70 Med Genet 1992; 29: 780-784

Presymptomatic diagnosis of myotonicdystrophy

Han G Brunner, Willy Nillesen, Bernard AHans-Hilger Ropers, Hubert J M Smeets

van Oost, Gert Jansen, Be Wieringa,

AbstractThe discovery of an expanded (CTG).repeat sequence in myotonic dystrophy(DM) has greatly improved our ability todetect DM gene carriers who have few ornone of the classical signs of this dis-order. We report here our experiencewith two such groups of gene carriers. Weused a PCR based protocol that should beespecially sensitive to small increases inCTG triplet number which might escapedetection by conventional Southern blotanalysis. Our analyses show that on 100non-DM chromosomes the number ofCTG triplets ranged from five to 37. Wethen studied 17 obligate gene carriersaged 55 years and over who showed nomuscle weakness. All of the gene carriersin this group showed a relatively smallincrease in the number of CTG triplets(52 to 90 CTG triplets) with limitedsomatic mosaicism. We subsequentlystudied 11 subjects (aged 19 to 36 years)who had previously been identified asgene carriers by genetic linkage studies,but who lacked diagnostic signs. In thisprospectively studied group, nine sub-jects showed an expanded allele, con-firming the earlier prediction fromlinked genetic markers. The other twosubjects had only two normal alleles andno expanded allele. Revision of theclinical data casts doubt on the originaldiagnosis of DM in their families. Pref-erential amplification of the normalnon-expanded allele was noted in threeasymptomatic gene carriers in this study(as well as in two of their clinically affec-ted relatives). We caution that, at least inour hands, the DM mutation can be con-fidently excluded by this PCR basedmethod only if both normal alleles havebeen identified. If an expanded allele isnot seen, and only a single normal alleleis visualised on 6% PAGE, confirmatorytesting with linked genetic markers orconventional Southern blotting of the(CTG). repeat is advocated.(J Med Genet 1992;29:780-4)

Myotonic dystrophy (DM) shows a wide rangeof clinical expression in affected subjects.'Severity of the disease is at least partly corre-lated with the number of CTG trinucleotiderepeat units found in the 3' untranslated regionof a protein kinase gene located in band q13.3of chromosome 19.2-10 There is a marked tend-ency for an increase in CTG triplet number

from one generation to the next, which isparalleled by earlier onset of the disease insuccessive generations,"1 12 a phenomenonknown as anticipation. Genealogical studies inDM families'"'5 suggest that in earlier genera-tions additional asymptomatic gene carriersmay have been present, presumably with onlya minor expansion of the CTG motif. How-ever, a premutation not involving expansion ofthe CTG trinucleotide repeat is another possi-bility. This situation is highly reminiscent ofthe CCG repeat expansion seen in the fragile Xsyndrome. 1-18 Our previous study using linkedDNA markers has suggested that a consider-able proportion of sibs and offspring of DMpatients are asymptomatic in early adult-hood.'9 Therefore, asymptomatic subjects maynot be confined to the early generations of aDM family.We studied these issues in detail by assaying

the CTG triplet number in 17 obligate carriersof the DM gene as defined by pedigree analysisand genealogical studies. We further studied agroup of asymptomatic offspring of DMpatients who had been identified previously asprobable gene carriers by the application ofclosely linked DNA markers.

MethodsFamilies were ascertained through the DNAdiagnostic service in our department. As fromJanuary 1992, this service has performed dia-gnosis with linked DNA markers in 164 DMfamilies, of which 76% were referred by othergenetic departments. Care was taken to ensurethat all families met previously defined dia-gnostic criteria.20 For the purposes of thisstudy, we selected 13 families that containedone or more obligate gene carriers aged 55 andover who showed no muscular weakness. Obli-gate gene carriers were identified by pedigreeanalysis or through genealogical studies thatlinked independently ascertained pedigrees.There were 14 males and three females in thisgroup, ranging in age from 55 to 92 years(mean 69-8 years). Among 17 obligate genecarriers, seven had undergone cataract sur-gery. The other 10 were considered asympto-matic. EMG and slit lamp examination hadbeen performed in six out of 10. Typicallenticular opacities were noted in three out ofsix. EMG was normal in all six. In addition, 11subjects from nine families were restudied, asour previous study19 had indicated a highprobability of carrying aDM mutation in spiteof normal results on clinical examination. Weused data from 50 normal Dutch subjects (100chromosomes) who had married into DM

Department ofHuman Genetics,University Hospitaland Medical Faculty,PO Box 9101, 6500 HBNijmegen, TheNetherlands.H G BrunnerW NillesenB A van OostH-H RopersH J M Smeets

Department of CellBiology and Histology,University Hospitaland Medical Faculty,Nijmegen, TheNetherlands.G JansenB Wieringa

Correspondence toDr Brunner.Received 8 June 1992.Revised version accepted30 July 1992.

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Presymptomatic diagnosis of myotonic dystrophy

76-

64 -

50 -

37 -

1 5 20 25Number of CTG triplets

........V

30 35

Figure 1 Distribution of allele sizes for 100 non-DM chromosomes. Allele sizes aregiven as the number of CTG triplets.

families to establish the normal range of CTGtriplet number for controls.Chromosomal DNA was isolated from peri-

pheral blood.2" Genetic marker systems flank-ing the DM mutation have been describedpreviously.22-26 Using a recently describedPCR assay,7 we tested the expansion of theCTG repeat in genomic DNA. The CTGrepeat was amplified with flanking primers 406and 409 reported by Mahadevan et al.7 Theresulting DNA fragments were resolved on

1% and 4% agarose gels. A Southern blot,made from the 1% gel on Gene Screen Plus,was screened with a 32P end labelled (CTG)IOoligonucleotide as a probe, and the hybridisingfragments were subsequently visualised byautoradiography. Alleles containing up to ap-proximately 140 CTG triplets could also beidentified on a 6% polyacrylamide/7 mol/l urea

sequencing gel after 32P end labelling of one ofthe amplification primers.

ResultsNORMAL CONTROLSData from 100 normal chromosomes are sum-marised in fig 1. Allele lengths were found torange from five to 37 CTG triplets. The mostcommon alleles in this sample were five, 13,and 14 CTG triplets (38, 22, and 15% respect-ively).

OBLIGATE CARRIERSAll of the 17 obligate gene carriers (14 malesand three females) showed an expanded allelewith multiple bands and weaker signals on 6%polyacrylamide gels, different from normalalleles at this locus (five to 37 CTG triplets).Allele size (estimated from the strongest signalamong these multiple bands) ranged from 52to 90 CTG triplets (fig 2). The apparent in-stability of the expanded allele could representan in vitro artefact of the PCR reaction. How-ever, in view of the strikingly different appear-ance of the 37 and 52 CTG triplet signals, it ismore likely that the CTG repeat shows soma-tic mosaicism in vivo above a threshold ofapproximately 40 to 50 CTG triplets. With

25 -

22 -20 -

14-13-12-

1 2 3 4 5 6 7 8~~w .~~~....u

3 10 12 14 13 - l 7

Figure 2 CTG repeat analysis of obligate DM genecarriers (lanes 1-5, 7, 8, 10-12, and 14-17) by 6%denaturing polyacrylamide gel electrophoresis. Expande.dPCR amplified alleles range from 52 to 76 CTGtriplets. The larger alleles show up less well, probablyowing to differences in the efficiency of the PCRreaction. Lane 7 represents a 72 year old male whoshows bilateral cataract as his only clinical feature. Thisman has an expanded allele of 74 CTG triplets and anormal allele of 37 CTG triplets.

one exception (II.7, fig 3), CTG repeats werefound to be larger in offspring than in theseobligate carriers, confirming that the expandedrepeat sequence was unstable.A striking example of anticipation is shown

in fig 3. In this family, two subjects (I.3 andII.7), aged 90 and 53 respectively, are asymp-tomatic on full examination, including EMGand slit lamp examination, except for a fewtypical lenticular opacities in I.3. A third sub-ject (I. 1) is also completely normal at the age of85, but no EMG has been performed. Theseobligate heterozygotes showed CTG tripletnumbers of 52, 60, and 52 respectively, basedon results of 6% PAGE (lanes 16, 15, and 17,fig 2). The 1% agarose Southern blot of thePCR product shows the expected band ofapproximately 0-2 kb for these subjects. Abovethis band, a smear can be detected, which isfrequently observed for alleles of this size, andwhich may be the result of heteroduplex for-mation of normal and expanded alleles, ratherthan somatic mosaicism. Symptomatic diseasehas been diagnosed in the descendants of theseobligate carriers (111.1, 2, 3, fig 3). These

30

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5 10

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Brunner, Nillesen, van Oost, J7ansen, Wieringa, Ropers, Smeets

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Figure 3 Southern blot analysis of PCR amplified C1TG repeat sequence

1% agarose gel in a DM family. Transmission ofDM through three cliniasymptomatic subjects (I.1, I.3, II.7), aged 90 years, 85 years, and 53 yshow only mild expansion in this assay. CTG triplet numbers for these ob,(determinedfrom 6% polyacrylamide gel electrophoresis) were 52, 52, an

respectively. Clinically affected subjects (II.4, II.5, III.I, III.2, III.3) s

extensive expansion of the CTG repeat.

subjects all have expanded alleles200 CTG triplets.

PROBABLE GENE CARRIERS IDENTIFIE]LINKAGE ANALYSISAn expanded CTG repeat was foun11 asymptomatic offspring, whohigh risk haplotype for closely litmarkers (table). These subjects rai

from 19 to 36 years. In four of the n

of the expanded allele fell withinpreviously observed for asymptcataract only) subjects aged 55 yearIn the other five asymptomatic DAriers, allele sizes were found to be cllarger, more than 200 CTG triplet

In the final two cases, a clinical c

DM in a key relative was not suppcDNA studies. In the first family,old proband had received a clinicLof myotonic dystrophy on the basismyographical myotonia, typical mucataract, and muscle weakness (aunusual distribution for DM). Tcarries two normal alleles for the Cleotide repeat. ElectromyographicLand multicoloured lens opacities wefor her father, who also showecnumber of CTG triplets on botsomes. Either this family showsform of the disease, not associatedsion of the CTG repeat, or the orig

diagnosis was incorrect. If the latter applies,the sister, who carries the same haplotype formarkers flanking the DM locus (subject 8 inthe table) is probably not at risk for the disease.In the other family, several affected subjectsshowed a characteristic smear of expandedrepeats. However, in a female in whom DMhad been diagnosed based on mild muscularweakness and electromyographical myotonia,an expanded allele was not found. This putsthe clinical diagnosis in doubt for this womanand indicates that her son (subject 6 in thetable) is probably not at risk for DM.

PREFERENTIAL AMPLIFICATION OF THENORMAL ALLELEAn expanded allele was not initially detected inthree subjects with the primer pair (406 and409 from Mahadevan et all) used in this studyin spite of multiple (up to five) attempts. Thesesubjects had previously been predicted to begene carriers by linkage analysis. Since only asingle normal allele was seen on 6% poylacry-lamide gel electrophoresis, we assumed that anabnormal allele was present but undetectable.By choosing another primer combination (primer406 from Mahadevan et aft and a new primerGGC.ACA.GAA.GCC.CGG.CCC.ACC, nt

e run on a position 11-32) the presence of an expandedically allele was confirmed in two subjects. In thePears, who third subject an expanded allele could only beligate carriers shown by direct restriction analysis andid 60show more Southern blotting of the genomic fragment

containing the CTG repeat sequence.

exceeding DiscussionWe show here that asymptomatic carriers ofthe DM gene mutation can be diagnosed byPCR based analysis of the CTG trinucleotide

D BY repeat. This method is especially helpful indetermining the origin of the mutation when

Id in nine of both parents are clinically unaffected and thecarried the family history is negative.27nked DNA Among 17 cases aged 55 years and overnged in age without muscle weakness, none showed anline the size allele exceeding 90 CTG triplets. Largerl the range alleles may not be compatible with absence ofomatic (or muscular symptoms in middle and old age.s and older. However, more data are needed before predic-A gene car- tions are possible for individual cases.onsiderably Our results confirm the paucity of new mu-:s). tations in DM suggested by genealogical stud-diagnosis of ies,' -15 since subjects identified as obligate)rted by the gene carriers always carried an expandedthe 34 year allele. This is consistent with the results ofal diagnosis studies showing linkage disequilibrium ins of electro- various populations.2>30 In fact, the finding ofilticoloured linkage disequilibrium in DM is difficult tolbeit in an explain, unless we assume that either the pene-his subject trance of the DM gene is virtually zero for:TG trinuc- many generations as suggested by the anticipa-al myotonia tion model, or that carriers of the DM muta-~re reported tion have increased fecundity, or both. TheI a normal finding that all of the asymptomatic obligateth chromo- carriers defined by pedigree analysis or genea-an unusual logical studies showed only a small increase inwith expan- CTG triplet number (52 to 90 CTG triplets),inal clinical supports the anticipation model of DM. In

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Presymptomatic diagnosis of myotonic dystrophy

Results of CTG analysis in 11 asymptomatic subjects considered to be at high risk of carrying a DM mutation bylinked DNA markers.

Subject* Age Sex Affected parent Clinical examinationt CTG analysis$

1 19 M Father Normal

2 19 M Father Atypical lens changes.Equivocal clinicalmyotonia/normal EMG

3 33 M Father Normal§

4 33 F Father Normal

5 24 F Mother Normal

6 29 M

7 36 F

8 28 F

Normal

Expanded smear(> 200 CTG triplets)Expanded smear(> 200 CTG triplets)

Expanded smear(> 200 CTG triplets)Approximately 74 CTGtripletsApproximately 60 CTGtripletsNormal

Father Slight ptosis. Expanded smearAtypical lens changes§ (> 200 CTG triplets)Normal Normal

9 33 M Mother Normal

10 38 M Mother Normal!l

11 21 M Father Normal

Approximately 61 CTGtripletsApproximately 88 CTGtripletsExpanded smear(> 200 CTG triplets)

Remarks

Mental retardation

Not at risk.Diagnosis in relatives in doubt

Not at risk.Diagnosis in relatives in doubt

* Subjects at high risk were identified in a previous study of 139 asymptomatic offspring of DM patients.'9t Clinical examination included EMG, neurological examination, and slit lamp examination. Non-specific signs (such as colourlessopacities or mental retardation) were found in three out of nine (33%), compared to 24 out of 128 (19%) at those at low risk.I Based on results of 1% agarose gel electrophoresis and 6% polyacrylamide gel electrophoresis of PCR amplified products.§ After DNA marker analysis, repeated EMG examination was performed, and myotonia was diagnosed.11 After DNA marker analysis, DM was diagnosed in a son.

spite of anticipation, the DM mutation mayoccasionally remain clinically silent, evenamong sibs and offspring of symptomaticcases. In five of our patients, (aged 19, 19, 21,33, and 36 respectively) careful clinical exami-nation including EMG and slit lamp examina-tion failed to show any definite sign of DM.This may appear surprising since each showedan expanded allele which clearly exceeded 200CTG triplets (subjects 1, 2, 3, 7, and 1 1 in thetable). Follow up studies are required to estab-lish whether these subjects will develop mus-cular symptoms in later life. So far, repeatedEMG studies (performed after the results ofthe DNA analysis were known) have shownmyotonic discharges in two of them (subjects 3and 7, table). Neither clinical myotonia norsignificant muscle weakness was detected onre-examination. In the other asymptomaticcases, CTG triplet numbers of 60, 61, 74, and88 were within the range found for obligategene carriers without muscular symptomsafter the age of 55.We currently estimate the chance that a

clinically normal sib or offspring of a DMpatient carries a mutated gene to be 7-8% forthose aged between 20 and 39 years of age.This figure is very similar to our previousestimate in the same sample of DM families(8 3%).19 We have now excluded two subjectsbecause of unreliable diagnosis, while we hadformerly anticipated the reclassification as lowrisk of two subjects because of genetic recom-bination with the marker loci.'9 In addition,our study confirms that at the molecular levelanticipation is the rule in DM, since in oursample of nine asymptomatic offspring wealways found a CTG triplet number equal toor exceeding that of the affected parent.Finally, we do not know the explanation forthe family in which expansion of the CTGrepeat was not detected in either clinicallyaffected member. Such families have beennoted by others.7 It remains to be seen whether

the results in these families are the result ofdiagnostic error or allelic mutation. The exis-tence of allelic mutations, not causing expan-sion of the CCG repeat sequence, has beensuggested for the fragile X syndrome.3

Finally, we want to stress that in subjectswith only a single normal band by PCR, theDM mutation should not be consideredexcluded, because of the possibility of prefer-ential amplification of the normal allele. Insuch instances, additional studies are required,using either alternative primer combinations,conventional Southern blotting of genomicfragments, or linked genetic markers. Onlywhen such studies confirm the presence ofnormal alleles only, can the consultand bereassured that future offspring will not be atrisk.

We wish to thank Drs van de Biezenbos, vanEssen, Snoek, Sterringa, Niermeijer, Busch,Howeler, De Die-Smulders, and Hennekamfor providing additional clinical information.We thank Marcel Nelen and Hennie Bruggen-wirth for skilful technical assistance. Thisstudy was supported by the American Muscu-lar Dystrophy Association, the Prinses Bea-trixfonds, Grant 87-2694, and by Grant Ro389/15-4 from the Deutsche Forschungs-gemeinschaft.

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3 Aslanidis C, Jansen G, Amemiya C, et al. Cloning of theessential myotonic dystrophy region and mapping of theputative defect. Nature 1992;355:549-51.

4 Buxton J, Shelbourne P, Davies J, et al. Detection of anunstable fragment of DNA specific to individuals withmyotonic dystrophy. Nature 1992;355:547-8.

5 Brook JD, McCurrach ME, Harley HG, et al. Molecularbasis of myotonic dystrophy: expansion of a trinucleotide(CTG) repeat at the 3' end of a transcript encoding aprotein kinase family member. Cell 1992;68:799-808.

6 Fu YH, Pizzuti A, Fenwick R, et al. A sequence scanningmethod identifies an unstable triplet repeat in myotonicdystrophy. Science 1992;255:1256-8.

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doi: 10.1136/jmg.29.11.780 1992 29: 780-784J Med Genet

 H G Brunner, W Nillesen, B A van Oost, et al. dystrophy.Presymptomatic diagnosis of myotonic

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