MYO1E mutations and childhood familial focal segmental glomerulosclerosis

Abstract

Background: Focal segmental glomerulosclerosis is a kidney disease that is manifested as the nephrotic syndrome. It is often resistant to glucocorticoid therapy and progresses to end-stage renal disease in 50 to 70% of patients. Genetic studies have shown that familial focal segmental glomerulosclerosis is a disease of the podocytes, which are major components of the glomerular filtration barrier. However, the molecular cause in over half the cases of primary focal segmental glomerulosclerosis is unknown, and effective treatments have been elusive.

Methods: We performed whole-genome linkage analysis followed by high-throughput sequencing of the positive-linkage area in a family with autosomal recessive focal segmental glomerulosclerosis (index family) and sequenced a newly discovered gene in 52 unrelated patients with focal segmental glomerulosclerosis. Immunohistochemical studies were performed on human kidney-biopsy specimens and cultured podocytes. Expression studies in vitro were performed to characterize the functional consequences of the mutations identified.

Results: We identified two mutations (A159P and Y695X) in MYO1E, which encodes a nonmuscle class I myosin, myosin 1E (Myo1E). The mutations in MYO1E segregated with focal segmental glomerulosclerosis in two independent pedigrees (the index family and Family 2). Patients were homozygous for the mutations and did not have a response to glucocorticoid therapy. Electron microscopy showed thickening and disorganization of the glomerular basement membrane. Normal expression of Myo1E was documented in control human kidney-biopsy specimens in vivo and in glomerular podocytes in vitro. Transfection studies revealed abnormal subcellular localization and function of the A159P-Myo1E mutant. The Y695X mutation causes loss of calmodulin binding and of the tail domains of Myo1E.

Conclusions: MYO1E mutations are associated with childhood-onset, glucocorticoid-resistant focal segmental glomerulosclerosis. Our data provide evidence of a role of Myo1E in podocyte function and the consequent integrity of the glomerular filtration barrier.

Figure 1

Panels A–B. Pedigrees of the families with MYO1E mutations. Panel A shows the pedigree of the index family and Panel B, the IST family. The probands are indicated with an arrow, plus indicates the wild-type allele; A159P and Y695X indicate the c.475G>C and the c.2085T>G mutations, in the transcript of MYO1E, corresponding to the amino acid changes Ala159Pro and Tyr695Stop, respectively. Squares indicate male family members, and circles, female; slashes indicate deceased persons. Solid symbols: persons with FSGS. The double horizontal bars indicate consanguinity. The parents of IST012 are from the same village. IST012 was homozygous in all SNPs in MYO1E (Supplementary Table 5) indicating consanguinity. Identification codes and ages at last observation (or at death) are shown when available. na: DNA not available. *Died at 13 years of age due to meningitides; °perinatal sudden death. Panels C–F. Ultrastructural changes of the glomerular capillary wall in patient IST012 with the homozygous Y695X Myo1E mutation (C and E) and in myo1e-knockout mice (D: 2 months, F: 4 months old). Both in the patient and in mice, the glomerular basement membrane (BM) is thickened and disorganized with multiple electron lucent areas and multilamination. Intramembranous granular material is seen in lucent areas between laminae. Foot processes are effaced. Intercellular contacts focally show increased density of plasma membranes (panel E). Podocytes show microvillous transformation (original magnification, C and D, ×12,000, scale bars are shown).

Figure 2. Identification of MYO1E mutations in FSGS

Panel A. Parametric multipoint LOD score profile across the human genome, calculated in the index family. Note the single peak of positivity on human chromosome 15. Panel B (Left). Sequence electropherograms of the index family. The affected child has a homozygous G>C substitution (arrow), while the unaffected father is heterozygous for the mutation. Panel B (Right). Sequence electropherograms of the IST family. The affected child has a homozygous T>G substitution (arrow), while the unaffected mother is heterozygous for the mutation. The bottom electropherograms are from healthy controls. Panel C. Functional domains in myosin 1E (Myo1E) protein. Myo1E contains a motor head domain (that includes a P-loop, a Switch-1 and a Switch-2 domains) that binds ATP and F-actin (Actin b.s.), a light-chain binding neck domain (IQ) that binds calmodulin and a tail, containing lipid binding tail homology-1 (TH1), proline-rich tail homology-2 (TH2) and Src homology-3 (SH3) domains. The Ala159Pro change is within the Switch-1 region. The Y695X causes a truncation at the beginning of the neck domain. Panel D. Sequence alignment of the Myo1E protein and homologues among various species. The red frame indicates the location of the highly conserved Ala159 amino acid that in the index family is changed to Pro (P). Sequences were from the National Center for Biotechnology Information.

Figure 3. Endogenous Myo1E Expression

Panels A–C. Immunofluorescence staining of normal human kidney with DAPI (blue) for cell nuclei and antibodies against myosin 1E (Myo1E, red, panel A) and the podocyte marker synaptopodin (Synpo, green, panel B) or the endothelial cell marker VE-cadherin (VE-Cadh, green, panel C). Myo1E staining is observed in the glomerulus. The yellow color shows the merge of the Myo1E and the synaptopodin stainings in the podocytes. Panels D–F. Staining of endogenous Myo1E in cultured undifferentiated (panel D), terminally differentiated non confluent (14 day culture at 37 °C, panel E) and confluent human podocytes (panel F). The Myo1E staining (green) shows predominant localization along the plasma membrane, mainly on cell lamellipodia both in undifferentiated and in fully differentiated podocytes and fainter staining in confluent podocytes. The white arrows show areas of colocalization between Myo1E and the F-actin tips (stained by rhodamine-phalloidin, red) (merge yellow). Panel G. Partial co-staining (yellow, white arrows) of endogenous Myo1E (red) and CD2AP (green) in cultured differentiated podocytes. Original magnification 400×. Panels H–I. Representative pattern of immunogold labelling for Myo1E in podocytes of control human glomeruli. Gold particles (arrows) are localized on the cytoplasmic side of the podocyte plasma membrane. Original magnification, ×44,000 (scale bars are shown).

Figure 4. Expression of recombinant Myo1E in cultured podocytes

Panels A–D. Undifferentiated human podocytes transfected with wild-type, E753K (control SNP, rs8024923) or mutant A159P GFP-tagged Myo1E (green, 1 µg plasmid) and analyzed by confocal microscopy, the nuclei were stained with DAPI (blue). Panel A. Untransfected human podocytes. Panels B–D. Human podocytes transfected with wild-type Myo1E (panel B), control E753 Myo1E (panel C), or mutant A159P Myo1E (panel D). Wild-type and control E753K Myo1E localize along the plasma membrane while the A159P mutant shows a cytoplasmic distribution. Tranfection efficiency was about 30% for the wild-type, E753K and A159P-GFP-tagged constructs (by calculating the ratio between the numbers of green cells and of blue nuclei x100 in 10–15 high power fields). Panels E–F. Staining of podocyte plasma membranes with rhodamine-labeled lectin (red). Wild-type Myo1E-GFP (panel E) partially colocalizes with lectin (yellow) while the A159P mutant does not (panel F, green). The mislocalization of the mutant protein is not attributable to instability or degradation as the sizes and the amounts of the wild-type and A159P-tagged proteins expressed in transfected podocytes were the same in Western blot (Supplementary Fig. 13A–B). Panels G–H. Staining of F-actin filaments (red) in transfected human podocytes. Wild-type Myo1E-GFP (panel G) partially colocalizes with F-actin (yellow) while the A159P mutant does not (panel H, green). Panels I–L, staining with an anti-CD2AP antibody (green). Wild-type Myo1E-MYC (panel I, red) partially colocalizes with CD2AP at the plasma membrane (yellow) while in cells transfected with the A159P mutant (panel L), Myo1E-MYC and CD2AP abnormally co-localize in the cytoplasm (yellow). Original magnification, 400×. Panel M. Scrape-wound migration assay of cultured human podocytes. Transfection with wild-type Myo1E-GFP (WT) increases podocyte migration vs. podocytes transfected with empty-GFP (Empty), while the A159P Myo1E-GFP (A159P) has no effect on migration. Knockdown of endogenous Myo1E by siRNA (Myo1E siRNA) reduced podocyte migration vs. podocytes transfected with universal scrambled siRNA (Scrambled siRNA). The numbers of podocytes that migrated into the wound track, 6 h after the podocyte layer was scraped, are shown (podocyte/field, one field=71500 µm²). Results are mean ±SE of 3 wells (n=5 fields counted/well). Images of representative fields are shown (the dashed lines indicate the left margin of the scrape-wound, the white arrows highlight migrating cells). *P<0.05 wild-type vs. empty-GFP and A159P (by one-way analysis of variance), °P<0.05 vs Scrambled siRNA (Student’s t-test).