Cowan Jason, Li Duanxiang, Gonzalez-Quintana Jorge, Morales Ana, Hershberger Ray E
Cardiovascular Division, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
Circ Cardiovasc Genet. 2010 Feb;3(1):6-14. doi: 10.1161/CIRCGENETICS.109.905422. Epub 2009 Nov 17.
Mutations in the LMNA gene, encoding lamins A/C, represent a significant cause of dilated cardiomyopathy. We recently identified 18 protein-altering LMNA variants in a cohort of 324 unrelated patients with dilated cardiomyopathy. However, at least one family member with dilated cardiomyopathy in each of 6 pedigrees lacked the LMNA mutation (nonsegregation), whereas small sizes of 5 additional families precluded definitive determinations of segregation, raising questions regarding contributions by those variants to disease.
We have consequently expressed, in COS7 cells, GFP-prelamin A (GFPLaA) fusion constructs incorporating the 6 variants in pedigrees with nonsegregation (R101P, A318T, R388H, R399C, S437Hfsx1, and R654X), the 4 variants in pedigrees with unknown segregation (R89L, R166P [in 2 families], I210S, R471H), and 3 additional missense variants (R190Q, E203K, and L215P) that segregated with disease. Confocal immunofluorescence microscopy was used to characterize GFP-lamin A localization and nuclear morphology. Abnormal phenotypes were observed for 10 of 13 (77%) variants (R89L, R101P, R166P, R190Q, E203K, I210S, L215P, R388H, S437Hfsx1, and R654X), including 4 of 6 showing nonsegregation and 3 of 4 with uncertain segregation. All 7 variants affecting coil 1B and the lamin A-only mutation, R654X, exhibited membrane-bound GFP-lamin A aggregates and nuclear shape abnormalities. Unexpectedly, R388H largely restricted GFP-lamin A to the cytoplasm. Equally unexpected were unique streaked aggregates with S437Hfsx1 and giant aggregates with both S437Hfsx1 and R654X.
This work expands the recognized spectrum of lamin A localization abnormalities in dilated cardiomyopathy. It also provides evidence supporting pathogenicity of 10 of 13 tested LMNA variants, including some with uncertain or nonsegregation.
编码核纤层蛋白A/C的LMNA基因突变是扩张型心肌病的一个重要病因。我们最近在324名无亲缘关系的扩张型心肌病患者队列中鉴定出18种导致蛋白改变的LMNA变异体。然而，6个家系中每个家系至少有一名扩张型心肌病家庭成员未携带LMNA突变（不分离），而另外5个家系规模较小，无法明确确定分离情况，这引发了关于这些变异体对疾病贡献的疑问。
因此，我们在COS7细胞中表达了绿色荧光蛋白-前体核纤层蛋白A（GFP-LaA）融合构建体，其中包含6个在不分离家系中的变异体（R101P、A318T、R388H、R399C、S437Hfsx1和R654X）、4个分离情况未知家系中的变异体（R89L、R166P[在2个家系中]、I210S、R471H）以及另外3个与疾病共分离的错义变异体（R190Q、E203K和L215P）。共聚焦免疫荧光显微镜用于表征GFP-核纤层蛋白A的定位和核形态。在13个变异体中的10个（77%）（R89L、R101P、R166P、R190Q、E203K、I210S、L215P、R388H、S437Hfsx1和R654X）中观察到异常表型，包括6个显示不分离的变异体中的4个以及4个分离情况不确定的变异体中的3个。所有7个影响1B卷曲螺旋区的变异体和仅影响核纤层蛋白A的突变R654X均表现出膜结合的GFP-核纤层蛋白A聚集物和核形状异常。出乎意料的是，R388H主要将GFP-核纤层蛋白A限制在细胞质中。同样出乎意料的是，S437Hfsx1出现独特的条纹状聚集物，S437Hfsx1和R654X同时出现巨大聚集物。
这项工作扩展了扩张型心肌病中已认识到的核纤层蛋白A定位异常的范围。它还提供了证据支持所测试的13个LMNA变异体中的10个具有致病性，包括一些分离情况不确定或不分离的变异体。
