Mohiddin Saidi A, Lu Shajia, Cardoso John-Paul, Carroll Stefanie, Jha Sanjaya, Horowits Robert, Fananapazir Lameh
National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Cell Motil Cytoskeleton. 2003 Jul;55(3):200-12. doi: 10.1002/cm.10123.
Linkage analysis identifies 10q24-26 as a disease locus for dilated cardiomyopathy (DCM), a region including the N-RAP gene. N-RAP is a nebulin-like LIM protein that may mediate force transmission and myofibril assembly in cardiomyocytes. We describe the sequence, genomic structure, and expression of human N-RAP, as well as an initial screen to determine whether N-RAP mutations cause cardiomyopathy. Human expressed sequence tag databases were searched with the published 3,528-bp mouse N-RAP open reading frame (ORF). Putative cDNA sequences were interrogated by direct sequencing from cardiac and skeletal muscle RNA. We identified two human N-RAP isoforms with ORFs of 5,085 bp (isoform C) and 5,190 bp (isoform S), encoding products of 193-197 kDa. Genomic database searches localize N-RAP to human chromosome 10q25.3 and match isoforms C and S to 41 and 42 exons. Only isoform C is detected in human cardiac RNA; in skeletal muscle, approximately 10% is isoform C and approximately 90% is isoform S. We investigated apparent differences between human N-RAP cDNA and mouse sequences. Two mouse N-RAP isoforms with ORFs of 5,079 and 5,184 bp were identified with approximately 85% similarity to human isoforms; published mouse sequences include cloning artifacts truncating the ORF. Murine and human isoforms have similar gene structure, tissue specificity, and size. N-RAP is especially conserved within its nebulin-like and LIM domains. We expressed both N-RAP isoforms and the previously described truncated N-RAP in embryonic chick cardiomyocytes. All constructs targeted to myofibril precursors and the cell periphery, and inhibited myofibril assembly. Several human N-RAP polymorphisms were detected, but none were unique to cardiomyopathy patients. N-RAP is highly conserved and exclusively expressed in cardiac and skeletal muscle. Genetic abnormalities remain excellent candidate causes for cardiac and skeletal myopathies.
连锁分析确定10q24 - 26为扩张型心肌病（DCM）的一个疾病位点，该区域包含N - RAP基因。N - RAP是一种类伴肌动蛋白的LIM蛋白，可能介导心肌细胞中的力传递和肌原纤维组装。我们描述了人类N - RAP的序列、基因组结构和表达情况，以及用于确定N - RAP突变是否导致心肌病的初步筛查。用已发表的3528 bp小鼠N - RAP开放阅读框（ORF）搜索人类表达序列标签数据库。通过对心脏和骨骼肌RNA进行直接测序来分析推定的cDNA序列。我们鉴定出两种人类N - RAP异构体，其ORF分别为5085 bp（异构体C）和5190 bp（异构体S），编码产物的分子量为193 - 197 kDa。基因组数据库搜索将N - RAP定位到人类染色体10q25.3，并将异构体C和S分别与41和42个外显子匹配。在人类心脏RNA中仅检测到异构体C；在骨骼肌中，约10%是异构体C，约90%是异构体S。我们研究了人类N - RAP cDNA与小鼠序列之间的明显差异。鉴定出两种小鼠N - RAP异构体，其ORF分别为5079和5184 bp，与人类异构体的相似性约为85%；已发表的小鼠序列包括截断ORF的克隆假象。小鼠和人类异构体具有相似的基因结构、组织特异性和大小。N - RAP在其类伴肌动蛋白和LIM结构域内特别保守。我们在胚胎鸡心肌细胞中表达了两种N - RAP异构体以及先前描述的截短型N - RAP。所有构建体均靶向肌原纤维前体和细胞周边，并抑制肌原纤维组装。检测到几种人类N - RAP多态性，但没有一种是心肌病患者特有的。N - RAP高度保守，且仅在心脏和骨骼肌中表达。遗传异常仍然是心脏和骨骼肌疾病的极佳候选病因。
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