Abstract:

Objective　To analyze the application value of high-throughput sequencing (NGS) in screening neonatal genetic metabolic diseases. Methods　This was a prospective study. One hundred and eighty-five critical cases of acidosis or encephalopathy of unknown causes hospitalized in Neonatal Intensive Care Unit (NICU), Huangshi Maternal and Child Health Hospital from January 2021 to June 2023 were selected, including 95 boys and 90 girls. The heel blood was collected from all the subjects. The biochemical screening (tandem mass spectrometry and time-resolved immunofluorescence) and NGS screening were performed. The NGS screening was designed to capture 202 genes related to genetic metabolism, and the mutation pathogenesis was interpreted according to the relevant guidelines. The positive abnormal sites were verified by the Sanger sequencing. Results　Of the 185 neonates, 7.57% (14/185) were preterm infants and 8.11% (15/185) were from assisted reproduction; 15 (8.10%) were positive in the biochemical screening, and 5 (2.70%) were positive in the re-detection after recall, including 1 case of decreased glucose-6-phosphate dehydrogenase (G6PD), 1 case of decreased 17-hydroxyprogesterone (17α-OHP), 1 case of decreased thyroid stimulating hormone (TSH), and 2 cases of increased phenylalanine. Among the 185 newborns, 6 cases (3.24%) were positive in NGS, including 4 cases of DOUX 2, MATIA, G6PD, CYP21A2 compound heterozygous mutations and 2 cases of PAH mutation. In addition, except for the positive ones, there were 45 newborns (24.32%) who had recessive mutations and 140 negative newborns (72.43%). The results of the two screens were consistent with each other in 5 cases and not in 1 case; 3 of the 6 patients were confirmed, including 1 case of hyperphenylalaninemia, 1 case of 21-hydroxylase deficiency congenital adrenocortical hyperplasia, and 1 case of congenital hypogonadism. Taking the clinical diagnosis as the reference, the medical records to be confirmed were included in the true positive; the biochemical screening sensitivity was 83.3%, the specificity was 100.0%, and the positive predictive value was 100.0%; the NGS screening's sensitivity was 100.0%, the specificity 100.0%, and the positive predictive value was 100.0%. Conclusion　The NGS screening accuracy is high, so it can comprehensively diagnose neonatal genetic metabolic diseases.

Key words:

High throughput sequencing, Newborns, Genetic screening, Metabolism, Sensitivity

摘要：

目的　分析高通量测序（NGS）在新生儿遗传代谢病筛查中的应用价值。方法　采用前瞻性研究方式，选取2021年1月至2023年6月黄石市妇幼保健院新生儿重症监护病房（NICU）住院的不明原因的酸中毒、脑病等185例危重新生儿病例为研究对象，其中男95例、女90例。所有受试者均采集足跟血，实施生化筛查（串联质谱技术、时间分辨免疫荧光法）、NGS筛查。NGS筛查设计与遗传代谢相关的202个基因捕获探针，依据相关指南进行变异致病判读，对阳性异位点采用Sanger测序验证。结果　185例新生儿中，早产儿占7.57%（14/185），辅助生殖占8.11%（15/185）；生化筛查初筛阳性者15例（8.10%），召回复测后阳性者5例（2.70%），其中5例复查阳性者分别为存在葡萄糖-6-磷酸脱氢酶（G6PD）降低1例、17α-羟孕酮（17α-OHP）升高1例、促甲状腺素（TSH）升高1例、苯丙氨酸（Phe）升高2例。NGS阳性筛查率为3.24%（6/185），分别为DOUX2、MATIA、G6PD、CYP21A2复合杂合突变各1例，PAH基因复合杂合突变2例。此外，除阳性者外，存在隐性遗传病变异携带的新生儿有45例（24.32%），阴性新生儿140例（72.43%）。两种筛查结果一致有5例，不一致有1例，6例患者中3例得到确诊，分别为高苯丙氨酸血症1例、21-羟化酶缺乏性先天性肾上腺皮质增生症1例、先天性甲腺功能减退症1例。以临床诊断为参考，将待确诊病例纳入真阳性中，生化筛查灵敏度为83.3%，特异度为100.0%，阳性预测值为100.0%；NGS筛查灵敏度为100.0%，特异度为100.0%，阳性预测值为100.0%。结论　NGS筛查准确度较高，可较为全面的诊断新生儿遗传代谢病。

关键词:

高通量测序, 新生儿, 遗传筛查, 代谢, 灵敏度