Laminin β2 variants associated with isolated nephropathy that impact matrix regulation.
ABSTRACT: Mutations in LAMB2, encoding laminin β2, cause Pierson syndrome and occasionally milder nephropathy without extrarenal abnormalities. The most deleterious missense mutations that have been identified affect primarily the N-terminus of laminin β2. On the other hand, those associated with isolated nephropathy are distributed across the entire molecule, and variants in the β2 LEa-LF-LEb domains are exclusively found in cases with isolated nephropathy. Here we report the clinical features of mild isolated nephropathy associated with 3 LAMB2 variants in the LEa-LF-LEb domains (p.R469Q, p.G699R, and p.R1078C) and their biochemical characterization. Although Pierson syndrome missense mutations often inhibit laminin β2 secretion, the 3 recombinant variants were secreted as efficiently as WT. However, the β2 variants lost pH dependency for heparin binding, resulting in aberrant binding under physiologic conditions. This suggests that the binding of laminin β2 to negatively charged molecules is involved in glomerular basement membrane (GBM) permselectivity. Moreover, the excessive binding of the β2 variants to other laminins appears to lead to their increased deposition in the GBM. Laminin β2 also serves as a potentially novel cell-adhesive ligand for integrin α4β1. Our findings define biochemical functions of laminin β2 variants influencing glomerular filtration that may underlie the pathogenesis of isolated nephropathy caused by LAMB2 abnormalities.
Project description:Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin β2 (LAMB2), a major component of the glomerular basement membrane (GBM). Pathogenic missense mutations in human LAMB2 cluster in or near the laminin amino-terminal (LN) domain, a domain required for extracellular polymerization of laminin trimers and basement membrane scaffolding. Here, we investigated an LN domain missense mutation, LAMB2-S80R, which was discovered in a patient with Pierson syndrome and unusually late onset of proteinuria. Biochemical data indicated that this mutation impairs laminin polymerization, which we hypothesized to be the cause of the patient's nephrotic syndrome. Testing this hypothesis in genetically altered mice showed that the corresponding amino acid change (LAMB2-S83R) alone is not pathogenic. However, expression of LAMB2-S83R significantly increased the rate of progression to kidney failure in a Col4a3-/- mouse model of autosomal recessive Alport syndrome and increased proteinuria in Col4a5+/- females that exhibit a mild form of X-linked Alport syndrome due to mosaic deposition of collagen α3α4α5(IV) in the GBM. Collectively, these data show the pathogenicity of LAMB2-S80R and provide the first evidence of genetic modification of Alport phenotypes by variation in another GBM component. This finding could help explain the wide range of Alport syndrome onset and severity observed in patients with Alport syndrome, even for family members who share the same COL4 mutation. Our results also show the complexities of using model organisms to investigate genetic variants suspected of being pathogenic in humans.
Project description:The importance of the glomerular basement membrane (GBM) in glomerular filtration is underscored by the manifestations of Alport and Pierson syndromes, caused by defects in type IV collagen α3α4α5 and the laminin β2 chain, respectively. Lamb2 null mice, which model the most severe form of Pierson syndrome, exhibit proteinuria prior to podocyte foot process effacement and are therefore useful for studying GBM permselectivity. We hypothesize that some LAMB2 missense mutations that cause mild forms of Pierson syndrome induce GBM destabilization with delayed effects on podocytes. While generating a CRISPR/Cas9-mediated analogue of a human LAMB2 missense mutation in mice, we identified a 44-amino acid deletion (LAMB2-Del44) within the laminin N-terminal domain, a domain mediating laminin polymerization. Laminin heterotrimers containing LAMB2-Del44 exhibited a 90% reduction in polymerization in vitro that was partially rescued by type IV collagen and nidogen. Del44 mice showed albuminuria at 1.8-6.0 g/g creatinine (ACR) at one to two months, plateauing at an average 200 g/g ACR at 3.7 months, when GBM thickening and hallmarks of nephrotic syndrome were first observed. Despite the massive albuminuria, some Del44 mice survived for up to 15 months. Blood urea nitrogen was modestly elevated at seven-nine months. Eight to nine-month-old Del44 mice exhibited glomerulosclerosis and interstitial fibrosis. Similar to Lamb2<sup>-/-</sup> mice, proteinuria preceded foot process effacement. Foot processes were widened but not effaced at one-two months despite the high ACRs. At three months some individual foot processes were still observed amid widespread effacement. Thus, our chronic model of nephrotic syndrome may prove useful to study filtration mechanisms, long-term proteinuria with preserved kidney function, and to test therapeutics.
Project description:The glomerular basement membrane (GBM) is an important component of the kidney's glomerular filtration barrier. Like all basement membranes, the GBM contains type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan. It is flanked by the podocytes and glomerular endothelial cells that both synthesize it and adhere to it. Mutations that affect the GBM's collagen ?3?4?5(IV) components cause Alport syndrome (kidney disease with variable ear and eye defects) and its variants, including thin basement membrane nephropathy. Mutations in LAMB2 that impact the synthesis or function of laminin ?5?2?1 (LM-521) cause Pierson syndrome (congenital nephrotic syndrome with eye and neurological defects) and its less severe variants, including isolated congenital nephrotic syndrome. The very different types of kidney diseases that result from mutations in collagen IV vs. laminin are likely due to very different pathogenic mechanisms. A better understanding of these mechanisms should lead to targeted therapeutic approaches that can help people with these rare but important diseases.
Project description:<h4>Background</h4>Both Pierson syndrome (PS) and isolated nephrotic syndrome can be caused by LAMB2 biallelic pathogenic variants. Only 15 causative splicing variants in the LAMB2 gene have been reported. However, the pathogenicity of most of these variants has not been verified, which may lead to incorrect interpretation of the functional consequence of these variants.<h4>Methods</h4>Using high-throughput DNA sequencing and Sanger sequencing, we detected variants in a female with clinically suspected PS. A minigene splicing assay was performed to assess the effect of LAMB2 intron 20 c.2885-9C>A on RNA splicing. We also performed the immunohistochemical analysis of laminin beta-2 in kidney tissues.<h4>Results</h4>Two novel LAMB2 heteroallelic variants were found: a paternally inherited variant c.2885-9C>A in intron 20 and a maternally inherited variant c. 3658C>T (p. (Gln1220Ter)). In vitro minigene assay showed that the variant c.2885-9C>A caused erroneous integration of a 7 bp sequence into intron 20. Immunohistochemical analysis revealed the absence of glomerular expression of laminin beta-2, the protein encoded by LAMB2.<h4>Conclusion</h4>We demonstrated the impact of a novel LAMB2 intronic variant on RNA splicing using the minigene assay firstly. Our results extend the mutational spectrum of LAMB2.
Project description:Mutations of LAMB2 typically cause autosomal recessive Pierson syndrome, a disorder characterized by congenital nephrotic syndrome, ocular and neurologic abnormalities, but may occasionally be associated with milder or oligosymptomatic disease variants. LAMB2 encodes the basement membrane protein laminin beta2, which is incorporated in specific heterotrimeric laminin isoforms and has an expression pattern corresponding to the pattern of organ manifestations in Pierson syndrome. Herein we review all previously reported and several novel LAMB2 mutations in relation to the associated phenotype in patients from 39 unrelated families. The majority of disease-causing LAMB2 mutations are truncating, consistent with the hypothesis that loss of laminin beta2 function is the molecular basis of Pierson syndrome. Although truncating mutations are distributed across the entire gene, missense mutations are clearly clustered in the N-terminal LN domain, which is important for intermolecular interactions. There is an association of missense mutations and small in frame deletions with a higher mean age at onset of renal disease and with absence of neurologic abnormalities, thus suggesting that at least some of these may represent hypomorphic alleles. Nevertheless, genotype alone does not appear to explain the full range of clinical variability, and therefore hitherto unidentified modifiers are likely to exist.
Project description:Pierson syndrome is a recently defined disease usually lethal within the first postnatal months and caused by mutations in the gene encoding laminin beta2 (LAMB2). The hallmarks of Pierson syndrome are congenital nephrotic syndrome accompanied by ocular abnormalities, including microcoria (small pupils), with muscular and neurological developmental defects also present. Lamb2(-/-) mice are a model for Pierson syndrome; they exhibit defects in the kidney glomerular barrier, in the development and organization of the neuromuscular junction, and in the retina. Lamb2(-/-) mice fail to thrive and die very small at 3 weeks of age, but to what extent the kidney and neuromuscular defects each contribute to this severe phenotype has been obscure, though highly relevant to understanding Pierson syndrome. To investigate this, we generated transgenic mouse lines expressing rat laminin beta2 either in muscle or in glomerular epithelial cells (podocytes) and crossed them onto the Lamb2(-/-) background. Rat beta2 was confined in skeletal muscle to synapses and myotendinous junctions, and in kidney to the glomerular basement membrane. In transgenic Lamb2(-/-) mice, beta2 deposition in only glomeruli prevented proteinuria but did not ameliorate the severe phenotype. By contrast, beta2 expression in only muscle restored synaptic architecture and led to greatly improved health, but the mice died from kidney disease at 1 month. Rescue of both glomeruli and synapses was associated with normal weight gain, fertility and lifespan. We conclude that muscle defects in Lamb2(-/-) mice are responsible for the severe failure to thrive phenotype, and that renal replacement therapy alone will be an inadequate treatment for Pierson syndrome.
Project description:Pierson syndrome is a congenital nephrotic syndrome with ocular and neurological defects caused by mutations in LAMB2, the gene encoding the basement membrane protein laminin ?2 (Lam?2). It is the kidney glomerular basement membrane (GBM) that is defective in Pierson syndrome, as Lam?2 is a component of laminin-521 (LM-521; ?5?2?1), the major laminin in the mature GBM. In both Pierson syndrome and the Lamb2(-/-) mouse model for this disease, laminin ?1 (Lam?1), a structurally similar homolog of Lam?2, is marginally increased in the GBM, but it fails to fully compensate for the loss of Lam?2, leading to the filtration barrier defects and nephrotic syndrome. Here we generated several lines of Lam?1 transgenic mice and used them to show that podocyte-specific Lam?1 expression in Lamb2(-/-) mice abrogates the development of nephrotic syndrome, correlating with a greatly extended lifespan. In addition, the more Lam?1 was expressed, the less urinary albumin was excreted. Transgenic Lam?1 expression increased the level of Lam?5 in the GBM of rescued mice, consistent with the desired increased deposition of laminin-511 (?5?1?1) trimers. Ultrastructural analysis revealed occasional knob-like subepithelial GBM thickening but intact podocyte foot processes in aged rescued mice. These results suggest the possibility that up-regulation of LAMB1 in podocytes, should it become achievable, would likely lessen the severity of nephrotic syndrome in patients carrying LAMB2 mutations.
Project description:Mutations in the laminin ?2 gene (LAMB2) cause Pierson syndrome, a severe congenital nephrotic syndrome with ocular and neurologic defects. LAMB2 is a component of the laminin-521 (?5?2?1) trimer, an important constituent of the glomerular basement membrane (GBM). The C321R-LAMB2 missense mutation leads to congenital nephrotic syndrome but only mild extrarenal symptoms; the mechanisms underlying the development of proteinuria with this mutation are unclear. We generated three transgenic mouse lines, in which rat C321R-LAMB2 replaced mouse LAMB2 in the GBM. During the first postnatal month, expression of C321R-LAMB2 attenuated the severe proteinuria exhibited by Lamb2(-/-) mice in a dose-dependent fashion; proteinuria eventually increased, however, leading to renal failure. The C321R mutation caused defective secretion of laminin-521 from podocytes to the GBM accompanied by podocyte endoplasmic reticulum (ER) stress, likely resulting from protein misfolding. Moreover, ER stress preceded the onset of significant proteinuria and was manifested by induction of the ER-initiated apoptotic signal C/EBP homologous protein (CHOP), ER distention, and podocyte injury. Treatment of cells expressing C321R-LAMB2 with the chemical chaperone taurodeoxycholic acid (TUDCA), which can facilitate protein folding and trafficking, greatly increased the secretion of the mutant LAMB2. Taken together, these results suggest that the mild variant of Pierson syndrome caused by the C321R-LAMB2 mutation may be a prototypical ER storage disease, which may benefit from treatment approaches that target the handling of misfolded proteins.
Project description:CONTEXT:Mutations in LAMB2, encoding the basement membrane protein, laminin ?2, are associated with an autosomal recessive disorder characterized by congenital nephrotic syndrome, ocular abnormalities, and neurodevelopmental delay (Pierson syndrome). CASE DESCRIPTION:This report describes a 12-year-old boy with short stature, visual impairment, and developmental delay who presented with macroscopic hematuria and albuminuria. He had isolated growth hormone deficiency, optic nerve hypoplasia, and a small anterior pituitary with corpus callosum dysgenesis on his cranial magnetic resonance imaging, thereby supporting a diagnosis of optic nerve hypoplasia syndrome. Renal histopathology revealed focal segmental glomerulosclerosis. Using next-generation sequencing on a targeted gene panel for steroid-resistant nephrotic syndrome, compound heterozygous missense mutations were identified in LAMB2 (c.737G>A p.Arg246Gln, c.3982G>C p.Gly1328Arg). Immunohistochemical analysis revealed reduced glomerular laminin ?2 expression compared to control kidney and a thin basement membrane on electron microscopy. Laminin ?2 is expressed during pituitary development and Lamb2-/- mice exhibit stunted growth, abnormal neural retinae, and here we show, abnormal parenchyma of the anterior pituitary gland. CONCLUSION:We propose that patients with genetically undefined optic nerve hypoplasia syndrome should be screened for albuminuria and, if present, screened for mutations in LAMB2.
Project description:Laminin ?2 is a component of laminin-521, which is an important constituent of the glomerular basement membrane (GBM). Null mutations in laminin ?2 (LAMB2) cause Pierson syndrome, a severe congenital nephrotic syndrome with ocular and neurologic defects. In contrast, patients with LAMB2 missense mutations, such as R246Q, can have less severe extrarenal defects but still exhibit congenital nephrotic syndrome. To investigate how such missense mutations in LAMB2 cause proteinuria, we generated three transgenic lines of mice in which R246Q-mutant rat laminin ?2 replaced the wild-type mouse laminin ?2 in the GBM. These transgenic mice developed much less severe proteinuria than their nontransgenic Lamb2-deficient littermates; the level of proteinuria correlated inversely with R246Q-LAMB2 expression. At the onset of proteinuria, expression and localization of proteins associated with the slit diaphragm and foot processes were normal, and there were no obvious ultrastructural abnormalities. Low transgene expressors developed heavy proteinuria, foot process effacement, GBM thickening, and renal failure by 3 months, but high expressors developed only mild proteinuria by 9 months. In vitro studies demonstrated that the R246Q mutation results in impaired secretion of laminin. Taken together, these results suggest that the R246Q mutation causes nephrotic syndrome by impairing secretion of laminin-521 from podocytes into the GBM; however, increased expression of the mutant protein is able to overcome this secretion defect and improve glomerular permselectivity.