LAMA2 Nonsense Variant in an Italian Greyhound with Congenital Muscular Dystrophy

A 4-month-old, male Italian Greyhound with clinical signs of a neuromuscular disease was investigated. The affected dog presented with an abnormal short-strided gait, generalized muscle atrophy, and poor growth since 2-months of age. Serum biochemistry revealed a marked elevation in creatine kinase activity. Electrodiagnostic testing supported a myopathy. Histopathology of muscle biopsies confirmed a dystrophic phenotype with excessive variability in myofiber size, degenerating fibers, and endomysial fibrosis. A heritable form of congenital muscular dystrophy (CMD) was suspected, and a genetic analysis initiated. We sequenced the genome of the affected dog and compared the data to that of 795 control genomes. This search revealed a private homozygous nonsense variant in LAMA2, XM_022419950.1:c.3285G>A, predicted to truncate 65% of the open reading frame of the wild type laminin α2 protein, XP_022275658.1:p.(Trp1095*). Immunofluorescent staining performed on muscle cryosections from the affected dog confirmed the complete absence of laminin α2 in skeletal muscle. LAMA2 loss of function variants were shown to cause severe laminin α2-related CMD in humans, mouse models, and in one previously described dog. Our data together with current knowledge on other species suggest the LAMA2 nonsense variant as cause for the CMD phenotype in the investigated dog.

Identification of a novel LAMA2 c.2217G > A, p.(Trp739*) mutation in a Moroccan patient with congenital muscular dystrophy: a case report

Background Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a rare autosomal recessive genetic condition caused by deleterious mutations in the LAMA2 gene encoding the laminin-α2 chain. It is the most frequent subtype of congenital muscular dystrophies (CMDs) characterized by total laminin-α2 deficiency with muscle weakness at birth or in the first six months of life. To the best of our knowledge, this study reports the first molecular diagnosis and genetic defect of this heterogeneous form of CMD performed in a Moroccan medical genetic center using next-generation sequencing (NGS). It allows us to expand the mutational spectrum of the LAMA2 gene. Case presentation We report the case of a female Moroccan child with clinical and paraclinical features in favor of a CMD. She has global congenital hypotonia with generalized muscle weakness, psychomotor retardation, increased serum creatine kinase, and normal brain scan at the age of six months. Targeted NGS leads to the identification of a novel homozygous nonsense mutation c.2217G > A, p.(Trp739*) in the exon 16 of LAMA2. Sanger sequencing confirmed this mutation in the affected patient and showed that her parents are heterozygous carriers. Conclusions A modern genetic analysis by NGS improves the genetic diagnosis pathway for adequate genetic counseling of affected families more precisely. An accession number from the National Center for Biotechnology Information (NCBI) ClinVar database was retrieved for this novel LAMA2 mutation.

The extracellular matrix complexity of idiopathic epiretinal membranes and the bilaminar arrangement of the associated internal limiting membrane in the posterior retina

Purpose To study the composition of the internal limiting membrane (ILM) of the retina, the extracellular matrix (ECM) of idiopathic epiretinal membranes (iERMs), and the relationships occurring between the two membranes. Methods Forty-six iERMs, 24 of them associated with the ILM, were collected and included in this study. The investigation has been carried out by immunofluorescence and confocal microscopy on glutaraldehyde- and osmium-fixed epon-embedded samples and on frozen samples. Sections were double or triple labelled with antibodies against vimentin; collagens I, III, IV, α5(IV), and VI; laminin 1 + 2; laminin α2-, α4-, α5-, β1-, β2-, β3-, γ1-, and γ2-chains; entactin; and fibronectin. Results iERM thickness was not uniform. Almost 14% of iERMs showed thickenings due to folding of their ECM component under the cell layer. The vitreal side of iERMs was often shorter than the attached ILM. In this case, the ILM resulted folded under the iERM. ILMs contained laminin 111; laminin α2-, α5-, β1-, β2-, and γ1-chains; entactin; collagens I; α5(IV); [α1(IV)]2α2(IV); and VI. Laminins, entactin, and α5(IV) were gathered on the retinal half of the ILM, whereas collagens [α1(IV)]2α2(IV) and I were restricted to the vitreal side. Collagen VI was detected on both sides of the ILM. iERMs expressed laminin 111, collagens III, [α1(IV)]2α2(IV) and VI, entactin, and fibronectin. Entactin co-localized with laminins and collagen IV. Conclusions Analysis of laminins and collagen chain expression indicates that ILM contains laminin 111 (former laminin 1), laminin 521 (former laminin 11), laminin 211 (former laminin 2), collagen [α1(IV)]2α2(IV), and collagen α3(IV)α4(IV)α5. In contrast, iERMs express only collagen [α1(IV)]2α2(IV) and laminin 111. In addition, both iERMs and ILMs contain entactin. The presence of three major constituents of the basement membranes co-localized together in iERMs is suggestive for a deranged process of basement membrane formation which fails to assemble properly. In view of the many interactions occurring among its proteins, the ECM of either the iERMs or the ILMs can account for their reciprocal adhesiveness. In addition, the peculiar deposition of the ECM observed in some samples of iERM is suggestive for its involvement in the formation of macular puckers.

MO033THE DUAL ETAR/AT1R BLOCKER SPARSENTAN SLOWS RENAL DISEASE, IMPROVES LIFESPAN, AND ATTENUATES HEARING LOSS IN ALPORT MICE: COMPARISON WITH LOSARTAN

Background and Aims In Alport syndrome (AS), endothelin type A receptor (ETAR) activation plays a role in both renal and inner ear pathologies. Currently, angiotensin converting enzyme inhibitors or angiotensin II type 1 receptor (AT1R) blockers are the standard of care for patients with AS; however, these drugs have not been shown to improve hearing. Previously, we showed that sparsentan (SP), a dual ETAR/AT1R inhibitor, prevented increases in proteinuria, fibrosis, glomerulosclerosis, and glomerular basement membrane dysmorphology. The AS mouse model lifespan was extended when SP treatment was started at 3 weeks (W) of age (pre-proteinuric). Here we compare the effect of SP and the AT1R blocker losartan (LS) on lifespan and proteinuria in AS mice treated from 4 W, and on hearing loss and associated inner ear pathology in AS mice treated from 3 W to 8.75 W. Method Wild type (WT) and AS mice were treated daily with vehicle (V), 60, 120, or 200 mg/kg SP (SP60, SP120, or SP200) by oral gavage, or 20 mg/kg LS by daily oral gavage from 3-4 W of age and in drinking water at 10 mg/kg from 4-8.75 W. Three studies were conducted: early intervention for hearing from 3-8.75 W (V, SP120, and LS, n=5) or for assessment of laminin α2, laminin α5, and collagen IVα1 expression in stria vascularis from 3-7 W (V, SP200, and LS, n=7), and for lifespan with treatment from 3 W (V, n=10) or from 4 W (LS, SP60, or SP120, n=5). Urinary protein/creatinine (UP/C) was assessed weekly during the lifespan study. In the hearing study, the auditory brainstem response (ABR) assessed hearing ability and sensitivity to noise at 8-9 W (n=5). The cochleae were preserved and transmission electron microscopy was used to assess severity of strial pathology and to measure strial capillary basement membrane (SCBM) thickness (n=5-7). Accumulation of extracellular matrix in SCBM was determined in mice treated with V, SP200, or LS by immunofluorescence microscopy using antibodies to laminin α2, laminin α5, or collagen IVα1. Results In AS mice treated with SP from 4 W, SP120 significantly (P<0.05) increased median lifespan by 34 days compared to LS-treated mice, and by 50.5 days compared to V-treated mice (Figure 1). AS mice treated with SP60 or LS initiated at 4 W did not have a significant difference in lifespan compared to V. At 8 W, SP120 but not SP60 or LS significantly (P<0.05) attenuated the increase in UP/C compared to V (UP/C mg/mg mean±SD 8 W: 47±16 V; 31±6 LS; 61±44 SP60; 20±3 SP120). Notably, development of UP/C at 11 W in AS-SP120 mice was significantly attenuated (P<0.05) compared to that in AS-LS mice (UP/C mg/mg mean±SD 11 W: 58±8 LS; 77±51 SP60; 18±10 SP120). In hearing studies, although both SP120 and LS significantly (P<0.05) prevented SCBM thickening compared to AS-V mice (mean SCBM width±SD nm: 57.8±2.1 WT-V; 67.6±5.5 AS-V; 54.7±2.4 AS-SP120; 55.0±5.9 AS-LS), post-noise ABR thresholds at 16 kHz were significantly improved only in AS-SP120 mice compared to AS-V mice (*P<0.05; Figure 2). AS-LS post-noise hearing did not differ from that of AS-V mice. Moreover, SP200 tended to ameliorate increases in expression levels of laminin α2, laminin α5, and collagen IVα1 to a greater extent than LS in the SCBM of AS mice. Finally, dysmorphology of the stria vascularis was noted in LS but not SP120-treated AS mice. Conclusion SP120 provided significant nephroprotection in AS mice when treatment was started at 4 W, significantly extending lifespan beyond that of mice treated with LS while delaying the development of proteinuria. SP120 treatment in AS mice reduced the SCBM width. Moreover, SP120 treatment significantly attenuated noise-induced hearing loss in AS mice whereas LS was without effect. These pre-clinical studies, if successfully translated to the clinic, may suggest that sparsentan offers a novel, dual-therapeutic approach in AS by reducing both renal injury and hearing loss.

Laminin-111 protein therapy after disease onset slows muscle disease in a mouse model of laminin-α2 related congenital muscular dystrophy

Laminin-α2 related congenital muscular dystrophy (LAMA2-CMD) is a fatal muscle disease caused by mutations in the LAMA2 gene. Laminin-α2 is critical for the formation of laminin-211 and -221 heterotrimers in the muscle basal lamina. LAMA2-CMD patients exhibit hypotonia from birth and progressive muscle loss that results in developmental delay, confinement to a wheelchair, respiratory insufficiency and premature death. There is currently no cure or effective treatment for LAMA2-CMD. Several studies have shown laminin-111 can serve as an effective protein-replacement therapy for LAMA2-CMD. Studies have demonstrated early treatment with laminin-111 protein results in an increase in life expectancy and improvements in muscle pathology and function. Since LAMA2-CMD patients are often diagnosed after advanced disease, it is unclear if laminin-111 protein therapy at an advanced stage of the disease can have beneficial outcomes. In this study, we tested the efficacy of laminin-111 protein therapy after disease onset in a mouse model of LAMA2-CMD. Our results showed laminin-111 treatment after muscle disease onset increased life expectancy, promoted muscle growth and increased muscle stiffness. Together these studies indicate laminin-111 protein therapy either early or late in the disease process could serve as an effective protein replacement therapy for LAMA2-CMD.

Human Laminin-111 and Laminin-211 protein therapy prevents muscle disease progression in an immune deficient mouse model of LAMA2-CMD

Background Laminin-α2 related Congenital Muscular dystrophy (LAMA2-CMD) is a devastating genetic disease caused by mutations in the LAMA2 gene. These mutations result in progressive muscle wasting and inflammation leading to delayed milestones, and reduced lifespan in affected patients. There is currently no cure or treatment for LAMA2-CMD. Preclinical studies have demonstrated that mouse Laminin-111 can serve as an effective protein replacement therapy in a mouse model of LAMA2-CMD. Methods In this study, we generated a novel immunocompromised dy W mouse model of LAMA2-CMD to study the role the immune system plays in muscle disease progression. We used this immune deficient dy W mouse model to test the therapeutic benefits of recombinant human laminin-111 and laminin-211 protein therapy on Laminin-α2 deficient muscle disease progression. Results We show that immune deficient Laminin-α2 null mice demonstrate subtle differences in muscle regeneration compared to immune competent animals during early disease stages, but overall exhibit a comparable muscle disease progression. We found human laminin-111 and laminin-211 could serve as effective protein replacement strategies with mice showing improvements in muscle pathology and function. We observed that human laminin-111 and laminin-211 exhibit differences on satellite and myoblast cell populations and differentially affect muscle repair. Conclusions This study describes the generation of a novel immune deficient mouse model that allows investigation of the role the immune system plays in LAMA2-CMD. This model can be used to assess the therapeutic potential of heterologous therapies that would illicit an immune response. Using this model, we show that recombinant human Laminin-111 can serve as effective protein replacement therapy for the treatment of LAMA2-CMD.

Antioxidants Reduce Muscular Dystrophy in the dy2J/dy2J Mouse Model of Laminin α2 Chain-Deficient Muscular Dystrophy

Congenital muscular dystrophy with laminin α2 chain-deficiency (LAMA2-CMD) is a severe neuromuscular disorder without a cure. Using transcriptome and proteome profiling as well as functional assays, we previously demonstrated significant metabolic impairment in skeletal muscle from LAMA2-CMD patients and mouse models. Reactive oxygen species (ROS) increase when oxygen homeostasis is not maintained and, here, we investigate whether oxidative stress indeed is involved in the pathogenesis of LAMA2-CMD. We also analyze the effects of two antioxidant molecules, N-acetyl-L-cysteine (NAC) and vitamin E, on disease progression in the dy2J/dy2J mouse model of LAMA2-CMD. We demonstrate increased ROS levels in LAMA2-CMD mouse and patient skeletal muscle. Furthermore, NAC treatment (150 mg/kg IP for 6 days/week for 3 weeks) led to muscle force loss prevention, reduced central nucleation and decreased the occurrence of apoptosis, inflammation, fibrosis and oxidative stress in LAMA2-CMD muscle. In addition, vitamin E (40 mg/kg oral gavage for 6 days/week for 2 weeks) improved morphological features and reduced inflammation and ROS levels in dy2J/dy2J skeletal muscle. We suggest that NAC and to some extent vitamin E might be potential future supportive treatments for LAMA2-CMD as they improve numerous pathological hallmarks of LAMA2-CMD.

Bioactive fragments of laminin and collagen chains: lesson from the testis

Recent studies have shown that the testis is producing several biologically active peptides, namely the F5- and the NC1-peptides from laminin-γ3 and collagen α3 (IV) chain, respectively, that promotes blood–testis barrier (BTB) remodeling and also elongated spermatid release at spermiation. Also the LG3/4/5 peptide from laminin-α2 chain promotes BTB integrity which is likely being used for the assembly of a ‘new’ BTB behind preleptotene spermatocytes under transport at the immunological barrier. These findings thus provide a new opportunity for investigators to better understand the biology of spermatogenesis. Herein, we briefly summarize the recent findings and provide a critical update. We also present a hypothetical model which could serve as the framework for studies in the years to come.

Laminin α2, α4, and α5 Chains Positively Regulate Migration and Survival of Oligodendrocyte Precursor Cells

In the developing central nervous system (CNS), oligodendrocyte precursor cells (OPCs) migrate along blood vessels and are widely distributed in the CNS. Meanwhile, OPCs require survival factors from the extracellular microenvironment. In other tissues, laminins, heterotrimetric (αβγ) extracellular matrix proteins, promote cell migration and survival. However, the expression pattern and functions of laminins in OPC development remain poorly understood. In the present study, we first investigated the expression of laminin α chains, which bind to cell surface receptors such as integrins, in the postnatal murine brain. We found that laminin α1, α2, α4, and α5 chains were expressed around blood vessels and OPCs attached the laminin α chain-positive vessels. We then evaluated the effect of these laminins on OPCs activity using recombinant laminin E8s (LME8s) that are minimally active fragments of the laminin isoforms. OPCs attached on LM211E8, LM411E8, and LM511E8, containing laminin α2, α4, and α5 chains, respectively, through integrin β1. Further, these three LME8s promoted migration of OPCs, and OPC survival was prolonged on either LM411E8 or LM511E8 via the activation of focal adhesion kinase. Together, our findings suggest that laminins expressed surrounding blood vessels positively regulate migration and survival of OPCs through the integrin β1-FAK pathway.