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Analysis of the CAG repeat region of the androgen receptor gene in a kindred with X-linked spinal and bulbar muscular atrophy
Journal of the Neurological Scie::ces, 112 (1992) 133-138
@ 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00
133
JNS 03830
Analysis of the CAG repeat region of the androgen receptor gene in a kindred with X-linked spinal and bulbar muscular atrophy Denise D. Belsham
a,
Woon-Chee Yee
b,1
Cheryl R. Greenberg a,c and Klaus Wrogemann a,d
Departments of "aHuman Genetics, b Medicine, c Pediatrics and Child Health, and d Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, Canada
(Received 5 February, 1992) (Accepted 27 March, 1992)
Key words: X-linked spinal and bulbar muscular atrophy; Kennedy's disease; Androgen receptor; Mutation; Carrier detection; Polymerase chain reaction; DNA sequencing
Summary Herein we describe a family with X-linked spinal and bulbar muscular atrophy (SBMA or Kennedy's disease), an adult onset neuromuscular disease characterized by slow progression, predominant proximal and bulbar muscle weakness. One frequent association is the appearance of gynecomastia. This disorder was previously shown to be linked to the locus DXYS1 on the proximal long arm of the X chromosome. Recently, a report implicated a mutation at the N-terminus of the androgen receptor gene involving amplification of CAG repeats as the cause of X-linked SBMA. We studied this region of the androgen receptor in a kindred clinically suspected but not confirmed of having X-linked SBMA by the polymerase chain reaction (PCR) followed by Southern analysis and DNA sequencing. The mutated allele was found to have an increased number of 51 CAG repeats confirming the clinical diagnosis of SBMA. Normal individuals revealed 23 repeat numbers" within the normal range, while another unrelated X-linked SBMA patient had an enlarged CAG repeat region. The carrier or disease status could be established or confirmed in 12 individuals of this family on the basis of detecting normal and disease alleles reflected by the number of CAG repeats.
Introduction
X-linked spinal and bulbar muscular atrophy (SBMA) was first described by Kennedy et al. (1968) in his report of two families in which 11 members, all male, were affected by an unusual, slowly progressive spinal and bulbar muscular atrophy affecting the anterior horn cells (Kennedy et al. 1968). The disease often presents in the third to fifth decades and initially involves degeneration of the anterior horn cells, leading to proximal muscle wastage. The life span of an affected individual is only slightly shortened, if at all (Harding et al. 1982). Since the initial description, more than 30 families have been reported to have this
I Present address: Dept. of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA. Correspondence to: Dr. K. Wrogemann, Department of Biochemistry and Molecular Biology, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba R3E 0W3, Canada.
condition (Harding et al. 1982; Arbizu et al. 1983; Fischbeck et al. 1991; La Spada et al. 1991; Warner et al. 1991). The chronic spinal muscular atrophies developing in the adult life are a clinically and genetically heterogeneous group of disorders (Harding et al. 1982). They show autosomal dominant, autosomal recessive and X-linked patterns of inheritance and are pathologically similar. Although clinical differences do exist between autosomal and X-linked forms, X-linked SBMA has been under-recognized in males without a fami'~ history suggesting X-linked inheritance, and it can be misdiagnosed. The clinical features of X-linked SBMA frequently include gynecomastia, often the earliest manifestation of the disease. When thoroughly examined some patients have also been noted to have testicular atrophy and azoospermia (Harding et al. 1982; Arhizu et al. 1983). Recently, a patient with SBMA was also shown to have decreased specific androgen-binding affinity (Warner et al. 1991), but according to La Spada (8th
134
Materials and methods
International Congress of Human Genetics, Washington, DC, October, 1991), this finding may be rare. These characteristics are similar to those found in patients with mild forms of androgen insensitivity syndrome (AIS). Fischbeck et al. (1986) found significant linkage between SBMA and DXYS1 on the proximal long arm of the X-chromosome, suggesting that the gene for SBMA mapped to the pericentric region of the X-chromosome. Concurrently close linkage was also shown between DXYS1 and AIS androgen insensitivity syndromes caused by defects of the androgen receptor (Wieacker et al. 1987). The androgen receptor itself was mapped to Xqll-12 on the long arm of the X-chromosome (Brown et al. 1989). These preliminary findings suggested that the gene encoding the androgen receptor may also be linked to SBMA (Fischbeck et al. 1986) and the possibility of a contiguous gene syndrome (Schmickel 1986) consisting of a series of contiguous genes involving androgen insensitivity and SBMA was raised, because of the overlap of clinical characteristics. Furthermore, an alternate hypothesis wL,,ld be that the genes encoding the mutant protein responsible for both disorders are identical, with allelic mutations responsible for the different phenotypes. Evidence for the latter hypothesis was reported by La Spada et al. (1991). In a study of 35 SBMA patients and 75 control subjects, an increased number of CAG repeats in exon 1 at the N-terminus of the androgen receptor gene was absolutely associated with the disease. The CAG (polyglutamine) repeats, double the average 21 repeats in normal controls, segregated with the disease phenotype in 15 characterized families with no recombination seen in the 61 meioses studied ( Z 13.2 at ~ ffi 0) (La Spada et al. 1991). It was now possible to test whether one could use these findings to confirm the clinical diagnosis in a large Manitoba kindred suspected of having SBMA, and to predict the carrier status in at risk females.
The subject and family The kindred is shown in Fig. 1. The proband (IV-14) is a 35-year-old male who presented with an insidious 10 year history of slowly progressive muscle weakness primarily involving shoulders and proximal lower limb muscles with mild muscle cramps noticed with exercise. There was also a recent onset of mild dysarthria and dysphagia. The family history revealed that his maternal grandfather (II-1) had retired in his mid-30's because of "muscular dystrophy" or a "neuropathy", but walked until his death from a myocardial infarction at 66 years of age. Physical examination of the proband (IV-14) revealed a nasal speech, generalized fasciculations in proximal, distal and in facial muscles, and symmetric muscle wasting and weakness also involving the bulbar and facial muscles. Sensory examination was normal. Reflexes were preserved. Coordination was normal and there was no percussion or action myotonia. There was neither gynecomastia nor evidence of hypogonadism. Electromyography showed evidence of widespread chronic denervation associated with fasciculations. All other investigations including CT of the head, CSF examination, myelogram, biochemical investigations, hexosaminidase A assay and DNA analysis of the dystrophin gene were normal, except for an elevated creatine kinase level of 571 U / I (normal, 52-175 U/l). A review of the maternal grandfather's medical records strongly suggested a similar clinical picture. His wife's history corroborated the impression that two maternal great-uncles (II-3 and II-5) were similarly afflicted, one of whom had choked to death. Adult onset X-linked spinal and bulbar muscular atrophy was the most likely clinical diagnosis. The maternal aunt (III-3) has a long-standing history of mild muscle aching with exertion. Her neurological examination was no~mal. However, her EMG
Spinal and Bulbar Muscular Atrophy
12
17
V
10
15
13
148
ZO
m x41nkedSBMA i
2
3
4
5
8
Personagy examk'Iod
Fig. 1. A complete pedigree of the X-linked spinal and bulbar muscular atrophy kindred. The proband is IV-14.
135 demonstrated mild chronic denervation in both uper and lower limb muscles. There is a history of vague complaints of muscle discomfort in individual IV-16, the brother of the proband, and in individual IV-7, his maternal cousin, but these individuals were not available for clinical evaluation. Neurological evaluation of individuals 111-7, III-11 and 111-14 was normal. Peripheral blood samples were obtained for DNA extraction from 14 family members (Fig. 2).
DNA analysis D N A was extracted according to established protocols (Greenberg et al. 1987). The PCR method was essentially the same as that described by Saiki et al. (1988) and amplification of the CAG repeat region was performed using the primer set described by La Spada et al. (1991). Each 100 /~1 PCR reaction contained PCR buffer containing 500 mM KCI, 100 mM Tris-HCl pH 8.3 (Perkin Elmer Cetus, Norwalk, CT), 20 nmol of each dNTP, 75 pmol of each primer, 1 # g DNA, 0.01% gelatin and 2.5 units of Taq polymerase covered with 100 /~1 of mineral oil. The reaction was carried out cyclically with denaturation at 95"C for 1 min, anneal-
Manitoba SBMA Family
ing at 67°C for 2 min, and extension at 72°C for 1.5 min. After 35 cycles, the final extension continued for another 8.5 min at 72°C.
Polyacrylamide or agarose gel electrophoresis The PCR amplified products were chloroform extracted and analyzed on either a 12% polyacrylamide gel using a mini-gel apparatus (Bio-Rad, Richmond, CA) or a 1.2% agarose gel. Gels were run at 150 V for 45 min, then the gels were stained in a 0.5 /zg/ml ethidium bromide solution and photographed with type 57 high-speed Polaroid film. Molecular weight markers used on each gel were the p G E M markers (Promega, Madison, WI).
Southern blot analysis After the PCR product was chloroform extracted, concentrated by ethanol precipitation and size-separated on 1.2% agarose gels, the DNA was transferred to Hybond-N membrane (Amersham, Arlington Heights, IL) (Southern, 1975). Prabes were labeled by random priming (Feinberg and Vogelstein, 1983) to a specific activity greater than 5 x 108 cpm//~g. The
CAG repeats, PCR, ARcDNA
~ N T
,NT
III IV I
I
I
--1 1;i ~'-Is--1 14 --re 1
I I_ ,
, ~ I
I
1-11100 I
I
i
I
A
-91 676 bp
iiiil
-41 222 bp
B
:~i
.,4
222 bp
Fig. 2. Analysisof the PCR products obtained by the amplification of the CAG repeat region in the individuals of the studied pedigree. (A) Agarose gel electrophoresis (1.2%) of PCR products in which the gel is stained with ethidium bromide. (B) Southern analysisof the gel in (A) probed with the h-AR cDNA defined by Smal 641-Stul 1153 containing the CAG repeat region (sequence according to Chang et al. 1988). Controls are in lanes from right to left: (1) partial androgen insensitivitysyndromepatient; (2) complete androgen insensitivitysyndromepatient; (3) unrelated SBMA patient; (4) control male. NT, not tested. Molecularweight markers indicated are pGEM markers.
136 probe used to recognize the CAG repeat region was a 512 bp fragment from the N-terminal region of the androgen receptor gene defined by SmaI 641 to StuI 1153 (sequence according to Chang et al. 1988).
Direct sequencing of the PCR amplified CAG repeat region For sequence analysis, a set of internal primers were synthesized (5'-TGGAAGATCAGCCAAGCTC-3'; 5'-TI'CCTCATCCAGGACCAGGT-3') for amplification of the CAG repeat region. Both the upper and lower bands were gel separated and cut out individually for purification by spinning through glass wooi at 6000 rpm for 10 min. The purified DNA was precipitated in 2.5 vol of ethanol, then resuspended in low TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0). The double-stranded DNA was sequenced with the dsDNA Cycle Sequencing Kit (BRL, Gaithersburg, MD) according to the manufacturer's recommendations using the primary method of Sanger et al. (1977). 7-DeazadGTP was used to avoid unnecessary secondary structure formation, particularly hydrogen bond formation, which causes band compressions during the sequencing run. One of the primers used to amplify the fragment for sequencing was end-labelled with [32p]dATP and used in the cycle sequencing reaction as the primer.
Results
The amplification of the CAG repeats or polyglutamine region located in exon 1 at the N-terminus of the androgen receptor gene was performed for the 14 available kindred members, The size of the fragment representing the normal allele from this region is predicted to be about 400-450 bp, while the putative
PROBAND A.A. $ 3 . SLLLLQQQQQQQQQQOOQQQQQQQQQQQQQQQQOOQQQQQQOQQQQQQQQQQOQQETSPR.
REPEAT
.-
REGION !
"
""
""
,.,, ~
,_ . .
'
t
I
ATG |
113
DNA
~ ",,, % %
IClOl
s
~
I STOP
" " " ~ -- "~' "-,.. •" --.
A.A. 53 - SLLI'~t.LQQQQQQQQQ
HOR/VIONE
( 2 3 kb) ~
"
ETSPR • B,~
CONTROL
S = SERINE; L = LEUCINE; O = GLUTAP~IINE; E = GLUTAMIC ACID; T = THREONINE; P = PROUNE; R = ARGININE
Fig. 3. Deduced amino acid numbers from the sequencing of the androgen receptor gene of the proband (IV-14) and his normal (control) brother (IV-16) at the CAG repeat region. The sequencing results indicate that the proband has nearly twice as many glutamine repeats as his normal brother (51 and 23 repeats, respectively).
3'
mutant allele, with its amplified region is expected to yield a fragment of about 500 bp or more. The two sets of bands could be easily detected, one 400 bp and one approximately 500 bp in length (Fig. 2(A)). DNA sequencing of the purified top band confirmed an increased number of CA(; repeats (51) (Fig. 3), very close to the predicted number by the size analysis of agarose and PAGE gels. The normal fragment has 23 CAG repeats (Fig. 3). These numbers are within the ranges for SBMA (40-52 repeats) and controls (17-26 repeats) reported by La Spada et al. (1991).
Discussion
We present a kindred with suspected X-linked SBMA in whom DNA analysis of the androgen receptor gene confirms this diagnosis in the affected proband, predicts his 3 sisters to be carriers of this X-linked recessive disease and his maternal aunt to be a manifesting carrier. Three maternal cousins are predicted to be non-carriers and the brother of the proband is predicted to be unaffected. Interestingly, the daughter (III-11) of II-3 would be predicted to be an obligate carrier, however her DNA revealed only a single band with the CAG repeat region within the normal range (lane 11, Fig. 2) as did her daughter (IV-20). Either the diagnosis of X-linked SBMA is wrong in individual II-3 or the possibility of nonpaternity exists. The latter possibility cannot be explored but personal discussion with III-11 indicates that this is the correct explanation. The diverse number of polyglutamine repeats or CAG repeats in the coding region in exon 1 at the N-terminus of the androgen receptor has been reported to range from 17 to 26 with an average of 21 in normal individuals (La Spada et al. 1991). The differences noted in this area were first noted upon the cloning and sequencing of the androgen receptor gene in different laboratories (Lubahn et al. 1988; Tilley et al. 1989). There is another region further downstream from this polyglutamine region in the androgen receptor gene known to contain varying numbers of G G T / G G C repeats, called the polyglycine region. It had been thought that perhaps patients with differing degrees of androgen insensitivity would have varying sizes of these repeats (Lubahn et al. 1988; Tilley et al. 1989). Upon analysis of a few patients with complete and partial AIS the number of repeats falls into the normal range, and therefore cannot be the sole explanation for varying degrees of androgen insensitivity. Patients with AIS are feminized but do not show any signs of muscle weakness, which is very different from patients with SBMA who have normal fetal sexual development, despite later signs of feminization. Specific androgen receptor binding studies in patients
137 with SBMA indicate that more often than not the binding activity is normal, although 2 patients have been found with decreased binding activity (La Spada, 8th International Congress of Human Genetics, Washington, DC, October, 1991). The insertional mutation probably does not affect the DNA- or androgen-binding characteristics of the AR, but possibly alters the function of the AR in motor neurons. Androgen receptors have been found to be concentrated in spinal and bulbar motor neurons, the cells that degenerate in SBMA (Sar and Stumpf 1977). In the rat, androgens are known to play an important role in normal spinal motor neuron growth, development and response to injury (Kurz et al. 1986; Yu 1989). It is known that the number of CAG repeats does not seem to correlate with the severity ~Jr onset of the disease (La Spada, 8th International Cbngress of Human Genetics, Washington, DC, October, 1991). In our study, the proband does not have gynecomastia, but the patient control, an unrelated SBMA patient has this variable characteristic of SBMA. The number of CAG repeats is smaller in our proband than in this control patient; thus, it would be interesting to search for a correlation between the number of CAG repeats and the presence of gynecomastia in other SBMA patients to determine if this finding is of any importance. The significance of the molecular finding has yet to be elucidated, but the increased number of CAG repeats has been implicated to be the cause of X-linked SBMA (La Spada et al. 1991). Yet, how this occurs is difficult to imagine. One hypothesis is that the Nterminus of the androgen receptor is required for an operational task associated with motor neuron function. The regulation of genes by the AR could be predicted to be altered in patients with SBMA (La Spada et al. 1991), as repeats of polyglutamine have been implicated in both developmental regulation and transcriptional regulation of other genes in different species (Wharton et al. 1985; Duboule et al. 1987; Mitchell and Tijan 1989; Kao et al. 1990). Alternatively, the CAG repeat may simply alter tissue specific expression of the AR. Yet another possibility could be that the AR gene encoded protein exerts in these motor neurons a function which is different from the conventionally understood role of this steroid receptor. It is truly remarkable to have different mutations of this gene responsible on one hand for the diverse spectrum of X-linked androgen insensitivity and on the other hand for this distinct motor neuron disease. Since the role of the N-terminal region of the androgen receptor is not well understood, one would believe that any study of its functionality would help to uncover the significance of the N-terminal region of not only the androgen receptor, but perhaps other members of the steroid/ thyroid/ retinoic acid supergene
family (Evans 1988; O'Malley 1990) due to the similar organization of these proteins. Acknowledgements This work was supported by the Muscular Dystrophy Association of Canada, the Medical Research Council of Canada and the Children's Hospital of Winnipeg Research Foundation. We thank Dr. K. Fischbeck, University of Pennsylvania, for providing a genital skin fibroblast cell strain from a patient with SBMA as a positive control.
References Arbizu, T., J. Santamaria, J.M. Gomez, A. Quilez and J.P. Serra (1983) A family with adult spinal and bulbar muscular atrophy, X-linked inheritance and associated testicular failure. J. Neurol. Sci., 59: 371-382. Brown, C.J., S.G. Goss, D.B. Lubahn, D.R. Joseph, E.M. Wilson, F.S. French and H.F. Willard (1989) Androgen receptor locus on the human X chromosome: Regional localization to Xq11-12 and description of a DNA polymorphism. Am. J. Hum. Genet., 44: 264-269. Chang, C., J. Kokontis and S. Liao (1988) Molecular cloning of human and rat complementary DNA encoding androgen receptot's. Science, 240: 324-326. Duboule, D., M. Haenlin, B. Galliot and E. Mohier (1987) DNA sequences homologous to the Drosophila opa repeat are present in murine mRNAs that are differentially expressed in fetuses and adult tissues. Mol. Ceil. Biol., 7: 2003-2006. Evans, R.M. (1988) The steroid and thyroid hormone receptor superfamily. Science, 240: 889-895. Feinberg, A.P. and B. Vogelstein (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132: 6-13. Fischbeck, K.H., V. Ionasescu, A.W. Ritter, R. Ionasescu, K. Davies, S. Ball, P. Bosch, T. Burns, I. Hausmanowa Petrusewicz, J. Borkowska et ai. (1986) Localization of the gene for X-linked spinal muscular atrophy. Neurology, 36: 1595-1598. Fischbeck, K.H., D. Souders and A. La Spada (1991) A candidate gene for X-linked spinal muscular atrophy. Adv. Neurol., 56: 209--.213. Greenherg, C.R., J.L. Hamerton, M. Nigli and K. Wrogemann (1987) DNA studies in a family with Duchenne muscular dystrophy and a deletion at Xp21. Am. J. Hum. Genet., 41: 128-137. Harding, A.E., P.K. Thomas, M. Baraitser, P.G. Bradbury, J.A. Morgan-Hughes and J.R. Ponsford (1982) X-linked recessive bulbospinal neuronopathy: a report of ten cases. J. Neurol. Neurosurg. Psychiat., 45: 1012-1019. Kao, C.C., P.M. Lieberman, M.C. Schmidt, Q. Zhou, R. Pei and A.J. Berk (1990) Cloning of a transcriptionally active human TATA binding factor. Science, 248: 1646-1650. Kennedy, W.R., M. Alter and J.H. Sung (1968) Progressive proximal spinal and bulbar muscular atrophy of late onset. Neurology, 18: 671-680. Kurz, E.M., D.R. Sengelaub and A.P. Arnold (1986) Androgens regulate the dendritic length of mammalian motorneurons 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 buibar muscular atrophy. Nature, 352: 77-79. Lubahn, D.B., D.R. Joseph, M. Sar, J. Tan, H.N. Higgs, R.E. Larson, F.S. French and E.M. Wilson (1988) The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. Mol. Endocrinol., 2: 12651275.
138 Mitchell, PJ. and R, Tijan (1989) Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science, 245: 371-398. O'Malley, B. (1990) The steroid receptor superfamily: more excitement for the future. Moi. Endocrinol., 4: 363-369. Saiki, R.K,, D.H. Gelfand, S. Stoffel, SJ. Scharf, R. Higuchi, G.T. Horn, K.B. Mullis and H.A. Ehrlich (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239: 487-491. Sanger, F., S. Nicklen and A.R. Coulson (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74: 5463-5467. Sar, M. and W.E. Stumpf (1977) Androgen concentration in motor neurons of cranial nerves and spinal cord. Science, 197: 77-80. Schmickel, R.D. (1986) Contiguous gene syndromes: A component of recognizable syndromes. `i. Pediatr., 109: 231-241. Southern, E. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. `i. Mol. Biol., 98: 503-517.
Tilley, W.D., M. MarceUi, J.D. Wilson and MJ. McPhaul (1989) C!~aracterization and expression of a cDNA encoding the human androgen receptor. Proc. Natl. Acad. Sci. USA, 86: 327-331. Warner, C.L., J.E. Griffin, J.D. Wilson, L.D. Jacobs, K. Murray, K.H. Fischbeck, D. Dickoff and R.C. Griggs (1991) X-linked spinomuscular atrophy: a kind~ed with associated abnormal androgen receptor binding. Neurology, 41 (Suppl. 1): 313. Wharton, K.A., B. Yedvobnick, V.G. Finnerty and S. ArtavanisTsakonas (1985) Opa: a novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. mei,~;,ogaster. Cell, 40: 55-62. Wieacker, P., J.E. Griffin, T. Wienker, J.M. Lopez, ,I.D. Wilson and M. Breckwoldt (1987) Linkage analysis with RFLPs in families with androgen resistance syndromes: evidence for close linkage between the androgen receptor locus and the DXS1 segment. Hum. Genet., 76: 248-252. Yu, W-b.A. (1989) Administration of testosterone attenuates neuronal loss following axotomy in the brain-stem motor nuclei of female rats. J. Neurosci., 9(11): 3908-3914.
@ 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00
133
JNS 03830
Analysis of the CAG repeat region of the androgen receptor gene in a kindred with X-linked spinal and bulbar muscular atrophy Denise D. Belsham
a,
Woon-Chee Yee
b,1
Cheryl R. Greenberg a,c and Klaus Wrogemann a,d
Departments of "aHuman Genetics, b Medicine, c Pediatrics and Child Health, and d Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, Canada
(Received 5 February, 1992) (Accepted 27 March, 1992)
Key words: X-linked spinal and bulbar muscular atrophy; Kennedy's disease; Androgen receptor; Mutation; Carrier detection; Polymerase chain reaction; DNA sequencing
Summary Herein we describe a family with X-linked spinal and bulbar muscular atrophy (SBMA or Kennedy's disease), an adult onset neuromuscular disease characterized by slow progression, predominant proximal and bulbar muscle weakness. One frequent association is the appearance of gynecomastia. This disorder was previously shown to be linked to the locus DXYS1 on the proximal long arm of the X chromosome. Recently, a report implicated a mutation at the N-terminus of the androgen receptor gene involving amplification of CAG repeats as the cause of X-linked SBMA. We studied this region of the androgen receptor in a kindred clinically suspected but not confirmed of having X-linked SBMA by the polymerase chain reaction (PCR) followed by Southern analysis and DNA sequencing. The mutated allele was found to have an increased number of 51 CAG repeats confirming the clinical diagnosis of SBMA. Normal individuals revealed 23 repeat numbers" within the normal range, while another unrelated X-linked SBMA patient had an enlarged CAG repeat region. The carrier or disease status could be established or confirmed in 12 individuals of this family on the basis of detecting normal and disease alleles reflected by the number of CAG repeats.
Introduction
X-linked spinal and bulbar muscular atrophy (SBMA) was first described by Kennedy et al. (1968) in his report of two families in which 11 members, all male, were affected by an unusual, slowly progressive spinal and bulbar muscular atrophy affecting the anterior horn cells (Kennedy et al. 1968). The disease often presents in the third to fifth decades and initially involves degeneration of the anterior horn cells, leading to proximal muscle wastage. The life span of an affected individual is only slightly shortened, if at all (Harding et al. 1982). Since the initial description, more than 30 families have been reported to have this
I Present address: Dept. of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA. Correspondence to: Dr. K. Wrogemann, Department of Biochemistry and Molecular Biology, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba R3E 0W3, Canada.
condition (Harding et al. 1982; Arbizu et al. 1983; Fischbeck et al. 1991; La Spada et al. 1991; Warner et al. 1991). The chronic spinal muscular atrophies developing in the adult life are a clinically and genetically heterogeneous group of disorders (Harding et al. 1982). They show autosomal dominant, autosomal recessive and X-linked patterns of inheritance and are pathologically similar. Although clinical differences do exist between autosomal and X-linked forms, X-linked SBMA has been under-recognized in males without a fami'~ history suggesting X-linked inheritance, and it can be misdiagnosed. The clinical features of X-linked SBMA frequently include gynecomastia, often the earliest manifestation of the disease. When thoroughly examined some patients have also been noted to have testicular atrophy and azoospermia (Harding et al. 1982; Arhizu et al. 1983). Recently, a patient with SBMA was also shown to have decreased specific androgen-binding affinity (Warner et al. 1991), but according to La Spada (8th
134
Materials and methods
International Congress of Human Genetics, Washington, DC, October, 1991), this finding may be rare. These characteristics are similar to those found in patients with mild forms of androgen insensitivity syndrome (AIS). Fischbeck et al. (1986) found significant linkage between SBMA and DXYS1 on the proximal long arm of the X-chromosome, suggesting that the gene for SBMA mapped to the pericentric region of the X-chromosome. Concurrently close linkage was also shown between DXYS1 and AIS androgen insensitivity syndromes caused by defects of the androgen receptor (Wieacker et al. 1987). The androgen receptor itself was mapped to Xqll-12 on the long arm of the X-chromosome (Brown et al. 1989). These preliminary findings suggested that the gene encoding the androgen receptor may also be linked to SBMA (Fischbeck et al. 1986) and the possibility of a contiguous gene syndrome (Schmickel 1986) consisting of a series of contiguous genes involving androgen insensitivity and SBMA was raised, because of the overlap of clinical characteristics. Furthermore, an alternate hypothesis wL,,ld be that the genes encoding the mutant protein responsible for both disorders are identical, with allelic mutations responsible for the different phenotypes. Evidence for the latter hypothesis was reported by La Spada et al. (1991). In a study of 35 SBMA patients and 75 control subjects, an increased number of CAG repeats in exon 1 at the N-terminus of the androgen receptor gene was absolutely associated with the disease. The CAG (polyglutamine) repeats, double the average 21 repeats in normal controls, segregated with the disease phenotype in 15 characterized families with no recombination seen in the 61 meioses studied ( Z 13.2 at ~ ffi 0) (La Spada et al. 1991). It was now possible to test whether one could use these findings to confirm the clinical diagnosis in a large Manitoba kindred suspected of having SBMA, and to predict the carrier status in at risk females.
The subject and family The kindred is shown in Fig. 1. The proband (IV-14) is a 35-year-old male who presented with an insidious 10 year history of slowly progressive muscle weakness primarily involving shoulders and proximal lower limb muscles with mild muscle cramps noticed with exercise. There was also a recent onset of mild dysarthria and dysphagia. The family history revealed that his maternal grandfather (II-1) had retired in his mid-30's because of "muscular dystrophy" or a "neuropathy", but walked until his death from a myocardial infarction at 66 years of age. Physical examination of the proband (IV-14) revealed a nasal speech, generalized fasciculations in proximal, distal and in facial muscles, and symmetric muscle wasting and weakness also involving the bulbar and facial muscles. Sensory examination was normal. Reflexes were preserved. Coordination was normal and there was no percussion or action myotonia. There was neither gynecomastia nor evidence of hypogonadism. Electromyography showed evidence of widespread chronic denervation associated with fasciculations. All other investigations including CT of the head, CSF examination, myelogram, biochemical investigations, hexosaminidase A assay and DNA analysis of the dystrophin gene were normal, except for an elevated creatine kinase level of 571 U / I (normal, 52-175 U/l). A review of the maternal grandfather's medical records strongly suggested a similar clinical picture. His wife's history corroborated the impression that two maternal great-uncles (II-3 and II-5) were similarly afflicted, one of whom had choked to death. Adult onset X-linked spinal and bulbar muscular atrophy was the most likely clinical diagnosis. The maternal aunt (III-3) has a long-standing history of mild muscle aching with exertion. Her neurological examination was no~mal. However, her EMG
Spinal and Bulbar Muscular Atrophy
12
17
V
10
15
13
148
ZO
m x41nkedSBMA i
2
3
4
5
8
Personagy examk'Iod
Fig. 1. A complete pedigree of the X-linked spinal and bulbar muscular atrophy kindred. The proband is IV-14.
135 demonstrated mild chronic denervation in both uper and lower limb muscles. There is a history of vague complaints of muscle discomfort in individual IV-16, the brother of the proband, and in individual IV-7, his maternal cousin, but these individuals were not available for clinical evaluation. Neurological evaluation of individuals 111-7, III-11 and 111-14 was normal. Peripheral blood samples were obtained for DNA extraction from 14 family members (Fig. 2).
DNA analysis D N A was extracted according to established protocols (Greenberg et al. 1987). The PCR method was essentially the same as that described by Saiki et al. (1988) and amplification of the CAG repeat region was performed using the primer set described by La Spada et al. (1991). Each 100 /~1 PCR reaction contained PCR buffer containing 500 mM KCI, 100 mM Tris-HCl pH 8.3 (Perkin Elmer Cetus, Norwalk, CT), 20 nmol of each dNTP, 75 pmol of each primer, 1 # g DNA, 0.01% gelatin and 2.5 units of Taq polymerase covered with 100 /~1 of mineral oil. The reaction was carried out cyclically with denaturation at 95"C for 1 min, anneal-
Manitoba SBMA Family
ing at 67°C for 2 min, and extension at 72°C for 1.5 min. After 35 cycles, the final extension continued for another 8.5 min at 72°C.
Polyacrylamide or agarose gel electrophoresis The PCR amplified products were chloroform extracted and analyzed on either a 12% polyacrylamide gel using a mini-gel apparatus (Bio-Rad, Richmond, CA) or a 1.2% agarose gel. Gels were run at 150 V for 45 min, then the gels were stained in a 0.5 /zg/ml ethidium bromide solution and photographed with type 57 high-speed Polaroid film. Molecular weight markers used on each gel were the p G E M markers (Promega, Madison, WI).
Southern blot analysis After the PCR product was chloroform extracted, concentrated by ethanol precipitation and size-separated on 1.2% agarose gels, the DNA was transferred to Hybond-N membrane (Amersham, Arlington Heights, IL) (Southern, 1975). Prabes were labeled by random priming (Feinberg and Vogelstein, 1983) to a specific activity greater than 5 x 108 cpm//~g. The
CAG repeats, PCR, ARcDNA
~ N T
,NT
III IV I
I
I
--1 1;i ~'-Is--1 14 --re 1
I I_ ,
, ~ I
I
1-11100 I
I
i
I
A
-91 676 bp
iiiil
-41 222 bp
B
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.,4
222 bp
Fig. 2. Analysisof the PCR products obtained by the amplification of the CAG repeat region in the individuals of the studied pedigree. (A) Agarose gel electrophoresis (1.2%) of PCR products in which the gel is stained with ethidium bromide. (B) Southern analysisof the gel in (A) probed with the h-AR cDNA defined by Smal 641-Stul 1153 containing the CAG repeat region (sequence according to Chang et al. 1988). Controls are in lanes from right to left: (1) partial androgen insensitivitysyndromepatient; (2) complete androgen insensitivitysyndromepatient; (3) unrelated SBMA patient; (4) control male. NT, not tested. Molecularweight markers indicated are pGEM markers.
136 probe used to recognize the CAG repeat region was a 512 bp fragment from the N-terminal region of the androgen receptor gene defined by SmaI 641 to StuI 1153 (sequence according to Chang et al. 1988).
Direct sequencing of the PCR amplified CAG repeat region For sequence analysis, a set of internal primers were synthesized (5'-TGGAAGATCAGCCAAGCTC-3'; 5'-TI'CCTCATCCAGGACCAGGT-3') for amplification of the CAG repeat region. Both the upper and lower bands were gel separated and cut out individually for purification by spinning through glass wooi at 6000 rpm for 10 min. The purified DNA was precipitated in 2.5 vol of ethanol, then resuspended in low TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0). The double-stranded DNA was sequenced with the dsDNA Cycle Sequencing Kit (BRL, Gaithersburg, MD) according to the manufacturer's recommendations using the primary method of Sanger et al. (1977). 7-DeazadGTP was used to avoid unnecessary secondary structure formation, particularly hydrogen bond formation, which causes band compressions during the sequencing run. One of the primers used to amplify the fragment for sequencing was end-labelled with [32p]dATP and used in the cycle sequencing reaction as the primer.
Results
The amplification of the CAG repeats or polyglutamine region located in exon 1 at the N-terminus of the androgen receptor gene was performed for the 14 available kindred members, The size of the fragment representing the normal allele from this region is predicted to be about 400-450 bp, while the putative
PROBAND A.A. $ 3 . SLLLLQQQQQQQQQQOOQQQQQQQQQQQQQQQQOOQQQQQQOQQQQQQQQQQOQQETSPR.
REPEAT
.-
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"
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,.,, ~
,_ . .
'
t
I
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113
DNA
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s
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" " " ~ -- "~' "-,.. •" --.
A.A. 53 - SLLI'~t.LQQQQQQQQQ
HOR/VIONE
( 2 3 kb) ~
"
ETSPR • B,~
CONTROL
S = SERINE; L = LEUCINE; O = GLUTAP~IINE; E = GLUTAMIC ACID; T = THREONINE; P = PROUNE; R = ARGININE
Fig. 3. Deduced amino acid numbers from the sequencing of the androgen receptor gene of the proband (IV-14) and his normal (control) brother (IV-16) at the CAG repeat region. The sequencing results indicate that the proband has nearly twice as many glutamine repeats as his normal brother (51 and 23 repeats, respectively).
3'
mutant allele, with its amplified region is expected to yield a fragment of about 500 bp or more. The two sets of bands could be easily detected, one 400 bp and one approximately 500 bp in length (Fig. 2(A)). DNA sequencing of the purified top band confirmed an increased number of CA(; repeats (51) (Fig. 3), very close to the predicted number by the size analysis of agarose and PAGE gels. The normal fragment has 23 CAG repeats (Fig. 3). These numbers are within the ranges for SBMA (40-52 repeats) and controls (17-26 repeats) reported by La Spada et al. (1991).
Discussion
We present a kindred with suspected X-linked SBMA in whom DNA analysis of the androgen receptor gene confirms this diagnosis in the affected proband, predicts his 3 sisters to be carriers of this X-linked recessive disease and his maternal aunt to be a manifesting carrier. Three maternal cousins are predicted to be non-carriers and the brother of the proband is predicted to be unaffected. Interestingly, the daughter (III-11) of II-3 would be predicted to be an obligate carrier, however her DNA revealed only a single band with the CAG repeat region within the normal range (lane 11, Fig. 2) as did her daughter (IV-20). Either the diagnosis of X-linked SBMA is wrong in individual II-3 or the possibility of nonpaternity exists. The latter possibility cannot be explored but personal discussion with III-11 indicates that this is the correct explanation. The diverse number of polyglutamine repeats or CAG repeats in the coding region in exon 1 at the N-terminus of the androgen receptor has been reported to range from 17 to 26 with an average of 21 in normal individuals (La Spada et al. 1991). The differences noted in this area were first noted upon the cloning and sequencing of the androgen receptor gene in different laboratories (Lubahn et al. 1988; Tilley et al. 1989). There is another region further downstream from this polyglutamine region in the androgen receptor gene known to contain varying numbers of G G T / G G C repeats, called the polyglycine region. It had been thought that perhaps patients with differing degrees of androgen insensitivity would have varying sizes of these repeats (Lubahn et al. 1988; Tilley et al. 1989). Upon analysis of a few patients with complete and partial AIS the number of repeats falls into the normal range, and therefore cannot be the sole explanation for varying degrees of androgen insensitivity. Patients with AIS are feminized but do not show any signs of muscle weakness, which is very different from patients with SBMA who have normal fetal sexual development, despite later signs of feminization. Specific androgen receptor binding studies in patients
137 with SBMA indicate that more often than not the binding activity is normal, although 2 patients have been found with decreased binding activity (La Spada, 8th International Congress of Human Genetics, Washington, DC, October, 1991). The insertional mutation probably does not affect the DNA- or androgen-binding characteristics of the AR, but possibly alters the function of the AR in motor neurons. Androgen receptors have been found to be concentrated in spinal and bulbar motor neurons, the cells that degenerate in SBMA (Sar and Stumpf 1977). In the rat, androgens are known to play an important role in normal spinal motor neuron growth, development and response to injury (Kurz et al. 1986; Yu 1989). It is known that the number of CAG repeats does not seem to correlate with the severity ~Jr onset of the disease (La Spada, 8th International Cbngress of Human Genetics, Washington, DC, October, 1991). In our study, the proband does not have gynecomastia, but the patient control, an unrelated SBMA patient has this variable characteristic of SBMA. The number of CAG repeats is smaller in our proband than in this control patient; thus, it would be interesting to search for a correlation between the number of CAG repeats and the presence of gynecomastia in other SBMA patients to determine if this finding is of any importance. The significance of the molecular finding has yet to be elucidated, but the increased number of CAG repeats has been implicated to be the cause of X-linked SBMA (La Spada et al. 1991). Yet, how this occurs is difficult to imagine. One hypothesis is that the Nterminus of the androgen receptor is required for an operational task associated with motor neuron function. The regulation of genes by the AR could be predicted to be altered in patients with SBMA (La Spada et al. 1991), as repeats of polyglutamine have been implicated in both developmental regulation and transcriptional regulation of other genes in different species (Wharton et al. 1985; Duboule et al. 1987; Mitchell and Tijan 1989; Kao et al. 1990). Alternatively, the CAG repeat may simply alter tissue specific expression of the AR. Yet another possibility could be that the AR gene encoded protein exerts in these motor neurons a function which is different from the conventionally understood role of this steroid receptor. It is truly remarkable to have different mutations of this gene responsible on one hand for the diverse spectrum of X-linked androgen insensitivity and on the other hand for this distinct motor neuron disease. Since the role of the N-terminal region of the androgen receptor is not well understood, one would believe that any study of its functionality would help to uncover the significance of the N-terminal region of not only the androgen receptor, but perhaps other members of the steroid/ thyroid/ retinoic acid supergene
family (Evans 1988; O'Malley 1990) due to the similar organization of these proteins. Acknowledgements This work was supported by the Muscular Dystrophy Association of Canada, the Medical Research Council of Canada and the Children's Hospital of Winnipeg Research Foundation. We thank Dr. K. Fischbeck, University of Pennsylvania, for providing a genital skin fibroblast cell strain from a patient with SBMA as a positive control.
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