Rho-GTPase Activating Protein myosin MYO9A identified as a novel candidate gene for monogenic focal segmental glomerulosclerosis

Abstract

Focal segmental glomerulosclerosis (FSGS) is a podocytopathy leading to kidney failure, whose molecular cause frequently remains unresolved. Here, we describe a rare MYO9A loss of function nonsense heterozygous mutation (p.Arg701^∗) as a possible contributor to disease in a sibling pair with familial FSGS/proteinuria. MYO9A variants of uncertain significance were identified by whole exome sequencing in a cohort of 94 biopsy proven patients with FSGS. MYO9A is an unconventional myosin with a Rho-GAP domain that controls epithelial cell junction assembly, crosslinks and bundles actin and deactivates the small GTPase protein encoded by the RHOA gene. RhoA activity is associated with cytoskeleton regulation of actin stress fiber formation and actomyosin contractility. Myo9A was detected in mouse and human podocytes in vitro and in vivo. Knockin mice carrying the p.Arg701^∗MYO9A (Myo9A^R701X) generated by gene editing developed proteinuria, podocyte effacement and FSGS. Kidneys and podocytes from Myo9A^R701X/+ mutant mice revealed Myo9A haploinsufficiency, increased RhoA activity, decreased Myo9A-actin-calmodulin interaction, impaired podocyte attachment and migration. Our results indicate that Myo9A is a novel component of the podocyte cytoskeletal apparatus that regulates RhoA activity and podocyte function. Thus, Myo9A^R701X/+ knock-in mice recapitulate the proband FSGS phenotype, demonstrate that p.R701X Myo9A is an FSGS-causing mutation in mice and suggest that heterozygous loss-of-function MYO9A mutations may cause a novel form of human autosomal dominant FSGS. Hence, identification of MYO9A pathogenic variants in additional individuals with familial or sporadic FSGS is needed to ascertain the gene contribution to disease.

Trial registration: ClinicalTrials.gov NCT00135811.

Keywords: FSGS; MYO9A variants; Myo9A-actin-calmodulin interaction; RhoA activity.

Figure 1.. Heterozygous nonsense mutation in MYO9A in a family with FSGS/proteinuria.

A) novel heterozygous single-bp mutation that leads to a stop codon in exon 14 of MYO9A c.C2101T: p.R701X (p.Arg701*) in a patient with proteinuria and FSGS. Nucleotide sequence traces show the C>T mutation shared by proband, sister and mother (top, indicated by asterisk) and wild type sequence from the father (bottom), based on human MYO9A reference sequence NM_006901.4. B) Kidney biopsy from the proband shows focal segmental glomerular sclerosis, mild interstitial infiltrate (PAS stain). C) Family pedigree shows genotype-phenotype segregation: individuals with proteinuria/FSGS and MYO9Ap.R701X variant (solid symbols), wild type (empty symbol), (#) indicates unknown MYO9A genotype. D) Wild type MYO9A (NP_008832.2) protein domains: motor domain (grey) includes loop 2 (yellow) and calmodulin binding site (red), neck domain (blue), tail domain (pink) including RhoGAP domain (green); domain boundaries are indicated by amino acid residue number; predicted p.R701X MYO9A terminates within calmodulin binding site in loop 2, lacking the neck and tail domains, asterisks indicate the location of missense MYO9A VUS identified. E) Evolutionary conservation of amino acid residues altered by MYO9A missense mutations identified in FSGS patients: MYO9A altered residues p.D156 (p.Asp156) and p.E765 (p.Glu765) (red arrows) are highly conserved through phylogeny. Accession numbers: H. sapiens (NP_008832.2), M. musculus (NP_766606.2), R. norvegicus (NP_599162.1), G. gallus (XP_015134553.1), D. rerio (E7EZG2.1), X. tropicalis (XP_002934596.2) were used for MYO9A multiple sequence alignment with Clustal Omega-EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Figure 2:. Glomerular and podocyte Myo9A expression.

A) Immunoblots show Myo9A in mouse kidney, immortalized mouse podocytes (1), undifferentiated (2) and differentiated (3) human podocytes; B) Immuno-fluorescence microscopy (IF) shows MYO9A in the cytoplasm of undifferentiated and differentiated human podocytes (green), partially co-localized with F-actin (red) in differentiated podocytes (yellow), scale bar=10μm; C) IF shows Myo9A (red) and podocin (green) partially co-localized in mouse glomeruli (merge=yellow), scale bars=50μm; D) Dual immunolabeling and confocal microscopy show Myo9A staining (red) on podocytes tightly contiguous to podocin (green) on its GBM side (arrowheads), insets show higher magnification; scale bar = 25μm. E) Confocal microscopy shows Myo9A staining closely contiguous or co-localized with laminin (green) along the outer aspect of the GBM (insets and arrowhead); scale bar = 25μm. F an G) Immuno-EM localization of Myo9A in glomeruli of wild type mouse kidneys: gold particles (black arrows) are observed in the podocyte cell body (pod) and the foot processes cytoplasm (fp), some gold particles are seen at the fp base next to the GBM but not at slit diaphragms; scale bars=500 nm.

Figure 3:. Glomerular Myo9A expression in glomerular disease mouse models.

A) developmental nephrotic syndrome induced by VEGF-A overexpression: IF shows Myo9A (red) in glomeruli from normal, uninduced 2 week old mice and clearly decreased Myo9A expression in glomeruli from nephrotic mice overexpressing VEGF-A, while podocin (green) is unchanged. B) Streptozotocin-induced diabetes with or without VEGF-A overexpression: IF detects Myo9A (green) in glomeruli from uninduced diabetic kidneys (DM control) and markedly decreased immunoreactive Myo9A in glomeruli from diabetic mice overexpressing VEGF-A that develop diabetic glomerulosclerosis (advanced DN), Dapi (blue) shows nuclei from both sections with outlined glomeruli (hatched white line). Scale bars= 50μm.

Figure 4:. Genotype and phenotype analysis of knock-in Myo9A^R701X mice at 2 weeks of age.

A) Sanger sequencing traces and deduced amino acid sequence from exon 14 are shown from mutant mice: heterozygous Myo9A^R701X/+ (top), homozygous Myo9A^R701X/R701X (middle) and wild type littermate (bottom); C>T mutation substitutes CGA encoding Arg (R) by TGA, a stop codon that predicts termination of translation at amino acid 701. B) Representative PAS stain from 2-week-old homozygous Myo9A^R701X/R701X mutant kidney shows abnormal glomeruli, tubular dilatation and protein casts. C) Representative PAS stain from 2-week-old heterozygous Myo9A^R701X/+ kidney shows normal histology. D) Representative TEM from 2-week-old Myo9A^R701X/R701X glomerulus shows massive foot process effacement (white arrows), absence of slit-diaphragms, replaced by adherens junctions (black arrows) and thickened, irregular GBM. E) Representative TEM from 2-week-old heterozygous Myo9A^R701X/+ glomerulus shows normal foot processes (black thin arrows), and normal slit-diaphragms (white thin arrows). F) TEM from 2-week-old wild type littermate (WT) shows normal glomerular filtration barrier: thin foot processes linked by slit-diaphragms, fenestrated endothelial cells and thin GBM. G) Quantitation of foot process effacement at 2 weeks of age: homozygous Myo9A^R701X/R701X kidneys show less than half foot processes/μm GBM than willd type or heterozygous Myo9A^R701X/+ littermates, indicating severe FPE , P<0.0001 (ANOVA). H) Coomassie blue stain of SDS-PAGE resolved urine samples shows albuminuria in both Myo9A^R701X/R701X (−/−) and Myo9A^R701X/+ (+/−), while WT urine is albumin free. BSA (10μg) serves as MW control. Scale bars: A and B= 20μm; C, D and E= 1μm.

Figure 5:. Adult heterozygous Myo9A^R701X/+ knock-in mice phenotype analysis.

A) Representative PAS stain from 4 months old Myo9A^R701X/+ kidney shows a glomerulus with extensive focal segmental glomerulosclerosis and mesangial proliferation. B) Representative TEM from 4 months old Myo9A^R701X/+ kidney shows extensive foot process effacement (white arrows), partial replacement of slit-diaphragms (thin black arrows) by adherens junctions (thick black arrows). C) Quantitation of foot process effacement (FPE) in adult Myo9A^R701X/+ mutant mice: 4 months of age heterozygous Myo9A^R701X/+ show 2.2±0.06 FP/μm, as compared to wild type 3.7±0.13 FP/μm, **** indicates P<0.0001. D) Urine albumin/creatinine ratio at 4 months of age is higher in Myo9A^R701X/+ (Het, red dots) than in wild type mice (Wt, black dots), *** indicates P>0.001. E) Urine volume (24 h) and F) body weight are not different in Wt (n=18) and Myo9A^R701X/+ (Het, n=16) mice. G and J) Representative PAS stain from Myo9A^R701X/+ (G) and Wt (J) kidneys: G) shows early focal sclerosis (arrows) and mesangial proliferation in 2 glomeruli, normal back to back tubules and no infiltrates; J) shows normal glomeruli and tubules in Wt kidney. H and K) Confocal microscopy of Myo9A/Megalin IF: H) Myo9A^R701X/+ kidney shows limited Myo9A (red) signal (arrowheads) in proximal tubular cells labeled with megalin (green), proximal tubules (PT) are not dilated; K) Wt kidney shows more abundant Myo9A (arrowheads) contiguous to megalin in PT cells. I and L) Confocal microscopy of Myo9A/Aquaporin2 (AQP2) IF: I) Myo9A^R701X/+ kidney shows limited Myo9A (red) in the glomerulus (arrowheads), Myo9A is not detected in distal nephron segments labeled with AQP2 (green), which are not dilated; L) Wt kidney shows Myo9A staining in glomerulus and AQP2 staining in distal nephron segments. Scale bars: A, G and J = 50μm; B= 2μm; H, I, K and L = 25μm.

Figure 6:. Myo9A expression and Myo9A-actin-calmodulin interaction knock-in Myo9A^R701X kidneys and podocytes.

A) IF detects glomerular Myo9A and podocin: Myo9A glomerular immunostaining (red) is strong in wild type kidneys, decreases significantly in Myo9A^R701X/+ glomeruli, and is not detected in Myo9A^R701X/R701X glomeruli; podocin (green) localizes to podocytes in all mice; podocyte-Myo9A partial co-localization is shown by merge (yellow). B) Immunoblotting: Intact Myo9A (280kDa) expression decreases to ~ half in Myo9A^R701X/+ (Het) kidneys and is not detected in Myo9A^R701X/R701X (Hom) kidneys, smaller bands (<75kDa) are considered non-specific, ~50 kDa bands correspond to IgG heavy chain in all genotypes. C) Myo9A/actin quantification (mean±SD), n=3 independent experiments, pool from 3 mice each, ** indicates P<0.001. D) qPCR: Myo9A mRNA expression normalized to GAPDH mRNA, fold change (mean±SD), n=3 independent experiments, n=3 individual mice each, **** indicates P<0.0001. E-F) co-IP of kidney lysates (E) and primary podocytes (F) show decreased Myo9A-actin-calmodulin interaction in Myo9A^R701X/+ (Het) proportionally to the reduced Myo9A expression (input), note input of actin and calmodulin is equal in both genotypes.

Figure 7:. Myo9A knockdown decreases Myo9A-actin-calmodulin interaction in immortalized mouse podocytes.

A) Myo9A siRNA [100nM] induces ~50% decrease in Myo9A mRNA, as compared to wild type control and non-targeting siRNA [100nM]. Myo9A mRNA normalized to GAPDH mRNA, expressed as fold change (mean±SD), n≥3 independent experiments,* indicates p<0.05. B) Representative IB shows that Myo9A siRNA induced ~50% reduction of Myo9A protein, as compared to Wt control and non-targeting siRNA; actin expression level was not altered by siRNAs; n≥3 independent experiments. C) Representative Myo9A IP shows decreased Myo9A-actin-calmodulin interaction in Myo9A^KD podocytes (siRNA-treated=KD) as compared to wild type immortalized mouse podocytes (Wt), proportionally to the decrease in Myo9A immunoprecipitated; n≥3 independent experiments.

Figure 8:. RhoA activity and podocyte function in Myo9A^R701X kidneys and Myo9A^KD podocytes.

A-C) Active RhoA (GTP-RhoA) is increased in Myo9A ^R701X mutants vs. wild type: >2-fold in whole kidneys (A), ~60% in isolated glomeruli (B), >2-fold in primary podocytes (C), Western blots representative of n=3–4 independent experiments, lysates pooled from 3 mice each, quantitation (mean±SD), ANOVA (A) or unpaired t-test (B and C), * P<0.05, ** P<0.001. D) Podocyte migration assay (wound assay): quantification of the migration area shows that Myo9A^R701X/+ (Het) podocyte migration is decreased; pre-incubation with Y26732 partially abrogates the migration defect, *** P<0.0005 (ANOVA),**** P<0.0001 (unpaired t-test). E) Podocyte attachment assay shows decreased attachment of Myo9A^R701X/+ (Het) podocytes; pre-incubation with Y26732 corrects the attachment defect, *** P<0.0005 (ANOVA). D-E) Data are mean±SD, n=3–4 independent experiments, n=3 mice/genotype/experiment. F) Podocyte migration assay: siRNA-mediated Myo9A knockdown (Myo9A^KD) decreases immortalized mouse podocytes migration (* P<0.05, ANOVA, ** P<0.01, unpaired t-test), whereas migration of non-targeting siRNA-treated podocytes (N-T) is not different from normal podocytes (Wt). G) Attachment assay: Myo9A^KD decreases immortalized mouse podocytes attachment, as compared to podocytes treated with non-targeting siRNA (N-T) and untreated Wt podocytes. Myo9A^KD data are mean±SD, n≥3 independent experiments. * indicates P<0.05, ** P<0.01, ns indicates no significant difference.