JYMed Genet 1998;35:778-783

Identical de novo mutation at the D4F104S1locus in monozygotic male twins affected byfacioscapulohumeral muscular dystrophy (FSHD)with different clinical expression

Rossella Tupler, Laura Barbierato, Mirella Memmi, Caroline A Sewry,Domenico De Grandis, Paola Maraschio, Luciano Tiepolo, Alessandra Ferlini

AbstractFacioscapulohumeral muscular dystro-phy (FSHD) is a progressive hereditaryneuromuscular disorder, transmitted inan autosomal dominant fashion. Its clini-cal expression is highly variable, rangingfrom almost asymptomatic subjects towheelchair dependent patients. The mo-lecular defect has been linked to chromo-some 4q35 markers and has been relatedto deletions of tandemly repeated se-quences located in the subtelomeric re-gion detected by probe pl3E-11(D4F104S1).We describe a pair ofmonozygotic male

twins affected by FSHD, carrying anidentical de novo pl3E-11 EcoRI frag-ment ofpaternal origin and showing greatvariability in the clinical expression ofthedisease, one being almost asymptomaticand the other severely affected. Theirmedical history was the same, with theexception of an anti-rabies vaccinationperformed at the age of 5 in the moreseverely affected twin. We hypothesise

that the vaccination might have triggeredan inflammatory immune reaction con-tributing to the more severe phenotype.(TMed Genet 1998;35:778-783)

Keywords: FSHD; monozygotic twins

Facioscapulohumeral muscular dystrophy(FSHD) is a neuromuscular disorder charac-terised by progressive weakness and atrophy ofthe facial and shoulder girdle muscles, withsubsequent involvement of the abdominal, footextensor, upper arm, and pelvic girdlemuscles.' 2 The incidence of the disease, whichis transmitted in an autosomal dominant fash-ion, is estimated as 1 in 20 000. Its penetranceis considered almost complete and in 95% ofpatients onset is by the age of 20 years. Itsclinical expression is highly variable evenwithin the same family, ranging from nearlyasymptomatic to wheelchair dependentpatients.3

Pathological alterations in muscle biopsiesare consistent with a primary defect of muscle

Biologia Generale eGenetica Medica,University ofPavia,CP 217, 27100 Pavia,ItalyR TuplerL BarbieratoM MemmiP MaraschioL Tiepolo

Neuromuscular Unit,HammersmithHospital, London, UKC A SewryA Ferlini

Divisione diNeurologia,Arcispedale S Anna,Ferrara, ItalyD De Grandis

Correspondence to:Dr Tupler.

Received 14 February 1997Revised version accepted forpublication 26 February1998

D4S171D4S1 63D4S1 39D4F1 04S1

I11161 143

2 29 5a a

1 EL }

153 1611 24 9a a

2 3

161 151 149 1572 5 4 69 4 7 3a a a a

1 C]III

-O 2

143 1512554a c

4 5 6 7

153 153 153 151 153 151 153 1539 8 1 5 1 5 3 67 9 4 4 4 4 2 1d a e c e c a a

2 3 4 5

151 157 151 153 153 153 151 153151 1535 6 5 9 1 9 5 3 5 64 3 4 7 4 7 4 2 4 1a a c d e d c a c a

Figure 1 Three generation pedigree of the family. The haplotypes obtained with four different 4q35 marker loci (D4S171,D4S163, D4S139, D4F104S1) are indicated below the symbols. A recombination event between loci D4S139 andD4F104S1 has occurred during maternal meiosis in II. 2. III. 3 shows the paternal haplotype carrying the de novo mutatedallele at the D4F104S1 locus and indicates that the mutational event has occurred during gametogenesis in the grandfatheror postzygotically, before the twinning, being associated with alleles 153, 1, and 4 at D4S171, D4S163, and D4S139,respectively. Locus D4F104S1 allele size range: a, larger than 48 kb; b, between 38 and 48 kb; c-d, between 33 and 38 kb;e, smaller than 23 kb. Closed symbols indicted the affected monozygotic twins (11.5 and II. 6).

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-t1 3 F i1

'-~~~~~~~~~~~~~~~~~~~. 0.I''s -L

Figure 2 Southern blot analysis ofEcoRI digested DNA with probe p1.presence of a de novo allele, shorter than 23 kb, in both twins (1. 4 and I

son of II. 4 (III. 2). Subjects affected by FSHD are indicated by closed syl

fibres, including variation of i

centrally placed nuclei, occasiomuscle fibres, and increased e:

perimysial connective tissue.mononuclear cells may beinflammatory changes have bee40-80% of FSHD patients.4The FSHD locus has bee:

genetic linkage analysis to the4q35-qter region."' Specific de]

p13Elz

CE

Figure 3 Southern blot analysis ofEcolEcoRIlBlnI digested DNA from the twinsaffected one, and CB, the almost asymptcprobe pl3E-11. Chromosome 4q35 allelesafter double digestion in both.

mosome 4q35 have been detected using probep13E-1 1 (D4F104S 1 ) .13 These deletions occurin highly polymorphic EcoRI fragments dis-playing a VNTR-like structure with a 3.3 kbtandem repeat unit."4 It has been postulated

D that deletions of the tandemly repeated unit,which have been localised to the subtelomericheterochromatin, may be responsible for the cisinactivation of the FSHD gene by a mechanismsimilar to the positional effect variegation.'5Alternatively this structural modification couldinfluence the regulation of gene transcription.However, no candidate gene has yet been iden-

-23 kLh tified in the FSHD region. Recently, probep1 3E-l 1 has shown that fragments with asimilar 3.3 kb repeat polymorphism occur onchromosome 1 0qter'6 and that 10% of the 1 0qlinked polymorphic fragments are smaller than35 kb, a size comparable to the 4q deleted frag-ments observed in FSHD patients.'7 The 10qlinked fragments are distinguishable from the4q linked fragments by double digestion of theDNA with EcoRI and BlnI.'8

9 kh To date three sets of monozygotic twinsaffected by FSHD have been described; onepair was discordant for the disease'9 and only

3E-11 shows the the affected twin showed a deleted EcoRI frag-1.5) and in one ment at the D4F104S1 locus.20 In the two otherwmbols. sets FSHD was familial and dominantly inher-

ibre diameter, ited in the concordant twins." Here wenal necrosis of describe two monozygotic male twins affectedndomysial and by FSHD, carrying an identical p1 3E-1 1 EcoRIOccasionally, fragment of "de novo" origin and showing a

detected' and great difference in the clinical expression of then described in disease.

n mapped by Materials and methodsE chromosome PATIENTSletions of chro- The twins, CE and CB, were born at term on

19 July 1952 after an uneventful pregnancy anda normal delivery. They were raised together

cn~ and their psychomotor development was nor-mal. The twin I.6 (fig 1) at the age of 5 years

L underwent a rabies vaccination, at that time(1957) performed with a nerve tissue vaccine(NTV). He had complained of weakness at theage of 12 years, followed by progressivedifficulty in his motor performance, hypotro-

- 23 kb, phy of the biceps, and upward slanting of thescapulae. At the age of 32, muscle weaknessextended to the lower limbs, mostly to the glu-teus muscles. He showed a marked lordoticposture and had difficulty in getting up fromthe floor. Neurological examination at the ageof 40 showed hypotonia and wasting in theupper limbs and quadriceps and a marked lor-dosis. His gait was unstable with a tendency tofall frequently. He was unable to walk ontiptoe. The facial muscles were involved withinability to blow out the cheeks and whistle.Distal power was conserved, although mildlyreduced in his hands. He has two sons (III.4

- 9 ki:L and III.5, fig 1), aged 15 and 9 years, who arecompletely asymptomatic with a normalneurological examination.Twin II.5 (fig 1) was almost asymptomatic.

RI and At the age of 15, neurological examinations (CE, the severely showed a mild hyperlordotic posture, upwardxmatic one) withsare preserved slanting of the scapulae, and mild hypotrophy

of the biceps. At the most recent examination

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in 1996, clinical progression had not occurred.No sign of muscular dystrophy was present inthe lower limbs. He was still able to whistle butshowed a mild inability to screw his eyes uptightly. He has two children (III.2 and III.3, fig1), aged 14 and 11 years, who are completelyasymptomatic, with the exception of mildwinging of the scapulae in both.The family history was negative for the pres-

ence of neuromuscular diseases on the basis ofclinical history. The twins' father (I.1) wasasymptomatic. His neurological examinationshowed mild winging of the scapulae withoutweakness. His father has been reported to showthe same posture and gait. No clinical recordsare available for him.

MOLECULAR TECHNIQUESDNA extracted from peripheral leucocytes ofall subjects was studied for allele segregation atloci D4S 163 (probe LILA5/PstI), D4S 139(probe pH30/TaqI), and D4F104S1 (pl3E-1 1/EcoRI) using standard electrophoresis andSouthern blot techniques. The size of the frag-ments at D4F104S1 was allocated by compari-son with high molecular weight DNA stand-

m1C; ards (Gibco BRL) of size 8.3, 8.6, 10.1, 12.2,15.0, 17.1, 19.4, 22.6, 24.8, 29.9, 33.5, 38.4,and 48.5 kb on the same gel. The de novopl3E-1 1 EcoRI fragments were assigned to

oins chromosome 4 by double digestion withgenetiC EcoRI/BlnI.

(CA)n dinucleotide Mfd22 (D4S 171 locus)was tested using published primer sequences.22PCR amplification was carried out in a totalvolume of 15 1d containing 100 ng of genomicDNA in 50 mmol/l KC1, 50 mmol/l Tris-HCl(pH 9.0), 7 mmol/l MgCl2, 5 pmol of eachprimer, 0.2 mmol/l dNTPs, 0.2 mg/ml BSA, 16mmol/l (NH4),SO4, and 1 U of Taq polymerase(SuperTaq, HT Biotechnology Ltd, Cam-bridge, UK). PCR conditions consisted of 27cycles of one minute at 950C, two minutes at55°C, and one minute at 72°C for each cycle.The PCR products were run on a denaturing6% polyacrylamide gel in 0.5 x TBE.Monozygosity was proven in the twins by

using DNA typing. Forty loci were analysedwith probes 33.6 and 33.15.23 24 Hybridisationwas carried out on HinfI digested DNA fromboth twins in a 0.5 mol/l NaPO4, 7% SDS, 1%albumin, and hybridisation mixture at 65°C.Washes were performed at 600C in 0.1 x SSC,0.1% SDS.Muscle biopsies were taken from the deltoid

muscle and frozen in liquid nitrogen. Cryostatsections were stained with standard histologicaltechniques and immunocytochemically withantibodies to a variety of proteins. Theseincluded dystrophin, spectrin, sarcoglycans,utrophin, fast, slow, and fetal isoforms ofmyosin, laminin, al, a2, (31, and y1 chains, andHLA class I antigens. Primary antibodies wereincubated for 30 minutes at room temperatureand visualised with a biotin-streptavidin-Texasred method, as previously described.25

ResultsMolecular analysis of the D4S 171, D4S 163,D4S139, and D4F104S1 loci was extended

over three generations in the family, includingthe parents and the children ofthe twins (fig 1).Probe p1 3E-1 1 showed a de novo fragment ofabout 23 kb at the D4F104S1 locus in bothtwins, which was transmitted to one child(III.2, fig 2). Haplotype segregation studies (fig1) indicated that the de novo fragmentoriginated from a mutational event occurringduring paternal gametogenesis, since in III.3the grandpaternal haplotype is associated withthe de novo fragment. Haplotype analysis alsoshowed that in subject II.2, a recombinationevent between D4S139 and D4F104S1 hadoccurred during maternal gametogenesis. Wewere able to confirm the origin of the de novop1 3E-1 1 EcoRI fragments at the chromosome4 D4F104S1 locus by double digestion withEcoRIIBlnI (fig 3). As reported by Deidda etal,'8 the 1Oq linked fragments aredistinguishable from the 4q linked fragmentsby double digestion of the DNA with EcoRIand BlnI. After this double digestion p1 3E-1 14q linked fragments appear 3 kb smaller thanthe EcoRI fragments, whereas the 1Oq linkedfragments are not detectable after electro-phoresis at the standard conditions, owing to ahigh number of BlnI restriction sites in thelOqrepeat units.Using the two minisatellite probes (33.15

and 33.6) monozygosity (fig 4) was confirmed,compared to a probability of chance associ-ation of <<5 x 10'-. Paternity was confirmedwith traditional and molecular markers (prob-ability of paternity W=99.999% according toEssen-Moller26) .The muscle biopsy of the asymptomatic twin

showed no significant features. There was noabnormality in fibre size, no increase inconnective tissue, and no structural changes.Immunocytochemistry also showed no abnor-malities and the expression of all proteins stud-ied was normal. Fetal myosin, usually associ-ated with regenerating fibres and thus anindicator of previous muscle damage, was notdetected in any fibres. Expression ofHLA classI antigens was also normal and confined to thevascular components (fig 5A, C, E).The biopsy of CE, in contrast, showed varia-

tion in fibre size with several very small fibres.Occasional necrotic fibres, a little connectivetissue, and a few infiltrating cells were alsoseen. Immunocytochemical labelling for spec-trin, dystrophin, the dystrophin associatedglycoproteins, and laminin chains was normal.Slight traces of utrophin and HLA class I anti-gens were detected in some mature fibres. Sev-eral of the small fibres also showed HLAexpression but these also expressed fetalmyosin and were probably immature. Fetalmyosin expression was mainly confined to thesmall fibres (fig 5B, D, F).

DiscussionWe have studied a pair of monozygotic twinsreferred to us for clinical and molecular assess-ment of FSHD. They are genetically identicaland both show a de novo p1 3E-1 1 EcoRI frag-ment associated with chromosome 4.Neurological examination of their parents wasnormal, with no sign of muscular dystrophy.

4' kb. -

Figure 4 DNAfingerprints of the tusing probes 33.6 a?33.15 confirm theiridentity.

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Figure 5 Immunolabelling of the biopsies from CB (A, C, E) and CE (B, D, F) withantibodies to C-terminal dystrophin (A, B), HLA class I antigens (C, D), andfetal myosin(E, F). Note the greater pathology in CE and the very smallfibres that express fetal myosinand HLA class I antigens, suggesting that they are regeneratingfibres. Note also the mildexpression ofHLA class I on mature fibres in CE but its absence from fibres in CB (the fineperipheral dots in E and F are the result of autofluorescence of lipofuscin). Bar=50 pm.

Haplotype analysis of the family showed thatthe de novo rearrangement in the D4F104S1locus had occurred in paternal gametogenesisor postzygotically in the paternal chromosome4 before the twinning and that the mutatedfragment was subsequently transmitted to one

of the grandchildren (III.3, fig 1). The geneticidentity of the two twins does not match theirclinical picture, with twin CB being almostasymptomatic and CE severely impaired.To our knowledge, three sets of monozygotic

twins affected by FSHD to a different extenthave been reported. In one pair, molecularanalysis of the D4F104S1 locus showed the

presence of a de novo short p1 3E- 11 EcoRIfragment only in the affected twin, presumablyowing to a postzygotic mutation and thusexplaining the phenotypic differences betweenthe two brothers.'9 20 The two other pairs weremonozygotic female twins with different clini-cal expression and carrying a similar EcoRIfragment at the D4F104S1 locus. Other chro-mosome 4q35 specific markers were not tested.No specific anamnestic records were reported.On account of the gender of the patients, theauthors considered that X inactivation mightpotentially influence the expression of the dis-ease in these subjects.2' This hypothesis doesnot apply to our cases as the monozygotic twinsdescribed here are male. They carry the samemutated fragment but show a different pheno-type.

Clinically discordant monozygotic twins forvarious diseases have often been reported. Dis-cordance is particularly evident, for example, inWiedeman-Beckwith syndrome (WBS); out ofthe eight cases of monozygotic twinsdescribed,27..9 seven were discordant for theexpression of this syndrome, showing one twinwith many classical manifestations of thedisease and the other having few or none.Minor anomalies observed in most of the nor-mal co-twins were ascribed to the presence ofmild forms ofWBS and have been explained asa consequence of either environmental factors27or somatic mosaicism.28 In our case a postzy-gotic mutation, occurring before the twinningand leading to a different extent of mosaicismin the two brothers, might account for the dif-ferences in the clinical manifestation of themuscular dystrophy. However, qualitative evi-dence of mosaicism, as shown by the presenceof an additional less intense band, was notdetected by probe p1 3E-1 1 .3"32

Considerable phenotypic variability is de-scribed among FSHD patients with wide vari-ation in the age of onset and in the rate of pro-gression as well as in the extent of specificmuscle involvement. In an attempt to explainthese clinical differences, Lunt et al3 studied alarge group of familial cases and found a corre-lation between the p1 3E-1 1 EcoRI fragmentsize and age at onset or progression of muscleimpairment. The smallest fragments wereassociated with the earliest age of onset andwith earlier use of a wheelchair. The authorsproposed that D4F104S1 fragment size deter-mines a part of the variance in age of onset andeventual severity of FSHD. Furthermore, theanalysis of the age at onset of clinical signs inpatients from multigenerational families sug-gested that anticipation could occur inFSHD.34 In standardised genotype-phenotypestudies it has been shown that the degree ofclinical severity present in the parent is signifi-cantly lower when compared to the offspring,supporting the hypothesis of anticipation inFSHD." Nevertheless, we have studied fami-lies in which almost asymptomatic subjects andwheelchair dependent patients were presentwithin the same generation, with affectedmembers carrying the identical EcoRI fragment(unpublished observations). In this respect,FSHD also differs from those hereditary

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diseases characterised by meiotic and mitoticinstability resulting in an expansion of tripletsinterfering with gene expression.36 In FSHDpatients, deletions of tandem repeats occur inthe heterochromatic subtelomeric region ofchromosome 4q. Structural changes of the het-erochromatin could interfere with the expres-sion of genes located proximally to this region.However, this hypothesis does not explain theclinical variability of the disease within thesame family, being the deleted fragmentstransmitted without any modification of theirlength. To clarify the basis of the great variancein the levels of expression of the disease, a morecomplex mechanism should be considered. Inthis respect, twins studies could be very usefulin order to separate the aspects linked to thegenetic background, and therefore to the natu-ral history of the disease, from those related toenvironmental factors that might have influ-enced the phenotype.There was no difference in the clinical

history of our patients, with the exception of ananti-rabies vaccination performed with a nervetissue vaccine (NTV) in CE at the age of 5years. Furthermore, their father and paternalgrandfather had sloping shoulders, causing anFSHD-like posture, suggestive of the presenceof an almost asymptomatic form of the diseasein the previous generations. If this were thecase, linkage analysis would confirm the segre-gation of FSHD with the putative 4q35 locus,while the de novo EcoRI fragment of paternalorigin present in the twins would represent amutational event independent of the transmis-sion of the disease and not interfering with itsclinical severity.Inflammatory pathological findings are com-

mon in several primitive muscular dystrophies.In view of a possible role of inflammation in theclinical picture of FSHD, Arahata et ar'analysed inflammatory mononuclear cells inFSHD muscle sections and in normal controls.They found that an increased number ofnecrotic fibres corresponds with an accumula-tion of inflammatory cells, and hypothesisedthat mononuclear cellular infiltrates may en-hance muscle fibre damage. The musclepathology in case CE shows features consistentwith FSHD and no specific defects in proteinexpression were seen. It is interesting to note,however, that there was some expression ofHLA class antigens in the muscle fibres. This isa common feature of inflammatory autoim-mune myopathies. In our case, the anti-rabiesvaccination administered to CE could havetriggered an inflammatory immune reactionthat may have contributed to the more severephenotype.

We thank Professor M Fraccaro and Dr G Perini for their help-ful suggestions, Dr C Previdere for the paternity testing, Profes-sor R R Frants for kindly providing the p 1 3E- 1 1 plasmid, and MGatti for her technical support. The financial support of theTelethon to project C18 (cell bank) is gratefully acknowledged.This work was supported by Telethon grants No 729. L Barbi-erato is supported for her doctorate by Telethon.

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7 Mathews KD, Mills KA, Bosch EP, et al. Linkagelocalization of facioscapulohumeral muscular dystrophy(FHSD) in 4q35. Am JHum Genet 1992;51:428-31.

8 Mills KA, Buetow KH, Xu Y, et al. Genetic and physicalmapping on chromosome 4 narrows the localization of thegene for facioscapulohumeral muscular dystrophy(FSHD). Am JfHum Genet 1992;51:432-9.

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22 Winokur ST, Schutte B, Weiffenbach B, et al. A radiationhybrid map of 15 loci on the distal long arm ofchromosome 4, the region containing the gene responsiblefor facioscapulohumeral muscular dystrophy. Am J HumGenet 1993;53:874-80.

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prenatal testing for facioscapulohumeral muscular 35 Tawil R, Forrester Y, Griggs RC, Mendell Y, Kissel Ydystrophy: diagnostic approach for sporadic and familial Evidence for anticipation and association of deletion sizecases. J Med Genet 1996;33:29-35. with severity in facioscapulohumeral muscular dystrophy.

33 Lunt P, Jardine PE, Koch MC, et al. Correlation between Ann Neurol 1996;39:744-8.fragments size at D4Fl04S1 and age at onset or at 36 La Spada AR, Paulson HL, Fischbeck KH. Trmucleotidewheelchair use, with a possible generational effect, accounts repeaSpada inneulso gicalFisease.Annucleotidefor much phenotypic variation in 4q35 facioscapulohumeral repeat expansion in neurological disease. Ann Neurol 1994;muscular dystrophy (FSHD). Hum Mol Genet 1995;4:951-8. 36:814-22.

34 Zatz M, Marie SK, Passos-Bueno MR, et al. High 37 Arahata K, Ishihara T, Fukunaga H, et al. Inflammatoryproportion of new mutations and possible anticipation in response in facioscapulohumeral muscular dystrophyBrazilian facioscapulohumeral muscular dystrophy fami- (FSHD): immunocytochemical and genetic analysis. Mus-lies. AmJ Hum Genet 1995;56:99-105. cle Nerve 1995;suppl 2:56-66.

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