PMC3976684_01
Fundus changes in the proband with the heterozygous c.1264G>A (p.A422T) mutation in Family 1. A and B: The fundus of Patient 1 showed a retinal fold of the right eye and dragged disc of the left eye. C-F: Fundus photos of the asymptomatic father. C and D: Fundus photos of the asymptomatic father showed normal posterior fundi. E and F: Fundus fluorescein angiography (FFA) showed that the father had nonperfusion areas in both eyes.
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The patient has been followed up for 2 years and the retinal folds remained stable (Figure 1A, B). His father had a good vision of 20/20 in both eyes, while FFA showed nonperfusion areas of both eyes (Figure 1C-F).
Fundus changes in the proband with the heterozygous c.1619T>C (p.L540P) mutation in Family 2. A and B: RetCam examination of both of Patient 2's eyes. The fundus of the left eye could not be viewed due to the opacity of the lens. C and D: Ultrasound B- scan of Patient 2. These photos demonstrate leukocoria and severe retinal detachment of both eyes. The left eye is more severely affected than the right eye. Posterior synechia is evident in the left eye. E: Fundus of Patient 2's right eye at the last follow-up after lensectomy and vitrectomy. The retina was mostly attached. F-I: Fundus photos and fundus fluorescein angiography (FFA) of the asymptomatic mother with c.1619T>C in Family 2. F and G: Fundus photos show normal posterior fundi. H and I: FFA shows that the mother has peripheral nonperfusion areas, increased ramification, and shunts of the peripheral retinal vessels in both eyes.
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However, the patient was capable of walking without aid at the last follow-up (Figure 2A-E). The mother had normal eyesight, while FFA showed nonperfusion areas, increased ramification, and shunts of the peripheral retinal vessels in both eyes (Figure 2F-I).
Pedigrees of families with autosomal dominant familial exudative vitreoretinopathy (FEVR) and the mutations identified in LRP5. A: In Family 1, Patient 1 had two LRP5 mutations, c.1264G>A (p.A422T) from the affected father and c.2447A>C (p.Q816P) from unaffected mother. B: In Family 2, Patient 2 also had two LRP5 mutations; c.1619T>C (p.L540P) was from the affected mother, but c.2555C>T (p.T852M) was not detected in either parent. The columns from left to right display the pedigree and the sequence chromatograms for these patients. Squares, male; circles, female; closed, affected; open, unaffected.
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DNA sequence analysis identified two novel mutations in LRP5, including c.1264G>A (p.A422T) in Patient 1 and his affected father and c.1619T>C (p.L540P) in Patient 2 and his affected mother (Figure 3). Moreover, beyond p.L540P, Patient 2 had an additional LRP5 sequence change (c.2555C>T, p.T852M); this was not detected in either parent, suggesting that it was a new mutation in Patient 2 (Figure 3).
Protein alignment of 10 (LRP5) orthologs demonstrates conservation in the regions with mutations. The 10 orthologs are from the following 10 species: Homo sapiens (NP_002326.2), Pan troglodytes (XP_508605.3), Canis lupus familiaris (XP_003432463.1), Bos taurus (XP_002699451.1), Macaca mulata [M. musculus] (NP_032539.2), Rattus norvegicus (NP_001099791.2), Gallus gallus (NP_001012915.1), Danio rerio (NP_001170929.1), Drosophila melanogaster (NP_524737.2), and Anopheles gambiae (XP_320740.4). The regions with the four missense mutations are highly conserved. A422T stands for Ala422Thr, L540P stands for Leu540Pro, Q816P stands for Gln816Pro, and T852M stands for Thr852Met.
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The two mutations involve a highly evolutionarily conserved residue (Figure 4). The two sequence changes also involved evolutionarily conserved residues (Figure 4).
Failure of mutant LRP5 in activation of the Norrin/beta-catenin pathway. SuperTopFlash (STF) cells cotransfected with LRP5-pRK5 and FZD4 constructs were treated with Norrin and assayed for luciferase reporter activity. Each assay was performed in triplicate at the same time and repeated three times. The results are an average of three measurements. Both of the novel LRP5 mutants failed to induce the luciferase reporter activity in STF cells in response to Norrin (87% reduction for p.A422T, 97% reduction for p.L540P). The additional LRP5 mutation in Patient 1 (p.Q816P) from the unaffected mother had similar luciferase reporter activity compared with the wild type, while the second mutation of Patient 2 (p.T852M), which was not detected in either parent, exhibited a 94.9% reduction. The luciferase intensities of the two combinations (p.A422T and p.Q816P, p.L540P and p.T852M) decreased significantly compared to the wild type, but no significant differences were observed compared with the mere mutation (p.A422T or p.L540P; p>0.05). The mutant p.R570Q (positive control) exhibited a 96% reduction of its wild-type activity, which was similar to the results of a previous report. Asterisks indicate significant differences between the mutant and wild type as judged by the Student two-tailed t test (p<0.05).
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It lost 96% of its wild-type activity, which was similar to the results of a previous report (Figure 5).