Genetic Medicine, Manchester Academic Health Science Centre, Central Manchester University Hospitals NHS Foundation Trust, St. Mary's Hospital, Manchester, United Kingdom.
Invest Ophthalmol Vis Sci. 2010 May;51(5):2356-62. doi: 10.1167/iovs.09-4620. Epub 2009 Dec 10.
AMD has a complex etiology with environmental and genetic risk factors. Ten fibulin 5 sequence variants have been associated with AMD and two other fibulin 5 mutations cause autosomal-recessive cutis laxa. Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Biophysical techniques were used to detect structural changes in the fibulin 5 mutants and to determine whether changes are predictive of pathogenicity.
Native PAGE, nonreduced SDS-PAGE, size-exclusion column multiangle laser light scattering, sedimentation velocity, and circular dichroism (CD) were used to investigate the mobility, hydrodynamic radii, folding, and oligomeric states of the fibulin 5 mutants in the absence and presence of Ca(2+).
CD showed that all mutants are folded, although perturbations to secondary structure contents were detected. Both cutis laxa mutants increased dimerization. Most other mutants slightly increased self-association in the absence of Ca(2+) but this was also demonstrated by G202R, a polymorphism detected in a control individual. The AMD-associated mutant G412E showed lower-than-expected mobility during native-PAGE, the largest hydrodynamic radius for the monomer form and the highest levels of aggregation in both the absence and presence of Ca(2+).
The results identified structural differences for the disease-causing cutis laxa mutants and for one AMD variant (G412E), suggesting that this may also be pathogenic. Although the other AMD-associated mutants showed no gross structural differences, they cannot be excluded as pathogenic by differences outside the scope of this study-for example, disruption of heterointeractions.
AMD 具有复杂的病因，包括环境和遗传风险因素。已经发现 10 个纤维连接蛋白 5 序列变异与 AMD 有关，另外两个纤维连接蛋白 5 突变导致常染色体隐性皮肤松弛症。纤维连接蛋白 5 是一种 52kDa 的钙结合表皮生长因子（cbEGF）丰富的细胞外基质蛋白，对于弹性组织的形成至关重要。使用生物物理技术来检测纤维连接蛋白 5 突变体的结构变化，并确定这些变化是否具有致病性。
使用天然 PAGE、非还原 SDS-PAGE、尺寸排阻柱多角度激光光散射、沉降速度和圆二色性（CD）来研究纤维连接蛋白 5 突变体在没有和存在 Ca2+的情况下的迁移率、流体力学半径、折叠和寡聚状态。
CD 表明所有突变体都是折叠的，尽管检测到二级结构含量的扰动。两个皮肤松弛症突变体都增加了二聚体形成。大多数其他突变体在没有 Ca2+的情况下略微增加了自身缔合，但这也被检测到在对照个体中存在的 G202R 多态性所证明。与 AMD 相关的突变体 G412E 在天然 PAGE 中表现出低于预期的迁移率、单体形式的最大流体力学半径和在没有和存在 Ca2+的情况下的最高聚集水平。
结果确定了致病皮肤松弛症突变体和一个 AMD 变体（G412E）的结构差异，表明这也可能具有致病性。尽管其他与 AMD 相关的突变体没有显示出明显的结构差异，但由于本研究范围之外的差异，例如异源相互作用的破坏，不能排除它们具有致病性。
