基因Panel诊断新生儿重症监护病房患儿基因变异的价值

Diagnostic value of targeted sequencing panel for genetic variation in neonates in neonatal intensive care unit

目的探讨基因Panel在诊断新生儿重症监护病房(neonatal intensive care unit,NICU)患儿的基因变异中的应用价值。方法前瞻性纳入2022年11月至2023年1月湖州市妇幼保健院NICU收治的所有日龄≤28 d的新生儿(病例组)以及在本院足月出生的200例表型无明显异常的健康新生儿作为对照组。以中国可防可治罕见病、中国新筛病种等为基础,筛选明确致病基因与变异位点为靶标,设计适合中国新生儿的基因Panel,针对542种疾病、601个基因。制备干血斑样本,采用该基因Panel进行检测,检出的致病位点采用Sanger测序验证。结合临床特征分析患儿基因检测结果,依据患儿的主要临床诊断(早产儿、新生儿高胆红素血症、出血性疾病、新生儿感染、室间隔缺损/动脉导管未闭和其他)数,分为1、2、3和≥4种诊断。采用χ^(2)检验及线性关联χ^(2)检验进行统计学分析。结果病例组共纳入173例患儿,致病变异检出率为30.6%(53/173),包括致病基因阳性52例及染色体拷贝数变异1例。病例组致病基因阳性检出率高于对照组[30.1%(52/173)与15.0%(30/200),χ^(2)=12.26,P<0.001];病例组共检出14个致病基因,分别为FLG、UGT1A1、G6PD、MYH7、AR、ABCC2、ACADS、C YP21A2、GJB2、MEFV、PAH、PKHD1、SCN4A及HBA。在病例组,致病变异检出率在黄疸新生儿中高于无黄疸者[35.2%(44/125)与18.8%(9/48),χ^(2)=4.42,P=0.036],但在男婴女婴间、母亲是否高龄及是否有妊娠期糖尿病、是否早产儿、是否发生出血性疾病、是否有新生儿感染、是否发生室间隔缺损/动脉导管未闭组间差异均无统计学意义(P值均>0.05)。临床诊断数目1、2、3和≥4种的患儿,致病变异检出率呈线性增长[21.1%(8/38)、25.4%(15/59)、38.2%(13/34)与40.5%(17/42),线性关联χ^(2)=4.84,P=0.028]。在病例组,变异(包含致病基因阳性及携带者)检出率较高的基因有7个,检出率最高的为UGT1A1[检出率为24.9%(43/173)],其他依次为GJB2、FLG、DUOX2、ABCA4、G6PD和MUT基因;7个高频变异位点为UGT1A1基因c.211G>A(p.Gly71Arg)、c.1091C>T(p.Pro364Leu)、c.-41_-40dupTA及c.686C>A(p.Pro229Gln),GJB2基因c.109G>A(p.Val37Ile),FLG基因c.12064A>T(p.Lys4022Ter)及c.3321del(p.Gly1109GlufsTer13)。结论本研究设计的基因Panel可以为部分NICU患儿提供有效的基因检测,尤其是临床诊断复杂的患儿。

ObjectiveTo investigate the clinical value of targeted sequencing panel in the detection of genetic variation in neonates in neonatal intensive care unit(NICU).MethodsAll neonates(≤28 d of age)admitted in the NICU(case group)and 200 full-term healthy neonates born with no obvious phenotypic abnormalities of Huzhou Maternity and Child Health Care Hospital were enrolled in this prospective study from November 2022 to January 2023.Based on a list of preventable and treatable rare diseases as well as newly screened diseases in China,a targeted sequencing panel suitable for Chinese newborns was designed to target the pathogenic genes and mutation sites associated with 601 genes and 542 diseases.Dried blood spot specimens were prepared and analyzed by the targeted sequencing panel.Pathogenic sites detected by the panel sequencing were verified using Sanger sequencing.The genetic testing results were analyzed according to the clinical features of the neonates.According to the number of primary clinical diagnosis index(including premature infants,neonatal hyperbilirubinemia,hemorrhagic diseases,neonatal infections,ventricular septal defect/patent ductus arteriosus,and others),these patients were divided into four groups with 1,2,3,and≥4 diagnosis index,respectively.Chi-square test and linear correlation Chi-square test were used for statistical analysis.ResultsThere were 173 patients in the case group and 30.6%(53/173)of them carried pathogenic variants,including 52 positive for pathogenic genes and one with chromosome copy number variant.The positive rate of pathogenic genes was significantly higher in the case group than in the control group[30.1%(52/173)vs.15.0%(30/200),χ^(2)=12.26,P<0.001].Fourteen pathogenic genes were detected in the case group,including FLG,UGT1A1,G6PD,MYH7,AR,ABCC2,ACADS,CYP21A2,GJB2,MEFV,PAH,PKHD1,SCN4A,and HBA.In the case group,the detection rate of pathogenic variants in jaundiced neonates was higher than that in non-jaundiced neonates[35.2%(44/125)vs.18.8%(9/48),χ^(2)=4.42,P=0.036].However,there were no statistically significant differences in the detection rates of pathogenic variants between male and female infants,infants born to mothers of advanced maternal age or not,infants born to mothers with or without gestational diabetes mellitus,premature and term infants,or infants with or without hemorrhagic disorders,neonatal infections,or ventricular septal defects/patent ductus arteriosus in the case group(all P>0.05).The detection rate of pathogenic variants showed a linear increase in infants with 1,2,3,and≥4 diagnosis index[21.1%(8/38),25.4%(15/59),38.2%(13/34),and 40.5%(17/42);linear correlationχ^(2)=4.84,P=0.028].In the case group,seven genes with a high detection rate of genetic variation(including positive pathogenic genes and carriers)were UGT1A1[had the highest detection rate,24.9%(43/173)],GJB2,FLG,DUOX2,ABCA4,G6PD,and MUT.Seven loci with higher mutation frequency were c.211G>A(p.Gly71Arg),c.1091C>T(p.Pro364Leu),c.-41_-40dupTA,and c.686C>A(p.Pro229Gln)in the UGT1A1 gene,c.109G>A(p.Val37Ile)in the GJB2 gene,and c.12064A>T(p.Lys4022Ter)and c.3321del(p.Gly1109GlufsTer13)in the FLG gene.ConclusionThis panel sequencing can provide effective genetic testing for neonates in NICU,especially in children with complex clinical diagnosis.