Dystrophinopathy in isolated cases of myopathy in females

expedited publication

Dystrophinopathy in isolated cases of myopathy in females

EP Hoffman PhD K Arahata MD2 C Minetti MD E Bonilla MD3 LP Rowland MD3 and Co-Authors

Article abstract-X-linked dystrophinopathy is the most common cause of isolated cases of myopathy in males To investigate dystrophin abnormalities as a cause of myopathy in girls and women we used dystrophin immunocytochemistry to study muscle biopsies from 505 girls and women with neuromuscular disease Forty-six muscle biopsies showed a combination of fibers containing or lacking dystrophin this mosaic immunostaining pattern denoted a carrier status Twenty-one of 46 (456) had a family history of Duchenne muscular dystrophy in males Twenty-five of 46 i5431 were isolated cases with no previous family history of neuromuscular disorder The laboratory findings of the isolated cases were consistent with the familial cases all showed myopathic histopathology and abnormal elevations of serum CK The clinical presentations of the isolated cases varied but were consistent with the familial cases 40 (1025) of isolated cases showed proximal limb weakness before age 10 24 (6425) presented with myalgias or cramps 24 (625) presented with incidental findings of grossly elevated CK levels 8 (225) noted easy fatigue and 4 (125) had slowly progressive proximal limb weakness beginning at age 45 From our data the clinical criteria for consideration of an underlying dystrophinopathy in isolated female cases of myopathy are CK levels greater than 1000 IUA and myopathic histopathology About 10 of the isolated cases of hyperCKemic myopathy (25210) were proven by dystrophin analysis to have a dystrophinopathy as the cause of their disease (manifesting carriers of Duchenne dystrophy) However we feel that this may be an underestimate The correct diagnosis in these patients is imperative for appropriate genetic counseling to the patients and their families

NEUROLOGY 199242967-975

Duchenne muscular dystrophy1 is found in a high incidence in all populations (1 in 3500 live-born males) Affected boys show proximal limb weakness before age 5 followed by progressive and debilitat- ing weakness and wasting that leads to death or respirator dependence before age 30 The disease is inherited as an X-linked recessive trait However many cases are isolated with no family history of neuromuscular disorder The high proportion of isolated cases is attributed to an unusually high mutation rate 10 times more than most other genetic disorders About 30 of all cases have been considered isolated new mutations2 However recent advances in prenatal diagnosis and genetic counseling have decreased the incidence of familial

cases The high mutation rate corresponds to the extremely large size of the Duchenne gene the 25 million base pairs (1 of the X chromosome) pro- vide a large target for random mutational events

The normal protein product of this gene is dystrophin and all cases of Duchenne muscular dystrophy result from marked reduction in the levels of this protein in skeletal Cloning of the dystrophin gene and the availability of antibodies against the dystrophin protein have created molecular diagnostic criteria for Duchenne dystrophy Becker dystrophy and related disorders A deletion of DNA can be identified in 55 of affected r n a l e ~ ~ and dystrophin protein abnormalities are found in nearly 100 by immunoblotting or immunocyto-

Co-Authors C Angelini MI) E Arikawa MI)2 C Baba MIgt5 PE Barkhaus MD SC Bauserman MD7 IJ Butler MDR JD Cook MD9 JG Chutkow MDO G Cordone MD OB Evans MD2 A Fideianska MD C Garcia MD JM Gilchrist MDI5 M Glasberg MD16 K Hamada MD17 T Ishihara AVMDlX N Ishikawa MDy SD Johnsen MI)2o K Kamakura MDzl 0 Kikumoto MDZZ M Kinoshita MDL K Kumagai H Marks MDL6 W hlarks MI)2a J Maytal MDZ hl Moggio MD2s E Moser MD MA Nigro MD W Noll hlD2y I Nonaka MD2 A Prelle MG Iieyes E Ricci MD3z AD Roses MDj R Sakuta MD34 E Satoyosbi IliDZ S Servidei hID3 A Smith MS36 M Steele MD3 SH Subramony

Iv Sunohara MD2 JZ Wang MD HB Wessel MD T Yanagawa MD3y T Munsat MI)) 1 Hausmanowa-Petrusewicz MDI4 and H Sugita MD2

t For all author affiliations see page 974 Please note that the superscripted numbers in the by-line and the co-author listing refer only to the numbers in the affiliations section on page 974 and not to reference numbers in the reference section of the article

Support for this work was provided hg the National Institute of Neurological Disorders and Stroke (NS29525 E Hoffman NS11766 E Bonilla the Ministry of Education Science and Culture and the National Center for Neurology and Psychiatry of the Ministry of Health and Kelfare of Japan (K Arahatai and the Muscular Dystrophy Association (E Hoffman E Bonilla and K Arahata) Received January 8 1992 Accepted for publication in final form January 22 1992

Address correspondence and reprint request to Dr EP Hoffman Department of Molecular Genetics and Biochemistry IJniversity of Pittshurgh School of Medicine Biomedical Science Tower Pittsburgh PA 15261 Dr K Arahata National Institute of Neuroscience 4-1- 1 Ogawahigashi-cho Kodaira Tokyo 187 Japan or Dr E Bonilla Department of Neurology Columbia-Presbyterian Medical Center New York hT 10032

May 1992 NEUROLOGY 42 967

Protein test results do not depend on the patientrsquos age or clinical severity and are therefore useful even before t h e r e are definite symptoms or signs

In familial cases of Duchenne dystrophy the disease is t r a n s m i t t e d by a female ca r r i e r of the mutated gene Carriers are heterozygotes with a normal dystrophin gene on one X chromosome and a mutated gene on the other X chromosome If the son of a car r ie r woman inherits the mutated X ch romosome D u c h e n n e d y s t r o p h y inev i t ab ly appears More than 90 of female carriers are

However some car r ie rs show calf hypertrophy or proximal limb weakness these women have been called ldquomanifestingrdquo or ldquosymp- tomaticrdquo carriers The traditional clinical criteria for identifying a manifesting carrier a re a family history of Duchenne dystrophy proximal l imb weakness high serum CK levels and m changes on both muscle biopsy and EMGll

The high frequency of isolated male Duchenne dystrophy patients implies that there are also isolated female dystrophinopathy patients Until the advent of molecular diagnostics however there was n o test t o iden t i fy s i m p l e x c a s e s of dys - t rophinopathy in females Immunocytochemical visualization of the dystrophin protein in manifesting carriers with an X-linked family history typical- ly shows a mosaic pattern of both dystrophin-posi- t ive (normal) myofibers and dystrophin-negative m y ~ f i b e r s rsquo ~ - - rsquo ~ This mosaic pa t te rn seems specific for carriers of Duchenne muscular dystrophy and this test has been used to identify six simplex manifesting carriers in the past year18-ldquo) DNA analysis can define manifesting carriers in 55 of patients but this analysis is technically problematic because of heterozygosity for gene deletions the accuracy of DNA analysis to identify female carriers generally depends on prior characterization of mutations in affected male relatives

To ascertain the frequency of isolated cases of dystrophinopathy in females we studied muscle biopsies from 505 girls and women with a neuromuscular disorder

Methods Patient population We studied muscle biopsies from 505 women and girls with a neuromuscular disorder These included all biopsies from female patients referred to K Arahata (National Ins t i tu te of Neuroscience 245 patients) E Hoffman (University of Pittsburgh School of Medicine 188 patients) and E Ronilla (Columbia-Presbyterian Medical Center 72 patients) Patients were selected for biopsy by the referring clinicians because of overt neuromuscular symptoms or hyperCKemia We have not analyzed chemical findings other than dystrophin and serum CK in patients with normal dystrophin As described below some patients with a dystrophinopathy presented with fatigue and myalgia These patients were biopsied because of the subsequent findings of elevations of serum CK and not because of fatigue and myalgia Clinical presentations of fatigue and myalgia are not specific symptoms for muscle disease and we do not know the frequency of these

symptoms in the large group of women who had normal dystrophin studies Also we do not know how often asymptomatic girls and women would have been identified as carriers by dystrophin studies

Asymptomatic obligate o r possible carr iers of Duchenne muscular dystrophy within X-linked pedigrees were excluded from this study the accuracy of dystrophin immunofluorescence in detecting nonmanifesting carriers with normal CK levels was not studied and is the focus of future research There was an ascertainment bias in the selection of familial cases of manifesting carriers both patients and clinicians were sensitized to possible neuromuscular symptoms and in some instances familial cases were specifically recruited for this study However this bias has no bearing on the conclusions of this study which focus upon isolated cases

Muscle biopsies obtained for diagnostic purposes were flash frozen in isopentane chilled in liquid nitrogen Biopsies were stored in air-tight containers below -70 OC and sent to the collaborating laboratory on dry ice

Dystrophin analysis Dystrophin immunofluorescence was done on cryosections of all 505 muscle biopsies as previously described by each participating l ab~ra to ry ~ ~ The histochemical diagnosis of carrier status was not checked for inter-observer reliability however all slides were reviewed by ei ther Dr Araha ta Bonilla o r Hoffman The specific anti-dystrophin antibody prepara- tions differed in the three laboratories but all have been shown to give similar results The antibodies used by K Arahata were rabbit polyclonal anti-DMDP I anti- DMDP 11 and mouse monoclonal 4CEiZ1 used on separate serial sections for each biopsy studied E Hoffman used sheep polyclonals directed against a region of the amino- terminus (60 kd)l and the carboxyl-terminus of dystrophin (d10P separately on each biopsy E Ronilla used either the sheep anti-60-kd polyclonal or a rabbit polyclonal raised against Torpedo dy~tr0phinl~ The percentage of dystrophin-negative fibers dystrophin-positive fibers and partially positivelpartially negative fibers was determined for 41 of the 46 biopsies showing multiple dystrophin-negative muscle fibers One-hundred to 500 fibers in single cryosections were scored for each biopsy This analysis is not quantitative the visual classification of fibers as positive negative or partially positive is sub- jective Also positive and negative fibers were often arranged in three-dimensional clonal groups particular- ly in patients older than 15 years (see Discussion) The small biopsy tested may not be representative of the patientrsquos muscle as a whole A few biopsies showed rare dystrophin-negative fibers that were overtly necrotic by parallel histopathology the lack of dystrophin in these fibers was attributed to necrosis and these patients were not considered to have a dystrophinopathy

Dystrophin immunoblotting was done on 136 of the 505 female muscle biopsies and for 21 of 25 isolated dystrophinopathy biopsies (table) as previously describedH The primary antibodies used were the same as for the immunofluorescence except that E Hoffman used the 30-kd central domain a n t i b ~ d y ~

The amount of dystrophin was quantitated densito- metrically from immunoblots for 17 dystrophinopathy patients and visually estimated from blots for an addi- tional 17 patients (table) The experimental methods used for densitometric quantitation of 11 patients is as follows Experimental samples were quantitated relative to two adjacent lanes of a control muscle that was taken from a woman with normal dystrophin but with myoneu- ropathic histopathologic changes oflsquo fiber group atrophy

968 NEUKO1OGY 42 May 1992

Table Clinical and biochemical features of female dystrophinopathy patients with previous family history of Duchenne dystrophy in males (Part A) and isolated cases with no family history of Duchenne dystrophy (Part B)

Pt

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21

22

23 24 25 26

27 28 29 3 0 31 32 32 3 4 35 36 37 38 39 40 41 42 43 44 45 46

Previous diagnosis

Manifesting carrierrsquordquo Manifesting carrierrsquoldquordquo Manifesting carrier Manifcsting carrier Manifesting carrier Manifesting carrierTs Manifesting carrierrsquordquo Manifesting carrierrsquoR Manifesting carrier1deg Manifesting ca r r i eP Manifesting carrierrdquo Manifesting carrierrsquordquo

Manifesting carrier Manifesting carrier Manifesting carrierrsquo Manifr-sting carrierlsquorsquo Manifesting carrierrsquordquo Manifpsting carrierrsquo

Translocation female DMD Muscular dystrophyrdquordquo Muscular dystrophyg 1 IyperCKmnia Translocation frmalt 1)MD AMuscnlar dystrophy Muscular dystrophy 1imbgirdk HypwCKemia Limh-giidleg Iimh-girdlersquog Irsquoolymyositis Limb-girdle IIyperCKemia Myopathy HyperC Kemia Iimli-girdliJ7 HyprrCKemia I fyperC Kemia Limb-girdle Iimh-girdlerdquo6 Limb-girdle HyperCKemia Iimh-gndle HyperCKemia

-

714 479 451 419 325 323 x22 283 271 224 187 182 88 82 31 19 07 SD XI) ND NI)

974

885 745 642 SI)

636 592 527 F24 Y21 513 507 449 421 369 314 100 203 169 168 165 120 67 62 21

Irsquoercenkdgc fibersT 9 eurolalalive +- + Bln t i Gene analysis9 Age clinical sevcrityS

22 39

159 222 175 84

221 84

103 31 14

16

167

174 257 143

363 318 238 296 196 69

130 2 13 149 165 87

101 132 300

264 482 391 358 500 594

69 I 834 866 950 978

1 o

191

234 283 333

130 234 342 334 489 632 667 619 682 670 792 829 806 683

( 100 33

(301 I 0 3 0 50 I 50

(50 ~1001

45 (801 57 NI) Nn 1101

0

19

(51 110rsquo

(101 70 (SO) 42 38

(100 140 3 7 33 150) 31 (100) 16 68 lsquo1001 1100rsquo 64

280 Soninfnimative C20

40 Noninformative 55

270

Noninforniative 630 Noninformative 510

460 430

Suninfmmative 520 460

xoxxxxx 560 40 280 220

Deletion 510 580 550

1)eletion 200 600

h l e t i o n 170

Translocation

Soninfmmatim Soninfoimrtive Translocation x5 Translocation

Soninformative Soninformative

4 7 m Deletion Drletion

1)nplicat ion

55

80 35 50 160

80

115 100 100 100 110 170 140 90 15

130

250 280 110 190 345 610 250 330 60

Mild Mild Irsquoreclinical Irsquorecliniral Severe Moderate Mild Mild Mild Mild Mild Mild Mild Irsquorerlinicill Seviw Mild Mild Modrrnte Mild Severe Mild

Severe

Severe Irsquorerlinical Preclinical Sewrt

Severe Severl Modtmte Preclinical Moderate Moderato Mild Mild Mild Moderac eurolsquoreclinical Modcriite Prerlinical Irsquorerlinical Mild Moderate Mild Mild Mild 1rsquorecliinerl

Range of serum CKX

781 571 1106-4680 3666-3796

495 2000 200 300 152 500 401 430-2818 3440-9060 5140 461 702 1215-4000 932 2630-5520 736

5880

7500-8000 lll60-17700

3315 000-24001)

10580 225n-i46oo 8060-12BBO 3650-9110

3000-6600 3156-7000 12800

1645 1672 802-1720 1195 1831 900-4284 8870

Age - presentation

- cardiac failurt 4 - prox weakncss 3 - prox weakness 8 - prox weaknrss 10 - prox weakness 43 - prox weakness 44 - prox weakness 23 - prox wrakness I prux weakness 25 - prox weakness 35 - prox wveakncw 52 - niyalgia 4 - calf hypertrophy 5 - prox weakness 18 - incidental CK 26 pmx weakness 30 - prox weakness 28 - proon weakness 5 - prox weakness 40 - prox weakness

5 - prox wrakness

6 - prox weaknoss 14 - prox weakness 5 - incidental CK 3 - prox veakness

4 - prox weakness 4 - prox weakness 8 - niyalgia 8 - incidental (K 4 - prox weakness 5 - prox weakness 5 - myalgia fatigue 14 - myocarditis CK 11 - cramps swdling 8 - cramps 12 - incidental CK 14 - myalgia cramps 28 - incidental CK I 1 - incidental CK 19 - incidental CK 5 - prox weakness 45 - prox weakness 20 - fatigue 20 - fatigue 5 - myalgia

+ Previous diagnosis is that which the patient was carrying hefore dyatrophin analysis of the musclr hiopsy Limb-g~rdle rc t o limb-girdle ninsrnlar dystrophy a poorly defined disorder that is often presumed to he inhwited as an autosomal recessive trait Previously reported pa s are indicatrd with the appropriate r i t f i~i nc~

rsquor Shown a i ~ percentages o f muscle fibers t h a t showed no dystrophin immnnostaining by immunofluorescence I- those tha t showed a region of peripheral immunostaining 4 +-I and thosa that showed a cornplrte ring of peripheral imnnimrstaining I+ This analysis is only semi-quantitative due to the suhjrctive and variable nature of the data ND not quantitated

to adjacent normal controls All n t h i ~ vilues are corrected densitnmotric calculations Standard

nut he unambiguously determined noti that only 55 of patients would be expected to bc heterozygous for a deletion niutation

PI The four categories eurolsquoriclinical Severe AVcderate and Mild art general descriptions that take into account thi age of the patient and tlie rlinical findings Severe = clinical progression similar to that of Iluchenne dystrophy in males Moderate = marked weakness in the teens or tw-enties although ambulatory Mild = clinically dctrctable although minor wfekness Preclinical = either the patient i tm young to a s s i p a relative clinical sevr~rity or no overt weakness Is as yet clinically dewctahle

CK levels are given in LX If mure than one CK level was available then the range of CK d u e s is shown otherwise single values are given The normal range varies from lahoratur to labnratory although the upper limit o f riormal is usually less than 200 11711

2 Valurs in parentheses arc visual (stiniates of immunohlot dat Cations for somr of drnsitonidric readings are en in ihe oninfnrmativerdquo indicates that heterozygosity for ii deleti

and focal fibrotic replacement similar to the findings of late-stage manifesting carriers Immunoblots were subjected to two-dimensional reflectance densitometry (BioRad Model 620 Video Densitometer) followed by 2-D

to 1-L) conversion of the digitized data The ratio of peak areas of the experimental biopsy relative to the two adjacent controls was calculated The post-transfer Coomasie blue-stained acrylamide gels corresponding to the quan-


Figure 1 Dystrophin immunofluorescence in a lfi-year- old girl with very high CK levels This baby girl (patient 37) was picked up on grossly elevated CK levels (9155 UII) upon a blood test incidental to hospitalization for fever and vomiting Subsequent CKs were 8448 and 14000 This girl shows no previous family history for any neuromuscular disorder Shown is dystrophin visualization (panel A) and corresponding Nomarski optics (panel B) The figure shows the nearly random distribution of positive and negative fibers characteristic of very young manifesting carriers of Duchenne muscular dystrophy Dystrophin-positive fibers accounted for 489 of fibers negative fibers 314 and partially positive fibers 196 Immunoblot quantitation was 33 f 3 Bar = 500 pm

titated blots were dried between sheets of dialysis membrane and subjected to two-dimensional transmission densitometry of the myosin heavy-chain protein followed by 2-D to l - D conversion of the digital information Again a ratio of experimental to controls for the myosin heavy-chain protein was calculated Finally the relative percentage of dystrophin in each experimental biopsy was adjusted for the muscle protein content of the lane by dividing the relative percentages of dystrophin from the immunoblot by the myosin heavy chain ratio from the post-transfer gel The resulting corrected values for the percentage of normal dystrophin in 11 of the manifesting carriers with the standard deviation for two separate measurements is as follows 703 f 48) (patient 291 682 f 46 (patient 42) 641 f 199 (patient 451

970 NEUROLOGY 42 May 1992

574 f 145 (patient 36) 571 f 163 (patient 17) 455 f 40 (patient 41) 453 f 21 (patient 15) 353 k 73 (patient 41 332 f 349 (patient 371 308 f 02 (patient 391 and 0 f 0 (patient 22) (there was no dystrophin sig- nal in this last patient)

Clinical data Clinical information obtained included the following date of birth previous clinical diagnosis current age family history of Duchenne dystrophy serum CK levels (three if available) and clinical presentation and progression (clinical findings of calf hypertrophy limb weakness myoglobinuria laboratory findings of serum CK EMG dystrophin gene analysis and histopathology) All information was entered into a flat- field database (QampA)

Patients were separated into two groups 21 with a family history of affected males (table part A) and 25 with no older affected relatives (table part B) More detailed clinical and histopathologic summaries on these patients are available upon request from E Hoffman

Genetic analysis Eleven of the 25 isolated cases of manifesting carriers were studied genetically Three were found to have Xautosome translocations (table) and one a 47XXX karyotype Seven others were studied by Southern blot analysis using the dystrophin cDNA7 (table) one had a duplication mutation two a deletion mutation and four were noninformative Of nine X- linked familial cases studied three were heterozygous for a deletion mutation five were noninformative and one had an XOxXXXX karyotype

Results Frequency of mosaic dystrophin immunostaining patterns in female neuromuscular disease patients Multiple non-necrotic dystrophin-negative fibers were seen in 46 of 505 muscle biopsies from female patients tested for dystrophin-negative myofibers (figures 1 to 4 table) The percentage of non-necrotic dystrophin-deficient fibers ranged from 21 t o 974 in the 46 biopsies (table) Twenty-one of the 46 (456) patients had a family history of Duchenne dystrophy in males All had elevated CK levels and myopathic muscle histology Twenty-five (543) were isolated cases (table part B) The 25 isolated cases were clinically and bio- chemically similar to and consistent with the 21 cases with a family history (see below) On the other hand 274 patients were studied who showed clinical features inconsistent with those of the known manifesting carriers with X-linked family history (normal CK neuropathic histopathology or distal limb weakness) None of the 274 showed multiple dystrophin-negative fibers

Cl in ica 1 features of fe m a 1 e d y s t rop h i nopa t h y patients Clinical manifestations varied in patients with dystrophin-negative fibers (table) but all had high CK levels and myopathic histopathology In those patients categorized as moderately or severely clinically affected (table) focal endomysial fibro- sis fiber atrophy (failed regeneration) and grouped degenerationhegeneration occurred Histopatho- logic features of asymptomatic or mildly affected patients were variation in fiber size and increased central nuclei ECGs were done on seven patients and five were considered abnormal Calf hypertrophy was observed in 28 of 36 patients (77)

Figure 2 Dystrophin immunoftuorescence in an isolated case of hyperCKemia and calf hypertrophy in an 8-year-old girl Calf hypertrophy and hyperCKemia was first observed in this girl (patient 30) at 8 years of age She could never run well CK determinations were 5120 IUll 3650 KJll and 9110 IU1 Verbal development was delayed Currently 10 years of age there has been little progression of her weakness Histopathology showed a chronic myopathy fpanel A) The dystrophin immunofluorescence (panel B) shows a mosaic pattern of positive and negative fibers indicating that she is an isolated manifesting carrier of Duchenne muscular dystrophy Totally positive fibers accounted for 333 of fibers totally negative were 524 and partially positive 143 Comparison of the immunofluorescence (panel B) with the parallel H-E histopathology (panel A) shows that the dystrophin-negative regions exhibit more severe pathology than do the positive regions with substantial fiber size variation and connective tissue proliferation There is also more grouping ofpositive and negative fibers than in the younger patient shown in figure 1 This suggests a gradual replacement of smaller clonal negative areas by a n expansion of positive areas Bar = 50 pm

Weakness was asymmetric in seven patients (15) Correlation of distribution of dystrophin-negative

and -positive fibers with patient age clinical seueri- ty and histopathology The distribution and percentage of negative fibers differed from patient to patient but there was a correlation between the distribution of negative and positive fibers and the patientrsquos age at time of biopsy In the youngest patient age 15 years dystrophin-positive and dystrophin-negative fibers showed a nearly random distribution (figure 1) After age 5 years positive and negative fibers were arranged in groups of varying size (figures 2 and 3) Patients with an age of onset later than 15 years and a mild clinical course generally showed more dystrophin-positive fibers and less dystrophin-negative fibers (figure 31 and all of these patients showed a very mild myopathy histopathologically

Quantitative comparisons between localized histopathology and localized dystrophin expression are difficult and beyond the scope of this paper

However dystrophin-negative regions often showed more pronounced histopathology than positive regions (figure 2 panels A and B) The correlation of dystrophin content and histopathology was not obvious in all biopsies studied but those biopsies showing large groups of positive and negative fibers frequently showed corresponding histopathology In severely affected girls who were biopsied older than age 10 the dystrophin-negative fiber groups were atrophic and fibrotic while the dystrophin-positive fiber groups appeared normal or showed minor histologic changes (mild variation in fiber size and some central nuclei)

There was a correlation between the number of dystrophin-negative fibers and clinical severity (table part B) In the isolated cases the four patients with a severe clinical picture showed an average of 77 dystrophin-negative fibers (range 592 to 974) The seven women with a mild phe- notype showed an average of 25 dystrophin-negative fibers (62 to 507))


Figure 3 qystrophin analysis in a 22-year-old woman with very mild proximal weakness This woman (patient 44) presented at 14 years of age with a chief complaint ofgeneral muscular fatigue A t 22 years the fatigue became problematic and she was admitted to the hospital for a neuromuscular evaluation Serum CK lecels of 1834 U I l were found along with mild proximal weakness and wasting of the legs A muscle biopsy (panel A) showed nearly normal histology with a slight inrrcase in internal nuclei (45) Dystrophin immunofluorescence (panel B) showed rare negativc fibers in isolation or in small groups Totally positive fibers accounted for 829 totally negative were 67 and partially positive 104 Bar = 50 pm

Discussion Our goal was to determine the frequency of dystrophinopathy as the cause of myopathy in girls and women with no family history of Duchenne dystrophy in males and to determine the typical clinical manifestations of these women We used dystrophin analysis of muscle biopsies to detect dystrophinopathy The accuracy of dystrophin analysis in detecting female dystrophinopathy is not known However our data suggest that there are few if any ldquofalse positiverdquo results of the 505 female biopsies studied 274 showed clinical features were inconsistent with the familial cases and none of these 274 showed any dystrophin-negative fibers On the other hand ldquofalse negativerdquo results the finding of normal dystrophin in a true dystrophinopathy patient are more likely X-inactivation may lead to some regions of fibers that are all dystrophin-positive and the relatively small region of muscle tested in each patient could result in significant sampling error Thus in non-Duchenne families the calcula- tion of dystrophinopathy frequency by dystrophin analysis must be an underestimate

The determination of frequency of dystrophinopathy in females could be invalidated by

972 NEIIZOIOGY 42 May 1992

ascertainment bias We did find evidence of substantial ascertainment bias in the selection of familial cases there were no correlations between clinical severity dystrophin expression and serum CK levels (table par t A) A family history of Duchenne dystrophy sensitizes the patient her family and her clinician to the presence of neuromuscular symptoms However we excluded familial cases from our calculations of dystrophinopathy frequency in isolated cases of myopathy The clinical and ethnic range of the isolated myopathy cases in this study was quite broad Viewed retrospec- tively only 55 of the 505 girls and women studied by dystrophin satisfied the clinical prerequisites for consideration of a dystrophinopathy derived from the familial cases (see below) Thus the range of patients studied was broad enough to minimize ascertainment bias in isolated cases

Analysis of the familial cases (table part A) showed two invariant features high serum CK activity and myopathic histopathology Two-hundred ten of the 505 females studied were isolated cases with these clinical features Twenty-five of the 210 had dystrophin-negative fibers and were classified as having a dystrophinopathy (table part

DYSTROPHIN

1 2 3 4 5 6 7 8 9 1011 121314151617

MYOSIN

Figure 4 D-ystrophin immunoblot analysis of muscle biopsies from female manifesting carriers of Duchenne dystrophy Shown is dystrophin immunoblot analysis (top panel) of control muscle containing normal dystrophin (odd-numbered lanes) and eight of the patients shown in the table (even-numbered lanes) The lower panel is the correspondingpost- transfer gel stained with Coomassie blue to visualize the myosin heavy-chain protein this serves as a control for the amount of muscle protein in each lane Patients are as follows lane 2 patient 17 lane 4 patient 15 lane 6 patient 28 lane 8 patient 41 lane 10 patient 37 lane 12 patient 29 lane 14 patient 36 lane 16 patient 25

B) Therefore approximately 10 (25210) of isolated female patients with high serum CK (gt1000 IUL) and myopathic histopathology were found to be true carriers of Duchenne dystrophy by dystrophin immunocytochemistry

All isolated female dystrophinopathy patients had had other diagnoses prior to dystrophin testing (table part B) The most common diagnosis was ldquolimb-girdle muscular dystrophyrdquo a disorder of unknown etiology that is undoubtedly heteroge- neous only some are clearly inherited as an auto- soma1 recessive trait Arikawa et allH found that 13 (215) of isolated female limb-girdle patients were Duchenne carriers and 31 of isolated male limb-girdle patients had Becker dystrophy another dystrophinopathy In our series nine patients had previously been diagnosed with limb-girdle dystrophy but actually had dystrophinopathy Together with other studies of limb-girdle dystrophy patients who proved to have denervation or a metabolic myopathy23 the category of limb-girdle dystrophy is being eroded

There was clinical histopathologic and biochemical variability in the 25 isolated cases of dystrophinopathy but there were some unifying features Most apparent was the striking elevations of serum CK activity in all carriers (gt1000 IUl) probably a consequence of the membrane instabili- ty or impaired function induced by dystrophin deficiency in the myofiber membrane24s2rdquo In all 25 histopathologic study showed variation of fiber size and an increase in the number of central nuclei In most subjects older than age 5 years the pathology

was focal and the regions of visible pathology often corresponded to dystrophin-negative regions (figure 2) Many (50) familial cases (table part A) had serum CK levels substantially lower than those in the isolated cases (table part B) probably because ascertainment bias in selecting familial cases identified more mildly affected girls and women

In the isolated carriers there was a correlation between the degree of dystrophin deficiency eleva- tion of serum CK activity and clinical severity (table part B) Patients with the highest CK levels generally showed the highest percentage of dystrophin-negative fibers and they were also the most clinically severe However this was not always true perhaps because CK levels are influ- enced by factors other than dystrophin levels or because the biopsy is not representative of the total muscle mass In either case dystrophin studies cannot be used to offer a prognosis

All of the cells in a human female display lsquoX- inactivationrdquo That is one of the two X-chromosomes is inactivated to leave only a single active X- chromosome In female carriers of the Duchenne gene one of the X-chromosomes encodes normal production of dystrophin in skeletal muscle but the other X-chromosome has lost that pr0perty~~~7 X- inactivation is a random event that occurs early in fetal development2H However muscle fibers are syncytial cells each myofiber contains thousands of nuclei that share a common cytoplasm and each nucleus derives from a different mononuclear myoblast In a carrier half of the nuclei in a fiber should direct production of dystrophin and half


should not depending on which X-chromosome was inactivated in the myoblast contributing the nucleus to the myofiber However dystrophin mRNA dystrophin protein or both diffuse within the cytoplasm in heterozygous mdx mice dystrophin-positive myonuclei can compensate for neighboring dystrophin-negative nuclei by overproducing dys- trophin29--ldquo1

The manifestations of muscle weakness in a Duchenne carrier probably depend on two contrast- ing variables the tendency for the muscle t o become dystrophin-positive (biochemical normal- ization ) and the progressive histopathologic changes of dystrophin-deficient muscle that lead to fiber loss and clinical weakness If in a carrier child a small region of a muscle fiber is dystrophin-negative intracellular diffusion of dystrophin from dystrophin-positive regions might compensate for the deficiency Alternatively dystrophin-negative fibers might degenerate and might then be regenerated by dystrophin-positive myoblasts On the other hand if the dystrophin- negative region of the muscle tissue exceeds some threshold level effective replacement by either intracellular diffusion o r regeneration may be impossible because these mechanisms depend on nearby dystrophin-positive nuclei or myoblasts Large dystrophin-negative regions would then show progressive histopathology and functional deterioration

Although the percentage of dystrophin-negative myofibers seems to be an important marker of clinical severity (table) the distribution of negative fibers may be equally important For example two different 1-year-old carrier girls might each have 25 dystrophin-negative fibers but one might have the negative fibers in a checkerboard pattern and the other might have all the negative fibers in a group or even in one leg The checkerboard carrier might fully compensate and remain asymptomatic while the ldquogrouped carrier might show focal weakness perhaps in only one leg In fact seven of our manifesting carriers had overt asymmetry of weakness This asymmetry was the first symptom of one 14-year-old girl who noticed that her left leg was thinner than the right

Previous diagnostic criteria for a manifesting carrier diagnosis included a family history of Duchenne dystrophy in males11-13 The new data show that there are isolated nonfamilial cases of dystrophinopathy in girls and women clinical criteria that implicate this diagnosis are serum CK levels in excess of 1000 IUA and myopathic histology About 10 of isolated cases of girls or women ful- filling these criteria will be shown to have a dystrophinopathy by dystrophin analysis

Acknowledgments

The authors thank Genica Pharmaceuticals (Worcester MA) for referring three of the patients in this study

974 NHJROLOGY 42 May 1992

Affiliations

1

2

3

4

5

6

7 8

9

Molecular Genetics and Biochemistry Human Genetics and Pediatrics University of Pittsburgh School of Medicine Pittsburgh PA 15261 Nationa1 Institute of Neuroscience 4-1-1 Ogawahigashi-cho Kodaira Tokyo 187 Japan Department of Neurology College of Physicians and Surgeons of Columbia University Columbia-Presbyterian Medical Center New York NY 10032 Clinica Neurologica University of Padova via Giustiniani 5 35128 Padua Italy Japanese Red Cross Nagasaki Atomic Bomb Hospital 3-15 Mori-machi Nagasaki City Nagasaki 852 Japan Neurology Service Veterans Administration Medical Center Minneapolis MN 55417 Neuropathology Scott and White Clinic Temple TX 76508 Neurology University of Texas Health Science Center 6431 Fannin Suite 7044 MSB Houston TX 77030 Department of Neurology Texas Scottish Rite Hospital 2222 Wellborn St Dallas TX

10 Department of Neurology State IJnivwsity of New York at

11 Pediatric Clinic Istituto G Gaslini Largo G Gaslini 5

12 Department of Pediatrics University Medical Center

13 Department of Neurology LSU Mrdical Center 1542

14 Neuromuscular Unit Polish Academy of Sciences ul

15 Neurology Rhode Island Hospital Providence RI 16 Neurology Henry Ford Hospital 2799 W Grand Blvd

Detroit MI 48202 17 Department of Pediatrics Miyazaki-kenritsu Hospital 5-30

Kita-takamatsu cho Miyazaki City Miyazaki 880 Japan 18 National Higashi-Saitama Hospital Saitama 349-01 Japan 19 Department of Pediatrics Kitami Red Cross Hospital

Higashi 2 Kita 6 Kitami-shi Hokkaidou 196 Japan 20 Barrow Neurological Institute St Josephrsquos Hospital and

Medical Center 350 West Thomas Road Phoenix AZ 85013 21 National Defence Medical College 3-2 Namiki Tokorozawa

City Saitama-ken 359 Japan 22 Department of Neuropsychiatry Hiroshima University

School of Medicine 1-2-3 Kasumi Minami-ku Hiroshima

Buffalo 426 Gardier St Buffalo NY 14215

16148 Genoa Italy

Jackson MS 39216

Tulane Ave New Orleans LA 70112

Banacha 02 097 Warsaw Poland

730 Japan

Memro-ku Tokvo 153 JaDan 23 Ohashi Hospital Toho Medical College 2-17-6 Ohashi

24 KaGagawarsquoRehabilitation- Hospital 516 Nanasawa Atsugi

25 Division of Neurology Alfred I DuPont Hospital 1600

26 Child Neurology Associates 709 Leuda Ft Worth TX

27 Schneider Childrenrsquos Hospital Long Island Jewish Medical

28 Is t i tuto di Clinica Neurologica Centro Dino Ferrar i

29 Department of Clinical Pathology Dartmouth-Hitchcock

30 Michigan Inst i tute for Neurological Disorders 28595

31 Division of Pathology Cook County Hospital 67 South Wood

32 Department of Neurology Universita Cattolica del Sacro

33 Department of Neurology Duke University Medical Center

34 Department of Pediatrics Numazu City Hospital 550


36 Division of Genetics Childrenrsquos Hospital Denver 1056 E

37 Childrenrsquos Hospital of Pittsburgh Pittsburgh PA 15213 38 Neurology University Hospital 2500 N State St Jackson

City Kanagawa-ken 243-01 Japan

Rockland Rd Wilmington DE 19899

76104

Center New Hyde Park NY 11402

Universita di Milano Via F Sforza 3520122 Milan Italy

Medical Center 2 Maynard St Hanover NH 03756

Orchard Lake Farmington Hills MI 48334

St Chicago IL

Cuore Rome Italy

NC

Harunoki Toushiji Numazu City Shizuoka Japan

Cuore Rome Italy

19th Ave Denver CO 80218

MI 39216

Wakayama-shi Wakayama 640 ltJapan

St Boston MA 02111

39 Department of Pediatrics Wakayama Medical College 27-7

40 Neurology New England Medical Center 750 Washington

References

1 Duchenne GB Recherches sur la paralysie musculaire pseu- dohypertrophique ou paralysie myosclerosique Arch Gen Med 1868115-25 179-209 305-321 421-443 552-588

2 Moser H Duchenne muscular dystrophy pathogenetic aspects and genetic prevention Hum Genet 19846617-40

3 Hoffman EP Kuiikel LM Dystrophin abnormalities in 1)uchenneiBecker muscular dystrophy Neuron 198921019- 1029

4 Hoffman EP Brown RH Kiinkel LM Dystrophin the protein product of the Duchenne muscular dystrophy locus Cell 198751919-928

5 Arahata K Ishiura S lshiguro T ct a] lmmunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide Nature

6 Bonilla E Samitt CE Miranda AF et al Duchenne muscular dystrophy deficiency of dystrophin at the muscle cell surface Cell 1988 54447-452

7 Koenig M Hoffman Elrsquo 13ertelson CJ Monaco AP Feener C Kunkel LM Complete cloning of the Duchenne muscular dystrophy (DMD cDNA and preliminary genomic organiza- tion of the DMIl gene In normal and affected individuals Cell 198750509-517

8 Hoffman EP Pischbeck KH Brown RH et al Dystrophin characterization in muscle biopsies from Duchenne and Becker muscular dystrophy pa t ien ts N Engl J Med

9 Nicholson L V H Johnson MA Gardner-Medwin U Bhattacharya S Harris JB Heterogeneity of dystrophin expression in patients with Duchenne and Hecker muscular dystrophy Acta Neuropathol 199080239-250

10 Emery AEH The iise of serum creatine kinase for detecting carriers of Duchenne muscular dystrophy In Milhorat AT ed Exploratory concepts in muscular dystrophy and related disorders Amsterdam Excerpta Medica 196790-97

11 Moser H Eniery AEH The manifesting carrier in Duchenne muscular dystrophy Chn Genet 19745271-284

12 Boyd Y Buckle V Holt S Munro E Hunter I) Craig I Muscular dystrophy in girls with Xautosome translocations J Med Genet 198623484 490

13 Harkhaus PE Gilchrist JM Duchenne muscular dystrophy manifesting carriers Arch Neurol 198946673-675

14 Yoshioka M Clinically manifesting carriers in Duchenne muscular dystrophy Cliii Genet 1981206-12

15 Arahata K Ishihara T Kaniakura K et al Mosaic expression of dystrophin in symptomatic carriers of Duchennersquos muscular dystrophy N Engl J Med 1989320138-142

16 Ronilla E Schmidt B Samitt CE et al Normal and dystrophin-deficient muscle fibers in carriers of the gene for Iluchenne muscular dystrophy Am J Pathol 1988133440-445

17 Morandi I Mora M Gussoni E Tedeschi S Cornelio F Dystrophin analysis in Iluchenne and Becker muscular dystrophy carriers correlation with intracellular calcium and albumin Ann Neurol 199028674-679

18 Arikawa E Hoffman EIrsquo Kaido M Nonaka I Sugita H Arahata K The frequency of patients with dystrophin abnormalities in a limb-girdle patient population Neurology 1991411491-1496

19 Minctti C Chang HW Medori R et al Dystrophin deficiency in young girls with sporadic myopathy and normal kary-

1988333861-863

1988318 1363- 1368

otgtpe Neurology 199141 1288-1292 20 Richards CS Watkins SC Hoffman Elrsquo e t a1 Skewed X

inactivation in a female MZ twin rwults in Duchenne muscular dystrophy Am J Hum Genet 199046 672-681

21 Arahata K Reggs AH Honda H c gt t a1 Irsquoreservation of the C- terminus of d j sti ophin molrcul(gt in the skeletal muscle from Becker muscular dystrophy J Neurol Sci 1991101 148-156

22 Koenig RI Kunkel LM Detailed analysis of the repeat domain of dystrophin reveals 4 potential hinge reBons that may confer flexibilitj ltJ Biol Chem 1990265 4560-4566

23 Romland LP Impact of molecular genetics on clinical neurol- ogj In DiDonato S DiMauro S Mamoli A Rowland LP eds Molecular genetics of neurological and ncuromuscular disease New York Raven Press 1988 1-15

24 Rowland LP Biochemistry of niuscle membranes i n Duchenne musculz dystrophy Muscle Nerve 1980J3-20

25 Hoffman EIrsquo Cforospe JR The animal models of Uuchenne muscular dystrophy windows on the pathophysiologcal con- sequences of dystrophin deficiency In Morrow J Mooseker M eds Current topic5 in membrnnes vol 38 New York Academic Press 1991 113-154

Irsquo McKee I Johns DIZ Kunkel LM in clonal rnyoblasts derived from a

Duchennc muscular dystrophy carrier Am J Hum Genet

27 Miranda AErsquo Prancke U Bonilla E e t al Dystrophin immunocytochemistry in muscle culture detection of a carri- e r of Duchennc muscular dystrophy Am J hled Genet

28 Nance WE Invited editorial do twin Lions have larger spots Am J FIum Genet 199046 646-648

29 Watkins SC Hoffman EP S lay ter ITS Kunkel L M Dystrophin distnbution in heteroLygote MDX mice Muscle Nen-e 198912 861-868

30 Karpati G Zubrzycka-Gaarn EE Carpenter S Bulman DE Ray PN Worton RG Age-related conversion of dystrophm- negative to -positive fibers segments of skeletal but not cardiac muscle fibers in heteroyygote mdx mice J Neuropathol Exp Neurol 199049 96-105

31 Cooper BJ Gallagher EA Smith CA Valentine BA Winand NJ Mosaic expression of dystrophin in carners of canine X- linked muscular dystrophy Lab Invest 199062 171-178

32 Kamakura K Kawai M Arahata K Kowumi euro1 Watanabe K Sugita II A manifesting carrier of Duchenne muscular dystrophy with severe myocardial symptoms J Neurol

33 Bonilla E Younger DS Chang HW et a1 Partial dystrophin deficiency in inonozygous twin carriers of the Duchenne gene discordant for clinical inyopathy Keurology 1990401267-1270

34 Chutkow JO H y s w CL Edwards cJA Heffner RR Jr Czyrny ltJltJ Monozygotic fc3malc twin carriers discordant for the clinical mani fwtations of T)uchmne muscular dystrophy Neurology 198737 1147- 115 1

35 Kinoshita M Ikeda K Yorhimura M Saku A Watanabe K Duchenne muscular dystrophy carrier presenting with mosaic X chromosome constitution and inuscular symptoms-with analysis of I3arr bodies in the muscle Itinsho Shinkeigaku 199OX) 343-346

36 Lupski JR Garcia CA Zoghbi l lY Hoffman EP Fenwick RG Discoi dance of muscular dystrophy in monozygotic female twins evidence supporting asymmetric splitting of the inner cell mass in a manifesting carner of Duchenne dystroph Am J Med Genet 199140 354-364

37 Kikumoto 0 Yoshiiiaga eJ Sdsaki T Ideshita H Hihji A Arahata K A manifesting carrier of Duchenne muscular d j strophy presenting mosaic distnbution of dystrophin negative and positive muscle fibers Itinsho Shinkeigaku 199030 107-109

198944 820-826

198932 268-273

1990237 483-485

May 1992 NEUI101OGY 42 975

DOI 101212WNL425967199242967 Neurology

E P Hoffman K Arahata C Minetti et al Dystrophinopathy in isolated cases of myopathy in females

This information is current as of May 1 1992

ServicesUpdated Information amp

httpwwwneurologyorgcontent425967fullhtmlincluding high resolution figures can be found at

Citations

shttpwwwneurologyorgcontent425967fullhtmlotherarticleThis article has been cited by 4 HighWire-hosted articles

Permissions amp Licensing

httpwwwneurologyorgmiscaboutxhtmlpermissionsor in its entirety can be found online atInformation about reproducing this article in parts (figurestables)

Reprints

httpwwwneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Communications Inc All rights reserved Print ISSN 0028-3878 Online ISSN 1526-632Xsince 1951 it is now a weekly with 48 issues per year Copyright copy 1992 by Advanstar

reg is the official journal of the American Academy of Neurology Published continuouslyNeurology

Protein test results do not depend on the patientrsquos age or clinical severity and are therefore useful even before t h e r e are definite symptoms or signs

In familial cases of Duchenne dystrophy the disease is t r a n s m i t t e d by a female ca r r i e r of the mutated gene Carriers are heterozygotes with a normal dystrophin gene on one X chromosome and a mutated gene on the other X chromosome If the son of a car r ie r woman inherits the mutated X ch romosome D u c h e n n e d y s t r o p h y inev i t ab ly appears More than 90 of female carriers are

However some car r ie rs show calf hypertrophy or proximal limb weakness these women have been called ldquomanifestingrdquo or ldquosymp- tomaticrdquo carriers The traditional clinical criteria for identifying a manifesting carrier a re a family history of Duchenne dystrophy proximal l imb weakness high serum CK levels and m changes on both muscle biopsy and EMGll

The high frequency of isolated male Duchenne dystrophy patients implies that there are also isolated female dystrophinopathy patients Until the advent of molecular diagnostics however there was n o test t o iden t i fy s i m p l e x c a s e s of dys - t rophinopathy in females Immunocytochemical visualization of the dystrophin protein in manifesting carriers with an X-linked family history typical- ly shows a mosaic pattern of both dystrophin-posi- t ive (normal) myofibers and dystrophin-negative m y ~ f i b e r s rsquo ~ - - rsquo ~ This mosaic pa t te rn seems specific for carriers of Duchenne muscular dystrophy and this test has been used to identify six simplex manifesting carriers in the past year18-ldquo) DNA analysis can define manifesting carriers in 55 of patients but this analysis is technically problematic because of heterozygosity for gene deletions the accuracy of DNA analysis to identify female carriers generally depends on prior characterization of mutations in affected male relatives

To ascertain the frequency of isolated cases of dystrophinopathy in females we studied muscle biopsies from 505 girls and women with a neuromuscular disorder

Methods Patient population We studied muscle biopsies from 505 women and girls with a neuromuscular disorder These included all biopsies from female patients referred to K Arahata (National Ins t i tu te of Neuroscience 245 patients) E Hoffman (University of Pittsburgh School of Medicine 188 patients) and E Ronilla (Columbia-Presbyterian Medical Center 72 patients) Patients were selected for biopsy by the referring clinicians because of overt neuromuscular symptoms or hyperCKemia We have not analyzed chemical findings other than dystrophin and serum CK in patients with normal dystrophin As described below some patients with a dystrophinopathy presented with fatigue and myalgia These patients were biopsied because of the subsequent findings of elevations of serum CK and not because of fatigue and myalgia Clinical presentations of fatigue and myalgia are not specific symptoms for muscle disease and we do not know the frequency of these

symptoms in the large group of women who had normal dystrophin studies Also we do not know how often asymptomatic girls and women would have been identified as carriers by dystrophin studies

Asymptomatic obligate o r possible carr iers of Duchenne muscular dystrophy within X-linked pedigrees were excluded from this study the accuracy of dystrophin immunofluorescence in detecting nonmanifesting carriers with normal CK levels was not studied and is the focus of future research There was an ascertainment bias in the selection of familial cases of manifesting carriers both patients and clinicians were sensitized to possible neuromuscular symptoms and in some instances familial cases were specifically recruited for this study However this bias has no bearing on the conclusions of this study which focus upon isolated cases

Muscle biopsies obtained for diagnostic purposes were flash frozen in isopentane chilled in liquid nitrogen Biopsies were stored in air-tight containers below -70 OC and sent to the collaborating laboratory on dry ice

Dystrophin analysis Dystrophin immunofluorescence was done on cryosections of all 505 muscle biopsies as previously described by each participating l ab~ra to ry ~ ~ The histochemical diagnosis of carrier status was not checked for inter-observer reliability however all slides were reviewed by ei ther Dr Araha ta Bonilla o r Hoffman The specific anti-dystrophin antibody prepara- tions differed in the three laboratories but all have been shown to give similar results The antibodies used by K Arahata were rabbit polyclonal anti-DMDP I anti- DMDP 11 and mouse monoclonal 4CEiZ1 used on separate serial sections for each biopsy studied E Hoffman used sheep polyclonals directed against a region of the amino- terminus (60 kd)l and the carboxyl-terminus of dystrophin (d10P separately on each biopsy E Ronilla used either the sheep anti-60-kd polyclonal or a rabbit polyclonal raised against Torpedo dy~tr0phinl~ The percentage of dystrophin-negative fibers dystrophin-positive fibers and partially positivelpartially negative fibers was determined for 41 of the 46 biopsies showing multiple dystrophin-negative muscle fibers One-hundred to 500 fibers in single cryosections were scored for each biopsy This analysis is not quantitative the visual classification of fibers as positive negative or partially positive is sub- jective Also positive and negative fibers were often arranged in three-dimensional clonal groups particular- ly in patients older than 15 years (see Discussion) The small biopsy tested may not be representative of the patientrsquos muscle as a whole A few biopsies showed rare dystrophin-negative fibers that were overtly necrotic by parallel histopathology the lack of dystrophin in these fibers was attributed to necrosis and these patients were not considered to have a dystrophinopathy

Dystrophin immunoblotting was done on 136 of the 505 female muscle biopsies and for 21 of 25 isolated dystrophinopathy biopsies (table) as previously describedH The primary antibodies used were the same as for the immunofluorescence except that E Hoffman used the 30-kd central domain a n t i b ~ d y ~

The amount of dystrophin was quantitated densito- metrically from immunoblots for 17 dystrophinopathy patients and visually estimated from blots for an addi- tional 17 patients (table) The experimental methods used for densitometric quantitation of 11 patients is as follows Experimental samples were quantitated relative to two adjacent lanes of a control muscle that was taken from a woman with normal dystrophin but with myoneu- ropathic histopathologic changes oflsquo fiber group atrophy

968 NEUKO1OGY 42 May 1992


Pt

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21

22

23 24 25 26

27 28 29 3 0 31 32 32 3 4 35 36 37 38 39 40 41 42 43 44 45 46

Previous diagnosis




-

714 479 451 419 325 323 x22 283 271 224 187 182 88 82 31 19 07 SD XI) ND NI)

974

885 745 642 SI)

636 592 527 F24 Y21 513 507 449 421 369 314 100 203 169 168 165 120 67 62 21


22 39

159 222 175 84

221 84

103 31 14

16

167

174 257 143

363 318 238 296 196 69

130 2 13 149 165 87

101 132 300

264 482 391 358 500 594

69 I 834 866 950 978

1 o

191

234 283 333

130 234 342 334 489 632 667 619 682 670 792 829 806 683

( 100 33

(301 I 0 3 0 50 I 50

(50 ~1001

45 (801 57 NI) Nn 1101

0

19

(51 110rsquo

(101 70 (SO) 42 38

(100 140 3 7 33 150) 31 (100) 16 68 lsquo1001 1100rsquo 64



270


460 430


xoxxxxx 560 40 280 220


1)eletion 200 600

h l e t i o n 170

Translocation




1)nplicat ion

55

80 35 50 160

80

115 100 100 100 110 170 140 90 15

130

250 280 110 190 345 610 250 330 60


Severe



Range of serum CKX

781 571 1106-4680 3666-3796

495 2000 200 300 152 500 401 430-2818 3440-9060 5140 461 702 1215-4000 932 2630-5520 736

5880

7500-8000 lll60-17700

3315 000-24001)

10580 225n-i46oo 8060-12BBO 3650-9110

3000-6600 3156-7000 12800

1645 1672 802-1720 1195 1831 900-4284 8870

Age - presentation


5 - prox wrakness



































DYSTROPHIN

1 2 3 4 5 6 7 8 9 1011 121314151617

MYOSIN













Acknowledgments



Affiliations

1

2

3

4

5

6

7 8

9















16148 Genoa Italy

Jackson MS 39216



730 Japan


















76104





St Chicago IL

Cuore Rome Italy

NC


Cuore Rome Italy


MI 39216


St Boston MA 02111



References




















1988333861-863

1988318 1363- 1368





















198944 820-826

198932 268-273

1990237 483-485

May 1992 NEUI101OGY 42 975






Citations




Reprints





Pt

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21

22

23 24 25 26

27 28 29 3 0 31 32 32 3 4 35 36 37 38 39 40 41 42 43 44 45 46

Previous diagnosis




-

714 479 451 419 325 323 x22 283 271 224 187 182 88 82 31 19 07 SD XI) ND NI)

974

885 745 642 SI)

636 592 527 F24 Y21 513 507 449 421 369 314 100 203 169 168 165 120 67 62 21


22 39

159 222 175 84

221 84

103 31 14

16

167

174 257 143

363 318 238 296 196 69

130 2 13 149 165 87

101 132 300

264 482 391 358 500 594

69 I 834 866 950 978

1 o

191

234 283 333

130 234 342 334 489 632 667 619 682 670 792 829 806 683

( 100 33

(301 I 0 3 0 50 I 50

(50 ~1001

45 (801 57 NI) Nn 1101

0

19

(51 110rsquo

(101 70 (SO) 42 38

(100 140 3 7 33 150) 31 (100) 16 68 lsquo1001 1100rsquo 64



270


460 430


xoxxxxx 560 40 280 220


1)eletion 200 600

h l e t i o n 170

Translocation




1)nplicat ion

55

80 35 50 160

80

115 100 100 100 110 170 140 90 15

130

250 280 110 190 345 610 250 330 60


Severe



Range of serum CKX

781 571 1106-4680 3666-3796

495 2000 200 300 152 500 401 430-2818 3440-9060 5140 461 702 1215-4000 932 2630-5520 736

5880

7500-8000 lll60-17700

3315 000-24001)

10580 225n-i46oo 8060-12BBO 3650-9110

3000-6600 3156-7000 12800

1645 1672 802-1720 1195 1831 900-4284 8870

Age - presentation


5 - prox wrakness



































DYSTROPHIN

1 2 3 4 5 6 7 8 9 1011 121314151617

MYOSIN













Acknowledgments



Affiliations

1

2

3

4

5

6

7 8

9















16148 Genoa Italy

Jackson MS 39216



730 Japan


















76104





St Chicago IL

Cuore Rome Italy

NC


Cuore Rome Italy


MI 39216


St Boston MA 02111



References




















1988333861-863

1988318 1363- 1368





















198944 820-826

198932 268-273

1990237 483-485

May 1992 NEUI101OGY 42 975






Citations




Reprints


























DYSTROPHIN

1 2 3 4 5 6 7 8 9 1011 121314151617

MYOSIN













Acknowledgments



Affiliations

1

2

3

4

5

6

7 8

9















16148 Genoa Italy

Jackson MS 39216



730 Japan


















76104





St Chicago IL

Cuore Rome Italy

NC


Cuore Rome Italy


MI 39216


St Boston MA 02111



References




















1988333861-863

1988318 1363- 1368





















198944 820-826

198932 268-273

1990237 483-485

May 1992 NEUI101OGY 42 975






Citations




Reprints

















DYSTROPHIN

1 2 3 4 5 6 7 8 9 1011 121314151617

MYOSIN













Acknowledgments



Affiliations

1

2

3

4

5

6

7 8

9















16148 Genoa Italy

Jackson MS 39216



730 Japan


















76104





St Chicago IL

Cuore Rome Italy

NC


Cuore Rome Italy


MI 39216


St Boston MA 02111



References




















1988333861-863

1988318 1363- 1368





















198944 820-826

198932 268-273

1990237 483-485

May 1992 NEUI101OGY 42 975






Citations




Reprints










DYSTROPHIN

1 2 3 4 5 6 7 8 9 1011 121314151617

MYOSIN













Acknowledgments



Affiliations

1

2

3

4

5

6

7 8

9















16148 Genoa Italy

Jackson MS 39216



730 Japan


















76104





St Chicago IL

Cuore Rome Italy

NC


Cuore Rome Italy


MI 39216


St Boston MA 02111



References




















1988333861-863

1988318 1363- 1368





















198944 820-826

198932 268-273

1990237 483-485

May 1992 NEUI101OGY 42 975






Citations




Reprints




DYSTROPHIN

1 2 3 4 5 6 7 8 9 1011 121314151617

MYOSIN













Acknowledgments



Affiliations

1

2

3

4

5

6

7 8

9















16148 Genoa Italy

Jackson MS 39216



730 Japan


















76104





St Chicago IL

Cuore Rome Italy

NC


Cuore Rome Italy


MI 39216


St Boston MA 02111



References




















1988333861-863

1988318 1363- 1368





















198944 820-826

198932 268-273

1990237 483-485

May 1992 NEUI101OGY 42 975






Citations




Reprints








Acknowledgments



Affiliations

1

2

3

4

5

6

7 8

9















16148 Genoa Italy

Jackson MS 39216



730 Japan


















76104





St Chicago IL

Cuore Rome Italy

NC


Cuore Rome Italy


MI 39216


St Boston MA 02111



References




















1988333861-863

1988318 1363- 1368





















198944 820-826

198932 268-273

1990237 483-485

May 1992 NEUI101OGY 42 975






Citations




Reprints




MI 39216


St Boston MA 02111



References




















1988333861-863

1988318 1363- 1368





















198944 820-826

198932 268-273

1990237 483-485

May 1992 NEUI101OGY 42 975






Citations




Reprints









Citations




Reprints




Documents

Dystrophinopathy in isolated cases of myopathy in females