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Exonic trinucleotide repeats and expression of androgen receptor gene in spinal cord from X-linked spinal and bulbar muscular atrophy
74
Journal o/'"the .,Veurologlcul &w:t~e,~, J22 ( 19t~41 7q : ~: 1994 Elsevier Science B.V. All rights reser~'ed t022-5 t~tX,'~,; $(~7 (~t
JNS 4226
Exonic trinucleotide repeats and expression of androgen receptor gene in spinal cord from X-linked spinal and bulbar muscular atrophy Masaaki Nakamura, Shuji Mita *, Tatsufumi Murakami, Makoto Uchino, Susumu Watanabe, Makoto Tokunaga, Toshihide Kumamoto and Masayuki Ando First Department of lnternal Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860, Japan (Received 19 April, 1993) (Revised, received 8 September, 1993) (Accepted 27 September, 1993)
Key words: X-linked spinal and bulbar muscular atrophy; Androgen receptor; Exonic trinucleotide repeats; Expression of androgen receptor gene; Reverse transcriptase-polymerase chain reaction; Western blot
Summary We studied exonic trinucleotide repeats and expression of androgen receptor (AR) gene in the spinal cord from an autopsied patient with X-linked spinal and bulbar muscular atrophy (SBMA). Forty-nine CAG triplet repeats were found in tissues from the spinal cord, cerebrum, cerebellum, cardiac muscle and bladder, while there were 20-24 CAG repeats in these tissues from control subjects, consisting of three patients with amyotrophic lateral sclerosis (ALS) and three patients with lung cancer. Thus, mitotic instability of the AR gene in SBMA may not occur at the level of somatic cells. To determine whether expression of the AR gene in the spinal cord of SBMA differs from that in control subjects, we used quantitative reverse transcriptase (RT)-PCR and Western blot. AR mRNA and protein were detected in the spinal cord from the patient with SBMA, but the levels of both AR mRNA and protein were less than those from the patients with ALS in whom the loss of motor neurons was similar to findings in the patient with SBMA. These findings suggest that structural alteration plus a reduced level of AR in the spinal cord are involved in the pathogenesis of SBMA, resulting in degeneration of motor neurons.
Introduction X-linked spinal and bulbar muscular atrophy (SBMA) is characterized by adult-onset, X-linked recessive inheritance, slowly progressive proximal spinal and bulbar muscular weakness and atrophy, and abundant fasciculations (Kennedy et al. 1968). Patients with S B M A frequently exhibit signs of gynecomastia, testicular atrophy and reduced fertility, evidence of partial androgen resistance (Arbizu et al. 1983). Sar and Stumpf (1977) found that androgens were concentrated in the neurons of the cranial nerves and spinal cord of male rats, with sparing of cranial nerves III, IV, and VI. The distribution of androgen correlated with that of neurons involved in SBMA. Androgens affect motor neuron growth, development, and regeneration (Yu and Srinivasan 1981; Kurz et al. 1986; Yu 1989). The
* Corresponding author. Tel.: + 81-96-344-2111 (Ext. 5615); Fax: + 81-96-371-0582.
SSDI 0 0 2 2 - 5 t 0 X ( 9 3 ) E 0 2 3 7 - 4
androgen receptor (AR) gene was m a p p e d to the long arm of the X chromosome (Fischbeck et al. 1986). These findings suggested a relationship between the A R gene and the progressive motor weakness associated with SBMA. La Spada et al. (1991) found that in all SBMA patients, the n u m b e r of C A G repeats in the first exon of the A R gene was approximately double that in normal subjects, and concluded that expansion of the C A G repeats in the A R gene may be the cause of SBMA. Expansion of exonic trinucleotide repeats has been found linked to fragile X syndrome (Verkerk et al. 1991; Kremer et al. 1991; Fu et al. 1991) and myotonic dystrophy (Brook et al. 1992: Fu et al. 1992: Mahadevan et al. 1992). Somatic heterogeneity of the amplified repeats due to mitotic instability is present in subjects with these disorders. The mechanism of expansion of the CAG repeats and the role of mutant A R gene have remained unknown. To clarify how the triplet repeats amplification leads to degeneration of motor neurons, direct inspection of organs is needed, however, autopsies on SBMA
75 patients are few as the disease is rare. We searched for somatic heterogeneity of the AR gene in tissues (especially in the spinal cord) from a patient with SBMA and investigated how the mutant AR gene is expressed in the spinal cord.
Materials and methods
Subjects A 66-year-old Japanese man had difficulty in running fast from age 29 years. Since age 36, he had difficulty in lifting heavy things. Weakness and atrophy of the limbs were slow and progressive, and dysarthria was present at age 39. Neurological examination revealed fasciculation and atrophy of the tongue, dysarthria and proximal muscular atrophy and weakness of the limbs. Deep tendon reflexes were decreased, but there was no sensory impairment. He had no clinical signs of androgen insensitivity. The mode of inheritance in this pedigree was X-linked recessive and a diagnosis of SBMA was made. At age 63, he complained of dysphagia and died at age 66 in Saishunso Hospital (Kumamoto), from respiratory failure. Within 6 h after death, various organs including the spinal cord were removed at autopsy. As controls, spinal cords from three patients with amyotrophic lateral sclerosis (ALS) and three patients with lung cancer were obtained by autopsy. Histochemistry (Kluver-Barrera stain) of the spinal cords from the patients with SBMA, ALS and lung cancer showed that the average number of anterior horn cells in the lateral nucleus in a slice of lower cervical segment was 10.7 + 2.1 (n = 1), 12.3 + 2.6 (n = 3), and 31.7 + 2.8 (n = 3), respectively when counted in three slides each, as shown in Table 1. The autopsies were carried out by licensed pathologists in Saishuns0 Hospital and our
TABLE 1 T H E A V E R A G E N U M B E R O F A N T E R I O R H O R N CELLS IN T H E L A T E R A L N U C L E U S IN A SLICE O F T H E L O W E R C E R VICAL SEGMENT Patients
No. of patient
Age (years)
Sex
N u m b e r of anterior horn cells a
SBMA
1
66
M
10.7:1:2.1
ALS
1 2 3
60 44 63
F M M
10.0 + 1.0 11.7+1.2 15.3 _+ 1.5
L u n g cancer
1 2 3
67 63 65
M M M
30.0 + 3.5 31.3+2.1 33.7+2.1
a Average n u m b e r of anterior horn cells per slice. Three slices in each patient were counted. Data are expressed as m e a n s + standard deviation.
university, following acquisition of all required permissions.
Amplification of DNA fragment of the AR gene by PCR Total genomic DNA was isolated from various tissues as described previously (Kunkel et al. 1977). Amplification of the DNA fragment of the AR gene was done in 30 cycles of PCR in a final volume of 100/zl containing 0.5/~g of genomic DNA, 50 pmol of primer pairs of the AR gene (ARI: Nos. 330-353; 5'-TCCAGAATCTGTTCCAGAGCGTGC-3'; AR2: Nos. 711-690; 5'-GCTGTGAAGGTTGCTGTTCCTC-3') (Lubahn et al. 1989; La Spada et al. 1991), 200 ~M each of dNTPs, 1 × PCR buffer (50 mM KCI, 10 mM Tris-HC1, pH 8.3, 1.2 mM MgC12, 0.01% gelatin, 0.1% NP40) and 2.5 units of Taq polymerase. Each cycle consisted of denaturation for 10 min at 94°C, primer annealing for 2 min at 57°C, and polymerization for 2 min at 72°C. Ten /zl of the amplified DNA for each sample was electrophoresed through 1.5% agarose gel containing ethidium bromide and photographed with Polaroid film.
Sequencing of PCR products The DNA fragments including the CAG triplet repeats of the AR gene in the spinal cord, cerebrum and cardiac muscle from the patient with SBMA and in the spinal cords from the patients with lung cancer and ALS were amplified by PCR, purified by electrophoresis through 4% Nusive GTG agarose gel, and subcloned into the pCR T M II vector (Invitrogen Corp., San Diego, CA). These recombinant DNA plasmids were transfected into host cells, INVaF'. Three clones containing the DNA fragment derived from each tissue were obtained. The recombinant DNA plasmid was extracted from the clones and the inserted DNA fragment was sequenced according to the dideoxy chain termination method with the Sequenase II sequencing kit (United States Biochemical Corp., Cleveland, OH).
Reverse transcriptase PCR (RT-PCR) Total RNA was prepared from a lower cervical portion of the spinal cords of the patient with SBMA, three patients with ALS and three with lung cancer, using the guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi 1987). Single strand cDNA synthesis was done using a final volume of 20 ~1 containing 1 /zg of total RNA, 1 × RT buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCI, 5 mM MgCI2), 10 mM dithiothreitol, I mM each of dNTPs, 20 units of RNasin (Promega Corp., Madison, WI), 100 pmol of random hexamer, 200 units of M-MLV reverse transcriptase (Gibco BRL, Gaithersburg, MD). The mixture was incubated at 37°C for 1 h. The resulting single strand cDNA was amplified by PCR in a final volume of 100 /xl containing 10 /zl of the mixture, 6/zl of 10 × PCR
76 buffer, 50 pmol of primer pairs of the A R gene (AR2; AR3: Nos. 286-306; 5 ' - G C C T G T T G A A C T C T T C T G A G C - 3 ' ) (Lubahn et al. 1989; La Spada et al. 1991), 5/xCi of [a-3zp]dCTP and 2.5 units of Taq polymerase as described above. Five pmol of primer pairs (AI: Nos. 222-241; 5 ' - A A G A G A G G C A T C C T C A C C C T - 3 '; A2: Nos. 439-420; 5 ' - T A C A T G G C T G G G G T G T T G A A - 3 ' ) (Ponte et al. 1984) for/3-actin m R N A were also added to the reaction mixture to serve as an internal control. Four /zl of the P C R products were removed every three cycles from 21 to 27 cycles. These P C R products were electrophoresed through 5% polyacrylamide gels. The radioactivity of the bands corresponding to D N A fragments of the A R and /3-actin genes amplified was counted using a BA100 Image Analyzer (Fuji Film, Tokyo) and the ratio of A R m R N A to /3-actin m R N A was estimated. Western blot Briefly, lower cervical portions of the spinal cords from the subjects were homogenized with 9 vol. of 20 m M Tris-HCl, p H 7.5, 2 m M dithiothreitol, 20% glycerol, 0.5 M NaCI and 0.1 m M phenylmethanesulfonyl fluoride, and then centrifuged at 10,000 x g for 15 min at 4°C (Mizokami et al. 1992). The protein concentrations were determined by the method of Lowry et al. (1951). The supernatants containing 100/zg of protein were electrophoresed through 4 - 1 2 % gradient gels. The proteins were transferred to nitrocellulose sheets according to the method of Towbin et al. (1979). The blots were incubated with phosphate-buffered saline (PBS) containing 10% non-fat milk for 1 h, washed 3 times for 5 min each with 0.05% Tween 20 in PBS (PBS-Tween 20), incubated with a h u m a n androgen receptor-specific polyclonal antibody (Mizokami et al. 1992) diluted 1:1000 in PBS-Tween overnight at 4°C, washed, and incubated for 1 h with a 1:200 dilution of biotinylated goat anti-rabbit l g G (H + L) antibody (Vector Laboratories, Burlingame, CA). After further washing, the reactior~ was visualized using a "Vectastain ABC Elite kit" (Vector Laboratories).
1
2
3
4
5
6
7
I i J
Fig. 1. Analyses of the PCR products obtained by ~hc amplification of the CAG repeat region in various tissues from the SBMA patient (lanes 2-6). The PCR products were visualized on a 1,5~i.~agarose gel containing ethidium bromide. The lanes arc identified as follows: lane 1, molecular weight marker (pGEM marker); lane 2. bladder; lane 3, cardiac muscle; lane 4, cerebrum; lane 5, cerebellum; lane 6, spinal cord; lane 7, spinal cord from a patient with lung cancer. PCR products in various tissues from the SBMA patient gave a 370 bp band (arrow), and are about 75-87 bp larger than those from control subjects.
2
3
Results
C A G repeats in tissues from an SBMA patient The P C R product containing the C A G repeats in various tissues (bladder, cardiac muscle, cerebrum, cerebellum and spinal cord) from the SBMA patient gave a 370-bp band, and was about 80 bp larger than those from control subjects (Fig. 1). Sequence analysis of the P C R products derived from tissues from the patient with SBMA revealed that the number of C A G repeats was 49, that is 75-87 bp larger than that from control subjects, including the ALS patients (20-24 repeats) (Fig. 2).
i I
,-/
Fig. 2. Sequencing of the PCR products obtained by the amplification of the CAG repeat region in various tissues from the SBMA patient. The lanes are identified as follows: lane 1, cardiac muscle; lane 2, cerebrum; lane 3, spinal cord. The same result was obtained in the bladder and cerebellum tissues. Arrows indicate start and end points of the CAG repeats. The number of CAG repeats in the tissues was 49.
77 1
2
3
4
5
6
7
~ - - 13- actin
ratio AR/~-actin
2.1
2.9
5.0
1,5
2.1
3.1
0.5
lung cancer, and the ratios of AR mRNA in the patients with ALS and the patients with lung cancer to that in the patient with SBMA were 4.5 5:1.6 and 6.7 + 3.0, respectively. The level of AR mRNA in the spinal cord tended to be a little lower in the patients with ALS than in those with lung cancer, however, there was no statistical significance (Fig. 3).
radioactivity Fig. 3. RT-PCR analyses of the AR gene expression in the spinal cord. RT-PCR products from AR mRNA and/3-actin mRNA in the patients with lung cancer (lanes 1-3), ALS (lanes 4-6) and SBMA (lane 7) were electrophoresed through 5% polyacrylamide gel and the radioactivity was counted using a BA100 Image Analyzer (Fuji Film). The band for AR mRNA in SBMA (AR*) was larger than that in the others. The ratio of AR mRNA to/3-actin mRNA was 0.5 in the patient with SBMA, 2.2+0.8 in the patients with ALS, 3.3 + 1.5 in the patients with lung cancer.
AR protein in SBMA To investigate AR expression in the spinal cords from the SBMA patient, ALS and lung cancer patients, Western blot analysis was performed. As shown in Fig. 4, AR protein was detected in the spinal cords from all subjects, however, the level of AR protein in the spinal cord of the SBMA patient was significantly less than that seen in the ALS and lung cancer patients. The difference between the patients with ALS and lung cancer was not significant (Fig. 4).
Quantitative RT-PCR for AR mRNA Quantitative RT-PCR was used to examine the influence of enlargement of the CAG repeat in the first exon of the AR gene on AR mRNA levels in the spinal cords. We confirmed that the radioactivity of PCR amplification for both AR mRNA and /3-actin mRNA increased exponentially during 27 cycles of the reaction (data not shown). Therefore we used 24 cycles under which linearization of PCR amplification for both AR mRNA and /3-actin mRNA was detected. We estimated the intensity of the bands for AR mRNA to /3-actin mRNA (used as an internal control). As shown in Fig. 3, the ratio of AR mRNA to fl-actin mRNA was 0.5 in the patient with SBMA, 2.2 _+0.8 in the patients with ALS, and 3.3 +_ 1.5 in the patients with
1
2
3
4 KDa 200
AR
115 97
66 Fig. 4. Western blot of AR protein in the spinal cords of the patients with SBMA, ALS, and lung cancer. One hundred ~g protein of tissue extracts in the spinal cord from the SBMA patient (lane 1), ALS patient (lane 2), and lung cancer patient (lane 3) were electrophoresed and subjected to Western blot. Lane 4 is for the molecular weight markers. The level of A R protein in the spinal cord from the patient with SBMA was significantly less than that of the patients with ALS and lung cancer.
Discussion AR protein is present in many tissues such as testis, prostate, brain, spinal cord and cardiac muscle, but not in peripheral blood mononuclear cells (PBMC) (Mooradian et al. 1987; Ruizeveld de Winter et al. 1991). The mutant AR gene with increased triplet repeats causes SBMA, a disorder in which spinal and bulbar motor neurons are mainly involved, while disturbance of AR function resulting from a certain point mutation of the AR gene causes infertility but no weakness (Griffin and Wilson 1989). Thus, different mutations of the AR gene lead to different diseases. One hypothesis is that enlargement of the CAG repeats may alter tissue specific expression of the AR gene. In the present study, we addressed directly alteration in the exonic trinucleotide repeats and expression of the AR gene in the spinal cord from a patient with SBMA. We investigated the number of CAG repeats in the AR gene in various tissues from the patient with SBMA, and from controls. Forty-nine CAG triplet repeats were found in the spinal cord, cerebrum, cerebellum, cardiac muscle and bladder from the patient with SBMA, while 20-24 CAG repeats were noted in these tissues from control subjects, including ALS. Biancalana et al. (1992) reported that the CAG repeats in the AR gene are highly pleomorphic and that a limited somatic instability of the CAG repeats in the abnormal SBMA allele was detected in variation by 5:3 repeats which exceeded that in the normal allele (in variation by 1 repeat). This variation of the CAG repeats was found in PBMC. In fragile X syndrome and myotonic dystrophy in which expansion of exonic trinucleotide repeats occurs, somatic heterogeneity of the amplified repeats due to mitotic instability was extensive (n > 100
78 for fragile X syndrome, n > 60 for myotonic dystrophy). These findings prompted us to investigate the possibility of expansion of the CAG repeats in involved organs, especially the spinal cord. Unlike findings in these two diseases, our observations suggest that the extensive mitotic instability of the AR gene in SBMA may not occur at the level of somatic cells. Using quantitative RT-PCR and Western blot analysis, we found that the level of both AR mRNA and protein in the spinal cord from the patient with SBMA was less than that from the patients with ALS in whom the loss of motor neurons was similar to that in the patient with SBMA. Pieretti et al. (1991) reported markedly reduced levels of FMR-1 mRNA in the majority of male fragile X patients, suggesting that the reduced expression of the gene was probably involved in the development of the syndrome. In the relationship between mutations of the AR gene and AR function, McPhaul et al. (1993) analyzed 31 unrelated subjects with androgen resistance syndromes, most of whom had point mutations of the AR gene, and showed that in most of the cases the phenotypic abnormalities are the result of impairment of receptor function or decreases in receptor abundance or both. All these findings taken together suggest that not only structural alteration of AR protein resulting from enlargement of the polyglutamine repeats but also a reduced level of AR protein resulting from a decreased expression of the AR gene in the spinal cord are involved in abnormal functions of AR leading to SBMA. Acknowledgements We thank Drs. K. Nanba (Saishunso Hospital) for providing material from the autopsied case of SBMA, A. Mizokami (University of Occupational and Environmental Health) for providing AR antibody, H. Ueyama (Kumamoto University) for technical assistance, and M. Ohara for helpful comments.
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Fischbeck, K.H., V. Ionasescu, A.W. Ritter, R. hmascsct~, K. l)avic~. S. Ball. P. Bosch, T. Burns, 1. fIausmanowa-Pelrusewicz..I Borkowska, S.P. Ringel and L.Z. Stern (1986) Localization ~1' lhc gene for X-linked spinal muscular atrophy. Neurology, 36:151;'5 1598. Fu, Y.H., D.P.A. Kuhl, A. Pizzuti, M. Pieretti. J.S. Sutctil'le. 8. Richards, A.J.M.H. Verkerk, J.J.A. Holden, R.G. Fenwick, J r . S.T. Warren, B.A. Oostra, D.L. Nelson and ('.F. Caskey (1991) Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell, 67: 1047-1058. Fu, Y.H., A. Pizzuti, R.G. Fenwick, Jr., J. King, S. Rajnarayan, P.W. Dunne, J. Dubel, G,A. Nasser, T. Ashizawa, P. de Jong, B. Wieringa, R. Korneluk, M.B. Perryman, H.F. Epstein and C.T. Caskey (1992) An unstable triplet repeat in a gene related Io myotonic muscular dystrophy. Science, 255: 1256-1258. Griffin, J.E. and J.D. Wilson (1989) The androgen resistance syndromes: 5a-reductase deficiency, testicular feminization, and related disorders. In: Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (Eds.), The Metabolic Basis of Inherited Disease, 6th ed., McGraw-Hill, New York, NY. pp. 1919-1944. Kennedy, W.R., M. Alter and J.H. Sung (1968) Progressive proximal spinal and bulbar atrophy of late onset. Neurology, 18: 671-680. Kremer, E.J., M. Pritchard, M. Lynch, S. Yu, K. Holman, E. Baker, S.T. Warren, D. Schlessinger, G.R. Sutheriand and R.I. Richards (1991) Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CGG)n. Science, 252:1711 - 1714. Kunkel, L.M., K.D. Smith, S.H. Boyer, D.S. Borgaonkar, S.S. Wachtel, O.J. Miller, W.R. Breg, H.W. Jones, Jr. and J.M. Rary (1977) Analysis of human Y-chromosome-specific reiterated DNA in chromosome variants. Proc. Natl. Acad. Sci. USA, 74: 1245-1249. Kurz, E.M., D.R. Sengelaub and A.P. Arnold (19861 Androgens regulate the dendritic length of mammalian motoneurons in adulthood. Science, 232: 395-398. La Spada, A.R., E.M. Wilson, D.B. Lubahn, A.E. Harding and K.H. Fischbeck (1991) Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature, 352: 77-79. Lowry, O.H,, N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275. Lubahn, D.B., D.R, Joseph, M. Sar, J. Tan, H.N. Higgs, R.E, Larson, F.S. French and E.M. Wilson (1989) The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. Mol. Endocr., 2: 1265-1275. Mahadevan, M., C. Tsilfidis, L. Sabourin, G. Shutler, C. Amemiya, G. Jansen, C. Neville, M. Narang, J. Barcelo, K. O'Hoy, S. Leblond, J. Earle-Macdonald, P.J. de Jong, B. Wieringa and R.G. Korneluk (1992) Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science. 255: 1253-1255. McPhaul, M.J., M. Marcelli, S. Zoppi, J.E. Griffin and J.D. Wilson (1993) Genetic basis of endocrine disease 4: The spectrum of mutations in the androgen receptor gene that causes androgen resistance. J. Clin. Endocr. Metab., 76: 17-23. Mizokami, A., H. Saiga, T. Matui, T. Mita and A. Sugita (1992) Regulation of androgen receptor by androgen and epidermal growth factor in a human prostatic cancer cell line, LNCaP. Endocr. Jap., 39: 235-243. Mooradian, A.D., J.E. Morley and S.G. Korenman (1987) Biological actions of androgens. Endocr. Rev., 8: 1-28. Pieretti, M., F. Zhang, Y.-H. Fu, S.T. Warren, B.A. Oostra, C,T. Caskey and D.L. Nelson (1991) Absence of expression of the FMR-I gene in fragile X syndrome. Cell, 66: 817-822. Ponte, P., S.-Y. Ng, J. Engel, P. Gunning and L. Kedes (19841 Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin eDNA. Nucleic Acids Res., 12: 1687-1696.
79 Ruizeveld de Winter, J.A., J. Trapman, M. Vermey, E. Mulder, N.D. Zegers and T.H. van der Kwast (1991) Androgen receptor expression in human tissues: an immunohistochemical study. J. Histochem. Cytochem., 39: 927-936. Sar, M. and W.E. Stumpf (1977) Androgen concentration in motor neurons of cranial nerves and spinal cord. Science, 197: 77-80. Towbin, H., T. Stanehelin and J. Gordon (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Verkerk, A.J.M.H., M. Pieretti, J.S. Sutcliffe, Y.H. Fu, D.P.A. Kuhi, A. Pizzuti, O. Reiner, S. Richards, M.F. Victoria, F. Zhang, B.E.
Eussen, G.-J.B. van Ommen, L.A.J. Blonden, G.J. Riggins, J.L. Chastain, C.B. Kunst, H. Galjaard, C.T. Caskey, D.L. Nelson, B.A. Oostra and S.T. Warren (1991) Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell, 65: 905-914. Yu, W.A. (1989) Administration of testosterone attenuates neuronal loss following axotomy in the brain-stem motor nuclei of female rats. J. Neurosci., 9: 3908-3914. Yu, W.A. and R. Srinivasan (1981) Effect of testosterone and 5a-dihydrotestosterone on regeneration of the hypoglossal nerve in rats. Exp. Neurol., 71: 431-435.
Journal o/'"the .,Veurologlcul &w:t~e,~, J22 ( 19t~41 7q : ~: 1994 Elsevier Science B.V. All rights reser~'ed t022-5 t~tX,'~,; $(~7 (~t
JNS 4226
Exonic trinucleotide repeats and expression of androgen receptor gene in spinal cord from X-linked spinal and bulbar muscular atrophy Masaaki Nakamura, Shuji Mita *, Tatsufumi Murakami, Makoto Uchino, Susumu Watanabe, Makoto Tokunaga, Toshihide Kumamoto and Masayuki Ando First Department of lnternal Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860, Japan (Received 19 April, 1993) (Revised, received 8 September, 1993) (Accepted 27 September, 1993)
Key words: X-linked spinal and bulbar muscular atrophy; Androgen receptor; Exonic trinucleotide repeats; Expression of androgen receptor gene; Reverse transcriptase-polymerase chain reaction; Western blot
Summary We studied exonic trinucleotide repeats and expression of androgen receptor (AR) gene in the spinal cord from an autopsied patient with X-linked spinal and bulbar muscular atrophy (SBMA). Forty-nine CAG triplet repeats were found in tissues from the spinal cord, cerebrum, cerebellum, cardiac muscle and bladder, while there were 20-24 CAG repeats in these tissues from control subjects, consisting of three patients with amyotrophic lateral sclerosis (ALS) and three patients with lung cancer. Thus, mitotic instability of the AR gene in SBMA may not occur at the level of somatic cells. To determine whether expression of the AR gene in the spinal cord of SBMA differs from that in control subjects, we used quantitative reverse transcriptase (RT)-PCR and Western blot. AR mRNA and protein were detected in the spinal cord from the patient with SBMA, but the levels of both AR mRNA and protein were less than those from the patients with ALS in whom the loss of motor neurons was similar to findings in the patient with SBMA. These findings suggest that structural alteration plus a reduced level of AR in the spinal cord are involved in the pathogenesis of SBMA, resulting in degeneration of motor neurons.
Introduction X-linked spinal and bulbar muscular atrophy (SBMA) is characterized by adult-onset, X-linked recessive inheritance, slowly progressive proximal spinal and bulbar muscular weakness and atrophy, and abundant fasciculations (Kennedy et al. 1968). Patients with S B M A frequently exhibit signs of gynecomastia, testicular atrophy and reduced fertility, evidence of partial androgen resistance (Arbizu et al. 1983). Sar and Stumpf (1977) found that androgens were concentrated in the neurons of the cranial nerves and spinal cord of male rats, with sparing of cranial nerves III, IV, and VI. The distribution of androgen correlated with that of neurons involved in SBMA. Androgens affect motor neuron growth, development, and regeneration (Yu and Srinivasan 1981; Kurz et al. 1986; Yu 1989). The
* Corresponding author. Tel.: + 81-96-344-2111 (Ext. 5615); Fax: + 81-96-371-0582.
SSDI 0 0 2 2 - 5 t 0 X ( 9 3 ) E 0 2 3 7 - 4
androgen receptor (AR) gene was m a p p e d to the long arm of the X chromosome (Fischbeck et al. 1986). These findings suggested a relationship between the A R gene and the progressive motor weakness associated with SBMA. La Spada et al. (1991) found that in all SBMA patients, the n u m b e r of C A G repeats in the first exon of the A R gene was approximately double that in normal subjects, and concluded that expansion of the C A G repeats in the A R gene may be the cause of SBMA. Expansion of exonic trinucleotide repeats has been found linked to fragile X syndrome (Verkerk et al. 1991; Kremer et al. 1991; Fu et al. 1991) and myotonic dystrophy (Brook et al. 1992: Fu et al. 1992: Mahadevan et al. 1992). Somatic heterogeneity of the amplified repeats due to mitotic instability is present in subjects with these disorders. The mechanism of expansion of the CAG repeats and the role of mutant A R gene have remained unknown. To clarify how the triplet repeats amplification leads to degeneration of motor neurons, direct inspection of organs is needed, however, autopsies on SBMA
75 patients are few as the disease is rare. We searched for somatic heterogeneity of the AR gene in tissues (especially in the spinal cord) from a patient with SBMA and investigated how the mutant AR gene is expressed in the spinal cord.
Materials and methods
Subjects A 66-year-old Japanese man had difficulty in running fast from age 29 years. Since age 36, he had difficulty in lifting heavy things. Weakness and atrophy of the limbs were slow and progressive, and dysarthria was present at age 39. Neurological examination revealed fasciculation and atrophy of the tongue, dysarthria and proximal muscular atrophy and weakness of the limbs. Deep tendon reflexes were decreased, but there was no sensory impairment. He had no clinical signs of androgen insensitivity. The mode of inheritance in this pedigree was X-linked recessive and a diagnosis of SBMA was made. At age 63, he complained of dysphagia and died at age 66 in Saishunso Hospital (Kumamoto), from respiratory failure. Within 6 h after death, various organs including the spinal cord were removed at autopsy. As controls, spinal cords from three patients with amyotrophic lateral sclerosis (ALS) and three patients with lung cancer were obtained by autopsy. Histochemistry (Kluver-Barrera stain) of the spinal cords from the patients with SBMA, ALS and lung cancer showed that the average number of anterior horn cells in the lateral nucleus in a slice of lower cervical segment was 10.7 + 2.1 (n = 1), 12.3 + 2.6 (n = 3), and 31.7 + 2.8 (n = 3), respectively when counted in three slides each, as shown in Table 1. The autopsies were carried out by licensed pathologists in Saishuns0 Hospital and our
TABLE 1 T H E A V E R A G E N U M B E R O F A N T E R I O R H O R N CELLS IN T H E L A T E R A L N U C L E U S IN A SLICE O F T H E L O W E R C E R VICAL SEGMENT Patients
No. of patient
Age (years)
Sex
N u m b e r of anterior horn cells a
SBMA
1
66
M
10.7:1:2.1
ALS
1 2 3
60 44 63
F M M
10.0 + 1.0 11.7+1.2 15.3 _+ 1.5
L u n g cancer
1 2 3
67 63 65
M M M
30.0 + 3.5 31.3+2.1 33.7+2.1
a Average n u m b e r of anterior horn cells per slice. Three slices in each patient were counted. Data are expressed as m e a n s + standard deviation.
university, following acquisition of all required permissions.
Amplification of DNA fragment of the AR gene by PCR Total genomic DNA was isolated from various tissues as described previously (Kunkel et al. 1977). Amplification of the DNA fragment of the AR gene was done in 30 cycles of PCR in a final volume of 100/zl containing 0.5/~g of genomic DNA, 50 pmol of primer pairs of the AR gene (ARI: Nos. 330-353; 5'-TCCAGAATCTGTTCCAGAGCGTGC-3'; AR2: Nos. 711-690; 5'-GCTGTGAAGGTTGCTGTTCCTC-3') (Lubahn et al. 1989; La Spada et al. 1991), 200 ~M each of dNTPs, 1 × PCR buffer (50 mM KCI, 10 mM Tris-HC1, pH 8.3, 1.2 mM MgC12, 0.01% gelatin, 0.1% NP40) and 2.5 units of Taq polymerase. Each cycle consisted of denaturation for 10 min at 94°C, primer annealing for 2 min at 57°C, and polymerization for 2 min at 72°C. Ten /zl of the amplified DNA for each sample was electrophoresed through 1.5% agarose gel containing ethidium bromide and photographed with Polaroid film.
Sequencing of PCR products The DNA fragments including the CAG triplet repeats of the AR gene in the spinal cord, cerebrum and cardiac muscle from the patient with SBMA and in the spinal cords from the patients with lung cancer and ALS were amplified by PCR, purified by electrophoresis through 4% Nusive GTG agarose gel, and subcloned into the pCR T M II vector (Invitrogen Corp., San Diego, CA). These recombinant DNA plasmids were transfected into host cells, INVaF'. Three clones containing the DNA fragment derived from each tissue were obtained. The recombinant DNA plasmid was extracted from the clones and the inserted DNA fragment was sequenced according to the dideoxy chain termination method with the Sequenase II sequencing kit (United States Biochemical Corp., Cleveland, OH).
Reverse transcriptase PCR (RT-PCR) Total RNA was prepared from a lower cervical portion of the spinal cords of the patient with SBMA, three patients with ALS and three with lung cancer, using the guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi 1987). Single strand cDNA synthesis was done using a final volume of 20 ~1 containing 1 /zg of total RNA, 1 × RT buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCI, 5 mM MgCI2), 10 mM dithiothreitol, I mM each of dNTPs, 20 units of RNasin (Promega Corp., Madison, WI), 100 pmol of random hexamer, 200 units of M-MLV reverse transcriptase (Gibco BRL, Gaithersburg, MD). The mixture was incubated at 37°C for 1 h. The resulting single strand cDNA was amplified by PCR in a final volume of 100 /xl containing 10 /zl of the mixture, 6/zl of 10 × PCR
76 buffer, 50 pmol of primer pairs of the A R gene (AR2; AR3: Nos. 286-306; 5 ' - G C C T G T T G A A C T C T T C T G A G C - 3 ' ) (Lubahn et al. 1989; La Spada et al. 1991), 5/xCi of [a-3zp]dCTP and 2.5 units of Taq polymerase as described above. Five pmol of primer pairs (AI: Nos. 222-241; 5 ' - A A G A G A G G C A T C C T C A C C C T - 3 '; A2: Nos. 439-420; 5 ' - T A C A T G G C T G G G G T G T T G A A - 3 ' ) (Ponte et al. 1984) for/3-actin m R N A were also added to the reaction mixture to serve as an internal control. Four /zl of the P C R products were removed every three cycles from 21 to 27 cycles. These P C R products were electrophoresed through 5% polyacrylamide gels. The radioactivity of the bands corresponding to D N A fragments of the A R and /3-actin genes amplified was counted using a BA100 Image Analyzer (Fuji Film, Tokyo) and the ratio of A R m R N A to /3-actin m R N A was estimated. Western blot Briefly, lower cervical portions of the spinal cords from the subjects were homogenized with 9 vol. of 20 m M Tris-HCl, p H 7.5, 2 m M dithiothreitol, 20% glycerol, 0.5 M NaCI and 0.1 m M phenylmethanesulfonyl fluoride, and then centrifuged at 10,000 x g for 15 min at 4°C (Mizokami et al. 1992). The protein concentrations were determined by the method of Lowry et al. (1951). The supernatants containing 100/zg of protein were electrophoresed through 4 - 1 2 % gradient gels. The proteins were transferred to nitrocellulose sheets according to the method of Towbin et al. (1979). The blots were incubated with phosphate-buffered saline (PBS) containing 10% non-fat milk for 1 h, washed 3 times for 5 min each with 0.05% Tween 20 in PBS (PBS-Tween 20), incubated with a h u m a n androgen receptor-specific polyclonal antibody (Mizokami et al. 1992) diluted 1:1000 in PBS-Tween overnight at 4°C, washed, and incubated for 1 h with a 1:200 dilution of biotinylated goat anti-rabbit l g G (H + L) antibody (Vector Laboratories, Burlingame, CA). After further washing, the reactior~ was visualized using a "Vectastain ABC Elite kit" (Vector Laboratories).
1
2
3
4
5
6
7
I i J
Fig. 1. Analyses of the PCR products obtained by ~hc amplification of the CAG repeat region in various tissues from the SBMA patient (lanes 2-6). The PCR products were visualized on a 1,5~i.~agarose gel containing ethidium bromide. The lanes arc identified as follows: lane 1, molecular weight marker (pGEM marker); lane 2. bladder; lane 3, cardiac muscle; lane 4, cerebrum; lane 5, cerebellum; lane 6, spinal cord; lane 7, spinal cord from a patient with lung cancer. PCR products in various tissues from the SBMA patient gave a 370 bp band (arrow), and are about 75-87 bp larger than those from control subjects.
2
3
Results
C A G repeats in tissues from an SBMA patient The P C R product containing the C A G repeats in various tissues (bladder, cardiac muscle, cerebrum, cerebellum and spinal cord) from the SBMA patient gave a 370-bp band, and was about 80 bp larger than those from control subjects (Fig. 1). Sequence analysis of the P C R products derived from tissues from the patient with SBMA revealed that the number of C A G repeats was 49, that is 75-87 bp larger than that from control subjects, including the ALS patients (20-24 repeats) (Fig. 2).
i I
,-/
Fig. 2. Sequencing of the PCR products obtained by the amplification of the CAG repeat region in various tissues from the SBMA patient. The lanes are identified as follows: lane 1, cardiac muscle; lane 2, cerebrum; lane 3, spinal cord. The same result was obtained in the bladder and cerebellum tissues. Arrows indicate start and end points of the CAG repeats. The number of CAG repeats in the tissues was 49.
77 1
2
3
4
5
6
7
~ - - 13- actin
ratio AR/~-actin
2.1
2.9
5.0
1,5
2.1
3.1
0.5
lung cancer, and the ratios of AR mRNA in the patients with ALS and the patients with lung cancer to that in the patient with SBMA were 4.5 5:1.6 and 6.7 + 3.0, respectively. The level of AR mRNA in the spinal cord tended to be a little lower in the patients with ALS than in those with lung cancer, however, there was no statistical significance (Fig. 3).
radioactivity Fig. 3. RT-PCR analyses of the AR gene expression in the spinal cord. RT-PCR products from AR mRNA and/3-actin mRNA in the patients with lung cancer (lanes 1-3), ALS (lanes 4-6) and SBMA (lane 7) were electrophoresed through 5% polyacrylamide gel and the radioactivity was counted using a BA100 Image Analyzer (Fuji Film). The band for AR mRNA in SBMA (AR*) was larger than that in the others. The ratio of AR mRNA to/3-actin mRNA was 0.5 in the patient with SBMA, 2.2+0.8 in the patients with ALS, 3.3 + 1.5 in the patients with lung cancer.
AR protein in SBMA To investigate AR expression in the spinal cords from the SBMA patient, ALS and lung cancer patients, Western blot analysis was performed. As shown in Fig. 4, AR protein was detected in the spinal cords from all subjects, however, the level of AR protein in the spinal cord of the SBMA patient was significantly less than that seen in the ALS and lung cancer patients. The difference between the patients with ALS and lung cancer was not significant (Fig. 4).
Quantitative RT-PCR for AR mRNA Quantitative RT-PCR was used to examine the influence of enlargement of the CAG repeat in the first exon of the AR gene on AR mRNA levels in the spinal cords. We confirmed that the radioactivity of PCR amplification for both AR mRNA and /3-actin mRNA increased exponentially during 27 cycles of the reaction (data not shown). Therefore we used 24 cycles under which linearization of PCR amplification for both AR mRNA and /3-actin mRNA was detected. We estimated the intensity of the bands for AR mRNA to /3-actin mRNA (used as an internal control). As shown in Fig. 3, the ratio of AR mRNA to fl-actin mRNA was 0.5 in the patient with SBMA, 2.2 _+0.8 in the patients with ALS, and 3.3 +_ 1.5 in the patients with
1
2
3
4 KDa 200
AR
115 97
66 Fig. 4. Western blot of AR protein in the spinal cords of the patients with SBMA, ALS, and lung cancer. One hundred ~g protein of tissue extracts in the spinal cord from the SBMA patient (lane 1), ALS patient (lane 2), and lung cancer patient (lane 3) were electrophoresed and subjected to Western blot. Lane 4 is for the molecular weight markers. The level of A R protein in the spinal cord from the patient with SBMA was significantly less than that of the patients with ALS and lung cancer.
Discussion AR protein is present in many tissues such as testis, prostate, brain, spinal cord and cardiac muscle, but not in peripheral blood mononuclear cells (PBMC) (Mooradian et al. 1987; Ruizeveld de Winter et al. 1991). The mutant AR gene with increased triplet repeats causes SBMA, a disorder in which spinal and bulbar motor neurons are mainly involved, while disturbance of AR function resulting from a certain point mutation of the AR gene causes infertility but no weakness (Griffin and Wilson 1989). Thus, different mutations of the AR gene lead to different diseases. One hypothesis is that enlargement of the CAG repeats may alter tissue specific expression of the AR gene. In the present study, we addressed directly alteration in the exonic trinucleotide repeats and expression of the AR gene in the spinal cord from a patient with SBMA. We investigated the number of CAG repeats in the AR gene in various tissues from the patient with SBMA, and from controls. Forty-nine CAG triplet repeats were found in the spinal cord, cerebrum, cerebellum, cardiac muscle and bladder from the patient with SBMA, while 20-24 CAG repeats were noted in these tissues from control subjects, including ALS. Biancalana et al. (1992) reported that the CAG repeats in the AR gene are highly pleomorphic and that a limited somatic instability of the CAG repeats in the abnormal SBMA allele was detected in variation by 5:3 repeats which exceeded that in the normal allele (in variation by 1 repeat). This variation of the CAG repeats was found in PBMC. In fragile X syndrome and myotonic dystrophy in which expansion of exonic trinucleotide repeats occurs, somatic heterogeneity of the amplified repeats due to mitotic instability was extensive (n > 100
78 for fragile X syndrome, n > 60 for myotonic dystrophy). These findings prompted us to investigate the possibility of expansion of the CAG repeats in involved organs, especially the spinal cord. Unlike findings in these two diseases, our observations suggest that the extensive mitotic instability of the AR gene in SBMA may not occur at the level of somatic cells. Using quantitative RT-PCR and Western blot analysis, we found that the level of both AR mRNA and protein in the spinal cord from the patient with SBMA was less than that from the patients with ALS in whom the loss of motor neurons was similar to that in the patient with SBMA. Pieretti et al. (1991) reported markedly reduced levels of FMR-1 mRNA in the majority of male fragile X patients, suggesting that the reduced expression of the gene was probably involved in the development of the syndrome. In the relationship between mutations of the AR gene and AR function, McPhaul et al. (1993) analyzed 31 unrelated subjects with androgen resistance syndromes, most of whom had point mutations of the AR gene, and showed that in most of the cases the phenotypic abnormalities are the result of impairment of receptor function or decreases in receptor abundance or both. All these findings taken together suggest that not only structural alteration of AR protein resulting from enlargement of the polyglutamine repeats but also a reduced level of AR protein resulting from a decreased expression of the AR gene in the spinal cord are involved in abnormal functions of AR leading to SBMA. Acknowledgements We thank Drs. K. Nanba (Saishunso Hospital) for providing material from the autopsied case of SBMA, A. Mizokami (University of Occupational and Environmental Health) for providing AR antibody, H. Ueyama (Kumamoto University) for technical assistance, and M. Ohara for helpful comments.
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