FRMD7 Mutations Disrupt the Interaction with GABRA2 and May Result in Infantile Nystagmus Syndrome

Abstract

Purpose: To identify the pathogenic gene of infantile nystagmus syndrome (INS) in three Chinese families and explore the potential pathogenic mechanism of FERM domain-containing 7 (FRMD7) mutations.

Methods: Genetic testing was performed via Sanger sequencing. Western blotting was used to analyze protein expression of FRMD7. Glutathione S-transferase pull-down and immunoprecipitation were conducted to investigate the proteins interacting with FRMD7. Rescue assays were performed in Caenorhabditis elegans to explore the potential role of FRMD7 in vivo.

Results: We recruited three Chinese families with X-linked INS and identified a duplication and two missense mutations in FRMD7: c.998dupA/p.His333Glnfs*2, c.580G>A/p.Ala194Thr, and c.973A>G/p.Arg325Gly (one in each family). Expression levels of three mutants were similar to that of wild-type FRMD7 in vitro. Interestingly, the mutant p.His333Glnfs*2 exhibited a predominantly nuclear location, whereas wild-type FRMD7 localized to the cytoplasm. In addition, we found FRMD7 to directly interact with the loop between transmembrane domains 3 and 4 of GABRA2, a type A gamma-aminobutyric acid (GABA) receptor (GABAARs) subunit critical for receptor transport and localization, whereas the mutants p.Ala194Thr and p.Arg325Gly exhibited decreased binding to GABRA2. In frm-3 (a nematode homologue of FRMD7) null C. elegans, we found that FRMD7 mutants exhibited a poor rescue effect on the defects of locomotion and fluorescence recovery after photobleaching of GABAARs.

Conclusions: Our findings identified three FRMD7 mutants in three Chinese families with X-linked INS and confirmed GABRA2 as a novel binding partner of FRMD7. These findings suggest that FRMD7 plays an important role by targeting GABAARs.

Figure 1.

Pedigree analysis and molecular genetic analysis of three Chinese families with INS. (A) Pedigree structure of family 1. A square represents a male individual, a circle represents a female individual, an unfilled symbol represents a normal phenotype, a filled symbol represents an affected phenotype, a symbol containing a dot represents a carrier, a symbol containing a question mark represents an uncertain genotype, a symbol with a slash represents a deceased individual. The proband is marked with an arrow. (B) Pedigree structure of family 2. (C) Pedigree structure of family 3. (D) A novel mutation, c.998dupA, of the FRMD7 gene was identified in family 1. The arrow indicates the nucleotide where the mutation occurs. (E) The novel mutation c.580G>A in the FRMD7 gene was identified in family 2. The arrow indicates the position where the mutation occurs. (F) A mutation c.973A>G of the FRMD7 gene was identified in family 3. The arrow indicates the nucleotide where the mutation occurs. (G) The mutated residues p.Ala194 and p.Arg325 are evolutionarily conserved from humans (Homo sapiens) to nematodes (C. elegans).

Figure 2.

Expression and localization of wild-type and three mutant FRMD7. (A) Exogenous (total, cytosol, nucleus) protein expression of GFP-tagged wild-type FRMD7 and three mutants in COS7 cells was assessed by Western blotting. Original films are shown in Supplementary Figure S5. (B) Quantification of the ratio of GFP-FRMD7/β-actin and GFP-FRMD7/FBL was detected by Western blotting and normalized to the value obtained from GFP-FRMD7. The data are presented as the mean ± SEM (***P < 0.001, ns = nonsignificant) using 1-way ANOVA with Bonferroni post hoc comparisons. (C) Localization of wild-type and mutant FRMD7 in N2A cells. Representative images demonstrate that the GFP-tagged wild-type FRMD7 (green), as well as the mutants p.Ala194Thr and p.Arg325Gly, are primarily located in the cytoplasm; the p.His333Glnfs*2 mutant is mainly located in the cell nucleus (Scale bar, 20 µm). FBL, fibrillarin; WT, wild-type.

Figure 3.

FRMD7 binds to the loop between TM3 and TM4 of GABRA2. (A, B) Interaction between GFP-FRMD7 (expressed in COS7 cells) and full-length GABRA2 (extracted from mouse retina) was verified through IP assays. Original films are shown in Supplementary Figures S6 and S7. (C) Direct interaction between FRMD7 and the TM3-TM4 loop of GABRA2 was detected by GST pull-down assays. Original films are shown in Supplementary Figure S8. (D) Colocalization of FRMD7 and GABRA2 in the IPL and INL of the mouse retina is demonstrated by immunofluorescent staining and confocal imaging. ONL is the outer nuclear layer, OPL is the outer plexiform layer, INL is the inner nuclear layer, IPL is the inner plexiform layer, and GCL is the ganglion cell layer (Scale bar, 20 µm). (E) Confocal images showing INL and IPL (Scale bar, 20 µm).

Figure 4.

FRMD7 mutations disrupt interaction with GABRA2. (A) Colocalization of GABRA2 and wild-type FRMD7, but not the mutant p.His333Glnfs*2, is observed in COS7 cells (Scale bar, 20 µm). (B) The mutations p.Ala194Thr and p.Arg325Gly decrease the binding affinity of FRMD7 to the TM3-TM4 loop of GABRA2. Original films are shown in Supplementary Figure S9. (C) Quantification of the binding affinity of FRMD7 bound to GST-GABRA2-LOOP, which is normalized to the value obtained from GFP-FRMD7. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01) using 1-way ANOVA with Bonferroni post hoc comparisons. WT, wild-type.

Figure 5.

FRMD7 mutations impair rescue of the locomotion defect in frm-3 null C. elegans. (A) Locomotion tracking of frm-3 null and transgenic worms on NGM plates for 1 minute. Traces of worm locomotion in 1 minute are outlined in red. The start and end represent the start and end positions of individual worms in the recording, respectively (Scale bar, 1 mm). (B) Schematic structure of GABRA2, UNC-49B, and the artificial protein UNC-49B-A2loop. The TM3-TM4 loop of GABRA2 (scarlet) was inserted into the upstream of the TM3-TM4 loop (blue) of UNC-49B, forming an artificial cytoplasmic loop. (C, D) Quantification of the thrashing and locomotion rates of worms with different genotypes, respectively. Data are the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ns = nonsignificant) using 1-way ANOVA with Bonferroni post hoc comparisons. WT, wild-type.

Figure 6.

FRMD7 mutants show decreased ability to rescue the FRAP defect of GABA_ARs in frm-3 null C. elegans. (A) Both FRMD7-GFP (green) and UNC-49B-A2loop-RFP (red) are expressed in GABAergic neuromuscular junction. Colocalization of fluorescence puncta was chosen for FRAP experiments. (B) Representative pictures of UNC-49B-A2loop-RFP FRAP. (C) Quantitative data for fluorescence recovery of UNC-49B-A2loop-RFP at 300 seconds after photobleaching. These data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ns = nonsignificant) using 1-way ANOVA with Bonferroni post hoc comparisons. (D) Representative scatter plots of fluorescence recovery. WT, wild-type.