希特林蛋白缺乏症新生儿早期血氨基酸谱特征及新生儿筛查评估

Newborn screening program and blood amino acid profiling in early neonates with citrin deficiency

目的观察早期发病的希特林蛋白缺乏症即Citrin缺陷致新生儿肝内胆汁淤积症(NICCD)患儿在新生儿早期血氨基酸及酰基肉碱谱特点及新生儿筛查敏感性,探讨NICCD新生儿筛查潜在生化指标。方法采用串联质谱技术对2015年1月1日—2019年6月30日在广州市出生的158651例新生儿进行NICCD筛查,回顾分析在广州市妇女儿童医疗中心经SLC25A13基因确诊的55例NICCD患儿的新生儿筛查干血斑氨基酸及酰基肉碱谱,以瓜氨酸≥30μmol/L为NICCD筛查阳性。采用t检验比较真阳性组及假阴性组血斑采集时间,采用F检验比较分析真阳性(瓜氨酸≥30μmol/L)、假阴性(瓜氨酸21-<30μmol/L、瓜氨酸<21μmol/L)及正常对照组之间氨基酸及酰基肉碱水平。结果在158651例新生儿筛查中,初筛阳性39例,确诊NICCD3例,发现假阴性4例,筛查阳性预测值7.7%,灵敏度约43.0%。回顾分析55例NICCD患儿的新生儿筛查干血斑氨基酸及酰基肉碱谱,NICCD真阳性组18例(18/55,32.7%),假阴性组37例(37/55,67.3%)。真阳性组采血时间为(4.28±1.60)d,与假阴性组(2.98±0.74)d比较差异有统计学意义(t=4.06,P<0.01)。在瓜氨酸≥30μmol/L(n=18)组中,血酪氨酸[(176.0±98.4)μmol/L]、甲硫氨酸[(37.0±26.9)μmol/L]、苯丙氨酸[(133.0±80.9)μmol/L]水平均明显高于其他3组,差异有统计学意义(F值分别为117.0、58.5、135.0,P<0.01)。血精氨酸[(10.0±9.2)、(11.0±9.3)、(9.0±17.8)μmol/L]、缬氨酸[(119.0±29.8)、(107.6±14.1)、(102±68)μmol/L]及亮氨酸[(167.0±37.1)、(161.0±37.7)、(163.5±180.6)μmol/L]水平在瓜氨酸≥30μmol/L(n=18)、瓜氨酸21-<30μmol/L(n=7)、瓜氨酸<21μmol/L(n=30)间差异均无统计学意义(P>0.05),但较正常对照组[(4±3)、(78±21)、(114.0±31.5)μmol/L,n=1000]差异均有统计学意义(F值分别为30.1、23.0、29.8,P<0.01)。丙氨酸在4组间[(150±50)、(156.0±30.2)、(168±105)、(152±52)μmol/L]差异无统计学意义(F=0.86,P>0.05),但丙氨酸/瓜氨酸降低在4组间(1.52±1.44、6.82±1.56、12.06±7.71、19.42±6.27)差异均具有统计学意义(F=69.0,P<0.05)。各组间血酰基肉碱谱差异均无统计学意义(P>0.05)。以瓜氨酸≥30μmol/L伴丙氨酸/瓜氨酸<7.5为筛查阳性切值,初筛阳性由39例减少至22例,未增加假阴性病例。以瓜氨酸≥21μmol/L伴丙氨酸/瓜氨酸<7.5为筛查阳性切值,初筛阳性数则增加至117例,确诊病例增加1例。结论NICCD患儿在新生儿早期即出现不同程度的瓜氨酸、酪氨酸、甲硫氨酸、苯丙氨酸等增高,丙氨酸/瓜氨酸降低的代谢谱特征,增加丙氨酸/瓜氨酸指标可提高NICCD筛查阳性预测值。NICCD新生儿筛查敏感性较低可能与采血时间过早其代谢谱特征尚未明显呈现有关。

Objective To investigate the profiles of blood amino acid and acylcarnitine in early neonates with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and the sensitivity of newborn screening, and to explore potential biochemical metabolic markers for newborn screening program. Methods Amino acid and acylcarnitine profiles in dried blood spots of newborn screening program were analyzed by tandem mass spectrometry (MS/MS). A total of 158 651 neonates born in Guangzhou from January 1, 2015 to June 30, 2019 were enrolled in this newborn screening program, and additionally 55 patients with NICCD confirmed by SLC25A13 gene analysis in Guangzhou Women and Children Medical Center were included in this study. NICCD screen-positive was defined as the cutoff value of citrulline (Cit)≥ 30 μmol/L. The values of blood sampling time of the true positive group and those of the false negative group were compared by t-test. The levels of amino acid and acylcarnitine among different groups, including true positive group (Cit≥30 μmol/L), false negative group (Cit 21-<30 μmol/L and Cit<21 μmol/L) and the normal control group, were analyzed by F test, respectively. Results Among 158 651 neonates, 39 neonates were positive for NICCD screening. Three of them were confirmed NICCD and 4 cases were found to be false negatives. The positive predictive value was 7.7% and the sensitivity was about 43.0%. Among 55 patients with NICCD, 18 cases (18/55, 32.7%) were true positives and 37 cases (37/55, 67.3%) were false negatives based on the cutoff value of citrulline in the dried blood spots for newborn screening. The blood sampling time was significantly different between true positive group and false negative group ((4.28±1.6) vs.(2.98±0.74) d, t=4.06, P<0.01). The increased levels of tyrosine((176.0±98.4)μmol/L), methionine ((37.0±26.9)μmol/L) and phenylalanine ((133.0±80.9)μmol/L) in Cit≥30 μmol/L group (n=18) were significantly different as compared with those in the other three groups, respectively (F=117.0, 58.5, 135.0, P<0.01). The levels of arginine ((10.0±9.2),(11.0±9.3),(9.0±17.8)μmol/L), valine ((119.0±29.8),(107.6±14.1),(102±68)μmol/L) and leucine ((167.0±37.1),(161.0±37.7),(163.5±180.6)μmol/L) were not statistically significant among groups of Cit≥30 μmol/L(n=18), Cit21-<30 μmol/L(n=7) and Cit<21μmol/L(n=30,P>0.05), but they were significantly higher than those of the normal control group ((4±3),(78±21),(114.0±31.5)μmol/L, n=1 000), respectively(F=30.1, 23.0, 29.8, P<0.01). Alanine (Ala)((150±50),(156.0±30.2),(168±105),(152±52)μmol/L) levels showed no significant difference (F=0.86, P>0.05) but the ratios of Ala/Cit (1.52±1.44, 6.82±1.56, 12.06±7.71, 19.42±6.27) decreased significantly among the four groups (F=69.0, P<0.05). The acylcarnitine levels showed no statistically significant results among the different groups (P>0.05). With Cit≥30 μmol/L and Ala/Cit<7.5 as cutoff values, the number of screen-positive cases reduced from 39 to 22 cases with no additional false negative case. With Cit≥21 μmol/L and Ala/Cit<7.5 as cutoff values the number of screen-positive cases increased to 117 cases with 1 additional true positive. Conclusions The profiles of blood amino acid in early neonates with NICCD present the increased levels of multiple amino acids including citrulline, tyrosine, methionine and phenylalanine, and decreased ratio of Ala/Cit. Taking citrulline and ratio of Ala/Cit as screening markers can improve the positive predictive value appropriately. The limited sensitivity of NICCD newborn screening may be related to early blood sampling time.