1种用于分析疾病相关ABO抗原丢失的染色体杂合性缺失方法

Analysis of ABO antigen variants related to hematological diseases using chromosomal loss of heterozygosity technique

目的建立1种9号染色体杂合性缺失(LOH)的分析方法,以解释因ABO基因附近染色体的LOH导致的疾病相关ABO抗原丢失现象。方法在9号染色体长臂和短臂上共选取13个微卫星位点,其中10个位点(D9S1677、D9S289、D9S1776、D9S1682、D9S290、D9S164、D9S1818、D9S1826、D9S158、D9S1838)位于ABO血型基因近端,平均间距5.52 cM;另3个位点(D9S1858、D9S171、D9S1843)位于ABO基因远端,平均间距39.3 cM。分别设计合成FAM和HEX荧光标记引物,优化PCR扩增后,以聚丙烯酰胺凝胶毛细管电泳,通过GeneMapper软件分析微卫星不稳定性,观察染色体LOH发生情况。结果 13个微卫星位点均得到了较好的PCR扩增效果,PCR产物长度在75～273 bp之间,各位点均有较高的杂合度,适合于微卫星片段长度分析。13个位点可在2个泳道中进行双荧光同步分析,各位点不同等位基因间互不干扰。分别对1例正常血型标本和2例血液病相关的ABO抗原变异标本(包括口腔黏膜DNA和外周血DNA)进行微卫星分析,正常血型标本的口腔DNA和外周血DNA未发现染色体LOH现象。而2例ABO抗原变异标本,与口腔黏膜DNA相比,外周血DNA均存在不同程度的LOH现象,表明ABO抗原丢失可能源于血液系统疾病相关的9号染色体LOH现象。结论建立了1种基于微卫星不稳定分析的9号染色体LOH方法,可用于血液系统疾病相关的ABO抗原丢失标本分子机制的研究。

Objective To establish a method of chromosomal loss of heterozygosity to explain the phenomenon of ABO antigen loss related to hematological diseases. Methods A total of 13 microsatellite loci were selected on the long and short arm of chromosome 9, in which 10 loci （ D9S1677, D9S289, D9S1776, D9S1682, D9S290, D9S164, D9S1818, Dgs1826,D9S158 and D9S1838） were near to the ABO gene and 3 loci （DgS1858,D9S171 and D9S1843） were far from the ABO gene,with interval of 5.52 and 39.3 centimorgan,respectively. The polymerase chain reaction amplification was performed using FAM or HEX fluorescence-labeled primers. The loss of heterozygosity on chromosome 9 was analyzed using the GeneMapper software after the capillary polyacrylamide gel electrophoresis. Results The amplifications were success- ful in all 13 loci, and the alleles had not interfered each other in 2 electrophoresis lines. The DNA from peripheral blood of 2 samples with hematological disease has loss of heterozygosity in some loci compared with the DNA from oral swabs, using a normal blood group sample as control. It suggests that the loss of ABO antigens maybe derive from the loss of heterozygosi- ty of chromosome 9 related to hematological diseases. Conclusion A method was established to analyze loss of heterozygos- ity of chromosome 9 based on microsatellite instability, and can be used to study the molecular mechanism of ABO antigen variants related to hematological diseases.