Genetics of laminin α2 chain (or merosin) deficient congenital muscular dystrophy: from identification of mutations to prenatal diagnosis

PERGAMON

Neuromuscular Disorders 7 (1997) 180-186

Genetics of laminin chain (or merosin) deficient congenital muscular dystrophy: from identification of mutations to prenatal diagnosis P a s c a l e G u i c h e n e y a'*, N i c o l a s V i g n i e r a, A n n e H e l b l i n g - L e c l e r c a, M a r j a N i s s i n e n b, X u Z h a n g c, C o r i n n e C r u a u d d, J e a n - C l a u d e L a m b e r t e, C h r i s t i a n R i c h e l m e e, H a l u k T o p a l o g l u f, L u c i a n o M e r l i n i g , A n n i e B a r o i s h, K e t t y S c h w a r t z a, F e r n a n d o M . S . T o m 6 a, K a r l T r y g g v a s o n c, M i c h e l F a r d e a u a "INSERM U153, Groupe Hospitalier Piti~-SalpOtribre, lnstitut de Myologie, 47 boulevard de l'H@ital, 75651 Paris Cedex 13, France bBiocenter Oulu and Department of Biochemistry, University of Oulu, 90570 Oulu, Finland CDepartment of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden aG~n~thon, 1 rue de l'Internationale, 91002 Evry, France eH6pital de l'Archet 2, Nice, France fDepartment of Paediatric Neurology, Hacettepe Children's Hospital, 06100 Ankara, Turkey gNeuromuscular Laborato~, Rizzoli Orthopaedic Institute, Bologna, Italy hService de Pddiatrie-R~animation infantile, H~pital Raymond-Poincar~, 92380 Garches, France Received 14 November 1996;received in revised form 28 January 1997; accepted 2l February 1997

Abstract

Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of muscle disorders, with autosomal recessive inheritance. Absence of the laminin c~2 chain in the skeletal muscle of patients with classical CMD has permitted the identification of a subgroup, referred to as 'merosin-deficient CMD or laminin a2 chain deficient CMD'. We first identified a nonsense and a splice site mutation in laminin a2 gene (LAMA2) (Glu1241stop, 4573-2A --->T). We report here new mutations: nonsense mutations (Glu210stop, Trp2316stop) and 1- and 2-bp deletions (2418AC, 6968ATA), which result in truncation of the protein either in the short arm domains or in the C terminal globular domain and complete merosin deficiency. Another subgroup, referred to as 'partially-deficient in laminin c~2 chain', has been identified recently, and a LAMA2 missense mutation (Cys996Arg) has been shown to cause this partial deficiency. The laminin c~2 chain, together with the/31 or ~2 and 3'1 chains forms either laminin-2 (ot2-/31-3,1)or laminin-4 (~2-/32-3,1). The LAMA2 mutations induce the formation of abnormal laminins which probably dramatically disturb the assembly and stability of the laminin network, one of the major components of the extracellular matrix in skeletal muscle. We report also the first prenatal diagnosis performed by direct mutation analysis. © 1997 Elsevier Science B.V.

Keywords: Congenital muscular dystrophy; Laminin ~ chain; Merosin

1. Introduction

Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of muscle disorders, with onset in early infancy, and autosomal recessive inheritance [1-3]. Several forms have been identified: classical or occidental CMD with normal or sub-normal intelligence; Fukuyama's CMD (FCMD), prevalent in Japan, characterized by severe mental retardation and major structural brain * Corresponding author. Tel.: +33 1 42165735; fax: 33 1 42165700; e-mail: pguichen @myologie.infobiogen.fr

0960-8966/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved Pll S0960- 8 9 6 6 ( 9 7 ) 0 0 4 6 0 - 4

abnormalities [4]; and the Walker-Warburg syndrome (WWS) and the muscle-eye-brain disease (MEB), both of which are associated with muscle, eye and brain abnormalities [5-7]. A major advance in the characterisation of CMDs was the finding that the laminin c~2 chain was absent in the skeletal muscle in about 50% of patients with classical CMD. This permitted the identification of a subgroup of CMD, referred to as 'merosin-deficient CMD' or 'laminin ~2 chain deficient CMD' [8], which was further shown to be due to mutations in the laminin c~2 chain gene [9]. The 'merosindeficient' CMD patients presented with hypotonia, contrac-

P. Guicheney et al. / Neuromuscular Disorders (1997) 180-186

tures and motor development delay which are generally more severe than in 'non-deficient' CMD [10] patients. The aims of the present paper are to review various aspects of laminin expression and structure, the laminin or2 chain gene (LAMA2) locus and the previously known mutations causing CMD with complete or partial merosin deficiency. We also present new mutations, as well as the first prenatal diagnosis by direct mutation analysis.

2. The laminin family: heterotrimeric molecules Laminins are a family of large, highly glycosylated extracellular matrix proteins. Each laminin molecule is composed of c~,/3 and 7 chains (Fig. 1), with molecular sizes ranging from 140-400 kD [11,12]. The three chains form a

i

o~2 chain

V

lllb

131 chain

IV,, ~

VI

V

IV

71 chain

Ilia

III

III

IV

V

VI

181

Table 1 Laminin isoforms Isoform

Chain composition

Laminin- 1 Laminin-2 Laminin-3 Laminin-4 Laminin-5 Laminin-6 Laminin-7 Laminin-8 Laminin-9 Laminin-10

~ 1fl 1T 1 c~2fl I Y 1 ~ 1fl2y 1 c~2B23,1 o~3f13"y2 ~3fl 1T 1 ~3fl271 c~4/3171 c~4fl271 c~5fllyl

heterotrimeric molecule through a coiled-coil ot helix at the carboxy terminal end, while the amino termini form distinct short arms, which give to most of the laminin isoforms the cruciform appearance as observed in electron microscopy [ 13-15]. Ten distinct laminin polypeptide chains have been described to date [11,12,16], forming different combinations and resulting in at least ten heterotrimeric isoforms (Table 1) [17-19]. The complexity of the laminin family justified the adoption of a clear nomenclature system to replace the previous names [18]. The chains are assigned to three laminin chain subgroups: a,/3 and 3' according to their sequence homologies and domain structures. The otl and et2 chains, previously known as A and M respectively, are structurally the most similar a chains [20,2l]. The laminin c~2 chain, together with the /31 and 3'1 chains, forms the laminin-2 (previously named merosin [18]), which is found in the basement membranes of the adult skeletal muscle fibers and Schwann cells, in placental trophoblasts [22] and in skin [23]. Laminin-4, an isoform containing laminin et2, /32 and 3'1 chains is present at the myotendinous [24] and at neuromuscular junctions [25]. Using immunostaining, the a l chain has been shown to be upregulated in CMD patients with partial or complete laminin ~2 chain deficiency [8], as determined with the 4C7 antibody [24], but recently the specificity of this antibody has been questioned.

3. Structural domains of the laminin chains

,5 Fig. 1. Organisation of laminin-2 polypeptide chains. The Roman numerals correspond to laminin domain numbers (modified from [12]). Domains V and III are cysteine-rich domains. Domains I and II contain numerous heptad repeats in which hydrophobic and charged amino acids are located at regular intervals and correspond to the s-helical region [62,63].

All laminin chains have a homologous domain structure, as initially pointed out by Sasaki et al. [26,27]. The amino terminus of each chain is composed of cysteine-rich repeating regions, interrupted by globular domains (Fig. 1). The carboxy termini of the chains form an a-helical coiled coil domain and all the e~ chains have also a large globular domain at the end of the carboxy terminus. The domains are named from I to VI, with the additional domains found in the c¢ and/3 chains being referred to as domains G and ~, respectively [15]. The amino termini (domains VI) of the c~, 13 and 3, chains are involved in the formation of the laminin network by self aggregation [28].

P. Guicheney et al. / Neuromuscular Disorders (1997) 180-186

182

6q

/

/

/

/

/

/

/

/

D6S1715 1 c~

-

D6S407 LAMA2 gene

3 cM -

D6S1620 D6S1705

-

D6S1572

Fig. 2. Localisation of the laminin c~2 chain gene on chromosome 6q22-23.

4. Localisation of the laminin c~2 chain deficient C M D locus Linkage studies on consanguineous laminin o~2 chain deficient CMD families localized the disease to a 16 cM region on chromosome 6q2 [29], where the LAMA2 gene is located [21] (Fig. 2). Refined mapping of the disease locus narrowed the localisation to the 3 cM region on chromosome 6, between the D6S407 and D6S1705 markers; and localisation of the LAMA2 gene to the same region was confirmed by radiation hybrid mapping [30]. This locus has been confirmed by subsequent studies on a large number of consanguineous families presenting either a complete or a partial laminin o~2 chain deficiency by our group and Naom et al. [31]. The gene was found to be located between the D6S407 and D6S1620 [31] markers, probably closer to D6S1620 than to D6S407 (Fig. 2) but the distance is still of 3 cM and no intermediate or intragenic microsatellites are known so far. In contrast to CMD with complete or partial laminin 0~2 chain deficiency, none of the consanguineous CMD patients with normal expression of the or2 chain and normal brain MRI studied so far have any chromosome 6q involvement (personal unpublished results) and they are thought to form an heterogeneous group [10].

5. LAMA2 mutations in laminin a2 chain deficient C M D families The involvement of the laminin c~2 chain in a subgroup of CMD was definitely confirmed by the finding of two mutations in the LAMA2 in consanguineous laminin c~2 chain deficient CMD families [9]. We first identified a splice site mutation, 4573-2 A ~ T, leading to the deletion of 193 nucleotides and resulting in a frameshift and premature stop codon at the beginning of domain II, and a nonsense mutation, Gln1241X, changing a glutamine codon to a stop codon in domain IVa [9] (Fig. 3). Using the same approach

\ \ \ \ + N \ \ \ L

%'

'~1

'~1 "..1

%'

--4

t"

r~

-~

~,1

"-4

^

Fig. 3. Schematic representation of laminin a2 chain cDNA, normal and

truncated laminin c~2 chains. Deletions, nonsense and splice mutations occurring in CMD patients with complete laminin c~2 chain deficiency are given with the resulting truncated laminin or2 chains. The mutations, Gln1241stop and 4573-2 A --~ T, have been described previously [9]. The four other novel mutations have been identified by SSCP and direct sequencing techniques as previously reported.

P. Guicheney et al. /Neuromuscular Disorders (1997) 180-186

as in [9], we have recently identified novel nonsense mutations in non-consanguineous patients. A single base change G to T at position 677 found in a Turkish family resulted in the replacement of the GAA codon for glutamic acid 210 by the TAA stop codon in domain VI, Glu210X (Fig. 3). In a family originating from Uruguay, a G to A transition at position 6997 resulted in a nonsense mutation in the Cterminal G globular domain, Trp2316X (TGG ~ TGA) (Fig. 3). One- or 2-bp deletions also cause laminin c~2 chain deficiencies. A cytosine deletion was identified in a consanguineous family (family 2324) originating from Tunisia at position 2418 in LAMA2 gene. This mutation (2418AC) resulted in a frameshift and a premature termination within 17 amino acid residues in domain IIIb (Fig. 3). In a non-consanguineous Italian family, a 2-bp deletion at position 6968, 6968ATA, induces a premature ten nination within two amino acids in the C-terminal G globular domain (Fig. 3). In these three non-consanguineous families, only one mutation has been found so far. Four of the mutations that we identified, Glu210X, 2418AC, Gln1241X and 4573-2 A --~ T, result in truncated proteins corresponding only to part of the short arm of the laminin ~2 chain (Fig. 3). These proteins lack the carboxy terminal G domain, and domains I and II of the long ann of the laminin a2 chain and therefore, cannot participate in the formation of laminin heterotrimers. No laminin-2 or laminin-4 molecules could be formed. The two other mutations, 6968ATA and Trp2316X, which occur at positions 6968 and 6997 respectively in the G1 repeat of C-terminal globular G domain, should allow synthesis of the ot2-chains with domains I and II, but lacking G2 to G5 repeats of the globular G domain (Fig. 3). The sequence essential for heterotrimer formation and stability - the critical 25-amino acid sequence at the C terminus of domain I [32] - is maintained in both cases. Trimeric laminin molecules may be formed. Nevertheless, the half-life of such truncated proteins is probably reduced since they cannot associate with sarcolemmal constituants, such as c~-dystroglycan [33] which could bind to G4 and G5 repeats by analogy with laminin1 [34,35] and integrins [36]. Previous studies with laminin-1 indicate the involvement of G 1 to G3 repeats and integrin c¢6/31 in cell adhesion [37], but whether laminin-2 and -4 have similar properties remains to be examined. Hayashi et al. described a patient with a complete deficiency of the ~2 chain protein and transcript [38]. The total absence of the transcript could suggest that the mutation has occurred in a region interfering with gene transcription, but the mutation has not yet been reported. The recessive dystrophic mouse mutant dy/dy has been shown to be caused by a laminin a2 chain deficiency [3941 ] but the nature of the mutation is still unknown. The lack of the laminin ~2 chain appears to cause the abnormal basement structure seen by electron microscopy [41], which is also observed in the muscle fibers of CMD patients [42,43]. Myelination of the peripheral nerves is also incomplete in dy/dy mice [44,45], and this has been attributed to the

183

absence of the laminin ~2 chain which is known to be implicated in Schwann cell migration [46]. Disruption of the linkage between the o~2 chain and the peripheral nerve a-dystroglycan may play a role in the pathogenesis of peripheral neuropathy in dy/dy mice [47], since ot-dystroglycan in the peripheral nerve is shown to bind the laminin or2 chain [48,49].

6. LAMA2 mutation in CMD with partial laminin ~2 chain deficiency We identified the first missense mutation in the LAMA2 gene causing partial laminin ~2 chain deficiency in a 5-year old boy belonging to a Turkish consanguineous family [50]. This child who never became ambulant had cerebral white matter changes on MRI, as observed in patients with complete laminin c~2 chain deficiency. The homozygous mutation, Cys996Arg, changes a conserved cysteine residue to arginine in one of the cysteine-rich laminin motifs of domain IIIb. This missense mutation should be consistent with the synthesis and incorporation of the c~2 chain into the laminin isoforms-2 and 4; this is supported by immunochemical analysis which reveals decreased levels of the laminin c¢2 chain in the muscle biopsy of the patient. Decreased expression of the c~2 chain has also been reported in dystrophic dy2J/dy2J mice, where a mutation in the LAMA2 gene causes truncation within the amino terminal domain VI also allowing assembly of the heterotrimeric molecule [51,52]. The effect of the Cys996Arg mutation at the protein level is not precisely known. It occurs in the sixth of the nine cysteine-rich motifs in domain IIIb, deleting the sixth of the eight cysteines in that repeat [50]. This repeat is highly conserved between the a l and c~2 chains in man and mouse, the c~ chain in Drosophila and the ~5 chain in the mouse [16,20,21,27,53,54]. The bond between the fifth and sixth cysteine in the mutant protein should have been destroyed, leaving one cysteine residue free which could then cause abnormal folding of the domain or form abnormal disulphide bond with other domains of the molecule, or even other matrix molecules. This mutation could also disturb an unknown binding function or change the proteolytic sensitivity of the chain. Other patients with partial ~2 chain deficiency became ambulant but the mutations are not reported [55,56].

7. Prenatal diagnosis by direct mutation analysis We report here the first prenatal diagnosis by direct mutation analysis in the consanguineous family 2324. The two affected children, who demonstrated a complete laminin a2 chain deficiency, were homozygous carriers for the cytosine deletion (2418AC) as described above. For a subsequent pregnancy, DNA issued from trophoblast was studied to detect the fetus genotype. Reported prenatal diagnoses of

[84

P. Guicheney et al. / Neuromuscular Disorders (1997) 180-186

2324

A

C

B

D6S407 D6S1620 D6S1705 1

2

3

4

5

1

9.

3

4

5

6

C

D6S1620

5

209 t~

D6S170~

4

l ~

6

MW

Fig. 4. (A) Affected individuals of Family 2324 are represented by filled symbols, the unaffected by open symbols and the fetus by diamond. DNA was extracted from blood and chorionic villus biopsy by standard methods. (CA)n microsatellite markers were genotyped for each family member as previously described. The disease haplotypes are boxed. (B) PCR-products spanning exon 16 of LAMA2 gene (209-bp) were amplified from family members (lines 1--4), chorionic villus (line 5) and control DNA (6). SSCP analysis revealed abnormal conformers for the two affected children and a combined pattern for the parents and fetus. (C) Digestion of control PCR-product by the enzyme Scrfl (normal: CCTGG) lead to the formation of two bands of 115 and 94 bp. The cytosine deletion at position 2418 induced the loss of this site (CTGG). Digestion of the CMD children PCR-products revealed homozygosity for the mutation, while both parents and the fetus were heterozygous carriers of the same mutation. MW, molecular weight marker.

at-risk fetuses have been done so far by genotype analysis with the chromosome 6q2 microsatellite markers or by immunocytochemical analysis of the trophoblast [30,31, 57]. In family 2324, three markers were genotyped to determine the disease haplotypes transmitted by the parents (Fig. 4A). It is noteworthy that, when the identical genotypes were found for maternal and fetus DNA samples (as is the case) the paternal background has to be verified in the fetus DNA with markers located on other chromosomes to exclude any risk of contamination of the trophoblast sample by maternal tissue. An alternative to linkage analysis is the direct mutation analysis of a known LAMA2 gene defect. In family 2324, the parents were heterozygous carriers for a cytosine deletion at position 2418, which induces the loss of a restriction site for the enzyme Scrfl. This was used for prenatal testing as illustrated on Fig. 4C. ScrfI digestion of PCR products revealed homozygosity for the mutation in the two affected children, while the parents and the fetus were heterozygous carriers of the mutation, which accords with the microsatellite analysis (Fig. 4A), the SSCP pattern (Fig. 4B) as well as with the normal trophoblast staining with an anti-laminin

o~2-chain antibody (Chemicon) (not shown). The fetus has inherited one copy of the disease allele and it was predicted that he would be healthy. This was confirmed at birth by a normal CK level and no hypotonia or other clinical symptoms of CMD.

8. Conclusions Mutations in the laminin a2 chain gene cause CMD with partial or complete laminin ~2 chain deficiency. The nonsense, splice site mutations and small deletions reported here result in premature stop codon and truncation of the c¢2 chain, thus missing the C-terminal part of the globular G domain, against which the commercial antibody is directed (Chemicon). Missense mutations, as well as, probably small in-frame deletions, can cause partial laminin ~2 chain deficiency. All the mutations induce the formation of abnormal heterotrimeric muscle-specific laminins which probably dramatically disturb the assembly and stability of the laminin network, one of the major components of the extracellular

P. Guicheney et al. / Neuromuscular Disorders (1997) 180-186

matrix in skeletal muscle. This implies a weaker linkage of the cytoskeleton to the extracellular matrix leading to muscle cell degeneration. The reliability of prenatal diagnosis by microsatellite analysis is dependent on the absence of locus heterogeneity for the disease. Concerning families with a CMD child having complete merosin deficiency, such analysis can be used for prenatal diagnosis with or without a direct assessment of the laminin c¢2 chain in fetal chorionic villous biopsies [57-59]. In contrast, partial laminin c~2 chain deficiency is heterogeneous. It is known that a secondary reduction of the muscle laminin 0~2 chain occurs in Fukuyama CMD, which is linked to chromosome 9q31-32 [60,61 ]. In index cases with partial laminin a2-chain-deficiency, the existence of white matter hypodensity has to be determined by brain imaging [50]. However, this would still not be sufficient to affirm a laminin a2-chain gene defect. In addition, microsatellite analysis can only give reliable information in consanguineous families. It is thus important to detect the laminin o~2-chain gene defects causing partial laminin et2 chain deficiency in order to perform prenatal diagnoses by direct mutation analysis. It is also essential to identify the other possible gene defects which may also induce secondary partial laminin c~2 chain deficiency.

Acknowledgements This work was supported by the Institut National de la Sant6 et de la Recherche Mrdicale, the Association Franc ,aise contre les Myopathies (AFM, France) and the French Research Ministry (Actions ConcertEes Coordonnres Sciences du vivant). We thank Dr B. Estournet, Dr J. A. Urtizberea and Dr M. Medici for referring several patients and Dr V. Paquis for DNA extraction. We also thank the patients and their families for their cooperation.

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[47]

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[53]

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[58] [59] [60]

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