Sasaki Y, Ueno I, Nakakoshi R, Ishikawa S, Hidaka E, Furuwatari C, Furihata K, Katsuyama T, Okumura N
Central Clinical Laboratories, Shinshu University Hospital, Matsumoto.
Rinsho Byori. 1997 Dec;45(12):1163-6.
Abnormal hemoglobin is one of the most frequent genetic disorder, of which about 700 variants have been reported in the world. Some abnormal hemoglobins such as HbS and HbC are endemic among limited human populations. We have found five subjects with abnormal hemoglobin in routine hematology analysis using an automated system Sysmex NE-7000. We isolated genomic DNA from the peripheral blood leukocyte of each subject, and amplified a 558 bp fragment of DNA including the entire exons 1 and 2 as well as intron 1 of the beta-globin gene. The amplified DNA fragments were subjected to direct sequencing by the dideoxy termination method on an automated DNA sequencer. The sequence analysis showed the abnormalities including HbA/C, HbS/C, HbE/E, and HbA/G-Szuhu. In the present study, we report a simple method for the mutual differentiation of these abnormal hemoglobins using restriction fragment length polymorphism (PCR-RFLP) method. Digestion of the amplified fragments described above with a restriction enzyme MnlI gave different RFLP patterns for HbA, HbC or S, HbE, and for HbG-Szuhu. RFLP using another enzyme, DdeI, could distinguish for HbS from HbC.
异常血红蛋白是最常见的遗传性疾病之一，全世界已报道约700种变体。一些异常血红蛋白，如HbS和HbC，在有限的人群中呈地方性流行。我们在使用Sysmex NE - 7000自动化系统进行常规血液学分析时发现了5名异常血红蛋白患者。我们从每个患者的外周血白细胞中分离出基因组DNA，并扩增了一个558 bp的DNA片段，该片段包含β - 珠蛋白基因的整个外显子1和2以及内含子1。扩增的DNA片段在自动DNA测序仪上通过双脱氧末端终止法进行直接测序。序列分析显示存在包括HbA/C、HbS/C、HbE/E和HbA/G - 苏胡在内的异常情况。在本研究中，我们报告了一种使用限制性片段长度多态性（PCR - RFLP）方法对这些异常血红蛋白进行相互鉴别的简单方法。用限制性酶MnlI消化上述扩增片段，可得到针对HbA、HbC或S、HbE以及HbG - 苏胡的不同RFLP模式。使用另一种酶DdeI进行RFLP分析，可以区分HbS和HbC。
