新生儿地中海贫血高通量测序分析及初筛方法探讨

High-throughput sequencing analysis of neonates with thalassemia and study on primary screening methods

目的通过检测新生儿地中海贫血(地贫)发生情况、基因分型以及评估平均红细胞体积(MCV)、平均红细胞血红蛋白含量(MCH),评估其在新生儿地贫初筛中的价值。方法收集2018年12月至2019年8月在深圳市宝安区妇幼保健院经阴道分娩的1127例新生儿的脐血干血片,通过缺口聚合酶链反应(GAP-PCR)、反向斑点杂交聚合酶链反应(RDB-PCR)、高通量测序(NGS)技术进行筛查;1127例新生儿中有392例在出生1周内进行了血常规检查,回顾性分析392例新生儿MCV、MCH等血液学指标。结果(1)常规实验室GAP-PCR法、PCRRDB法检测结果与NGS检测结果一致。NGS技术在1127例新生儿中检测出128例地贫阳性,其中α-地贫85例,β-地贫40例,α&β复合型地贫3例,其中HBA2:c.217_218insC基因型α-地贫为世界上首次发现。此外还发现异常血红蛋白突变5例。(2)地贫新生儿MCV、MCH均明显低于对照组新生儿[(93.50±8.38)fl vs.(103.00±4.59)fl,(31.10±3.29)pg vs.(35.20±1.68)pg],两组差异具有统计学意义(P<0.001);α-地贫新生儿MCV、MCH均明显低于β-地贫新生儿[(89.70±7.57)fl vs.(101.00±4.76)fl,(29.50±3.01)pg vs.(34.00±1.42)pg](P<0.001);运用MCV、MCH筛选新生儿地贫ROC曲线下面积分别为0.837和0.872,截断值分别为95.5fl和33.5pg。结论经GAP-PCR法、PCR-RDB法验证,NGS技术可精准检测出新生儿地贫的类型,MCV和MCH指标在新生儿地贫初筛中具有重要参考价值。

Objective To investigate the occurrence and genotype,and to evaluate the value of mean corpuscular volume(MCV)and mean corpuscular hemoglobin(MCH)in the primary screening of neonatal thalassemia.Methods The dried blood tablets of cord blood of 1127 neonates of vaginal delivery from December 2018 to August 2019 were collected in Shenzhen Baoan Women’s and Children’s Hospital,and were screened by gap polymerase chain reaction(GAP-PCR),reverse dot blot hybridization polymerase chain reaction(RDB-PCR)and high-throughput sequencing(NGS).Among them,392 neonates had blood routine examination within one week after birth and the hematological indexes such as MCV and MCH of 392 neonates were analyzed retrospectively.Results(1)Among 1127 neonates,therer were 128 positive cases of thalassemia;85 cases wereα-thalassemia and 40 cases wereβ-thalassemia;3 cases wereα&βcomplex thalassemia;5 cases were abnormal hemoglobin mutation.Moreover,we identified a novel mutation at HBA2:c.217_218insC heterozygosity for the first time.(2)Both MCV and MCH of neonatal thalassemia were significantly lower than those of control newborns,and the difference between the two groups was statistically significant[(93.50±8.38)fl vs.(103.00±4.59)fl,(31.10±3.29)pg vs.(35.20±1.68)pg](P<0.001).MCV and MCH ofα-thalassemia newborns were significantly lower than those ofβ-thalassemia newborns[(89.70±7.57)fl vs.(101.00±4.76)fl,(29.50±3.01)pg vs.(34.00±1.42)pg](P<0.001).The area under the ROC curve for screening neonatal thalassemia using MCV and MCH were 0.837 and 0.872,and the cut-off values were 95.5fl and 33.5pg,respectively.Conclusion NGS technology can accurately detect the type of thalassemia in neonates.MCV and MCH have important reference value for early diagnosis of neonatal thalassemia.