Leiden Muscular Dystrophy pages

Laminin-alpha 2 (LAMA2)

( MDC1A )

(last modified June 28, 2003)



Contents




Summary


Laminins are a family of large trimeric basement membrane glyco-proteins composed of 3 non-identical chains, the alpha-, beta- and gamma-chains. The three chains associate at their C-terminal end in a coiled coil. They form a cruciform structure consisting of 3 short arms, each formed by a different chain, and a long arm composed of all 3 chains. The long arm is formed by the coiled coil and the short arms by the N-terminal ends. Each laminin chain is a multi-domain protein encoded by a distinct gene. Several isoforms of each chain have been described. In human, five different alpha, three beta and two gamma chains have been identified, their genes dispersed throughout the genome. The biological functions of the different chains and trimer molecules are largely unknown. Some of the chains have been shown to differ extensively with respect to their tissue distribution, presumably reflecting diverse functions in vivo.

chain gene localisation protein
sequence
alpha LAMA1 18p11.3 CAA41418  (laminin A)
LAMA2 6q22-23 NP_000417  (laminin M)
LAMA3 18q11.2 NP_000218  (epiligrin)
LAMA4 6q21 NP_002281  (LAMA3)
LAMA5 20q13.2-q13.3 NP_005551
beta LAMB1 7q22 NP_002282
LAMB2 3p21 NP_002283  (laminin S)
LAMB3 1q32 NP_000219  (nicein, kalinin)
gamma LAMC1 1q25-31 NP_002284  (formerly LAMB2)
LAMC2 NP_005553 (nicein, kalinin)

The different laminin isoforms consist of different combinations of the alpha-, beta- and gamma-chains which are designated by Arabic numerals in the order of their discovery. Laminin-1 (alpha-1 + beta-1 + gamma-1), laminin-2 (merosin, alpha-2 + beta-1 + gamma-1), laminin-3 (alpha-1 + beta-2 + gamma-1), laminin-4 (alpha-2 + beta-2 + gamma-1), laminin-5 (alpha-3 + beta-3 + gamma-2), laminin-6 (alpha-3 + beta-1 + gamma-1), laminin-7 (alpha-3 + beta-2 + gamma-1), laminin-8 (alpha-4 + beta-1 + gamma-1), laminin-9 (alpha-4 + beta-2 + gamma-1) and laminin-10 (alpha-5 + beta-1 + gamma-1) (Miner et al., 1995). Laminin-2 and -4 are specifically enriched in the basement membranes surrounding skeletal muscle fibers and peripheral nerves. While the beta-1, beta-2 and gamma-1 chains are ubiquitously expressed, tissue-specificity is provided only by the alpha-2 chain, formerly described as merosin.

Laminin-alpha 2 is widely expressed in the basement membrane of skeletal muscle, at neuromuscular synpases, extra-synaptically and in the myotendinous junctions. LAMA2 has been shown to bind to dystroglycan, which interacts with dystrophin and extra-cellular laminin.


The LAMA2 gene


Links to other databases:
Gene Symbol nomenclature   LocusLink 

The laminin alpha-2 gene (Gene Symbol LAMA2, alias lamin-M, merosin, LAMM) has been characterized by Zhang et al. (1996). The gene, localised to 6q22-23 spans some 633 kb of genomic DNA and contains 65 exons (NOTE: Zhang report 64 exons but missed one intron in exon 5). Two of the exons are extremely small, measuring 6 (exon 44) and 12 (exon 53) base pairs only. The introns vary from ... to ...

Exon Exon size
(in bp)
Intron size
(in kb)
5' cDNA
position
Splice after EcoRI
(in kb)
Remarks
1 (161) 138,249 (-49) 1 7.8 5' UTR (49 bp), 
protein domain VI
2 171 ? 113 1 2.8
3 113 ?   3 3.0
4 243 ?   3 3.3
5   2,878 640 3 5.5...
6   0.75 820 3 5.5 protein 
domain V
7 118 5.7     2.6
8 179 3.8     3.4
9 100 0.6     5.8
10 161 5     8.0
11 141 2.2     protein 
domain V / IVb
12 174 7     protein 
domain IVb
13 102 2     4.2
14 212 1.1     5.4
15 112 4     protein domain 
IVb / IIIb
16 114 3.5     2.1 protein 
domain IIIb
17 128 6.5     2.7
18 87 5.1     2.5
19 212 3.7     5.0
20 107 13    
21 181 3.1     9.5
22 137 ?     1.7
23 237 1.6     17
24 144 1.1     protein domain 
IIIb / IVa
25 180 0.15     protein 
domain IVa
26 189 1.1    
27 134 4.4    
28 118 8.5     protein domain 
IVa / IIIa
29 135 8     protein 
domain IIIa
30 125 3.9     3.4
31 87 6.3     6.5
32 194 0.3    
33 143 3.9     6.8 protein domain 
I + II
34 99 8     6.5
35 112 3.7     6.5 + 6.5
36 163 1.5     1.9
37 211 7.5     7.8
38 117 1.1     1.4
39 164 1.5     9.0
40 139 7    
41 103 2.2     6.4
42 117 10     15
43 183 2     2.4
44 6 2.5     3.2
45 155 5.5    
46 144 1     2.8 protein 
domain I / G
47 134 1.9     1.4 protein domain G
48 160 3.7     3.2
49 125 3.9     2.1
50 163 0.65     2.6
51 145 0.7     4.0 + 0.4
52 139 1.8     4.0
53 12 2.2    
54 121 2.3     4.6
55 177 4.7     13
56 148 9    
57 178 0.3    
58 169 8.5     4.6
59 113 0.45     6.8
60 190 1.9    
61 156 2.2     6.8 + 1.4
62 154 4.9     2.6
63 131 1.9     2.6 + 1.2
64 223 1.4     2.6
65 375 -     4.0 end protein domain 
G, 3' UTR (217 bp)

Legend:
Exon: numbering of exons and intron/exon boundaries largely as determined by Zhang, with their exon 5 split by an additional intron. The first base of the Met-codon is counted as position 1 (see LAMA2 cDNA Reference Sequence). Exon size: size of exon indicated in basepairs. Intron size: size of intron indicated in basepairs. 5' cDNA position: first base of the exon (according to LAMA2 cDNA Reference Sequence). Splice after: splicing occurs in between of two coding triplets (0), after the first (1) or the second (2) base of a triplet. EcoRI = size EcoRI fragment containing the exon in kilo basepairs (Zhang, 1996), exons 35, 51, 61 and 63 contain an EcoRI-site. Remarks: 5'UTR = 5' untranslated region, 3'UTR = 3' untranslated region.

primers for DNA-amplification


The LAMA2 mRNA


Links to other databases:     RefSeq: NM_024301     UniGene: Hs.75279

The LAMA2 (merosin) RNA was first described by Ehrig et al. (1990) as a ~10 kb transcript. It was fully characterised by Vuolteenaho (1994) using human placental cDNA-libraries. The transcript measures 9.6 kb and contains a 49 bp 5' UTR, a 9,333 bp open reading frame and a 217 bp 3' UTR. Differential splicing of the LAMA2-transcript was reported by Pegoraro et al.; the second half of exon 32 was spliced out of some 5% of all mRNA's yielding a 46 amino acid shorter LAMA2-protein. 

Northern blot analysis of human fetal tissues showed that LAMA2 was expressed in most tissues such as cardiac muscle, pancreas, lung, spleen, kidney, adrenal gland, skin, testis, meninges, choroid plexus and some other regions of the brain (Vuolteenaho [1994]). LAMA2 was not expressed in liver, thymus, and bone. In situ hybridization localized LAMA2 expression to cells of mesenchymal origin (in contrast, expression of the A chain is only in kidney, testis, neuroretina and some region of brain). Epithelial and endothelial cells were negative for both LAMA2 and LAMA1 transcripts.

primers for RNA-amplification


The LAMA2 protein


Links to other databases:    RefSeq: NP_077277    SwissProt: P24043

LAMA2 is cleaved at amino acid 2580 into a 300 kDa N-terminal and a 80 kDa C-terminal segment (Ehrig, 1990). The LAMA2 protein consist of 3110 amino acids; a 22-residue signal peptide, amino acids 1-22, and a 3,088 amino acid mature protein. It has a calculated MW of 400 kDa and consists of 6 domains, going from N- to C-terminus, domain VI (264 residues, amino acids 23-286), domain V (241 residues, aa ), domain VIb (196 residues, aa 528-723), domain IIIb (452 residues), domain IVa (204 residues, aa 1176-1379), domain IIIa (194 residues), domain II + I (571 residues, aa 1574-2144) and a C-terminal G domain (957 residues). Domains I and II are part of the long arm. Domains IIIa, IIIb and V contain cystein-rich EGF like repeats (17 in total) and are predicted to have rigid rod-like structures. Domains IV1, IVb and VI are predicted to form globular structures. The globular C-terminal region (G-domain) contains five internally homologous repeats with a conserved LFVGGLP-motif followed 17-21 and 40-43 residues later by a Cystein, mostly precede by Glycines (aa 2145-2328, 2340-2521, 2526-2710, 2763-2934 and 2939-3110). It also contains the interaction sites for membrane receptors, in striated muscle alpha-dystroglycan and integrin alpha-7/beta-1. LAMA2 contains 28 potential N-linked glycosylation sites.

LAMA2 has a domain structure similar to that of the human and mouse A chains. The homology between the two human laminin heavy chains is highest in the short arm region and lowest in the long arm helical domain I + II. 

In laminins, domains I and II and the C-terminal G domain of the three chains (alpha-2, beta-1 and gamma-1 for laminin-2) participate in the formation of the long-arm coiled coil. In muscle, this heterotrimer attaches through the globular G-domain of the alpha-2 chain with alpha-dystroglycan (Gee et al. [1993], Sunada et al. [1994]). Through this interaction, a link is provided between the extra-cellular matrix and the sarcolemmal cytoskeleton. The absence of LAMA2 in patients with congenital muscular dystrophy (CMD) has been suggested to weaken the anchorage of muscle fibers to the extra-cellular matrix, resulting in muscular dysfunction and dystrophy.

LAMA2 antibodies

...

The LAMA2 protein of Drosophila is encoded by a much compacter gene (14 kb) which contains only 15 exons (Zhang, 1996). Between human and Drosophila, only the location of introns 3 and 6 are conserved (2 and 3 in Drosophila).


LAMA2 and disease: MDC-1A


Homozygosity mapping and linkage analysis localised the MDC-1A (musuclar dystrophy, congenital type 1A) locus to 6q2 (Hillaire et al., 1994, Helbling-Leclerc et al. 1995). Since this region also contained the LAMA2 gene (localised to 6q22-23, Vuolteenaho et al., 1994), Helbling-Leclerc et al. (1995) analysed the LAMA2 gene as a candidate for MDC-1A. Using RT-PCR on lymphoblastoid cell lines truncating, disease-causing mutations were found in two families.

The Congenital Muscular Dystrophies (CMD) are a group of neuromuscular disorders with severe muscle hypotonia (at birth or within the first months of life), generalised muscle weakness, contractures of variable severity and delayed motor milestones. Histological changes in muscle biopsies consist of marked connective tissue proliferation and a large size variation of muscle fibres. In early stages of the disease some necrotic and regenerating fibres can be seen.

Merosin-deficient CMD (for a recent review see Allamand & Guicheney, 2002) is a severe form presenting with an absence of laminin alpha-2 (merosin) around muscle fibres, especially in the early months of life an elevated serum creatine kinase (CK), no independent ambulation due to weakness and contractures and a respiratory insufficiency which could need tracheotomy. Most patients have normal intelligence but some show moderate mental retardation and epilepsy.  In contrast to partial merosin deficiency cases (less frequent), merosin deficient CMD forms a clinically homogeneous subgroup. The use of several antibodies directed against different regions of lLAMA2 allows a precise distinction between complete and partial deficiency (He et al. [2001]).

Diagnosis is usually made by the clinical features and a muscle biopsy examination (complete LAMA2 deficiency can also be determined using a skin biopsy). Immuno-histochemically, at the basal lamina surrounding myofibres, LAMA2 is deficient, while LAMA4 and LAMA5 are overexpressed. Integrin alpha-7/beta-1 and alpha-dystroglycan (functional laminin receptors) show secondary reduction (Hayashi, Muntoni & Guicheney). Diffuse brain white matter changes in LAMA2-deficient patients, detected by magnetic resonance imaging (MRI), appear after the first 6-12 months of life. Prenatal diagnosis can be made by determination of at-risk haplotypes using intra- and extra-genic markers, and also by immuno-histochemical analysis of trophoblasts (complete deficiency only).

 In a sample from north-east Italy, the prevalence of CMDs has been estimated to be 0.7/100,000 (Mostacciuolo, 1996). LAMA2-deficient CMD accounts for about 30% of the CMD cases in European countries, but only 6% in Japan.

No treatment is available to date but the condition of life is  improved by physiotherapy to reduce contractures and arthrodesis to limit deformation. Ventilatory support and tracheotomy (when necessary) have contributed to a marked increase in life expectancy for the most severely affected patients.

Animal models

Several murine models for autosomal recessive LAMA2-deficient CMD have been described. These include the spontaneous mouse strains dy (dystrophia muscularis) and dy2J, and two knockout lines (dy3K [Miyagoe, 1997] and dyW [Straub, 1997]) which present a severe phenotype and a reduced life span. The dy and dy2J-mouse present a muscle pathology and a dysmyelination of the peripheral nervous system due to a complete and partial LAMA2 deficiency, respectively. The dy mouse is the most severely affected. It lacks LAMA2 in skeletal muscle and peripheral nerve and has severely defective basement membranes in muscle (Sunada et al. 1994). The mutation responsible for the dy phenotype is unknown, LAMA2 can not be detected on Western and Northern blots. The phenotype of the dy2J-mouse is less severe with antibodies against the 300 kDa C-terminal segment showing near normal staining patterns (Hu et al.). On SDS-PAGE, the N-terminal segment from muscle extracts is 10-50 kDa smaller than normal. LAMA2 mRNA is smaller in size compared to normal LAMA2 transcripts. The dy2J-mouse contains a G>A exon 2 splice donor site mutation (283+1G>A in relation to the human cDNA Reference Sequence). This change leads to abnormal splicing and expression of five mRNA species (Hu et al.); 

The predominant transcript is translated into a protein lacking 55 amino acids in protein domain VI (Val78_Gln132del). 


Disease-causing LAMA2 mutations


Links to other databases:   OMIM: ....

In 1995, Helbling-Leclerc et al. identified the first mutations in LAMA2. Analysis of the LAMA2 cDNA or gene revealed that nucleotide substitutions and small deletions / insertions may induce complete LAMA2-deficiency. Most pathogenic changes are localised in the N-terminal domain (exons 1-32) and are predicted to produce truncated proteins. Most changes reported are unique, but a 2 bp deletion (2098delAC) and nonsense mutation 2901C>A (Cys967X, identified in several Italian families) have been found in several studies. Some larger deletions have also been reported.

LAMA2 sequence variations
(mutations and polymorphisms)


Miscellaneous


DNA-primers

Amplified Length
(in bp)
Reference forward primer reverse primer Name
           

Legend:
Exonic sequences are in upper case, intronic and gene flanking sequences in lower case and added primer tails in italics. Amplified: region amplified. Numbering of exons is according to .... Length: length of PCR-product in basepairs. Reference: publication describing the primer(s). Forward primer: sequence of forward primer. Reverse primer: sequence of reverse primer. Name: name of the primers.


RNA-primers


LAMA2 sequences


Similar sequences



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