American Journal of Medical Genetics 40354-364 (1991)

Discordance of Muscular Dystrophy in Monozygotic Female Twins: Evidence Supporting Asymmetric Splitting of the Inner Cell Mass in a Manifesting Carrier of Duchenne Dystrophy James R. Lupski, Carlos A. Garcia, Huda Y. Zoghbi, Eric P. Hoffman, and Raymond G. Fenwick Institute for Molecular Genetics (J.R.L., H.Y.Z., R.G.F.) and Department of Pediatrics (J.R.L., H.Y.Z.), Baylor College of Medicine, Houston, Texas; Department of Neurology and Pathology, L.S.U. Medical Center, New Orleans, Louisiana (C.A.G.); The Children's Hospital, Boston, Massachusetts (E.P.H.) Eric P. Hoffman is now at the Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA. In 1990, Richards et al. reported dramatically skewed lyonization in a set of female monozygotic twins heterozygous for Duchenne muscular dystrophy (DMD).The skewed inactivation pattern was symmetrical in opposite directions, one twin being affected with DMD, the other one being normal. Here, we report an additional set of female monozygotic twins heterozygous for a mutation at the dystrophin locus. Similarly, one shows a manifesting carrier phenotype while one is normal. However, unlike the previous report, we find a skewed X inactivation pattern only in the affected twin, while the normal twin showed a random X inactivation pattern. Our results lend considerable experimental support for the models of twinning and X inactivation recently outlined by Nance in 1990,in that these twins probably represent asymmetric splitting of the inner cell mass (ICM):The affected twin likely arose when a small proportion of the ICM split off after lyonization had occurred. In this situation, the original ICM could give rise to the normal twin with random lyonization, while the newly split cells would experience catchup growth and lead to the affected twin. Genetic studies of this family showed that the specific dystrophin gene mutation was an exon duplication that arose sporadically in the paternally derived X chromosome.

KEY WORDS: Duchennemuscular dystrophy, duplication mutation, X-inactivation, discordant MZ twins, dystrophin

INTRODUCTION Female manifesting carriers of Duchenne muscular dystrophy (DMD) have occasionally been reported [Moser and Emery, 1974; Hazama et al., 1979; Isaacs and Badenhorst, 19871. Cytogenetic analysis of these rare individuals has sometimes shown X-autosome translocations with breakpoints involving Xp21 [Greenstein et al., 1977; Ray et al., 1985; Boyd et al., 19861. Other manifesting carriers have been demonstrated to have Ullrich-'hrner syndrome where a second X chromosome is not available to compensate for the DMD mutation [Ferrier et al., 19651. However, there are reports of manifesting carriers with normal chromosomes [Moser and Emery, 1974; Isaacs and Badenhorst, 19871. Interestingly, several reports have described a manifesting carrier phenotype in only one monozygotic twin and in every case the twins have been female [Gomez et al., 1977;Burn et al., 1986;Pena et al., 1987;Richards et al., 19901. To our knowledge, discordance of the DMD phenotype has never been described in male monozygotic twins. A postulated mechanism for the manifesting carrier phenotype in only one female monozygotic twin has been either non-random X chromosome inactivation leading to heterochromatization of the normal X chromosome with expression of the DMD mutation from the only active X chromosome or preferential survival of cells carrying the X chromosome with the DMD mutation after random X inactivation [Gomez et al., 1977; Received for publication October 11,1990;revision received De- Burn et al., 1986;Pena et al., 19871. Consistent with this model, all manifesting carriers show both dystrophincember 12, 1990. Address reprint requests to James R. Lupski, M.D., Ph.D., Insti- positive and dystrophin-negative fibers in their muscle tute for Molecular Genetics, Baylor College of Medicine, One which appear to be arranged in clonal groups [Bonilla et al., 1988; Arahata et al., 1989; Miranda et al., 19891. Baylor Plaza, Room T-905,Houston, TX 77030. 0 1991 Wiley-Liss, Inc.

Discordant MZ Twins Restriction fragment length polymorphisms (RFLPs) can be used in conjunction with methylation-sensitive restriction enzymes and X chromosome specific DNA probes such as the phosphoglycerate kinase gene (PGK), the hypoxanthine phosphoriboryltrderase gene (HPRT), and the variable number tandem repeat (VNTR) probe M27P to determine X chromosome inactivation patterns and to test this hypothesis. Unfortunate lyonization or a non-random X inactivation pattern leading to the DMD phenotype was described recently in a female monozygotic twin who presented with muscular dystrophy [Richards et al., 19901. It has been postulated that if X inactivation occurs before MZ twinning, the discordant expression of X-linked traits in heterozygous female pairs can be explained by the sampling process associated with the twinning event [Nance, 19901. A prediction from this hypothesis was that if the number of precursor cells is not equal, an extreme X inactivation profile will be even more likely in the twin arising from the fewest cells [Nance, 19901. Here, we report a newly identified set of female monozwotic twins where onlv one of the twins has clinical ebydence of muscular distrophy. Both twins had the same new mutation within the DMD gene resulting in duplication of exons 42 and 43. This mutation occurred in the paternally derived X chromosome and resulted in the muscular dystrophy phenotype in one of the female twins and in the male offspring of the clinically unaffected carrier twin. The muscular dystrophy phenotype in the affected female twin is associated with a non-random X chromosome inactivation pattern as demonstrated by muscle histochemical staining using dystrophin antibody and by the bias in the methylation patterns using an X chromosome-specific polymorphic probe. The data support the hypothesis that twinning may occur after lyonization and that a non-random X inactivation pattern may result from the sampling process associated with twinning.

MATERIALS AND METHODS Patient Material Peripheral blood was drawn, after informed consent was obtained, from all living members in the family except 111-3 (Fig. 1).High molecular weight DNA was obtained from peripheral lymphocytes by the procedure of Miller et al. [19881. Epstein-Barr-virus-transformed lymphoblasts were established by the procedure of Anderson and Gusella [1984]. Muscle tissue was obtained by biopsy from patients 11-3 and 111-2, and a portion was frozen in liquid nitrogen, as part of the clinical workup for muscular dystrophy. Dystrophin Immunofluorescence and Immunoblotting Studies Cryosections (8 p,m) were cut from a flash-frozen muscle biopsy from the affected twin, and the sections thawed on gelatin-coated slides and then processed for dystrophin immunofluorescenceas previously described [Bonilla et al., 1988; Hoffman et al., 19901. Dystrophin antibodies used were affinity-purified anti-60kd [Hoffman et al., 19871 directed against the amino-terminal portion of the central rod domain and affinity purified

355

anti-l0kd [Koenig and Kunkel, 1990; Hoffman et al., 1989al directed against the penultimate carboxyl-terminus domain. Dystrophin immunoblotting was done as previously described [Hoffman et al., 19881 using post-transfer myosin heavy chain staining as a control for muscle content of individual lanes [Hoffman et al., 1989bl. The antibodies used for blotting were affinity purified anti-30kd [Hoffman et al., 19871 directed against the central portion of the rod domain.

Multiplex DNA Amplification The multiplex PCR for nine exons within the dystrophin gene [Chamberlain et al., 1988,19901was carried out using reaction mixes obtained from J.S. Chamberlain and stored a t - 70" C. Amplifications were initiated by adding 5 p,l of DNA diluted to 20 ng/p,l, 2.5 units of AmpliTaq DNA polymerase (Cetus) and 50 pl of paraffin oil to thawed reaction mixes and incubating the samples in a DNA Thermalcycler (Perkin-Elmer Cetus) as described previously [Chamberlain et al., 1988, 19901. Probes The cDNA probes for the dystrophin gene (1-2,2-3, 4-5a, 5b-7,8, and 9-14) were described by Koenig et al. [19871 and were a gift from L.M. Kunkel. They were prepared for use as described by Baumbach et al. [19891. Assignments for the exons detected by those probes are reported by Koenig et al. [19891. Intragenic and flanking probes used for linkage and haplotype analyses of the dystrophin locus were gifts from L.M. Kunkel, pERT87.1 and pERT87.15 [Kunkel et al., 19861, J . Bir [Monacoet al., 19871,99.6[Aldridge et al., 19841,and C7 [de Martinville et al., 19851; R.G. Worton, XJ1.l [Ray et al., 19851; P.L. Pearson, 754 [Hofker et al., 19851; and G.J.B. van Ommen, J66 [van Ommen et al., 19871. Southern Analysis and Densitometry Southern transfer [Southern, 19751was carried out by standard protocols [Maniatis et al., 19821. Probes were labeled with [32Pl-dCTPby the random primer method of Feinberg and Vogelstein [1983] using kits from Boehringer Mannheim. Hybridizations were performed using conditions similar to those described by Church and Gilbert [1984]. Densitometry of the autoradiographs was conducted in an LKB UltraScan XL laser densitometer using LK 2400 GelScan XL software to integrate the areas under peaks and thus determine the intensity of each hybridization band (Table I). RFLP-Methylation Studies To identify polymorphisms at differentially methylated loci, we screened DNAs from this family using probes from the PGK gene and HPRT gene (kindly provided by Bert Vogelstein,Johns Hopkins) and the M27p [Abramson et al., 19901 probe at the DXS255 locus (kindly provided Ian Craig, Oxford, England). Polymorphisms were not detected using the PGK and HPRT probes, but were detected using M27p. Therefore, the RFLP-methylation analysis was carried out using the M27P probe and peripheral blood leukocyte DNA. The

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I

XJ1.l Taql..........(3.813.1) 87.1 Xmnl ...........(8.717.5) 87.15 BamH............(l0n) 87.15 Xmnl ........(2311.6) 87.15 Taqi .........(3.613.4) 87.J.Bir BamHl.....(2115) cDNA8 Taql ......(6.7/5.7) cDNA8 Pstl........(1113.4) J66 Pstl. ..( 1.5/1.45/1.3)

2

1

11 3.8 0.7 7 1.6 DUP-;'

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11 3.0 0.7 7 1.6 3.4 21 5.7 3.4 1.45 7.8 22

t1 3.1 0.7 7 1.6 3.6 5 5.7 3.4 1.45 7.0 22

11 3.1 8.7 7 1.6 3.6 5 5.7 3.4 1.45 7.0

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3.0

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7

7

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3.4 21

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DYSTROPHIN GENE MAP Probe 1 9 11 3.0 3.0 0 . 7 0.7 7 7 1.61.6 3.4 3.6 5 5 -DUP 5.7 6.7 3.4 3.4 1.45 1.45 7.8 7.0 22 22

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Approximate Scale (Mb)

4 4 48 45 51

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Fig. 1. Haplotype analysis at or near the DMD locus in the pedigree containing monozygotic twins discordant for the muscular dystrophy phenotype. Clinically unaffected males and females are denoted by open boxes and circles, respectively.The clinically affected female manifesting carrier is denoted by a filled circle, while the male who clinically demonstrates a muscular dystrophy phenotype 111-2 is depicted by a filled square. Asymptomatic carrier females are denoted by a dot within a circle. In the left upper corner of the figure, the probes utilized in the haplotype analysis are shown with the allele sizes, measured in kilobase pairs, given in parentheses. The approximate locations within the DMD gene of the probes that were used for RFLP analysis are shown a t the bottom of the figure. Also shown are the sites for the primers utilized in multiplex PCR.

DNA was digested with PstI, and the sample was subsequently divided into three aliquots; the first aliquot (PstI alone) was used to identify the polymorphic bands, the second aliquot was further digested with MspI to identify the methylation sensitive sites, and the third aliquot was further digested with HpaII to assess the methylation patterns.

RESULTS Clinical Manifestations The pedigree is shown in Figure 1.Monozygotic twinning was supported by HLA typing (identical for HLA Al, A3, B49, B40, Cw3, Cw7, Bw4, Bw6) and DNA typing with polymorphic probes. The twins (11-2 and 11-3)were identical for several alleles on the X chromosome (Fig. 1) and at the following highly polymorphic

variable number tandem repeat (VNTR) loci: YNZ22 (D17S5) [Nakamura et al., 19871MCT-118 (DlS80) "akamura et al., 19881, and APOB [Boerwinkel et al., 19891 (data not shown). Birth records did not document placental structure, thus not allowing a n approximation of the cell stage a t which the twinning process took place. However, by the twins' mother's account, "both girls were in one sac when born." Twin A (11-2), who weighed 2,440 g a t birth, has a normal neurological status and normal electromyography, but her CK has been above 1,000 IU/L on three different occasions. She refused a muscle biopsy. Twin B (11-3)weighed 2,270 g a t birth and walked at around age 12 months. She could not run or jump like the other children of her age, including her twin sister. At age 8, she began to fall, and a few months later, she had diffi-

Discordant MZ Twins

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TABLE I. Identification of a Dystrophin Duplication Mutation and Female Carriers of That Mutation by Densitometry of Southern Analysis Data Relative intensityb

Hybridization intensity for exons' Individuald Experiment A a b c = 111-2 d e f = control male Experiment B 1-2 11-2 11-3 111-1 111-2 Control female

42 30-33

f

43

+ 38-41

Number of exons 42 and 43' per genome equivalent

30-33

38-41

42

43

3.75 2.58 4.04 1.72 1.16 1.25

3.44 2.96 3.97 1.73

1.15 0.81 2.28 0.50

2.31 1.99 4.21 0.88

0.48 0.51 0.81 0.40

1.2 1.3 2.0 1.0

1.54

0.62

0.48

0.39

1.o

2.98 2.43 2.92 2.58 1.26 0.73

3.93 2.79 3.10 3.21 1.29 1.26

1.03 1.49 1.72 1.93 0.88 0.37

0.74 0.93 1.20 1.17 0.75 0.23

0.26 0.46 0.49 0.53 0.64 0.30

1.8 3.0 3.2 3.6 2.1 2.0

-

-

-

-

"Hybridization intensities for HindIII-digested DNAs probed with the dystrophin cDNA5b-6 probe were measured by densitometry as illustrated in Figure 5 and integration of the areas under the peaks was used to derive quantitative values (absorbance units x mm) for the exons listed. bIntensity values for exons 42 plus 43 were divided by those for exons 30-33 plus 38-41 to measure the relative concentrations of exons 42 and 43 in those individuals tested. 'The number of exons 42 and 43 per genome equivalent was calculated by dividing the relative intensity for those hybridization signals (b)by the value for the control individual in each experiment and multiplying that quotient by the number of X chromosomes carried by the individual. dExperiment A is the experiment illustrated in Figures 4 and 5, and the same designations are used here for the males who were tested. Experiment B involved densitometry of Southern analysis data from a similar experiment (not shown)in which members ofthe pedigree illustrated in Figure 1 were studied

culty climbing stairs and getting up from chairs. Early in the course of her disease, her arms had normal strength, but after a few months, she noticed difficulty getting her arms up to comb her hair. The weakness has progressed slowly but relentlessly. At age 7, she needed elongation of the right Achilles' tendon. At age 17, she had an exaggerated lumbar lordosis, a waddling gait, and enlarged rubbery calfs. There was weakness of the neck flexor muscles and proximal shoulder muscle weakness with some preservation of deltoid muscle strength but no winging of the scapulae. She had to push herself up with her hands to get up from a sitting position. During her last clinic visit, at age 34, she could not get up from a chair without the help of another person. She can only raise her hands to her mouth with some difficulty. When assisted, she can only walk a few steps with a slow waddling gait. There is no cardiac involvement, and her intellectual functions are normal. She finished two semesters of college and works as a secretary. Her CK has been between 802 and 1,700 IU/L (normal < 200 IU/L). The EMG has shown myopathic changes. The muscle histochemistry a t age 29 shows fatty infiltration and a marked increase of the endomysial connective tissue. All of the remaining muscle fibers are round in shape and vary in size from small atrophic fibers to large hypertrophic fibers. There are split fibers and an increased number of central nuclei. There is no active degeneration or regeneration of muscle fibers. There is type 1fiber predominance, but large and small fibers are of both fiber types. Twin A (11-2) has a boy (111-2) who is 8 years old. He walked a t 13 months, but falls easily and freuuentls, and since age I , has had to use his hands to get ;p from

the floor. At age 8, he has an exaggerated lumbar lordosis and a mild waddling gait. He can run short distances. He has no joint contractures. His neck and shoulder muscles have normal strength. He has enlarged rubbery calves and has a mild Gower sign when attempting to get up from the floor. His CK remained around 8,000 IU/L. The EMG showed myopathic changes. The nerve conduction velocities and latencies are normal. The muscle biopsy of the right vastus lateralis muscle, done at age 2 years, showed a mild increase of endomysial connective tissue. There is a slight variation of the muscle fiber size but no hypertrophic fibers. There are several dark fibers. There is an increase in the number of central nuclei, and there are split fibers. This muscle biopsy showed signs of a dystrophic myopathy, and DMD was suspected. Though the fibrotic replacement of the muscle was minimal, the fibrosis characteristic of DMD muscle is progressive, and the amount found was not inconsistent for that of a 2-year-old boy. The boy is currently 8 years old and has experienced a clinical course considered milder than that of typical DMD in that he shows little overt weakness. However, the clinical progression of DMD in the 7 to 9 year range can be quite variable. Thus, the clinical progression of the patient is not inconsistent with a diagnosis of DMD. High-resolution chromosome analysis of both twins showed normal chromosomes (46,XX) in 50 cells analyzed (data not shown). Uniparental disomy [Spence et al., 19881 was ruled out as a mechanism for disease expression in the affected twin by demonstrating that she has two different X chromosomes as evidenced by heterozygosity with X chromosome-specific probes (Fig. 1).

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Dystrophin Protein Analysis A small portion of the diagnostic muscle biopsy of the affected twin (11-3)was tested for dystrophin content by immunoblotting. This analysis showed dystrophin of normal size, yet of dramatically reduced quantities (approximately 20%of normal) (data not shown). This pattern in females is considered diagnostic of manifesting carriers of DMD [Hoffman et al., 1988; Richards et al., 19901. To verify this result, the biopsy was retested by dystrophin immunofluorescence of cryosections, a method which should show populations of dystrophin-negative and dystrophin-positive fibers in true manifesting carriers [Arahata et al., 19891. As shown in Figure 2, the manifesting-carrier diagnosis of the proposita was confirmed Distinct populations of dystrophin-negative fibers were found adjacent to dystrophin-positive populations. Each type of fiber population appeared grouped, suggesting a clonal derivation of each region of the muscle, again consistent with the skewed X chromosome

Fig. 2. Dystrophin immunofluorescenceof the affected twin’s (11-3) muscle. Panels A and B are corresponding fluorescence and phase micrographs. Dystrophin is visualized at the periphery of dystrophinpositive myofibers. Neighboring dystrophin-positive and dystrophinnegative fibers can be seen. This mosaic pattern is characteristic of manifesting carriers of Duchenne dystrophy. Bar = 130 k. Panel C shows a different field of the same biopsy, at lower magnification. Both the dystrophin-positive and dystrophin-negativefibers are arranged in foci, with the large dystrophin-negative region to the left largely replaced by fibro-fatty tissue. The dystrophin-positive foci appear to have little overt pathology, with the fibers relatively homogeneous in size and closely packed. Bar = 260 p.

inactivation pattern characteristic of manifesting carriers of DMD. The dystrophin-positive fiber groups predominated in the biopsy fragments tested, with only minimal abnormality evident in the dystrophin-positive groups. The dystrophin-negative groups were quite atrophic and largely replaced by fibro-fatty connective tissue. This suggested that the clinical phenotype would be expected to be less severe than that expected for the previously reported affected twin [Richards et al., 19901, an expectation that was verified by the milder clinical phenotype of the proposita. To determine if the affected boy did indeed have DMD, portions of the original muscle biopsy were subjected to dystrophin immunofluorescence and immunoblotting. By immunoblotting, a faint immunoreactive band at 250 kd was observed, (normal dystrophin molecular weight = 400 kd) (data not shown). The very small size andlow amounts (- 10%)ofthis immunoreactive protein suggested that it might represent either an immunological artefact of the blotting procedure or a degradation fragment of dystrophin. Immunofluorescence analyses using affinity-purified polyclonal antibodies to both the amino-terminus (60 kd) [Hoffman et al., 19871 and carboxyl-terminus (d10) [Koenig and Kunkel, 19901, were then performed. There was a complete absence of immunostaining using the carboxyl-terminus antibodies (Fig. 3, panel DJ, though occasional revertant fibers were found [Hoffman et al., 19901. Such a result is diagnostic of DMD. The amino-terminal antibodies recognized a very faint, sarcolemmal staining (Fig. 3, panel F), which was more characteristic of a very severe Becker dystrophy clinical progression. Given the gene study data, described below, we think that the 250 kd protein observed by immunoblot and the faint immunofluorescence signal with the amino-terminal antibodies could represent a truncated, presumably nonfunctional form of dystrophin produced by the mutated dystrophin gene. These data, taken together, indicate that the boy has DMD and that his maternal aunt (11-3) is a manifesting carrier of DMD.

Mutation Analysis Given the fact that the twins have five unaffected brothers, this suggested that the phenotype was likely due t o a new mutation present in the sperm cell that produced the twins. The initial studies utilized multiplex PCR [Chamberlain et al., 19881 to screen for a deletion in the DMD gene since deletions represent the most common genetic alteration leading to DMD and have been detected in approximately 60%of all patients [Monaco et al., 1985; Darras et al., 1988; Lindlof et al., 1988; Wapenaar et al., 1988; Baumbach et al., 1989; Gilliard et al., 1989; Koenig et al., 19891. Multiplex PCR can detect approximately 80% of deletion mutations [Chamberlain et al., 1988,19901.The multiplex pattern in patient 111-2 was normal when compared to controls, suggesting that the mutation was not a deletion involving the exons found to be involved frequently in deletion mutations (data not shown). Analysis of DNA from patient 111-2 was carried out using dystrophin probes 754, XJ1.l, 87.1, 87.15, 87.JBir, 44-1 (cDNA 8),J66, C7, and 99-6 (Fig. 11, and

Discordant MZ Twins

60kd IF

359

60kd IF

Fig. 3. Dystrophin immunofluorescence analysis of the affected male (111-2).Shown is the visualization of dystrophin in the muscle biopsies of an adult male with limb-girdle dystrophy (normal control) (panels A,B,E) and the affected male (111-2) at 2 years of age (panels C,D,F). Antibodies to both the aminoterminus (60kd; panels E,F) and the carboxyl-terminus (d10;panels B,D) of dystrophin were used. Panels A and C are the differential interference contrast micrographs (DIC) corresponding to the immunofluorescence (IF) shown in panels B and D. All immunofluorescencephotographs were from the same experiment and were photographed and printed using identical exposures. Bar = 500 k. Dystrophin is visualized as a continuous ring of immunostaining at the plasma membrane in the normal male using either antibody. In the affected male’s muscle, no dystrophin immunostaining is seen using the carboxyl-terminal antibody (panel D),while a very faint and variable peripheral staining is detected using the amino-terminal antibody (panel F). Also shown in panel F is two revertant fibers where isolated nuclei have sustained a phenotypic reversion enabling them to produce dystrophin in an otherwise dystrophin-deficient muscle [Hoffman et al., 19901.The same revertant fibers were seen by both antibodies. Such revertants are present at very low levels in approximately half of Duchenne dystrophy patients and do not alter the expected clinical progression of the disease. The data shown, taken together with the molecular analysis and immunoblot data of this patient, indicate that the affected male in this family has Duchenne muscular dystrophy.

cDNA probes 1-14 (including 5b-6; Fig. 4). DNA from patient 111-2 was subjected to Southern analysis using the cDNA probes from regions 1-14 and compared to the patterns obtained from other DMD affected males (Fig. 4). The only alteration noted was obtained with cDNA probe 5b-6. This analysis showed increased intensity from the hybridizing bands corresponding to exons 42

and 43 and suggested a duplication mutation including these exons. A quantitative measure of band intensity of the Southern blot shown in Figure 4 was obtained by a densitometric scan of the individual lanes. As shown in Figure 5c, patient 111-2has twice the intensity (relative absorbance) for the bands correspondingto exons 42 and 43 when compared t o four other DMD or control males

Lupski et al.

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B n

p

1811-

P

a I

b I

C I

d I

e I

f

I

- 30-33 - 43 C 0

5

2

6.26.1-

- 40-41

4.2 -

- 42

- 38-39

Fig. 4. Identification of a dystrophin duplication mutation in affected male 111-2 by Southern analysis. As part of more extensive studies, HindIII-digested DNAs from males affected with Duchenne muscular dystrophy were probed with the cDNA probe 5b-6. Pictured in the autoradiograph are the larger restriction fragments identified by that probe. The exons of the dystrophin gene contained in the various bands are designated in the figure. Lane h contains HindIII-digested lambda phage DNA as size standards, and lane f contains DNA from a normal male. Lanes a and b are DNAs from affected males for whom no mutations have been identified. Lane c contains the DNA of patient 111-2.Lane d is a DNA from an individual who has been found to have a deletion of exons 48-50 as detected with the cDNA8 probe (data not shown). Lane e identifies a deletion mutation of exons 38-43 in another patient. By comparison, the results for patient 111-2 show that he has abnormally strong signals for the hybridizing bands from exons 42 and 43, suggesting that he has a duplication mutation which includes those exons.

(Fig. 5a,b,d,f) who are not duplicated for this region of the DMD gene. The relative intensity for the bands corresponding to exons 42 and 43 was determined by dividing the intensity signal obtained (absorbance units x mm) from bands corresponding to these exons, by the intensity obtained for other bands in the same lane. In this manner, the patient’s own DNA is used as an internal control for normalization. The number of exons per genome equivalent was then calculated by normalizing the patients’ results to those of control individuals and factoring in the number of X chromosomes carried by the individuals. The data shown in Table I (Experiment A) obtained from Figures 4 and 5 clearly demonstrate that patient 111-2 is duplicated for exons 42 and 43. In a similar manner, densitometric studies were performed on all the at-risk females in the pedigree. Table I (Experiment B) demonstrates that the unaffected sister (111-11, unaffected mother (11-21, and affected monozygotic twin (11-3) aunt of patient 111-2 all harbor the duplication mutation. The maternal grandmother (1-2)does not contain the duplication, and therefore it is concluded that the DMD phenotype is associated with a de nouo duplication mutation in the sperm that led to the monozygotic twins 11-2 and 11-3. A duplication of exons 42 and 43 would cause a shift in the reading frame of dystrophin mRNA [Koenig et al., 1989;Gillard et al., 19891.This shift would cause an opal termination codon (UGA) to be encountered eight codons into the second copy of exon 42 [Koenig et al., 19891. This would result in a protein containing 2,104 amino

I

I I

I I

4 2 6 1 6 2 11 18 Size (kb)

I

I I

Exon 42 38- 40- 43 3039 41 33

Fig. 5. Densitometry of Southern analysis data confirms the presence of a duplication mutation in patient 111-2. The strengths of the hybridization signals on the autoradiograph illustrated in Figure 4 were measured with a laser densitometer. The results for patient 111-2 are illustrated in panel c, and those for an unaffected male in panel f. As described in Figure 4, the remaining panels are results for affected males from other families, only one of whom (panel e) has a deletion mutation detected by probe 5b-6. Comparison of the results demonstrates that the signals for exons 42 and 43 (marked by the arrows) for patient 111-2are clearly increased relative to those ofthe other exons in the scan, thus confirming a duplication mutation in this patient.

acids and having a molecular weight of approximately 243,000 daltons, a value very similar to that estimated for patient 111-2 by dystrophin immunoblotting.

Parental Origin of the New Mutation To determine the parental origin of the new mutation in the monozygotic twins 11-2 and 11-3, a haplotype had to be derived for the X chromosome of the deceased father. This was performed by determining the DNA haplotypes of the five unaffected brothers using the following probes: 754, XJ1.l, 87.1, 87.15, 87.J.Bir, cDNA 8,J66, C7, and 99-6 (Fig. 1).Individuals 11-4,II-5, 11-6,and 11-8have inherited one dystrophin haplotype as evidenced by the informative 3.8 kb TuqI polymorphism at XJ1.l. Individual 11-7has inherited the other maternal haplotype as shown by the 3.1 kb TuqI polymorphism using the XJ1.l probe. Individual 11-5 has a recombination event between probes 87.15 and 87.J.Bir. As expected, the monozygotic twins have identical haplotypes for each dystrophin allele. The X chromosomeof maternal origin is readily established from the results of studies in the unaffected brothers. This allows one to derive a putative haplotype for the paternal (1-1)allele. The dystrophin haplotype derived from the father of the twins is identical to the haplotype inherited by the af-

Discordant MZ Twins fected son (111-2) of the phenotypically normal twin (11-2). Since her sister (11-3) is a manifesting carrier of DMD, the mutation in this family is a new mutation in the paternal allele inherited by the twins.

1-2

11-2

11-3

361

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DNA Methylation Patterns in the Monozygotic Twins To investigate the hypothesis of a non-random X chromosome inactivation pattern being responsible for the disease phenotype in the affected monozygotic twin, we used a technique that relies on differential methylation of active and inactive X chromosomal genes. Specifically, we used a probe (M27p) that detects RFLPs and differential methylation patterns a t the DXS255 locus. At this locus, the DNA is methylated on the active X chromosome and unmethylated on the inactive X chromosome [Abramson et al., 19901.White blood cells from the monozygotic twins were examined for DNA methylation patterns at the M27p locus. The results shown in Figure 6 demonstrate that the affected monozygotic twin (11-3) shows a biased methylation pattern consistent with a non-random X chromosome inactivation pattern. In this patient, only the 7.4 kb allele is digested with HpaII, while the 8.4 kb allele was not digested with HpaII at all, suggesting non-random methylation of this allele. In the unaffected twin, both alleles are further digested with HpaII, indicating random methylation and suggesting that each X chromosome is randomly inactivated. Since the mother is homozygous for the 7.4 kb allele, it is clear that both twins inherited the 7.4 kb allele from her and the 8.4 kb allele from their father. Since the 8.4 kb allele is methylated, therefore indicating that it is from the active X chromosome in the symptomatic twin (11-31,this shows that the active X chromosome is of paternal origin. These data support the hypothesis that 11-3is a manifesting carrier while 11-2is asymptomatic, because the former has a larger fraction of cells in which the mutant allele is active.

Fig. 6. RFLP methylation analysis using peripheral blood leukocyte DNA and the M27P probe. Individual 11-3and 11-2 are the monozygotic female twins carrying the new DMD mutation. 11-3is the twin manifesting muscular dystrophy, and 11-2is the phenotypically normal twin. 1-2 is their normal mother, and 111-1is the phenotypically normal offspring of 11-2, who is also carrier of the same DMD mutation. The DNA was digested withPst1, and the sample was subsequently divided into three equal aliquots. The first aliquot (PstI-digested) was run in lanes 1, 4, 7, and 10 to identify the polymorphic bands. The second aliquot is further digested with MspI and results in the reduction of the size of the original alleles (lanes 2, 5, 8, 11).The third aliquot was digested withHpaI1 (lanes 3,6,9,12). After HpaII digestion, normally each band is split into two bands if random X chromosome inactivation is the case (11-2 and 111-1).(Individual 1-2 is not informative for this analysis.) In individual 11-3, only the 7.4 kb allele was digested with HpaII, while the 8.4 kb allele remained uncut, demonstrating a nonrandom X inactivation pattern. At this locus, the active allele is methylated, therefore demonstrating that the 8.4 kb allele (paternal allele) is the only active allele in this patient. This finding provides an explanation for the DMD phenotype, particularly since it has been shown that the mutation occurred on the paternal X chromosome (Fig. 1).

DISCUSSION In this paper we demonstrate that monozygotic (MZ) twins discordant for muscular dystrophy carry the same mutation involving duplication of exons 42 and 43 of the dystrophin gene. A series of experiments involving dystrophin gene and protein studies firmly establish the affected twin as being a manifesting carrier of DMD. This is a new mutation that most likely occurred in the sperm of their father. Evidence for new mutation is inferred by absence of the DMD phenotype in five sons of 1-2 who were demonstrated by linkage analysis to have inherited alternate maternal alleles at the dystrophin locus. Direct physical evidence for absence of mutation in 1-2was obtained using densitometry. Indeed, we were able to demonstrate that in this pedigree carrier status can be ascertained using densitometry to determine the number of exons 42 and 43 per genome equivalent. RFLP analysis with polymorphic markers spanning the DMD locus enabled us to derive a haplotype for the paternal (1-1)X chromosome. By determining the haplotypes of all of the relatives in the family, it was shown that the exon duplication in the twins and in the DMD affected male (111-2) originated in the X chromosome

from 1-1.Thus, this new mutation in the twins is an exon duplication of paternal origin. Many new mutations in the DMD gene which are of maternal origin are deletion mutations. A recent study of unrelated nondeletion DMD patients demonstrated that partial DMD gene duplication occurred in 14% of nondeletion cases or 6% of all cases [Hu et al., 19901. In that study, 80% of the duplication mutations, four out of five tested, were demonstrated to be of grandpaternal origin and unequal sister chromatid exchange was proposed to be the mechanism for the formation of these duplications [Hu et al., 19901. The MZ twins described in this study have inherited the same duplication mutation involving exons 42 and 43, yet they display very different phenotypes. Individual 11-2 is clinically normal, while 11-3 is clinically a manifesting carrier of DMD. Using a probe that detects RFLPs and DNA methylation patterns a t the DXS255 locus, the expected biased methylation pattern was observed in the affected twin. The apparent preferential inactivation was of the maternally derived X chromosome. Thus, by two methods utilizing two different tis-

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sue types, muscle histochemical staining with dystrophin antibody and RFLP analysis of DNA from white blood cells with methylation-sensitive enzymes, a nonrandom X inactivation pattern is observed in the manifesting carrier twin. Recently, Richards et al. [19901 reported a thorough characterization of a set of MZ twin females discordant for DMD and X inactivation patterns. The twins described in the present report are different from these previously reported twins in a number of ways. First, the previous report showed that both had tremendously skewed X inactivation patterns, with the normal twin having nearly all the normal X active (> 99%),while the affected twin had nearly all the mutant X active (>99%). The twins in this report, on the other hand, showed a skewed X inactivation pattern only in the affected twin. The clinical phenotypes of the two unrelated affected twins correlate quite well with the degree of skewed inactivation; the previously reported twin was phenotypically quite similar to a true DMD male and was wheelchair-bound in the early teens, while the affected twin, in this report was still ambulatory a t age 34. The histological patterns also correlated with the extent of skewed X inactivation pattern: The more severely affected girl showed small islands of dystrophin-positive myofibers in a dystrophin-negative background, while the affected twin in this report showed small islands of dystrophin-negative myofibers (largely replaced by fibro-fatty connective tissue) in a dystrophin-positive background. The most significant and informative difference between these studies is the pattern of X inactivation in each of the unaffected girls. In the previous report, the unaffected twin showed tremendously skewed inactivation in favor of the normal X chromosome (> 99%).Thus, the skewing of inactivation was symmetric between the two twin sisters. This suggested that the twinning process probably split the inner cell mass (ICM) equally and that the twinning happened soon after X inactivation when there were still relatively few cells in the ICM [Nance, 19901. Given the existing data concerning the mechanisms driving the twinning process, Nance suggested that there should be cases where twinning happens a t later time points, where a relatively small proportion of the ICM can be extruded through a break in the zona pellucida. Such small portions ofthe ICM would be expected to have skewed inactivation patterns, as they represent a small sample size from a probable clonal area of the parent ICM. The new ICM would then experience catch-up growth, giving rise to a second embryo with apparently skewed X inactivation. The original ICM, on the other hand, would be expected to have normal, random X inactivation, as it lost only a small portion of its ICM to the newly formed twin and would not have to experience catch-up growth. The twins described in this report represent a n example of such asymmetric, later-stage twinning as hypothesized by Nance [1990]. The normal twin likely arose from the original ICM, while the affected twin may have arisen from a n extruded small part of the ICM which then formed a new embryo through catch-up growth. The fact that the birth weight of the clinically affected twin

(111-3)was 170 g less than the unaffected twin (111-2) may also reflect this catch up growth. In addition, the likelihood that there was a monochorionic-monoamniotic placentation (“one sac”) supports later splitting of the inner cell mass (ICM). All the clinical, genetic, and biochemical analyses presented in this report can be considered consistent with a n asymmetric twinning mechanism in this particular set of twins. Recently, a n additional set of female MZ twins discordant for a muscular dystrophy phenotype has been reported in which molecular analysis and dystrophin staining were performed [Bonilla et al., 19901. The muscular dystrophy phenotype in the manifesting carrier female appears to result from mosaic Ullrich-Turner syndrome, where, in the affected twin, 18%of cells were 45,X, while in the asymptomatic twin, the peripheral blood lymphocyte karyotype was 46,XX. Thus, in the three pairs of MZ twins studied by molecular methods, three different molecular and developmental processes appear to be responsible for expression of the disease phenotype. Duchenne muscular dystrophy has been described in karyotypic normal female carriers [Moser and Emery, 1974; Isaacs and Badenhorst, 19871. A genetic mechanism to explain this was based on non-random X inactivation [Moser and Emery, 19741,and in this paper we demonstrate at the molecular level that a non-random X inactivation pattern is associated with a discordant muscular dystrophy phenotype in one set of MZ twins. The fact that the non-random X inactivation pattern does not occur in individuals 11-2 and 111-1suggests that this process is not inherent to the mutation itself, but that it may be associated with the twinning process or the timing of twinning with respect to X inactivation. This is supported by multiple clinical reports of a muscular dystrophy phenotype in only one female of several sets of MZ twins [Richards et al., 1990; Gomez et al., 1977; Burn et al., 1986; Pena et al., 19871and factor IX deficiency in one female in two pairs of MZ twins [Revesz et al., 1972; Kitchens, 19871.Since a significant proportion of twin conceptions result in singleton births secondary to fetal loss early in gestation, one hypothesis that may explain some females with DMD is that they were initially one of a twin gestation and the twinning process resulted in a non-random distribution of cells containing either a n active or inactive X chromosome. In conclusion, the MZ twins described in this report add new data that should contribute to our understanding of discordant expression of DMD in twins and the twinning process in general.

ACKNOWLEDGMENTS We thank Linda Haway for the preparation of the manuscript and Carolyn Fill and Pam Watson for their technical assistance. We are grateful to the patients and families for their cooperation, to the Muscular Dystrophy clinics of New Orleans and Lafayette, and to the numerous investigators who provided probes. We thank Dr. Jeffrey Chamberlain for providing multiplex PCR reaction mixes and Dr. David Ledbetter for the karyotype analysis. This work was supported in part by separate Muscular Dystrophy Association Task Force on Ge-

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netics grants to J.R.L. and R.G.F. and by grant 1 PO1 HD2 4234 from the National Institutes of Health to H.Y.Z. J.R.L. acknowledges support from the PEW Scholars Program in Biomedical Sciences.

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