滤纸片干血斑酶活性检测用于新生儿黏多糖贮积症Ⅱ型筛查的初步研究

doi:10.12372/jcp.2025.24e0514

摘要/Abstract

摘要：

目的探索滤纸片干血斑（DBS）检测艾杜糖醛酸-2-硫酸酯酶（IDS）活性用于筛查新生儿黏多糖贮积症Ⅱ型（MPSⅡ）的可行性。方法采用荧光定量法检测DBS的IDS活性，对医院新生儿疾病筛查中心2021年2月至4月出生的2 202例男性新生儿进行MPSⅡ筛查。将2012年3月至2020年12月儿内分泌遗传科收集的已确诊的51例MPSⅡ患儿、15例MPSⅡ女性携带者及32例非MPSⅡ患儿（对照组，均为男性）作为试筛人群。初步拟定筛查阳性切值，阳性者召回后复查酶活性，并进行尿液黏多糖和IDS基因检测。结果 51例MPSⅡ患儿中位年龄4.2岁，15例MPSⅡ女性携带者中位年龄27.5岁，32例对照组中位年龄4.5岁，绘制ROC曲线在98例试筛人群中获得DBS法检测血IDS活性的最佳阈值为9.59ρmol/(punch·20h)，灵敏度为100%，特异度为22.7%，AUC为0.962（0.928~0.995）；并进行白细胞法与DBS法的一致性检验分析，Kappa=0.96（P<0.001）。以DBS法检测2 202名新生儿IDS活性水平，中位数为58.22ρmol/(punch·20h)，中位数的20%为11.64ρmol/(punch·20h)。无论以DBS IDS活性≤9.59ρmol/(punch·20h)，还是≤11.64ρmol/(punch·20h) 作为阳性切值，均为6例（0.27%）阳性。6例阳性患儿全部召回，筛查阳性者中，虽有3例尿黏多糖检测定性阳性，但电泳仅显示硫酸软骨素区带，未见硫酸皮肤素及硫酸类肝素区带；基因检测发现5例携带母亲来源的IDS错义突变c.851C>T(p.P284L)，另有1例携带c.1499C>T(p.T500I)错义突变，均被归为良性变异。结论 DBS IDS活性检测能够有效地快速识别出MPSⅡ可疑患儿，可尝试用于新生儿MPSⅡ的筛查，但仍需大样本量研究确定准确的阳性切值。

关键词: 黏多糖贮积症Ⅱ型, 艾杜糖醛酸-2-硫酸酯酶, 滤纸片干血斑, 新生儿筛查

Abstract:

Objective To explore the feasibility of detecting iduronate 2-sulfatase (IDS) activity in dried blood spot (DBS) for screening of mucopolysaccharidosis type Ⅱ (MPS Ⅱ) in newborns. Methods The IDS activity of DBS was detected by fluorescence quantification method, and MPS Ⅱ screening was performed on 2202 male newborns born from February to April 2021 in the Neonatal Disease Screening Center of the hospital. The 51 confirmed children with MPS Ⅱ, 15 female carriers of MPS Ⅱ and 32 non-MPS Ⅱ children (the control group, all male) collected by the Department of Pediatric Endocrinology and Genetic Metabolism from March 2012 to December 2020 were taken as the pre-screened population. A preliminary screening cut-off value for positive cases has been established, and those who test positive will be recalled for retesting of enzyme activity and urine mucopolysaccharides and IDS gene testing. Results The median age of 51 children with MPS Ⅱ was 4.2 years, the median age of 15 female carriers with MPS Ⅱ was 27.5 years, and the median age of 32 control patients was 4.5 years. ROC curve was drawn, and the optimal threshold of DBS for detection of blood IDS activity was 9.59 ρmol/(punch‧20h) in 98 pre-screened patients, with sensitivity of 100%, specificity of 22.7% and AUC of 0.962 (0.928-0.995). The consistency analysis between the white blood cell method and DBS method showed that Kappa=0.96 (P<0.001). The activity level of IDS in 2202 neonates was detected by DBS method. The median value of IDS activity was 58.22 ρmol/(punch‧20h) and 20% of the median value was 11.64 ρmol/(punch‧20h). Whether the DBS IDS activity ≤9.59 ρmol/(punch‧20h) or≤11.64 ρmol/(punch‧20h) was taken as the positive cut-off value, 6 cases (0.27%) were detected as positive and recalled. Among the initial screening positive cases, although 3 patients had a qualitatively positive urine mucopolysaccharides, the electrophoresis showed only chondroitin sulfate band without dermatan sulfate and heparan sulfate bands. Genetic testing found five cases carrying the mother- derived IDS missense variant of c.851C>T (p.P284L) and one case carrying c.1499C>T (p.T500I), all of which were classified as benign variants. Conclusions The IDS activity test on DBS can effectively and promptly identify suspected patients with MPS Ⅱ and can be used for newborn screening of MPS Ⅱ. However, a large sample size study is still needed to determine the accurate positive cut-off value.

Key words: mucopolysaccharidosis type Ⅱ, iduronate-2-sulfatase, dried blood spot, newborn screening

高晓岚, 梁欢, 陈国庆, 张惠文, 韩连书, 邱文娟, 顾学范. 滤纸片干血斑酶活性检测用于新生儿黏多糖贮积症Ⅱ型筛查的初步研究[J]. 临床儿科杂志, 2025, 43(3): 191-198.

GAO Xiaolan, LIANG Huan, CHEN Guoqing, ZHANG Huiwen, HAN Lianshu, QIU Wenjuan, GU Xuefan. Exploring the feasibility of enzyme activity assay of dried blood spots for newborn screening of mucopolysaccharidosis type Ⅱ[J]. Journal of Clinical Pediatrics, 2025, 43(3): 191-198.

图/表 8

表1

图1

表2

表3

51例MPSⅡ患儿IDS酶活性、尿液MPS定性和电泳及IDS基因型"

患儿	性别	IDS白细胞法/nmol·4 h^-1·mg蛋白^-1	IDS DBS法/ρmol·punch^-1·20 h^-1	尿MPS定性	尿MPS电泳	基因型
例1	男	0.08	1.08	阳性	DS	c.781delC
例2	男	0.20	2.16	阳性	CS, DS	c.1122C>T
例3	男	0.00	0.00	阳性	DS	c.678_679insCAT
例4	男	0.15	2.08	阳性	DS	c.262C>T
例5	男	0.16	2.31	－	－	A型携带者
例6	男	0.23	1.71	阳性	DS	c.187A>G
例7	男	1.53	9.23	阳性	DS	c.442G>T
例8	男	0.20	1.93	阳性	CS, DS	c.262C>T
例9	男	0.29	0.58	阳性	DS	c.1270_1271 insC
例10	男	0.00	2.54	阳性	DS	c.401G>T
例11	男	0.05	2.56	－	－	c.416delC
例12	男	0.15	1.68	阳性	CS, DS	c.1122C>T
例13	男	0.01	2.89	阳性	DS	c.253G>A
例14	男	0.21	2.60	阳性	DS	C型携带者
例15	男	0.02	1.73	阳性	DS	c.1028G>T
例16	男	0.07	1.44	阳性	DS	C型携带者
例17	男	0.03	2.48	－	－	A型携带者
例18	男	0.30	2.20	阳性	DS	c.1270delG
例19	男	0.00	2.83	阳性	DS	delE1~E6
例20	男	0.10	1.64	阳性	CS, DS	c.418G>A
例21	男	0.23	1.85	阳性	DS	c.702C>G
例22	男	0.00	2.04	阳性	DS	c.1454T>A
例23	男	0.00	1.28	阳性	CS, DS	c.241-2A>G
例24	男	0.00	2.52	阳性	DS	c.401G>T
例25	男	0.13	2.87	阳性	CS, DS	C型携带者
例26	男	0.09	1.93	－	－	c.1480_148(ins37)
例27	男	0.01	3.54	－	－	c.283A>G
例28	男	0.11	0.29	－	－	A型携带者
例29	男	0.22	1.64	阳性	CS, DS	c.1506G>T
例30	男	0.31	1.89	阳性	DS	c.361C>T
例31	男	0.89	3.52	阳性	CS, DS	c.801G>A
例32	男	0.19	3.14	阳性	DS	c.1119_1120delAG
例33	男	0.15	0.10	阳性	CS, DS	c.908_909delCT
例34	男	0.01	3.93	阳性	CS, DS	－
例35	男	0.65	2.44	阳性	CS, DS, HS	c.1403G>A
例36	男	0.27	3.02	阳性	CS, DS	delE4~E7
例37	男	0.64	5.30	阳性	DS	c.1050T>G
例38	男	0.14	1.22	阳性	CS, DS	－
例39	男	0.14	1.15	－	－	c.685C>T
例40	男	0.45	4.76	阳性	CS, DS	－
例41	男	0.34	4.70	阳性	CS, DS	c.940delC
例42	男	0.55	4.60	阳性	CS, DS	c.748_749insG
例43	男	0.23	2.99	阳性	DS	－
例44	男	0.67	4.64	阳性	CS, DS	－
例45	男	0.00	1.97	阳性	CS, DS	－
例46	男	0.01	1.09	阳性	CS, DS	－
例47	男	0.09	2.20	阳性	CS, DS	－
例48	男	0.26	4.60	阳性	CS, DS	－
例49	男	0.46	5.37	阳性	DS	大片段插入
例50	男	0.06	2.18	阳性	CS, DS	c.281G>T
例51	男	0.17	0.67	阳性	CS, DS	－

表3

图2

表4

图3

图4

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实验室编号	性别	年龄/d	尿GAGs定量/ mg·mmoL^-1	尿GAGs电泳	DBS IDS活性/ ρmol·punch^-1·20 h^-1	碱基改变	ACMG致病	变异位置
10738	男	3	－	－	1.60	c.851C>T(p.P284L)	临床意义未明	E6
11401	男	5	413.77	CS	2.72	c.851C>T(p.P284L)	临床意义未明	E6
11824	男	3	321.07	CS	2.33	c.851C>T(p.P284L)	临床意义未明	E6
11840	男	3	211.00	CS	6.84	c.851C>T(p.P284L)	临床意义未明	E6
18835	男	3	－	－	4.57	c.1499C>T(p.T500I)	临床意义未明	E9
19290	男	3	－	－	0.10	c.851C>T(p.P284L)	临床意义未明	E6