运用PCR-DGGE技术检测葡萄糖-6-磷酸脱氢酶缺乏的初步研究

目的：运用多聚酶链反应-变性梯度凝胶电泳（PCR-DGGE）技术检测葡萄糖-6-磷酸脱氢酶（G-6-PD）缺乏患者及基因携带者基因变异,探讨其对该病的诊断和研究价值。方法：提取G-6-PD缺乏症患者及其家系（患者父亲和/或母亲等）的外周血RNA,逆转录合成cDNA后,选取第11至12外显子部分cDNA片段进行PCR-DGGE,观察其电泳行为,将电泳行为异常的标本进行基因测序,最后做出基因诊断。结果:：36个家系中33个家系发现G-6-PD基因在1304至1520片段出现PCR-DGGE多种异常电泳区带。9例母亲G-6-PD/6-PGD比值低于1.00,其中3例比值低于0.50,而且PCR-DGGE电泳行为一致,基因测序发现为双重杂合子;比值正常的G-6-PD缺乏基因携带者母亲均为单杂合子。该片段基因测序发现3个突变位点分别为：C1311T,G1376T,G1388A。各基因突变的位点有其特殊的电泳行为。结论：PCR-DGGE技术是一种敏感性高、可靠性强的筛查基因突变的方法。在临床研究G-6-PD缺乏,特别是常规诊断技术不能发现的女性G-6-PD缺乏基因携带者的检测中具有很强的应用价值。［中国当代儿科杂志,2007,9（6）：529-532］     

Neonatal screening for Glucose-6-Phosphate dehydrogenase deficiency

Objective : This study was carried out to detect the incidence of erythrocytic Glucose-6-Phosphate dehydrogenase (G-6-PD) deficiency, to compare the incidence of hyperbilirubinernia in G-6-PD deficient neonates as compared to G-6-PD normal neonates and to asses the usefulness of neonatal screening for G-6-PD deficiency.Method : In a retrospective hospital based study 2,479 male and female neonates consecutively born at Indraprastha Apollo hospital between July 1998 to June 2003 who were screened for G-6-PD levels were evaluated for the incidence of G-6-PD deficiency.Results : Incidence of G-6-PD deficiency was found to be 2.0%. Incidence in males was 283% and femle was 1.05%. The incidence of hyperbilirubinemia was found to be 32% in G-6-PD deficient neonates which was significantly higher than the incidence of hyperbilirubinemia in neonates with normal G-6-PD, which was 12.3% (P<0.001).Conclusion : Our data suggests that neonatal screening for G-6-PD deficiency is a useful test for preventing and early treatment of complications associated with it.     

葡萄糖-6-磷酸脱氢酶缺乏症患儿G-6-PDmRNA表达的研究

目的：检测葡萄糖-6-磷酸脱氢酶(G-6-PD)缺乏症患者及其家系成员的G-6-PDmRNA表达水平,从转录水平探讨其可能的发病机制。方法：提取G-6-PD缺乏症患者及其直系家属(患者父亲和/或母亲等)外周血RNA,采用逆转录方法形成cDNA后,运用逆转录实时定量PCR(QuantitativeReal-TimePCR,QRT-PCR)技术,测定G-6-PDmRNA的表达量。使用SPSS10.0统计分析软件将3组进行组间两两比较。结果：G-6-PD缺乏症患儿组mRNA表达量为0.57±0.19,父系组为0.74±0.21,母系组为0.67±0.21,患儿组与父系组比较t=-3.18,(P<0.01);与母系组比较t=-2.54,(P<0.05)。结论：G-6-PD缺乏症患者的G-6-PD基因发生突变后其G-6-PDmRNA表达量发生了改变,提示该病的发生与在转录水平上发生变化有关,在G-6-PD缺乏症的发病过程中起到一定的作用。     

不同G-6-PD活性新生儿光疗致溶血机制及其预防

目的探讨不同G6PD活性新生儿光疗溶血机制及预防。方法将G6PD正常与缺陷光疗患儿随机分为维生素E干预组和对照组,测定比较超氧化物歧化酶(SOD)、丙二醛(MDA)、活性氧(ROS)、总胆红素(TB)、血红蛋白(Hb)及光疗指数。结果光疗前G6PD缺陷组比正常组SOD和Hb低,ROS高;光疗中G6PD缺陷干预组比正常干预组SOD高,MDA低,光疗指数小,G6PD缺陷对照组比正常对照组ROS、MDA高,光疗指数大(各组比较均P<0.01或P<0.05)。光疗后G6PD缺陷对照组Hb下降,并比干预组低,G6PD正常两组Hb均下降,干预组比对照组高(各组比较均P<0.01或P<0.05)。结论光疗可致抗氧化能力下降,脂质过氧化损伤致G6PD缺陷光疗者溶血更突出,维生素E干预更有效。     

Effect of phenobarbital treatment on erythrocyte glucose-6-phosphate dehydrogenase in human newborns

The effect of phenobarbital (PB) treatment on erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) levels was studied in normal and G-6-PD-deficient human newborns. An increase of erythrocyte G-6-PD levels was observed in normal G-6-PD patients, while increase in the enzymatic activity was not observed in G-6-PD-deficient neonates.     

广西恭城县瑶族和汉族居民G-6-PD缺乏症发病率及基因频率的调查

目的研究广西恭城县瑶族和汉族居民的G-6-PD缺乏症发病率及基因频率。方法使用G-6-PD试纸法初筛,四氮唑蓝定量法测定确认的方法调查对2050名(男1126,女1124)瑶族和874名(男481,女393)汉族初中学生进行G-6-PD缺乏症的调查。结果瑶族男缺乏率5·75%(显著缺乏4·87%,中度缺乏0·97%),瑶族女性缺乏率1.95%(显著0·59%,中度1·36%),瑶族男女合并总缺乏率为3·85%;瑶族男性基因频率为:0·057,瑶族女性杂合子的估计值为10·84%:汉族男性缺乏率7·06%(显著缺乏6·03%,中度缺乏1·04%),汉族女性缺乏率3·56%(显著0·76%,中度2·80%),汉族男女合并总缺乏率为5·49%;汉族男性基因频率为0·0706,汉族女性杂合子的估计值为13·12%;全县瑶族和汉族合并缺乏率为4·34%。结论恭城县G-6-PD缺乏发病率,瑶族比汉族的稍低,但民族间的差异比地域间的差异相对要小。     

遗传因素在广西新生儿高胆红素血症中的作用

目的探讨UGT1A1 G71R突变、OATP2A388G突变和G-6-PD缺乏对在广西新生儿高胆红素血症发病的作用。方法用四氮唑蓝定量法（NBT法）测定G-6-PD酶活性。聚合酶链反应-等位基因特异性寡核苷酸探针点杂交（PCR-ASO）法确定G71R基因型。限制性片段长度多态性分析（RFLP）检测A388G基因型。测定109例新生儿脐血的G-6-PD活性及G71R基因型,其中101例同时检测了A388G基因型。据G-6-PD活性及G71R或A388G基因型分组,分析UGT1A1G71R突变、OATP2A388G突变和G-6-PD缺乏与足月新生儿高胆红素血症之间关系。结果G71R等位基因频率在G-6-PD缺乏组为22．03％,在G-6-PD正常组为28．00％。G-6-PD缺乏共存有G71R突变纯合子或杂合子的新生儿高胆红素血症发生率（95．50％）高于G-6-PD正常且G71R为野生型的新生儿（53．90％）,x^2=10．45,P=0．0012,前者发生高胆红素血症的机会比（95％可信区间）[OR（95％CI）]为18．00（2．12,152．9）。A388G等位基因频率在G-6-PD缺乏组为20．O％,在G-6-PD正常组为18．5％。G-6-PD缺乏共存有A388G突变新生儿的高胆红素血症发生率（90．0％）高于G-6-PD正常且A388G为野生型的新生）L（44．80％）,X2=10．39,P=0．0013,前者发生高胆红素血症的伽（95％CT）为11．08（2．15,56．48）。结论G71R突变与G-6-PD缺乏共存或A388G突变与G-6-PD缺乏共存对广西足月新生儿高胆红素血症的发生有协同作用。     

广西南宁地区G6PD基因突变与新生儿黄疸的关系

目的：分析本地区最常见的三种基因突变型G1388A、G1376T和A95G与葡萄糖-6-磷酸脱氢酶（G-6-PD）活性之间的相关性,并探讨G-6-PD基因突变对新生儿黄疸的影响。方法：124例广西南宁的高胆红素血症新生儿为研究对象。应用突变特异性扩增系统法检测G-6-PD基因突变,应用硝基四氮唑蓝（NBT）定量法检测G-6-PD活性。比较G-6-PD不同基因突变型之间以及与正常组之间胆红素脑病发生率、出生72 h后血清胆红素峰值组间的差异。采用非条件logistic回归分析血清胆红素值>340 μmol/L的危险度。结果：124例中有37例G-6-PD 基因突变（G1388A 20例,G1376T 14例,A95G 4例,1例同时存在G1388A与A95G突变）。20例G1388A突变者中5例（25%）G-6-PD酶活性正常,14例G1376T突变者中4例（29%）G-6-PD酶活性正常,4例A95G突变者G-6-PD 酶活性均缺乏。G1388A与G1376T组胆红素脑病发生率及出生72 h后血清胆红素峰值差异无显著性。G-6-PD 突变组出生72 h后血清胆红素峰值、胆红素脑病发生率及血清胆红素>340 μmol/L的危险度与G-6-PD正常组相比,差异无显著性。结论：广西南宁地区G-6-PD突变仍常见G1388A、G1376T和A95G基因型。NBT法诊断G-6-PD缺乏存在假阴性。不同基因型对出生72 h后血清胆红素峰值、胆红素脑病发生率的影响无差异。单独的G-6-PD基因突变对生后72 h血清胆红素峰值、急性胆红素脑病发生率及血清胆红素大于340 μmol/L危险性均无影响。［中国当代儿科杂志,2009,11（12）：970-972］     

Cord plasma alpha-fetoprotein values and neonatal jaundice

Umbilical cord plasma alpha-fetoprotein (AFP) values were determined in 127 infants with hyperbilirubinemia (56 glucose-6-phosphate dehydrogenase (G-6-PD) deficient and 71 G-6-PD normal) and 136 control subjects (73 G-6-PD deficient and 63 G-6-PD normal). The mean alpha-fetoprotein value of 173 +/- 35.2 (SD) mg/L for the group of infants with hyperbilirubinemia was significantly greater than that (122 +/- 21.7 mg/L) for the control infants (P less than .001). G-6-PD status and sex did not significantly affect the alpha-fetoprotein values. Using an alpha-fetoprotein level of 130 mg/L as a "cut-off" value, the incidence of false-positive results was 25.5% and the incidence of false-negative results was 11.8%. This test can be used as a screening procedure to detect infants at high risk for hyperbilirubinemia.     

G-6-PD缺陷与新生儿高胆、婴幼儿贫血及婴儿肝炎综合征关系的调查

广西是我国Ｇ－６－ＰＤ缺陷高发区。Ｇ－６－ＰＤ缺陷发病的主要表现为急慢性溶血性贫血和由此而产生的高间胆红素血症及胆汁郁积。近十年我院门诊及病房４４０例患儿：２３７例新生儿高间胆包括核黄症后遗症患儿。Ｇ－６－ＰＤ缺陷检出率达６９．６％，其中显著缺陷４３．９％（１０４／２３７），中间值２５．７％（６１／２３７）；１０３例３个月以下的婴儿贫血原因待查患儿，Ｇ－６－ＰＤ缺陷检出率达５９．２２％（３６／１０３）；１００例婴儿肝炎综合征患儿，Ｇ－６－ＰＤ缺陷检出率达１８％（１８／１００）。Ｇ－６－ＰＤ缺陷男女经例为２．３７：１。６７例进行家系调查，２８例Ｇ－６－ＰＤ缺陷男婴，母亲检出率为６７．８％（１８／２８）；１４例Ｇ－６－ＰＤ缺陷女婴，父亲检出率４３％（６／１４），母亲检出率５０％（７／１４）。说明了在我区Ｇ－６－ＰＤ缺陷是引起新生儿高间胆以及核黄痊后遗症、婴幼儿贫血较常见而且重要原因之一。     

Neonatal screening for glucose-6-phosphate dehydrogenase deficiency: sex distribution.

Eight hundred and six newborn infants at high risk for glucose-6-phosphate dehydrogenase (G-6-PD) deficiency were screened; 30.2% of the boys and 10.4% of the girls had severe G-6-PD deficiency. Surprisingly, 14% of the enzyme deficient girls had a father from a low risk ethnic group. Girls of high risk mothers should be screened for G-6-PD deficiency regardless of paternal origin.     

Glucose-6-Phosphate Dehydrogenase Deficiency in Female Heterozygoses and the X-Inactivation Hypothesis

The levels of G-6-PD activity in 75 female heterozygotes detected by pedigree analysis are reported, together with biochemical and genetic data of a pedigree in which a wide variation in G-6-PD activity was revealed in heterozygotes. The results strongly favour the hypothesis that one X-chromosome is inactivated during early embryonic development in the human female, at least as far as the locus of G-6-PD is concerned.     

Glucose-6-phosphate dehydrogenase deficiency and kernicterus of South-East anatolia

OBJECTIVE: We aimed to investigate the rate of kernicterus, and physical and laboratory examination findings in hyperbilirubinemic infants with glucose-6-phosphate dehydrogenase (G-6-PD) deficiency. MATERIALS AND METHODS: This study was carried out in the Dicle University Hospital Neonatal Intensive Care Unit between June 2005 and June 2006. Out of 56 male neonates who needed an exchange transfusion due to hyperbilirubinemia, 10 with G-6-PD deficiency were included in the study. Maternal age, gestational age, route of delivery, birth weight, age at the time of admission, and treatment and outcome were recorded. Laboratory investigations included determination of direct and indirect serum bilirubin concentrations, blood group typing, direct Coomb test, complete blood count, blood smear, thyroid-stimulating hormone, T4, C-reactive protein, urine analysis, and G-6-PD level. RESULTS: Out of 56 male neonates requiring exchange transfusion, 10 had G-6-PD deficiency (18%). In G-6-PD deficient neonates, other factors known to cause hyperbilirubinemia were excluded. The mean gestational age and the mean maternal age was 38.2+/-1.0 weeks and 31.3+/-5.9 years, respectively. The mean bilirubin level was 42.1+/-13.7 mg/dL. Four patients required a second exchange transfusions, and only 1 transfusion was sufficient for the remaining patients. Five patients (55%) developed kernicterus. CONCLUSIONS: Early detection of G-6-PD deficiency in the affected newborns may be important for reducing the risk of severe hyperbilirubinemia, kernicterus, and the need for exchange transfusion.     

血液学参数的变化与红细胞葡萄糖-6-磷酸脱氢酶活性关系的研究

本文报道１６４例住院的高胆红素血症患儿（贫血６６例，无贫血９８例）进行Ｇ－６－ＰＤ活性和几个血液学参数的测定，并作了相关性、回归分析和显著性检验。结果：Ｇ－６－ＰＤ活性与白细胞、网织红细胞、血小板数、血清钠及微量元素锌、硒含量呈正相关；与血红蛋白、红细胞和红细胞压积呈负相关，两者均有显著性意义（Ｐ＜０．０５～０．０１）。而与血清钾、氯、钙、白蛋白、球蛋白及胆红素含量无相关性。通过本实验结果分析，作者认为高胆红素血症患儿，伴有贫血或溶血时，由于骨髓增生，网织红细胞和年幼的红细胞数量增加，或者伴随感染时因白细胞明显增加等因素，致Ｇ－６－ＰＤ活性含量升高而掩盖了Ｇ－６－ＰＤ缺陷。因此，评价Ｇ－６－ＰＤ活性时，除了考虑以上几个血液学参数影响造成的误差，对贫血患儿的血样常规先行调整血浆和血细胞的比例外，白细胞增加者若能进行离心除去白细胞层后进行测定；或待感染控制后，有贫血者待贫血改善后；有溶血者待溶血停止后２—４个月左右，再复查Ｇ－６－ＰＤ活性，对Ｇ－６－ＰＤ缺陷才能作出正确的诊断。     

Salicylamide glucuronide formation in newborn babies with G-6-PD deficiency

Salicylamide glucuronide formation has been studied in 23 newborn babies with erythrocyte G-6-PD deficiency and in 15 normal newborns on the first day of life. Glucuronide formation was significantly lower (p less than 0.001) in the former in comparison with the controls. In the newborns with G-6-PD deficiency who subsequently became hyperbilirubinemic an even lower mean glucuronide formation was observed (p less than 0.01) in respect to the non-jaundiced G-6-PD-deficient newborns.     

Leucocyte glucose-6-phosphate dehydrogenase (G-6-PD) activity in G-6-PD deficient subjects

The activity of glucose-6-phosphate dehydrogenase (G-6-PD) in leucocytes was studied in the following groups of Greek people.Group 1: 43 male children and 16 male students with mean values of enzyme activity of 27.7±16.6 units and 24.6±5.6 units, respectively.Group 2: 15 G-6-PD deficient male children who had never experienced an acute haemolytic episode with a mean value of 10.8±4.6 units.Group 3: 19 G-6-PD deficient male children during favism and 3 months after the haemolytic crisis with mean values of 8±4 units and 9.2±1.9 units, respectively.Group 4: 19 mothers of children from group 3 who by definition were carriers of G-6-PD deficiency had a mean value of 18.2±8.2 units.The difference between means for group 1 and groups 2, 3 and 4 is highly significant (P<0.001).Therefore the enzymatic defect in Greek people is not limited to the erythrocytes but can be also demonstrated in leucocytes.     

Glucose-6-phosphate dehydrogenase,reduced glutathione and heinz bodies in congenital haemolytic anaemia

Summary Glucose-6-phosphate dehydrogenase (G-6-PD), glutathione instability (GSH) and heinz body formation were studied in ten children suffering from congenital haemolytic anaemia and in fifteen healthy subjects of similar age group. In congenital haemolytic anaemia, dye decolorisation time for assessing G-6-PD activity was within the normal range. Unstable GSH was less than 20% in four cases and more than 20% in the other four cases. In two cases no unstable GSH was observed. Heinz bodies, more than 50%, were present in nine of ten cases. Glutathione instability and increased percentage of heinz body formation with normal G-6-PD activity probably occurs due to glutathione reductase or TPNH (NADPH) deficiency. From the Department of Pathology and Microbiology, Sandar Patel Medical College, Bikaner, Rajasthan.     

G 6 PD deficiency induced hemolysis in diabetic twins

We report hemolysis due to glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in two Egyptian monozygotic brothers, at the onset of diabetes type 1. Hemolysis occurred following the treatment of hyperglycemia and ketoacidosis (one twin). It was related to unknown G-6-PD deficiency. The fall in glucose availability after the treatment of hyper glycemia is proposed as a possible etiology for hemolysis.     

THE RISK OF JAUNDICE IN GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENT BABIES EXPOSED TO MENTHOL

Abstract. Olowe, S. A. and Ransome-Kuti, O. (Department of Paediatrics, College of Medicine of the University of Lagos, Lagos, Nigeria). The risk of jaundice in glucose-6-phosphate dehydrogenase babies exposed to menthol. Acta Paediatr Scand, 69:341, 1980.—A major cause of neonatal morbidity and mortality in Lagos, Nigeria, is severe neonatal jaundice seen in G-6-PD deficient babies. The observation that the jaundice is more severe in outpatient than in inpatient babies suggests that its cause is exogenous. "Mentholated" powder which is commonly used in many clinics and at home to dress umbilical cords was suspected to be the offending agent. A controlled study of the effects of one of these powders was carried out on 60 consecutive G-6-PD deficient babies. In 30 of them the umbilical cords were dressed daily with the powder while the remaining half who were untreated served as controls. The treated babies developed statistically more significant jaundice than the controls. Inability of neonates to conjugate menthol in this power is probably responsible for the jaundice developed by these G-6-PD deficient babies. It is concluded that the use of menthol and/or camphor-containing commercial products on neonates be discontinued, especially in communities where the incidence of G-6-PD deficiency is high as the use of such products may be contributing to the severity of neonatal jaundice.     

β-Thalassemia trait and hyperbilirubinemia in G-6-PD deficient newborn infants

Hb A₂ was determined in 50 subjects with erythrocyte G-6-PD deficiency who presented with hyperbilirubinemia in the neonatal period and in 100 non-hyperbilirubinemic G-6-PD deficient newborn infants, at the age of 12 months or more. Six subjects in the first group and 13 in the second were found to be carriers of the -thalassemia trait. Statistical analysis of the data did not show any significant difference between the two groups. It seems that the -thalassemia trait does not provide any protection against neonatal hyperbilirubinemia associated with G-6-PD deficiency.