The field of pharmacogenetics is an exciting field with great potential to alter the landscape of modern healthcare. With the realisation that androgenic effects are modulated by the length of the androgen receptor, CAG has led to the speculation that pharmacogenetics can be applied to the ever-growing field of testosterone replacement therapy. Applications of pharmacogenetics include a novel method to screen for hypogonadism not detected by traditional means and to create tailored TRT dosing to provide safe and effective dosing. This paper will provide a review on the studies carried out to date on the effect that the polymorphic AR CAG has on men administered testosterone replacement therapy for the symptoms of late-onset hypogonadism. The study hopes to determine if there is scope for screening individuals for the repeat length of their AR CAG both to diagnose and to provide a safe and effective TRT dose.
I would like to thank my tutor, Dr Sudesna Chatterjee, for all the help and support.
ADAM= Androgen deficiency in the ageing male AR= Androgen receptor
ED= Erectile dysfunction EF= Erectile function
FSH= Follicle-stimulating hormone
GnRH= gonadotropin releasing hormone (GnRH) hCG= Human chorionic gonadotropin
HPG= hypothalamic-pituitary-gonadal (HPG) HH= hypogonadotropic hypogonadism IIEF=International Index of Erectile Function IS= Intercourse satisfaction
LOH= Late-onset hypogonadism LH= Luteinizing hormone
MMAS= Massachusetts Male Aging Study (MMAS). NO = Nitric oxide
NTD=N terminal domain OF= Orgasmic function OS = Overall satisfaction
PolyQ= Polyglutamine repeat PSHH=post pubertal hypogonadism PRHH= pre-pubertal hypogonadism SD = Sexual desire
TRT= Testosterone Replacement Therapy TU= Testosterone undecanoate
VEGF = vascular endothelial growth factor (VEGF)
A working definition of pharmacogenetics could be the effect that an individual’s genome has on a therapeutic drug. With the realisation that a person’s genes could influence the effects of the particular drug, the era of pharmacogenetics was born. Pharmacogenetics was first mentioned by Sir Archibald Garrod (Garrod AE, 1931) in his book “Inborn Factors in Disease.” Dr Motulsky continued studies in this field and in 1957 was the first to expound a hypothesis that variation among individuals in drug response may be caused by genetic variation.
Mahgoup A et al made an important contribution to the field of pharmacogenetics in 1977 with the identification of the hepatic cytochrome P450 oxidase responsible for the metabolism of debrisoquine and sparteine. It was found that the primary metabolite of debrisoquine, 4- hydroxydebrisoquine, was present in differing levels in the urine samples of 94 volunteers after the administration of a 10mg dose of debrisoquine. Further family studies revealed that alicyclic 4-hydroxylation of debrisoquine is controlled by a single autosomal gene. A defect in this metabolic step is caused by a recessive allele.
The work in pharmacogenetics initially focused on the drug metabolism enzymes and further drug metabolism phenotypes were discovered. One of these was the enzyme CYP2D6 of which there around 80 different alleles and it is now thought to be involved with the metabolism of about 25% of commonly used drugs (Maraz D et al.1997). Further studies revealed four general metabolism phenotypes associated with CYP2D6: ultra-rapid, extensive, intermediate, and poor. Clinical based testing of a patients CYP2D6 could then be done to determine the type of metabolic profile and drugs could subsequently be prescribed in doses that would compliment this.
Two other noteworthy discoveries include the two enzymes from the CYP2C subfamily: CYP2C19 and CYP2C9. Recent studies have demonstrated an association of the common CYP2C19*2 allele with reduced active clopidogrel metabolites. This can become clinically significant when increased platelet aggregation can occur when compared to non-carriers causing negative clinical consequences (Shuldiner AR et al. 2009).
Variant CYP2C9 alleles have demonstrated a strong correlation with inter-individual warfarin dosing variability (Klein TE et al. 2009). This work has pioneered the initiation of pharmacogenetic-guided warfarin dosing in the University of Maryland Medical Centre, which implemented a Personalized Anti‐platelet Pharmacogenetics Program (PAP3) for cardiac catheterization patients. Patients’ are offered CYP2C19 genetic testing with the subsequent results guiding the prescribing recommendations for antiplatelet therapy (Shuldiner AR et al. 2014).
Molecular Biology of the Androgen Receptor (AR)
The majority of the effects of androgens, such as testosterone (T), occur after binding to the androgen receptor (AR). However, the androgens are also able to exert a role independently of the AR. For example, testosterone (T) can cause a vasodilatory effect non-genomically through either activation of K+ channels or blockade of Ca 2+ channels in vascular muscle cells (Yildiz, O. and Seyrek, M., 2007). AR are found in most parts of the body except the spleen (Gelmann EP, 2002) and are present especially in the androgen-target tissues (Ruizeveld de Winter et al.1991, Kimura et al., 1993, Ruizeveld de Winter 1994).
Problems with the functioning of the AR can result in a number of clinical symptoms. For example, mild spermatogenic defects (Hiort and Holterhus 2003, Davis-Dao et al., 2007) to complete androgen insensitivity syndrome (CAIS), causing the development of the female phenotype in individuals with the XY karyotype (Hughes and Deeb 2006). The AR has also been implicated in the progression of several cancers. (Catalano et al., 2000; Heinlein and Chang 2004; Dehm and Tindall 2007; Vastermark et al., 2011).
A fully functioning AR is necessary for normal virilization during embryological development, during puberty and for the ongoing maintenance of the male phenotype. The profound importance of the AR explains why the region where the AR is coded, the Xq11-12 region, has been highly conserved for over 150 million ago (Spencer JA, et al., 1991).
Mutation in the gene that codes for the AR can result in structural modifications that can lead to malfunctioning of the AR. The CAG codes for the amino acid glutamine, which after transcription results in chains of varying length of this amino acid, hence, the term polyglutamine or poly Q. The effects of the subsequent polyQ can affect the AR activity, concentration and binding of co-activators, which in turn can result on the extent of androgenic action produced.
The AR Gene and Protein
The AR is a nuclear receptor and its official designation is N3C4; nuclear receptor subfamily 3, group C and belongs to the intracellular family of structurally related steroid hormone receptor. (Mangelsdorf DJ, et al 1995).
The AR gene is located on the X-chromosome (Migeon et al., 1981, Kuiper et al., 1989; Lubhan et al., 1989) and is made up of eight exons spanning more than 90kB. Carolyn J. Brown, et al 1989, Migeon et al. 1981and Lubahn et al. 1988 identified the exact location for the gene as chromosome Xq11-12.
The mRNA codes for a protein of 98.8kDA, made up of approximately 919 amino acids. The precise number can vary depending on the corresponding number of the polymorphic glutamine repeat chain located in the amino terminal domain (NTD) which can stretch from 9 to 39 residues in normal individuals (Giovannucci E, et al. 1997).
The functional domains of the AR and mechanism of action of the AR can be found in the appendix.
Pharmacogenetics and its application to Androgen Receptor PolyQ Repeat
In the normal population the number of polymorphic CAG repeats located on exon one on the NTD stretches from 9 to 39 (Giovannucci E et al. 1997). An inverse relationship exists between the number of CAG repeats in the AR gene and the subsequent AR protein transcriptional ability. Shorter repeats result in greater transcriptional activity and increased androgenicity while the reverse is the case in individuals with longer repeat tracts (Zitzmann, M. and Nieschlag, E. (2003). When the CAG repeat number extends beyond 39 it can lead to the neuromuscular disorder X‐linked spinal bulbar muscular atrophy (XSBMA), a condition in which hypogonadic symptoms are observed (La Spada, A. R et al 1991).
In vitro studies have demonstrated that the modulatory effect caused by the AR CAG repeat length on transcription is linear over a range from 0 to 200 repeats given a fixed concentration (Nakajima H et al, 1996). Hsaio PW et al 1999 first identified the coactivator ARA24 while Irvine RA et al 2000, studied the effects of the other coactivator, p160 both of which have been identified as the causes for the attenuation of transcriptional activity caused by extended CAG repeats. This modulatory effect is thought to be mediated by a differential affinity of coactivator proteins ARA24 and p160 to the polyglutamine stretch. These proteins although widely present are not uniformly expressed in different tissues. This could help to explain why AR expression varies from tissue to tissue.
The differing levels of androgenicity witnessed in individuals could be caused by this polymorphic CAG repeat found on the AR gene. This could lead to the exciting possibility of exploiting this genetic variability to tailor individual hormonal replacement therapy to hypogonadal men.
Late Onset Hypogonadism (LOH) refers to the collection of signs and symptoms accompanying androgen deficiency in aging males. (Wang et al 2008). With aging serum T levels decrease progressively in men. However the rate and extent of this decrease in T levels varies from one individual to the next. Kazi M, el al postulated that serum T decreases by about 1% each year with a corresponding increase in SHBG which results in lower free T or bioavailable T as men age. The symptoms of LOH include a decrease in general well-being, mood changes, a decrease in energy and virility, erectile dysfunction, a decrease in muscular mass and strength and an increase in upper and central body fat. Quantitatively it is defined by total testosterone 12 nmol l−1 and free testosterone 220 pmol l−1 and at least three sexual symptoms (Buvat J et al.2013). In the aging male, total testosterone and free testosterone levels decrease owing to defects at all levels of the
hypothalamic-pituitary-testicular axis, whilst at the same time the levels of sexual hormone binding globulin increase (Araujo AB et al. 2011).
In the case of classical hypogonadism the almost complete breakdown of the hypothalamic-pituitary-gonadal (HPG) axis can be subsequent to either primary or secondary origin. (Zitzmann 2009). In primary the testes are non- functioning while in secondary their function is preserved.
Testosterone Replacement Therapy
The aim of testosterone replacement therapy (TRT) is to restore depleted T levels in the aging or hypogonadic male back to physiological testosterone levels in order to improve their symptoms. Studies have demonstrated that TRT can improve the sexual and erectile dysfunctions seen in LOH (Boloña ER, et al. 2007, Isidori AM et al. 2005). Other studies have shown the positive effects on muscle mass and strength with the administration of TRT. (Bhasin S et al. 2001, Caminiti G, et al 2009) Similarly other clinical improvements associated with TRT are increases in lumbar bone mass (Tracz MJ et al 2006) and with depressive thoughts (Zarrouf FA, et al 2009, Dennis CL et al. 2006)
A stigma still remains attached to TRT as a concept due to the damaging effects witnessed when supra physiological doses are used (Pirompol, P, et al, 2016). A recent interventional study of frail hypogonadal men who were administered supra physiological doses of testosterone in an attempt to improve muscle strength had to be stopped prematurely due to the excess of cardiovascular side effects (Basaria et al.2010). This study added to the stigma and caution when approaching TRT, however when critically analysed this study several flaws become apparent. These include the use of immobile, frail elderly men who had pre-existing heart symptoms and the use of supra physiological doses. Since this study numerous other studies have demonstrated the safety and utility of TRT (Calof OM et al 2005). It is the author’s hope that this review will help further the implementation of TRT by allowing bespoke pharmacogenetic regimens to be adopted thus maximising an individual’s response to TRT, which will entail using lower doses and hence improving the safety profile of TRT. To date there have only been a handful of studies investigating how the AR CAG impacts on TRT in subjects with LOH. This had led to the stagnation of testing for LOH, with reliance on old references values and responses from questionnaires such as the ADAM questionnaire. As will be discussed later many men may experience hypogonadism due to their increased length of the AR CAG repeat but nonetheless will present with normal testosterone levels. The author will attempt to address this issue by using a screening process that incorporates the AR CAG length to determine a diagnosis of LOH. In addition there is no guidance on a safe dosing regimen in individuals with varying AR CAG repeats, at present TRT is administered and then the dose adjusted after further blood analysis, which could be months. Using the concept of pharmacogenetics it would allow a safe dosing from initiation thus preventing a higher or lower physiological response from occurring.
AR-CAG Repeat and Age-Related Decline
The subject area for the present discussion is the value of screening for CAG repeat length in men suffering from LOH to allow pharmacogenetic tailoring of their subsequent TRT regimens. It follows that some research should be conducted into the effects that occur due to aging, and more specifically how these are related to the CAG polymorphism. This would establish whether the premise is viable option to pursue in this cohort of subjects. Krithivas K et al (1999) determined the number of CAG repeats for 882 men aged between 40 and 70 years from the Massachusetts Male Aging Study (MMAS). The MMAS was a landmark study following a sample of the adult male population in Massachusetts. At follow up the authors investigated whether the length of the CAG repeat would be predictive of hormone levels. The study found that the AR-CAG repeat length was significantly associated with T (p=0.04), albumin- bound T (p=0.025) and free T (P=0.03) when controlled for age, baseline hormone levels and anthropometrics. T levels at follow up decreased by 0.74% +/- 0.36 per CAG repeat decrement. In a similar fashion free T decreased by 0.93% +/- 0.31 and albumin bound T by 0.71% +/- 0.32 per CAG decrement. This study found that individuals with shorter CAG repeats will have lower T, %free T and % albumin bound T at follow up, representing faster rates of decline. It is known that there is a decline in serum T, free-T, and albumin-bound T levels with advancing age in men, however it is not known what the speed of the decline will be in men with differing CAG repeats. The regulation of serum androgen levels occurs via the hypothalamic– pituitary–testicular axis. The observed reduction in androgen levels in the aging male is probably due to impaired functioning at all three levels. Krithivas K et al (1999) proposed an additional level of control for age- associated androgen declines via modulation of the strength of the hypothalamic negative feedback. The suppressive effects of androgens is mediated by the ARs in the hypothalamus it follows that the strength of this negative feedback is related to the length of the CAG repeat length, with shorter repeats causing greater negative feedback than the longer repeats.
Hence individuals with fewer CAG repeats in their AR gene could have an increased response to androgen negative feedback and in turn a greater age- associated androgen decline. The work by Krithivas K et al (1999) further supports the biologically link between CAG repeat length in the AR gene with androgen/AR signalling. The findings of Sobue et al. (1994) coupled with these data suggest that longer AR CAG repeats decrease AR activity in the hypothalamus resulting in decreased negative feedback and increased serum androgen levels.
Cardiac and skeletal muscles both contain lower concentrations of the AR than the accessory sex organs. This is compounded by the reduced levels of testosterone with aging. However, along with exercise, TRT can help to maintain the condition and mass of both types of muscle as well as help to prevent osteoporosis. TRT can also help to reverse the shift of cellular metabolism from aerobic to anaerobic due to changes such as reduced tissue oxygenation resulting from aging. (Moller J, Einfeldt H, 1984).
Read the full essay here: The Effect of the AR CAG Repeat on TRT