Brain, Vol. 124, No. 4, 698-704,
April 2001
© 2001 Oxford University Press
Mild muscular dystrophy due to a nonsense mutation in the LAMA2 gene resulting in exon skipping
1 Department of Neuromuscular Diseases, Istituto Nazionale Neurologico C. Besta, Milano, Italy and 2 Unité INSERM U523, Institut de Myologie, IFR ‘Coeur, Muscle et Vaisseaux’ N.14, Groupe Hospitalier Pitié-Salpêtrière, Paris, France
Correspondence to:
Marina Mora, Department of Neuromuscular Diseases, Istituto Nazionale Neurologico ‘C. Besta’, Via Celoria 11, 20133 Milano, Italy Email: mmora{at}tin.it
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Abstract |
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Nonsense mutations outside the splicing consensus sequence have been reported to cause skipping of the nonsense-containing exon in several human diseases. We describe, for the first time, nonsense-mediated exon skipping in the laminin
2 (LAMA2) gene. Two siblings from a consanguineous family had altered expression of the laminin 2 chain and moderate clinical manifestations. In both we identified the new nonsense mutation Arg744Stop, which we expected to result in a totally non-functional polypeptide. However, analysis of the transcript revealed skipping of exon 15, containing the mutation, even though the consensus sequences for splicing at both ends of the exon and the beginning of intron 15 were unaltered. Exon skipping restored the open reading frame of the mutant transcript and resulted in a truncated protein. In cases where the genetic findings do not elucidate the phenotype, mRNA analysis is necessary to clarify the primary effect of mutations. Our findings also point to the necessity of immunochemical screening for expression of laminin 2 chain in atypical dystrophic adults as well as children.
congenital muscular dystrophy; LAMA2; laminin 2 chain; merosin
EPs = evoked potentials; LAMA2 = laminin 2 chain gene; PCR = polymerase chain reaction
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Introduction |
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Laminins are heterotrimeric proteins, consisting of a heavy
chain, and light ß and 
chains, which assemble into cross-shaped molecules (Engel, 1992
; Tryggvason, 1993; Timpl and Brown, 1994). Laminin-2 (merosin) is the isoform of laminin present in the basal lamina of skeletal muscle fibres and elsewhere (Leivo et al., 1988; Engvall et al., 1990; Tryggvason, 1993; Sewry et al., 1996). It contains the 2 chain encoded by the laminin 2 (LAMA2) gene on chromosome 6q22 (Vuolteenaho et al., 1994). Deficient expression of laminin 2 chain is found in many patients with the classic occidental form of congenital muscular dystrophy (CMD) (Tomé et al., 1994). CMD due to complete lack of laminin 2 chain is characterized by severe hypotonia at birth associated with an inability to walk unsupported, delayed motor development, high creatine kinase levels and a clinically asymptomatic abnormality of the central white matter on MRI (Tomé et al., 1994; Dubowitz and Fardeau, 1995; Philpot et al., 1995).
Milder phenotypes have been described in several young patients (Herrmann et al., 1996; Mora et al., 1996; Nissinen et al., 1996; Allamand et al., 1997; Hayashi et al., 1997; Sewry et al., 1997; Tan et al., 1997; Cohn et al., 1998; Naom et al., 1998, 2000; Morandi et al., 1999), although a genetic defect, usually resulting in partial expression of laminin 2 chain, was characterized in only a few (Nissinen et al., 1996; Allamand et al., 1997; Hayashi et al., 1997; Naom et al., 2000). We recently described an adult patient with a myopathy resembling inclusion body myositis and a partial laminin 2 chain deficiency due to mutations in the LAMA2 gene (Di Blasi et al., 2000).
We describe here, for the first time, exon skipping in the LAMA2 gene mediated by a new nonsense mutation. The mutation is homozygous and occurs in two adult siblings from a consanguineous family. Exon skipping restores the open reading frame of the mutant transcript to produce a truncated protein and a mild clinical phenotype.
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Subjects
We studied members of a consanguineous family partially described in Morandi et al. (1999). The patients gave their informed consent and the study was approved by the Ethics Committee of the Instituto Nazionale Neurologico C, Besta, Milan. The proband (Patient 1) was a 39-year-old man with mild limb girdle weakness. His symptoms, some difficulty in running and jumping, first appeared at about the age of 15 years and worsened very slowly. He was first examined at age 32 years when he complained of limb weakness causing difficulty in getting up from the floor, climbing stairs and holding heavy weights. Neurological examination disclosed mild scapular, pelvic girdle, and proximal limb muscle weakness [score 3 on the MRC (Medical Research Council) scale]. The most characteristic features were severe contractures of neck and arm muscles, preventing head rotation or bending, and forearm supination. Elbow, Achilles tendon and quadriceps retractions were also present. The man walked with a broad base and flexed elbows, used Gower's manoeuvre to get up from the floor, and climbed stairs with the aid of a hand rail.
Subsequent clinical examinations remained unchanged until age 39, when greater difficulty in walking, rising from the floor and climbing stairs became evident. ECG and echocardiogram were normal, but respiratory function was mildly compromised (forced vital capacity was 81% of that expected). Creatine kinase was about four times above normal. EMG from biceps showed myogenic potentials, and electroneurography revealed motor and sensory nerve conduction velocities in the lower normal range, and a slightly longer F-wave. Visual and brainstem evoked potentials (EPs) were normal, while somato-sensory EPs showed mild abnormalities, suggesting peripheral neuropathy. Motor EPs from lower limbs showed mild changes in central motor conduction. Brain MRI revealed cerebellar hypotrophy and, in T2 weighted images, diffuse white matter hyperintensity in periventricular and subcortical areas.
One of the two siblings (Patient 2) of the proband was similarly affected when first seen at 36 years. She had difficulty in walking due to congenital bilateral hip dislocation, which had been treated surgically. However, neurological examination revealed similar clinical findings to her brother, although they were less severe. Her creatine kinase was about twice normal. Myopathic changes were detected by EMG, and peripheral nerve involvement was absent. EP studies revealed only a mild reduction in the amplitude of visual EPs. Brain MRI showed a few punctate areas of increased signal in peritrigonal and periventricular regions, and slight hyperintensity of the cerebellar white matter.
Immunohistochemistry
Muscle (quadriceps) and skin biopsies were frozen in isopentane cooled in liquid nitrogen and stored in liquid nitrogen until use. Immunohistochemical analysis of laminin 2 chain was performed as described previously (Morandi et al., 1999) on 6 µm thick cryosections using two commercial monoclonal antibodies: one recognizing the 300 kDa fragment towards the N-terminus (Alexis, San Diego, Calif., USA), and the other the 80 kDa fragment towards the C-terminus (Chemicon, Temecula, Calif., USA).
Immunoblots
Expression of laminin 2 chain was evaluated by immunoblotting as described previously (Morandi et al., 1999) using the monoclonal antibody recognizing the 80 kDa fragment. The other monoclonal antibody, raised against the 300 kDa fragment of laminin 2 chain from mouse heart (Schuler and Sorokin, 1995), does not detect a fragment on immunoblots of human tissue.
Molecular analysis
Genomic DNA, extracted from peripheral blood lymphocytes, was amplified by the polymerase chain reaction (PCR) using oligonucleotide primers flanking the intron–exon junctions of all 64 LAMA2 exons. Amplifications were performed using the PCR touchdown method and conditions were as described by Guicheney et al. (1998). Each amplification employed 100 ng of genomic DNA in a 50 µl reaction. The amplification products were resolved electrophoretically on 2% agarose gels and purified by the QIAquick PCR Purification Kit (QIAGEN GmbH, Germany).
For SSCP (single strand conformation polymorphism) analysis, an aliquot of the PCR product was denatured and separated on a 10% polyacrylamide (acrylamide x 2: bisacrylamide x 1, 37.5 : 1) mini-gel at 7 and 20°C. Electrophoresis was performed at 8 mA using Hoefer apparatus. A silver-staining kit (Biorad, Hercules, Calif., USA) was used to visualize the DNA bands on the gels. Aberrant conformers were sequenced on both strands using an Applied Biosystems automated sequencer (Perkin Elmer, Foster City, Calif., USA) and the same primers as for the PCR amplification.
RNA isolation and complementary DNA (cDNA) amplification
Total RNA was prepared from skeletal muscle using RNAWIZTM (Ambion, Austin, Tex., USA). The isolated RNA was reverse transcribed using a First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Rainham, UK) and the resulting cDNA amplified by reverse transcriptase PCR. The purified PCR products were subcloned into a pMOSBlue vector (Promega, Madison, Wis., USA) and the DNA was sequenced using appropriate primers.
Restriction site analysis
The mutation does not introduce a new restriction site in the genomic DNA. To determine whether the known HindII sites were all present in the patients we performed restriction nuclease (Boehringer Mannheim GmbH, Mannheim, Germany) digestion of the region spanning exons 11–16 of LAMA2 cDNA. Ten-microlitre aliquots of the PCR product of the two patients and of several unaffected controls were digested in a total volume of 20 µl as described by the manufacturers. The products were run on a 3% agarose gel.
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Results |
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Muscle biopsy revealed dystrophic changes in both patients, although the muscle specimen from the sister was more severely affected, being characterized by conspicuous fibrotic and fatty connective tissue proliferation and few surviving fibres.
Immunohistochemical analysis of muscle sections disclosed reduced expression of laminin 2 chain at the periphery of muscle fibres in Patient 1 (Fig. 1C and D) and almost normal expression in the sister (Fig. 1E and F). However, the sister's skin biopsy showed reduced positivity to laminin 2 chain immunostaining, similar to that in the brother's muscle (see Morandi et al., 1999).
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On electrophoresis, laminin
2 chain usually runs as 80 and 300 kDa fragments (Ehrig et al., 1990). Immunoblotting using the monoclonal antibody against the 80 kDa fragment revealed a normal sized fragment of altered intensity in both patients, as expected (data not shown). Unfortunately it was not possible, due to lack of material, to quantitate the amount of the 80 kDa fragment in relation to a contractile protein.
SSCP analysis revealed abnormal conformers in exon 15 in the patients and in heterozygous family members compared with controls (Fig. 2). Direct sequencing of this aberrant PCR product revealed a new homozygous mutation, a C
T transition at position 2230 of the cDNA sequence, resulting in a stop codon (Arg744Stop) (Fig. 3). There were no other alterations in the sequence of exon 15 of the patients or family members compared with controls. Sequencing of the beginning of intron 15 and of the flanking splice sites of exon 15 excluded the presence of additional mutations (data not presented).
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Amplification of the region spanning exons 11–16 from skeletal muscle cDNA showed two bands, one of the expected size [663 bp (base pair)] and one slightly smaller (Fig. 4
). Automated sequencing of the two subcloned PCR products demonstrated a 114 bp in-frame deletion in the smaller fragment due to in-frame skipping of the entire exon 15, which contains the nonsense mutation (Fig. 3
).
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HindII restriction analysis of the wild-type exon 11–16 region produces four fragments of 304, 160, 126 and 73 bp; both patients were shown to have these fragments and an additional one of 172 bp, consistent with exon skipping (data not shown).
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Partial laminin
2 chain deficiency has been reported in several cases of CMD, and the clinical picture is usually milder than in patients totally lacking the protein (Herrmann et al., 1996; Mora et al., 1996; Allamand et al., 1997; Hayashi et al., 1997; Sewry et al., 1997; Tan et al., 1997; Cohn et al., 1998; Naom et al., 1998, 2000; Morandi et al., 1999; Di Blasi et al., 2000). In the few cases characterized, the LAMA2 gene defects were missense mutations, which were homozygous (Nissinen et al., 1996) or associated with nonsense mutations (Di Blasi et al., 2000), splice site mutations (Allamand et al., 1997; Naom et al., 2000), or a heterozygous insertion in one allele and a deletion in the other, both of which caused frame shifts and stop codons downstream (Hayashi et al., 1997).
In our two cases, we found a new homozygous nonsense mutation in exon 15 and, unexpectedly, partial expression of laminin 2 chain. RNA analysis showed, in addition to the expected transcript, that there was a smaller transcript which arose due to skipping of exon 15, but retained the open reading frame. The smaller RNA was detected exclusively in Patients 1 and 2, and not in the 10 controls we tested, including one LAMA2-mutated patient. Other studies on the full-length LAMA2 cDNA in controls and LAMA2-mutated patients have not reported the presence of this fragment (Pegoraro et al., 1996; Allamand et al., 1997; Mendell et al., 1998; Pegoraro et al., 1998). We therefore conclude that the alteration is not a polymorphism.
The smaller transcript was presumably translated to produce a truncated protein lacking 38 amino acids in the IIIb domain. We detected the 80 kDa fragment of the protein on a Western blot and it was of normal molecular weight as expected. The missing portion of the laminin 2 chain, towards the N-terminus, should show up as a reduced molecular weight larger fragment, but the antibody against this portion did not detect this fragment on immunoblots.
In some genes, stop mutations have been shown to affect mRNA metabolism and to reduce the amount of detectable mRNA (Satoh et al., 1988; Kadowaki et al., 1990; Baserga et al., 1992; Belgrader et al., 1994). Less commonly, mRNA containing stop mutations is translated, which results in a truncated protein (Lehrmann et al., 1987). A third possible outcome is that the exon containing the stop mutation is spliced out of the mature mRNA, which has been demonstrated in several human genetic diseases (Dietz et al., 1993; Hull et al., 1994; Mazoyer et al., 1998; Ars et al., 2000). This is the first example of nonsense-mediated exon skipping to be described in the LAMA2 gene.
Exon skipping is a common mutational mechanism, usually caused by changes in consensus sequences at splice sites or lariat branch-point regions. Sequences outside these regions can also affect the inclusion or exclusion of exons and may be located in introns or exons (Green et al., 1986; Watakabe et al., 1993; Tanaka et al., 1994). Alternative splicing patterns are determined by the presence of proteins that bind these regulatory sequences (Liu et al., 1997). Sometimes the quantity of mutant transcript is reduced, which may be due to abnormal splice site selection (Dietz et al., 1994).
We suggest that the exon skipping in our family may be due to disruption of the purine-rich splicing enhancer sequence by the nonsense mutation. This mechanism has been demonstrated in the dystrophin gene (Shiga et al., 1997) by experimentally substituting a nucleotide of the purine-rich splicing enhancer sequence with a thymine, creating a nonsense mutation. In our patients, the CT transition takes place in a purine-rich sequence, which is very likely to be the splicing enhancer sequence. As well as in the dystrophin gene (Shiga et al., 1997), exon skipping mediated by a nonsense mutation disrupting the splicing enhancer sequence has been described in the 3-hydroxy-3-methylglutaryl-CoA lyase gene (Pié et al., 1997) and in the adenosine deaminase gene (Santisteban et al., 1995). Exon skipping within the LAMA2 gene has been reported previously in two siblings from a consanguineous family (Allamand et al., 1997): a mutation in the splice consensus sequence resulted in the skipping of exon 25, so that the predicted protein lacked 63 amino acids of domain IVa. The patients partially expressed laminin 2 chain and were mildly affected. The in-frame deletions found in our patients and in those of Allamand et al. occur in domains not involved in laminin trimer formation, and, therefore, it is not surprising that in both cases a semi-functional protein is produced, resulting in mild phenotypes.
To conclude, our data indicate that mRNA analysis is necessary to clarify the primary effect of mutations that alter mRNA processing and which would not be predicted by characterization solely at the genomic level. Our findings also point to the necessity of immunochemical screening for laminin 2 chain expression in adults as well as children, particularly those with limb girdle muscle weakness and severe contractures associated with high creatine kinase and dystrophic features on muscle biopsy, but with normal dystrophin and dystrophin-associated proteins.
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Acknowledgments |
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The authors thank Don Ward for help with the English. This work was supported by Telethon, Italy (grant No. 1093 to L.M.), by ARIN (Associazione per la promozione delle ricerche neurologiche), AFM (Association Franciaise contre les Myopathies) and the European Commission (grant No. QLG1-1999-00870 to P.G.).
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Received August 3, 2000. Revised October 30, 2000. Second revision on December 4, 2000. Accepted December 4, 2000.
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