DOI 101212WNL55811282000551128-1134 Neurology

E Pegoraro M Fanin CP Trevisan et al congenital muscular dystrophy

2 deficientα2 isoform in severe laminin αA novel laminin

This information is current as of October 24 2000

httpwwwneurologyorgcontent5581128fullhtmllocated on the World Wide Web at

The online version of this article along with updated information and services is

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

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

data from a small community living in the Reunion IslandBrain 1996119295ndash308

16 Eymard B Romero NB Leturcq F et al Primary adhalinopa-thy (a-sarcoglycanopathy) clinical pathologic and geneticcorrelation in 20 patients with autosomal recessive musculardystrophy Neurology 1997481227ndash1234

17 Linssen WHJP Notermans NC Van der Graaf Y et alMiyoshi-type distal muscular dystrophy Clinical spectrum in24 Dutch patients Brain 19971201989ndash1996

18 Keunen RWM Lambregts PCLA Op de Coul AAW JoostenEMG Respiratory failure as initial symptom of acid maltasedeficiency J Neurol Neurosurg Psychiatry 198447549ndash552

19 Lightman NI Shovley RT Adult-onset acid maltase defi-ciency Case report of an adult with severe respiratory diffi-culty Chest 197772250ndash252

20 Newson Davis J Goldman M Loh L Casson M Diaphragmfunction and alveolar hypoventilation Q J Med 197617787ndash100

21 Rosenow EC Engel AG Acid maltase deficiency in adultspresenting as respiratory failure Am J Med 197864485ndash491

22 Sivak ED Salanga VD Wilbourn AJ Mitsumoto H Golish JAdult-onset acid maltase deficiency presenting as diaphrag-matic paralysis Ann Neurol 19819613ndash615

23 Raben N Nichols RC Boerkoel C Plotz P Genetic defects inpatients with glycogenosis type II (acid maltase deficiency)Muscle Nerve 1995suppl 3S70ndashS74

24 Hirschhorn R Huie ML Frequency of mutations for glycogenstorage disease type II in different populations the D525Tand Dexon 18 mutations are not generally ldquocommonrdquo in whitepopulations J Med Genet 19993685ndash86

A novel laminin a2 isoform in severelaminin a2 deficient congenital

muscular dystrophyE Pegoraro MD PhD M Fanin MS CP Trevisan MD C Angelini MD and EP Hoffman PhD

Article abstractmdashObjectives Laminin a2 deficiency presents at birth with muscle weakness hypotonia and usuallyasymptomatic white matter signal on MRI Few patients with laminin a2 deficiency have been described with seizuresand structural brain abnormalities The reason for the variation in the severity of the clinical phenotype in congenitalmuscular dystrophy (CMD) with laminin a2 deficiency is not known Methods A patient with CMD with partial laminina2 presenting with brain structural abnormalities and untreatable generalized and partial complex seizure was studiedAlternative laminin a2 splicing was studied by single-strand conformational polymorphismsequencing analysis ResultsA novel laminin a2 isoform was identified Nonsense laminin a2 mutations (stop codons) were inherited from bothparents however one of the nonsense mutations was in a region of exon 31 which is alternatively spliced The alterna-tively spliced isoform excluded one of the stop codon mutations and was thus able to produce normal laminin a2corresponding to this isoform Laminin a2 immunofluorescence showed that this isoform was not evenly distributed at themuscle fiber basal lamina but preferentially localized in discrete areas Laminin a5 b1 g1 and nidogen showeddecreased expression by immunofluorescence Conclusions The severity of this patientrsquos phenotype may be due tooverexpression of the exon 31ndashspliced laminin a2 isoform Exon 31 lies in the IIIA domain of the laminin a2 protein justproximal to the triple coil-coiled region It is possible that chain assembly is impaired by this isoform resulting in a loss ofpossible rescue mechanisms

NEUROLOGY 2000551128ndash1134

Congenital muscular dystrophies (CMD) are geneti-cally and clinically heterogeneous Total laminin a2deficiency due to mutations of the laminin a2 geneaccounts for about 40 to 50 of cases of the classicform of CMD1-3

Laminins are extracellular matrix glycoproteinsresulting from the assembly of one heavy chain (a)and two light chains (b and g) Laminin-2 results

from the assembly of a2 with b1 and g1 and it is themajor laminin complex in muscle where it is local-ized at the basal lamina4-6

The clinical phenotype in complete laminin a2ndashdeficient CMD is relatively homogeneous7 althoughepilepsy89 and brain migration abnormalities1011

have been reported in a subset of patientsPartial laminin a2 deficiency underlies a broad

From the Departments of Neurological and Psychiatric Sciences (Drs Pegoraro Trevisan and Angelini and M Fanin) University of Padova Italy and theCenter for Genetic Medicine (Dr Hoffman) Childrenrsquos Research Hospital Washington DCSupported by grants from the NIH National Institute of Neurological Disorders and Stroke (R0I 29525) University of Padova (1462) and Italian Telethon(C41) (CA) (688) (CPT) The C4 hybridoma was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacol-ogy and Molecular Sciences Johns Hopkins University School of Medicine Baltimore MD and the Department of Biological Sciences University of IowaIowa City under contract N01-HD-6-2915 from the NICHDEPH is an Established Investigator of the American Heart AssociationReceived April 12 2000 Accepted in final form June 23 2000Address correspondence and reprint requests to Dr Elena Pegoraro Department of Neurological and Psychiatric Sciences University of Padova viaGiustiniani 5 35128 Padova Italy e-mail elenapux1unipdit

1128 Copyright copy 2000 by AAN Enterprises Inc

spectrum of primary and secondary defects Second-ary partial laminin a2 deficiency has been reportedin Fukuyama-type CMD12 in musclendasheyendashbrain dis-ease13 and in WalkerndashWarburg disease1415 Both pri-mary and secondary partial laminin a2 deficiencyoccur in classic CMD Recently in few patients diag-nosed with CMD whose muscle biopsy showed partiallaminin a2 deficiency the disease was not linked tochromosome 6q where the laminin a2 gene (LAMA2)maps1617 However most partial laminin a2ndashdefi-cient CMD are either linked to the LAMA2 locus18-20

or show primary laminin a2 mutations21-25

The clinical phenotype in primary partial laminina2 deficiency CMD varies from severe21 to mild22-25

but abnormal white matter signal on brain MRI hasbeen reported in all patients

Here we report a patient with laminin a2 defi-ciency but foci of laminin a2 staining in muscleThese foci corresponded to a laminin a2 isoform gen-erated by alternative splicing in all human muscletested We show data consistent with the hypothesisthat sole expression of this isoform may result in adestabilization of laminin chain assembly and thusin a loss of possible rescue mechanisms leading toan unusually severe phenotype

Patient and methods Patient The clinical data fromthis the patient were partially previously reported26

Briefly this 14-year-old girl with CMD was born at termafter an uneventful pregnancy She presented at birth withsevere hypotonia and joint contractures A diagnosis ofCMD was made based on elevated creatine kinase levelselectromyographic results indicative of myopathy and amuscle biopsy done at 10 days of age consistent with adystrophic process Motor milestones were severely de-layed and the maximal motor skill achieved was the abil-ity to walk a few steps with bilateral support at age 6years At age 7 years the patient began to have absenceseizures with myoclonic jerks of the upper limbs and eye-lids At age 9 years the seizures became the partial com-plex type Brain MRI study revealed tetraventriculardilatation severe and widespread white matter alter-

ations more evident in the frontal lobes pachygyria of theoccipital cortex and some small areas of cerebellar polymi-crogyria (figure 1) IQ study showed moderate mental re-tardation On examination the patient was wheelchairbound She presented severe hypotonia and hypotrophy inthe four limbs Scoliosis and multiple contractures werealso evident The patientrsquos second muscle biopsy done atage 3 years was recently included in a large screening forlaminin a2 abnormalities in CMD Based on immunofluo-rescence results a diagnosis of partial laminin a2 defi-ciency was made

Immunofluorescence and immunoblotting analysisFour-micron-thick cryostat muscle biopsy sections were in-cubated for 2 hours with mouse monoclonal antibody di-rected against both fragments in which laminin a2 isprocessed the 80-kD carboxyl-terminus (15000) (Chemi-con Temecula CA) and the 320-kD fragment (150) (clone2D9)27 The antibody directed against the 320-kD frag-ment of laminin a2 recognizes 229 amino acids in the G2to G3 region of the laminin a2 G-domain27 Anti-laminina5 (13500) (Gibco-BRL Gaithersburg MD) laminin b1(11500) (Gibco-BRL) laminin g1 (1100) (Gibco-BRL)laminin b2 (1500) (clone C4 from the Developmental Stud-ies Hybridoma Bank Johns Hopkins University Balti-more MD) and embryonic myosin heavy chain (11000)28

mouse monoclonal antibodies and anti-dystrophin (6-10)(12000)29 and anti-nidogen (11000) (donated by R Nis-cht Department of Dermatology University of CologneGermany) rabbit polyclonal antibodies were also used

Laminin a5 is recognized by an anti-human lamininclone III A-chain antibody (Gibco-BRL) previously thoughtto recognize laminin a1 Because laminin a1 is not ex-pressed in muscle at any age we considered all stainingobserved with this antibody to represent laminin a530

Anti-mouse and anti-rabbit Cy-3 conjugated secondaryantibody were used to visualize the immune complexesTetramethylrhodamine a-bungarotoxin (11000) (Molecu-lar Probes Eugene OR) was used to visualize neuromus-cular junctions Laminin a2 immunoblot was performedas previously described using antibody directed againstboth the 80-kD (150000) and 320-kD (1300) laminin a2fragments31

Figure 1 Axial and coronal MRI in thepatient (A) Axial T2-weighted imagedemonstrating severe white matter hy-perintensity more evident in the frontallobes and ventricular dilatation In theoccipital cortex diffuse pachygyria alsoevident in the T1-weighted coronal sec-tion (B) is seen

October (2 of 2) 2000 NEUROLOGY 55 1129

DNA analysis Blood was collected in ethylenediami-netetraacetic acid (EDTA) tubes from the patient and herparents DNA was isolated from peripheral blood as previ-ously described32

Laminin a2 mutations and alternative splicing studyRNA extraction complementary DNA (cDNA) synthesisand reverse transcription PCR (RT-PCR) were performedas previously described31 Single-strand conformationalpolymorphism analysis of the entire 10 kb of the laminina2 gene was done as previously described31

Confirmation of nucleotide changes Nucleotide changesand an autosomal recessive pattern of inheritance wereconfirmed in the genomic DNA of the patient and herparents The paternal laminin a2 mutation created anNlaIII restriction-enzyme site The appropriate PCR prod-uct obtained with primers specific for laminin a2 exon4933 was digested with NlaIII The maternal laminin a2mutation did not change a restriction-enzyme site so anamplification-refractory mutation system test was de-signed using genomic primers34

Quantitation of the exon 31ndashspliced isoform and full-length laminin a2 RNA by quantitative multiplex reversetranscription PCR RNA extraction and cDNA synthesiswere done as previously described31 Biopsies studied werefrom the patient six patients with partial laminin a2 defi-ciency one patient with complete laminin a2 deficiencyand one patient with undetermined myopathy (laminin a2and dystrophin immunofluorescence were normal)

Primer sets for coamplification of cDNA correspondingto a-sarcoglycan full-length laminin a2 and exon 31ndashspliced laminin a2 were designed as follows ADH3F59fluorescein-CTG CTC AAC GTC ACC TCT G-39 ADH3R59-CCG GCA CTG ACT TAT CCA C-39 for a-sarcoglycanMer 4476F 59-CCA GTA ACA ATT TCA GCC CCT CTTG-39 Mer 4784R 59fluorescein-TGC ACT CAT CTC CACAAA AAA-39 for full-length laminin a2 Mer 4476F andMer 4784Rspliced 59fluorescein-TGC ACT CAT CTC CACAAA ATT-39 for exon 31ndashspliced laminin a2 The laminina2 reverse primers and the a-sarcoglycan forward primerwere synthesized with fluorescein-linked 59 ends (Gibco-BRL) Quantitative multiplex RT-PCR35 was done using125 ng of each primer 15 mL of cDNA (corresponding toabout 20 ng of total RNA) 25 mM dNTPs 1 3 PCR reac-tion buffer and 25 U Ampli Taq DNA polymerase (PerkinndashElmer Foster City CA) Cycling conditions weredenaturation at 94 degC for 3 minutes 94 degC for 1 minute55 degC for 1 minute and 72 degC for 1 minute for 22 cyclesand extension at 72 degC for 10 minutes Quantitation offluorescent PCR products of exon 31ndashspliced laminin a2and full-length laminin a2 relative to a-sarcoglycan wasdone as previously described36 Briefly fluorescent RT-PCR products were denatured and loaded on an ABI 373DNA automated sequencer using a ROX-fluorescein matrixstandard (PE Biosystems Foster City CA) ROX-labeledmarkers (PE Biosystems) were used as internal size stan-dards Lanes were manually assigned from completed im-age files and peak heights and peak areas were analyzedusing GeneScan software (PE Biosystems) Peak areas cor-responding to the laminin a2 full-length and exon 31ndashspliced isoforms and the a-sarcoglycan internal controlwere determined and the quantitative results transferredto Excel software Two separate RT-PCR measurementswere done for each sample The ratios between full-length

laminin a2 versus a-sarcoglycan and exon 31ndashspliced lami-nin a2 versus a-sarcoglycan were calculated separately forthe patient and the control subjects as average with SEThe average ratios were compared and expressed as per-centage of control Statistical significance was calculatedusing analysis of variance (ANOVA) using all calculatedratios

Results Immunofluorescence and immunoblotting analy-ses of the muscle biopsy A panel of different laminin anti-bodies was used to characterize expression of differentlaminin subunits in the patientrsquos muscle biopsy done at age3 In parallel immunostaining was done with a biopsy show-ing no histopathology (normal control) a laminin a2ndashnega-tive patient with CMD and a laminin a2ndashpositive patientwith CMD All three chains of the muscle laminin-2 andlaminin-4 heterotrimeric complexes (laminin a2 b1 g1b2) were studied Laminin a2 immunofluorescence usingantibodies directed against both the 80-kD and 320-kDfragments of laminin a2 showed a barely detectable im-munoreactivity in some of the patientrsquos muscle fibers (fig-ure 2H) However some scattered myofibers showedpatchy discrete areas of highly positive immunostaining(see figure 2H) Laminin a2 was normal in the control (seefigure 2E) and the laminin a2ndashpositive CMD muscle biopsy(see figure 2G) and completely deficient in the laminina2ndashnegative CMD muscle biopsy (see figure 2F)

Laminin a5 was overexpressed in the laminin a2ndashnegative CMD muscle biopsy (see figure 2B) relative to thenormal control and laminin a2ndashpositive patient with CMDConversely laminin a5 did not appear to be overexpressedin the patient (see figure 2D)

Laminin b1 (see figure 2 I through N) and g1 (seefigure 2 O through R) showed immunostaining similar tocontrol tissue and laminin a2ndashnegative and ndashpositive CMDmuscle biopsies In the patientrsquos muscle biopsy laminin b1showed a markedly decreased immunostaining aroundmyofibers whereas capillaries and small vessels werestrongly stained (see figure 2N) Laminin g1 appearedslightly reduced relative to all controls (see figure 2R)Interestingly no positive laminin a2 immunostaining wasdetected in a large nerve twig included in the patientrsquosmuscle biopsy where both laminin b1 and laminin g1 werestrongly expressed (data not shown)

In order to determine if the laminin a2ndashpositive immu-nostaining in the patientrsquos muscle biopsy was localized atthe neuromuscular junctions laminin b2 and a-bungaro-toxin immunostaining were performed No neuromuscularjunctions were seen with either marker Also we did notobserve any colocalization of double-staining experimentswith laminin a2ndashlaminin b2 or laminin a2ndasha-bungarotoxin(data not shown) These data suggest that neuromuscularjunctions were not present in the biopsy and that thepositive laminin a2 regions did not correspond to neuro-muscular junctions

Double immunostaining with laminin a2ndashdystrophinand laminin a2ndashnidogen was done in order to determinethe localization of the laminin a2ndashpositive areas in thepatientrsquos muscle biopsy Laminin a2 was consistently local-ized outside the plasma membrane (externally to dystro-phin) and staining was colocalized with nidogen Todetermine if the laminin a2ndashpositive regions were fiber-type specific skeletal muscle cryosections were incubatedwith ATPase (pH 43) and then immunostained with lami-

1130 NEUROLOGY 55 October (2 of 2) 2000

Figure 2 Immunofluorescence study of laminin subunit expression in the patient muscle biopsy relative to normal con-trol laminin a2ndashnegative and laminin a2ndashpositive congenital muscular dystrophy (CMD) muscle biopsies (A E I andO) Normal muscle biopsy (B F L and P) Patient with laminin a2ndashnegative CMD (C G M and Q) Patient with laminina2ndashpositive CMD (D H N and R) Muscle biopsy from our reported patient (E through H) Laminin a2 immunostainingusing an antibody directed against the 80-kD carboxyl-terminus of the protein shows discrete areas of highly positive im-munostaining in few myofibers in the patientrsquos muscle biopsy whereas the majority of myofibers are largely laminin a2negative (H) Laminin a2 was normal in the control (E) and laminin a2ndashpositive CMD muscle biopsy (G) and completelynegative in the laminin a2ndashdeficient CMD muscle biopsy Study of the expression of a5 b1 and g1 chains showed thatour patientrsquos muscle does not overexpress laminin a5 (D) as in the patient with laminin a2ndashnegative CMD (B) Our pa-tient also shows a marked decrease in b1 and to a lesser degree g1 immunostaining (N and R) relative to control andlaminin a2ndashnegative and ndashpositive CMD muscle biopsies

Figure 3 Identification of two non-sense mutations in the laminin a2complementary DNA in the patient(Top) Schematic of the exon structureof the laminin a2 gene Exons are rep-resented as boxes introns as linesThe sequencer traces corresponding totwo aberrant conformers show two nu-cleotide changes C4694T in exon 31and C7196T in exon 49 Both result ina stop codon (R1549X and R2383X)(Bottom) The maternal laminin a2gene with a stop codon in exon 31 andthe paternal laminin a2 gene with astop codon in exon 49 are representedShown in the insert is immunostain-ing of the patientrsquos muscle biopsy withlaminin a2 Both paternal and mater-

nal laminin a2 genes are transcribed or translated in a truncated unstable protein as proven by the laminin a2ndashnegativemyofibers in the patientrsquos muscle biopsy The laminin a2ndashpositive immunostaining in the patientrsquos muscle biopsy is due toa novel alternatively spliced exon 31 laminin a2 isoform that skips the nonsense mutation in exon 31

October (2 of 2) 2000 NEUROLOGY 55 1131

nin a2 Laminin a2ndashpositive areas were present in associ-ation with both fiber types I and II (data not shown) Tocorrelate the regeneration status of myofibers with expres-sion of laminin a2 serial cryosections of the patientrsquos mus-cle biopsy done at age 3 were stained with monoclonalantibodies against embryonic myosin heavy chain and the80-kD carboxyl-terminus region of the laminin a2 geneThe frequency of regenerating myofibers in the patientrsquosmuscle biopsy was 2 (532719) of total fibers countedRegenerating fibers were laminin a2 negative Laminin a2immunoblotting using antibodies directed against both the80-kD and 320-kD fragments of laminin a2 failed to showany detectable laminin in the patientrsquos muscle biopsy (datanot shown)

Study of laminin a2 mutations Single-strand confor-mational polymorphism analysis was used to screen forlaminin a2 mutations using muscle RNA from the pa-tientrsquos biopsy The coding sequence of the laminin a2 genewas analyzed using 20 sets of overlapping primer pairsAberrant conformers were identified with primer setsMer4476FndashMer4981R and Mer7015FndashMer7541R Directsequencing of these conformers revealed two nonsense mu-tations C to T at nucleotide position 4694 (R1549X) and Cto T at nucleotide position 7196 (R2383X) of the laminin a2coding sequence (figure 3)

Both mutations were confirmed at the genomic DNAlevel and the inheritance pattern of the mutations deter-mined The C4649T nucleotide change did not cause anyrestriction-enzyme site change thus the amplification-refractory mutation system test was used Primers weredesigned in exon 31 of the laminin a2 gene and mutatedPCR products were present in the patient and her motherNucleotide change C7196T resulted in a gain of an NlaIIIrestriction-enzyme site Primers were designed in exon 49

of the laminin a2 gene and the PCR product was digestedwith NlaIII Digestion fragments were present in the pa-tient and her father

Characterization of an exon 31ndashspliced laminin a2 iso-form The appearance of laminin a2 protein in the patientrsquosmuscle despite the two nonsense mutations suggested thatsome transcripts were excluding one of the two mutationsConsistent with this hypothesis we detected a smallerRT-PCR product over the region of the C4694T mutationin both the patient and control subjects This smallerRT-PCR product was present in the patient and in 10 of 10normal control subjects run in parallel Direct sequencingof the smaller band showed a 138-bp in-frame deletionfrom nucleotides 4629 to 4766 of the laminin a2 codingsequence This deletion corresponded to about 70 of theexon 31 sequence of the laminin a2 gene (figure 4)

Quantitation of the exon31ndashspliced laminin isoformRNA was isolated from the patientrsquos muscle biopsy andfrom eight control muscle biopsies (six with partial laminina2 deficiency one with complete laminin a2 deficiencyand one undetermined myopathy) For each RNA sample100 ng of total RNA underwent RT into cDNA using oligodT10 primers A fluorescent multiplex RT-PCR ampli-fication containing three different PCR products (full-length laminin a2 exon31ndashspliced laminin a2 isoform anda-sarcoglycan) was done in duplicate for each samplePeak heights and areas were calculated and results tabu-lated as ratios between full-length laminin a2 versusa-sarcoglycan and exon 31ndashspliced laminin a2 versusa-sarcoglycan Mean and SE for each ratio were calculatedfor the patient and the control group (figure 5)

The patientrsquos muscle biopsy showed a significant de-crease in full-length laminin a2 RNA relative to the con-trol subjects 24 6 7 versus 100 6 27 ( p 5 0003

Figure 4 Identification of a novelexon 31ndashspliced laminin a2 isoformFull-length laminin a2 and exon 31ndashspliced laminin a2 are shown Thefull-length laminin a2 is the majortranscript in muscle and results fromthe expected splicing between exon 31and 32 Reverse transcription PCRamplification using a reverse comple-mentary DNA primer that spans exon31 and exon 32 (4786R) and a for-ward primer starting at nucleotide4476 shows an reverse transcriptionPCR product of the appropriate size(upper arrow 4476F to 4786R) Theexon 31ndashspliced laminin a2 isoformderives from the use of a cryptic splicesite 57-bp into exon 31 (4629 bp ofRNA) This results in a 138-bp in-frame deletion of the remainder ofexon 31 Sequencing of exon 31 at thegenomic DNA level showed no suchdeletion and 10 of 10 control subjects

showed the same deleted product proving that this was due to normal alternative splicing Reverse transcription PCRamplification using a reverse primer that spanned the spliced region (4784R) with the common 4476F forward primershowed a smaller PCR fragment (lower arrow 4476F to 4784R) corresponding to the 138-bp deletion In both the full-length and exon 31ndashspliced laminin a2 isoforms the nucleotides at the donor splice site are shown Both isoforms show agood match to consensus splicing sequences

1132 NEUROLOGY 55 October (2 of 2) 2000

when ratios were calculated using peak height p 5 0013when ratios were calculated using peak area) The alterna-tively spliced isoform was at similar levels in all musclebiopsies Our patient showed 65 6 8 of control levels ofexon 31ndashspliced laminin a2 RNA (100 6 38)

Discussion Here we describe a novel alterna-tively spliced isoform of the laminin a2 gene in apatient with CMD who was a compound heterozy-gote for two laminin a2 nonsense mutations Onemutation was contained within the 46 amino acidsdeleted in the alternatively spliced isoform Becausethe alternatively spliced isoform removed one of hernonsense mutations this patient was able to produceonly the alternatively spliced isoform with no evi-dence of the full-length isoform This alternativelyspliced product was found in all individuals testedboth control subjects and patients with neuromuscu-lar disease The isoform involved the recognition of acryptic splice site within exon 31 such that the ma-jority of exon 31 was skipped and directly spliced toexon 32 This splicing event removed 46 amino acidsin the cysteine-rich domain IIIa just proximal to thetriple coil-coiled region that associates with the b1and g1 chains of laminin The alternatively splicedisoform removes eight cysteines which are thoughtto participate in the formation of inter- and intramo-lecular disulfide bonds in the basal lamina No sub-function has been assigned to this region

This isoform was localized extracellularly pre-dominantly to discrete highly positive areas in iso-lated myofibers We were unable to determine anycharacteristics of these myofibers that distinguishedthem from the remainder Fiber typing showed thatboth type I (oxidative) and type II (glycolytic) fibersstained highly positively and a neuromuscular junc-

tion localization was ruled out No regenerating fi-bers were laminin a2 positive

Immunostaining for other subunits of lamininshowed a unique downregulation of laminin b1 andg1 and a failure of the upregulation of laminin a5typically seen in patients lacking laminin a2 Thisfinding suggests that the small amount of the novelisoform present in muscle fibers acts as a ldquodominantnegativerdquo protein it prevents the upregulation oflaminin a5 via its presence in muscle yet does notassociate well with either laminin b1 or g1 resultingin their downregulation This suggests that thecysteine-rich region of laminin a2 is important forinteraction with the b and g subunits and that thisnovel isoform alters the binding characteristics Al-ternatively the cysteine-rich region of laminin a2might be crucial for a proper chaperonndashlaminin in-teraction involved in the intracellular transport andassembly of the laminin chains Because b and gchains cannot be delivered to the extracellular ma-trix without their assembly in a abg heterotrimer37

the lack of a proper transport of the a chain in theintracellular milieu results in defective secretion ofthe entire heterotrimer

Our patient showed CNS involvement that wasconsiderably more severe than typically seen in pa-tients with complete laminin a2 deficiency Her MRIwas highly abnormal with tetraventricular dilata-tion white matter alterations pachygyria of the oc-cipital cortex and some small areas of cerebellarpolymicrogyria findings not seen in any other pa-tient with laminin a2 deficiency studied to dateAlso no other patient with laminin a2 deficiency hasbeen reported with mental retardation our patientshowed a moderately low IQ with progressive mentalretardation

It is important to explain the severe CNS involve-ment in this patient Laminin a2 is normally ex-pressed in the inner part of the basal lamina (laminadensa) of striated muscle fibers and in Schwann cellsof peripheral nerves Expression in the CNS is stillnot well studied particularly in regard to neural de-velopment In a laminin a2ndashdeficient patient withCMD the absence of laminin a2 results in a severederangement of the muscle basal lamina Electronmicroscopy shows a discontinuous thinner basallamina with occasional holes in skeletal muscle fi-bers in this category of patients3839 but a normalwell-preserved basement membrane in the intramus-cular nerves39 No EM studies of the basal lamina inbrain of laminin a2ndashdeficient patients with CMDhave been reported therefore there are still too fewdata concerning the role of laminin a2 in brain devel-opment and homeostasis to derive adequate modelsthat explain the severe CNS involvement in thispatient

AcknowledgmentThe authors thank Dr Eva Engvall for critical review of themanuscript

Figure 5 Normal expression of the exon 31ndashspliced lami-nin a2 isoform and reduced expression of the full-lengthlaminin a2 in the patientrsquos muscle Shown are examples ofautomated sequencer traces of quantitative multiplex fluo-rescent reverse transcription PCR of the exon 31ndashsplicedlaminin a2 and full-length laminin a2 isoforms relative toa-sarcoglycan RNA isolated from the patientrsquos muscle bi-opsy consistently showed lower levels of full-length lami-nin a2 RNA relative to control subjects The level of exon31ndashspliced laminin a2 RNA was similar in the patient rel-ative to control subjects

October (2 of 2) 2000 NEUROLOGY 55 1133

References1 Tome FM Evangelista T Leclerc A et al Congenital muscu-

lar dystrophy with merosin deficiency M CR Acad Sci Paris1994317351ndash357

2 HelblingndashLeclerc A Zhang X Topaloglu H et al Mutations inthe laminin a2-chain gene (LAMA2) cause merosin-deficientcongenital muscular dystrophy Nat Genet 199511216ndash218

3 Pegoraro E Marks H Garcia CA et al Laminin a2 musculardystrophy genotypephenotype studies of 22 patients Neurol-ogy 199851101ndash110

4 Ehrig K Leivo I Argraves WS Ruoslahti E Engvall E Mer-osin a tissue-specific basement membrane protein is a lami-nin like protein Proc Natl Acad Sci USA 1990873264ndash3268

5 Voulteenaho R Nissinen M Sainio K et al Human lamininM chain (merosin) complete primary structure chromosomalassignment and expression of the M and A chain in humanfetal tissues J Cell Biol 1994124381ndash394

6 Patton B Miner JH Chiu AY Sanes JR Distribution andfunction of laminins in the neuromuscular system of develop-ing adult and mutant mice J Cell Biol 19971391507ndash1521

7 Dubowitz V Workshop Report 22nd ENMC sponsored work-shop on congenital muscular dystrophy Baarn the Nether-lands May 1993 Neuromuscul Disord 1994475ndash81

8 Reed UC Marie SK Vainzof M et al Congenital musculardystrophy with cerebral white matter hypodensity Correla-tion of clinical features and merosin deficiency Brain Dev19961853ndash58

9 Pini A Merlini L Tome FM Chevallay M Gobbi G Merosin-negative congenital muscular dystrophy occipital epilepsywith periodic spasms and focal cortical dysplasia Report ofthree Italian cases in two families Brain Dev 199618316ndash322

10 Sunada Y Edgar TS Lotz BP Rust RS Campbell KPMerosin-negative congenital muscular dystrophy associatedwith extensive brain abnormalities Neurology 1995452084ndash2089

11 Van der Knaap MS Smit LME Barth PG et al Magneti-cresonance imaging in classification of congenital musculardystrophies with brain abnormalities Ann Neurol 19974250ndash59

12 Hayashi YK Engvall E ArikawandashHirasawa E et al Abnor-mal localization of laminin subunits in muscular dystrophiesJ Neurol Sci 199311953ndash64

13 Haltia M Leivo I Somer H et al Musclendasheyendashbrain diseasea neuropathological study Ann Neurol 199741173ndash180

14 Kanoff RJ Curless RG Petito C Falcone S Siatkowski RMPegoraro E WalkerndashWarburg syndrome neurological featuresand muscle membrane structure Ped Neurol 19981876ndash80

15 Villanova M Sabatelli P He Y et al Immunofluorescencestudy of a muscle biopsy from a 1-year-old patient with Walk-erndashWarburg syndrome Acta Neuropathol 199896651ndash654

16 Topaloglu H Talim B Vignier N et al Merosin-deficient con-genital muscular dystrophy with severe mental retardationand normal cranial MRI a report of two siblings Neuromus-cul Disord 19988169ndash174

17 Brockington M Sewry CA Hermann R et al Assignment of aform of congenital muscular dystrophy with secondary mer-osin deficiency to chromosome 1q42 Am J Hum Genet 200066428ndash435

18 Sewry CA Naom I DrsquoAlessandro M et al Variable clinicalphenotype in merosin-deficient congenital muscular dystrophyassociated with differential immunolabelling of two fragmentsof the laminin a2 chain Neuromuscul Disord 19977169ndash175

19 Tan E Topaloglu H Sewry C et al Late onset musculardystrophy with cerebral white matter changes due to partialmerosin deficiency Neuromuscul Disord 1997785ndash89

20 Naom I DrsquoAlessandro M Topaloglu H et al Refinement ofthe laminin a2 chain locus to human chromosome 6q2 insevere and mild merosin deficient congenital muscular dystro-phy J Med Genet 19973499ndash104

21 Nissinen M HelblingndashLeclerc A Zhang X et al Substitutionof a conserved cysteine-996 in a cysteine-rich motif of thelaminin a2-chain in congenital muscular dystrophy with par-tial deficiency of the protein Am J Hum Genet 1996581177ndash1184

22 Allamand V Sunada Y Salih MAM et al Mild congenitalmuscular dystrophy in two patients with an internally deletedlaminin a2-chain Hum Mol Genet 19976747ndash752

23 Hayashi YK Ishihara T Domen K Hori H Arahata K Abenign allelic form of laminin a2 chain deficient musculardystrophy Lancet 19973491147

24 Naom I DrsquoAlessandro M Sewry CA et al Laminin a2-chaingene mutations in two siblings presenting with limb-girdlemuscular dystrophy Neuromuscul Disord 19988495ndash501

25 Naom I DrsquoAlessandro M Sewry CA et al Mutations in thelaminin a2-chain gene in two children with early-onset mus-cular dystrophy Brain 200012331ndash41

26 Trevisan CP Martinello F Fanin M et al Merosin expressionin muscle of western case with Fukuyama-like congenitalmuscular dystrophy Basic Appl Myol 19966101ndash106

27 Hori H Kanamori T Mizuta T Yamaguchi N Liu Y Nagai YHuman laminin M chain epitope analysis of its monoclonalantibodies by immunoscreening of cDNA clones and tissueexpression J Biochem 19941161212ndash1219

28 Schiaffino S Gorza L Pitton G et al Embryonic and neonatalmyosin heavy chain in denervated and paralyzed rat skeletalmuscle Dev Biol 19881271ndash11

29 Byers TJ Kunkel LM Watkins SC The subcellular distribu-tion of dystrophin in mouse skeletal cardiac and smoothmuscle J Cell Biol 1991115411ndash421

30 Tiger CF Champliaud MF Pedrosadomellof F Thornell LEEkblom P Gullberg D Presence of laminin alpha-5 chain andlack of laminin alpha-1 chain during human muscle develop-ment and in muscular dystrophies J Biol Chem 199727228590ndash28595

31 Pegoraro E Mancias P Swerdlow SH et al Laminin a2 (mer-osin) gene mutations cause congenital muscular dystrophy(CMD) Ann Neurol 199640782ndash791

32 Miller SA Dykes DD Polesky HF A simple salting out proce-dure for extracting DNA from human nucleated cells NucleicAcids Res 1988161215

33 Zhang X Vuolteenaho R Tryggvason K Structure of the hu-man laminin a2-chain gene (LAMA2) which is affected incongenital muscular dystrophy J Biol Chem 199627127664ndash27669

34 Little S Amplification-refractory mutation system (ARMS)analysis of point mutations In Dracopoli NC Haines JL KorfBR et al eds Current protocols in human genetics NewYork John Wiley 1994981ndash985

35 Zhou J Hoffman EP Pathophysiology of sodium channelopa-thies Studies of sodium channel expression by quantitativemultiplex fluorescence polymerase chain reaction J BiolChem 199426918563ndash18571

36 Morrone A Pegoraro E Angelini C Zammarchi E Marconi GHoffman EP RNA metabolism in myotonic dystrophy Patientmuscle shows decreased insulin receptor RNA and proteinconsistent with abnormal insulin resistance J Clin Invest1997991691ndash1698

37 Yurchenco PD Quan Y Colognato H et al The alpha chain oflaminin-1 is independently secreted and drives secretion of itsbeta- and gamma-chain partners Proc Natl Acad Sci USA19979410189ndash10194

38 Minetti C Bado M Morreale G Pedemonte M Cordone GDisruption of muscle basal lamina in congenital muscular dys-trophy with merosin deficiency Neurology 1996461354ndash1358

39 Osari S Kobayashi O Yamashita Y et al Basement mem-brane abnormality in merosin-negative congenital musculardystrophy Acta Neuropathol 199691332ndash336

1134 NEUROLOGY 55 October (2 of 2) 2000

DOI 101212WNL55811282000551128-1134 Neurology

E Pegoraro M Fanin CP Trevisan et al dystrophy

2 deficient congenital muscularα2 isoform in severe laminin αA novel laminin

This information is current as of October 24 2000

ServicesUpdated Information amp

httpwwwneurologyorgcontent5581128fullhtmlincluding high resolution figures can be found at

References

httpwwwneurologyorgcontent5581128fullhtmlref-list-1at This article cites 37 articles 16 of which you can access for free

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spectrum of primary and secondary defects Second-ary partial laminin a2 deficiency has been reportedin Fukuyama-type CMD12 in musclendasheyendashbrain dis-ease13 and in WalkerndashWarburg disease1415 Both pri-mary and secondary partial laminin a2 deficiencyoccur in classic CMD Recently in few patients diag-nosed with CMD whose muscle biopsy showed partiallaminin a2 deficiency the disease was not linked tochromosome 6q where the laminin a2 gene (LAMA2)maps1617 However most partial laminin a2ndashdefi-cient CMD are either linked to the LAMA2 locus18-20

or show primary laminin a2 mutations21-25

The clinical phenotype in primary partial laminina2 deficiency CMD varies from severe21 to mild22-25

but abnormal white matter signal on brain MRI hasbeen reported in all patients

Here we report a patient with laminin a2 defi-ciency but foci of laminin a2 staining in muscleThese foci corresponded to a laminin a2 isoform gen-erated by alternative splicing in all human muscletested We show data consistent with the hypothesisthat sole expression of this isoform may result in adestabilization of laminin chain assembly and thusin a loss of possible rescue mechanisms leading toan unusually severe phenotype

Patient and methods Patient The clinical data fromthis the patient were partially previously reported26

Briefly this 14-year-old girl with CMD was born at termafter an uneventful pregnancy She presented at birth withsevere hypotonia and joint contractures A diagnosis ofCMD was made based on elevated creatine kinase levelselectromyographic results indicative of myopathy and amuscle biopsy done at 10 days of age consistent with adystrophic process Motor milestones were severely de-layed and the maximal motor skill achieved was the abil-ity to walk a few steps with bilateral support at age 6years At age 7 years the patient began to have absenceseizures with myoclonic jerks of the upper limbs and eye-lids At age 9 years the seizures became the partial com-plex type Brain MRI study revealed tetraventriculardilatation severe and widespread white matter alter-

ations more evident in the frontal lobes pachygyria of theoccipital cortex and some small areas of cerebellar polymi-crogyria (figure 1) IQ study showed moderate mental re-tardation On examination the patient was wheelchairbound She presented severe hypotonia and hypotrophy inthe four limbs Scoliosis and multiple contractures werealso evident The patientrsquos second muscle biopsy done atage 3 years was recently included in a large screening forlaminin a2 abnormalities in CMD Based on immunofluo-rescence results a diagnosis of partial laminin a2 defi-ciency was made

Immunofluorescence and immunoblotting analysisFour-micron-thick cryostat muscle biopsy sections were in-cubated for 2 hours with mouse monoclonal antibody di-rected against both fragments in which laminin a2 isprocessed the 80-kD carboxyl-terminus (15000) (Chemi-con Temecula CA) and the 320-kD fragment (150) (clone2D9)27 The antibody directed against the 320-kD frag-ment of laminin a2 recognizes 229 amino acids in the G2to G3 region of the laminin a2 G-domain27 Anti-laminina5 (13500) (Gibco-BRL Gaithersburg MD) laminin b1(11500) (Gibco-BRL) laminin g1 (1100) (Gibco-BRL)laminin b2 (1500) (clone C4 from the Developmental Stud-ies Hybridoma Bank Johns Hopkins University Balti-more MD) and embryonic myosin heavy chain (11000)28

mouse monoclonal antibodies and anti-dystrophin (6-10)(12000)29 and anti-nidogen (11000) (donated by R Nis-cht Department of Dermatology University of CologneGermany) rabbit polyclonal antibodies were also used

Laminin a5 is recognized by an anti-human lamininclone III A-chain antibody (Gibco-BRL) previously thoughtto recognize laminin a1 Because laminin a1 is not ex-pressed in muscle at any age we considered all stainingobserved with this antibody to represent laminin a530

Anti-mouse and anti-rabbit Cy-3 conjugated secondaryantibody were used to visualize the immune complexesTetramethylrhodamine a-bungarotoxin (11000) (Molecu-lar Probes Eugene OR) was used to visualize neuromus-cular junctions Laminin a2 immunoblot was performedas previously described using antibody directed againstboth the 80-kD (150000) and 320-kD (1300) laminin a2fragments31

Figure 1 Axial and coronal MRI in thepatient (A) Axial T2-weighted imagedemonstrating severe white matter hy-perintensity more evident in the frontallobes and ventricular dilatation In theoccipital cortex diffuse pachygyria alsoevident in the T1-weighted coronal sec-tion (B) is seen

October (2 of 2) 2000 NEUROLOGY 55 1129

DNA analysis Blood was collected in ethylenediami-netetraacetic acid (EDTA) tubes from the patient and herparents DNA was isolated from peripheral blood as previ-ously described32

Laminin a2 mutations and alternative splicing studyRNA extraction complementary DNA (cDNA) synthesisand reverse transcription PCR (RT-PCR) were performedas previously described31 Single-strand conformationalpolymorphism analysis of the entire 10 kb of the laminina2 gene was done as previously described31

Confirmation of nucleotide changes Nucleotide changesand an autosomal recessive pattern of inheritance wereconfirmed in the genomic DNA of the patient and herparents The paternal laminin a2 mutation created anNlaIII restriction-enzyme site The appropriate PCR prod-uct obtained with primers specific for laminin a2 exon4933 was digested with NlaIII The maternal laminin a2mutation did not change a restriction-enzyme site so anamplification-refractory mutation system test was de-signed using genomic primers34

Quantitation of the exon 31ndashspliced isoform and full-length laminin a2 RNA by quantitative multiplex reversetranscription PCR RNA extraction and cDNA synthesiswere done as previously described31 Biopsies studied werefrom the patient six patients with partial laminin a2 defi-ciency one patient with complete laminin a2 deficiencyand one patient with undetermined myopathy (laminin a2and dystrophin immunofluorescence were normal)

Primer sets for coamplification of cDNA correspondingto a-sarcoglycan full-length laminin a2 and exon 31ndashspliced laminin a2 were designed as follows ADH3F59fluorescein-CTG CTC AAC GTC ACC TCT G-39 ADH3R59-CCG GCA CTG ACT TAT CCA C-39 for a-sarcoglycanMer 4476F 59-CCA GTA ACA ATT TCA GCC CCT CTTG-39 Mer 4784R 59fluorescein-TGC ACT CAT CTC CACAAA AAA-39 for full-length laminin a2 Mer 4476F andMer 4784Rspliced 59fluorescein-TGC ACT CAT CTC CACAAA ATT-39 for exon 31ndashspliced laminin a2 The laminina2 reverse primers and the a-sarcoglycan forward primerwere synthesized with fluorescein-linked 59 ends (Gibco-BRL) Quantitative multiplex RT-PCR35 was done using125 ng of each primer 15 mL of cDNA (corresponding toabout 20 ng of total RNA) 25 mM dNTPs 1 3 PCR reac-tion buffer and 25 U Ampli Taq DNA polymerase (PerkinndashElmer Foster City CA) Cycling conditions weredenaturation at 94 degC for 3 minutes 94 degC for 1 minute55 degC for 1 minute and 72 degC for 1 minute for 22 cyclesand extension at 72 degC for 10 minutes Quantitation offluorescent PCR products of exon 31ndashspliced laminin a2and full-length laminin a2 relative to a-sarcoglycan wasdone as previously described36 Briefly fluorescent RT-PCR products were denatured and loaded on an ABI 373DNA automated sequencer using a ROX-fluorescein matrixstandard (PE Biosystems Foster City CA) ROX-labeledmarkers (PE Biosystems) were used as internal size stan-dards Lanes were manually assigned from completed im-age files and peak heights and peak areas were analyzedusing GeneScan software (PE Biosystems) Peak areas cor-responding to the laminin a2 full-length and exon 31ndashspliced isoforms and the a-sarcoglycan internal controlwere determined and the quantitative results transferredto Excel software Two separate RT-PCR measurementswere done for each sample The ratios between full-length

laminin a2 versus a-sarcoglycan and exon 31ndashspliced lami-nin a2 versus a-sarcoglycan were calculated separately forthe patient and the control subjects as average with SEThe average ratios were compared and expressed as per-centage of control Statistical significance was calculatedusing analysis of variance (ANOVA) using all calculatedratios

Results Immunofluorescence and immunoblotting analy-ses of the muscle biopsy A panel of different laminin anti-bodies was used to characterize expression of differentlaminin subunits in the patientrsquos muscle biopsy done at age3 In parallel immunostaining was done with a biopsy show-ing no histopathology (normal control) a laminin a2ndashnega-tive patient with CMD and a laminin a2ndashpositive patientwith CMD All three chains of the muscle laminin-2 andlaminin-4 heterotrimeric complexes (laminin a2 b1 g1b2) were studied Laminin a2 immunofluorescence usingantibodies directed against both the 80-kD and 320-kDfragments of laminin a2 showed a barely detectable im-munoreactivity in some of the patientrsquos muscle fibers (fig-ure 2H) However some scattered myofibers showedpatchy discrete areas of highly positive immunostaining(see figure 2H) Laminin a2 was normal in the control (seefigure 2E) and the laminin a2ndashpositive CMD muscle biopsy(see figure 2G) and completely deficient in the laminina2ndashnegative CMD muscle biopsy (see figure 2F)

Laminin a5 was overexpressed in the laminin a2ndashnegative CMD muscle biopsy (see figure 2B) relative to thenormal control and laminin a2ndashpositive patient with CMDConversely laminin a5 did not appear to be overexpressedin the patient (see figure 2D)

Laminin b1 (see figure 2 I through N) and g1 (seefigure 2 O through R) showed immunostaining similar tocontrol tissue and laminin a2ndashnegative and ndashpositive CMDmuscle biopsies In the patientrsquos muscle biopsy laminin b1showed a markedly decreased immunostaining aroundmyofibers whereas capillaries and small vessels werestrongly stained (see figure 2N) Laminin g1 appearedslightly reduced relative to all controls (see figure 2R)Interestingly no positive laminin a2 immunostaining wasdetected in a large nerve twig included in the patientrsquosmuscle biopsy where both laminin b1 and laminin g1 werestrongly expressed (data not shown)

In order to determine if the laminin a2ndashpositive immu-nostaining in the patientrsquos muscle biopsy was localized atthe neuromuscular junctions laminin b2 and a-bungaro-toxin immunostaining were performed No neuromuscularjunctions were seen with either marker Also we did notobserve any colocalization of double-staining experimentswith laminin a2ndashlaminin b2 or laminin a2ndasha-bungarotoxin(data not shown) These data suggest that neuromuscularjunctions were not present in the biopsy and that thepositive laminin a2 regions did not correspond to neuro-muscular junctions

Double immunostaining with laminin a2ndashdystrophinand laminin a2ndashnidogen was done in order to determinethe localization of the laminin a2ndashpositive areas in thepatientrsquos muscle biopsy Laminin a2 was consistently local-ized outside the plasma membrane (externally to dystro-phin) and staining was colocalized with nidogen Todetermine if the laminin a2ndashpositive regions were fiber-type specific skeletal muscle cryosections were incubatedwith ATPase (pH 43) and then immunostained with lami-

1130 NEUROLOGY 55 October (2 of 2) 2000

Figure 2 Immunofluorescence study of laminin subunit expression in the patient muscle biopsy relative to normal con-trol laminin a2ndashnegative and laminin a2ndashpositive congenital muscular dystrophy (CMD) muscle biopsies (A E I andO) Normal muscle biopsy (B F L and P) Patient with laminin a2ndashnegative CMD (C G M and Q) Patient with laminina2ndashpositive CMD (D H N and R) Muscle biopsy from our reported patient (E through H) Laminin a2 immunostainingusing an antibody directed against the 80-kD carboxyl-terminus of the protein shows discrete areas of highly positive im-munostaining in few myofibers in the patientrsquos muscle biopsy whereas the majority of myofibers are largely laminin a2negative (H) Laminin a2 was normal in the control (E) and laminin a2ndashpositive CMD muscle biopsy (G) and completelynegative in the laminin a2ndashdeficient CMD muscle biopsy Study of the expression of a5 b1 and g1 chains showed thatour patientrsquos muscle does not overexpress laminin a5 (D) as in the patient with laminin a2ndashnegative CMD (B) Our pa-tient also shows a marked decrease in b1 and to a lesser degree g1 immunostaining (N and R) relative to control andlaminin a2ndashnegative and ndashpositive CMD muscle biopsies

Figure 3 Identification of two non-sense mutations in the laminin a2complementary DNA in the patient(Top) Schematic of the exon structureof the laminin a2 gene Exons are rep-resented as boxes introns as linesThe sequencer traces corresponding totwo aberrant conformers show two nu-cleotide changes C4694T in exon 31and C7196T in exon 49 Both result ina stop codon (R1549X and R2383X)(Bottom) The maternal laminin a2gene with a stop codon in exon 31 andthe paternal laminin a2 gene with astop codon in exon 49 are representedShown in the insert is immunostain-ing of the patientrsquos muscle biopsy withlaminin a2 Both paternal and mater-

nal laminin a2 genes are transcribed or translated in a truncated unstable protein as proven by the laminin a2ndashnegativemyofibers in the patientrsquos muscle biopsy The laminin a2ndashpositive immunostaining in the patientrsquos muscle biopsy is due toa novel alternatively spliced exon 31 laminin a2 isoform that skips the nonsense mutation in exon 31

October (2 of 2) 2000 NEUROLOGY 55 1131

nin a2 Laminin a2ndashpositive areas were present in associ-ation with both fiber types I and II (data not shown) Tocorrelate the regeneration status of myofibers with expres-sion of laminin a2 serial cryosections of the patientrsquos mus-cle biopsy done at age 3 were stained with monoclonalantibodies against embryonic myosin heavy chain and the80-kD carboxyl-terminus region of the laminin a2 geneThe frequency of regenerating myofibers in the patientrsquosmuscle biopsy was 2 (532719) of total fibers countedRegenerating fibers were laminin a2 negative Laminin a2immunoblotting using antibodies directed against both the80-kD and 320-kD fragments of laminin a2 failed to showany detectable laminin in the patientrsquos muscle biopsy (datanot shown)

Study of laminin a2 mutations Single-strand confor-mational polymorphism analysis was used to screen forlaminin a2 mutations using muscle RNA from the pa-tientrsquos biopsy The coding sequence of the laminin a2 genewas analyzed using 20 sets of overlapping primer pairsAberrant conformers were identified with primer setsMer4476FndashMer4981R and Mer7015FndashMer7541R Directsequencing of these conformers revealed two nonsense mu-tations C to T at nucleotide position 4694 (R1549X) and Cto T at nucleotide position 7196 (R2383X) of the laminin a2coding sequence (figure 3)

Both mutations were confirmed at the genomic DNAlevel and the inheritance pattern of the mutations deter-mined The C4649T nucleotide change did not cause anyrestriction-enzyme site change thus the amplification-refractory mutation system test was used Primers weredesigned in exon 31 of the laminin a2 gene and mutatedPCR products were present in the patient and her motherNucleotide change C7196T resulted in a gain of an NlaIIIrestriction-enzyme site Primers were designed in exon 49

of the laminin a2 gene and the PCR product was digestedwith NlaIII Digestion fragments were present in the pa-tient and her father

Characterization of an exon 31ndashspliced laminin a2 iso-form The appearance of laminin a2 protein in the patientrsquosmuscle despite the two nonsense mutations suggested thatsome transcripts were excluding one of the two mutationsConsistent with this hypothesis we detected a smallerRT-PCR product over the region of the C4694T mutationin both the patient and control subjects This smallerRT-PCR product was present in the patient and in 10 of 10normal control subjects run in parallel Direct sequencingof the smaller band showed a 138-bp in-frame deletionfrom nucleotides 4629 to 4766 of the laminin a2 codingsequence This deletion corresponded to about 70 of theexon 31 sequence of the laminin a2 gene (figure 4)

Quantitation of the exon31ndashspliced laminin isoformRNA was isolated from the patientrsquos muscle biopsy andfrom eight control muscle biopsies (six with partial laminina2 deficiency one with complete laminin a2 deficiencyand one undetermined myopathy) For each RNA sample100 ng of total RNA underwent RT into cDNA using oligodT10 primers A fluorescent multiplex RT-PCR ampli-fication containing three different PCR products (full-length laminin a2 exon31ndashspliced laminin a2 isoform anda-sarcoglycan) was done in duplicate for each samplePeak heights and areas were calculated and results tabu-lated as ratios between full-length laminin a2 versusa-sarcoglycan and exon 31ndashspliced laminin a2 versusa-sarcoglycan Mean and SE for each ratio were calculatedfor the patient and the control group (figure 5)

The patientrsquos muscle biopsy showed a significant de-crease in full-length laminin a2 RNA relative to the con-trol subjects 24 6 7 versus 100 6 27 ( p 5 0003

Figure 4 Identification of a novelexon 31ndashspliced laminin a2 isoformFull-length laminin a2 and exon 31ndashspliced laminin a2 are shown Thefull-length laminin a2 is the majortranscript in muscle and results fromthe expected splicing between exon 31and 32 Reverse transcription PCRamplification using a reverse comple-mentary DNA primer that spans exon31 and exon 32 (4786R) and a for-ward primer starting at nucleotide4476 shows an reverse transcriptionPCR product of the appropriate size(upper arrow 4476F to 4786R) Theexon 31ndashspliced laminin a2 isoformderives from the use of a cryptic splicesite 57-bp into exon 31 (4629 bp ofRNA) This results in a 138-bp in-frame deletion of the remainder ofexon 31 Sequencing of exon 31 at thegenomic DNA level showed no suchdeletion and 10 of 10 control subjects

showed the same deleted product proving that this was due to normal alternative splicing Reverse transcription PCRamplification using a reverse primer that spanned the spliced region (4784R) with the common 4476F forward primershowed a smaller PCR fragment (lower arrow 4476F to 4784R) corresponding to the 138-bp deletion In both the full-length and exon 31ndashspliced laminin a2 isoforms the nucleotides at the donor splice site are shown Both isoforms show agood match to consensus splicing sequences

1132 NEUROLOGY 55 October (2 of 2) 2000

when ratios were calculated using peak height p 5 0013when ratios were calculated using peak area) The alterna-tively spliced isoform was at similar levels in all musclebiopsies Our patient showed 65 6 8 of control levels ofexon 31ndashspliced laminin a2 RNA (100 6 38)

Discussion Here we describe a novel alterna-tively spliced isoform of the laminin a2 gene in apatient with CMD who was a compound heterozy-gote for two laminin a2 nonsense mutations Onemutation was contained within the 46 amino acidsdeleted in the alternatively spliced isoform Becausethe alternatively spliced isoform removed one of hernonsense mutations this patient was able to produceonly the alternatively spliced isoform with no evi-dence of the full-length isoform This alternativelyspliced product was found in all individuals testedboth control subjects and patients with neuromuscu-lar disease The isoform involved the recognition of acryptic splice site within exon 31 such that the ma-jority of exon 31 was skipped and directly spliced toexon 32 This splicing event removed 46 amino acidsin the cysteine-rich domain IIIa just proximal to thetriple coil-coiled region that associates with the b1and g1 chains of laminin The alternatively splicedisoform removes eight cysteines which are thoughtto participate in the formation of inter- and intramo-lecular disulfide bonds in the basal lamina No sub-function has been assigned to this region

This isoform was localized extracellularly pre-dominantly to discrete highly positive areas in iso-lated myofibers We were unable to determine anycharacteristics of these myofibers that distinguishedthem from the remainder Fiber typing showed thatboth type I (oxidative) and type II (glycolytic) fibersstained highly positively and a neuromuscular junc-

tion localization was ruled out No regenerating fi-bers were laminin a2 positive

Immunostaining for other subunits of lamininshowed a unique downregulation of laminin b1 andg1 and a failure of the upregulation of laminin a5typically seen in patients lacking laminin a2 Thisfinding suggests that the small amount of the novelisoform present in muscle fibers acts as a ldquodominantnegativerdquo protein it prevents the upregulation oflaminin a5 via its presence in muscle yet does notassociate well with either laminin b1 or g1 resultingin their downregulation This suggests that thecysteine-rich region of laminin a2 is important forinteraction with the b and g subunits and that thisnovel isoform alters the binding characteristics Al-ternatively the cysteine-rich region of laminin a2might be crucial for a proper chaperonndashlaminin in-teraction involved in the intracellular transport andassembly of the laminin chains Because b and gchains cannot be delivered to the extracellular ma-trix without their assembly in a abg heterotrimer37

the lack of a proper transport of the a chain in theintracellular milieu results in defective secretion ofthe entire heterotrimer

Our patient showed CNS involvement that wasconsiderably more severe than typically seen in pa-tients with complete laminin a2 deficiency Her MRIwas highly abnormal with tetraventricular dilata-tion white matter alterations pachygyria of the oc-cipital cortex and some small areas of cerebellarpolymicrogyria findings not seen in any other pa-tient with laminin a2 deficiency studied to dateAlso no other patient with laminin a2 deficiency hasbeen reported with mental retardation our patientshowed a moderately low IQ with progressive mentalretardation

It is important to explain the severe CNS involve-ment in this patient Laminin a2 is normally ex-pressed in the inner part of the basal lamina (laminadensa) of striated muscle fibers and in Schwann cellsof peripheral nerves Expression in the CNS is stillnot well studied particularly in regard to neural de-velopment In a laminin a2ndashdeficient patient withCMD the absence of laminin a2 results in a severederangement of the muscle basal lamina Electronmicroscopy shows a discontinuous thinner basallamina with occasional holes in skeletal muscle fi-bers in this category of patients3839 but a normalwell-preserved basement membrane in the intramus-cular nerves39 No EM studies of the basal lamina inbrain of laminin a2ndashdeficient patients with CMDhave been reported therefore there are still too fewdata concerning the role of laminin a2 in brain devel-opment and homeostasis to derive adequate modelsthat explain the severe CNS involvement in thispatient

AcknowledgmentThe authors thank Dr Eva Engvall for critical review of themanuscript

Figure 5 Normal expression of the exon 31ndashspliced lami-nin a2 isoform and reduced expression of the full-lengthlaminin a2 in the patientrsquos muscle Shown are examples ofautomated sequencer traces of quantitative multiplex fluo-rescent reverse transcription PCR of the exon 31ndashsplicedlaminin a2 and full-length laminin a2 isoforms relative toa-sarcoglycan RNA isolated from the patientrsquos muscle bi-opsy consistently showed lower levels of full-length lami-nin a2 RNA relative to control subjects The level of exon31ndashspliced laminin a2 RNA was similar in the patient rel-ative to control subjects

October (2 of 2) 2000 NEUROLOGY 55 1133

References1 Tome FM Evangelista T Leclerc A et al Congenital muscu-

lar dystrophy with merosin deficiency M CR Acad Sci Paris1994317351ndash357

2 HelblingndashLeclerc A Zhang X Topaloglu H et al Mutations inthe laminin a2-chain gene (LAMA2) cause merosin-deficientcongenital muscular dystrophy Nat Genet 199511216ndash218

3 Pegoraro E Marks H Garcia CA et al Laminin a2 musculardystrophy genotypephenotype studies of 22 patients Neurol-ogy 199851101ndash110

4 Ehrig K Leivo I Argraves WS Ruoslahti E Engvall E Mer-osin a tissue-specific basement membrane protein is a lami-nin like protein Proc Natl Acad Sci USA 1990873264ndash3268

5 Voulteenaho R Nissinen M Sainio K et al Human lamininM chain (merosin) complete primary structure chromosomalassignment and expression of the M and A chain in humanfetal tissues J Cell Biol 1994124381ndash394

6 Patton B Miner JH Chiu AY Sanes JR Distribution andfunction of laminins in the neuromuscular system of develop-ing adult and mutant mice J Cell Biol 19971391507ndash1521

7 Dubowitz V Workshop Report 22nd ENMC sponsored work-shop on congenital muscular dystrophy Baarn the Nether-lands May 1993 Neuromuscul Disord 1994475ndash81

8 Reed UC Marie SK Vainzof M et al Congenital musculardystrophy with cerebral white matter hypodensity Correla-tion of clinical features and merosin deficiency Brain Dev19961853ndash58

9 Pini A Merlini L Tome FM Chevallay M Gobbi G Merosin-negative congenital muscular dystrophy occipital epilepsywith periodic spasms and focal cortical dysplasia Report ofthree Italian cases in two families Brain Dev 199618316ndash322

10 Sunada Y Edgar TS Lotz BP Rust RS Campbell KPMerosin-negative congenital muscular dystrophy associatedwith extensive brain abnormalities Neurology 1995452084ndash2089

11 Van der Knaap MS Smit LME Barth PG et al Magneti-cresonance imaging in classification of congenital musculardystrophies with brain abnormalities Ann Neurol 19974250ndash59

12 Hayashi YK Engvall E ArikawandashHirasawa E et al Abnor-mal localization of laminin subunits in muscular dystrophiesJ Neurol Sci 199311953ndash64

13 Haltia M Leivo I Somer H et al Musclendasheyendashbrain diseasea neuropathological study Ann Neurol 199741173ndash180

14 Kanoff RJ Curless RG Petito C Falcone S Siatkowski RMPegoraro E WalkerndashWarburg syndrome neurological featuresand muscle membrane structure Ped Neurol 19981876ndash80

15 Villanova M Sabatelli P He Y et al Immunofluorescencestudy of a muscle biopsy from a 1-year-old patient with Walk-erndashWarburg syndrome Acta Neuropathol 199896651ndash654

16 Topaloglu H Talim B Vignier N et al Merosin-deficient con-genital muscular dystrophy with severe mental retardationand normal cranial MRI a report of two siblings Neuromus-cul Disord 19988169ndash174

17 Brockington M Sewry CA Hermann R et al Assignment of aform of congenital muscular dystrophy with secondary mer-osin deficiency to chromosome 1q42 Am J Hum Genet 200066428ndash435

18 Sewry CA Naom I DrsquoAlessandro M et al Variable clinicalphenotype in merosin-deficient congenital muscular dystrophyassociated with differential immunolabelling of two fragmentsof the laminin a2 chain Neuromuscul Disord 19977169ndash175

19 Tan E Topaloglu H Sewry C et al Late onset musculardystrophy with cerebral white matter changes due to partialmerosin deficiency Neuromuscul Disord 1997785ndash89

20 Naom I DrsquoAlessandro M Topaloglu H et al Refinement ofthe laminin a2 chain locus to human chromosome 6q2 insevere and mild merosin deficient congenital muscular dystro-phy J Med Genet 19973499ndash104

21 Nissinen M HelblingndashLeclerc A Zhang X et al Substitutionof a conserved cysteine-996 in a cysteine-rich motif of thelaminin a2-chain in congenital muscular dystrophy with par-tial deficiency of the protein Am J Hum Genet 1996581177ndash1184

22 Allamand V Sunada Y Salih MAM et al Mild congenitalmuscular dystrophy in two patients with an internally deletedlaminin a2-chain Hum Mol Genet 19976747ndash752

23 Hayashi YK Ishihara T Domen K Hori H Arahata K Abenign allelic form of laminin a2 chain deficient musculardystrophy Lancet 19973491147

24 Naom I DrsquoAlessandro M Sewry CA et al Laminin a2-chaingene mutations in two siblings presenting with limb-girdlemuscular dystrophy Neuromuscul Disord 19988495ndash501

25 Naom I DrsquoAlessandro M Sewry CA et al Mutations in thelaminin a2-chain gene in two children with early-onset mus-cular dystrophy Brain 200012331ndash41

26 Trevisan CP Martinello F Fanin M et al Merosin expressionin muscle of western case with Fukuyama-like congenitalmuscular dystrophy Basic Appl Myol 19966101ndash106

27 Hori H Kanamori T Mizuta T Yamaguchi N Liu Y Nagai YHuman laminin M chain epitope analysis of its monoclonalantibodies by immunoscreening of cDNA clones and tissueexpression J Biochem 19941161212ndash1219

28 Schiaffino S Gorza L Pitton G et al Embryonic and neonatalmyosin heavy chain in denervated and paralyzed rat skeletalmuscle Dev Biol 19881271ndash11

29 Byers TJ Kunkel LM Watkins SC The subcellular distribu-tion of dystrophin in mouse skeletal cardiac and smoothmuscle J Cell Biol 1991115411ndash421

30 Tiger CF Champliaud MF Pedrosadomellof F Thornell LEEkblom P Gullberg D Presence of laminin alpha-5 chain andlack of laminin alpha-1 chain during human muscle develop-ment and in muscular dystrophies J Biol Chem 199727228590ndash28595

31 Pegoraro E Mancias P Swerdlow SH et al Laminin a2 (mer-osin) gene mutations cause congenital muscular dystrophy(CMD) Ann Neurol 199640782ndash791

32 Miller SA Dykes DD Polesky HF A simple salting out proce-dure for extracting DNA from human nucleated cells NucleicAcids Res 1988161215

33 Zhang X Vuolteenaho R Tryggvason K Structure of the hu-man laminin a2-chain gene (LAMA2) which is affected incongenital muscular dystrophy J Biol Chem 199627127664ndash27669

34 Little S Amplification-refractory mutation system (ARMS)analysis of point mutations In Dracopoli NC Haines JL KorfBR et al eds Current protocols in human genetics NewYork John Wiley 1994981ndash985

35 Zhou J Hoffman EP Pathophysiology of sodium channelopa-thies Studies of sodium channel expression by quantitativemultiplex fluorescence polymerase chain reaction J BiolChem 199426918563ndash18571

36 Morrone A Pegoraro E Angelini C Zammarchi E Marconi GHoffman EP RNA metabolism in myotonic dystrophy Patientmuscle shows decreased insulin receptor RNA and proteinconsistent with abnormal insulin resistance J Clin Invest1997991691ndash1698

37 Yurchenco PD Quan Y Colognato H et al The alpha chain oflaminin-1 is independently secreted and drives secretion of itsbeta- and gamma-chain partners Proc Natl Acad Sci USA19979410189ndash10194

38 Minetti C Bado M Morreale G Pedemonte M Cordone GDisruption of muscle basal lamina in congenital muscular dys-trophy with merosin deficiency Neurology 1996461354ndash1358

39 Osari S Kobayashi O Yamashita Y et al Basement mem-brane abnormality in merosin-negative congenital musculardystrophy Acta Neuropathol 199691332ndash336

1134 NEUROLOGY 55 October (2 of 2) 2000

DOI 101212WNL55811282000551128-1134 Neurology

E Pegoraro M Fanin CP Trevisan et al dystrophy

2 deficient congenital muscularα2 isoform in severe laminin αA novel laminin

This information is current as of October 24 2000

ServicesUpdated Information amp

httpwwwneurologyorgcontent5581128fullhtmlincluding high resolution figures can be found at

References

httpwwwneurologyorgcontent5581128fullhtmlref-list-1at This article cites 37 articles 16 of which you can access for free

Citations

cleshttpwwwneurologyorgcontent5581128fullhtmlotherartiThis article has been cited by 5 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

DNA analysis Blood was collected in ethylenediami-netetraacetic acid (EDTA) tubes from the patient and herparents DNA was isolated from peripheral blood as previ-ously described32

Laminin a2 mutations and alternative splicing studyRNA extraction complementary DNA (cDNA) synthesisand reverse transcription PCR (RT-PCR) were performedas previously described31 Single-strand conformationalpolymorphism analysis of the entire 10 kb of the laminina2 gene was done as previously described31

Confirmation of nucleotide changes Nucleotide changesand an autosomal recessive pattern of inheritance wereconfirmed in the genomic DNA of the patient and herparents The paternal laminin a2 mutation created anNlaIII restriction-enzyme site The appropriate PCR prod-uct obtained with primers specific for laminin a2 exon4933 was digested with NlaIII The maternal laminin a2mutation did not change a restriction-enzyme site so anamplification-refractory mutation system test was de-signed using genomic primers34

Quantitation of the exon 31ndashspliced isoform and full-length laminin a2 RNA by quantitative multiplex reversetranscription PCR RNA extraction and cDNA synthesiswere done as previously described31 Biopsies studied werefrom the patient six patients with partial laminin a2 defi-ciency one patient with complete laminin a2 deficiencyand one patient with undetermined myopathy (laminin a2and dystrophin immunofluorescence were normal)

Primer sets for coamplification of cDNA correspondingto a-sarcoglycan full-length laminin a2 and exon 31ndashspliced laminin a2 were designed as follows ADH3F59fluorescein-CTG CTC AAC GTC ACC TCT G-39 ADH3R59-CCG GCA CTG ACT TAT CCA C-39 for a-sarcoglycanMer 4476F 59-CCA GTA ACA ATT TCA GCC CCT CTTG-39 Mer 4784R 59fluorescein-TGC ACT CAT CTC CACAAA AAA-39 for full-length laminin a2 Mer 4476F andMer 4784Rspliced 59fluorescein-TGC ACT CAT CTC CACAAA ATT-39 for exon 31ndashspliced laminin a2 The laminina2 reverse primers and the a-sarcoglycan forward primerwere synthesized with fluorescein-linked 59 ends (Gibco-BRL) Quantitative multiplex RT-PCR35 was done using125 ng of each primer 15 mL of cDNA (corresponding toabout 20 ng of total RNA) 25 mM dNTPs 1 3 PCR reac-tion buffer and 25 U Ampli Taq DNA polymerase (PerkinndashElmer Foster City CA) Cycling conditions weredenaturation at 94 degC for 3 minutes 94 degC for 1 minute55 degC for 1 minute and 72 degC for 1 minute for 22 cyclesand extension at 72 degC for 10 minutes Quantitation offluorescent PCR products of exon 31ndashspliced laminin a2and full-length laminin a2 relative to a-sarcoglycan wasdone as previously described36 Briefly fluorescent RT-PCR products were denatured and loaded on an ABI 373DNA automated sequencer using a ROX-fluorescein matrixstandard (PE Biosystems Foster City CA) ROX-labeledmarkers (PE Biosystems) were used as internal size stan-dards Lanes were manually assigned from completed im-age files and peak heights and peak areas were analyzedusing GeneScan software (PE Biosystems) Peak areas cor-responding to the laminin a2 full-length and exon 31ndashspliced isoforms and the a-sarcoglycan internal controlwere determined and the quantitative results transferredto Excel software Two separate RT-PCR measurementswere done for each sample The ratios between full-length

laminin a2 versus a-sarcoglycan and exon 31ndashspliced lami-nin a2 versus a-sarcoglycan were calculated separately forthe patient and the control subjects as average with SEThe average ratios were compared and expressed as per-centage of control Statistical significance was calculatedusing analysis of variance (ANOVA) using all calculatedratios

Results Immunofluorescence and immunoblotting analy-ses of the muscle biopsy A panel of different laminin anti-bodies was used to characterize expression of differentlaminin subunits in the patientrsquos muscle biopsy done at age3 In parallel immunostaining was done with a biopsy show-ing no histopathology (normal control) a laminin a2ndashnega-tive patient with CMD and a laminin a2ndashpositive patientwith CMD All three chains of the muscle laminin-2 andlaminin-4 heterotrimeric complexes (laminin a2 b1 g1b2) were studied Laminin a2 immunofluorescence usingantibodies directed against both the 80-kD and 320-kDfragments of laminin a2 showed a barely detectable im-munoreactivity in some of the patientrsquos muscle fibers (fig-ure 2H) However some scattered myofibers showedpatchy discrete areas of highly positive immunostaining(see figure 2H) Laminin a2 was normal in the control (seefigure 2E) and the laminin a2ndashpositive CMD muscle biopsy(see figure 2G) and completely deficient in the laminina2ndashnegative CMD muscle biopsy (see figure 2F)

Laminin a5 was overexpressed in the laminin a2ndashnegative CMD muscle biopsy (see figure 2B) relative to thenormal control and laminin a2ndashpositive patient with CMDConversely laminin a5 did not appear to be overexpressedin the patient (see figure 2D)

Laminin b1 (see figure 2 I through N) and g1 (seefigure 2 O through R) showed immunostaining similar tocontrol tissue and laminin a2ndashnegative and ndashpositive CMDmuscle biopsies In the patientrsquos muscle biopsy laminin b1showed a markedly decreased immunostaining aroundmyofibers whereas capillaries and small vessels werestrongly stained (see figure 2N) Laminin g1 appearedslightly reduced relative to all controls (see figure 2R)Interestingly no positive laminin a2 immunostaining wasdetected in a large nerve twig included in the patientrsquosmuscle biopsy where both laminin b1 and laminin g1 werestrongly expressed (data not shown)

In order to determine if the laminin a2ndashpositive immu-nostaining in the patientrsquos muscle biopsy was localized atthe neuromuscular junctions laminin b2 and a-bungaro-toxin immunostaining were performed No neuromuscularjunctions were seen with either marker Also we did notobserve any colocalization of double-staining experimentswith laminin a2ndashlaminin b2 or laminin a2ndasha-bungarotoxin(data not shown) These data suggest that neuromuscularjunctions were not present in the biopsy and that thepositive laminin a2 regions did not correspond to neuro-muscular junctions

Double immunostaining with laminin a2ndashdystrophinand laminin a2ndashnidogen was done in order to determinethe localization of the laminin a2ndashpositive areas in thepatientrsquos muscle biopsy Laminin a2 was consistently local-ized outside the plasma membrane (externally to dystro-phin) and staining was colocalized with nidogen Todetermine if the laminin a2ndashpositive regions were fiber-type specific skeletal muscle cryosections were incubatedwith ATPase (pH 43) and then immunostained with lami-

1130 NEUROLOGY 55 October (2 of 2) 2000

Figure 2 Immunofluorescence study of laminin subunit expression in the patient muscle biopsy relative to normal con-trol laminin a2ndashnegative and laminin a2ndashpositive congenital muscular dystrophy (CMD) muscle biopsies (A E I andO) Normal muscle biopsy (B F L and P) Patient with laminin a2ndashnegative CMD (C G M and Q) Patient with laminina2ndashpositive CMD (D H N and R) Muscle biopsy from our reported patient (E through H) Laminin a2 immunostainingusing an antibody directed against the 80-kD carboxyl-terminus of the protein shows discrete areas of highly positive im-munostaining in few myofibers in the patientrsquos muscle biopsy whereas the majority of myofibers are largely laminin a2negative (H) Laminin a2 was normal in the control (E) and laminin a2ndashpositive CMD muscle biopsy (G) and completelynegative in the laminin a2ndashdeficient CMD muscle biopsy Study of the expression of a5 b1 and g1 chains showed thatour patientrsquos muscle does not overexpress laminin a5 (D) as in the patient with laminin a2ndashnegative CMD (B) Our pa-tient also shows a marked decrease in b1 and to a lesser degree g1 immunostaining (N and R) relative to control andlaminin a2ndashnegative and ndashpositive CMD muscle biopsies

Figure 3 Identification of two non-sense mutations in the laminin a2complementary DNA in the patient(Top) Schematic of the exon structureof the laminin a2 gene Exons are rep-resented as boxes introns as linesThe sequencer traces corresponding totwo aberrant conformers show two nu-cleotide changes C4694T in exon 31and C7196T in exon 49 Both result ina stop codon (R1549X and R2383X)(Bottom) The maternal laminin a2gene with a stop codon in exon 31 andthe paternal laminin a2 gene with astop codon in exon 49 are representedShown in the insert is immunostain-ing of the patientrsquos muscle biopsy withlaminin a2 Both paternal and mater-

nal laminin a2 genes are transcribed or translated in a truncated unstable protein as proven by the laminin a2ndashnegativemyofibers in the patientrsquos muscle biopsy The laminin a2ndashpositive immunostaining in the patientrsquos muscle biopsy is due toa novel alternatively spliced exon 31 laminin a2 isoform that skips the nonsense mutation in exon 31

October (2 of 2) 2000 NEUROLOGY 55 1131

nin a2 Laminin a2ndashpositive areas were present in associ-ation with both fiber types I and II (data not shown) Tocorrelate the regeneration status of myofibers with expres-sion of laminin a2 serial cryosections of the patientrsquos mus-cle biopsy done at age 3 were stained with monoclonalantibodies against embryonic myosin heavy chain and the80-kD carboxyl-terminus region of the laminin a2 geneThe frequency of regenerating myofibers in the patientrsquosmuscle biopsy was 2 (532719) of total fibers countedRegenerating fibers were laminin a2 negative Laminin a2immunoblotting using antibodies directed against both the80-kD and 320-kD fragments of laminin a2 failed to showany detectable laminin in the patientrsquos muscle biopsy (datanot shown)

Study of laminin a2 mutations Single-strand confor-mational polymorphism analysis was used to screen forlaminin a2 mutations using muscle RNA from the pa-tientrsquos biopsy The coding sequence of the laminin a2 genewas analyzed using 20 sets of overlapping primer pairsAberrant conformers were identified with primer setsMer4476FndashMer4981R and Mer7015FndashMer7541R Directsequencing of these conformers revealed two nonsense mu-tations C to T at nucleotide position 4694 (R1549X) and Cto T at nucleotide position 7196 (R2383X) of the laminin a2coding sequence (figure 3)

Both mutations were confirmed at the genomic DNAlevel and the inheritance pattern of the mutations deter-mined The C4649T nucleotide change did not cause anyrestriction-enzyme site change thus the amplification-refractory mutation system test was used Primers weredesigned in exon 31 of the laminin a2 gene and mutatedPCR products were present in the patient and her motherNucleotide change C7196T resulted in a gain of an NlaIIIrestriction-enzyme site Primers were designed in exon 49

of the laminin a2 gene and the PCR product was digestedwith NlaIII Digestion fragments were present in the pa-tient and her father

Characterization of an exon 31ndashspliced laminin a2 iso-form The appearance of laminin a2 protein in the patientrsquosmuscle despite the two nonsense mutations suggested thatsome transcripts were excluding one of the two mutationsConsistent with this hypothesis we detected a smallerRT-PCR product over the region of the C4694T mutationin both the patient and control subjects This smallerRT-PCR product was present in the patient and in 10 of 10normal control subjects run in parallel Direct sequencingof the smaller band showed a 138-bp in-frame deletionfrom nucleotides 4629 to 4766 of the laminin a2 codingsequence This deletion corresponded to about 70 of theexon 31 sequence of the laminin a2 gene (figure 4)

Quantitation of the exon31ndashspliced laminin isoformRNA was isolated from the patientrsquos muscle biopsy andfrom eight control muscle biopsies (six with partial laminina2 deficiency one with complete laminin a2 deficiencyand one undetermined myopathy) For each RNA sample100 ng of total RNA underwent RT into cDNA using oligodT10 primers A fluorescent multiplex RT-PCR ampli-fication containing three different PCR products (full-length laminin a2 exon31ndashspliced laminin a2 isoform anda-sarcoglycan) was done in duplicate for each samplePeak heights and areas were calculated and results tabu-lated as ratios between full-length laminin a2 versusa-sarcoglycan and exon 31ndashspliced laminin a2 versusa-sarcoglycan Mean and SE for each ratio were calculatedfor the patient and the control group (figure 5)

The patientrsquos muscle biopsy showed a significant de-crease in full-length laminin a2 RNA relative to the con-trol subjects 24 6 7 versus 100 6 27 ( p 5 0003

Figure 4 Identification of a novelexon 31ndashspliced laminin a2 isoformFull-length laminin a2 and exon 31ndashspliced laminin a2 are shown Thefull-length laminin a2 is the majortranscript in muscle and results fromthe expected splicing between exon 31and 32 Reverse transcription PCRamplification using a reverse comple-mentary DNA primer that spans exon31 and exon 32 (4786R) and a for-ward primer starting at nucleotide4476 shows an reverse transcriptionPCR product of the appropriate size(upper arrow 4476F to 4786R) Theexon 31ndashspliced laminin a2 isoformderives from the use of a cryptic splicesite 57-bp into exon 31 (4629 bp ofRNA) This results in a 138-bp in-frame deletion of the remainder ofexon 31 Sequencing of exon 31 at thegenomic DNA level showed no suchdeletion and 10 of 10 control subjects

showed the same deleted product proving that this was due to normal alternative splicing Reverse transcription PCRamplification using a reverse primer that spanned the spliced region (4784R) with the common 4476F forward primershowed a smaller PCR fragment (lower arrow 4476F to 4784R) corresponding to the 138-bp deletion In both the full-length and exon 31ndashspliced laminin a2 isoforms the nucleotides at the donor splice site are shown Both isoforms show agood match to consensus splicing sequences

1132 NEUROLOGY 55 October (2 of 2) 2000

when ratios were calculated using peak height p 5 0013when ratios were calculated using peak area) The alterna-tively spliced isoform was at similar levels in all musclebiopsies Our patient showed 65 6 8 of control levels ofexon 31ndashspliced laminin a2 RNA (100 6 38)

Discussion Here we describe a novel alterna-tively spliced isoform of the laminin a2 gene in apatient with CMD who was a compound heterozy-gote for two laminin a2 nonsense mutations Onemutation was contained within the 46 amino acidsdeleted in the alternatively spliced isoform Becausethe alternatively spliced isoform removed one of hernonsense mutations this patient was able to produceonly the alternatively spliced isoform with no evi-dence of the full-length isoform This alternativelyspliced product was found in all individuals testedboth control subjects and patients with neuromuscu-lar disease The isoform involved the recognition of acryptic splice site within exon 31 such that the ma-jority of exon 31 was skipped and directly spliced toexon 32 This splicing event removed 46 amino acidsin the cysteine-rich domain IIIa just proximal to thetriple coil-coiled region that associates with the b1and g1 chains of laminin The alternatively splicedisoform removes eight cysteines which are thoughtto participate in the formation of inter- and intramo-lecular disulfide bonds in the basal lamina No sub-function has been assigned to this region

This isoform was localized extracellularly pre-dominantly to discrete highly positive areas in iso-lated myofibers We were unable to determine anycharacteristics of these myofibers that distinguishedthem from the remainder Fiber typing showed thatboth type I (oxidative) and type II (glycolytic) fibersstained highly positively and a neuromuscular junc-

tion localization was ruled out No regenerating fi-bers were laminin a2 positive

Immunostaining for other subunits of lamininshowed a unique downregulation of laminin b1 andg1 and a failure of the upregulation of laminin a5typically seen in patients lacking laminin a2 Thisfinding suggests that the small amount of the novelisoform present in muscle fibers acts as a ldquodominantnegativerdquo protein it prevents the upregulation oflaminin a5 via its presence in muscle yet does notassociate well with either laminin b1 or g1 resultingin their downregulation This suggests that thecysteine-rich region of laminin a2 is important forinteraction with the b and g subunits and that thisnovel isoform alters the binding characteristics Al-ternatively the cysteine-rich region of laminin a2might be crucial for a proper chaperonndashlaminin in-teraction involved in the intracellular transport andassembly of the laminin chains Because b and gchains cannot be delivered to the extracellular ma-trix without their assembly in a abg heterotrimer37

the lack of a proper transport of the a chain in theintracellular milieu results in defective secretion ofthe entire heterotrimer

Our patient showed CNS involvement that wasconsiderably more severe than typically seen in pa-tients with complete laminin a2 deficiency Her MRIwas highly abnormal with tetraventricular dilata-tion white matter alterations pachygyria of the oc-cipital cortex and some small areas of cerebellarpolymicrogyria findings not seen in any other pa-tient with laminin a2 deficiency studied to dateAlso no other patient with laminin a2 deficiency hasbeen reported with mental retardation our patientshowed a moderately low IQ with progressive mentalretardation

It is important to explain the severe CNS involve-ment in this patient Laminin a2 is normally ex-pressed in the inner part of the basal lamina (laminadensa) of striated muscle fibers and in Schwann cellsof peripheral nerves Expression in the CNS is stillnot well studied particularly in regard to neural de-velopment In a laminin a2ndashdeficient patient withCMD the absence of laminin a2 results in a severederangement of the muscle basal lamina Electronmicroscopy shows a discontinuous thinner basallamina with occasional holes in skeletal muscle fi-bers in this category of patients3839 but a normalwell-preserved basement membrane in the intramus-cular nerves39 No EM studies of the basal lamina inbrain of laminin a2ndashdeficient patients with CMDhave been reported therefore there are still too fewdata concerning the role of laminin a2 in brain devel-opment and homeostasis to derive adequate modelsthat explain the severe CNS involvement in thispatient

AcknowledgmentThe authors thank Dr Eva Engvall for critical review of themanuscript

Figure 5 Normal expression of the exon 31ndashspliced lami-nin a2 isoform and reduced expression of the full-lengthlaminin a2 in the patientrsquos muscle Shown are examples ofautomated sequencer traces of quantitative multiplex fluo-rescent reverse transcription PCR of the exon 31ndashsplicedlaminin a2 and full-length laminin a2 isoforms relative toa-sarcoglycan RNA isolated from the patientrsquos muscle bi-opsy consistently showed lower levels of full-length lami-nin a2 RNA relative to control subjects The level of exon31ndashspliced laminin a2 RNA was similar in the patient rel-ative to control subjects

October (2 of 2) 2000 NEUROLOGY 55 1133

References1 Tome FM Evangelista T Leclerc A et al Congenital muscu-

lar dystrophy with merosin deficiency M CR Acad Sci Paris1994317351ndash357

2 HelblingndashLeclerc A Zhang X Topaloglu H et al Mutations inthe laminin a2-chain gene (LAMA2) cause merosin-deficientcongenital muscular dystrophy Nat Genet 199511216ndash218

3 Pegoraro E Marks H Garcia CA et al Laminin a2 musculardystrophy genotypephenotype studies of 22 patients Neurol-ogy 199851101ndash110

4 Ehrig K Leivo I Argraves WS Ruoslahti E Engvall E Mer-osin a tissue-specific basement membrane protein is a lami-nin like protein Proc Natl Acad Sci USA 1990873264ndash3268

5 Voulteenaho R Nissinen M Sainio K et al Human lamininM chain (merosin) complete primary structure chromosomalassignment and expression of the M and A chain in humanfetal tissues J Cell Biol 1994124381ndash394

6 Patton B Miner JH Chiu AY Sanes JR Distribution andfunction of laminins in the neuromuscular system of develop-ing adult and mutant mice J Cell Biol 19971391507ndash1521

7 Dubowitz V Workshop Report 22nd ENMC sponsored work-shop on congenital muscular dystrophy Baarn the Nether-lands May 1993 Neuromuscul Disord 1994475ndash81

8 Reed UC Marie SK Vainzof M et al Congenital musculardystrophy with cerebral white matter hypodensity Correla-tion of clinical features and merosin deficiency Brain Dev19961853ndash58

9 Pini A Merlini L Tome FM Chevallay M Gobbi G Merosin-negative congenital muscular dystrophy occipital epilepsywith periodic spasms and focal cortical dysplasia Report ofthree Italian cases in two families Brain Dev 199618316ndash322

10 Sunada Y Edgar TS Lotz BP Rust RS Campbell KPMerosin-negative congenital muscular dystrophy associatedwith extensive brain abnormalities Neurology 1995452084ndash2089

11 Van der Knaap MS Smit LME Barth PG et al Magneti-cresonance imaging in classification of congenital musculardystrophies with brain abnormalities Ann Neurol 19974250ndash59

12 Hayashi YK Engvall E ArikawandashHirasawa E et al Abnor-mal localization of laminin subunits in muscular dystrophiesJ Neurol Sci 199311953ndash64

13 Haltia M Leivo I Somer H et al Musclendasheyendashbrain diseasea neuropathological study Ann Neurol 199741173ndash180

14 Kanoff RJ Curless RG Petito C Falcone S Siatkowski RMPegoraro E WalkerndashWarburg syndrome neurological featuresand muscle membrane structure Ped Neurol 19981876ndash80

15 Villanova M Sabatelli P He Y et al Immunofluorescencestudy of a muscle biopsy from a 1-year-old patient with Walk-erndashWarburg syndrome Acta Neuropathol 199896651ndash654

16 Topaloglu H Talim B Vignier N et al Merosin-deficient con-genital muscular dystrophy with severe mental retardationand normal cranial MRI a report of two siblings Neuromus-cul Disord 19988169ndash174

17 Brockington M Sewry CA Hermann R et al Assignment of aform of congenital muscular dystrophy with secondary mer-osin deficiency to chromosome 1q42 Am J Hum Genet 200066428ndash435

18 Sewry CA Naom I DrsquoAlessandro M et al Variable clinicalphenotype in merosin-deficient congenital muscular dystrophyassociated with differential immunolabelling of two fragmentsof the laminin a2 chain Neuromuscul Disord 19977169ndash175

19 Tan E Topaloglu H Sewry C et al Late onset musculardystrophy with cerebral white matter changes due to partialmerosin deficiency Neuromuscul Disord 1997785ndash89

20 Naom I DrsquoAlessandro M Topaloglu H et al Refinement ofthe laminin a2 chain locus to human chromosome 6q2 insevere and mild merosin deficient congenital muscular dystro-phy J Med Genet 19973499ndash104

21 Nissinen M HelblingndashLeclerc A Zhang X et al Substitutionof a conserved cysteine-996 in a cysteine-rich motif of thelaminin a2-chain in congenital muscular dystrophy with par-tial deficiency of the protein Am J Hum Genet 1996581177ndash1184

22 Allamand V Sunada Y Salih MAM et al Mild congenitalmuscular dystrophy in two patients with an internally deletedlaminin a2-chain Hum Mol Genet 19976747ndash752

23 Hayashi YK Ishihara T Domen K Hori H Arahata K Abenign allelic form of laminin a2 chain deficient musculardystrophy Lancet 19973491147

24 Naom I DrsquoAlessandro M Sewry CA et al Laminin a2-chaingene mutations in two siblings presenting with limb-girdlemuscular dystrophy Neuromuscul Disord 19988495ndash501

25 Naom I DrsquoAlessandro M Sewry CA et al Mutations in thelaminin a2-chain gene in two children with early-onset mus-cular dystrophy Brain 200012331ndash41

26 Trevisan CP Martinello F Fanin M et al Merosin expressionin muscle of western case with Fukuyama-like congenitalmuscular dystrophy Basic Appl Myol 19966101ndash106

27 Hori H Kanamori T Mizuta T Yamaguchi N Liu Y Nagai YHuman laminin M chain epitope analysis of its monoclonalantibodies by immunoscreening of cDNA clones and tissueexpression J Biochem 19941161212ndash1219

28 Schiaffino S Gorza L Pitton G et al Embryonic and neonatalmyosin heavy chain in denervated and paralyzed rat skeletalmuscle Dev Biol 19881271ndash11

29 Byers TJ Kunkel LM Watkins SC The subcellular distribu-tion of dystrophin in mouse skeletal cardiac and smoothmuscle J Cell Biol 1991115411ndash421

30 Tiger CF Champliaud MF Pedrosadomellof F Thornell LEEkblom P Gullberg D Presence of laminin alpha-5 chain andlack of laminin alpha-1 chain during human muscle develop-ment and in muscular dystrophies J Biol Chem 199727228590ndash28595

31 Pegoraro E Mancias P Swerdlow SH et al Laminin a2 (mer-osin) gene mutations cause congenital muscular dystrophy(CMD) Ann Neurol 199640782ndash791

32 Miller SA Dykes DD Polesky HF A simple salting out proce-dure for extracting DNA from human nucleated cells NucleicAcids Res 1988161215

33 Zhang X Vuolteenaho R Tryggvason K Structure of the hu-man laminin a2-chain gene (LAMA2) which is affected incongenital muscular dystrophy J Biol Chem 199627127664ndash27669

34 Little S Amplification-refractory mutation system (ARMS)analysis of point mutations In Dracopoli NC Haines JL KorfBR et al eds Current protocols in human genetics NewYork John Wiley 1994981ndash985

35 Zhou J Hoffman EP Pathophysiology of sodium channelopa-thies Studies of sodium channel expression by quantitativemultiplex fluorescence polymerase chain reaction J BiolChem 199426918563ndash18571

36 Morrone A Pegoraro E Angelini C Zammarchi E Marconi GHoffman EP RNA metabolism in myotonic dystrophy Patientmuscle shows decreased insulin receptor RNA and proteinconsistent with abnormal insulin resistance J Clin Invest1997991691ndash1698

37 Yurchenco PD Quan Y Colognato H et al The alpha chain oflaminin-1 is independently secreted and drives secretion of itsbeta- and gamma-chain partners Proc Natl Acad Sci USA19979410189ndash10194

38 Minetti C Bado M Morreale G Pedemonte M Cordone GDisruption of muscle basal lamina in congenital muscular dys-trophy with merosin deficiency Neurology 1996461354ndash1358

39 Osari S Kobayashi O Yamashita Y et al Basement mem-brane abnormality in merosin-negative congenital musculardystrophy Acta Neuropathol 199691332ndash336

1134 NEUROLOGY 55 October (2 of 2) 2000

DOI 101212WNL55811282000551128-1134 Neurology

E Pegoraro M Fanin CP Trevisan et al dystrophy

2 deficient congenital muscularα2 isoform in severe laminin αA novel laminin

This information is current as of October 24 2000

ServicesUpdated Information amp

httpwwwneurologyorgcontent5581128fullhtmlincluding high resolution figures can be found at

References

httpwwwneurologyorgcontent5581128fullhtmlref-list-1at This article cites 37 articles 16 of which you can access for free

Citations

cleshttpwwwneurologyorgcontent5581128fullhtmlotherartiThis article has been cited by 5 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

Figure 2 Immunofluorescence study of laminin subunit expression in the patient muscle biopsy relative to normal con-trol laminin a2ndashnegative and laminin a2ndashpositive congenital muscular dystrophy (CMD) muscle biopsies (A E I andO) Normal muscle biopsy (B F L and P) Patient with laminin a2ndashnegative CMD (C G M and Q) Patient with laminina2ndashpositive CMD (D H N and R) Muscle biopsy from our reported patient (E through H) Laminin a2 immunostainingusing an antibody directed against the 80-kD carboxyl-terminus of the protein shows discrete areas of highly positive im-munostaining in few myofibers in the patientrsquos muscle biopsy whereas the majority of myofibers are largely laminin a2negative (H) Laminin a2 was normal in the control (E) and laminin a2ndashpositive CMD muscle biopsy (G) and completelynegative in the laminin a2ndashdeficient CMD muscle biopsy Study of the expression of a5 b1 and g1 chains showed thatour patientrsquos muscle does not overexpress laminin a5 (D) as in the patient with laminin a2ndashnegative CMD (B) Our pa-tient also shows a marked decrease in b1 and to a lesser degree g1 immunostaining (N and R) relative to control andlaminin a2ndashnegative and ndashpositive CMD muscle biopsies

Figure 3 Identification of two non-sense mutations in the laminin a2complementary DNA in the patient(Top) Schematic of the exon structureof the laminin a2 gene Exons are rep-resented as boxes introns as linesThe sequencer traces corresponding totwo aberrant conformers show two nu-cleotide changes C4694T in exon 31and C7196T in exon 49 Both result ina stop codon (R1549X and R2383X)(Bottom) The maternal laminin a2gene with a stop codon in exon 31 andthe paternal laminin a2 gene with astop codon in exon 49 are representedShown in the insert is immunostain-ing of the patientrsquos muscle biopsy withlaminin a2 Both paternal and mater-

nal laminin a2 genes are transcribed or translated in a truncated unstable protein as proven by the laminin a2ndashnegativemyofibers in the patientrsquos muscle biopsy The laminin a2ndashpositive immunostaining in the patientrsquos muscle biopsy is due toa novel alternatively spliced exon 31 laminin a2 isoform that skips the nonsense mutation in exon 31

October (2 of 2) 2000 NEUROLOGY 55 1131

nin a2 Laminin a2ndashpositive areas were present in associ-ation with both fiber types I and II (data not shown) Tocorrelate the regeneration status of myofibers with expres-sion of laminin a2 serial cryosections of the patientrsquos mus-cle biopsy done at age 3 were stained with monoclonalantibodies against embryonic myosin heavy chain and the80-kD carboxyl-terminus region of the laminin a2 geneThe frequency of regenerating myofibers in the patientrsquosmuscle biopsy was 2 (532719) of total fibers countedRegenerating fibers were laminin a2 negative Laminin a2immunoblotting using antibodies directed against both the80-kD and 320-kD fragments of laminin a2 failed to showany detectable laminin in the patientrsquos muscle biopsy (datanot shown)

Study of laminin a2 mutations Single-strand confor-mational polymorphism analysis was used to screen forlaminin a2 mutations using muscle RNA from the pa-tientrsquos biopsy The coding sequence of the laminin a2 genewas analyzed using 20 sets of overlapping primer pairsAberrant conformers were identified with primer setsMer4476FndashMer4981R and Mer7015FndashMer7541R Directsequencing of these conformers revealed two nonsense mu-tations C to T at nucleotide position 4694 (R1549X) and Cto T at nucleotide position 7196 (R2383X) of the laminin a2coding sequence (figure 3)

Both mutations were confirmed at the genomic DNAlevel and the inheritance pattern of the mutations deter-mined The C4649T nucleotide change did not cause anyrestriction-enzyme site change thus the amplification-refractory mutation system test was used Primers weredesigned in exon 31 of the laminin a2 gene and mutatedPCR products were present in the patient and her motherNucleotide change C7196T resulted in a gain of an NlaIIIrestriction-enzyme site Primers were designed in exon 49

of the laminin a2 gene and the PCR product was digestedwith NlaIII Digestion fragments were present in the pa-tient and her father

Characterization of an exon 31ndashspliced laminin a2 iso-form The appearance of laminin a2 protein in the patientrsquosmuscle despite the two nonsense mutations suggested thatsome transcripts were excluding one of the two mutationsConsistent with this hypothesis we detected a smallerRT-PCR product over the region of the C4694T mutationin both the patient and control subjects This smallerRT-PCR product was present in the patient and in 10 of 10normal control subjects run in parallel Direct sequencingof the smaller band showed a 138-bp in-frame deletionfrom nucleotides 4629 to 4766 of the laminin a2 codingsequence This deletion corresponded to about 70 of theexon 31 sequence of the laminin a2 gene (figure 4)

Quantitation of the exon31ndashspliced laminin isoformRNA was isolated from the patientrsquos muscle biopsy andfrom eight control muscle biopsies (six with partial laminina2 deficiency one with complete laminin a2 deficiencyand one undetermined myopathy) For each RNA sample100 ng of total RNA underwent RT into cDNA using oligodT10 primers A fluorescent multiplex RT-PCR ampli-fication containing three different PCR products (full-length laminin a2 exon31ndashspliced laminin a2 isoform anda-sarcoglycan) was done in duplicate for each samplePeak heights and areas were calculated and results tabu-lated as ratios between full-length laminin a2 versusa-sarcoglycan and exon 31ndashspliced laminin a2 versusa-sarcoglycan Mean and SE for each ratio were calculatedfor the patient and the control group (figure 5)

The patientrsquos muscle biopsy showed a significant de-crease in full-length laminin a2 RNA relative to the con-trol subjects 24 6 7 versus 100 6 27 ( p 5 0003

Figure 4 Identification of a novelexon 31ndashspliced laminin a2 isoformFull-length laminin a2 and exon 31ndashspliced laminin a2 are shown Thefull-length laminin a2 is the majortranscript in muscle and results fromthe expected splicing between exon 31and 32 Reverse transcription PCRamplification using a reverse comple-mentary DNA primer that spans exon31 and exon 32 (4786R) and a for-ward primer starting at nucleotide4476 shows an reverse transcriptionPCR product of the appropriate size(upper arrow 4476F to 4786R) Theexon 31ndashspliced laminin a2 isoformderives from the use of a cryptic splicesite 57-bp into exon 31 (4629 bp ofRNA) This results in a 138-bp in-frame deletion of the remainder ofexon 31 Sequencing of exon 31 at thegenomic DNA level showed no suchdeletion and 10 of 10 control subjects

showed the same deleted product proving that this was due to normal alternative splicing Reverse transcription PCRamplification using a reverse primer that spanned the spliced region (4784R) with the common 4476F forward primershowed a smaller PCR fragment (lower arrow 4476F to 4784R) corresponding to the 138-bp deletion In both the full-length and exon 31ndashspliced laminin a2 isoforms the nucleotides at the donor splice site are shown Both isoforms show agood match to consensus splicing sequences

1132 NEUROLOGY 55 October (2 of 2) 2000

when ratios were calculated using peak height p 5 0013when ratios were calculated using peak area) The alterna-tively spliced isoform was at similar levels in all musclebiopsies Our patient showed 65 6 8 of control levels ofexon 31ndashspliced laminin a2 RNA (100 6 38)

Discussion Here we describe a novel alterna-tively spliced isoform of the laminin a2 gene in apatient with CMD who was a compound heterozy-gote for two laminin a2 nonsense mutations Onemutation was contained within the 46 amino acidsdeleted in the alternatively spliced isoform Becausethe alternatively spliced isoform removed one of hernonsense mutations this patient was able to produceonly the alternatively spliced isoform with no evi-dence of the full-length isoform This alternativelyspliced product was found in all individuals testedboth control subjects and patients with neuromuscu-lar disease The isoform involved the recognition of acryptic splice site within exon 31 such that the ma-jority of exon 31 was skipped and directly spliced toexon 32 This splicing event removed 46 amino acidsin the cysteine-rich domain IIIa just proximal to thetriple coil-coiled region that associates with the b1and g1 chains of laminin The alternatively splicedisoform removes eight cysteines which are thoughtto participate in the formation of inter- and intramo-lecular disulfide bonds in the basal lamina No sub-function has been assigned to this region

This isoform was localized extracellularly pre-dominantly to discrete highly positive areas in iso-lated myofibers We were unable to determine anycharacteristics of these myofibers that distinguishedthem from the remainder Fiber typing showed thatboth type I (oxidative) and type II (glycolytic) fibersstained highly positively and a neuromuscular junc-

tion localization was ruled out No regenerating fi-bers were laminin a2 positive

Immunostaining for other subunits of lamininshowed a unique downregulation of laminin b1 andg1 and a failure of the upregulation of laminin a5typically seen in patients lacking laminin a2 Thisfinding suggests that the small amount of the novelisoform present in muscle fibers acts as a ldquodominantnegativerdquo protein it prevents the upregulation oflaminin a5 via its presence in muscle yet does notassociate well with either laminin b1 or g1 resultingin their downregulation This suggests that thecysteine-rich region of laminin a2 is important forinteraction with the b and g subunits and that thisnovel isoform alters the binding characteristics Al-ternatively the cysteine-rich region of laminin a2might be crucial for a proper chaperonndashlaminin in-teraction involved in the intracellular transport andassembly of the laminin chains Because b and gchains cannot be delivered to the extracellular ma-trix without their assembly in a abg heterotrimer37

the lack of a proper transport of the a chain in theintracellular milieu results in defective secretion ofthe entire heterotrimer

Our patient showed CNS involvement that wasconsiderably more severe than typically seen in pa-tients with complete laminin a2 deficiency Her MRIwas highly abnormal with tetraventricular dilata-tion white matter alterations pachygyria of the oc-cipital cortex and some small areas of cerebellarpolymicrogyria findings not seen in any other pa-tient with laminin a2 deficiency studied to dateAlso no other patient with laminin a2 deficiency hasbeen reported with mental retardation our patientshowed a moderately low IQ with progressive mentalretardation

It is important to explain the severe CNS involve-ment in this patient Laminin a2 is normally ex-pressed in the inner part of the basal lamina (laminadensa) of striated muscle fibers and in Schwann cellsof peripheral nerves Expression in the CNS is stillnot well studied particularly in regard to neural de-velopment In a laminin a2ndashdeficient patient withCMD the absence of laminin a2 results in a severederangement of the muscle basal lamina Electronmicroscopy shows a discontinuous thinner basallamina with occasional holes in skeletal muscle fi-bers in this category of patients3839 but a normalwell-preserved basement membrane in the intramus-cular nerves39 No EM studies of the basal lamina inbrain of laminin a2ndashdeficient patients with CMDhave been reported therefore there are still too fewdata concerning the role of laminin a2 in brain devel-opment and homeostasis to derive adequate modelsthat explain the severe CNS involvement in thispatient

AcknowledgmentThe authors thank Dr Eva Engvall for critical review of themanuscript

Figure 5 Normal expression of the exon 31ndashspliced lami-nin a2 isoform and reduced expression of the full-lengthlaminin a2 in the patientrsquos muscle Shown are examples ofautomated sequencer traces of quantitative multiplex fluo-rescent reverse transcription PCR of the exon 31ndashsplicedlaminin a2 and full-length laminin a2 isoforms relative toa-sarcoglycan RNA isolated from the patientrsquos muscle bi-opsy consistently showed lower levels of full-length lami-nin a2 RNA relative to control subjects The level of exon31ndashspliced laminin a2 RNA was similar in the patient rel-ative to control subjects

October (2 of 2) 2000 NEUROLOGY 55 1133

References1 Tome FM Evangelista T Leclerc A et al Congenital muscu-

lar dystrophy with merosin deficiency M CR Acad Sci Paris1994317351ndash357

2 HelblingndashLeclerc A Zhang X Topaloglu H et al Mutations inthe laminin a2-chain gene (LAMA2) cause merosin-deficientcongenital muscular dystrophy Nat Genet 199511216ndash218

3 Pegoraro E Marks H Garcia CA et al Laminin a2 musculardystrophy genotypephenotype studies of 22 patients Neurol-ogy 199851101ndash110

4 Ehrig K Leivo I Argraves WS Ruoslahti E Engvall E Mer-osin a tissue-specific basement membrane protein is a lami-nin like protein Proc Natl Acad Sci USA 1990873264ndash3268

5 Voulteenaho R Nissinen M Sainio K et al Human lamininM chain (merosin) complete primary structure chromosomalassignment and expression of the M and A chain in humanfetal tissues J Cell Biol 1994124381ndash394

6 Patton B Miner JH Chiu AY Sanes JR Distribution andfunction of laminins in the neuromuscular system of develop-ing adult and mutant mice J Cell Biol 19971391507ndash1521

7 Dubowitz V Workshop Report 22nd ENMC sponsored work-shop on congenital muscular dystrophy Baarn the Nether-lands May 1993 Neuromuscul Disord 1994475ndash81

8 Reed UC Marie SK Vainzof M et al Congenital musculardystrophy with cerebral white matter hypodensity Correla-tion of clinical features and merosin deficiency Brain Dev19961853ndash58

9 Pini A Merlini L Tome FM Chevallay M Gobbi G Merosin-negative congenital muscular dystrophy occipital epilepsywith periodic spasms and focal cortical dysplasia Report ofthree Italian cases in two families Brain Dev 199618316ndash322

10 Sunada Y Edgar TS Lotz BP Rust RS Campbell KPMerosin-negative congenital muscular dystrophy associatedwith extensive brain abnormalities Neurology 1995452084ndash2089

11 Van der Knaap MS Smit LME Barth PG et al Magneti-cresonance imaging in classification of congenital musculardystrophies with brain abnormalities Ann Neurol 19974250ndash59

12 Hayashi YK Engvall E ArikawandashHirasawa E et al Abnor-mal localization of laminin subunits in muscular dystrophiesJ Neurol Sci 199311953ndash64

13 Haltia M Leivo I Somer H et al Musclendasheyendashbrain diseasea neuropathological study Ann Neurol 199741173ndash180

14 Kanoff RJ Curless RG Petito C Falcone S Siatkowski RMPegoraro E WalkerndashWarburg syndrome neurological featuresand muscle membrane structure Ped Neurol 19981876ndash80

15 Villanova M Sabatelli P He Y et al Immunofluorescencestudy of a muscle biopsy from a 1-year-old patient with Walk-erndashWarburg syndrome Acta Neuropathol 199896651ndash654

16 Topaloglu H Talim B Vignier N et al Merosin-deficient con-genital muscular dystrophy with severe mental retardationand normal cranial MRI a report of two siblings Neuromus-cul Disord 19988169ndash174

17 Brockington M Sewry CA Hermann R et al Assignment of aform of congenital muscular dystrophy with secondary mer-osin deficiency to chromosome 1q42 Am J Hum Genet 200066428ndash435

18 Sewry CA Naom I DrsquoAlessandro M et al Variable clinicalphenotype in merosin-deficient congenital muscular dystrophyassociated with differential immunolabelling of two fragmentsof the laminin a2 chain Neuromuscul Disord 19977169ndash175

19 Tan E Topaloglu H Sewry C et al Late onset musculardystrophy with cerebral white matter changes due to partialmerosin deficiency Neuromuscul Disord 1997785ndash89

20 Naom I DrsquoAlessandro M Topaloglu H et al Refinement ofthe laminin a2 chain locus to human chromosome 6q2 insevere and mild merosin deficient congenital muscular dystro-phy J Med Genet 19973499ndash104

21 Nissinen M HelblingndashLeclerc A Zhang X et al Substitutionof a conserved cysteine-996 in a cysteine-rich motif of thelaminin a2-chain in congenital muscular dystrophy with par-tial deficiency of the protein Am J Hum Genet 1996581177ndash1184

22 Allamand V Sunada Y Salih MAM et al Mild congenitalmuscular dystrophy in two patients with an internally deletedlaminin a2-chain Hum Mol Genet 19976747ndash752

23 Hayashi YK Ishihara T Domen K Hori H Arahata K Abenign allelic form of laminin a2 chain deficient musculardystrophy Lancet 19973491147

24 Naom I DrsquoAlessandro M Sewry CA et al Laminin a2-chaingene mutations in two siblings presenting with limb-girdlemuscular dystrophy Neuromuscul Disord 19988495ndash501

25 Naom I DrsquoAlessandro M Sewry CA et al Mutations in thelaminin a2-chain gene in two children with early-onset mus-cular dystrophy Brain 200012331ndash41

26 Trevisan CP Martinello F Fanin M et al Merosin expressionin muscle of western case with Fukuyama-like congenitalmuscular dystrophy Basic Appl Myol 19966101ndash106

27 Hori H Kanamori T Mizuta T Yamaguchi N Liu Y Nagai YHuman laminin M chain epitope analysis of its monoclonalantibodies by immunoscreening of cDNA clones and tissueexpression J Biochem 19941161212ndash1219

28 Schiaffino S Gorza L Pitton G et al Embryonic and neonatalmyosin heavy chain in denervated and paralyzed rat skeletalmuscle Dev Biol 19881271ndash11

29 Byers TJ Kunkel LM Watkins SC The subcellular distribu-tion of dystrophin in mouse skeletal cardiac and smoothmuscle J Cell Biol 1991115411ndash421

30 Tiger CF Champliaud MF Pedrosadomellof F Thornell LEEkblom P Gullberg D Presence of laminin alpha-5 chain andlack of laminin alpha-1 chain during human muscle develop-ment and in muscular dystrophies J Biol Chem 199727228590ndash28595

31 Pegoraro E Mancias P Swerdlow SH et al Laminin a2 (mer-osin) gene mutations cause congenital muscular dystrophy(CMD) Ann Neurol 199640782ndash791

32 Miller SA Dykes DD Polesky HF A simple salting out proce-dure for extracting DNA from human nucleated cells NucleicAcids Res 1988161215

33 Zhang X Vuolteenaho R Tryggvason K Structure of the hu-man laminin a2-chain gene (LAMA2) which is affected incongenital muscular dystrophy J Biol Chem 199627127664ndash27669

34 Little S Amplification-refractory mutation system (ARMS)analysis of point mutations In Dracopoli NC Haines JL KorfBR et al eds Current protocols in human genetics NewYork John Wiley 1994981ndash985

35 Zhou J Hoffman EP Pathophysiology of sodium channelopa-thies Studies of sodium channel expression by quantitativemultiplex fluorescence polymerase chain reaction J BiolChem 199426918563ndash18571

36 Morrone A Pegoraro E Angelini C Zammarchi E Marconi GHoffman EP RNA metabolism in myotonic dystrophy Patientmuscle shows decreased insulin receptor RNA and proteinconsistent with abnormal insulin resistance J Clin Invest1997991691ndash1698

37 Yurchenco PD Quan Y Colognato H et al The alpha chain oflaminin-1 is independently secreted and drives secretion of itsbeta- and gamma-chain partners Proc Natl Acad Sci USA19979410189ndash10194

38 Minetti C Bado M Morreale G Pedemonte M Cordone GDisruption of muscle basal lamina in congenital muscular dys-trophy with merosin deficiency Neurology 1996461354ndash1358

39 Osari S Kobayashi O Yamashita Y et al Basement mem-brane abnormality in merosin-negative congenital musculardystrophy Acta Neuropathol 199691332ndash336

1134 NEUROLOGY 55 October (2 of 2) 2000

DOI 101212WNL55811282000551128-1134 Neurology

E Pegoraro M Fanin CP Trevisan et al dystrophy

2 deficient congenital muscularα2 isoform in severe laminin αA novel laminin

This information is current as of October 24 2000

ServicesUpdated Information amp

httpwwwneurologyorgcontent5581128fullhtmlincluding high resolution figures can be found at

References

httpwwwneurologyorgcontent5581128fullhtmlref-list-1at This article cites 37 articles 16 of which you can access for free

Citations

cleshttpwwwneurologyorgcontent5581128fullhtmlotherartiThis article has been cited by 5 HighWire-hosted articles

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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

nin a2 Laminin a2ndashpositive areas were present in associ-ation with both fiber types I and II (data not shown) Tocorrelate the regeneration status of myofibers with expres-sion of laminin a2 serial cryosections of the patientrsquos mus-cle biopsy done at age 3 were stained with monoclonalantibodies against embryonic myosin heavy chain and the80-kD carboxyl-terminus region of the laminin a2 geneThe frequency of regenerating myofibers in the patientrsquosmuscle biopsy was 2 (532719) of total fibers countedRegenerating fibers were laminin a2 negative Laminin a2immunoblotting using antibodies directed against both the80-kD and 320-kD fragments of laminin a2 failed to showany detectable laminin in the patientrsquos muscle biopsy (datanot shown)

Study of laminin a2 mutations Single-strand confor-mational polymorphism analysis was used to screen forlaminin a2 mutations using muscle RNA from the pa-tientrsquos biopsy The coding sequence of the laminin a2 genewas analyzed using 20 sets of overlapping primer pairsAberrant conformers were identified with primer setsMer4476FndashMer4981R and Mer7015FndashMer7541R Directsequencing of these conformers revealed two nonsense mu-tations C to T at nucleotide position 4694 (R1549X) and Cto T at nucleotide position 7196 (R2383X) of the laminin a2coding sequence (figure 3)

Both mutations were confirmed at the genomic DNAlevel and the inheritance pattern of the mutations deter-mined The C4649T nucleotide change did not cause anyrestriction-enzyme site change thus the amplification-refractory mutation system test was used Primers weredesigned in exon 31 of the laminin a2 gene and mutatedPCR products were present in the patient and her motherNucleotide change C7196T resulted in a gain of an NlaIIIrestriction-enzyme site Primers were designed in exon 49

of the laminin a2 gene and the PCR product was digestedwith NlaIII Digestion fragments were present in the pa-tient and her father

Characterization of an exon 31ndashspliced laminin a2 iso-form The appearance of laminin a2 protein in the patientrsquosmuscle despite the two nonsense mutations suggested thatsome transcripts were excluding one of the two mutationsConsistent with this hypothesis we detected a smallerRT-PCR product over the region of the C4694T mutationin both the patient and control subjects This smallerRT-PCR product was present in the patient and in 10 of 10normal control subjects run in parallel Direct sequencingof the smaller band showed a 138-bp in-frame deletionfrom nucleotides 4629 to 4766 of the laminin a2 codingsequence This deletion corresponded to about 70 of theexon 31 sequence of the laminin a2 gene (figure 4)

Quantitation of the exon31ndashspliced laminin isoformRNA was isolated from the patientrsquos muscle biopsy andfrom eight control muscle biopsies (six with partial laminina2 deficiency one with complete laminin a2 deficiencyand one undetermined myopathy) For each RNA sample100 ng of total RNA underwent RT into cDNA using oligodT10 primers A fluorescent multiplex RT-PCR ampli-fication containing three different PCR products (full-length laminin a2 exon31ndashspliced laminin a2 isoform anda-sarcoglycan) was done in duplicate for each samplePeak heights and areas were calculated and results tabu-lated as ratios between full-length laminin a2 versusa-sarcoglycan and exon 31ndashspliced laminin a2 versusa-sarcoglycan Mean and SE for each ratio were calculatedfor the patient and the control group (figure 5)

The patientrsquos muscle biopsy showed a significant de-crease in full-length laminin a2 RNA relative to the con-trol subjects 24 6 7 versus 100 6 27 ( p 5 0003

Figure 4 Identification of a novelexon 31ndashspliced laminin a2 isoformFull-length laminin a2 and exon 31ndashspliced laminin a2 are shown Thefull-length laminin a2 is the majortranscript in muscle and results fromthe expected splicing between exon 31and 32 Reverse transcription PCRamplification using a reverse comple-mentary DNA primer that spans exon31 and exon 32 (4786R) and a for-ward primer starting at nucleotide4476 shows an reverse transcriptionPCR product of the appropriate size(upper arrow 4476F to 4786R) Theexon 31ndashspliced laminin a2 isoformderives from the use of a cryptic splicesite 57-bp into exon 31 (4629 bp ofRNA) This results in a 138-bp in-frame deletion of the remainder ofexon 31 Sequencing of exon 31 at thegenomic DNA level showed no suchdeletion and 10 of 10 control subjects

showed the same deleted product proving that this was due to normal alternative splicing Reverse transcription PCRamplification using a reverse primer that spanned the spliced region (4784R) with the common 4476F forward primershowed a smaller PCR fragment (lower arrow 4476F to 4784R) corresponding to the 138-bp deletion In both the full-length and exon 31ndashspliced laminin a2 isoforms the nucleotides at the donor splice site are shown Both isoforms show agood match to consensus splicing sequences

1132 NEUROLOGY 55 October (2 of 2) 2000

when ratios were calculated using peak height p 5 0013when ratios were calculated using peak area) The alterna-tively spliced isoform was at similar levels in all musclebiopsies Our patient showed 65 6 8 of control levels ofexon 31ndashspliced laminin a2 RNA (100 6 38)

Discussion Here we describe a novel alterna-tively spliced isoform of the laminin a2 gene in apatient with CMD who was a compound heterozy-gote for two laminin a2 nonsense mutations Onemutation was contained within the 46 amino acidsdeleted in the alternatively spliced isoform Becausethe alternatively spliced isoform removed one of hernonsense mutations this patient was able to produceonly the alternatively spliced isoform with no evi-dence of the full-length isoform This alternativelyspliced product was found in all individuals testedboth control subjects and patients with neuromuscu-lar disease The isoform involved the recognition of acryptic splice site within exon 31 such that the ma-jority of exon 31 was skipped and directly spliced toexon 32 This splicing event removed 46 amino acidsin the cysteine-rich domain IIIa just proximal to thetriple coil-coiled region that associates with the b1and g1 chains of laminin The alternatively splicedisoform removes eight cysteines which are thoughtto participate in the formation of inter- and intramo-lecular disulfide bonds in the basal lamina No sub-function has been assigned to this region

This isoform was localized extracellularly pre-dominantly to discrete highly positive areas in iso-lated myofibers We were unable to determine anycharacteristics of these myofibers that distinguishedthem from the remainder Fiber typing showed thatboth type I (oxidative) and type II (glycolytic) fibersstained highly positively and a neuromuscular junc-

tion localization was ruled out No regenerating fi-bers were laminin a2 positive

Immunostaining for other subunits of lamininshowed a unique downregulation of laminin b1 andg1 and a failure of the upregulation of laminin a5typically seen in patients lacking laminin a2 Thisfinding suggests that the small amount of the novelisoform present in muscle fibers acts as a ldquodominantnegativerdquo protein it prevents the upregulation oflaminin a5 via its presence in muscle yet does notassociate well with either laminin b1 or g1 resultingin their downregulation This suggests that thecysteine-rich region of laminin a2 is important forinteraction with the b and g subunits and that thisnovel isoform alters the binding characteristics Al-ternatively the cysteine-rich region of laminin a2might be crucial for a proper chaperonndashlaminin in-teraction involved in the intracellular transport andassembly of the laminin chains Because b and gchains cannot be delivered to the extracellular ma-trix without their assembly in a abg heterotrimer37

the lack of a proper transport of the a chain in theintracellular milieu results in defective secretion ofthe entire heterotrimer

Our patient showed CNS involvement that wasconsiderably more severe than typically seen in pa-tients with complete laminin a2 deficiency Her MRIwas highly abnormal with tetraventricular dilata-tion white matter alterations pachygyria of the oc-cipital cortex and some small areas of cerebellarpolymicrogyria findings not seen in any other pa-tient with laminin a2 deficiency studied to dateAlso no other patient with laminin a2 deficiency hasbeen reported with mental retardation our patientshowed a moderately low IQ with progressive mentalretardation

It is important to explain the severe CNS involve-ment in this patient Laminin a2 is normally ex-pressed in the inner part of the basal lamina (laminadensa) of striated muscle fibers and in Schwann cellsof peripheral nerves Expression in the CNS is stillnot well studied particularly in regard to neural de-velopment In a laminin a2ndashdeficient patient withCMD the absence of laminin a2 results in a severederangement of the muscle basal lamina Electronmicroscopy shows a discontinuous thinner basallamina with occasional holes in skeletal muscle fi-bers in this category of patients3839 but a normalwell-preserved basement membrane in the intramus-cular nerves39 No EM studies of the basal lamina inbrain of laminin a2ndashdeficient patients with CMDhave been reported therefore there are still too fewdata concerning the role of laminin a2 in brain devel-opment and homeostasis to derive adequate modelsthat explain the severe CNS involvement in thispatient

AcknowledgmentThe authors thank Dr Eva Engvall for critical review of themanuscript

Figure 5 Normal expression of the exon 31ndashspliced lami-nin a2 isoform and reduced expression of the full-lengthlaminin a2 in the patientrsquos muscle Shown are examples ofautomated sequencer traces of quantitative multiplex fluo-rescent reverse transcription PCR of the exon 31ndashsplicedlaminin a2 and full-length laminin a2 isoforms relative toa-sarcoglycan RNA isolated from the patientrsquos muscle bi-opsy consistently showed lower levels of full-length lami-nin a2 RNA relative to control subjects The level of exon31ndashspliced laminin a2 RNA was similar in the patient rel-ative to control subjects

October (2 of 2) 2000 NEUROLOGY 55 1133

References1 Tome FM Evangelista T Leclerc A et al Congenital muscu-

lar dystrophy with merosin deficiency M CR Acad Sci Paris1994317351ndash357

2 HelblingndashLeclerc A Zhang X Topaloglu H et al Mutations inthe laminin a2-chain gene (LAMA2) cause merosin-deficientcongenital muscular dystrophy Nat Genet 199511216ndash218

3 Pegoraro E Marks H Garcia CA et al Laminin a2 musculardystrophy genotypephenotype studies of 22 patients Neurol-ogy 199851101ndash110

4 Ehrig K Leivo I Argraves WS Ruoslahti E Engvall E Mer-osin a tissue-specific basement membrane protein is a lami-nin like protein Proc Natl Acad Sci USA 1990873264ndash3268

5 Voulteenaho R Nissinen M Sainio K et al Human lamininM chain (merosin) complete primary structure chromosomalassignment and expression of the M and A chain in humanfetal tissues J Cell Biol 1994124381ndash394

6 Patton B Miner JH Chiu AY Sanes JR Distribution andfunction of laminins in the neuromuscular system of develop-ing adult and mutant mice J Cell Biol 19971391507ndash1521

7 Dubowitz V Workshop Report 22nd ENMC sponsored work-shop on congenital muscular dystrophy Baarn the Nether-lands May 1993 Neuromuscul Disord 1994475ndash81

8 Reed UC Marie SK Vainzof M et al Congenital musculardystrophy with cerebral white matter hypodensity Correla-tion of clinical features and merosin deficiency Brain Dev19961853ndash58

9 Pini A Merlini L Tome FM Chevallay M Gobbi G Merosin-negative congenital muscular dystrophy occipital epilepsywith periodic spasms and focal cortical dysplasia Report ofthree Italian cases in two families Brain Dev 199618316ndash322

10 Sunada Y Edgar TS Lotz BP Rust RS Campbell KPMerosin-negative congenital muscular dystrophy associatedwith extensive brain abnormalities Neurology 1995452084ndash2089

11 Van der Knaap MS Smit LME Barth PG et al Magneti-cresonance imaging in classification of congenital musculardystrophies with brain abnormalities Ann Neurol 19974250ndash59

12 Hayashi YK Engvall E ArikawandashHirasawa E et al Abnor-mal localization of laminin subunits in muscular dystrophiesJ Neurol Sci 199311953ndash64

13 Haltia M Leivo I Somer H et al Musclendasheyendashbrain diseasea neuropathological study Ann Neurol 199741173ndash180

14 Kanoff RJ Curless RG Petito C Falcone S Siatkowski RMPegoraro E WalkerndashWarburg syndrome neurological featuresand muscle membrane structure Ped Neurol 19981876ndash80

15 Villanova M Sabatelli P He Y et al Immunofluorescencestudy of a muscle biopsy from a 1-year-old patient with Walk-erndashWarburg syndrome Acta Neuropathol 199896651ndash654

16 Topaloglu H Talim B Vignier N et al Merosin-deficient con-genital muscular dystrophy with severe mental retardationand normal cranial MRI a report of two siblings Neuromus-cul Disord 19988169ndash174

17 Brockington M Sewry CA Hermann R et al Assignment of aform of congenital muscular dystrophy with secondary mer-osin deficiency to chromosome 1q42 Am J Hum Genet 200066428ndash435

18 Sewry CA Naom I DrsquoAlessandro M et al Variable clinicalphenotype in merosin-deficient congenital muscular dystrophyassociated with differential immunolabelling of two fragmentsof the laminin a2 chain Neuromuscul Disord 19977169ndash175

19 Tan E Topaloglu H Sewry C et al Late onset musculardystrophy with cerebral white matter changes due to partialmerosin deficiency Neuromuscul Disord 1997785ndash89

20 Naom I DrsquoAlessandro M Topaloglu H et al Refinement ofthe laminin a2 chain locus to human chromosome 6q2 insevere and mild merosin deficient congenital muscular dystro-phy J Med Genet 19973499ndash104

21 Nissinen M HelblingndashLeclerc A Zhang X et al Substitutionof a conserved cysteine-996 in a cysteine-rich motif of thelaminin a2-chain in congenital muscular dystrophy with par-tial deficiency of the protein Am J Hum Genet 1996581177ndash1184

22 Allamand V Sunada Y Salih MAM et al Mild congenitalmuscular dystrophy in two patients with an internally deletedlaminin a2-chain Hum Mol Genet 19976747ndash752

23 Hayashi YK Ishihara T Domen K Hori H Arahata K Abenign allelic form of laminin a2 chain deficient musculardystrophy Lancet 19973491147

24 Naom I DrsquoAlessandro M Sewry CA et al Laminin a2-chaingene mutations in two siblings presenting with limb-girdlemuscular dystrophy Neuromuscul Disord 19988495ndash501

25 Naom I DrsquoAlessandro M Sewry CA et al Mutations in thelaminin a2-chain gene in two children with early-onset mus-cular dystrophy Brain 200012331ndash41

26 Trevisan CP Martinello F Fanin M et al Merosin expressionin muscle of western case with Fukuyama-like congenitalmuscular dystrophy Basic Appl Myol 19966101ndash106

27 Hori H Kanamori T Mizuta T Yamaguchi N Liu Y Nagai YHuman laminin M chain epitope analysis of its monoclonalantibodies by immunoscreening of cDNA clones and tissueexpression J Biochem 19941161212ndash1219

28 Schiaffino S Gorza L Pitton G et al Embryonic and neonatalmyosin heavy chain in denervated and paralyzed rat skeletalmuscle Dev Biol 19881271ndash11

29 Byers TJ Kunkel LM Watkins SC The subcellular distribu-tion of dystrophin in mouse skeletal cardiac and smoothmuscle J Cell Biol 1991115411ndash421

30 Tiger CF Champliaud MF Pedrosadomellof F Thornell LEEkblom P Gullberg D Presence of laminin alpha-5 chain andlack of laminin alpha-1 chain during human muscle develop-ment and in muscular dystrophies J Biol Chem 199727228590ndash28595

31 Pegoraro E Mancias P Swerdlow SH et al Laminin a2 (mer-osin) gene mutations cause congenital muscular dystrophy(CMD) Ann Neurol 199640782ndash791

32 Miller SA Dykes DD Polesky HF A simple salting out proce-dure for extracting DNA from human nucleated cells NucleicAcids Res 1988161215

33 Zhang X Vuolteenaho R Tryggvason K Structure of the hu-man laminin a2-chain gene (LAMA2) which is affected incongenital muscular dystrophy J Biol Chem 199627127664ndash27669

34 Little S Amplification-refractory mutation system (ARMS)analysis of point mutations In Dracopoli NC Haines JL KorfBR et al eds Current protocols in human genetics NewYork John Wiley 1994981ndash985

35 Zhou J Hoffman EP Pathophysiology of sodium channelopa-thies Studies of sodium channel expression by quantitativemultiplex fluorescence polymerase chain reaction J BiolChem 199426918563ndash18571

36 Morrone A Pegoraro E Angelini C Zammarchi E Marconi GHoffman EP RNA metabolism in myotonic dystrophy Patientmuscle shows decreased insulin receptor RNA and proteinconsistent with abnormal insulin resistance J Clin Invest1997991691ndash1698

37 Yurchenco PD Quan Y Colognato H et al The alpha chain oflaminin-1 is independently secreted and drives secretion of itsbeta- and gamma-chain partners Proc Natl Acad Sci USA19979410189ndash10194

38 Minetti C Bado M Morreale G Pedemonte M Cordone GDisruption of muscle basal lamina in congenital muscular dys-trophy with merosin deficiency Neurology 1996461354ndash1358

39 Osari S Kobayashi O Yamashita Y et al Basement mem-brane abnormality in merosin-negative congenital musculardystrophy Acta Neuropathol 199691332ndash336

1134 NEUROLOGY 55 October (2 of 2) 2000

DOI 101212WNL55811282000551128-1134 Neurology

E Pegoraro M Fanin CP Trevisan et al dystrophy

2 deficient congenital muscularα2 isoform in severe laminin αA novel laminin

This information is current as of October 24 2000

ServicesUpdated Information amp

httpwwwneurologyorgcontent5581128fullhtmlincluding high resolution figures can be found at

References

httpwwwneurologyorgcontent5581128fullhtmlref-list-1at This article cites 37 articles 16 of which you can access for free

Citations

cleshttpwwwneurologyorgcontent5581128fullhtmlotherartiThis article has been cited by 5 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

when ratios were calculated using peak height p 5 0013when ratios were calculated using peak area) The alterna-tively spliced isoform was at similar levels in all musclebiopsies Our patient showed 65 6 8 of control levels ofexon 31ndashspliced laminin a2 RNA (100 6 38)

Discussion Here we describe a novel alterna-tively spliced isoform of the laminin a2 gene in apatient with CMD who was a compound heterozy-gote for two laminin a2 nonsense mutations Onemutation was contained within the 46 amino acidsdeleted in the alternatively spliced isoform Becausethe alternatively spliced isoform removed one of hernonsense mutations this patient was able to produceonly the alternatively spliced isoform with no evi-dence of the full-length isoform This alternativelyspliced product was found in all individuals testedboth control subjects and patients with neuromuscu-lar disease The isoform involved the recognition of acryptic splice site within exon 31 such that the ma-jority of exon 31 was skipped and directly spliced toexon 32 This splicing event removed 46 amino acidsin the cysteine-rich domain IIIa just proximal to thetriple coil-coiled region that associates with the b1and g1 chains of laminin The alternatively splicedisoform removes eight cysteines which are thoughtto participate in the formation of inter- and intramo-lecular disulfide bonds in the basal lamina No sub-function has been assigned to this region

This isoform was localized extracellularly pre-dominantly to discrete highly positive areas in iso-lated myofibers We were unable to determine anycharacteristics of these myofibers that distinguishedthem from the remainder Fiber typing showed thatboth type I (oxidative) and type II (glycolytic) fibersstained highly positively and a neuromuscular junc-

tion localization was ruled out No regenerating fi-bers were laminin a2 positive

Immunostaining for other subunits of lamininshowed a unique downregulation of laminin b1 andg1 and a failure of the upregulation of laminin a5typically seen in patients lacking laminin a2 Thisfinding suggests that the small amount of the novelisoform present in muscle fibers acts as a ldquodominantnegativerdquo protein it prevents the upregulation oflaminin a5 via its presence in muscle yet does notassociate well with either laminin b1 or g1 resultingin their downregulation This suggests that thecysteine-rich region of laminin a2 is important forinteraction with the b and g subunits and that thisnovel isoform alters the binding characteristics Al-ternatively the cysteine-rich region of laminin a2might be crucial for a proper chaperonndashlaminin in-teraction involved in the intracellular transport andassembly of the laminin chains Because b and gchains cannot be delivered to the extracellular ma-trix without their assembly in a abg heterotrimer37

the lack of a proper transport of the a chain in theintracellular milieu results in defective secretion ofthe entire heterotrimer

Our patient showed CNS involvement that wasconsiderably more severe than typically seen in pa-tients with complete laminin a2 deficiency Her MRIwas highly abnormal with tetraventricular dilata-tion white matter alterations pachygyria of the oc-cipital cortex and some small areas of cerebellarpolymicrogyria findings not seen in any other pa-tient with laminin a2 deficiency studied to dateAlso no other patient with laminin a2 deficiency hasbeen reported with mental retardation our patientshowed a moderately low IQ with progressive mentalretardation

It is important to explain the severe CNS involve-ment in this patient Laminin a2 is normally ex-pressed in the inner part of the basal lamina (laminadensa) of striated muscle fibers and in Schwann cellsof peripheral nerves Expression in the CNS is stillnot well studied particularly in regard to neural de-velopment In a laminin a2ndashdeficient patient withCMD the absence of laminin a2 results in a severederangement of the muscle basal lamina Electronmicroscopy shows a discontinuous thinner basallamina with occasional holes in skeletal muscle fi-bers in this category of patients3839 but a normalwell-preserved basement membrane in the intramus-cular nerves39 No EM studies of the basal lamina inbrain of laminin a2ndashdeficient patients with CMDhave been reported therefore there are still too fewdata concerning the role of laminin a2 in brain devel-opment and homeostasis to derive adequate modelsthat explain the severe CNS involvement in thispatient

AcknowledgmentThe authors thank Dr Eva Engvall for critical review of themanuscript

Figure 5 Normal expression of the exon 31ndashspliced lami-nin a2 isoform and reduced expression of the full-lengthlaminin a2 in the patientrsquos muscle Shown are examples ofautomated sequencer traces of quantitative multiplex fluo-rescent reverse transcription PCR of the exon 31ndashsplicedlaminin a2 and full-length laminin a2 isoforms relative toa-sarcoglycan RNA isolated from the patientrsquos muscle bi-opsy consistently showed lower levels of full-length lami-nin a2 RNA relative to control subjects The level of exon31ndashspliced laminin a2 RNA was similar in the patient rel-ative to control subjects

October (2 of 2) 2000 NEUROLOGY 55 1133

References1 Tome FM Evangelista T Leclerc A et al Congenital muscu-

lar dystrophy with merosin deficiency M CR Acad Sci Paris1994317351ndash357

2 HelblingndashLeclerc A Zhang X Topaloglu H et al Mutations inthe laminin a2-chain gene (LAMA2) cause merosin-deficientcongenital muscular dystrophy Nat Genet 199511216ndash218

3 Pegoraro E Marks H Garcia CA et al Laminin a2 musculardystrophy genotypephenotype studies of 22 patients Neurol-ogy 199851101ndash110

4 Ehrig K Leivo I Argraves WS Ruoslahti E Engvall E Mer-osin a tissue-specific basement membrane protein is a lami-nin like protein Proc Natl Acad Sci USA 1990873264ndash3268

5 Voulteenaho R Nissinen M Sainio K et al Human lamininM chain (merosin) complete primary structure chromosomalassignment and expression of the M and A chain in humanfetal tissues J Cell Biol 1994124381ndash394

6 Patton B Miner JH Chiu AY Sanes JR Distribution andfunction of laminins in the neuromuscular system of develop-ing adult and mutant mice J Cell Biol 19971391507ndash1521

7 Dubowitz V Workshop Report 22nd ENMC sponsored work-shop on congenital muscular dystrophy Baarn the Nether-lands May 1993 Neuromuscul Disord 1994475ndash81

8 Reed UC Marie SK Vainzof M et al Congenital musculardystrophy with cerebral white matter hypodensity Correla-tion of clinical features and merosin deficiency Brain Dev19961853ndash58

9 Pini A Merlini L Tome FM Chevallay M Gobbi G Merosin-negative congenital muscular dystrophy occipital epilepsywith periodic spasms and focal cortical dysplasia Report ofthree Italian cases in two families Brain Dev 199618316ndash322

10 Sunada Y Edgar TS Lotz BP Rust RS Campbell KPMerosin-negative congenital muscular dystrophy associatedwith extensive brain abnormalities Neurology 1995452084ndash2089

11 Van der Knaap MS Smit LME Barth PG et al Magneti-cresonance imaging in classification of congenital musculardystrophies with brain abnormalities Ann Neurol 19974250ndash59

12 Hayashi YK Engvall E ArikawandashHirasawa E et al Abnor-mal localization of laminin subunits in muscular dystrophiesJ Neurol Sci 199311953ndash64

13 Haltia M Leivo I Somer H et al Musclendasheyendashbrain diseasea neuropathological study Ann Neurol 199741173ndash180

14 Kanoff RJ Curless RG Petito C Falcone S Siatkowski RMPegoraro E WalkerndashWarburg syndrome neurological featuresand muscle membrane structure Ped Neurol 19981876ndash80

15 Villanova M Sabatelli P He Y et al Immunofluorescencestudy of a muscle biopsy from a 1-year-old patient with Walk-erndashWarburg syndrome Acta Neuropathol 199896651ndash654

16 Topaloglu H Talim B Vignier N et al Merosin-deficient con-genital muscular dystrophy with severe mental retardationand normal cranial MRI a report of two siblings Neuromus-cul Disord 19988169ndash174

17 Brockington M Sewry CA Hermann R et al Assignment of aform of congenital muscular dystrophy with secondary mer-osin deficiency to chromosome 1q42 Am J Hum Genet 200066428ndash435

18 Sewry CA Naom I DrsquoAlessandro M et al Variable clinicalphenotype in merosin-deficient congenital muscular dystrophyassociated with differential immunolabelling of two fragmentsof the laminin a2 chain Neuromuscul Disord 19977169ndash175

19 Tan E Topaloglu H Sewry C et al Late onset musculardystrophy with cerebral white matter changes due to partialmerosin deficiency Neuromuscul Disord 1997785ndash89

20 Naom I DrsquoAlessandro M Topaloglu H et al Refinement ofthe laminin a2 chain locus to human chromosome 6q2 insevere and mild merosin deficient congenital muscular dystro-phy J Med Genet 19973499ndash104

21 Nissinen M HelblingndashLeclerc A Zhang X et al Substitutionof a conserved cysteine-996 in a cysteine-rich motif of thelaminin a2-chain in congenital muscular dystrophy with par-tial deficiency of the protein Am J Hum Genet 1996581177ndash1184

22 Allamand V Sunada Y Salih MAM et al Mild congenitalmuscular dystrophy in two patients with an internally deletedlaminin a2-chain Hum Mol Genet 19976747ndash752

23 Hayashi YK Ishihara T Domen K Hori H Arahata K Abenign allelic form of laminin a2 chain deficient musculardystrophy Lancet 19973491147

24 Naom I DrsquoAlessandro M Sewry CA et al Laminin a2-chaingene mutations in two siblings presenting with limb-girdlemuscular dystrophy Neuromuscul Disord 19988495ndash501

25 Naom I DrsquoAlessandro M Sewry CA et al Mutations in thelaminin a2-chain gene in two children with early-onset mus-cular dystrophy Brain 200012331ndash41

26 Trevisan CP Martinello F Fanin M et al Merosin expressionin muscle of western case with Fukuyama-like congenitalmuscular dystrophy Basic Appl Myol 19966101ndash106

27 Hori H Kanamori T Mizuta T Yamaguchi N Liu Y Nagai YHuman laminin M chain epitope analysis of its monoclonalantibodies by immunoscreening of cDNA clones and tissueexpression J Biochem 19941161212ndash1219

28 Schiaffino S Gorza L Pitton G et al Embryonic and neonatalmyosin heavy chain in denervated and paralyzed rat skeletalmuscle Dev Biol 19881271ndash11

29 Byers TJ Kunkel LM Watkins SC The subcellular distribu-tion of dystrophin in mouse skeletal cardiac and smoothmuscle J Cell Biol 1991115411ndash421

30 Tiger CF Champliaud MF Pedrosadomellof F Thornell LEEkblom P Gullberg D Presence of laminin alpha-5 chain andlack of laminin alpha-1 chain during human muscle develop-ment and in muscular dystrophies J Biol Chem 199727228590ndash28595

31 Pegoraro E Mancias P Swerdlow SH et al Laminin a2 (mer-osin) gene mutations cause congenital muscular dystrophy(CMD) Ann Neurol 199640782ndash791

32 Miller SA Dykes DD Polesky HF A simple salting out proce-dure for extracting DNA from human nucleated cells NucleicAcids Res 1988161215

33 Zhang X Vuolteenaho R Tryggvason K Structure of the hu-man laminin a2-chain gene (LAMA2) which is affected incongenital muscular dystrophy J Biol Chem 199627127664ndash27669

34 Little S Amplification-refractory mutation system (ARMS)analysis of point mutations In Dracopoli NC Haines JL KorfBR et al eds Current protocols in human genetics NewYork John Wiley 1994981ndash985

35 Zhou J Hoffman EP Pathophysiology of sodium channelopa-thies Studies of sodium channel expression by quantitativemultiplex fluorescence polymerase chain reaction J BiolChem 199426918563ndash18571

36 Morrone A Pegoraro E Angelini C Zammarchi E Marconi GHoffman EP RNA metabolism in myotonic dystrophy Patientmuscle shows decreased insulin receptor RNA and proteinconsistent with abnormal insulin resistance J Clin Invest1997991691ndash1698

37 Yurchenco PD Quan Y Colognato H et al The alpha chain oflaminin-1 is independently secreted and drives secretion of itsbeta- and gamma-chain partners Proc Natl Acad Sci USA19979410189ndash10194

38 Minetti C Bado M Morreale G Pedemonte M Cordone GDisruption of muscle basal lamina in congenital muscular dys-trophy with merosin deficiency Neurology 1996461354ndash1358

39 Osari S Kobayashi O Yamashita Y et al Basement mem-brane abnormality in merosin-negative congenital musculardystrophy Acta Neuropathol 199691332ndash336

1134 NEUROLOGY 55 October (2 of 2) 2000

DOI 101212WNL55811282000551128-1134 Neurology

E Pegoraro M Fanin CP Trevisan et al dystrophy

2 deficient congenital muscularα2 isoform in severe laminin αA novel laminin

This information is current as of October 24 2000

ServicesUpdated Information amp

httpwwwneurologyorgcontent5581128fullhtmlincluding high resolution figures can be found at

References

httpwwwneurologyorgcontent5581128fullhtmlref-list-1at This article cites 37 articles 16 of which you can access for free

Citations

cleshttpwwwneurologyorgcontent5581128fullhtmlotherartiThis article has been cited by 5 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

References1 Tome FM Evangelista T Leclerc A et al Congenital muscu-

lar dystrophy with merosin deficiency M CR Acad Sci Paris1994317351ndash357

2 HelblingndashLeclerc A Zhang X Topaloglu H et al Mutations inthe laminin a2-chain gene (LAMA2) cause merosin-deficientcongenital muscular dystrophy Nat Genet 199511216ndash218

3 Pegoraro E Marks H Garcia CA et al Laminin a2 musculardystrophy genotypephenotype studies of 22 patients Neurol-ogy 199851101ndash110

4 Ehrig K Leivo I Argraves WS Ruoslahti E Engvall E Mer-osin a tissue-specific basement membrane protein is a lami-nin like protein Proc Natl Acad Sci USA 1990873264ndash3268

5 Voulteenaho R Nissinen M Sainio K et al Human lamininM chain (merosin) complete primary structure chromosomalassignment and expression of the M and A chain in humanfetal tissues J Cell Biol 1994124381ndash394

6 Patton B Miner JH Chiu AY Sanes JR Distribution andfunction of laminins in the neuromuscular system of develop-ing adult and mutant mice J Cell Biol 19971391507ndash1521

7 Dubowitz V Workshop Report 22nd ENMC sponsored work-shop on congenital muscular dystrophy Baarn the Nether-lands May 1993 Neuromuscul Disord 1994475ndash81

8 Reed UC Marie SK Vainzof M et al Congenital musculardystrophy with cerebral white matter hypodensity Correla-tion of clinical features and merosin deficiency Brain Dev19961853ndash58

9 Pini A Merlini L Tome FM Chevallay M Gobbi G Merosin-negative congenital muscular dystrophy occipital epilepsywith periodic spasms and focal cortical dysplasia Report ofthree Italian cases in two families Brain Dev 199618316ndash322

10 Sunada Y Edgar TS Lotz BP Rust RS Campbell KPMerosin-negative congenital muscular dystrophy associatedwith extensive brain abnormalities Neurology 1995452084ndash2089

11 Van der Knaap MS Smit LME Barth PG et al Magneti-cresonance imaging in classification of congenital musculardystrophies with brain abnormalities Ann Neurol 19974250ndash59

12 Hayashi YK Engvall E ArikawandashHirasawa E et al Abnor-mal localization of laminin subunits in muscular dystrophiesJ Neurol Sci 199311953ndash64

13 Haltia M Leivo I Somer H et al Musclendasheyendashbrain diseasea neuropathological study Ann Neurol 199741173ndash180

14 Kanoff RJ Curless RG Petito C Falcone S Siatkowski RMPegoraro E WalkerndashWarburg syndrome neurological featuresand muscle membrane structure Ped Neurol 19981876ndash80

15 Villanova M Sabatelli P He Y et al Immunofluorescencestudy of a muscle biopsy from a 1-year-old patient with Walk-erndashWarburg syndrome Acta Neuropathol 199896651ndash654

16 Topaloglu H Talim B Vignier N et al Merosin-deficient con-genital muscular dystrophy with severe mental retardationand normal cranial MRI a report of two siblings Neuromus-cul Disord 19988169ndash174

17 Brockington M Sewry CA Hermann R et al Assignment of aform of congenital muscular dystrophy with secondary mer-osin deficiency to chromosome 1q42 Am J Hum Genet 200066428ndash435

18 Sewry CA Naom I DrsquoAlessandro M et al Variable clinicalphenotype in merosin-deficient congenital muscular dystrophyassociated with differential immunolabelling of two fragmentsof the laminin a2 chain Neuromuscul Disord 19977169ndash175

19 Tan E Topaloglu H Sewry C et al Late onset musculardystrophy with cerebral white matter changes due to partialmerosin deficiency Neuromuscul Disord 1997785ndash89

20 Naom I DrsquoAlessandro M Topaloglu H et al Refinement ofthe laminin a2 chain locus to human chromosome 6q2 insevere and mild merosin deficient congenital muscular dystro-phy J Med Genet 19973499ndash104

21 Nissinen M HelblingndashLeclerc A Zhang X et al Substitutionof a conserved cysteine-996 in a cysteine-rich motif of thelaminin a2-chain in congenital muscular dystrophy with par-tial deficiency of the protein Am J Hum Genet 1996581177ndash1184

22 Allamand V Sunada Y Salih MAM et al Mild congenitalmuscular dystrophy in two patients with an internally deletedlaminin a2-chain Hum Mol Genet 19976747ndash752

23 Hayashi YK Ishihara T Domen K Hori H Arahata K Abenign allelic form of laminin a2 chain deficient musculardystrophy Lancet 19973491147

24 Naom I DrsquoAlessandro M Sewry CA et al Laminin a2-chaingene mutations in two siblings presenting with limb-girdlemuscular dystrophy Neuromuscul Disord 19988495ndash501

25 Naom I DrsquoAlessandro M Sewry CA et al Mutations in thelaminin a2-chain gene in two children with early-onset mus-cular dystrophy Brain 200012331ndash41

26 Trevisan CP Martinello F Fanin M et al Merosin expressionin muscle of western case with Fukuyama-like congenitalmuscular dystrophy Basic Appl Myol 19966101ndash106

27 Hori H Kanamori T Mizuta T Yamaguchi N Liu Y Nagai YHuman laminin M chain epitope analysis of its monoclonalantibodies by immunoscreening of cDNA clones and tissueexpression J Biochem 19941161212ndash1219

28 Schiaffino S Gorza L Pitton G et al Embryonic and neonatalmyosin heavy chain in denervated and paralyzed rat skeletalmuscle Dev Biol 19881271ndash11

29 Byers TJ Kunkel LM Watkins SC The subcellular distribu-tion of dystrophin in mouse skeletal cardiac and smoothmuscle J Cell Biol 1991115411ndash421

30 Tiger CF Champliaud MF Pedrosadomellof F Thornell LEEkblom P Gullberg D Presence of laminin alpha-5 chain andlack of laminin alpha-1 chain during human muscle develop-ment and in muscular dystrophies J Biol Chem 199727228590ndash28595

31 Pegoraro E Mancias P Swerdlow SH et al Laminin a2 (mer-osin) gene mutations cause congenital muscular dystrophy(CMD) Ann Neurol 199640782ndash791

32 Miller SA Dykes DD Polesky HF A simple salting out proce-dure for extracting DNA from human nucleated cells NucleicAcids Res 1988161215

33 Zhang X Vuolteenaho R Tryggvason K Structure of the hu-man laminin a2-chain gene (LAMA2) which is affected incongenital muscular dystrophy J Biol Chem 199627127664ndash27669

34 Little S Amplification-refractory mutation system (ARMS)analysis of point mutations In Dracopoli NC Haines JL KorfBR et al eds Current protocols in human genetics NewYork John Wiley 1994981ndash985

35 Zhou J Hoffman EP Pathophysiology of sodium channelopa-thies Studies of sodium channel expression by quantitativemultiplex fluorescence polymerase chain reaction J BiolChem 199426918563ndash18571

36 Morrone A Pegoraro E Angelini C Zammarchi E Marconi GHoffman EP RNA metabolism in myotonic dystrophy Patientmuscle shows decreased insulin receptor RNA and proteinconsistent with abnormal insulin resistance J Clin Invest1997991691ndash1698

37 Yurchenco PD Quan Y Colognato H et al The alpha chain oflaminin-1 is independently secreted and drives secretion of itsbeta- and gamma-chain partners Proc Natl Acad Sci USA19979410189ndash10194

38 Minetti C Bado M Morreale G Pedemonte M Cordone GDisruption of muscle basal lamina in congenital muscular dys-trophy with merosin deficiency Neurology 1996461354ndash1358

39 Osari S Kobayashi O Yamashita Y et al Basement mem-brane abnormality in merosin-negative congenital musculardystrophy Acta Neuropathol 199691332ndash336

1134 NEUROLOGY 55 October (2 of 2) 2000

DOI 101212WNL55811282000551128-1134 Neurology

E Pegoraro M Fanin CP Trevisan et al dystrophy

2 deficient congenital muscularα2 isoform in severe laminin αA novel laminin

This information is current as of October 24 2000

ServicesUpdated Information amp

httpwwwneurologyorgcontent5581128fullhtmlincluding high resolution figures can be found at

References

httpwwwneurologyorgcontent5581128fullhtmlref-list-1at This article cites 37 articles 16 of which you can access for free

Citations

cleshttpwwwneurologyorgcontent5581128fullhtmlotherartiThis article has been cited by 5 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

DOI 101212WNL55811282000551128-1134 Neurology

E Pegoraro M Fanin CP Trevisan et al dystrophy

2 deficient congenital muscularα2 isoform in severe laminin αA novel laminin

This information is current as of October 24 2000

ServicesUpdated Information amp

httpwwwneurologyorgcontent5581128fullhtmlincluding high resolution figures can be found at

References

httpwwwneurologyorgcontent5581128fullhtmlref-list-1at This article cites 37 articles 16 of which you can access for free

Citations

cleshttpwwwneurologyorgcontent5581128fullhtmlotherartiThis article has been cited by 5 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