Brain, Vol. 123, No. 1, 31-41,
January 2000
© 2000 Oxford University Press
Mutations in the laminin 
2-chain gene in two children with early-onset muscular dystrophy
1 Neuromuscular Unit, Department of Paediatrics and Neonatal Medicine, 2 MRC Muscle Cell Biology Group, Hammersmith Hospital, London, Departments of 3 Paediatrics and 4 Neuropathology, Frenchay Hospital, Bristol, UK
Correspondence to:
Francesco Muntoni, MD, Professor and Consultant in Paediatric and Neonatal Medicine, Department of Paediatrics and Neonatal Medicine, Imperial College School Of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK E-mail: fmuntoni{at}rpms.ac.uk
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We investigated two children who presented with delayed motor milestones. The first was a girl who was referred at 20 months because of developmental delay. She walked at 28 months and currently, aged 5 years, is independently mobile but has difficulty rising from the floor or going upstairs. The second was also a girl who presented at 6 weeks of age with hypotonia. Her motor milestones were delayed and she walked at the age of 2 years and 8 months and is currently independently mobile at the age of 3 years. Serum creatine kinase was elevated and a muscle biopsy showed dystrophic changes in both children. Immunohistochemistry of the laminin
2 chain of merosin was very similar in both cases: using a C-terminal antibody that recognizes an 80 kDa fragment, there was a mild reduction in expression on most fibres, while the staining with another antibody that recognizes a 300 kDa fragment showed a very marked reduction. Mutational analysis of the laminin 2 chain gene in the first patient showed that one of the two alleles had a de novo single nucleotide deletion at position 5702, causing a frameshift. In the other allele, we identified two point mutations present in cis; one was a G
C transition at position +5 while the second was a TC transition at position +6 of the conserved donor splicing consensus sequence of introns 37 and 63, respectively. Transcription analysis of the corresponding cDNA region did not show any alternative splicing occurring as a result of these splice site mutations. Therefore, these mutations probably affect the splicing efficiency. Interestingly, the second child carried in both alleles the same two splicing consensus sequence mutations found in cis in the first patient. Our data provide further evidence that mutations in the laminin 2 chain gene are responsible not only for the severe form of congenital muscular dystrophy with onset at birth, but also for milder phenotypes, with later onset, in which the synthesis of a partially functional protein, or of a normal protein but in reduced quantity, is possible. The finding that these two unrelated patients had the same unusual mutation in common might suggest that this is a relatively commonly allele responsible for partial merosin deficiency in the UK.
lamina 2 chain; merosin; partial deficiency; muscular dystrophy
CK = creatine kinase; DAGs = dystrophin-associated glycoproteins; LAMA2 = laminin 2 chain gene; PCR = polymerase chain reaction; SSCP = single strand conformation polymorphism
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Introduction |
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Over the past few years, there has been a remarkable advance in our understanding of the molecular basis of muscular dystrophies. Dystrophin was the first protein found to be responsible for a muscular dystrophy (Koenig et al., 1987
). In Duchenne muscular dystrophy, no dystrophin is detected, usually as a result of mutations causing frameshifts in the corresponding gene, whilst in Becker muscular dystrophy, a milder allelic disease variant, mutations usually maintain the reading frame of dystrophin and an abnormal protein is produced (Monaco et al., 1988). Studies on the subcellular localization of dystrophin led to the discovery of a group of dystrophin-associated glycoproteins (DAGs), and the absence of each of four components of this complex recently has been found to be responsible for four types of autosomal recessive limb girdle muscular dystrophy (Campbell, 1995; Nigro et al., 1996). One protein of the DAG complex, the -dystroglycan, is one of the native ligands for the laminin 2 chain of merosin, an extracellular matrix protein involved in another form of muscular dystrophy (Yamada et al., 1994). In particular, a total absence of the laminin 2 chain of merosin has been observed in a significant proportion of children affected by congenital muscular dystrophy, a severe neuromuscular condition characterized by weakness and/or contractures at birth or in the first 6 months of life (Tomé et al., 1994; Dubowitz and Fardeau, 1995; Philpot et al., 1995).
The primary role of the laminin 2 chain gene (LAMA2) in these cases has been demonstrated not only by linkage to the corresponding locus on chromosome 6q2 in informative families (Hillaire et al., 1994; Helbling-Leclerc et al., 1995a; Naom et al., 1997), but also by the detection of intragenic mutations in several patients. The mutations responsible for this severe phenotype usually produce a frameshift and this is the reason for the lack of protein production in these patients (Helbling-Leclerc et al., 1995b; Pegoraro et al., 1996; Guicheney et al., 1998).
Interestingly, there have been a few recent reports on the occurrence of partial deficiency of laminin 2 chain in children who were either as severely affected as children with total absence of the protein (Nissinen et al., 1996) or had a significantly milder phenotype (Herrmann et al., 1996; Mora et al., 1996; Allamand et al., 1997; Hayashy et al., 1997; Naom et al., 1997; Sewry et al., 1997; Tan et al., 1997; Cohn et al., 1998). While in the studies of both Mora and Herrmann the evidence for the involvement of the LAMA2 gene came from the protein studies (Herrmann et al., 1996; Mora et al., 1996), in previous reports we showed that these families with a milder disease course and partial protein expression were linked to the LAMA2 gene (Naom et al., 1997; Sewry et al., 1997; Tan et al., 1997). Recently, Allamand reported a LAMA2 mutation in a consanguineous family with a mild form of congenital muscular dystrophy (Allamand et al., 1997), while we presented two siblings with a form of limb-girdle muscular dystrophy in whom we could identify two mutations in the same gene (Naom et al., 1998). Here we report two further case of mild muscular dystrophy due to mutations in the LAMA2 gene. These two unrelated children of British origin shared the mutations found in one allele, suggesting that this might be relatively common in our population.
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Case reports
Case 1
This is a 6-year-old girl, born to non-consanguineous Caucasian parents. She has a healthy older brother and there is no history of a neuromuscular condition in the family. She was born at 36 weeks of gestation following a normal pregnancy and a spontaneous vaginal delivery. She was well in the neonatal period and was discharged home on day 3. She was breast-fed and remained well in the first year of life. She sat at 8 months, crawled at 15 months, stood unaided at 22 months and started walking at 28 months.
Her parents started to be worried at 20 months because she had difficulty in pulling herself up to standing and did not stand unsupported.
She was referred for investigation at 20 months, and mild proximal muscle weakness and tightness of her ankles was noted. Her serum creatine kinase (CK) level was found to be moderately elevated at 723 IU/l (normal <200 IU/l). Serum CK at 25 months, when a needle muscle biopsy was obtained, was also elevated (928 IU/l).
Since then, her motor skills have improved and in particular she is able to rise to standing from lying down; she can walk quickly and is able to stand on either leg. However, she falls more frequently than other children and continues to have difficulties going upstairs.
On examination at the age of 4 years, she had a normal muscle build and facial expression and her gait was characterized by a slight widening of the base. However, when attempting to run, a mild waddle became evident. She rose from the floor with minimal difficulties (one-hand Gowers). She had a mild Achilles tendon tightness. Her muscle ultrasound showed a mild increase in echogenicity in both her calf and leg muscles; her motor nerve conduction velocity (ulnar nerve, 44 m/s) was at the lower end of normal values.
Brain T2-weighted MRI showed a striking increased signal of the white matter, involving the frontal and the occipital lobes, progressively increasing in a caudocephalic direction. The corpus callosum, basal ganglia, internal capsule, brain stem and cerebellum were unaffected (data not shown).
Case 2
This female child was born at 39 weeks gestation following an uncomplicated pregnancy. Her parents were unrelated and there was no family history of neuromuscular disorders. She was in good condition at birth, with Apgar scores of 9 at 1 min and 10 at 5 min. There were no neonatal problems. At 6 weeks of age, however, she was noted to be hypotonic. On examination at 7 months of age, there was limb and truncal hypotonia with marked facial weakness. Anti-gravity power was present in the upper and lower limbs but she was unable to sit or bear weight on her legs. Chewing and swallowing were difficult and there was slow weight gain. She walked at 2 years and 8 months, although there was still significant proximal muscle weakness. Intellectual and speech development was normal. There were no hearing or vision difficulties. Investigation at 7 months of age showed a serum CK of 1761 IU/l (24–170). Motor and sensory nerve conduction studies were normal. Needle electromyography suggested a myopathic process.
MRI brain scan demonstrated a bilateral symmetrical high signal in the periventricular white matter.
Muscle biopsy
A muscle biopsy of the quadriceps was obtained from both children, frozen in isopentane cooled in liquid nitrogen, and stained histochemically according to standard procedures (Dubowitz, 1985).
Cryostat sections were immunolabelled with a panel of monoclonal antibodies as previously described (Sewry et al., 1997). This included antibodies to dystrophin, ß-spectrin, -sarcoglycan (all Novocastra), laminin 2, ß1, 
1, a laminin chain recognized by clone 4C7 (all Chemicon) and a rat monoclonal to a 300 kDa fragment of laminin 2 (Sewry et al., 1997). Antibodies were detected using relevant biotynilated secondary antibodies followed by Texas Red conjugated to streptavidin.
No material was available for Western blot analysis.
CA repeat genotyping
The genomic DNA of the individuals was extracted from peripheral blood by the salt–chloroform method (Mullenbach et al., 1989). Five highly informative microsatellite markers spanning the laminin 2 chain locus on chromosome 6q2, D6S407, D6S1620, D6S1705, D6S1572 and D6S262, were analysed in both families as described previously (Naom et al., 1997).
Southern blot analysis
Genomic DNA of individuals in family 1 was digested with the restriction enzyme HindIII, electrophoresed on 1% agarose gel at 38 V over night, and transferred onto Gene Screen Plus membranes (NEN) on 10x SSC (standard saline citrate) buffer. Membranes were hybridized with [-32P]dATP radiolabelled probes (Megaprime kit Amersham, UK) using the Church and Gilbert method (Church and Gilbert, 1984) under high competitive conditions (300 mg of herring sperm DNA). The probe used was an amplification product obtained using a forward primer located in exon 36 (5'-TGATGCTTGGGACCTTTTGAGAG-3') and a reverse primer located in exon 40 (5'-CATTTGCTAACTTCT- TGGCTTGC-3') (see below).
High stringency conditions were used (hybridization and washing at 64°C). Membranes were exposed for 1.5 days at –80°C for autoradiography.
RNA isolation and cDNA amplification
Total cellular RNA was isolated from skeletal muscle of the two individuals by homogenization in a 4 M solution of guanidinium thiocyanate (Chomczynski and Sacchi, 1987). Total cDNA was synthesized from 2.5 µg of RNA in a final volume of 50 µl, using M-MulV Reverse Transcriptase (Pharmacia, St Albans, Herts, UK) and oligonucleotides of a random priming reaction (PdN6). Polymerase chain reaction (PCR) was performed on 1 µl of cDNA at 35 cycles of 94°C for 30 s, 65°C for 30 s and 72°C for 3 min in a final volume of 12.5 µl containing 0.5 mM dNTPs, 0.5 U of Taq Pfu (Stratagene, Amsterdam, Holland), 1x cloned Pfu buffer [10 mM KCl; 10 mM (NH4)2SO4; 20 mM Tris–HCl, pH 8.8; 2 mM MgSO4; 1% Triton X-100; and 10 g/ml BSA (bovine serum albumin)], and 5 pmol of each primer. The two primers were the same as those mentioned in the Southern blot section. cDNA primers for amplifying the exon 63 region were forward (5'-TTCGCGACAACTACAACGACTGGAG3') and reverse (5'-CTCGGAACGGAATACTGGTTGTTAGG-3'). The cDNA amplification products were first analysed in 2.5% agarose gels (Stratagene) and then stained with 0.1 µg/ml of ethidium bromide.
SSCP analysis
Single strand conformation polymorphism (SSCP) analysis of 62 exons was performed using a designed set of primers (Zhang et al., 1996). To analyse exons 37, 38 and 63 and the corresponding splice sites, the following forward primers were designed: 5'-GATGTCTTGTTCATAATGGTC, 5'-CTT-AAAATGCCCTCTTCTCTAC and 5'-CTGTGACTGTTCTATTTCC, and the reverse primers: 5'-CTCTCTTTTGAGTTTTACCCA, 5'-CACAAACATTTCCAAAATG and 5'-GAAATTGTTGCTGGGGTAG, respectively. PCR was performed in a total volume of 12.5 µl containing 75 ng of genomic DNA, 20 pmol of each primer, 5 mM dATP, dGTP and dTTP, and 1.25 mM dCTP, 6.7 mM MgCl2 and 0.75 U of Taq DNA polymerase (Promega, Southampton, UK). The genomic PCR was labelled by incorporation of [-32P]dCTP. After 5 min denaturation at 94°C, the PCRs were subjected to 27 cycles of 94°C for 1 min, 61°C annealing for 45 s and 72°C extension for 1 min, followed by 5 min elongation at 72°C. Samples were denatured in formamide buffer, with 5 min boiling before electrophoresis on non-denaturing 0.5x MDE gel (mutation detection enhancement solution containing acrylamide for mutation detection by SSCP and heteroduplex) (Flowgen, Leicestershire, UK). The electrophoresis was carried out in a Model S2 sequencing apparatus (BRL, Edinburgh, UK) for 16 h run at 4 W and 4°C. Gels were then dried and processed for autoradiography. The SSCP analysis of the cDNA was performed in 1 µl of cDNA, and PCR products were incorporated with the isotope in a second round of PCR.
DNA cloning and sequencing
A fragment of 1425 bp comprising both exons 37 and 38 and the entire intron 37 was amplified in case 1 by 30 cycles of PCR of 94°C for 1 min, 61°C for 1 min, 72°C for 3 min. The PCR products were cloned using the Stratagene pCR-Script+© Amp SK(+) cloning kit. A 100 µl aliquot of the PCRs was gene-cleaned using the QIAquick PCR purification kit (Qiagen, Crawley, Sussex, UK), polished with Pfu DNA polymerase and inserted into PCR-Script+© vector (as indicated by the supplier). Clones that carried the insert, indicated by double digest with KpnI and SacI, were digested with HindIII before sequencing with the Taq sequencing kit (Perkin Elmer, Warrington, UK) using fluorescent dideoxynucleotides, and both T7 and T3 primers to determine the sequence of both ends. Reactions were run on an automated DNA sequencer (Perkin Elmer). Sequencing was also performed for the PCR products of exons 37, 38 and 63 after purification with the QIAquick PCR purification kit. The sequencing reaction was carried out using the AmpliCycle sequencing kit (Perkin Elmer) and amplified for 27 cycles, after 2 min denaturing at 95°C. Cycles were as follows: 60 s at 95°C, 60 s at 68°C and 60 s at 72°C. Sequencing products were denatured and resolved in 6% polyacrylamide denaturing gels.
Sequencing using an ABI-377 automatic sequencer was repeated on patient 1 to confirm the mutations identified with the manual sequencing, and on the patient 2 to demonstrate the mutations in introns 37 and 63.
Restriction site analysis
The presence of mutations was confirmed by gain or loss of restriction sites in the first family analysed. The mutation found in exon 38 abolished a HindIII site, and its loss was confirmed after digestion of exon 38 and PCR amplification using the primer combination indicated above.
The intron 37 mutation present in the other allele did not occur within a restriction site. Therefore, we created a restriction site in the PCR product by designing a mismatch reverse primer of exon 37: 5'-CTCTTTTGAGTTTTACCCAcTA, which has a C nucleotide instead of a T at position 20, to introduce an SpeI restriction enzyme site (TGATCA). These primers were used in the following three-step cycles: one cycle of 95°C for 5 min, 57°C for 1 min and 72°C for 1 min; five cycles of 95°C for 1 min, 56°C for 1 min and 72°C for 1 min; and 24 cycles of 95°C for 1 min, 55°C for 70 s and 72°C for 90 s. A 7 µl aliquot of the PCR product of the affected individual and unaffected controls was digested in a 10 µl digest (as described by the suppliers). The products were then run on 3% agarose gels.
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Muscle biopsy
The muscle biopsy showed dystrophic changes in both children, with variation in fibre size, an increase in connective tissue, an increase in internal nuclei and occasional splitting and hypercontracted and whorled fibres.
Immunochemistry showed normal expression of dystrophin and -sarcoglycan in patient 1. Expression of ß-spectrin was also normal in this patient except for reduced expression on some regenerating fibres.
Dystrophin was normal using antibodies to the C- and N-terminal domains of dystrophin but very weak using an antibody to the dystrophin rod domain in case 2.
Labelling of laminin 2 with the antibody to the 80 kDa fragment showed reduced labelling on some fibres compared with controls, but with the 300 kDa antibody there was a marked reduction on most fibres (Fig. 1A, B, E and F). The laminin detected by the 4C7 antibody (previously thought to be laminin 1) was slightly overexpressed, whilst the expression of laminin ß1 and 1 was normal (Fig. 1C, D, G and H). The findings were almost identical in the muscle biopsy of the second child (result not shown).
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Haplotype analysis
Genotyping the microsatellite markers spanning the LAMA2 locus in family 1 suggested that the proband was heterozygous for the two at-risk haplotypes. This was expected since the family is not consanguineous; the unaffected brother was found to be a carrier, having inherited the maternal at-risk haplotype (Fig. 2
). In the second family, only the propositus and her mother were available for haplotype analysis. The patient was found to be homozygous for the LAMA2 markers studied, suggesting a distant level of consanguinity of her parents.
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Comparison of the haplotypes of the propositi in the two families showed that they did not share a common haplotype.
Southern blot analysis
Southern blot analysis using a cDNA probe encompassing exons 36–40 revealed an abnormal fragment (of ~1.8 kb) that was present only in the affected child in family 1 (not shown). This fragment was not present in the two parents, nor in 17 unrelated controls.
Mutation analysis
Family 1
SSCP analysis was carried out in all exons in family 1. Analysis of exon 38 and flanking splice sites (Zhang et al., 1996) revealed an abnormal conformer in the proband compared with controls (Fig. 3C). Direct sequence analysis of the aberrant PCR product revealed a single nucleotide deletion of an A at position 5702 of the cDNA sequence (Fig. 4), numbered according to Vuolteenaho (Vuolteenaho et al., 1994). This was confirmed with automatic sequencing and resulted in a frameshift leading to a stop codon 48 nucleotides downstream of the deletion. These results were confirmed on a separate PCR with sequencing. This deletion abolished a HindIII site in the LAMA2 gene and therefore corresponded to the junction fragment seen with the Southern blot.
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We also carried out a restriction digest of PCR products in this family. HindIII restriction analysis of wild-type exon 38 results in a 119 and a 112 bp fragment, while the affected child had, in addition to these two normal fragments, a 231 bp uncut fragment (Fig. 5A
). Interestingly, this uncut 231 bp DNA fragment was not present in either parent, confirming that this is a de novo nucleotide deletion. In addition to the 17 controls studied by Southern blot analysis, we performed restriction digest PCR on a total of 50 unrelated controls who also failed to show this fragment (data not shown).
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This de novo single nucleotide deletion apparently occurred in the paternal haplotype of the affected girl in family 1 (see below).
SSCP analysis of exon 37, exon 63 and flanking splice sites showed an abnormal conformer in the proband (Fig. 3A and B
). A similar band was never observed in 50 controls (exon 37) and 90 controls (exon 63). It was similarly not seen in an additional 15 patients with merosin-deficient congenital muscular dystrophy (not shown). Linkage and sequence analysis (see below) confirmed that these two mutations were both in cis; they were, however, in trans compared with the exon 38 frameshift mutation described above.
Direct nucleotide sequencing of the two regions demonstrated TC and GC substitutions at position +6 and +5 of the consensus donor splice site of introns 63 and 37, respectively (Fig. 6B and D). The mutation in intron 37 was confirmed by generating an SpeI restriction enzyme site (see Material and methods), and digesting the PCR product of the affected girl and other family members (Fig. 5B). The digestion results in shortening of the wild-type fragment of exon 37 (171 bp) to one 151 and one 20 bp fragment. The co-existence of the 171 bp (wild-type) and 151 bp (mutated) fragments was seen not only in the affected child, but also in her mother and the unaffected brother, suggesting that all these individuals were heterozygous for this mutation; this was in agreement with the inheritance of the haplotypes (Figs 2 and 5B). Ninety-four chromosomes of non-related individuals were also tested for the GC mutation by restriction digest, and this was never found (data not shown).
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As the single nucleotide deletion in exon 38 occurred de novo, and therefore it was not possible to tell by linkage analysis whether it was in cis or in trans compared with the consensus splice site mutation found in intron 37, we amplified a fragment of 1425 bp comprising exon 37, intron 37 and exon 38 from the patient and subcloned it. Clones were digested with HindIII in order to recognize the clones which had lost the normal restriction site (i.e. which carried the deleted allele). Sequence analysis showed that clones that had lost the restriction site did not carry the splice site G
C mutation, and vice versa (not shown). These results suggest that these two mutations are in trans. Segregation analysis of the consensus donor splice site mutation affecting intron 63 showed that this was inherited from the mother, confirming therefore that it was in cis with the other splice site mutation, and in trans with the frameshift mutation found in the paternal allele.
Transcription analysis.
Amplification of exons 36–40 and 62–64 from skeletal muscle cDNA in patient 1 resulted in the amplification of one single, expected fragment in each case (data not shown). There were no higher or lower molecular weight fragments suggestive of exon skipping or of other splicing abnormalities. The abundance of the laminin 2 transcript was considerably lower compared with control, as judged by semi-quantitative PCR analysis (data not shown).
Family 2
SSCP analysis of exons 63 and 37 was performed in this family. Analysis of exons 63 (Fig. 3B) and 37 (Fig. 3A) and flanking splice sites revealed an abnormal conformer in the proband compared with controls. Interestingly, this affected girl was found not to have any wild-type sequence for these two regions, suggesting that she is homozygous for both mutations. Haplotype analysis for LAMA2 markers confirmed that she is indeed homozygous.
Direct sequence analysis of the aberrant PCR product demonstrated TC and GC substitutions at position +6 and +5 of the consensus donor splice site of introns 63 (Fig. 6C) and 37 (data not shown), respectively. Unfortunately, there was no muscle available for transcriptional analysis in this case.
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Discussion |
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In this study, we describe two unrelated children affected by an early-onset muscular dystrophy with two novel mutations in the LAMA2 gene. The mutations allowed the production of sufficient protein in both girls to result in a considerably milder phenotype compared with children affected by the severe form of congenital muscular dystrophy. In this form, manifest at birth or in the first months of life, no expression of the protein can be observed (Tomé et al., 1994
; Dubowitz and Fardeau, 1995; Philpot et al., 1995; Sewry et al., 1995). The mutation found in one of the two alleles in family 1 was a single nucleotide deletion causing a frameshift and a stop codon in exon 38. Interestingly, this was a de novo mutation that arose in the paternal haplotype and, as far as we are aware, it represents the first evidence for a de novo mutation in the LAMA2 gene.
The mutations in the other allele were a GC point mutation at position +5 in the conserved donor splicing consensus sequence of intron 37, and a TC mutation at position +6 in the conserved donor splicing consensus sequence of intron 63.
Interestingly, these two mutations present in cis in one of the two alleles in family 1 were found also to be homozygous in family 2, further suggesting a role for at least one of them in determining the phenotype. As we were unable to identify exon skipping as a result of these mutations, we hypothesize that either one or both of them affect splicing efficiency.
Computer analysis of the effect that these mutation might have on splicing (Allikments et al., 1998; Kannabiran et al., 1998; O'Neill et al., 1998) suggests that the GC variant in intron 37 is a fairly severe mutation. In particular, this mutation is predicted to have a 15-fold reduction in binding to the splicesome (O'Neill et al., 1998). Similar analysis of the TC variant in intron 63 shows that its effect on splicing is less dramatic compared with the previous mutation: in particular, this variation is presumed to give rise to only a 2.6-fold difference in binding (Allikments et al., 1998; Kannabiran et al., 1998; O'Neill et al., 1998). As both variations are in cis in the same haplotype, it is not possible to tell conclusively from our study which one is causative or whether they both contribute to the abnormal splicing of the laminin 2 chain. The fact that they were not found in >100 controls but were found in two unrelated patients with partial laminin 2 chain deficiency does, however, suggest that they are not rare polymorphisms. Interestingly, linkage analysis suggested that the two affected children do not have a haplotype in common, as might have been predicted on the basis of having inherited the same unusual mutations in cis in the same allele. However, the possibility of a double recombinant event between the markers used (3 cM for the two markers that flank the LAMA2 chain gene) and the gene cannot be excluded.
Other studies have reported similar causative mutations in the pro-2 chain of type 1 collagen in a patient with severe dentinogenesis imperfecta (Nicholls et al., 1996). Transcription analysis of the pro-2 chain of type 1 collagen showed that the mutation induced exon skipping in that patient (Nicholls et al., 1996). Reduced splicing efficiency, and not exon skipping, has, however, already been documented for mutations occurring in the consensus sequence of splice sites (Vreken et al., 1995; Ainsworth et al., 1996; Raben et al., 1996; Tumer et al., 1997). Our results suggest that the two mutations present in this allele allowed the production of a normal LAMA2 mRNA, but in reduced amount. The semi-quantitative PCR analysis we performed in case 1 showed low levels of LAMA2 mRNA in skeletal muscle, further suggesting that the mutations in this patient affect RNA quantity and/or stability.
Two of the mutations identified in family 1 (exon 38 and intron 37) lie in domain I + II of the laminin 2 chain protein, while the second splice site mutation lies in the G domain. They are all located in a different region from the previously reported cases with residual expression of the protein. These cases had mutations affecting domain IVa, a region known to be involved in the formation of the laminin network (Nissinen et al., 1996; Allamand et al., 1997).
Regarding the protein studies performed in these children, the pattern of immunocytochemical expression of the laminin 2 chain was very different with the two antibodies used. While only minimal abnormalities were noted with the antibody that recognizes the C-terminal 80 kDa fragment, a significant reduction of protein expression was detected using the antibody that recognized a 300 kDa fragment. We have reported a similar pattern of laminin 2 chain expression in other patients with a milder disease course (Sewry et al., 1997; Tan et al., 1997; Naom et al., 1998). It has also been observed in two siblings with an internally deleted protein, in whom a splice site mutation induced an in-frame deletion of exon 25 (Allamand et al., 1997), and in all patients with partial laminin 2 chain deficiency reported by Cohn and colleagues (Cohn et al., 1998). The reason for the discrepancy of the staining obtained using these two different antibodies is not clear, and neither epitope recognized by these antibodies is known. The precise epitope recognized by the 300 kDa antibody is unknown; the fact that a residual staining was present when this antibody was used does not support the idea that this epitope was removed by a deletion. A possible explanation for the differential expression of the two fragments is that these mutations disrupt the formation of the extracellular matrix network and result in increased proteolytic degradation of the protein. Alternatively, the domain against which the 300 kDa antibody is raised might be the binding site for a protein with a very high affinity for the laminin 2 chain, and this results in `sequestration' of the epitope by this putative binding protein. This might result in significant reduction of epitope exposure when a mutation significantly reduces the quantity of the laminin 2 chain.
Our data reinforce the view that the spectrum of phenotypes observed as a result of mutations in the LAMA2 gene is wider than initially appreciated. Mutations that severely affect the expression or structure of this gene (Helbling-Leclerc et al., 1995a; Nissinen et al., 1996; Pegoraro et al., 1996; Guicheney et al., 1998; Pegoraro et al., 1998) result in the severe form of congenital muscular dystrophy with onset at birth or within the first few months of life, while in-frame deletions (Allamand et al., 1997) or mutations that are compatible with the production of low levels of a normal protein (Naom et al., 1998; this report) can give rise to a significantly milder phenotype, characterized by childhood-onset muscular dystrophy. On the basis of our results, we suggest that the involvement of the laminin 2 chain of merosin, using antibodies raised against several domains of the protein, should be excluded in undiagnosed cases of childhood-onset muscular dystrophies, especially if other suggestive features, such as white matter abnormalities, can be demonstrated on brain MRI.
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Acknowledgments |
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The authors wish to thank Dr J. Lowe, who initially referred the biopsy of A.M., Dr D. Mellor who referred A.M. to our Centre for further analysis, Dr N. Gilbertson for referring patient J.B. to Bristol, and Professor P. Rogan for helpful comments on the effect on splicing efficiency of the mutations we identified. This work was very generously supported by the Muscular Dystrophy Campaign of Great Britain and Northern Ireland. M.D. is an Italian Telethon Ph.D. student.
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Received May 18, 1999. Revised July 16, 1999. Accepted July 30, 1999.
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