Partial Laminin Research Articles

CONGENITAL MUSCULAR DYSTROPHIES: EP.69 LAST STRONG: LAMA2 and SELENON to study trial readiness, outcome measures and natural history

SELENON (SEPN1)-related myopathy (SELENON-RM) is a rare congenital myopathy characterized by slowly progressive proximal muscle weakness, early onset rigidity of the spine and respiratory insufficiency. Patients with mutations in LAMA2 gene causing merosin-deficient congenital muscular dystrophy (LAMA2-MD) have a similar clinical phenotype, with a heterogenous disease spectrum ranging from a severe, early-onset congenital (complete laminin α2 deficiency) to a mild, childhood-onset limb-girdle type muscular dystrophy (partial laminin α2 deficiency). For both SELENON-RM and LAMA2-MD, no curative treatment options exist, yet promising preclinical studies are ongoing. Currently, there is a paucity on natural history data. Appropriate clinical and functional outcome measures need to be selected to reach trial readiness. We are performing a natural history study in Dutch-speaking patients diagnosed with SELENON-RM (n=10, mean age=18 (3-50) years, F=3, M=7) and LAMA2-MD (n=20, mean age=19 (3-50) years, F=15; M=5). Patients have four visits over a period of 1.5 year, with an interval of six months. At all visits, they undergo standard neurological examination, hand-held dynamometry (age≥5 years), functional measurements, questionnaires (patient report and/or parent proxy; age≥2 years), muscle ultrasound including ultrasound of the diaphragm, pulmonary function tests (spirometry, maximal inspiratory and expiratory pressure, sniff nasal inspiratory pressure; age≥5 years), and accelerometry for eight days; at visit one and three, they undergo cardiac evaluation (electrocardiogram, echocardiography), X-ray of the spine (age≥2 years), Dual-energy X-ray absorptiometry (DEXA-)scan (age≥2 years) and full body magnetic resonance imaging (MRI) (age≥10 years). Hereby we aim to describe the natural history of patients diagnosed with SELENON-RM or LAMA2-MD and to select outcome measures for future clinical trials. Our natural history study is an essential step to reach trial readiness.

Natural history, outcome measures and trial readiness in LAMA2-related muscular dystrophy and SELENON-related myopathy in children and adults: protocol of the LAST STRONG study

BackgroundSELENON (SEPN1)-related myopathy (SELENON-RM) is a rare congenital myopathy characterized by slowly progressive proximal muscle weakness, early onset spine rigidity and respiratory insufficiency. A muscular dystrophy caused by mutations in the LAMA2 gene (LAMA2-related muscular dystrophy, LAMA2-MD) has a similar clinical phenotype, with either a severe, early-onset due to complete Laminin subunit α2 deficiency (merosin-deficient congenital muscular dystrophy type 1A (MDC1A)), or a mild, childhood- or adult-onset due to partial Laminin subunit α2 deficiency. For both muscle diseases, no curative treatment options exist, yet promising preclinical studies are ongoing. Currently, there is a paucity on natural history data and appropriate clinical and functional outcome measures are needed to reach trial readiness.MethodsLAST STRONG is a natural history study in Dutch-speaking patients of all ages diagnosed with SELENON-RM or LAMA2-MD, starting August 2020. Patients have four visits at our hospital over a period of 1.5 year. At all visits, they undergo standardized neurological examination, hand-held dynamometry (age ≥ 5 years), functional measurements, questionnaires (patient report and/or parent proxy; age ≥ 2 years), muscle ultrasound including diaphragm, pulmonary function tests (spirometry, maximal inspiratory and expiratory pressure, sniff nasal inspiratory pressure; age ≥ 5 years), and accelerometry for 8 days (age ≥ 2 years); at visit one and three, they undergo cardiac evaluation (electrocardiogram, echocardiography; age ≥ 2 years), spine X-ray (age ≥ 2 years), dual-energy X-ray absorptiometry (DEXA-)scan (age ≥ 2 years) and full body magnetic resonance imaging (MRI) (age ≥ 10 years). All examinations are adapted to the patient’s age and functional abilities. Correlation between key parameters within and between subsequent visits will be assessed.DiscussionOur study will describe the natural history of patients diagnosed with SELENON-RM or LAMA2-MD, enabling us to select relevant clinical and functional outcome measures for reaching clinical trial-readiness. Moreover, our detailed description (deep phenotyping) of the clinical features will optimize clinical management and will establish a well-characterized baseline cohort for prospective follow-up.ConclusionOur natural history study is an essential step for reaching trial readiness in SELENON-RM and LAMA2-MD.Trial registrationThis study has been approved by medical ethical reviewing committee Region Arnhem-Nijmegen (NL64269.091.17, 2017–3911) and is registered at ClinicalTrial.gov (NCT04478981).

Laminin α1 reduces muscular dystrophy in dy2J mice

Muscular dystrophies, including laminin α2 chain-deficient muscular dystrophy (LAMA2-CMD), are associated with immense personal, social and economic burdens. Thus, effective treatments are urgently needed. LAMA2-CMD is either a severe, early-onset condition with complete laminin α2 chain-deficiency or a milder, late-onset form with partial laminin α2 chain-deficiency. Mouse models dy3K/dy3K and dy2J/dy2J, respectively, recapitulate these two forms of LAMA2-CMD very well. We have previously demonstrated that laminin α1 chain significantly reduces muscular dystrophy in laminin α2 chain-deficient dy3K/dy3K mice. Among all the different pre-clinical approaches that have been evaluated in mice, laminin α1 chain-mediated therapy has been shown to be one of the most effective lines of attack. However, it has remained unclear if laminin α1 chain-mediated treatment is also applicable for partial laminin α2 chain-deficiency. Hence, we have generated dy2J/dy2J mice (that express a substantial amount of an N-terminal truncated laminin α2 chain) overexpressing laminin α1 chain in the neuromuscular system. The laminin α1 chain transgene ameliorated the dystrophic phenotype, restored muscle strength and reduced peripheral neuropathy. Thus, these findings provide additional support for the development of laminin α1 chain-based therapy for LAMA2-CMD.

Muscle MR pattern of partial Laminin α2 deficient patients and its role in diagnostic algorithms

Objective: Laminin α2 (merosin) deficient muscular dystrophy (MDC1A) is one of the most frequent types of congenital muscular dystrophy (CMD) and it is due to LAMA2 gene mutations. The suspicion of classical phenotype with complete merosin deficiency is easy, based in a homogeneous and typical severe phenotype including floppy baby, hyperCKemia, not acquisition of walking ability and very striking abnormal white matter changes in brain MRI. Merosin deficiency is complete in muscle. Differential diagnosis may be difficult in partial deficiency patients who present later and with milder symptoms, often overlapping dystroglycanopathies and limb girdle muscular dystrophies. Methods and results: The authors present the cohort of pelvic girdle and lower limb muscle MRI findings from 7 patients with partial Laminin α2 deficiency, most of them ambulatory. The most affected muscles were gluteus medium and minimum, adductor magnus, soleus and gastrocnemios muscles. Relatively spared muscles were gracilis and tibialis posterior muscles. The pattern was similar for all patients except one. There was one interesting feature: the rim of peripheral involvement between vastus lateralis and intermedius muscles, and between gastrocnemius and soleus muscles (COL6 –like sign). Conclusion: The results of our cohort showed that muscle MRI of Laminin α2 deficient patients has a homogeneous and recognizable pattern, although sharing similar features in the lower limbs to another CMD with extracellular matrix abnormalities (collagen 6 –related myopathies such as Ullrich CMD or Bethlem myopathy). Further studies in whole-body MRI will be useful to search for selective or more specific involvement of merosin deficient patients. The results support the recommendation to use muscle MRI in diagnostic algorithm of early onset myopathies. This work was supported by COST ACTION project BM 1304.

Bortezomib Does Not Reduce Muscular Dystrophy in the dy2J/dy2J Mouse Model of Laminin α2 Chain-Deficient Muscular Dystrophy.

Congenital muscular dystrophy with laminin α2 chain-deficiency, also known as MDC1A, is a severe neuromuscular disorder for which there is no cure. Patients with complete laminin α2 chain-deficiency typically have an early onset disease with a more severe muscle phenotype while patients with residual laminin α2 chain expression usually have a milder disease course. Similar genotype-phenotype correlations can be seen in the dy3K/dy3K and dy2J/dy2J mouse models of MDC1A, respectively, with dy3K/dy3K mice presenting the more severe phenotype. Recently, we demonstrated that the proteasome inhibitor bortezomib partially improves muscle morphology and increases lifespan in dy3K/dy3K mice. Here, we explore the use of bortezomib in dy2J/dy2J animals. However, bortezomib neither improved histological hallmarks of disease nor increased muscle strength and locomotive activity in dy2J/dy2J mice. Altogether our data suggest that proteasome inhibition does not mitigate muscle dysfunction caused by partial laminin α2 chain-deficiency. Still, it is possible that proteasome inhibition could be useful as a supportive therapy in patients with complete absence of laminin α2 chain.

Laminin α2 Deficiency-Related Muscular Dystrophy Mimicking Emery-Dreifuss andCollagen VI related Diseases.

Background: Laminin α2 deficient congenital muscular dystrophy, caused by mutations in the LAMA2 gene, is characterized by early muscle weakness associated with abnormal white matter signal on cerebral MRI. Objective: To report on 4 patients with LAMA2 gene mutations whose original clinical features complicated the diagnosis strategy. Methods: Clinical, electrophysiological, muscle imaging and histopathological data were retrospectively collected from all patients. DNA samples were analysed by next-generation sequencing or direct gene sequencing. Laminin α2 was analysed by western-blot and immunohistochemistry. Results: The four patients achieved independent walking. All had proximal muscle weakness with scapular winging and prominent joint contractures without peripheral neuropathy. During follow-up, two patients suffered from refractory epilepsy associated with brain leukoencephalopathy in one, polymicrogyria and lissencephaly without white matter changes in the other. In two patients, the distribution of fatty infiltration resembles that of collagen-VI related myopathies. Dilated cardiomyopathy contstartabstractwith conduction defects, suggestive of Emery-Dreifuss myopathy, emerged in two of them within the 4th decade. Molecular diagnosis remained elusive for many years. Finally, targeted capture-DNA sequencing unveiled the involvement of the LAMA2 gene in two families, and led us to further identify LAMA2 mutations in the remaining family using Sanger sequencing.Conclusions: This report extends the clinical and radiological features of partial Laminin α2 deficiency since patients showed atypical manifestations including dilated cardiomyopathy with conduction defects in 2, epilepsy in 2, one of whom also had sole cortical brain abnormalities. Importantly, clinical findings and muscle imaging initially pointed to collagen-VI related disorders and Emery-Dreifuss muscular dystrophy.

LAMA2-related congenital muscular dystrophy complicated by West syndrome

BackgroundMutations in the LAMA2 gene cause autosomal recessive laminin α2 related congenital muscular dystrophy. In patients with partial laminin α2 deficiency the phenotype is usually milder than in those with absent protein. Apart from the typical white matter abnormalities, there is an increased risk of cerebral complications such as epilepsy and mental retardation, despite a structurally normal brain. Methods/resultsWe present a patient with primary partial laminin α2 deficiency due to a homozygous novel LAMA2 missense mutation who developed West syndrome in his first year of life. To our knowledge, this combination has not previously been reported. A 5 year-old boy exhibited global hypotonia with generalized muscle weakness from birth. At 8 months of age he presented infantile spasms and an EEG finding of hypsarrhythmia. Seizures were controlled in a few weeks with intramuscular synthetic ACTH, followed by valproic acid. Two years later antiepileptic medication was withdrawn. He achieved unsupported walking at the age of 4, but his cognitive status corresponded to a 2 year-old child. Epilepsy has not recurred and brain MRI showed the typical white matter abnormalities without associated neuronal migration defects. ConclusionThis report widens the clinical spectrum of cerebral manifestations related with mutations in LAMA2. The beginning of a severe epileptic encephalopathy modifies the natural history of the disease.

High creatine kinase levels and white matter changes: Clinical and genetic spectrum of congenital muscular dystrophies with laminin alpha-2 deficiency

Primary deficiency of laminin alpha-2 due to mutations in the LAMA2 gene accounts for 30% of all patients with congenital muscular dystrophy. Here, we present seven patients with partial or total laminin alpha-2 deficiency (MDC1A) with a wide clinical spectrum, ranging from ambulant patients to patients who were never able to stand or sit. We identified two pathogenic mutations in the LAMA2 gene in all patients except for one patient in whom only one mutation was found. Six of the mutations were previously undescribed. In some of the milder cases, laminin alpha-2 expression in the muscle biopsy was only slightly reduced. These findings emphasize that analysis of the LAMA2 gene might be necessary in patients with muscle weakness, cerebral white matter changes and high creatine kinase levels, even in the presence of laminin alpha-2 in the muscle biopsy.

Clinical and molecular study in congenital muscular dystrophy with partial laminin alpha 2 (LAMA2) deficiency.

Complete laminin alpha2 (LAMA2) deficiency causes approximately half of congenital muscular dystrophy (CMD) cases. Many loss-of-function mutations have been reported in these severe, neonatal-onset patients, but only single missense mutations have been found in milder CMD with partial laminin alpha2 deficiency. Here, we studied nine patients diagnosed with CMD who showed abnormal white-matter signal at brain MRI and partial deficiency of laminin alpha2 on immunofluorescence of muscle biopsy. We screened the entire 9.5 kb laminin alpha2 mRNA from patient muscle biopsy by direct capillary automated sequencing, single strand conformational polymorphism (SSCP), or denaturing high performance liquid chromatography (DHPLC) of overlapping RT-PCR products followed by direct sequencing of heteroduplexes. We identified laminin alpha2 sequence changes in six of nine CMD patients. Each of the gene changes identified, except one, was novel, including three missense changes and two splice-site mutations. The finding of partial laminin alpha2 deficiency by immunostaining is not specific for laminin alpha2 gene mutation carriers, with only two patients (22%) showing clear causative mutations, and an additional three patients (33%) showing possible mutations. The clinical presentation and disease progression was homogeneous in the laminin alpha2-mutation positive and negative CMD patients.

Structure and function of an early divergent form of laminin in hydra: a structurally conserved ECM component that is essential for epithelial morphogenesis.

As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin alpha chain and the full-length hydra laminin beta chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin alpha and beta subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin beta chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin beta chain, in situ hybridization experiments revealed that hydra laminin alpha chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin alpha chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin alpha chain is required for head regeneration.

A novel laminin alpha2 isoform in severe laminin alpha2 deficient congenital muscular dystrophy.

Laminin alpha2 deficiency presents at birth with muscle weakness, hypotonia, and usually asymptomatic white matter signal on MRI. Few patients with laminin alpha2 deficiency have been described with seizures and structural brain abnormalities. The reason for the variation in the severity of the clinical phenotype in congenital muscular dystrophy (CMD) with laminin alpha2 deficiency is not known. A patient with CMD with partial laminin alpha2 presenting with brain structural abnormalities and untreatable generalized and partial complex seizure was studied. Alternative laminin alpha2 splicing was studied by single-strand conformational polymorphism/sequencing analysis. A novel laminin alpha2 isoform was identified. Nonsense laminin alpha2 mutations (stop codons) were inherited from both parents; however, one of the nonsense mutations was in a region of exon 31, which is alternatively spliced. The alternatively spliced isoform excluded one of the stop codon mutations, and was thus able to produce normal laminin alpha2 corresponding to this isoform. Laminin alpha2 immunofluorescence showed that this isoform was not evenly distributed at the muscle fiber basal lamina, but preferentially localized in discrete areas. Laminin alpha5, beta1, gamma1, and nidogen showed decreased expression by immunofluorescence. The severity of this patient's phenotype may be due to overexpression of the exon 31-spliced laminin alpha2 isoform. Exon 31 lies in the IIIA domain of the laminin alpha2 protein, just proximal to the triple coil-coiled region. It is possible that chain assembly is impaired by this isoform, resulting in a loss of possible rescue mechanisms.

Partial laminin ?2 chain deficiency in a patient with myopathy resembling inclusion body myositis

It is becoming evident that clinical phenotypes associated with partial laminin alpha2 chain deficiency are variable. We recently observed a 29-year-old man with leukoencephalopathy and vacuolar myopathy resembling inclusion body myositis. Laminin alpha2 immunohistochemical analysis showed reduction of the protein on muscle fiber surfaces. Molecular analysis revealed two novel compound heterozygous mutations in the LAMA2 gene. This is the first report linking a mutation in the LaMA2 gene with leukoencephalopathy and inclusion body-like myositis.

Partial laminin α2 chain deficiency in a patient with myopathy resembling inclusion body myositis

Partial laminin α2 chain deficiency in a patient with myopathy resembling inclusion body myositis

Laminin α2-chain gene mutations in two siblings presenting with limb-girdle muscular dystrophy

We report two siblings, an 11-year-old boy and his 7-year-old sister, referred to us with a diagnosis of muscular dystrophy. The boy presented at 22 months with delay in walking. A very high serum creatine kinase (CK) level and a dystrophic muscle biopsy lead to a diagnosis of Duchenne muscular dystrophy prior to the identification of the dystrophin gene. Two years later his sister presented with similar problems. A diagnosis of limb-girdle muscular dystrophy was made when they were shown to have inherited different X-chromosomes and normal expression of dystrophin and all sarcoglycans. Their conditions remained static. Recently a slowing of the peripheral motor nerve conduction velocities and T2-weighted brain magnetic resonance imaging showed increased signal of the white matter, both of which are features of merosin-deficient congenital muscular dystrophy. Immunolabelling using a C-terminal laminin α2 chain antibody showed a reduction in expression, while labelling with another antibody that recognises a 300-kDa fragment showed a very significant reduction. Mutational analysis of the LAMA2 gene showed two mutations: one was a G→C point mutation at position −1 of intron 28 acceptor splicing site. This mutation induced activation of a cryptic splice at nucleotide 4429 of exon 29 and partial skipping of this exon, with conservation of the open reading frame. The other was a nonsense mutation due to a C_T transition at position 5525 of the cDNA sequence (exon 37), resulting in a stop codon. These data confirm that mutations of the LAMA2 gene that do not completely disrupt the production of the protein can give rise to phenotypes considerably milder than classical merosin-deficient congenital muscular dystrophy. Partial laminin α2 deficiency should be considered in the differential diagnosis of limb-girdle muscular dystrophy.

Laminin alpha2 chain-deficient congenital muscular dystrophy: variable epitope expression in severe and mild cases.

To characterize the expression of distinct fragments of laminin alpha2 chain in patients with partial laminin alpha2 chain deficiency and variable clinical severity. Deficiency of laminin alpha2 chain caused by mutations of the LAMA2 gene on chromosome 6q2 account for approximately 50% of cases of congenital muscular dystrophy (CMD) in white patients. The complete absence of laminin alpha2 is usually associated with a severe phenotype affecting skeletal muscle and the peripheral and central nervous systems. Quantitative assessment of immunofluorescence to study the expression of C- and N-terminal portions of laminin alpha2 chain in five patients with partial laminin alpha2 chain deficiency and variable phenotype. All five patients showed abnormal T2 signal on brain MRI. Immunohistochemistry of muscle specimens showed preserved or minimally reduced expression of the C-terminal region of the laminin alpha2 chain (67 to 74%), but a marked reduction of the N-terminal region in four patients (13 to 19%). One patient with a mild phenotype had a partial reduction (45%) of the C-terminal and the N-terminal (51%) portions of the laminin alpha2 chain. Two patients were unable to walk or sit, although the C-terminal portion of the laminin alpha2 chain was expressed at significant levels (67 to 74%). In contrast, two patients with a similar expression of the C-terminus (67 to 70%) had a milder phenotype and became ambulatory. It was impossible to predict the phenotypes in these four patients with a strong expression of the C-terminus and with low levels of the N-terminus based on the amount of protein expressed. In addition, the laminin beta2 chain was moderately reduced (54 to 75%) in all patients with laminin alpha2 chain deficiency. A strong correlation between the amount of the C-terminus but not for the N-terminus and laminin beta2 reduction could be observed. N-terminal antibodies to the laminin alpha2 chain provide a more precise immunohistochemical detection of partially laminin alpha2 chain-deficient CMD. The secondary reduction of laminin beta2 chain may better define laminin alpha2 chain-deficient CMD. More data are needed to predict which portions of C-terminus and midrod region of the laminin alpha2 chain result in a semifunctional protein and a milder phenotype.

A benign allelic form of laminin α2 chain deficient muscular dystrophy

A deficiency of laminin α2 chain of merosin (laminin 2 and 4) in the muscle fibre basal lamina has been found in patients with the classic form of autosomal recessive congenital muscular dystrophy (CMD), 1 Tomé FMS Evangelista T Leclerc A et al. Congenital muscular dystrophy with merosin deficiency. C R Acad Sci. 1994; 317: 351-357 PubMed Google Scholar along with mutations in the α2 chain gene (LAMA2) on chromosome 6q2. 2 Helbling-Leclerc A Zhang X Topaloglu H et al. Mutations in the laminin α2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy. Nat Genet. 1995; 11: 216-218 Crossref PubMed Scopus (559) Google Scholar The patients show rather homogeneous clinical features with severe muscular dystrophy, difficulty in walking, demyelinating neuropathy, white matter changes in the brain, and poor prognosis. 3 Philpot J Sewry C Pennock J Dubowitz V Clinical phenotype in congenital muscular dystrophy: correlation with expression of merosin in skeletal muscle. Neuromusc Disord. 1995; 5: 301-305 Summary Full Text PDF PubMed Scopus (169) Google Scholar A few CMD patients with linkage to 6q2 who showed partial laminin α2 chain expression in the muscle have been reported. 4 Nissinen M Helbling-Leclerc A Zhang X et al. Substitution of a conserved cystein-996 in a cystein-rich motif of the laminin α2-chain in congenital muscular dystrophy with partial deficiency of the protein. Am J Hum Genet. 1996; 58: 1177-1184 PubMed Google Scholar , 5 Naom IS D'Alessandro M Topaloglu H et al. Refinement of laminin α2 chain locus to human chromosome 6q2 in severe and mild merosin deficient congenital muscular dystrophy. J Med Genet. 1997; 34: 99-104 Crossref PubMed Google Scholar These patients had either severe or milder clinical phenotypes. However, little is known about the clinical and molecular biological diversity of the disease. Here we report an adult patient with a benign allelic variant of the α2 chain deficient CMD who had novel compound heterozygote mutations in LAMA2. Importantly, the patient showed partially positive immunoreaction in the muscle fibre basal lamina but negative immunoreaction in the peripheral nerve Schwann cell basal lamina.

The role of immunocytochemistry and linkage analysis in the prenatal diagnosis of merosin-deficient congenital muscular dystrophy.

Complete or partial deficiency of the laminin alpha2 chain of merosin has been demonstrated in a proportion of children with classical congenital muscular dystrophy and linkage to the laminin alpha2 chain gene (LAMA2) on chromosome 6q2 has been established. As the laminin alpha2 chain is also expressed in the trophoblast, its detection and linkage analysis are useful tools for prenatal diagnosis. We report our experience of seven prenatal diagnoses in families with partial deficiency or total absence of the laminin alpha2 chain in the muscle of the propositi. In five instances, expression of the laminin alpha2 chain in the trophoblast was normal and linkage data suggested that the fetuses were unaffected. In one family, the immunocytochemical studies of the trophoblast showed the absence of laminin alpha2, suggesting that the fetus was affected. Linkage analysis confirmed that the fetus had inherited the two at-risk haplotypes. In one family with partial laminin alpha2 chain deficiency, the haplotype analysis was hampered by maternal DNA contamination. Immunocytochemical analysis of chorionic villus sampling showed a reduction in laminin alpha2 expression. The pregnancy was presumed to be at high-risk and terminated. However, subsequent analysis of fetal DNA indicated that the fetus was probably heterozygous. Our data suggest that immunocytochemical analysis of the trophoblast can detect abnormalities in affected fetuses and gives normal results in unaffected and carrier fetuses. Nevertheless, we recommend that linkage analysis to the LAMA2 locus is also studied in all cases.

Refinement of the laminin alpha2 chain locus to human chromosome 6q2 in severe and mild merosin deficient congenital muscular dystrophy.

About half of the children with classical congenital muscular dystrophy (CMD) show an absence in their skeletal muscle of laminin alpha2 chain, one of the components of the extracellular matrix...

Distinct roles of mouse laminin β1 long arm domains for α1β1γ1 trimer formation

Mouse embryonal carcinoma F9 cells expressing partial mouse laminin β1 covering either the C-terminal end (Δβ1S) or the whole (Δβ1L) of the long arm were established to study the assembly and interchain disulfide-bonding of β1 to endogenous laminin α1 and γ1. Both Δβ1S and Δβ1L were disulfide-bonded to γ1 but only Δβ1Lγ1 dimer formed a disulfide-bonded α1Δβ1Lγ1 trimer which was actively secreted into the medium. Meanwhile, in the cells producing Δβ1Sγ1 dimer, the level of endogenous α1β1γ1 was reduced but the level of monomeric α1 was increased, suggesting that α1 was recruited to trimer formation with the Δβ1Sγ1 dimer without disulfide-bonding. This shows that the Δβ1Sγ1 dimer can associate with α1 but not support the disulfide-bonding at the N-terminus of the long arm of α1. While control cells secrete neither monomeric α1 nor the β1γ1 dimer into the medium, the Δβ1Sγ1 producing cells probably do as α1Δβ1γ1 trimer. We thus propose that the N- and C-termini of the long arm of laminin β1 have distinct roles for trimer formation.

Partial laminin alpha2 chain restoration in alpha2 chain-deficient dy/dy mouse by primary muscle cell culture transplantation.

Laminin-2 is a component of skeletal and cardiac basal lamina expressed in normal mouse and human. Laminin alpha2 chain (LAMA2), however, is absent from muscles of some congenital muscular dystrophy patients and the dystrophia muscularis (dy/dy) mouse model. LAMA2 restoration was investigated following cell transplantation in vivo in dy/dy mouse. Allogeneic primary muscle cell cultures expressing the beta-galactosidase transgene under control of a muscular promoter, or histocompatible primary muscle cell cultures, were transplanted into dy/dy mouse muscles. FK506 immunosuppression was used in noncompatible models. All transplanted animals expressed LAMA2 in these immunologically-controlled models, and the degrees of LAMA2 restoration were shown to depend on the age of the animal at transplantation, on muscle pretreatment, and on duration time after transplantation in some cases. LAMA2 did not always colocalize with new or hybrid muscle fibers formed by the fusion of donor myoblasts. LAMA2 deposition around muscle fibers was often segmental and seemed to radiate from the center to the periphery of the injection site. Allogeneic conditionally immortalized pure myogenic cells expressing the beta-galactosidase transgene were characterized in vitro and in vivo. When injected into FK506-immunosuppressed dy/dy mice, these cells formed new or hybrid muscle fibers but essentially did not express LAMA2 in vivo. These data show that partial LAMA2 restoration is achieved in LAMA2-deficient dy/dy mouse by primary muscle cell culture transplantation. However, not all myoblasts, or myoblasts alone, or the muscle fibers they form are capable of LAMA2 secretion and deposition in vivo.