45 s denaturation at 96 °C, followed by a 2 min combined annealing and extension step at 72 °C, were performed in an Eppendorf Mastercycler
Results and discussion
This is the first in vitro study comparing the transactivating activity of the AR with the two most common GGN alleles in combination with the median CAG length. Current work shows that GGN repeat lengths other than the most frequent length of 23 are associated with lower AR transcriptional activity, both with testosterone and DHT as ligands.
We have recently reported an alteration in the distribution of the GGN lengths among cases with these congenital malformations (Aschim et al., 2004). In
Disclosure statement
None of the authors have anything to declare.
Acknowledgements
We wish to thank Åke Lundwall and Yvonne Olsson for kindly providing us with the vector harbouring the PSA promoter together with the Luciferase reporter gene, and Margareta Persson for valuable technical assistance.
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2017, Frontiers in NeuroendocrinologyPolymorphism of CAG and GGN repeats of androgen receptor gene in women with polycystic ovary syndrome
2015, Reproductive BioMedicine OnlineCitation Excerpt :However, the effect of GGN polymorphism on AR activity remains unclear, as there were discrepancies in previous studies (Jaaskelainen, 2012). One in-vitro study indicated that in response to both testosterone and 5α-dihydrotestosterone, those AR with GGN10, GGN27 and GGN24 in the N-terminal showed significantly lower AR activities than the AR with GGN23 (Lundin et al., 2007). However, there were little epidemiological investigations on the association between GGN repeat length and female infertility (Panda et al., 2011).
No effects of androgen receptor gene CAG and GGC repeat polymorphisms on digit ratio (2D: 4D): A comprehensive meta-analysis and critical evaluation of research
2014, Evolution and Human BehaviorCitation Excerpt :In this latter context, it should be noted that the evidence concerning the linearity of the effect and its direction is inconsistent for the less polymorphic and moreover non-normally distributed (Bogaert et al., 2009) GGC polymorphism. Whereas some studies support an inverse relationship between GGC repeat-length and AR transcriptional activity (Ding et al., 2005), others report direct (i.e., directionally opposite) effects or evidence for nonlinear effects (Lundin et al., 2007). These peculiarities or uncertainties may have consequences for analyzing associations of GGC repeat-number with other variables, as they would call for detailed distributional, instead of merely correlational (Zhang et al., 2013), data analysis.
Non-linear association between androgen receptor CAG and GGN repeat lengths and reproductive parameters in fertile European and Inuit men
2013, Molecular and Cellular EndocrinologyCitation Excerpt :One study reported no difference in transactivation between AR receptors with short and long repeats (Ding et al., 2005), whereas others reported an inverse (Werner et al., 2006) or a positive relationship (Brockschmidt et al., 2007). Most recently, a non-linear receptor transactivation potential was shown by Lundin et al. (2007) with highest activity in receptors harbouring 23 repeats. Interestingly, a similar non-linear relationship with risk of prostate cancer has been reported with highest risk in men with 23 repeats compared to men with either shorter or longer lengths (Platz et al., 1998).
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The study was supported by grants from The Swedish Research Council (Grant No. 521-2004-6072 and Grant No K2005–72X-14545-03A), The Swedish Cancer Society (Grant No: 4857-B05-03XCC) The Swedish Childhood Cancer Society (Grant No RKT04/001and 05/056), Gunnar Nilsson Cancer Fund and Crafoords Foundation.