Sexual Reassignment Fails to Prevent Kennedy’s Disease

Abstract

Spinal and bulbar muscular atrophy is caused by polyglutamine expansion in the androgen receptor. As an X-linked disease dependent on androgens, symptoms and findings are only fully manifest in males. Here we describe a 40-year-old male-to-female transgender SBMA patient who developed full disease manifestations despite undetectable levels of androgens. We used cell culture and animal models to show that spironolactone, the anti-androgen she had taken for 15 years, promotes nuclear localization and toxicity of the mutant protein, which may explain the disease manifestations in this patient.

Introduction

Kennedy’s disease, or spinal and bulbar muscular atrophy (SBMA), is an X-linked neuromuscular disorder. Affected males have muscle weakness and atrophy, bulbar dysfunction, dysphagia, and often signs of androgen insensitivity including gynecomastia and infertility. Females with the mutation are unaffected or have only mild disease manifestations [1]. Here we describe an SBMA patient who is biologically male but was feminized with spironolactone therapy for 15 years to become a transgender woman. It has been reported that androgen reduction rescues the SBMA phenotype in mice [2]. However, this patient’s disease manifestations are similar to those of her affected brother and other SBMA patients despite androgen levels in the female range. Spironolactone decreases 17α-hydroxylase activity and cytochrome P-450 content, effectively reducing testosterone production. It also competes with testosterone for active sites on the androgen receptor (AR) [3]. In order to investigate why our patient has features of SBMA, we tested whether spironolactone induces AR nuclear localization and toxicity in model systems.

MATERIALS AND METHODS

Clinical data

Clinical testing was performed at the National Institutes of Health (NIH) Clinical Center with the patient’s consent in an IRB approved protocol.

Cell culture experiments: Immunocytochemistry

Rat pheochromocytoma (PC12) cells expressing full length human AR with a 112 glutamine repeat and a tetracycline-inducible promoter were treated with dihydrotestosterone (DHT) or spironolactone. Spironolactone concentrations were chosen from pharmacokinetic data showing peak plasma concentrations of the compound reaching 200 nM in human subjects with tissue-to-plasma concentration ratios of 0.50, 0.79, and 11.0 in the brain, skeletal muscle, and adrenals, respectively [4, 5]. The number of cells with AR-immunoreactive nuclear inclusions and the total number of cells were counted in a blinded fashion. Three images per well were analyzed, and experiments were done in triplicate. A dual luciferase assay to assess AR function was done in PC12 cells co-transfected with a luciferase reporter under the control of androgen-response elements.

Drosophila model

To determine whether spironolactone induces a degenerative phenotype in a fly model of SBMA, we determined the viability of transgenic flies that were reared on food containing increasing concentrations of spironolactone (Sigma Aldrich) or 1% ethanol vehicle. Virgin female homozygous Elav >Gal4 flies were mated to male transgenic fliescarrying the UAS-hAR52Q transgene and Cyo-GFP second chromosome balancer to produce either Elav >AR52Q (SBMA fly model) or Elav;Cyo-GFP (control) progeny [6]. Upon the presence of pupae within the mating vial containing spironolactone, parental flies were removed and emerging adult progeny were scored for the presence or absence of the Cyo-GFP phenotypic marker over the course of 10 days.

RESULTS

The patient is a 40 year old biological male on androgen-reduction therapy for 15 years for gender reassignment. She noted gynecomastia before starting the therapy. At age 25, she took progesterone for one year and started spironolactone 100 mg daily. Her testosterone levels were checked annually to ensure that they were <20 ng/dL. Initially, she also took estrogen orally to achieve a target level of 29 ng/dL, and she has been receiving weekly intramuscular estrogen injections for the past 5 years. At age 29, four years after starting androgen reduction therapy, she developed general weakness and fatigue, and this led to the diagnosis of SBMA at age 31. Over the course of the disease, her symptoms have included muscle cramps, tremor, nasal voice, and throat spasms, all of which have worsened despite her hormonal therapy regimen. She has difficulty climbing stairs and reported five to six falls in the previous year.

On physical examination, the patient had moderate weakness of the face, bulbar, and extremity muscles. Atrophy was noted in the tongue and thenar muscles bilaterally. A resting tremor was present in the upper extremities, and fasciculations were seen around the chin. Her voice had increased nasal tone. She had decreased temperature sensation distally in her arms and legs and reduced vibratory sensation in her fingertips and toes. She had mild difficulty squatting and rising to a stand, consistent with proximal weakness. The patient has a family history of SBMA with an affected brother, maternal grandfather, and first cousin once removed. The patient’s brother is similarly affected by SBMA and noticed weakness starting at age 34, accompanied by fatigue and cramping, with diagnosis at age 36. The brother also notes sensory abnormalities and tremorbilaterally.

Clinical analysis (Table 1)

Genetic testing on the patient’s blood showed a 49 CAG repeat expansion in the AR gene. Hormone levels were consistent with complete feminization. Her creatine kinase was increased in a range typical of SBMA and comparable to her affected brother. The patient’s Adult Myopathy Assessment Tool (AMAT) score, a measure of SBMA disease manifestations and progression [7], was 36/45 total (18/21 functional tasks, 18/24 endurance tasks), equal to her brother’s total score of 36/45 (19/21 functional, 17/24 endurance). Quantitative muscle testing confirmed that her strength was diminished compared to healthy controls and commensurate with SBMA patients (Table 1). Electromyography and nerve conduction studies were abnormal with diffuse neurogenic changes and low sensory nerve amplitudes, indicative of a motor neuron disorder with sensory neuropathy, as is typical of SBMA.

Molecular and functional analyses

Given the reported effects of spironolactone on AR function [8, 9], we sought to evaluate its effects on translocation to the nucleus, transactivation, and formation of inclusions by the mutant protein [1]. In PC12 cells expressing the expanded AR112Q, spironolactone was shown to induce all three responses (Fig. 1A-C). To assess toxicity in vivo, a fly model of SBMA, where expression of human 52Q AR is specifically driven in neuronal tissues, was evaluated. Spironolactone exposure at ≥1000 μM resulted in a significant decrease in viability in the flies. Since the absolute numbers of control flies were not reduced, even in the presence of high doses of spironolactone, it is apparent that spironolactone-induced lethality in SBMA flies is specific to the mutant AR and not a generalized toxic effect (Fig. 1D).

DISCUSSION

Here we report an SBMA patient who has undergone androgen reduction therapy to transition from male to female. As the AR toxicity responsible for the SBMA phenotype is activated by androgens, androgen reduction has been investigated as a potential treatment for this disease. This strategy is beneficial in animal models of SBMA [2], but randomized, placebo-controlled clinical trials of dutasteride and leuprorelin did not result in significant effects on primary outcome measures. This could be because the treatment was not started early enough or continued long enough. Our patient provides insight into the efficacy of early and prolonged treatment with an androgen-reducing drug. After evaluating the patient, we confirmed that she has findings similar to her brother and other affected individuals with SBMA despite fully feminizing hormone levels for 15 years, starting before the onset of muscleweakness.

The patient’s disease manifestations could be related to the specific androgen-reducing agent used. Although spironolactone is a known anti-androgen, several reports suggest that the drug also has pro-androgenic activity [8, 9]. Spironolactone was found to increase levels of prostate specific antigen [9], a target of AR transcriptional regulation. The findings in our patient provide further evidence that spironolactone may act as a partial agonist or selective AR modulator, as well as an androgen-reducingagent.

We found that, similar to DHT, spironolactone induces nuclear uptake, transactivation, and inclusion formation by the mutant protein. When tested in a SBMA fly model, spironolactone induces significant lethality. The findings in cell and fly models confirm that spironolactone has pro-androgenic properties in addition to its ability to reduce androgen levels. Thus, spironolactone may be responsible for the disease manifestations of SBMA in this patient. We do not recommend use of this drug in patients with the disease.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.