1tMed Genet 1995;32:14-18

Myotonic dystrophy: genetic, clinical, andmolecular analysis of patients from 41 Brazilianfamilies

Maria Rita Passos-Bueno, Antonia Cerqueira, Mariz Vainzof, Suely K Marie,Mayana Zatz

AbstractResults of genealogical, DNA, and clinicalfindings in 41 families with 235 patientsaffected with myotonic dystrophy (DM)led to the following observations. (1) Therelative proportion of affected patientsamong blacks is apparently lower thanamong whites or orientals. (2) A significantexcess ofmales was observed. (3) The fre-quency of DM patients who did not re-produce was similar for males andfemales; however, female patients had onaverage 25% fewer children than malepatients. (4) There was a significant in-tergenerational increase in the meanlength of the CTG repeat which was alsocorrelated with the severity of the pheno-type. (5) No significant difference was ob-served in the mean size of the CTG repeatin offspring ofmale as compared to femaletransmitters. (6) With the exception ofthecongenital cases of maternal origin, thelargest expansions were paternally in-herited, but did not lead to congenital DM.

(J Med Genet 1995;32:14-18)

Centro de Miopatias,Departamento deBiologia, Instituto deBioci6ncias,Universidade de SaoPaulo, Rua do Matao277, Sao Paulo, SPBrazil 05508-900M R Passos-BuenoA CerqueiraM VainzofM Zatz

Departamento deNeurologia, Hospitaldas Clinicas,Universidade de SaoPaulo, Sao Paulo,BrazilS K Marie

Correspondence to:Dr Zatz.

Received 22 April 1994Revised version accepted forpublication 24 August 1994

Myotonic dystrophy (DM) is an autosomaldominant disorder, with an incidence of 1 in7000 to 8000 births, and represents the com-

monest neuromuscular disorder in adulthood.Its symptoms may include muscle weaknessand atrophy, myotonia, cataract, frontal bald-ness with a characteristic facial appearance,cardiac arrhythmias, infertility, and endocrinedisorders.' It is characterised by an extremelyvariable clinical course, ranging from personsin whom the only clinical sign may be a cataractor frontal baldness to severely affected patients.In addition, there is a severe congenital form,which is frequently fatal and with rare

exceptions23 is transmitted by affected moth-ers.4An interesting feature in DM is the phe-

nomenon of anticipation, that is, an increase

in severity and earlier age of onset in successive556generations.

Following the characterisation ofthe molecu-lar defect responsible for myotonic dystrophyin 1992 by three independent groups,7 9 manyinvestigations have been published worldwide.It was found that the genetic mechanism re-

sponsible for DM is an expansion of a CTGrepeat in the 3' untranslated region of themyotonin protein kinase gene.'`-`2 In normal

persons, the number of CTG repeats rangesfrom five to about 37 copies while in affectedpersons it varies between 50 to thousands ofcopies.71013 The increase in size of the DNAfragment containing the repeat from one gen-eration to another provides the molecular basisfor anticipation.' 1'7A relationship between the size of the CTG

repeat and disease severity has been reportedin several studies,'6 l8-20 although there is anoverlap between clinical groups.'4"Other features which have been investigated

include: the influence of the sex of the trans-mitting parent as well as of parental allele sizeon the CTG expansion1920; the proportion ofaffected male versus female patients in multi-generation families20; the mechanism and thefrequency of intergenerational CTG con-traction21; the association between the presenceof smear and the patients' age as well as theCTG repeat size.'7-'9 All these investigationshave been done in DM families from Europe,United States, Canada, orJapan but apparentlynone has been reported so far for the populationof South America.The purpose of the present study is to report

results of genealogical, DNA, and clinical find-ings in 235 DM patients, belonging to 41 Bra-zilian families of different racial backgrounds,with the following purposes; (1) to verity if therelative frequency of affectedDM families fromdifferent racial backgrounds (white, black, ori-ental) is in accordance with the frequency ofeach of these racial groups in our population;(2) to investigate the sex ratio and the relativereproductive performance of DM males ascompared toDM females from multigenerationfamilies; (3) to correlate the size of the CTGrepeat from leucocyte DNA with the phenotypein patients with different degrees of clinicalseverity; (4) to analyse the influence of sex aswell as the size of the parental CTG repeat inthe CTG expansion of affected offspring; (5)to estimate the frequency of contraction of theCTG repeat in this sample of families.

Patients and methodsA total of 235 DM patients, belonging to 41families, have been ascertained at the Centrode Miopatias, Departamento de Biologia,Universidade de Sao Paulo during the last10 years. Of them, 111 patients (aged 1 to 78years) and 120 normal relatives had their DNAanalysed.Based on the severity of the phenotype and

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Myotonic dystrophy: genetic, clinical, and molecular analysis ofpatients from 41 Brazilian families

age of onset, patients were classified into fourgroups'9: (A) the mildest form, in which theonly clinical sign may be cataract or frontalbaldness, with little or no muscle involvement;(B) the classical form, with onset in adolescenceor early adult life, characterised by myotoniaand progressive muscle weakness; (C) the rarecongenital severe form, with early onset, gen-eralised muscular hypoplasia, and mentalretardation, which is almost invariably trans-mitted through carrier mothers; (D) the earlychildhood form, which shows some featurescomparable to those seen in the congenitalform, but with no documented abnormality atthe time of birth.DNA was extracted from whole blood ac-

cording to the method of Miller et al.22 Mo-lecular analyses were performed according tomethods described previously.710 The DNAwas digested with EcoRI and electrophoresedin a 0 7% agarose gel. In all cases that showedsmall or undetectable expansions,DNA analysiswas repeated after digestion with PstI.'9For estimating the size of the repeat the most

prominent band was taken into considerationor the average size of the smear.2' Samples fromall affected members belonging to each familywere run on the same gel. All molecular resultsare expressed in terms of kilobases (kb) ofadditional DNA.

Statistical analysis included x' analysis andthe usual paired and unpaired tests for com-paring means (t tests and one way analysis ofvariance).

ResultsClinical and DNA findings are summarised inthe table.

ANALYSIS OF PEDIGREESThe affected patients belonged to 41 unrelatedfamilies of the following racial background: 35white, three black, and three oriental (Jap-anese).

Analysis of the sex ratio showed an excessof DM males (n = 153) as compared to DMfemales (n = 82), which was statistically sig-nificant (p<0 00 1). This included patients clin-ically affected as well as non-manifestingtransmitters of the DM mutation from the41 families. A statistically significant (p<0 01)disproportion between the sexes was also ob-served among the probands (29 males: 12 fe-males). When the analysis was repeated withoutthe probands, there were still significantly moremale (n = 124) than female (n = 70) patients(p<0-01). However, the sex ratio among un-affected adult relatives (107 males and 98 fe-males) did not differ from 1:1.

In order to compare the reproductive per-formance between affected males and femaleswe considered the total number of childrenborn to DM males versus those born to femaleDM patients who were older than 25. It wasobserved that of 110 DM males, 36 did notreproduce and the remaining had a total of 250children (250/110, average 2-27 per affectedmale). On the other hand, of 68 DM females,

22 did not reproduce and the remainder hada total of 116 children (116/68, average 1-70per affected female). Although DM males hadon average more children than DM females,the distribution ofthe mean number ofchildrenper affected patient did not differ significantlybetween the two sexes (p>005).

ANALYSIS OF THE CTG REPEATConsidering all affected patients, the size ofthe repeat varied from 0 1 to 9 0 kb with nostatistically significant difference between sexes(mean=1-6kb, SD 1 3, n=62 for males;mean=20kb, SD 1-7, n=49, for females;p>0.05) (fig 1).

In 13 patients (seven males and six females),the CTG repeats were very small or un-detectable (less than 05 kb) following EcoRIdigestion but were confirmed with PstI. Thelargest CTG repeats (ranging from 5*0 to5 2 kb) were observed in three congenital cases(fig 2), in four patients from group D who werementally retarded (3 4, 4 0, 5 0, and 5-3 kb,respectively), and in three female patients (in-cluding two sisters) classified in group B, whohad severe muscle weakness (3 5, 5 0, and 9 kb,respectively).

Five children (aged 1 to 14) who had in-herited theDM allele from their affected parent(two mothers and three fathers), with repeatsranging from 0-2 to 1-0 kb, were clinicallyasymptomatic.Comparison of the CTG repeat size among

the four clinical groups of patients (A to D)showed that, on average, the size of the repeatwas larger in the groups of more severely affec-ted patients, although there was an overlapbetween them (table). The mean size of theCTG repeat was similar in patients from groupC and group D, but the number of personswas too small for statistical analysis.A wider variability in the size of the CTG

repeat (from 0 2 to 9 0 kb) was observed amongpatients classified as classical DM (group B).Interestingly, in this group, the mean size of theCTG repeat was significantly greater (p<005)among affected females (2 3 kb, SD 1 -6, n=32) than among affected males (1 -6 kb, SDIO0, n=45).

SIZE OF REPEAT AND SEX OF TRANSMITTINGPARENTAnalysis of 48 pairs of affected parent/childshowed a highly significant intergenerationalincrease in the mean size of the CTG repeat(mean= 1-46 kb, SD 0-20, p<0-001).No statistically significant difference between

the CTG expansion in maternally as comparedto paternally transmitted alleles (p>005) wasobserved (for 33 pairs of affected DM fathers/offspring: mean= 1-39 kb, SD 1-17; for 15 pairsof affected DM mothers/offspring: mean =1-63 kb, SD 1-24).

Interestingly, although not statistically sig-nificant, the analysis of the distribution of theCTG intergenerational expansion according tothe sex of the parent showed a higher pro-portion of fathers (68%) than mothers (47%)

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Passos-Bueno, Cerqueira, Vainzof, Marie, Zatz

45

40 -M A Father/offspring

:>35-

FZ B Mother/offspring030-

ID 25 -

CD 20-U- 15

10

5

0

0.5 1.0 1.5 2.0 25 3.0 3.5 4.0 4.5 >5Size of CTG expansion (Kb)

Figure 1 Comparison of the frequency of intergenerational CTG expansion in paternallytransmitted alleles (n = 33) (A) and in maternally transmitted alleles (n = 15) (B).

, i_

t7X1-.'.I N ...mm*14"

Figure 3 Intergenerational CTG contraction in a femalewho inherited the DM allele from her father (arrow). Heraffected sister had an expansion.

transmitting small expansions (up to 1-5 kb)(fig 1). In order to assess if the lack of sig-nificance is because of the sample size it wouldbe important to verify if this difference is main-tained in a larger sample.A contraction in the size of the trinucleotide

repeat was observed in only one affected female(fig 3). The abnormal allele was transmittedby her DM father, who had a normal son andthree affected daughters, one of whom had a

child with the severe congenital form.

DiscussionRACIAL DISTRIBUTIONThe incidence of DM has been estimated as1 in 8000 in western European and NorthAmerican populations' and as 1 in 20 000 inJapan.23 However, there are apparently no re-ports ofDM in African populations.24The relatively low frequency of DM families

of black racial background (three of 41) in the

Clinical and DNAfindings in 111 DM patients

Clinical No of Age Mean size ofclassification patients in range the CTG

each group (y) repeat (kb)*

Group A 27 36-76 0 49 SD 0-35Group B 77 17-66 1-88 SD 1-32Group C 3 1-10 5-20 SD 0 00Group D 4 8-15 4 08 SD 1-49

*Analysis of variance among the four groups: F=25-19,p<O-OO1

present sample attracted our attention, sincethe population in the city of Sao Paulo is madeup of about 50% white, 40% black, and 10%oriental (mostly of Japanese ancestry). There-fore we would expect a proportional dis-tribution of these racial groups among the DMfamilies ascertained. One possibility that wecannot rule out would be a bias in as-certainment, although, in our experience of761 families with Duchenne muscular dys-trophy, the proportion of affected patients fromdifferent racial backgrounds is in accordancewith that expected (unpublished observations).On the other hand, several independent stud-

ies suggest that DM is a consequence of one ora few ancestral mutations.71923 If the ancestralmutation in the DM gene had occurred afterdivergence of the black from other racialgroups, we would expect to find this conditionamong blacks only through miscegenation,which would explain its relatively lower in-cidence in our population. One possible ex-planation is that the DM gene would be morestable in this racial group, that is, less prone toexpansion. The recent report of Goldman etal,24 in which they show that non-DM SouthAfrican blacks have a significantly lower fre-quency of large CTG repeat alleles than inpopulations in which the disease occurs rel-atively frequently, supports this hypothesis.

PROPORTION OF MALE VERSUS FEMALEAFFECTED PATIENTSThe significantly greater proportion of affectedmale patients in the families ascertained is inaccordance with the recent report of Brunneret al'0 and other previous studies,25.28 who ob-served a disproportionate excess of males intheir families. However, in the study ofBrunneret al20 the disproportionate excess of maleswas more evident in the last asymptomaticgeneration while in the present report suchdisproportion was found in all generations,including the index cases. We did not observea significant difference in the proportion ofpatients classified as asymptomatic or mini-mally affected among males as compared to

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DM females (45/153=28-1% for males; 21/82=25-6% for females; p>005).We have no explanation for the excess of

affected DM males except for ascertainmentbias, that is, families in which the proband isa male or with multiple affected males would bemore often referred to genetic services because:(1) owing to the early frontal baldness, malesmore often have the characteristic facial ap-pearance ofDM at an earlier age than females;(interestingly this has been observed by usamong affected sibs who carry the same sizeCTG repeat in leucocyte DNA); (2) manyphysicians still believe that muscular dystrophyis restricted to males.

SIZE OF THE CTG REPEAT IN SUCCESSIVEGENERATIONSAs reported in numerous publications,'929 wealso observed a significant increase in the av-erage size of the CTG repeat in successivegenerations, which constitutes the biologicalexplanation for anticipation. There was also arelationship between the size ofthe CTG repeatand severity of the phenotype in affectedpatients. Although there was some overlap inthe mean CTG size among the four clinicalgroups, the larger ones were observed inpatients from groups C and D (congenital andearly childhood) and in three females who wereclassified in group B who had severe muscleweakness.

In the present sample, the mean size of theCTG repeat did not differ significantly in theoffspring of affected mothers as compared tothe offspring of affected fathers, in accordancewith Harley et aL.'9 No statistically significantdifference between the average repeat ex-pansion for maternally and paternally inheritedalleles was also reported by Redman et al,'4'7but, contrary to our data, only when the con-genital cases were excluded.The severe congenital form ofDM has been

associated with large CTG expansions whichare transmitted almost exclusively through thematernal line. However, interestingly, in thepresent sample, excluding the three congenitalcases, the largest CTG repeats (ranging from3-4 to 9 kb) were paternally transmitted in fiveof six unrelated families. Such expansions werefound in four children in group D (who werealso mentally retarded) as well as in three fe-males (including two sisters) who had the adultform but with severe muscular weakness. Infour families the leucocyte DNA of the trans-mitting father could be analysed: three of themhad CTG repeat sizes ranging from 02 to0-9 kb and one had a repeat size of 1-3 kb. Inthe fifth family with paternal inheritance, inwhich the female proband had the largest ex-pansion in the present study (9 kb), the trans-mitting father had only cataract of late onset.Paternal transmission of large expansions veryrarely leads to congenital DM.2" However, suchexpansions maternally transmitted would prob-ably result in the severe form.A greater tendency to initial instability in

male meiosis has been previously suggested.'9Such increased instability associated with male

transmission could reflect the larger numberof cells divisions in spermatogenesis than inoogenesis or that expansion is predominantlya postzygotic event which would be influenceddifferentially by maternal or paternal geneticimprinting.20An intergenerational decrease in the size of

the fragment, more often of paternal origin,has been reported in a small proportion ofcases.'4 19-21 3032 The mechanism of this phe-nomenon is still unknown, and several hy-potheses such as meiotic instability, geneconversion, or deletion of the expanded repeathave been suggested."-3The only contraction of the CTG repeat in

the present sample was observed in an affectedfemale and was transmitted, as the majorityof the previously reported cases, through thepaternal line. Since the affected father had lateonset of clinical symptoms and the affecteddaughter is still young, it was not possible toassess if her clinical picture will be milder thanthe one observed in her DM father. However,interestingly, recent results of a large mul-ticentre study have shown that in approximately50% of the cases there was clinical anticipationdespite the contraction of the CTG repeat sizein the leucocytes of the offspring.2' Althoughthis could not be assessed in the present series,we observed in one pair of affected mother/sonthat although both had the same size of theCTG repeat (1 5 kb), the mother was onlyminimally affected while her son had a severephenotype with early onset. This observationsupports the hypothesis that some maternalfactor may be involved in the pathophysiologyof anticipation.2

RELATIVE MALE VERSUS FEMALE FERTILITY ANDCONGENITAL MYOTONIC DYSTROPHYThe three congenital cases (two males and onefemale), belonging to two unrelated families,were transmitted through an affected mother,who had inherited the DM gene, in both cases,from their minimally affected father. Althoughthe sample was small, an excess of transmittinggrandfathers has also been reported for con-genital cases by Harley et al. 9A hypothesis to explain the predominantly

maternal transmission of congenital cases isthat large expansions would have a detrimentaleffect on male fertility but apparently not on'female fertility. 19 34 35 In a study of 101 kindreds,Harley et al'9 found only four males in whichthe repeat size was greater than 2-0 kb.

Interestingly, when we compared the pro-portions of patients older than 25 who had notreproduced, they were very similar for bothsexes: 36/110 or 32-7% among males and 22/68 or 32-3% among females. In addition, theanalysis of the mean size of the CTG repeat in23 males and 15 females who had not re-produced (23 males and 15 females) did notdiffer significantly between sexes (mean=1-83 kb, SD 1-23 for males and 2-5 kb, SD 2-1for females; p>0 05).

It is important to bear in mind that theseexpansions were assessed in leucocyte DNAand might be different in germline tissues."

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18

However, our data suggest that although the

size of the CTG repeats might not be a direct

cause of sterility in females there is reduced

fertility in both sexes, probably associated with

other factors such as the severity of the

condition.

ConclusionsIn summary, the results of the present study led

to the following observations. (1) The relative

proportion of affected DM families among 41

pedigrees was apparently lower in black than

white or oriental populations suggesting that

the DM mutation might be rarer in this racial

group. (2) A significant excess of affected males

was observed in our families including the pro-

bands. (3) The distribution in the number of

children and the frequency ofDM patients who

did not reproduce was similar for males and

females; however, female patients had on av-

erage 25% fewer children than male patients.

(4) There was a significant intergenerational

increase in the mean length of the CTG repeat

which was also correlated with the severity of

the phenotype. (5) No significant difference

was observed in the mean size of the CTG

repeat in the offspring of male transmitters as

compared to female transmitters. (6) With the

exception of the congenital cases of maternal

origin, the largest expansions were paternally

inherited, but did not lead to congenital DM.

(7) Only one CTG contraction of paternal

origin was observed in a female from a three

generation family.

The collaboration of the following persons is gratefully ac-

knowledged: Dr Helen Harley, Dr Duncan Shaw, and Professor

Peter Harper for sending us the DM probes, which made this

work possible; Dr Paulo A Otto for his help with the statistical

analysis; Dr Rita de Cassia M Pavanello, Dr Jose Antonio Levy,

Dr Lucia MendonSa, and all the physicians who referred affected

patients; Simone Campiotto, Marta Canovas, Reinaldo I Tak-

ata, Sabine Eggers, Maria Cecilia Siqueira de Mello, and

the staff from ABDIM for their invaluable help. This work was

supported by grants from FAPESP, CNPq, and ABDIM.

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