Proc. Natl. Acad. Sci. USAVol. 89, pp. 623-627, January 1992Genetics

Possible influences on the expression of X chromosome-linkeddystrophin abnormalities by heterozygosity for autosomalrecessive Fukuyama congenital muscular dystrophy

(Duchenne muscular dystrophy/Becker muscular dystrophy/gene interaction/unlinked noncomplementation)

ALAN H. BEGGS*, PAUL E. NEUMANNt, KIICHI ARAHATAt, ERI ARIKAWAt, IKUYA NONAKAt,MARYDILYS S. ANDERSON*, AND Louis M. KUNKEL*§*Howard Hughes Medical Institute and Genetics Division, Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, MA 02115;tDepartment of Neurology, Children's Hospital and Harvard Medical School, Boston, MA 02115; and tNational Institute of Neuroscience, NationalCenter of Neurology and Psychiatry, Kodaira, Tokyo 187, Japan

Contributed by Louis M. Kunkel, October 23, 1991

ABSTRACT Abnormalities of dystrophin, a cytoskeletalprotein of muscle and nerve, are generally considered specificfor Duchenne and Becker muscular dystrophy. However,several patients have recently been identified with dystrophindeficiency who, before dystrophin testing, were considered tohave Fukuyama congenital muscular dystrophy (FCMD) on thebasis of clinical findings. Epidemiologic data suggest that only1/3500 males with autosomal recessive FCMD should haveabnormal dystrophin. To explain the observation of 3/23FCMD males with abnormal dystrophin, we propose thatdystrophin and the FCMD gene product interact and that theearlier onset and greater severity of these patients' phenotype(relative to Duchenne muscular dystrophy) are due to theirbeing heterozygous for the FCMD mutation in addition to beinghenizygous for Duchenne muscular dystrophy, a genotype thatis predicted to occur in 1/175,000 Japanese males. This modelmay help explain the genetic basis for some of the clinical andpathological variability seen among patients with FCMD, andit has potential implications for understanding the inheritanceofother autosomal recessive disorders in general. For example,sex ratios for rare autosomal recessive disorders caused bymutations in proteins that interact with X chromosome-linkedgene products may display predictable deviation from 1:1.

Interactions between different genetic loci have long beenappreciated and studied in experimental animal models.Modification (e.g., suppression or enhancement) (1, 2) andunlinked noncomplementation (3-7) are examples of geneinteraction that have been defined in yeast, Drosophila, andmice, to name a few. In humans, on the other hand, mostgenetic diseases appear to follow simple Mendelian inheri-tance patterns [e.g., Duchenne muscular dystrophy (DMD),Becker muscular dystrophy (BMD), cystic fibrosis, etc.] orare clearly multifactorial in nature (e.g., cancer, cardiovas-cular disease). Owing to the rarity of most human geneticdiseases, as well as the obvious inability to perform classicalgenetic breeding experiments, interactions between differenthuman disease genes are difficult to study. Here we presentevidence for gene interactions between mutant dystrophinalleles, which generally cause DMD or BMD, and the as yetunidentified mutant allele for Fukuyama congenital musculardystrophy (FCMD).DMD is one of the most common human neuromuscular

genetic diseases of young males with an incidence of 1/3500(8, 9). Both DMD and the milder allelic variant BMD arecaused by mutations in the X chromosome-linked gene fordystrophin, a membrane-associated cytoskeletal protein that

is generally undetectable in boys with DMD and is altered inpatients with BMD (reviewed in ref. 10). Dystrophin isexpressed in skeletal, cardiac, and smooth muscle as well asin Purkinje cells in the cerebellar cortex and cerebral corticalpyramidal neurons (11, 12). Based on sequence homology toa-actinin and the spectrins, dystrophin has a putative actin-binding domain at the N terminus (13) and a C-terminaldomain that may also interact with other components of themembrane cytoskeleton (14).Dystrophin abnormalities are considered specific for

DMD/BMD; however, in the process of screening patientswith various neuromuscular diseases, a few unusual cases ofdystrophin abnormalities were found (15-17). One patientwith a clinical diagnosis of FCMD was previously shown tohave no detectable dystrophin in his skeletal muscle biopsy(16). Since then, two more FCMD patients with dystrophinabnormalities have been found (ref. 17; this work), suggestingthat these coincident findings are not merely the result ofchance.FCMD is a rare autosomal recessive disorder endemic to

Japan with an incidence of 6.9-11.9 per 100,000 births(reviewed in ref. 18). Both FCMD and DMD result inmuscular weakness with dystrophic muscle pathology; how-ever, the phenotypes of these diseases are quite distinct. Theformer presents with hypotonia and weakness at birth orearly infancy and patients often never walk well (18). Incontrast, DMD usually first manifests between the ages of 4and 5 years and patients usually walk until they are 11-13years old (8). Furthermore, FCMD is characterized by pro-found mental retardation and polymicrogyria ofthe cerebrumand cerebellum. In contrast, while up to one-third of DMDpatients suffer from some cognitive deficits (8), no consistentcentral nervous system (CNS) pathology has been detected(19) despite the presumed absence of dystrophin in thepostsynaptic membranes of cerebral and cerebellar corticalneurons (12).

In this report, we present a hypothesis to explain why somepatients with the clinical phenotype of FCMD have under-lying dystrophin abnormalities. We propose that these pa-tients are FCMD heterozygotes who also have DMD andthat, in the absence of dystrophin, reduced levels or activityof the FCMD gene product are detrimental, resulting in aphenotype more severe than expected for this genotype.

MATERIALS AND METHODSPatient Selection and Analysis. FCMD patients in this study

were referred to the National Institutes of Neuroscience,

Abbreviations: FCMD, Fukuyama congenital muscular dystrophy;DMD, Duchenne muscular dystrophy; BMD, Becker muscular dys-trophy; CNS, central nervous system; SMA, spinal muscular atro-why.To whom reprint requests should be addressed.

623

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Proc. Natl. Acad. Sci. USA 89 (1992)

Japan, where clinical diagnoses were made by standardcriteria (18, 20). Significant features include either sporadicoccurrences or a family history compatible with autosomalrecessive inheritance; CNS manifestations, including mod-erate to severe mental retardation and occasionally seizures(=50% of patients); and muscle hypotonia and weaknesssince early infancy with delayed motor milestones and dys-trophic muscle pathology, including fiber necrosis and re-generation with dense fibrosis. Facial muscles are frequentlyinvolved and joint contractures appear in -70% of cases. Of>3500 biopsies available, samples from 37 patients with anoriginal clinical diagnosis of FCMD (23 male, 14 female) wereassayed for dystrophin by indirect immunofluorescenceand/or Western blotting as described (15-17). DNA analysiswas performed by polymerase chain reaction (21) and/orSouthern blotting and hybridization with dystrophin cDNAprobes (22, 23).Case Histories of Dystrophin-Negative FCMD Patients. Pa-

tient 52 is a 5-year-old male product of a normal pregnancywho presented with hypotonia at birth and growth anddevelopmental delay in the postnatal period. There was nofamily history of neuromuscular disease. He first walked at2.8 years of age, at which time his developmental quotientwas 65. Currently, he is mildly hypotonic with facial muscleinvolvement consistent with FCMD, has hip contractures,and cannot walk or sit alone. His serum creatine kinase levelwas 8387 units/liter (normal = 12-75), an electromyogramwas consistent with myopathy, and muscle biopsy showeddystrophic changes. A computerized tomography scan re-vealed diffuse corticomedullary atrophy and ventricular di-latation. Polymicrogyria, agyria, and pachygyria were notidentified on a magnetic resonance imaging scan, and anelectroencephalogram demonstrated background slowing.

Patient 109 is a 6-year-old male with no family history ofneuromuscular disease who was noted at birth to be hypo-tonic. Good head control was delayed until 9 months of age,standing was at 18 months, and walking was at 20 months. Helost the ability to walk by 5 years of age due to muscularweakness and hip and ankle contractures. Currently, he ishypotonic, unable to stand, has facial muscle involvement,and is unable to speak in sentences. His serum creatinekinase was 9650 units/liter, an electromyogram was consis-tent with myopathy, and muscle biopsy showed features ofmuscular dystrophy. A computerized tomography scan re-vealed diffuse cortical atrophy, and an electroencephalogramrevealed background slowing but no epileptiform activity.

Patient 137 is a 15-year-old male who was hypotonic atbirth with subsequent developmental delay. Walking wasdelayed until 3 years of age and he required a wheelchair bythe time he was 7 years old. His first words were at 3.5 yearsand he used sentences at 4.5 years of age. Currently, he hasgeneralized hypotonia, muscular atrophy and weakness withslight facial muscle involvement and multiple joint contrac-tures. Serum creatine kinase levels were 799 and 1061 units/liter, an electromyogram was consistent with myopathy, anda biopsy (at 13 years of age) showed severe dystrophicchanges. A computerized tomography scan revealed corti-comedullary atrophy and ventricular dilatation. Magneticresonance imaging showed no evidence of polymicrogyria,agyria, or pachygyria, and an electroencephalogram revealeda slow background. An elder brother with similar signs andsymptoms died at 14.3 years of age. Postmortem examinationshowed no polymicrogyria, agyria, or pachygyria. Althoughthese brothers fit the clinical criteria for FCMD, their patho-logical diagnosis is atypical FCMD or some other congenitalmuscular dystrophy with CNS manifestations.

Incidence Calculations. Frequencies of alleles, genotypes,phenotypes, and sex ratios were calculated assuming Hardy-Weinberg equilibrium, an incidence of dystrophin abnormal-ities of 1/3500 males (9), and a FCMD incidence of 1/10,000

(18). These calculations disregard the observation of a highrate of consanguineous marriage among the parents of pa-tients with FCMD (18), which would cause us to slightlyoverestimate the allele frequency and carrier rate in thegeneral population. Assuming interaction between an auto-somal and an X-linked locus, the frequency (incidence) of adisease phenotype (fD) that is presumed to have an autosomalrecessive mode of inheritance is equal to the sum of thefrequency of homozygosity at the autosomal locus and thefrequency of heterozygosity at the autosomal locus andhemizygosity for the X-linked disease-associated allele.

Therefore,

fD =fa + 2fa(1 -fa)(fx/2),

wherefa is the frequency of the autosomal disease-associatedallele and fx is the frequency of the X-linked disease-associated allele. For example, given that the incidence ofFCMD (fD) is 1 x 10-' and the incidence of DMD in males(fx) is 2.86 x 10-4, the allele frequency for the FCMDmutation (fa) is estimated to be 9.86 x 10-3. The proportionof male FCMD patients who are affected because they areFCMD heterozygotes and DMD hemizygotes is

2fa(l -fa(fx/2)/fD -fa/2) ,

or =5.4%.

RESULTSDystrophin Abnormalities in Patients with FCMD. In the

course of screening patients with various neuromusculardiseases for dystrophin abnormalities, it was discovered thata single patient (patient 52), of five with clinical diagnoses ofFCMD, had no detectable dystrophin by either indirectimmunofluorescence or Western blotting of his muscle bi-opsy (16). Subsequently, biopsies from 31 more patients wereexamined and another (patient 109) was found to have nodetectable dystrophin (17). We have since examined onemore patient (patient 137) who also had no detectable dys-trophin on Western blots despite having clearly detectablemyosin levels on the posttransfer gels (Fig. 1). Indirectimmunofluorescence for dystrophin in patient 137 was re-markable for low levels of patchy, staining with antibodiesagainst the central portion of the protein and no detectablestaining when antibodies against the C terminus were used(data not shown). Due to the extensive fibrosis that is ahallmark of FCMD (18), biopsies from many patients withFCMD appear to have reduced levels of dystrophin, but afternormalizing for muscle-specific myosin, most FCMD pa-tients clearly have significant levels on Western blots (e.g.,patients 53 and 54 in Fig. 1). Furthermore, immunocytochem-ical analysis of these dystrophin-positive biopsies demon-strated that both dystrophin and spectrin were present in an

N 53; .4 \N \ IBO ( N I \If\lu . - ,t. w

IM~~~~~~~~~~S.-W

FIG. 1. Western blot analysis of dystrophin in muscle biopsiesfrom selected patients with FCMD. Numbers refer to patient num-bers from text; N, normal controls. (Upper) Western blot stainedwith anti-30-kDa antibodies (11). The band corresponding to dystro-phin is indicated (Dys.). (Lower) Portion of the posttransfer poly-acrylamide gel illustrating relative levels of muscle-specific myosin.

624 Genetics: Beggs et al.

Proc. Natl. Acad. Sci. USA 89 (1992) 625

occasionally patchy and fluffy pattern with many positive andsome negative fibers, suggesting nonspecific alterations dueto general membrane defects (17). All the dystrophin-deficient patients are male (e.g., 3/23), while none of the 14females studied had any detectable dystrophin abnormalities.To examine whether the lack of dystrophin in these pa-

tients was a primary or secondary effect of their disease, westudied their dystrophin genes directly. Patient 52 has anintragenic deletion ofexons 51-54 (Fig. 2), which is predictedto cause a frameshift of protein translation resulting in nodetectable dystrophin (23). No deletion was detected bypolymerase chain reaction analysis of DNA from patient 137(data not shown); however, this is not surprising in light ofthefact that only 65% ofDMD patients have detectable deletions(22, 24). DNA from patient 109 was unavailable for study.

DISCUSSIONThe absence of dystrophin in some patients with clinicaldiagnoses of FCMD was intriguing because dystrophin defi-ciency is considered specific for DMD and BMD (reviewedin ref. 10). The simplest explanation for these findings is thatthe three dystrophin-deficient patients have both DMD andFCMD. This might be expected to occur with a frequency of1 in 3.5 x 107 males in the Japanese population or 1 in 3500males with FCMD. However, the observed frequency ofdystrophin abnormalities in our sample (3/23 males withFCMD) appears to be too high to accept this proposal. Theprobability of obtaining 3 or more dystrophin abnormalities in23 FCMD patients by chance alone is ~1 in 2.1 x 107.A second possible explanation involves the difficulty of

establishing an accurate diagnosis without a biochemical orgenetic marker for FCMD. Maternal, environmental, ge-netic, or chance factors could cause variable expressivity ofdystrophin abnormalities such that our three patients repre-sent extremes in the DMD/BMD spectrum. Although diffi-cult to address, we believe this hypothesis unlikely given thehigh observed incidence of unusual presentations in oursample (3/37 FCMD patients) and the lack of similar findingsamong non-Japanese patients with congenital muscle disor-ders (ref. 15; E. Hoffman and L.M.K., unpublished data).

1 2 3 4

-52 5jft

-50

-51 =

-5055

BALA4

RE -51

FIG. 2. Dystrophin gene deletion analysis in patient 52. Southern

blots of genomic DNA from a control (lanes 1 and 3) and patient 52

(lanes 2 and 4) digested with Hind111 and probed with dystrophincDNA probes 7-8 (lanes 1 and 2) and 9-10 (lanes 3 and 4). Relevant

exon numbers are indicated on the right.

Since the clinical presentations of patients 52, 109, and 137bear a striking resemblance to FCMD, a disorder that isrelatively common among the Japanese, yet have the bio-chemical features ofDMD, we suggest a third explanation-namely, that they may be heterozygous for FCMD andhemizygous for dystrophin abnormalities. This genotypecould conceivably result in a phenotype that has an earlieronset and is more severe than that usually associated withdystrophin deficiency such that it resembles FCMD. Givenan incidence of 10-4 for FCMD, the carrier frequency ispredicted to be 2 x 10-2 (or -1.3 x 10-2 when consanguinityis accounted for) (18). Thus, as many as 1/50 Japanese maleswith DMD/BMD will also be carriers for FCMD. If weassume that FCMD heterozygotes with DMD/BMD have thephenotype of FCMD, then -5.4% of male patients withFCMD will have dystrophin abnormalities (see Materials andMethods and Fig. 3A). Given our observation that 3/23FCMD males have abnormal dystrophin, the relative odds infavor of this frequency (i.e., 5.4% or 1/18.5) over the nullhypothesis (1/3500) are -2.2 x 106:1. If the high rate ofconsanguinity [up to 27% (18)] is taken into account, theestimated carrier rate is lower, thus increasing the estimate ofthe proportion of male FCMD patients with dystrophinabnormalities.The three patients with dystrophin abnormality all carried

an original clinical diagnosis of FCMD, yet two have somefeature that is atypical for FCMD. Patient 52 has an atrophicbrain, as assessed by computerized tomography scan; how-ever, magnetic resonance imaging examination did not revealevidence of corticogyral defects. Patient 137 had a similarlyaffected brother whose brain did not have pachygyria orpolymicrogyria on autopsy. Although the muscular dystro-phy was congenital in these cases, the CNS involvement isapparently milder than typical FCMD, yet it is clearly moresevere than found in DMD. Furthermore, a previous report(25) described a Japanese family in which one brother hadclassical FCMD and another had DMD with unusually severeCNS manifestations, including severe mental retardation,slight cortical atrophy on computed tomography scan, andrare sporadic polyspike wave complexes in both frontal areason electroencephalogram. These observations are consistentwith the idea that dystrophin deficiency, combined withheterozygosity for FCMD, might yield an intermediate phe-notype. Thus, the presence of these dystrophin-deficientFCMD patients (estimated to be -1/175,000 Japanese males)may explain some of the clinical and pathological variabilityamong patients with presumptive diagnoses of FCMD (18).Our explanation for the high frequency of dystrophin

abnormalities in patients with FCMD is somewhat analogousto the phenomenon of unlinked noncomplementation that hasbeen demonstrated in Drosophila melanogaster (5, 6), Cae-norhabditis elegans (3), Saccharomyces cerevisiae (4), and,most recently, Mus musculus (7). In both Drosophila andyeast, one mechanism for noncomplementation involvesinteracting proteins as proven by the demonstration thatrecessive mutations in a-tubulin fail to complement recessive,B-tubulin mutations in double heterozygotes (4, 5). In otherwords, the combination of heterozygosity at both loci givesa phenotype similar to that caused by homozygosity at eitherlocus. Johnson (26) has also proposed a similar mechanism inhumans, termed metabolic interference, to explain unusualpatterns of inheritance. In this model, heterozygosity at oneor more loci may be more harmful than homozygosity be-cause of deleterious interactions between normal and mutantproteins. One well characterized example of gene interactionin human disease is the effect that a-globin gene mutationshave on patients with 3-globin mutations. ,B-Thalassemiahomozygotes who are heterozygous for a-thalassemia have amore balanced ratio of globin chain synthesis and a milderphenotype (27), while a-globin gene triplications exacerbate

Genetics: Beggs et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

-10 -8 -6 -4Log recessive disease frequency

0,000CD4 a

O ,

OCd

0,-0

A4 >

-2 -10 -8 -6 -4 -2Log recessive disease frequency

FIG. 3. Relationship between disease frequency of an apparent autosomal recessive disorder and the proportion of male patients who areaffected because they are heterozygous for the recessive disorder and hemizygous for an X-linked defect (assuming a model of intergenicinteraction as described in the text). (A) The frequency ofX-linked abnormalities in males affected with an apparent autosomal recessive disorderis inversely related to the frequency of the recessive disorder. Shown is the relationship assuming the frequency of the X-linked disorder to be1/3500 male births as in DMD. (B) Varying the frequency of the X-linked disorder demonstrates that the proportion of males affected becauseof gene interaction is directly related to the incidence of the X-linked disorder. The four curves represent different X-linked disease frequenciesin males chosen to be comparable to representative diseases; o, 1/60,000 (factor IX deficiency); A, 1/7500 (factor VIII deficiency); *, 1/3500(DMD); o, 1/1500 (fragile X).

the otherwise benign phenotype of 8-thalassemia heterozy-gotes (28).By analogy to the unlinked noncomplementation seen for

a- and f3-tubulin alleles (4, 5), we suggest that dystrophin(also a cytoskeletal protein) and the normal FCMD geneproduct may physically interact in the myofibrillar and/orneuronal cytoskeleton. Dystrophin is localized at the innersurface of the plasma membrane and is associated with an

integral membrane glycoprotein complex (14). In light of ourhypothesis, it is interesting that freeze-fracture electronmicroscopy has shown that orthogonal arrays in the plasmamembranes are smaller and contain fewer subunit particles inboth DMD and FCMD (29, 30). Thus, both dystrophin andthe FCMD gene product apparently play a role in maintainingthese subcellular structures.One interesting implication ofour model is that phenotypes

produced by other X-linked recessive genes may be modifiedby heterozygosity for mutations in any autosomally encodedproteins that interact with them, and, in fact, this may be onemechanism contributing to the clinical heterogeneity oftenattributed to differences in genetic background. The fre-

1.0*

A

X 0.9-

4 0.8-

0 0.7

-10 -8 -6 -4Log recessive disease frequency

quency of this phenomenon is related to the frequencies ofthe interacting genes. The proportion of males who areaffected because they are hemizygous for an X-linked defectand heterozygous for an autosomal mutation is inverselyproportional to the frequency of the autosomal disease anddirectly proportional to the frequency ofthe X-linked disease(Fig. 3B).Another prediction is that these interactions will skew the

sex ratio for disorders that are considered autosomal reces-sive traits. For example, for a relatively common disorder,such as FCMD in Japan, with an incidence of 1/10,000, wewould predict that approximately 51.4% of the FCMD pa-tients would be male (Fig. 4A), and, in fact, this proportionwould be higher if the high rate of consanguinity were takeninto account (18). The reported male/female ratio of 1.1:1(18) is therefore consistent with our hypothesis. For rarerdisorders, we would expect a larger proportion ofthe patientsto show X-linked defects and, therefore, more unbalancedsex ratios. For instance, if an autosomal recessive disorderwith a frequency of 1/106 interacts with DMD, we wouldexpect 62% of these patients to be male. As the frequency of

1.0

0.9 rCe

0

0

0

0.6-

0.5-

-2 -10 -8 -6 -4 -2

Log recessive disease frequency

FIG. 4. Effect of disease frequency on the sex ratio of the affected population assuming a model of intergenic interaction as discussed inthe text. (A) The degree of male preponderance is inversely related to the incidence of the recessive disorder. The X-linked disease frequencyis 1/3500 as in DMD. (B) The degree of male preponderance is directly related to the incidence ofthe X-linked disorder. The four curves representdifferent X-linked disease frequencies as described in Fig. 3B.

1.0

-, 0.8

0.6E

c:4r0.4

0.0

626 Genetics: Beggs et al.

Proc. Natl. Acad. Sci. USA 89 (1992) 627

the X-linked disorder increases, the proportion of affectedmales also goes up (Fig. 4B). This model also implies that ifmore than one autosomal locus is associated with the auto-somal recessive phenotype, a larger proportion of patientswould be expected to be male, and more ofthese males wouldhave dystrophin abnormalities.There have been suggestions of a male predominance in

some forms of autosomal recessive spinal muscular atrophy(SMA) (31, 32), a disease with an incidence of4-6 x 1lo- (9).Further support for the idea that there may be some inter-action between dystrophin abnormalities and the SMA geneproduct is our previous observation of a smaller dystrophinprotein in a male patient with an ambiguous clinical pictureintermediate between SMA type III and BMD (patient 4 inref. 15). This patient had an older brother who carried thediagnosis ofclassic SMA type III, suggesting that one brotherwas homozygous for SMA while the other may have been aheterozygote with BMD.

Finally, our hypothesis that dystrophin and other disease-related gene products (e.g., FCMD) may interact can betested by mating dystrophin-deficient mice with variousautosomal recessive mouse neuromuscular mutants. Thisapproach has already been successfully used to identifyinteracting muscle genes in Drosophila (33). In this way, wehope to identify mice with mutations in proteins that interactwith dystrophin by the increased severity of the phenotypeassociated with a hemizygous dystrophin mutation. Hope-fully, the eventual identification of the FCMD gene defectwill allow direct confirmation of our hypothesis.

We thank Dr. Eric Hoffman for Western blot analysis of dystro-phin; Drs. Hideo Sugita and Barbara Pober for helpful advice anddiscussions; Drs. Nishimura, Nomura, and Sakuta for providingclinical material and information; and Drs. Hart Lidov, VictorMcKusick, and David Botstein for critical reading of this manuscript.This work was supported by grants from the National Institutes ofHealth (NS 23740 and HD 18658) and the Muscular DystrophyAssociation of America. L.M.K. is an Associate Investigator of theHoward Hughes Medical Institute.

1. Rutledge, B. J., Mortin, M. A., Schwarz, E., Thierry-Mieg, D.& Meselson, M. (1988) Genetics 119, 391-397.

2. Moore, K. J., Seperack, P. K., Strobel, M. C., Swing, D. A.,Copeland, N. G. & Jenkins, N. A. (1988) Proc. Natl. Acad.Sci. USA 85, 8131-8135.

3. Kusch, M. & Edgar, R. S. (1986) Genetics 113, 621-639.4. Stearns, T. & Botstein, D. (1988) Genetics 119, 249-260.5. Hays, T. S., Deuring, R., Robertson, B., Prout, M. & Fuller,

M. T. (1989) Mol. Cell. Biol. 9, 875-884.6. Regan, C. L. & Fuller, M. T. (1988) Genes Dev. 2, 82-92.7. Sweet, H. O., Cook, S. & Davison, M. T. (1990) Mouse

Genome 86, 239.8. Emery, A. E. H. (1988) Duchenne Muscular Dystrophy (Ox-

ford Univ. Press, New York), pp. 1-317.9. Emery, A. E. H. (1991) Neuromuscular Disord. 1, 19-29.

10. Hoffman, E. P. & Kunkel, L. M. (1989) Neuron 2, 1019-1029.

11. Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987)Cell 51, 919-928.

12. Lidov, H. G. W., Byers, T. J., Watkins, S. C. & Kunkel,L. M. (1990) Nature (London) 348, 725-728.

13. Hammonds, R. G., Jr. (1987) Cell 51, 1.14. Ervasti, J. M. & Campbell, K. P. (1991) Cell 66, 1121-1131.15. Hoffman, E. P., Fischbeck, K. H., Brown, R. H., Johnson,

M., Medori, R., Loike, J. D., Harris, J. B., Waterston, R.,Brooke, M., Specht, L., Kupsky, W., Chamberlain, J., Caskey,C. T., Shapiro, F. & Kunkel, L. M. (1988) N. Engl. J. Med.318, 1363-1368.

16. Arahata, K., Hoffman, E. P., Kunkel, L. M., Ishiura, S.,Tsukahara, T., Ishihara, T., Sunohara, N., Nonaka, I., Ozawa,E. & Sugita, H. (1989) Proc. Nail. Acad. Sci. USA 86,7154-7158.

17. Arikawa, E., Ishihara, T., Nonaka, I., Sugita, H. & Arahata, K.(1991) J. Neurol. Sci. 105, 79-87.

18. Fukuyama, Y., Osawa, M. & Suzuki, H. (1981) Brain Dev. 3,1-29.

19. Dubowitz, V. & Crome, L. (1969) Brain 92, 805-808.20. Nonaka, I. & Chou, S. M. (1979) in Handbook of Clinical

Neurology, Vol. 41, Part II, eds. Vinken, P. J. & Bruyn, G. W.(North-Holland, Amsterdam), pp. 27-50.

21. Beggs, A. H., Koenig, M., Boyce, F. M. & Kunkel, L. M.(1990) Hum. Genet. 86, 45-48.

22. Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P.,Feener, C. & Kunkel, L. M. (1987) Cell 50, 509-517.

23. Koenig, M., Beggs, A. H., Moyer, M., Scherpf, S., Heindrich,K., Bettecken, T., Meng, G., Muller, C. R., Lindlof, M.,Kaariainen, H., de la Chapelle, A., Kiuru, A., Savontaus,M.-L., Gilgenkrantz, H., Recan, D., Chelly, J., Kaplan, J.-C.,Covone, A. E., Archidiacono, N., Romeo, G., Liechti-Gallati,S., Schneider, V., Braga, S., Moser, H., Darras, B. T., Mur-phy, P., Francke, U., Chen, J. D., Morgan, G., Denton, M.,Greenberg, C. R., van Ommem, G. J. B. & Kunkel, L. M.(1989) Am. J. Hum. Genet. 45, 498-506.

24. den Dunnen, J. T., Grootscholten, P. M., Bakker, E.,Blonden, L. A., Ginjaar, H. B., Wapenaar, M. C., van Paas-sen, H. M., van Broeckhoven, C., Pearson, P. L. & vanOmmen, G. J. (1989) Am. J. Hum. Genet. 45, 835-847.

25. Itagaki, Y., Sakamoto, Y. & Nishitani, Y. (1980) Clin. Neurol.20, 897-903.

26. Johnson, W. G. (1980) Am. J. Hum. Genet. 32, 374-386.27. Wainscoat, J. S., Kanavakis, E., Wood, W. G., Letsky, E. A.,

Huehns, E. R., Marsh, G. W., Higgs, D. R., Clegg, J. B. &Weatherall, D. J. (1983) Br. J. Haematol. 53, 411-416.

28. Kulozik, A. E., Thein, S. L., Wainscoat, J. S., Gale, R., Kay,L. A., Wood, J. K. & Weatherall, D. J. (1987) Br. J. Haematol.66, 109-112.

29. Wakayama, Y., Okayasu, H., Shibuya, S. & Kumagai, T.(1984) Neurology 34, 1313-1317.

30. Wakayama, Y., Kumagai, T. & Jimi, T. (1986) Acta Neuro-pathol. 72, 130-133.

31. Kugelberg, E. & Welander, L. (1956) Arch. Neurol. Psychiatry75, 500-509.

32. Namba, T., Aberfeld, D. C. & Grob, D. (1970) J. Neurol. Sci.11, 401-423.

33. Homyk, T., Jr., & Emerson, C. P., Jr. (1988) Genetics 119,105-121.

Genetics: Beggs et al.