PMC3742126_01
Pedigree of the Chinese family with ICN. The squares and the circles represent males and females, respectively. The index patient is marked with an arrow. The black-filled symbols indicate patients with idiopathic congenital nystagmus, the dotted circles represent female carriers, and a diagonal line symbol indicates a deceased family member.
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The family pedigree is shown in Figure 1.
DNA sequence chromatograms of the FRMD7 gene for affected and unaffected family members. The affected and unaffected family members have been shown in Table 1. Affected family members refers to II:1, III:3, III:5, IV:3, IV:4, IV:6 and IV:7. Unaffected family members refers to III:1, III:2, III:4, III:6, III:7, III:8, IV:1, IV:2, and IV:5.
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Obligate female carriers were heterozygous in these mutations and the affected males were homozygous, consistent with XL- inheritance (Figure 2).
Novel mutation of FRMD7 downregulates the activation of Rac1 signaling HA-tagged human Rac1 was co-transfected into HEK293T cells with Flag-tagged wild-type (Wild) or mutant-type (c.635T>C) FRMD7. The supernatant of cell lysates was incubated with GST-PAK2 protein immobilized on glutathione-sepharose beads, where bound GTP-Rac1 proteins were detected by Western blotting using anti-HA monoclonal antibody. The amount of input HA-Rac1 and Flag-FRMD7 detected by anti-HA or anti-Flag monoclonal antibody. Extracts of HEK293T cells transfected with wild-type FRMD7 could detect the PAK2 precipitation GTP-Rac1 band, however mutant-type FRMD7 contained decreased amounts of GTP-Rac1 compared with the wild-type (A). (Wild: wild-type FRMD7+Rac1; C635: mutant-type (c.635T>C) FRMD7+Rac1; Control: empty vector+Rac1). The experiments were repeated five times, and the graphs represent the average of five independent experiments (B) (Columns, mean; bars, SEM; *p<0.05).
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The quantity of activated Rac1 induced by this novel missense mutant FRMD7 (c.635T>C) protein was obviously decreased compared with WT (p<0.05; Figure 3).