瘢痕疙瘩CAMTA1基因G65551片段突变的研究

Study of mutation in G65551 fragment of CAMTA1 genes in keloid

目的利用变性高效液相色谱法（DHPLC）结合DNA直接测序法，筛选和鉴定瘢痕疙瘩CAMTAI基因G65551片段突变情况。方法PCR扩增13例瘢痕疙瘩患者的瘢痕组织和血液标本1p36区域CAMTA1基因G65551片段。采用DHPLC技术，对扩增片断进行基因变异检测，随机选取不同类型PCR片断，进行全序列测定并对照分析。结果DHPLC筛查显示，在扩增片段中出现单个色谱峰，提示为纯合链；双峰则表示是杂合异源双链。瘢痕疙瘩组双峰出现率92．3％（12／13）；对照组为38．5％（5／13）；DNA测序发现2个突变位点，其中第54位碱基G／T的颠换突变率：瘢痕疙瘩组为69．2％（9／13），对照组为38．5％（5／13），第412位碱基T的缺失突变率：瘢痕疙瘩组为92．3％（12／13），对照组为15．4％（2／13）。经酽检验（确切概率法），第54位点突变差异无统计学意义（P〉0．05）；第412位点突变差异有统计学意义（P〈0．05）。结论DHPLC结合DNA直接测序是一种高效、经济、简便、可靠的筛选碱基位点突变的方法；CAMTA1基因G65551片段突变与瘢痕疙瘩的发生有关，CAMTA1基因可能是瘢痕抑制基因。

Objective To study the somatic mutation in G65551 fragment of CAMTA1 genes at lp36 among the Chinese patients with keloid by denaturing high-performance liquid chromatography （DHPLC） and DNA sequencing. Methods The fragment of CAMTAI genes at lp36 isolated from the keloid tissue and blood samples from 13 patients were amplified by Polymerase Chain Reaction （PCR）, and the products were analyzed by DH- PLC. Some samples of interests were sequenced and compared with the sequences between keloid tissue and blood sample of the same patient. Results By DHPLC, the results of the majority of the blood samples showed single chromatographic peak indicating homoduplexes, meanwhile the results of keloid tissue samples showed double peak indicating heteroduplexes. Through sequencing analysis, 2 somatic mutation sites were identified in according with DHPLC. They were respectively base G/T in 54th place and base T deletion in 412th place. The base G/T rate was 69.2% （9/13）, while the control group＇s mutation rate was 38.5% （5/13）. The base T deletion rate was 92.3% （12/13）; while the control group＇s mutation rate was 15.4% （2/13）. Significance difference was identified in the 412th sites. Conclusion DHPLC combined with DNA sequencing is an effective, economical, and simple method with reliability for the screening somatic mutations of a large number of samples. There was a strong correlation between the CAMTA1 gene mutation and Keloid. CAMTAlgene could be possibly a scar suppressor gene （SSG）.