PMC8378307_01
Clinical and neuroimaging features of patients with LAMA1 mutations. (A) Clinical features of patients showing distinct ophthalmologic characteristics. Patients 1 (A), 2 (B) and 3 (C) had unremarkable facial features. Note residual strabismus of patient 1 after corrective surgery. (D) Composite colour fundus photography of patient 1's right eye demonstrating a dysplastic optic nerve with nasalised retinal vasculature (arrow) and diffuse chorioretinal atrophy involving the macula and midperiphery. (E) Composite fundus autofluorescence of the same eye, showing a 'cobblestone' pattern (arrow) indicating chorioretinal atrophy. (F) Composite colour fundus photograph of patient 2, the older sister of patient 1, showing moderate chorioretinal atrophy (arrow). (G) Colour fundus photo of patient 3 showing normal-appearing posterior pole and peripheral lattice degeneration (arrow), despite only mild myopia. (H-S) Brain MRI demonstrates cerebellar dysplasia associated with cysts. Brain MRI findings of patient 1 (H-J), patient 2 (K-M), patient 3 (N-P) and a healthy control (Q-S) are shown. (H) Patient 1 at 7 months of age has multiple cortical/subcortical cysts in the cerebellar vermis (arrow), dysplastic cerebellar vermis and enlarged fourth ventricle (arrowhead) with an abnormal rectangle-like shape (midsagittal-T1). The midbrain is mildly elongated, and the pons is mildly reduced in size. (I) Subcortical cysts (arrows) and dysplasia are also seen in the cerebellar hemispheres (axial-T1). (J) At 10 years 7 months, dysplasia in cerebellar vermis and hemispheres with multiple cortical and subcortical cysts (arrow) remain visible. (K) Patient 2 has hypoplasia and dysplasia of the cerebellar vermis with cortical and subcortical cysts, enlarged fourth ventricle (asterisk), elongated midbrain and pons appearing small (midsagittal-T1). (L) Cerebellar hemispheres are dysplastic and with multiple cortical and subcortical cysts in patient 2 (axial-T2). (M) Cortical and subcortical cysts within the left cerebellar hemisphere of patient 2 (parasagittal-T1) are shown. Images (K-M) were taken at age 6 years and 10 months. (N) Patient 3 has a normal sized but severely dysplastic vermis (white dot) with lack of separation of the vermian lobules (midsagittal-T1). Axial (O) and coronal (P) T2-weighted images of patient 3 show a markedly dysplastic appearance of the cerebellar hemispheres and the vermis with a diffusely abnormal configuration of the cerebellar folia and sulci (black arrows). Multiple small cysts are noted in the cerebellar hemispheres bilaterally (white arrows). Images (N-P) were taken at 4 years 3 months. (Q) A healthy 12-year-old control with normal anatomy of the posterior fossa structures (midsagittal-T1) and a normal triangle-shaped fourth ventricle is shown. Normal morphology of the cerebellum with an 'onion-like' foliation/fissuration pattern and white matter arborisation in the cerebellar hemispheres seen in axial (R) and coronal (S) T2 weighted images.
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Patient 3 (figure 1C), an 8.5-year-old female born to non-consanguineous parents Caucasian and Native American descent, had mild myopia associated with peripheral lattice degeneration of retina (figure 1G). She also had a severely dysplastic cerebellar vermis and multiple small cysts in the cerebellar hemispheres (figure 1N-P). ID: 284217) spans ~176 kb, and is divided into 63 exons. Its mRNA is 9657 bp and encodes a 3075 amino acid protein, laminin alpha1, which is selectively expressed in tissues (see online supplementary figure S1A). We noticed a significant decrease of the mRNA expression of these three subunits in both patient 2 and patient 3 fibroblasts (see online supplementary figure S1B).
PMC8378307_02
Clinical and neuroimaging features of patients with LAMA1 mutations. (A) Clinical features of patients showing distinct ophthalmologic characteristics. Patients 1 (A), 2 (B) and 3 (C) had unremarkable facial features. Note residual strabismus of patient 1 after corrective surgery. (D) Composite colour fundus photography of patient 1's right eye demonstrating a dysplastic optic nerve with nasalised retinal vasculature (arrow) and diffuse chorioretinal atrophy involving the macula and midperiphery. (E) Composite fundus autofluorescence of the same eye, showing a 'cobblestone' pattern (arrow) indicating chorioretinal atrophy. (F) Composite colour fundus photograph of patient 2, the older sister of patient 1, showing moderate chorioretinal atrophy (arrow). (G) Colour fundus photo of patient 3 showing normal-appearing posterior pole and peripheral lattice degeneration (arrow), despite only mild myopia. (H-S) Brain MRI demonstrates cerebellar dysplasia associated with cysts. Brain MRI findings of patient 1 (H-J), patient 2 (K-M), patient 3 (N-P) and a healthy control (Q-S) are shown. (H) Patient 1 at 7 months of age has multiple cortical/subcortical cysts in the cerebellar vermis (arrow), dysplastic cerebellar vermis and enlarged fourth ventricle (arrowhead) with an abnormal rectangle-like shape (midsagittal-T1). The midbrain is mildly elongated, and the pons is mildly reduced in size. (I) Subcortical cysts (arrows) and dysplasia are also seen in the cerebellar hemispheres (axial-T1). (J) At 10 years 7 months, dysplasia in cerebellar vermis and hemispheres with multiple cortical and subcortical cysts (arrow) remain visible. (K) Patient 2 has hypoplasia and dysplasia of the cerebellar vermis with cortical and subcortical cysts, enlarged fourth ventricle (asterisk), elongated midbrain and pons appearing small (midsagittal-T1). (L) Cerebellar hemispheres are dysplastic and with multiple cortical and subcortical cysts in patient 2 (axial-T2). (M) Cortical and subcortical cysts within the left cerebellar hemisphere of patient 2 (parasagittal-T1) are shown. Images (K-M) were taken at age 6 years and 10 months. (N) Patient 3 has a normal sized but severely dysplastic vermis (white dot) with lack of separation of the vermian lobules (midsagittal-T1). Axial (O) and coronal (P) T2-weighted images of patient 3 show a markedly dysplastic appearance of the cerebellar hemispheres and the vermis with a diffusely abnormal configuration of the cerebellar folia and sulci (black arrows). Multiple small cysts are noted in the cerebellar hemispheres bilaterally (white arrows). Images (N-P) were taken at 4 years 3 months. (Q) A healthy 12-year-old control with normal anatomy of the posterior fossa structures (midsagittal-T1) and a normal triangle-shaped fourth ventricle is shown. Normal morphology of the cerebellum with an 'onion-like' foliation/fissuration pattern and white matter arborisation in the cerebellar hemispheres seen in axial (R) and coronal (S) T2 weighted images.
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F1
Patient 3 (figure 1C), an 8.5-year-old female born to non-consanguineous parents Caucasian and Native American descent, had mild myopia associated with peripheral lattice degeneration of retina (figure 1G). She also had a severely dysplastic cerebellar vermis and multiple small cysts in the cerebellar hemispheres (figure 1N-P). ID: 284217) spans ~176 kb, and is divided into 63 exons. Its mRNA is 9657 bp and encodes a 3075 amino acid protein, laminin alpha1, which is selectively expressed in tissues (see online supplementary figure S1A). We noticed a significant decrease of the mRNA expression of these three subunits in both patient 2 and patient 3 fibroblasts (see online supplementary figure S1B).
LAMA1 null mutations in patients with lamininopathy. (A) Pedigrees of the two families including chromatograms of their identified LAMA1 variants. (B) Quantitative real-time PCR results for LAMA1 mRNA expression in fibroblast from patient 2 and patient 3 compared with control using five different probes spanning LAMA1 cDNA. Values are percentage expression of LAMA1 in patient cells compared with control cells, normalised to ACTB (error bars represent S.D., n=4 from three independent experiments; **p<0.01, Mann-Whitney test); under the histogram, localisation of the different probes on LAMA1 transcript (NM_005559.3) is shown. (c) Immunoblot of fibroblast lysates of patient 2, patient 3 and control, showing absence of LAMA1 in the patients. Loading was controlled by vinculin (VCL).
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Pro2234Leufs*9), presumably inherited from their unavailable father, since it was absent from the mother's DNA (figure 2A). For patient 3, Sanger sequencing of the LAMA1 gene identified a nonsense mutation in exon 15 (NM_005559.3:c.2160T>A; p.Cys720*) inherited from her mother and a 7 bp deletion in exon 42 (NM_005559.3:c.5985_5991del; p.Ile1996Glufs*7), presumably inherited from her unavailable father (figure 2A). LAMA1 mRNA transcript was absent from cultured fibroblasts of patients 2 and 3 (figure 2B), presumably due to nonsense-mediated decay. Indeed, no LAMA1 protein was produced, as confirmed by immunoblotting (figure 2C). As the identified mutations lead to null alleles and no expression of LAMA1 protein (figure 2B), we tested the expression of the laminin subunits that can assemble together with the alpha1 chain (LAMB1, encoding laminin beta1, MIM 150240; LAMB2, encoding laminin beta2, MIM 150325 and LAMC1, encoding laminin gamma1, MIM 150290). Knockdown efficiency was evaluated using 0.1, 0.2 and 0.6 muM, and we used 0.6 muM for the rest of the study since it gave the most robust knockdown (~80%, see online supplementary figure S2). Stable overexpression of pLenti-LAMA1 in patients' fibroblasts led to an increase in the amount of LAMA1 protein by immunoblot (see online supplementary figure S3A; image of full blots are shown in see online supplementary figure S4), compared with absent LAMA1 protein in patients' cells without rescue (figure 2C).
LAMA1-deficient cells exhibit impaired adhesion and migration. (A) Equal numbers of control and patient 2 fibroblasts were plated on wells coated with either bovine serum albumin (BSA), collagen-I (Col-I) or Laminin-111 and allowed to attach. OD values represent amounts of DNA in cells that remain attached to the wells after 90 min of incubation. Bar graph gives mean and SD of five replicates from three independent experiments; *p<0.05; **p<0.01 (Mann-Whitney test). (B) Equal numbers of control and patient 2 fibroblasts were seeded on culture wells and allowed to form a monolayer and grow to confluence, after which a 0.9 mm scratch was made. 'Wound' closure was monitored by imaging cells stained with Calcein AM after 12, 24, 36, 48 and 60 h. Percentage closure and migration rates were calculated from six experiments. Scale bar represents 100 mum. Bar graph gives mean and SD of three replicates from three independent experiments; *p<0.05; **p<0.01 (Mann-Whitney test).
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LAMA1 is a component of the basement membrane, we evaluated the functional consequences of absent LAMA1 by measuring the adhesion response of trypsinised fibroblasts to different ECM components (collagen-1, laminin-111) (figure 3A). LAMA1-deficient cells showed significantly decreased adhesion to non-coated and coated wells when compared with control fibroblasts (figure 3A). Patient's LAMA1-deficient fibroblasts demonstrated less migration into the cell-denuded gap than did control fibroblasts (figure 3B). Stable overexpression of pLenti-LAMA1 in patients' fibroblasts led to an increase in the amount of LAMA1 protein by immunoblot (see online supplementary figure S3A; image of full blots are shown in see online supplementary figure S4), compared with absent LAMA1 protein in patients' cells without rescue (figure 2C). Patients' fibroblasts stably expressing LAMA1 showed improvement in cell adhesion (see online supplementary figure S3B), increased migration (see online supplementary figure S3C) and reduced apoptosis (see online supplementary figure S3D) compared with patient fibroblasts cells stably expressing pLenti-empty lentiviral vector. In addition, patient cells transduced with pLenti-LAMA1 exhibited an increase in GTP-bound Cdc42 (see online supplementary figure S3E), indicating that LAMA1 is important in filopodia formation and, consequently, cell adhesion and migration.
LAMA1 deficiency disrupts cell architecture for focal adhesion in fibroblasts. Equal numbers of cells from control and patient 2 fibroblasts were plated on non-coated or laminin-111-coated coverslips. Laminin-111 is a heterotrimer complex composed of LAMA1, LAMB1 and LAMC1. (A) Cells were imaged after staining with phalloidin (red) that labels actin filaments, and DAPI (blue) that labels the nuclei. On uncoated coverslips, the control cells show intact, highly organised cytoskeletal organisation, and numerous filopodia. In cells of patient 2 and patient 3, there is less complexity in cytoskeletal network, and filopodia structures are remarkably reduced or absent, indicating the importance of LAMA1 in the focal adhesion complex. On laminin-111-coated coverslips, control cells appear to spread more than on non-coated coverslips, while the filopodia length is increased. Cells of patient 2 and patient 3 show increased number of filopodia on laminin-coated coverslips. Scale bar represents 20 mum. (B) On scanning electron microscopy, control cells on uncoated slides appear thoroughly spread and flat with well-formed filopodia structures at the edges, as expected in normal control fibroblasts. Patient 2 and patient 3 cells on uncoated slides generally appear 'lifted', creating a three-dimensional appearance. The number of filopodia in patient 2 cells are markedly lower as compared with control cells; this number normalised to almost similar to control cells when patient 2 and patient 3 cells were grown on laminin-coated slides. In addition, microvilli protrusions, which are presumably unattached filopodia or lamellipodia, are seen on the surface of the cells. Scale bars in (B) represent 50 mum in B1-2 and B4-5, 10 mum in B3, B6, B7 and B9 and 25 mum in B8. (C) GTPase activation assay of Cdc42, Rac1 and RhoA was performed in control and patient 2 fibroblasts. Results show a significant decrease of the activated form (GTP-bound) of Cdc42 in patient 2 and patient 3 fibroblasts compared with control; no difference was observed for Rac1 and RhoA (error bars represent SD, n=6; *p<0.05, Mann-Whitney test).
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To visualise the effect of LAMA1 deficiency on cell migration, we analysed cell membrane projections (lamellipodia and filopodia) using immunohistochemistry (figure 4A) and scanning electron microscopy (figure 4B). In contrast, LAMA1-deficient cells from patient 2 appear less stretched out, with short and stubby lamellipodia, and increased microvillus projection on the cell surface (figure 4A). When grown on laminin-111-coated coverslips, patient 2 cell processes increased in number and length with broader lamellipodia, but less visible focal adhesion points, indicating partial rescue in cell architecture (figure 4B). We tested the activation of different GTPases using G-Lisa (figure 4C); Cdc42 was significantly decreased in patients' fibroblasts compared with control, whereas other GTPases (RhoA and Rac1) were normal. Stable overexpression of pLenti-LAMA1 in patients' fibroblasts led to an increase in the amount of LAMA1 protein by immunoblot (see online supplementary figure S3A; image of full blots are shown in see online supplementary figure S4), compared with absent LAMA1 protein in patients' cells without rescue (figure 2C).
Effects of LAMA1 knockdown in neuronal cells (A-C); LAMA1 knockdown in neuronal cells reduced the number of filopodia. Non-targeting (control) siRNA and siRNA to LAMA1 (LAMA1-siRNA) were introduced to neuronal cells differentiated from Neural Stem Cells (NSC). Immunohistochemistry with phalloidin (A) showed that LAMA1-siRNA reduced the number of neurites and branching, in addition to reduction of filopodia (red, phalloidin; blue, DAPI stain for nucleus). Scanning electron microscopy (SEM) (B,C) confirmed that the number of filopodia is reduced when LAMA1 is knocked down (LAMA1-siRNA) as compared with control. Filopodia were partially recovered when wild-type hLAMA1 was reintroduced to cells (LAMA1-siRNA+Rescue). Magnified images of (B) are shown in (C). Scale bars represent 100 mum in (A), 10 mum in (B) and 2 mum in (C). (D) LAMA1 deficiency increases susceptibility to apoptosis. To quantify apoptosis, caspase-3/7 activity was measured at 12 and 30 h after plating control (white bars), patient 2 (light grey bars) and patient 3 cells, with or without the addition of the known apoptosis inducer staurosporine. Results show an increased susceptibility of patient's cells to apoptosis. Bars represent SD for three replicates of three experiments; *p<0.05; **p<0.01 (Mann-Whitney test).
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In contrast, LAMA1 knockdown resulted in reduced branching of processes (figure 5A) and disappearance of filopodia (figure 5B,C), supporting the importance of LAMA1 in neuronal cell filopodia formation and cell migration. At baseline, a significantly greater number of LAMA1-deficient cells underwent apoptosis compared with control cells, demonstrated by increased Annexin V expression (data not shown). After treatment with staurosporine, a known apoptosis inducer, LAMA1-deficient cells show markedly increased susceptibility to cell death, demonstrated by significantly increased caspase 3/7 activity (figure 5D).