[Evaluations of newborn screening program performance and enzymatic diagnosis of glucose-6-phosphate dehydrogenase deficiency in Guangzhou].

Objective: To reveal the molecular epidemiologic characteristics of glucose-6-phosphate dehydrogenase (G6PD) gene and to evaluate based on the genetic analysis the newborn screening program performance and enzymatic diagnosis of G6PD deficiency in Guangzhou. Methods: G6PD enzyme activities were measured by quantitative fluorescence assay in dry blood spots of 16 319 newborns(8 725 males, 7 594 females) 3-7 days after birth in Guangzhou Newborn Center. They were born in Guangzhou form Oct. 1 to 20, 2016. The cutoff value of G6PD was less than 2.6 U/g Hb in dry blood spots. G6PD deficiency was diagnosed when G6PD<1 700 U/L or G6PD/6PGD<1 in red blood cells. Genetic analysis of G6PD gene was performed on the dry blood spot samples of 823 newborns (including positive 346, negative 477)with various levels of G6PD enzyme activities through fluorescence PCR melting curve analysis(FMCA) to detect 15 kinds of mutations reported to be common among Chinese.G6PD gene Sanger sequency was performed in seven highly suspicious patients with negative results by FMCA. Results: (1) Using the cutoff value of G6PD< 2.6 U/g Hb , a total of 687(4.2%) newborns showed positive screening results, including 560 (6.4%) males and 127(1.7%) females. (2) Among the newborns with positive screening results, 214 males and 122 females were randomly chosen for G6PD gene analysis. The results showed that 197 (92.1%) males were hemizygote and 108(88.6%) females were mutation carriers with one to four alleles. Among the newborns with negative screening results, 41 males with G6PD 2.6-2.8 U/g Hb and 436 females with G6PD 2.6-4.5 U/g Hb were chosen for genetic analysis.Mutations were detected in 5(12.2%)boys, and 226(51.8%) girls were carriers.G6PD gene Sanger sequency of seven highly suspicious patients showed that c.406C>T, c.551C>T, c.835A>T hemizygote were found in 3 male's samples, respectively. (3) The estimated prevalence of harboring mutation was 6.0% in males and 13.5% in females according to rates of mutation in samples with various levels of G6PD enzyme activities. Six common mutations were c.1388G>A、c.1376G>T, c.95A> G, c.871G>A, c.1024C>T, c.392G>T, accounting for 95.5% of detected alleles .(4) based on results of G6PD gene analysis, the newborn scereening of G6PD deficiency with cutoff value G6PD<2.6 U/g Hb yielded a positive predict value(PPV) of 93.5%, a false-positive rate of 0.5%, and a sensitivity of 99.0% for males. A PPV of 88.5%, a false-positive rate of 0.2% . The prevalence of severe type G6PD deficiency in females was about 1.5%. Compared with to genetic analysis, the sensitivity and PPV of G6PD activity assay in red blood cells were 95.5%, 97.2%, respectively. Conclusions: The prevalence of G6PD deficiency in males was 6.0% in Guangzhou. Six mutations c.1388G>A, c.1376G>T, c.95A>G, c.871G>A, c.1024C>T, c.392G>T accounted for 95.5%. The cutoff value of G6PD<2.6 U/g Hb innewborn screening program and the criteria of biochemical diagnosis could accurately identify G6PD deficiency . Combined with biochemical and molecular analysis will improve the accuracy of diagnosis of G6PD deficiency and detect more heterozygous females.目的： 探讨广州市葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症分子遗传学特征，基于分子诊断评估现有的G6PD缺乏症新生儿筛查及酶学诊断准确性。 方法： 采用干血斑G6PD荧光定量法，对广州市妇女儿童医疗中心广州市新生儿筛查中心辖区145家助产机构2016年10月1—20日出生、生后3~7 d的16 319例（男8 725例，女7 594例）新生儿进行G6PD缺乏症筛查，并采用荧光PCR熔解曲线法，对823例（筛查阳性346例及筛查阴性477例）不同水平的G6PD酶活性样本进行G6PD基因15种常见变异定量分析。对筛查阳性而基因热点变异分析阴性的高度疑似样本进行G6PD基因测序。以干血斑G6PD<2.6 U/g血红蛋白为筛查阳性切值，当红细胞G6PD<1 700 U/L或G6PD/6-磷酸葡萄糖脱氢酶<1诊断为G6PD缺乏症。 结果： （1）以G6PD< 2.6 U/g血红蛋白为切值，在16 319例新生儿中，筛查阳性687例，其中男560例，女127例，筛查阳性率为4.2%，其中男6.4%，女1.7%。（2）选取214例男性及122例女性筛查阳性样本，进行G6PD基因热点变异检测，其中197例（92.1%）男性为半合子，108例（86.5%）女性检出1~ 4个位点变异。对筛查阴性、G6PD 2.6~2.8 U/g血红蛋白的41例男性样本及G6PD 2.6~4.5 U/g血红蛋白的436例女性样本进行基因分析，5例（12.2%）男性及226例（51.8%）女性检出变异。对7例高度疑似患儿的样本进行G6PD基因测序，发现3例男性分别有c.406C>T、c.551C>T、c.835A>T半合子变异。（3）根据干血斑不同水平G6PD酶活性样本对应的基因变异检出率推测，广州市G6PD基因变异检出率男性约6.0%，女性约13.5%。6种常见变异c.1388G>A、c.1376G> T、c.95A>G、c.871G>A、c.1024C>T、c.392G>T占检出变异位点的95.5%。（4）以G6PD基因诊断为依据，G6PD<2.6 U/g血红蛋白为切值，G6PD缺乏症筛查阳性预测值男性为93.5% （200/214 ），敏感度为99.0% （5例假阴性），假阳性率0.5%；女性阳性预测值为88.5% （108/122），假阳性率0.2%。女性重型G6PD缺乏症（筛查阳性伴基因变异阳性）患病率约1.5%；G6PD酶学诊断的阳性预测值为97.2%，敏感度为95.5%。 结论： 基于G6PD基因分析结果，广州市G6PD缺乏症男性患病率约为6.0%，女性重型G6PD缺乏症患病率约1.5%，6种常见变异为c.1388G>A、c.1376G>T、c.95A>G、c.871G>A、c.1024C>T、c.392G>T，占检出变异位点的95.5%。现有的G6PD缺乏症筛查切值及酶学诊断标准可检出绝大多数男性患儿及女性重型患儿。酶学结合基因热点变异分析可提高G6PD缺乏症诊断准确度及女性杂合子检出率。.