Pathogenicity of a Human Laminin β 2 Mutation Revealed in Models of Alport Syndrome

Abstract

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.

Keywords: Alport syndrome; glomerular basement membrane; laminin; nephrotic syndrome.

Figure 1.

LAMB2-S83R protein resulting from CRISPR/Cas9 mediated gene editing accumulates in the GBM but is not pathogenic. (A) Sanger sequencing shows the heterozygous A to C mutation (asterisk) resulting in an Ser to Arg conversion; the boxed sequence is the WT, and the lower sequence is the mutant. (B) SDS-PAGE analysis of urine shows the lack of elevated albuminuria in Lamb2^−/S83R mice versus the WT at up to 13 months (n=3–5). (C) Immunofluorescence analysis of LAMB2 in the GBMs of Lamb2^+/+ (10 months), Lamb2^+/S83R (10 months), Lamb2^S83R/S83R (9 months), and Lamb2^−/S83R (10 months) mice. LAMB2 levels in any S83R-containing mice were similar to the WT at any age up to 13 months; n=3–4 for each genotype.

Figure 2.

LAMB2-S83R impairs LM polymerization on Schwann cells in vitro. (A) Schwann cells were incubated with LM-111 or LM-121 heterotrimers (either the WT or with the indicated mutations), collagen IV, and nidogen with or without the LM polymerization competitor netrin-4 (concentrations shown in B). Immunofluorescence for LAMC1 was performed to assess retention of LM heterotrimers. (B) The immunofluorescence intensity of the anti-LAMC1 signal was normalized to the number of nuclei and calculated as the net summed intensity per cell of the signal detected on Schwann cells cultured with the indicated basement membrane constituents; n=3–5. P values were determined by one-way ANOVA followed by Holm–Sidak pairwise comparisons. *Denotes the representation of 2 independent comparisons by the adjacent bar, whereas every other bar indicates single comparison between 2 experimental groups.

Figure 3.

LAMB2-S83R worsens the Alport syndrome (Col4a3^−/−) phenotype. (A) The age at ESRD of Lamb2^+/+; Col4a3^−/− and Lamb2^+/S83R; Col4a3^−/− mice was followed over the indicated time. Lamb2^+/S83R; Col4a3^−/− mice reached ESRD significantly faster than Lamb2^+/+; Col4a3^−/− mice; P<0.01 by log rank test and n=8–9. (B) Urinary ACRs were calculated at the indicated time points in control, Lamb2^+/+; Col4a3^−/−, and Lamb2^+/S83R; Col4a3^−/− mice; n=4–7. (C) BUN was measured in control, Lamb2^+/+; Col4a3^−/−, and Lamb2^+/S83R; Col4a3^−/− mice at the indicated time points; n=4–7. *P<0.05 by t test; ^#P<0.01 by t test.

Figure 4.

LAMB2-S83R incorporation into the GBM is unimpaired in Alport mice. Immunofluorescence assay for LAMB2 in Lamb2^+/S83R; Col4a3^−/− mice was compared with control and Lamb2^+/+; Col4a3^−/− littermates at P7–P11. Similar levels of LAMB2 were observed in each genotype as well as clear colocalization with the ubiquitous basement membrane protein nidogen in the GBM; n=3–4. Scale bar, 50 μm.

Figure 5.

The LAMB2-S83R allele dramatically worsened Alport phenotype histopathology. (A) PAS staining of P7–P11 control, Lamb2^+/+; Col4a3^−/−, and Lamb2^+/S83R; Col4a3^−/− kidney sections revealed no pathology; n=3–5. (B) PAS staining of kidneys at 6 weeks revealed significant pathologic features in Lamb2^+/S83R; Col4a3^−/− mice, including glomerulosclerosis, crescents, and tubular protein casts, but no severe lesions in Lamb2^+/+; Col4a3^−/− littermates; n=3–4. (C) Widespread and severe pathology in a P7 Lamb2^S83R/S83R; Col4a3^−/− double-homozygous kidney shows a very early effect on the Alport phenotype; n=3. (D) Quantification of glomerulosclerosis in multiple kidneys representative of those shown in A–C. The indicated P values were calculated by t test. C, control; Col, Col4a3; LM, Lamb2; +/+, WT; +/S, Lamb2^+/S83R; S/S, Lamb2^S83R/S83R.

Figure 6.

LAMB2-S83R increases Alport-associated deposition of ectopic laminins into the GBM. Frozen sections of kidneys from 6- to 10-week-old mice of the indicated genotypes were stained for LAMB2 (6 weeks shown), LAMB1 (10 weeks shown), and LAMA2 (10 weeks shown for control and Lamb2^+/+; Col4a3^−/− and 8 weeks shown for Lamb2^+/S83R; Col4a3^−/−). LAMB2 was detected in the GBM in all cases. LAMB1 and LAMA2 are normally in the mesangial matrix but were detected ectopically in the Alport GBM regardless of Lamb2 genotype, although the levels appeared slightly increased in some Lamb2^+/S80R; Col4a3^−/− versus Lamb2^+/+; Col4a3^−/− glomeruli. Colocalization with GBM agrin is shown in Supplemental Figures 3 and 4; n=4–5.

Figure 7.

The Lamb2-S83R allele enhances Alport-associated GBM abnormalities and foot process effacement. (A) Transmission electron micrographs of glomeruli from mice of the indicated genotypes at the indicated ages reveal GBM splitting by P7–P11 and depict the progression of foot process effacement and sclerosis; n=3–5. Note the unusually thick and electron-lucent GBM in the Lamb2^+/S80R; Col4a3^−/− kidney at 7 weeks. (B) The number of foot processes per 1 μm of GBM was counted for each genotype at each time point; n=3–5. *P<5×10⁻⁴ by t test; **P<6×10⁻⁵ by t test; ***P<2×10⁻⁷ by t test.

Figure 8.

LAMB2-S83R accelerates disease in Col4a5^+/− females. (A) LAMB2 and COL4Α345 were detected by immunofluorescence in 8-week-old tissues. Anti-COL4A345 NC1 domain staining revealed the expected mosaic expression pattern mediated by naturally random inactivation of either the WT or the Col4a5 mutant X chromosome in each podocyte. LAMB2 was detected in both COL4Α345-negative and -positive regions of the GBM but exhibited increased intensity in COL4Α345-negative capillary loops (red in the merged images); n=4. (B) Urinalysis revealed significantly higher ACRs in Lamb2^+/S83R; Col4a5^+/− mice versus Col4a5^+/− littermates between 2 and 6 weeks of age and at 12 weeks of age but with similar ratios at week 8; n=4–7 at each time point. Urine volumes normalized to 20 mg/dl creatinine are shown on a representative SDS-PAGE gel for Lamb2^+/+; Col4a5^+/− and Lamb2^+/S83R; Col4a5^+/− littermates at 12 weeks. (C) PAS staining indicated accelerated glomerulosclerosis, inflammation, and protein casts in 8-week-old Lamb2^+/S83R; Col4a5^+/− females versus Col4a5^+/− females; n=3–5. *P<0.05 by t-test.