American Journal of Medical Genetics 117A:161–163 (2003)

Somatic Instability of the Androgen Receptor CAGRepeat in a Normal Female

Andrew Sharp1* and Jane Hurst2

1Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, Wiltshire SP2 8BJ, United Kingdom2Department of Clinical Genetics, Oxford Regional Genetics Service, Oxford Radcliffe Hospital Trust, The Churchill,Oxford OX3 7LJ, United Kingdom

The polyglutamine repeat disorders repre-sent a family of degenerative neurologicaldiseases which are characterized by expan-sions of tandemly repeated CAG repeats.Investigations have demonstrated that inHuntington disease, dentatorubral pallido-luysian atrophy, and the spinocerebellarataxias type 1, 2, and 3, the polyglutamineexpansions show both meiotic and mito-tic instability. However, previous studieshave suggested that the polyglutamine motifwithin exon 1 of the androgen receptor gene(AR) which expands in cases of spinobulbarmuscular atrophy differs in that it is appar-ently mitotically stable. During linkage anal-ysis in a family with FG syndrome, a rarecondition mapped to Xq12-q22.1, we detectedthe presence of an unusually small AR allelesegregating within the pedigree. Sequen-cing, cytogenetic analysis, and PCR of flank-ing markers indicate that this allele arose bya somatic contraction of seven CAG repeatsin the proband’s mother, representing thefirstreport ofmitotic instability ofanARCAGrepeat of normal size. � 2003 Wiley-Liss, Inc.

KEY WORDS: instability; androgen recep-tor; SBMA; trinucleotiderepeat; mosaicism

INTRODUCTION

The androgen receptor gene (AR) at Xq12 contains a(CAG)n tandem repeat which is highly polymorphic(n¼9–36) in the normal population. Expansions of this

repeat (n�38) cause spinobulbar muscular atrophy(SBMA), a degenerative motor-neuron disease thatshows features typical of other trinucleotide repeatexpansion diseases [Andrew et al., 1997].

CAG repeat expansions have also been associatedwith Huntington disease (HD), dentatorubral pallido-luysian atrophy (DRPLA), and the spinocerebellarataxias type 1, 2, and 3 (SCA1, 2, and 3) [Andrew et al.,1997]. The expansions in these latter polyglutaminedisorders show moderate to high meiotic instability[Huntington’s Disease Collaborative Research Group,1993; Orr et al., 1993; Kawaguchi et al., 1994; Koideet al., 1994; Igarashi and Tsuji, 1999], and mitoticvariations in repeat length have also been demonstratedin HD [Telenius et al., 1994], DRPLA, SCA1, and SCA3[Hashida et al., 1997]. However, while limited meioticinstability has been demonstrated for the expanded ARCAG repeat [Biancalana et al., 1992; Zhang et al., 1995],several studies have failed to detect somatic variation ofrepeat length in SBMA patients [Spiegel et al., 1996;Tanaka et al., 1996; Watanabe et al., 1996; Jedele et al.,1998]. Thus, the AR CAG repeat differs from otherknown polyglutamine motifs in that it is apparentlymitotically stable.

Because the length of AR CAG repeat is highlyvariable in the normal population, it is commonly usedas a polymorphic marker. During linkage analysis in afamily with FG syndrome, a rare condition mapped toXq12-q22.1 characterized by mental retardation andspecific physical abnormalities [Graham et al., 1998], wedetected the presence of an unusually small AR allelesegregating within the pedigree. Further investigationsrevealed that this was caused by a mitotic contraction ofthe AR CAG repeat in the proband’s mother, represent-ing the first report of somatic instability at this locus in anormal individual.

CLINICAL REPORT

The proband GM, now 22 years of age, has a clinicaldiagnosis of FG syndrome with severe learning difficul-ties, epilepsy, dysmorphic features, and agenesis of thecorpus callosum, as described previously [Thompsonet al., 1985; Romano et al., 1994]. His sister SM has aseizure disorder, and is of normal intelligence. A pedi-gree is shown in Figure 1.

Grant sponsor: Wellcome Trust Prize Studentship; Grantnumber: 058387.

*Correspondence to: Andrew Sharp, Wessex Regional GeneticsLaboratory, Salisbury District Hospital, Salisbury, Wiltshire SP28BJ, United Kingdom. E-mail: asharp@hgmp.mrc.ac.uk

Received 16 May 2002; Accepted 15 July 2002

DOI 10.1002/ajmg.a.10897

� 2003 Wiley-Liss, Inc.

CYTOGENETIC AND MOLECULAR ANALYSIS

Samples of peripheral blood were obtained from eachmember of the pedigree, and high-resolution G-bandingperformed. Genomic DNA was extracted and used forfluorescent PCR analyses of polymorphic markers con-tained within or flanking the FG syndrome criticalregion [Graham et al., 1998] by standard protocols. TheCAG repeat in exon 1 of theAR gene was amplified usingprimers AR2for/rev, as described previously [Sharpet al., 2000]. In order to sequence each AR allele, PCRproducts were resolved by electrophoresis in 3% agaroseand individual bands excised into 100 ml of 1�TE bufferand stored overnight to allow the elution of DNA. Onemicroliter of this was then used as template in a secondidenticalPCRreactiontoreamplifyeachindividualallele.Reactions were then cleaned using ExoSAP-IT (USB,Cleveland, OH), sequenced with BigDye Terminators(PE Biosystems, Foster City, CA), and purified throughDyeEx Spin columns (Qiagen, Crawley, West Sussex,UK) according to manufacturer’s protocols, prior toelectrophoresis using an ABI310 Sequencer.

RESULTS

Cytogenetic analyses revealed an apparently normalkaryotype in all 30 cells examined in each individual.Examination of an additional 30 cells in LM also yieldeda normal 46,XX karyotype. Results of PCR using pri-mers AR2for/rev which span the AR CAG repeat areshown in Figure 2. In track 2, the proband’s mother LMhas two alleles of normal intensity (228 and 234 bp) inaddition to a weak third allele (207 bp), which has beenpassed on to her daughters CM and SM. The reducedintensity of this 207 bp allele in LM indicates that it ispresent in a minority of cells. Sequencing of the 207 bpallele showed that it contains 11 CAG repeats with noevidence of any deletion in the regions flanking therepeat, compared to 18 repeats in the 228 bp allele(Fig. 3), thus indicating that the 207 bp allele originatedby a somatic contraction of the CAG repeat in LM.

Results of PCR analyses using the microsatellitemarkers DXS1204, DXS8092, and DXS6803 which flankthe AR gene are summarized in Table I. A normal pat-tern of inheritance was observed at each locus, with noevidence of any additional alleles in the proband’smother LM. The proband GM shares the same allele as

his sisters CM and SM at each of these loci. Therefore,barring a rare double-recombination event (P<0.01),this indicates that the 207 bpAR allele originated from acontraction of the 228 bp AR allele in the proband’smother LM, representing the somatic loss of seven CAGrepeats.

DISCUSSION

We have observed somatic variation in CAG repeatlength at the AR locus in a normal female (LM). Whileprevious reports have demonstrated contractions of thisrepeat in tumors [Schoenberg et al., 1994; Ferro et al.,2000], this represents the first known mitotic instabilityof the SBMA repeat in normal tissue. Our results indi-cate that in LM, a normal AR allele composed of 18 CAGrepeats has somatically contracted to 11 repeats, re-presenting the mitotic loss of seven CAG repeats suchthat it is present in a mosaic form in her peripherallymphocytes. As both the 11 repeat and 18 repeat alleleshave been passed on to the offspring of LM, mosaicismfor the contracted allele must also extend to her germcells, and thus it seems likely that the mitotic event

Fig. 1. Pedigree of the family with FG syndrome.

Fig. 2. Results of PCR using primers AR2for/rev. In track 2, theproband’s mother LM has two alleles of normal intensity (228 and 234 bp)in addition to a weak third allele (207 bp).

162 Sharp and Hurst

which gave rise to the contraction must have occurredrelatively early during embryonic development. In thisregard, mitotic instabilities of the CGG and CTG re-peats in the FMR1 and DMPK genes, respectively, havesimilarly been shown to be limited to early embryonicdivisions [Wohrle et al., 1993; Jansen et al., 1994].

Although we detected this instability during linkageanalyses of an FG syndrome pedigree, a condition whichhas been mapped to Xq12-q22.1 including the AR gene[Graham et al., 1998], as the proband GM has not inhe-rited the contracted allele it appears to be a coincidentalfinding unconnected to the occurrence of FG syndrome.

In summary, contrary to previous reports which haveindicated somatic stability of the expanded AR repeat inSBMA patients, we have shown that even normal ARalleles are capable of relatively large mitotic variation,suggesting that similar somatic variations in repeatlength may also exist in SBMA patients.

ACKNOWLEDGMENTS

We would like to thank Prof. Patricia Jacobs forhelpful discussions during the preparation of thismanuscript. Andrew Sharp is supported by a WellcomeTrust Prize Studentship (Ref. 058387).

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Fig. 3. Results of sequencing the CAG repeat region of the 207 bp allelein CM (track a) and 228 bp allele in GM (track b). The 207 bp allele contains11 CAG repeats compared to 18 repeats in the 228 bp allele.

TABLE I. Summary of PCR Analyses of Polymorphic MarkersFlanking the AR Gene

MarkerDistance from

Xpter, cMa GM LM JM CM SM

DXS1204 89.34 2 1,2 3 2,3 2,3AR 89.85 2 (1),2,3 4 1,4 1,4DXS8092 97.81 1 1,2 2 1,2 1,2DXS6803 99.24 2 1,2 2 2 2

Numbers correspond to allele scores, those present at reduced intensity arein parentheses.aMapping data obtained from The Linkage Data Base (http://cedar.genetics.soton.ac.uk/public_html/ldb.html).

Instability of the AR Repeat 163