377I Med Genet 1994;31:377-382

Instability of CAG repeats in Huntington'sdisease: relation to parental transmission and ageof onset

Yvon Trottier, Valerie Biancalana, Jean-Louis Mandel

AbstractHuntington's disease (HD) has recentlybeen found to be caused by expansion of atrinucleotide (CAG) repeat within theputative coding region of a gene with an

unknown function. We report here an

analysis ofHD mutation and the charac-teristics of its transmission in 36 HDfamilies. CAG repeats on HD chromo-somes were unstable when transmittedfrom parent to offspring. Instabilityappeared more frequent and strongerupon transmission from a male thanfrom a female, with a clear tendencytowards increased size. We have alsofound a significant inverse correlation(p=000001) between the age of onset andthe CAG repeat length. The observedscatter would, however, not allow an ac-

curate individual prediction of age of on-set. Three juvenile onset cases analysedhad an HD mutation of paternal origin.In at least two of these cases a largeexpansion of the HD allele upon paternaltransmission may explain the major an-

ticipation observed. Our results suggestthat several features of the expansionmutation in HD are similar to those pre-

viously observed for mutations ofsimilarsize in spinobulbar muscular atrophyand in myotonic dystrophy, and to thoseobserved more recently in spinocerebel-lar ataxia type 1 and in dentatorubropal-lidoluysian atrophy, four diseases alsocaused by expansion of CAG repeats.

(I Med Genet 1994;31:377-382)

LGME/CNRS andINSERM U184,Institut de ChimieBiologique, Faculte deMkdecine et CentreHospitalier RegionalUniversitaire,Strasbourg Cedex67085, FranceY TrottierV BiancalanaJ-L Mandel

Correspondence toDr Mandel.Received 6 October 1993Revised version accepted forpublication 23 December1993

Huntington's disease (HD) is a severe neuro-

degenerative disorder characterised by pro-

gressive motor disturbance, cognitive loss, andpsychological changes.' It is an autosomaldominantly inherited disease occurring with a

frequency of 1 in 10 000 persons in most whitepopulations.2 Although the disorder typicallyhas insidious onset around middle age

(mean= 38-3 years), a marked variation isobserved in age of onset and clinical manifesta-tions.3 Recently, the defective gene in Hunt-ington's disease was identified and was foundto contain a trinucleotide repeat (CAG) in theputative coding region.4 The repeat is poly-morphic in the normal population (11 to 34CAG repeats) but was found to be expandedbeyond 42 units in HD chromosomes.Thus, HD is the fourth disease found to be

caused by expansion of a trinucleotide repeat,

after the fragile X syndrome (CGG repeats inthe 5' untranslated region (UTR) of the FMR-1 gene57), spinobulbar muscular atrophy(SBMA) (CAG repeats in the coding region ofthe androgen receptor gene8), and myotonicdystrophy (DM) (CTG repeats in the 3' UTRof a gene coding for a putative protein kin-asel"). Since completion of the present study,three other unstable trinucleotide repeats havebeen characterised, associated with spino-cerebellar ataxia type 1 (SCA1)12 and withdentatorubropallidoluysian atrophy (DRPLA)'3(CAG repeats in both cases), and with theFRAXE fragile site (GCC repeats'4). At leasttwo of these diseases are characterised byincreased severity or penetrance of the clinicalphenotype in successive generations (calledanticipation in DM and 'Sherman paradox' infragile X syndrome). 15'7 These phenomenaresult from the tendency for amplification ofthe repeat when transmitted from parent tochild.7111819 However, parental sex biases areobserved with respect to the severity of thedisease. The congenital form of DM and thetransition from premutation to full mutation inthe fragile X syndrome both occur only uponmaternal transmission.71819 In HD it has alsobeen reported that important anticipationoccurs in a minority of cases, predominantlywhen the mutation is transmitted by thefather.20 This effect was previously suggestedto be caused by genomic imprinting.202' SCAIshows a similar pattern of inheritance.22

In the initial report of the expansion muta-tion in HD,4 it was suggested that age of onsetcould be inversely correlated with the CAGrepeat number. We report here an analysis ofHD mutations and the characteristics of theirtransmission in 36 families, and these featuresare compared to those observed in the otherdiseases caused by the expansion of CAG/CTG repeats.

Materials and methodsFAMILIESBlood samples have been obtained since 1986through French clinical geneticists, in generalfor families where an adult at risk had asked forpresymptomatic diagnosis or prenatal exclu-sion diagnosis by linkage analysis. In a veryfew cases, children were included, only to helpcharacterise parental haplotypes. For somefamilies, blood samples had been conserved forpossible later analysis. In all cases, informedconsent had been obtained by a clinical geneti-cist for the use of samples in linkage analysis.Individual results from the present study are

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communicated to clinical geneticists only ifspecifically requested for adults at risk who askfor presymptomatic diagnosis after proper pre-paration and information. Our laboratory hasno direct contact with families.

PCR DNA ANALYSISGenomic DNA was extracted from peripheralblood samples using standard procedures.PCR amplification across the CAG repeat inthe HD gene was carried out using the set ofprimers described previously,4 but other con-ditions were modified. Reaction mixturescontained 10 mmol/l Tris-HCl (pH 8 3),50 mmol/l KCI, 2 mmol/l MgCl2, 10% DMSO,200 pmol/I dATP, 200 jimol/l dCTP,200 gmol/l dTTP, 50 imol/l dGTP, 150 ztmol/lC7-deaza-dGTP and 1 pmol of a 5' end32P labelled primer (10 ng), 10 pmol of theopposite unlabelled primer, 200 ng genomicDNA, and 2 units Ampli Taq Polymerase(Perkin-Elmer, Norwalk, CT) in a totalvolume of 25 pl. Samples were overlayed withmineral oil and subjected to 35 temperaturecycles consisting of one minute at 95°C, oneminute at 65°C, two minutes at 72°C, followedby a final 10 minute extension at 72°C. Five klof each reaction mixture were analysed on a0 4mm thick 6% w/v acrylamide-8 mol/l ureadenaturing gel. PCR products were sized bycomparing with the sequence of a fragmentcontaining 45 CAG repeats, cloned from anSBMA patient. For analysis of stability oftransmission, samples from the same familywere analysed in adjacent lanes of the same gel.Absolute size measurements of HD allelesfrom different gels may be affected by anuncertainty of one or two repeats. The numberof CAG repeats was deduced from the lengthof the PCR product using the published se-quence4 and thus reflecting the recently dis-covered CGG repeat polymorphism.23

STATISTICAL ANALYSISThe association between HD allele size andage of onset were examined using linear andpolynomial regression and linear regression

with logarithmic transformation of the age ofonset. Although all analyses yielded highlysignificant (p=0 0001) association, log trans-formation of the age of onset appeared to give abetter fit based on analysis of the variance andregression coefficients.

ResultsDISTRIBUTION OF CAG REPEAT LENGTH ONNORMAL AND HD CHROMOSOMESTo compare the number of CAG repeats at theHD locus in normal and HD chromosomes,PCR DNA analysis was performed on mem-bers of 36 unrelated HD families using aprotocol modified from the original one4 (seeMethods). The distribution of 145 indepen-dent normal chromosomes and 85 HD chro-mosomes showed different ranges of CAGrepeat length with no overlap (fig 1). Normalchromosomes displayed at least 18 alleles, con-taining between 13 and 37 repeats with amedian of 20 units (mean 20-1, SD 3 5). Six-teen discrete HD sized alleles ranging from 40to 87 repeats were found and the median was45 repeats (mean 46-7, SD 6 7). Of the 85 HDchromosomes, 47 were derived from patientswith an HD phenotype and 38 were frompersons who had no clinical symptoms at thetime of blood sampling. This latter group hasbeen included in the HD alleles based onfamily data (individual results are communi-cated to genetic counsellors only for thoseadults at risk who request presymptomatictesting after proper preparation and informa-tion (see Methods)). All except four are mem-bers of HD families with affected relativeshaving both a clear HD phenotype and an HDallele. In most cases, previous linkage analysis,notably with VNTRs at D4S43 and D4S125,had shown that they carried an HD mutation.The other four are from families with affectedrelatives for whom no DNA was available. Thepersons still free of clinical symptoms are onaverage younger (mean 26 5, SD 8 2, n = 32)than the mean age of onset of HD. No sexdifference was found for the size distributionof normal or HD alleles (not shown).The 40 paternally derived HD chromo-

somes had 40 to 87 CAG repeats with a medianof 44 (mean 47 9, SD 8 9) and the 34 mater-nally derived ones displayed between 40 and54 CAG repeats with a median of 46 (mean45 9, SD 3 7). Notably, the three largest alleles(66, 72, 87 units) were derived from the father,suggesting a paternal effect for transmission ofsuch alleles.

EFFECT OF PARENTAL TRANSMISSION ONi [ STABILITY OF CAG REPEATS

0 20 70 80

Normal CAG alleles were transmitted fromI ________________ l__ nlllIL ,lDll I n n ° parent to offspring without any alteration ino 10 20 30 40 50 60 70 80 size in the 135 cases analysed. However, trans-

Normal and HD CAG allele mission of HD alleles was associated with a

1 Distribution of allele size on normal and HD chromosomes. One hundred considerable degree of instability (table 1).,ty five independent normal chromosomes (filled bars) and 85HD (open bars) Among 35 transmissions of an HD allele fromPsomes were derived from 36 HD families. Number of repeats was calculated by parent to offspring, 14 (40%) were found to beng that allele size is dependent only on CAG repeats. Recent observations . .e that variation of up to three trinucleotides may be because of the adjacent unstable. In 30 additional transmissions, the,epeat.23 size of the parental HD allele could not be

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Instability of CAG repeats in Huntington's disease: relation to parental transmission and age of onset

Table I Effect of parental transmission on stability ofHD allele

Transmission

Total Stable

Observed parent-child pairs 35 21 (0 60)Maternal 21 14 (0 67)Paternal 14 7 (0 50)

Deduced parent-child pairs* 30 20 (067)Maternal 10 8 (0 80)Paternal 20 12 (0 60)

Cumulative transmission 65 41 (063)* The degree of stability of CAG alleles was deduced by comparing CAG a]offspring of a missing parent when the family contained two or more childreconsidered stable if all children had the same allele length.

a)0)0.0U)CU

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40 45 50Parental CAG repeat number

Figure 2 Change in CAG repeat number on HD chromosomes betwparent and progeny. The alteration of HD allele size in progeny is siof the sex and repeat number of the affected parent. Twenty seven mltransmissions (circle) are indicated including six (open circle) trans,the maternal allele was deducedfrom the repeat number observed in ITwenty three paternal transmissions (square) are shown including ni(open square) for which the paternal allele was deduced from that otoffspring.

7O r

60 II

50 I

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30 F*. *-*00 0

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20 1-

I

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10 Fo40 45 50 55 60 65 70 75

CAG repeat number

Figure 3 Relationship between CAG repeat number and age of onse

30 HD patients is shown as a function of the repeat number observedchromosome.

directly examined but the degree of stability ofthe transmitted alleles was deduced by com-paring the HD allele observed in the offspring

Unstable when it had been transmitted to two or more

14 (040) children. Even in these additional transmis-7 (0.53) sions, an instability was observed with 10 of 3010 (0-33) (33%) transmissions showing an alteration of2 (0°20) allele size. Thus, the overall allele alteration on8 (0-40)

24 (0 37) HD chromosomes was 37% (24/65) of alltransmissions analysed. Table 1 also shows the

lleles observed in then. Transmission was influence of the parental origin of the HD

chromosome on the degree of stability of theHD allele. The instability was more frequentlyseen in paternal transmission (overall, 15 of 34(45%) transmissions showed instability) thanin maternal transmission (overall, 9 of 31

t 20 (29%) transmissions were unstable).The comparison of the CAG repeat number

of the HD mutation between affected parentand corresponding progeny is shown in fig 2.Six of 27 maternal transmissions showed al-teration (three increases and three decreases)of only one unit between the mother and her

* child, and one transmission showed a decreaseof two units. The average alteration in CAG

.- - - - - repeat number in the 27 female meioses was adecrease of 0-1 unit. On 23 paternally derivedHD alleles, all the eight alterations of repeatnumber observed corresponded to an increasein length. Six were of more than one repeat,

.- -.-- including two dramatic increases of 20 and 39units. The average change in CAG repeatnumber among 23 transmissions from father tochild was an increase of 3-2 units. There wasno obvious correlation between instability andthe size of the parental allele. However, it canbe noted that the two largest increases concern

55 parental alleles of 48 and 52 repeats which areabove the median value.

)een affected An inverse correlation between age of onset

heown as a function and number of repeats was observed (fig 3),aternal which was highly significant (p=0-0001) onmissions for which regression analysis. The best fit was foundthetr affspring. with linear regression analysis with logar-bserved in their ithmic transformation of the age of onset

(r2 = 0-79). Three of the four cases with onsetbefore 20 years had alleles of 66, 72, and 87repeats respectively and were of paternal ori-gin. In the latter, the child with 87 units wasaffected at the age of 8 years, while the father(48 units) was affected at the age of 44. Thefourth case, with 48 repeats, had a very slowlyprogressive course of the disease. The inversecorrelation was still significant when the threelargest alleles (66, 72, and 87 units) wereremoved from the analysis (r2= 0-51,p= o0001).One family appeared to present a new muta-

tion since the grandparents died unaffectedaged 92 and 94 years, while in generation II asingle child (dead) manifested the disease at 53years, and his son was affected at the age of 34

* and carried an allele of 49 repeats. In thisfamily, two sisters in generation II are now

' ' ' ~aged 75 and 76, have no HD symptoms, and80 85 90 carry an allele with 37 repeats, which is at theupper end of the distribution ofnormal alleles.

t. Age of onset of This allele might represent the original length'on their HD before the mutation occurred in this family.

Analysis with the D4S127 microsatellite and

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Table 2 Comparison of allele distribution and stability of CAG repeats at the HD, AR, SCAI, and DRPLA loci

Locus Normal alleles Mutated alleles Transmission instability

Mean (SD) Range Mean (SD) Range % (instabilitiesjtransmissions observed) Average variation

Total Female Male Female Male

HD gene 201 (35) 13-37 467 (67) 40-87 37% (24/65) 29% (9/31) 45% (15'34) -01 32AR gene 20-22* 12-30* 47 (4)t 40-62t 32% (20/'62)t 20% (9144)+ 64% (7 11)+ 0 1+ 12-1-4+SCAI gene -30 19-36 43-81 Some instability through paternal transmissionDRPLA gene - 15 8-25 54-68 100% (7 7) - 10 42

* Data from ref 28; t data from ref 29; * cumulative data from ref 29 and 30; SCAI data from ref 12; DRPLA data from ref 13.

the D4S95 TaqI RFLP (these markers are theclosest to the CAG repeat4), and the moreproximal D4S125 VNTR was consistent with,but could not prove, a common origin betweenthe HD mutation and the large normal allele.The more distal marker, D4S93 VNTR, onthe other hand, suggested either a differentorigin or a meiotic recombination (results notshown). The structure of the family (three keypersons had died) did not allow us to reach anunequivocal conclusion.

DiscussionHuntington's disease is the fourth reportedexample of a genetic disorder that results fromexpansion of an unstable trinucleotide repeat.It is of interest to analyse transmission of theHD mutation and genotype-phenotype corre-lations, both to understand the genetic featuresof Huntington's disease better, and also forcomparison with the observations in the otherexpansion diseases, in particular those wherethe mutation involves a CAG/CTG repeat unit(myotonic dystrophy,9-" spinobulbar muscu-lar atrophy,8 spinocerebellar ataxia type 1,12and, most recently, hereditary dentatorubral-pallidoluysian atrophy'3). Such comparisonsmay clarify those features which are an intrin-sic property of the repeat itself, and showwhether other factors, such as the flankingsequence or chromosomal environment of themutation target, or the function of the mutatedgene in germ cells or in other cells may influ-ence stability and parental sex biases of trans-mission.The distribution of allele size on normal and

HD chromosomes in the population westudied is in very good agreement with theresults presented in the original report.4 Inparticular, we found no overlap between thesizes of normal and HD alleles, although thedifference between the longest normal allele(n = 37) and the smallest HD allele found in apatient (n = 40) is only three repeats (this inter-val was from n= 34 to n =42 in the previousreport). Since completion of this study, threereports have been published on sizing of CAGrepeat length at the HD locus in normal per-sons and HD patients.2>26 The largest normalalleles (L) and the smallest disease alleles (S)observed by the three groups were respectivelyL=34 and S=37,24 L=37 and S=38,26 andL = S = 34 and three possible HD alleles withn = 30-31.25 Because of the rare apparent over-lap between large normal and small HD alleles,one should stress the necessity of very accuratesizing of alleles at that locus for diagnosticpurposes. All these analyses included a small

stretch of CCG repeats flanking the CAGmotif, which was recently shown to be poly-morphic, with major alleles that differ by threerepeats.23 It will be important to determinewhether this polymorphism contributes to theapparent overlap between normal and HDalleles.While normal alleles were found to be com-

pletely stable, limited instability of the HDmutation was found in maternal transmission(in most case a change of a single repeatunit) and a more pronounced instability wasobserved in paternal transmission, with allchanges being increases in size (3 2 repeatunits on average). Two of 23 paternal trans-missions had much larger expansions (20 and39 units), and the three largest alleles (n 66)were all paternally derived. This correlateswell with the notion that juvenile onset cases,who make up 5 to 10% of all HD patients, haveinherited the disease from their father in mostcases, and that 8% of paternally inherited casesshow major anticipation of 243 + 92 years.327These observations are consistent with theinverse correlation between age of onset andmutation size (fig 3).42s26 The three largestalleles were indeed found in juvenile onsetcases. On the other hand, in the n = 40-52range, while there is a statistically significantcorrelation between allele size and age of onset,the distribution of the latter is wide for ident-ical or very similar allele length (fig 3).2>26Analysis of HD allele size will thus be oflimited value for individual prediction of ageof onset. The size distribution of normal anddisease alleles in the HD gene resembles strik-ingly that found in the androgen receptor genein normal and SBMA patients respectively,and that observed most recently at the SCAland DRPLA loci (table 2). In the four dis-eases, 21329-30 a greater instability in paternaltransmission is present (or probable in the caseof DRPLA). The lack of alleles larger than 62in SBMA is in contrast with those observed atthe HD locus here and in the initial report4 andat the SCAl locus.'2 However, this lack oflarge alleles in SBMA may be because ofsample size as the cumulative number ofSBMA persons reported29-3' is about 120.Other explanations could be that such largemutations do not arise at the androgen recep-tor locus, or that they would lead to a differentphenotype from SBMA (for instance, testicu-lar feminisation).'3

It is more difficult to compare the instabilityofHD, SBMA, SCA1, and DRPLA mutationsto that of similar sized alleles at the DM locus.Disease alleles in DM may reach about 1000repeats, while alleles in the 50-80 range (called

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Instability of CAG repeats in Huntington's disease: relation to parental transmission and age of onset

pre- or protomutations) are associated with atmost minimal and late onset manifestationsand are found in general in parents of affectedchildren with larger mutations, giving a biastowards apparently higher instability. Singlestep enlargements of such small mutations upto 600 repeats have been observed in a fewcases.'932 In one study where such protomu-tations were analysed in extended DM fami-lies, they could be found transmitted with noor minimal amplification (in 20 of 60 transmis-sions) and a trend was noted for more frequentand larger instability in male transmission (upto parental protomutations of n = 80). On theother hand, instability started to be more pro-minent in the maternal line for parental allelesover 80 repeats.32 It is likely that HD allelesmuch larger than n = 100 would be lethal earlyin life, as the few cases of alleles in the 80-100range reported up to now lead to very earlyonset of the disease.We have observed a new mutation in a

family where two members carry an allele atthe upper extreme of normal distribution(n=37), although the common origin of themutated allele and the large normal one couldnot be established. In the original HD report,4two new mutation events were shown to occurfrom alleles with n= 33 and 36 repeats. This isconsistent with the conclusions, based on link-age disequilibrium studies in fragile X syn-drome and myotonic dystrophy, that thesediseases are maintained in the population byrecurrent mutations occurring on large normalalleles.33-35 From our data and those of theinitial report,4 the frequency of such largealleles at the HD locus can be estimated atabout 1% (for n=33 to 35). Given the veryrare occurrence of new mutations in HD(direct estimated range between 0-5 to5 x 10-62) and assuming that the disease fre-quency is in a state of equilibrium and thatpatients who have severe juvenile HD (about5% of all patients) will not reproduce, one canderive a maximum mutation frequency of5 x 10- for HD alleles ofn= 33-35; thus thesewould not pose a real genetic counsellingproblem.

Interestingly, a similar frequency of alleleswith 30-37 CAG repeats was observed at theDM locus in white populations3'37 and thefrequencies ofHD and DM are comparable inthis population. Although the mean and rangeof normal alleles at the AR locus is very similarto that found at the HD locus, large alleles inthe 33-36 range appear extremely rare (-1/10002830), and this could account for the lowerfrequency of SBMA (together with significantlydecreased fitness owing to reduced fertility ofaffected males). A similar explanation is likelyto account for the rarity of DRPLA13 (table 2).Although the frequency of alleles in the 30-36range is very high at the SCAl locus, itsinterruption by the CAT motif is likely to havea stabilising effect,'839 which could account forthe low frequency of SCAI.'2

In conclusion, the present results suggestthat the behaviour of the CAG repeat in theHD gene is quite similar to that in the andro-gen receptor, SCA1, and DRPLA genes, and

perhaps also to that of alleles of comparablesize in DM.We would like to thank Drs M L Briard, C Marescaux, E Flori,C Franconnet, S Gilgenkrantz, J C Lambert, A Nivelon-Chevallier, M 0 Peter, and H Puissant for contributing to thefamily analysis, collection of blood samples, and clinical exami-nations. We thank A Staub for oligonucleotide synthesis. Weare grateful to D Kauffmann for help in preparation of themanuscript. YT was supported by a fellowship from theMedical Research Council of Canada.

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