红细胞葡萄糖6磷酸脱氢酶缺乏症

干血纸片法用于葡萄糖-6-磷酸脱氢酶基因突变型的检测

更新时间:2011-10-21 12:31:41 | fx_d2a6e834

  干血纸片法用于葡萄糖-6-磷酸脱氢酶基因突变型的检测

  中华儿科杂志 2000年第7期第38卷 论著

  作者:区小冰 佟莉贞 张力 余一平

  单位:区小冰(510120 广州市儿童医院检验科血液室);佟莉贞(510120 广州市儿童医院检验科血液室);张力(510120 广州市儿童医院检验科血液室);余一平(510120 广州市儿童医院检验科血液室)

  关键词:葡糖磷酸脱氢酶缺乏;基因型;突变;聚合酶链反应;dna限制酶类

  【摘要】 目的 了解广东人葡萄糖-6-磷酸脱氢酶(g-6-pd)缺陷的基因突变型。探讨干血纸片法用于基因突变型诊断的效果。方法 采用专用滤纸干血斑,运用5对特异性引物进行dna直接扩增、结合限制性内切酶分析技术对87例g-6-pd缺陷的住院患儿进行检测。结果 87例患儿中30例(35%)为g1376t,28例(32%)为g1388a,6例(7%)为a95g,1例(1%)为g392t,未检出c1024t,尚有22例未能定型。结论 g1376t、g1388a是广东人最常见的g-6-pd突变型。对急性溶血患儿,高铁血红蛋白还原率和g-6-pd酶活性(nbt法)假阴性时,干血纸片法可提高检出率。滤纸干血斑标本采集容易,直接进行dna扩增,方法简单,准确度高,值得在基因研究和临床诊断中应用推广。

  examination of glucose-6-phosphate dehydrogenase mutation types with a method of dried blood spots on filter paper

  ou xiaobing, tong lizhen, zhang li, et al. laboratorial department, guangzhou children′s hospital, guangzhou 510120, china

  【abstract】 objective to understand gene mutant types of glucose-6-phosphate dehydrogenase (g-6-pd) deficiency among local guangdong children and to evaluate the method using dried blood spots on filter paper in examination of patients suffered from acute hemolytic anemia caused by g-6-pd deficiency. methods the subjects of this study were 87 hospitalized children suffering from acute hemolytic anemia episodes between 1993 and 1997. among these children, 76 were male and 11 were female. the age of these cases ranged from 1.5 mo to 10 years and 7 mo. all the patients were diagnosed as having g-6-pd deficient disease on the basis of clinical symptoms and the methemoglobin reducing test or the activity of g-6-pd enzymes (nbt method). all these patients were native guangdong people without blood relation to each other. peripheral blood specimens were collected from the finger tips of the patients, then the blood was dropped on a sheet of special filter paper (s%26s) and the detection was performed after dry blood spot formed. direct pcr amplification of the target gene segment was performed by using 5 pairs of special primers designed against 5 g-6-pd mutant types: g1376t, g1388a, a95g, g392t, and c1024t from dried blood spot on filter paper. the amplified products were added with related 5 different restriction endonucleases: bfrⅰ, ndeⅰ, miuⅰ, banⅰ, mboⅱ, and were digested at 37℃ overnight. the products obtained after digestion were analyzed with polyacrylamide gel electrophoresis. results the detection rates of different gene mutant types were as follows: g1376t 35% (30 cases), g1388a 32% (28 cases), a95g 7% (6 cases), g392t 1% (1 case); c1024t was not found. in 22 cases the mutant types could not be defined. of 7 cases with acute hemolytic anemia who had normal g-6-pd enzyme activity (nbt method), 5 were g1376t-positive, one was g1388a-positive and the remaining one was a95g-positive. conclusion g1376t and g1388a seemed to be the most common mutant types of g-6-pd among the subjects studied. the method of using dried blood spots on filter paper was useful for the patients suffered from acute hemolytic anemia caused by g-6-pd deficient disease. this method may have higher detection rates than methemoglobin reducing test and the g-6-pd enzyme activity tests which often show false negative results. the dry blood spot test has the advantages of easy to perform, using small quantity of blood sample, convenient to transport and store, which would make direct dna amplification simple and accurate. this method appears to be suitable for mass screening for g-6-pd deficiency.

  【key words】 glucosephosphate dehydrogenase deficiency; genotype; mutation; polymerase chain reaction; dna restriction enzymes

  目前国内对葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase, g-6-pd)基因突变型的检测方法有寡核苷酸探针斑点杂交技术,pcr产物dna单链构象多态技术、等位基因特异性扩增和内切酶分析法等。而干血纸片法检测只用于新生儿筛查[1]。我们运用5种特异性引物,采用滤纸干血斑直接dna扩增结合内切酶分析技术,对87例g-6-pd缺陷症引起急性溶血的患儿进行了检测。

  对象和方法

  一、对象

  87例均为我院1993~1997年因急性溶血性贫血发作而住院的患儿,其中男性76人,女性11人,年龄从1个半月~10岁7个月,经临床症状和高铁血红蛋白还原率或g-6-pd酶活性测定(nbt法)确诊为g-6-pd缺陷症[2],相互间无亲缘关系,广东籍人。

  二、方法

  1.标本采集:采患儿手指尖血,直接滴在新生儿筛查用滤纸(s%26s)上,形成约1 cm直径的血斑,每个患儿取6个血斑备用,待血斑自然干燥后用塑料袋装好,放-20℃冰箱内保存(滤纸由台北荣民总医院医学研究部萧广仁博士提供)。

  2.引物及限制性内切酶:针对5种不同的点突变所设计的5对特异性引物:g1376t,g1388a,a95g,g392t,c1024t(序列从略[3,4],由萧广仁博士提供),及相应的5种内切酶:bfrⅰ,ndeⅰ,mluⅰ,banⅰ,mboⅱ(均为美国promega公司产品)(表1)。

  表1 5种突变类型相应的内切酶及酶切结果

  突变类型限制性内切酶切点酶切片段大小(bp)

  正常突变

  g1376tbfrⅰc/ttaag169,12153,16,12

  g1388andeⅰca/tatg220200,20

  a95gmluⅰa/cgcgt126,13 99,27,13

  g392tbanⅰg/g pypucc262,59237,59,25

  c1024tmboⅱgaaga(n8)/162,22125,37,22

  3.聚合酶链反应:取直径3 mm的滤纸干血斑,先经甲醇∶丙酮(1∶1)混合液处理,置60℃烤箱8~10 min待溶剂挥发,然后加入pcr反应所需的各种试剂(taqdna聚合酶除外),95℃水浴10 min,60℃水浴20 min,最后加入taqdna聚合酶进行pcr循环,反应总体积30 μl。各组分的终浓度为:datp、dgtp、dctp、dttp各0.2 mmol/l, mgcl2:2 mmol/l,上下游引物各0.2 μmol/l,1× 缓冲液(taqdna聚合酶厂家提供),1u taqdna聚合酶(promega公司产品)。pcr反应在dna扩增仪(hema 480)上自动进行:95℃、1 min,59℃、1 min,74℃、30 s,1个循环;95℃、1 min,58℃、1 min,74℃、30 s,1个循环;95℃、30 s,58℃、50 s,74℃、30 s,33个循环;最后74℃延伸5 min。结果观察:取pcr产物5 μl与1 μl上样液混合,2%琼脂糖电泳,溴化乙锭染色,紫外灯下观察结果。

  4.限制性内切酶分析:取pcr产物5 μl,用相应的内切酶4 u 及相应缓冲液,总体积20 μl,在酗37℃消化过夜,消化产物用8%聚丙烯酰胺凝胶电泳,溴化乙锭染色后置紫外灯下观察结果。结果

  一、各基因突变型的检出率

  在检测的87例患儿滤纸干血斑中,有65例(75%)分别属于下列4种不同的基因突变型:g1376t,g1388a,a95g,g392t,未发现c1024t,有22例(25%)尚未能定型。结果见表2。

  表2 87例g-6-pd缺陷症患儿基因突变类型

  突变类型氨基酸置换  例数 百分比(%)

  g1376targ→leu

  30

  35

  g1388aarg→his2832

  a95ghis→arg67

  g392tgly→val11

  c1024tleu→phe00

  未知未知2225

  合计

  87

  100

  二、患儿高铁血红蛋白还原率或g-6-pd酶活性(nbt法)的检测情况

  本组7例因药物或进食蚕豆诱发急性溶血,临床诊断为 g-6-pd缺陷的患儿中,2例高铁血红蛋白还原率正常,g-6-pd酶活性(nbt法)低下;1例高铁血红蛋白还原率低下,g-6-pd酶活性(nbt法)正常,经检测证实均属g1376t突变型。另4例高铁血红蛋白还原率和g-6-pd酶活性(nbt法)均正常,分别为g1376t 2 例,g1388a和a95g各1例。

  三、c392t突变型酶切结果

  我们采用滤纸干血斑直接dna扩增,限制性内切酶分析法是根据点突变性质[3,4]应用了不完全配对的pcr引物,扩增反应后使其形成一个新的内切酶位点和一个内质控切点,经相应的内切酶切割后,形成与正常的pcr产物被切割后不同的片段(图1)。

  讨论

  本组结果表明:87例g-6-pd缺陷患儿中,分别属于4种突变型的有65例,未定型有22例,可能是其他的突变型不在本组检测范围,因此我们认为此法亦适合用于g-6-pd缺陷引起急性溶血的患儿。本组中c1376t占35%,g1388a占32%,两种共占67%,与杜传书、谢建生、何永蜀等[5-7]学者用不同的方法在不同的地区检出的52.8%、62%和68%,台湾人中占71.3%的结果基本相同[3],都超过半数以上。也说明这两种基因突变型是中国人g-6-pd缺陷的主要突变型。本组未发现c1024t,据报道其发生率是1.8%和5.6%[1,5],提示可能g-6-pd基因突变点不同,其诱发溶血因素可能也不同,前两种突变型易发生溶血,而后者却少见。

  m:dna分子量标准;1: pcr产物未与内切酶反应;2:正常pcr产物与内切酶反应;3:g392t突变型pcr产物与内切酶反应

  1 g392t突变型酶切分析电泳图谱

  从本组结果可看出,在临床发生急性溶血的病例,如实验室检查高铁血红蛋白还原率和g-6-pd酶活性(nbt法)出现假阴性,或两种方法检查结果不一致而不能确诊时,运用此法进行dna检测,可提高g-6-pd缺陷症的检出率。

  用滤纸干血斑直接进行dna扩增的优点是:采集标本容易,无需抽血,仅用指尖几滴血,用血量少,可在不同的时间收集标本储藏起来,在适当的时候进行检测。滤纸干血斑标本运输极为方便,操作时无需dna抽提的繁锁步骤,避免了交叉污染。适用于大量的普查工作。滤纸干血斑标本直接dna扩增,结合内切酶分析法具有方法简单易掌握,准确性好,灵敏度高,结果易判断,值得在基因研究和临床诊断方面应用和推广。

  参考文献

  1,唐东江,马燮琴,宋诚燕,等. 干血纸片法用于广东人g-6-pd基因点突变筛查研究. 中华血液学杂志,1998,19:189-191.

  2,杜传书,芮琳. 红细胞葡萄糖-6-磷酸脱氢酶活性四氮唑蓝定量法. 中华血液学杂志,1981,2:188.

  3,chang jg,chiou ss,perng li,et al. molecular characterization of glucose-6-phosphate dehydrogenase (g-6-pd) deficiency by natural and amplification created restriction sites:five mutations account for most g-6-pd deficiency cases in taiwan. blood,1992,80:1079-1082.

  4,tang kt,huang cs,huang mj,et al.diverse point mutations result in glucose-6-phosphate dehydrogenase(g-6-pd)polymorphism in taiwan. blood, 1992,79:2135-2140.

  5,杜传书,王青,陈路明,等. 中国人所见的六种葡萄糖-6-磷酸脱氢酶基因的点突变.中华血液学杂志,1993,14:395-398.

  6,谢建生,龙桂芳,蒋南,等. 两种常见葡萄糖-6-磷酸脱氢酶基因突变检测及临床分析.中华血液学杂志,1995,16:179-182.

  7,何永蜀,杜传书,蒋玮莹,等.云南省几个民族葡萄糖-6-磷酸脱氢酶基因突变型分析.中华血液学杂志,1997,18:193-196.

  (收稿日期:1999-07-23)

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