Effect of Newborn Genomic Screening for Lysosomal Storage Disorders: A Randomized Clinical Trial in China

doi:10.21203/rs.3.rs-4877360/v1

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Effect of Newborn Genomic Screening for Lysosomal Storage Disorders: A Randomized Clinical Trial in China

https://doi.org/10.21203/rs.3.rs-4877360/v1

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Background

Lysosomal storage disorders (LSDs) have a relatively high incidence among rare diseases and can lead to severe consequences if not treated promptly. However, many countries and regions have not included these disorders in their newborn screening programs, resulting in missed early detection, underdiagnosis, and delayed treatment. Newborn genomic screening has shown good screening effectiveness for traditional biochemical screening diseases; however, its effectiveness for LSDs has not yet been evaluated in the general newborn population.

Methods

To evaluate the outcome of newborn genomic screening for LSDs, a cohort study was conducted involving newborns recruited from Nanjing Women and Children’s Healthcare Hospital in China from March 18, 2022, to September 21, 2023. All participants underwent genomic screening of 15 LSDs (18 genes) via dried blood spots, with enzyme activity testing of positive samples. The study calculated the prevalence and carrier rates for each LSD, as well as the positive screening rate, the false positive rate and the positive predictive value of the screening process.

Results

This study prospectively recruited 22,687 newborns (11,996 males [52.88%]). The mean (SD) gestational week was 39.2 (1.08) weeks, and the mean (SD) birth weight was 3369.7 (412.95) g. Genomic screening identified 1,344 carriers (6.0%) and 30 (0.13%) initial positive newborns for LSDs. Among the 30 initial positive newborns, 4 were excluded, 15 newborns were diagnosed, and 11 newborns were followed-up. The combined birth incidence of LSDs in Nanjing, Jiangsu Province, is 1/1,512, mainly Fabry disease, Krabbe disease, glycogen storage disease type II, Niemann-Pick disease and mucopolysaccharidosis type II. Compared with enzyme activity detection, newborn genomic screening had greater positive predictive value (P < 0.05) and a lower false positive rate (P < 0.05).

Conclusions

This study emphasizes the clinical utility of incorporating newborn genomic screening for LSDs into routine newborn screening, offering a proactive approach for early detection and intervention, ultimately enhancing public health and the well-being of newborns and their families.

Lysosomal storage disorder

Newborn screening

Newborn genomic screening

Rare diseases

Newborn screening is a highly effective public health initiative that involves widespread screening for specific genetically or congenitally harmful diseases during the newborn phase. The objective is to achieve early diagnosis and treatment, aiming to prevent or mitigate the negative impact of these diseases and enhance the quality of life for families affected by them¹. Presently, newborn screening predominantly focuses on genetic metabolic disorders due to limitations in detection methodologies^2–5.

Among rare diseases, lysosomal storage disorders (LSDs) have a relatively high overall incidence and can lead to severe consequences. Early detection, diagnosis, and intervention play crucial roles in the clinical management of LSDs. However, only a handful of regions, such as specific areas in the United States⁶, northern Italy⁷, Japan⁸, and Shanghai and Taiwan in China^{9, 10}, have introduced newborn screening for some LSDs, and the screening methodology in these regions involves measuring lysosomal enzyme activity.

Research conducted in various countries and regions indicates that newborn genomic screening (NBGS) has multiple benefits. These include a significant reduction in the false-positive rate associated with traditional newborn screening (tNBS), broader disease coverage, more detailed risk information, and enhanced support for future genetic counseling^11–13. Moreover, prospective parents generally strongly support the implementation of the NBGS^14–17. Collectively, these findings suggest that the NBGS has the potential to represent a significant advancement in the field of newborn screening. However, the potential of NBGS for newborn LSDs remains inadequately explored. Consequently, we plan to analyze samples from more than 22,000 newborns to assess the clinical application value of integrating LSDs genomic screening with newborn screening. This study aims to establish a foundation for the clinical screening, diagnosis, and genetic counseling of LSDs.

Study population and data collection

This investigation was conducted at Nanjing Women and Children’s Healthcare Hospital in Jiangsu Province, China. From March 18, 2022, to September 21, 2023, a total of 30 440 infants were born at Nanjing Women and Children’s Healthcare Hospital, 22,687 of whom participated in the combined screening approach.

Ethical approval

for the study was granted by the Ethics Committee of the Women’s Hospital of Nanjing Medical University (2021KY-071), and written informed consent was obtained from all participants.

Heel blood collection

When the newborn was 48–72 hours old and fully lactating, a 200 µL heel blood sample was obtained to produce a dried blood filter paper for the detection of pathogenic genes through targeted capture-based next-generation sequencing (NGS).

NBGS based on targeted capture-based NGS

A detailed list of 15 lysosomal diseases and the corresponding 18 genes identifiable through targeted capture-based NGS is provided in Table S1 in the Supplement. Genomic DNA was extracted via the QIAamp DNA Blood Midi Kit (51185, Qiagen, Hilden, Germany), followed by fragmentation via a Covaris LE220 ultrasonic instrument. Magnetic beads were used to isolate fragments (150–200 bp), which were subjected to purification, end repair, A-tailing, and adapter ligation for DNA library construction. Library quantification was performed with an Agilent Bioanalyzer 2100. After A-tailing and ligation, a custom IDT xGen Lockdown probe captured the target sequences, and the hybridization library was sequenced via a high-throughput gene sequencer (MGISEQ-2000) following dynabead creation with a PE100 + 10 sequencing type.

LSDs can be included in newborn screening according to the principle of early disease detection ^18–20, which meets the following criteria: (1) the disease can be seriously harmful, and its early symptoms might not be obvious; (2) we have a good understanding of the disease, it occurs somewhat frequently, and without timely intervention, it can lead to poor outcomes; and (3) there are effective treatments available for the disease.

Priority was given to variants meeting either of the following criteria: (1) identified as pathogenic or likely pathogenic in ClinVar; (2) categorized as nonsense, frameshift, or canonical variants affecting splice sites or the initiation codon in genes known for loss-of-function mechanisms, with an allele frequency of 1% or less in population databases (GnomAD, ESP6500, and 1000 Genomes). Interpretation followed American College of Medical Genetics and Genomics guidelines, with only curated pathogenic or likely pathogenic variants included in the report. A genetically positive result was defined by the presence of pathogenic or likely pathogenic variants matching the inheritance pattern.

Bioinformatics analysis

The Illumina bcl2fastq tool was used to convert raw high-throughput sequencing data from the Bcl format to the Fastq format. After low-quality reads were filtered out, the sequencing reads were aligned to the NCBI human reference genome (hg19/GRCh37). GATK software was used for the detection of single nucleotide variants and insertions/deletions (indels). Variant site frequencies in the normal population were obtained from dbSNP (http://www.ncbi.nlm.nih.gov/snp), the 1000 Genomes Project (http://browser.1000genomes.org), and the Exome Aggregation Consortium (http://exac.broadinstitute.org/).

The interpretation of variants followed the guidelines of the American College of Medical Genetics²¹ and literature searches^22–24, considering the level of evidence supporting pathogenicity. Pathogenic genes and sites were identified and correlated with diseases via databases such as OMIM (http://www.omim.org), ClinVar (http://www.ncbi.nlm.nih.gov/clinvar), and the Human Gene Variant Database (http://www.hgmd.org). Predictions of the biological functions affected by the variants were made via software, including SIFT (http://sift.jcvi.org), Variant Taster (http://www.varianttaster.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2), and PROVEAN (http://provean.jcvi.org/index.php).

Sanger sequencing

Positive variants identified by NGS were validated by Sanger sequencing. Genomic DNA extracted from dried blood spots was used to amplify the target region with specific primers. The amplification reaction employed Phanta Max Master Mix (Vazyme, China). The purified PCR products were subjected to sequencing and analysis through capillary electrophoresis, which was conducted with an ABI Prism 3500XL Genetic Analyzer (Thermo Fisher, Waltham, MA, USA).

Lysosomal enzyme activity detection

The NeoLSD™ MSMS kit (PerkinElmer, Massachusetts, USA) was used for the quantitative measurement of the activity of the lysosomal enzymes acid-β-glucocerebrosidase (ABG), acid-sphingomyelinase (ASM), acid-α-glucosidase (GAA), β-galactocerebrosidase (GALC), α-galactosidase A (GLA) and α-L-iduronidase (IDUA). A 3.2 mm dried blood spot was punched into a 96-well plate and treated with a 30 µL assay cocktail (3093–0020, PerkinElmer, USA). After sealing, the plate was incubated for 18 hours at 37°C with orbital shaking. Following incubation, 100 µL of quenching solvent (methanol/ethyl acetate, 1:1, v/v) was added, and the mixture was transferred to a deep well plate. Liquid‒liquid extraction was performed by adding ethyl acetate and water to each well. After centrifugation, 50 µL of the ethyl acetate phase was transferred to a new plate, and the solvent was evaporated with nitrogen gas. The sample was reconstituted with 100 µL of mobile phase solution (3093–0020, PerkinElmer, USA) before being subjected to LC‒MS/MS analysis via a Waters 1525µ HPLC PUMP and Xevo TQD instrument equipped with a Waters 2777 sample manager.

Statistical analysis

Data are expressed as the median ± standard deviation. Ordinary one-way ANOVA, the Kruskal‒Wallis test, multiple t tests or paired t tests were used for statistical comparisons. Two-sided P values < 0.05 were considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001). GraphPad version 6.02 was used for statistical analysis.

1. Study population

A total of 30,440 newborns were delivered at Nanjing Women and Children’s Healthcare Hospital. Among them, 22,687 individuals (11,996 males [52.88%]) underwent NBGS, resulting in a genomic screening rate of 74.53%. The mean (SD) number of gestational weeks was 39.2 (1.08) weeks, the mean (SD) birth weight was 3369.7 (412.95) g, and 9456 (41.69%) of them had a cesarean delivery (Table 1).

Table 1

Characteristics of the newborns in the study cohort.
	NBGS	NBGS + Enzyme activity detection
	No. (%) (N = 22256)	All (N = 900)	Negative (N = 290)	Carrier (N = 543)	Positive (N = 29)	Other Positive (N = 38)
Sex
Male	11996 (52.88)	490 (54.44)	158 (54.48)	290 (53.41)	14 (48.28)	28 (73.68)
Female	10691 (47.12)	410 (45.56)	132 (45.52)	253 (46.59)	15 (51.72)	10 (26.32)
Unknown	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Gestation age
Preterm (< 37wk)	844 (3.79)	25 (2.78)	2 (0.69)	19 (3.50)	3 (10.34)	1 (2.63)
Full term (37-40wk)	16178 (72.69)	661 (73.44)	220 (75.86)	396 (72.93)	19 (65.52)	26 (68.42)
Late term (40.1-42wk)	5223 (23.47)	214 (23.78)	68 (23.45)	128 (23.57)	7 (24.14)	11 (28.95)
Post term (≥ 42wk)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Unknown	11 (0.05)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Birth weight
<2750 g	1342 (6.03)	44 (4.89)	6 (2.07)	34 (6.26)	3 (10.34)	1 (2.63)
2750–4000 g	19810 (89.01)	816 (90.67)	276 (95.17)	483 (88.95)	24 (82.76)	33 (86.84)
>4000 g	1094 (4.92)	40 (4.44)	8 (2.76)	26 (4.79)	2 (6.90)	4 (10.53)
Unknown	10 (0.04)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Delivery
Eutocia	13224 (58.29)	500 (55.56)	142 (48.97)	325 (59.85)	11 (37.93)	22 (57.89)
Cesarean	9456 (41.71)	400 (44.44)	148 (51.03)	218 (40.15)	18 (62.07)	16 (42.11)
Unknown	7 (0.03)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)

2. Screening status for LSDs genes

Among the 22,687 individuals screened, 21,296 tested negatives for LSD gene screening, 1,361 were carriers (24 carriers with two different disease variants), and 30 had initial positive results (Fig. 1). Among the initial positive cases, Fabry disease had the highest proportion (50.00%) and the highest initial positive rate (0.066%, 1/1,512), followed by Krabbe disease (26.67% and 0.035%, 1/2,836), glycogen storage disease type II (GSD II) (13.33% and 0.018%, 1/5,672), Niemann‒Pick disease (NPD) (6.66% and 0.009%, 1/11,344) and mucopolysaccharidosis type II (MPS II) (3.33% and 0.004%, 1/22,687). The overall initial positive rate for LSDs was approximately 0.13% (1/756) (Table 2).

Table 2

Results from the NBGS of the LSDs
Disease	Negative screening result	Positive screening result	Positive screening rate (1/n)	Diagnosed	Prevalence (1/n)	Excluded (Carrier)	Follow-up	FPR	PPV
Fabry	22672	15	1/1 512	5	1/4537	0	10	NA	NA
Krabbe	22679	8	1/2 836	8	1/2 836	0	0	0%	100.00%
GSD II	22683	4	1/5 672	1	1/22 687	3	0	0.0132%	25.00%
NPD	22685	2	1/11 344	1	1/22 687	1	0	0.0044%	50.00%
MPS II	22686	1	1/22 687	0	NA	0	1	NA	NA
All	22657	30	1/756	15	1/1 512	4	11	0.0177%	50.00%

FPR, false positive rate = false positives/(false positives + true negatives) = newborns without the disease but with positive NBGS results in the related gene/all newborns without the disease; PPV, positive predictive value = true positives/(true positives + false positives) = patients with the disease, and positive NBGS results in related genes/all newborns with positive NBGS results in the disease-related gene. NA, not applicable.

NBGS identified a total of 1,367 pathogenic variants for LSDs carried by 1,344 newborns. The overall carrier rate was approximately 6.03% (1/17), with Krabbe disease having the highest carrier rate (2.56%, 1/39), followed by NPD (1.05%, 1/95) and GSD II (0.86%, 1/116). Among Krabbe disease carriers, the c.1901T > C variant is highly prevalent, accounting for up to 86.38%. The c.955C > G variant is more prevalent at 65.19% in carriers of the NPD-A/B type, and the most prevalent variant observed in the GSD II type is c.2132_2133delinsGG, accounting for approximately 23.08%. (Table S2).

3. Enzyme activity detection for LSDs.

The lysosomal enzyme assay kit is limited to detecting GALC, GAA, ASM, IDUA, and GLA. Lysosomal enzyme activity was detected in a total of 900 samples. Among these, 290 healthy newborns with negative results in traditional newborn screening were utilized to establish high-risk cutoff values (0.2 or 0.3 multiples of the median)^{7, 25} and 0.5–99.5 percentile reference ranges for LSDs enzyme activity (Table S3). The histogram and statistical data illustrating the distribution of enzymatic activity in healthy newborns are displayed in Figure S1. No pathogenic variants in the LSDs genes were detected in these samples. A cutoff value lower than the high-risk cutoff value with relevant gene variants confirmed the diagnosis.

There were 29 genomic screening positive samples for LSDs (excluding one case of MPS II without corresponding enzyme activity detection) (Fig. 1 and Table 3) and 38 positive samples for other diseases (Table S4). Additionally, 543 samples were LSDs carriers (220 Krabbe carriers, 153 glycogen GSD II carriers, 117 NPD-A/B carriers, 45 mucopolysaccharidosis type I (MPS I) carriers, and 8 individuals with more than one gene carrier). The characteristics of these newborns are shown in Table 1. The enzyme activity results for individuals with negative, carrier, and positive genomic screening results are illustrated in Fig. 2, which shows a distinct decrease in enzyme activity in positive samples compared with that in negative and carrier samples. Additionally, the carriers presented significantly lower enzyme activity than did the negative samples. There were no statistically significant differences in gestational age or birth weight (Figure S2).

Table 3

Genomic screening positive samples for LSDs.
No.	Disease	Gene	Genotype	Location	Gender	Pathogenetic	Enzyme activity	Status
1	Fabry disease	GLA	c.640-801G > A/.	IVS4/.	Male	P	1.91	Diagnosed
2			c.640-801G > A/.	IVS4/.	Male	P	1.34	Diagnosed
3			c.640-801G > A/.	IVS4/.	Male	P	1.41	Diagnosed
4			c.1067G > A/.	EX7E/.	Male	LP	1.57	Diagnosed
5			c.1067G > A/.	EX7E/.	Male	LP	1.03	Diagnosed
6			c.911G > C/.	EX6/.	Male	LP	4.76	Follow-up
7			c.911G > C/.	EX6/.	Male	LP	4.27	Follow-up
8			c.640-801G > A/-	IVS4/-	Female	P	3.38	Follow-up
9			c.640-801G > A/-	IVS4/-	Female	P	4.32	Follow-up
10			c.640-801G > A/-	IVS4/-	Female	P	8.62	Follow-up
11			c.640-801G > A/-	IVS4/-	Female	P	3.43	Follow-up
12			c.911G > C/-	EX6/-	Female	LP	9.75	Follow-up
13			c.911G > C/-	EX6/-	Female	LP	8.58	Follow-up
14			c.1072_1074delGAG/-	EX7E/-	Female	LP	4.41	Follow-up
15			c.593T > C/-	EX4/-	Female	VUS	2.62	Follow-up
16	Krabbe	GALC	c.1901T > C/ c.1912G > A	EX16/EX17E	Male	LP/LP	0.74	Diagnosed
17			c.1901T > C/ c.2041G > A	EX16/EX17E	Male	LP/LP	0.21	Diagnosed
18			c.1901T > C/ c.1901T > C	EX16/ EX16	Male	LP/LP	0.29	Diagnosed
19			c.1901T > C/ c.1901T > C	EX16/ EX16	Male	LP/LP	0.34	Diagnosed
20			c.1901T > C/ c.1901T > C	EX16/ EX16	Female	LP/LP	0.17	Diagnosed
21			c.1901T > C/ c.1901T > C	EX16/ EX16	Female	LP/LP	0.24	Diagnosed
22			c.1901T > C/ c.1901T > C	EX16/ EX16	Female	LP/LP	0.31	Diagnosed
23			c.1901T > C/ c.1901T > C	EX16/ EX16	Female	LP/LP	0.95	Diagnosed
24	GSD II	GAA	c.2237G > A, c.503G > A /-	EX16, EX2/-	Male	P/LP	3.51	Excluded
25			c.2132_2133delinsGG, c.1669A > T/-	EX11/EX20E	Male	LP/P	3.80	Excluded
26			c.1634C > T/ c.2815_2816delGT	EX15, EX12/-	Male	LP/LP	0.63	Excluded*
27			c.2132_2133delinsGG, c.1669A > T/-	EX15, EX12/-	Female	LP/LP	0.36	Diagnosed
28	NPD-A/B	SMPD1	c.995C > G/ c.995C > G	EX2/EX2	Female	LP/LP	0.26	Diagnosed
29	NPD-C	NPC1	c.1351G > A, c.3734_3735delCT/-	EX9/EX24	Female	LP/LP	14.74	Excluded
30	MPS II	IDS	c.817C > T/.	EX6/.	Male	LP/.	-	Follow-up

*, The pseudo-defective alleles c.1726G > A and c.2065G > A were detected.

4. Prevalence and genetic profiles of LSDs.

Among the 30 newborns with positive results from the genomic screening for lysosomal diseases, 4 were excluded after familial verification and enzyme activity testing. Fifteen newborns were diagnosed, and 11 newborns were followed-up (Table 2 and Table 3). The combined birth incidence rate, which included Fabry disease, Krabbe disease, GSD II, NPD and MPS II, was 1/1,512, and the diagnosis rate was 0.066%.

There were 15 newborns identified as potential Fabry patients, with an overall incidence rate of 1/1,512. Among them, seven were male, with a male incidence rate of approximately 1/1,590, and eight were female, with a female incidence rate of approximately 1/1,391. Enzyme activity detection of GLA was detected in only five male patients, yielding an incidence rate of approximately 1/2,226, which is lower than the rate determined by the NBGS. The primary GLA variant was c.640-801G > A (7/15), followed by c.911G > C (4/15). Eight newborns were diagnosed with Krabbe disease, with an incidence rate of 1/2,782. The primary genotypes included homozygous variations of c.1901T > C (3/4) and compound heterozygous variations with c.1901T > C (1/4). There is one potential patient each for GSD II, NPD, and MPS II, leading to an incidence rate of 1/22,256 for each of these three diseases.

5. NBGS is more efficient than enzyme activity detection for LSDs.

After our statistical investigation, the false positive rates (FPRs) and positive predictive values (PPVs) for NBGSs of LSDs such as Krabbe disease, GSD II, and NPD were obtained, as shown in Table 2. Enzyme activity results in a lack of representativeness in female patients with Fabry disease^{26, 27}, preventing the evaluation of PPV and FRP. Compared with previous studies, NBGS shows better potential than enzyme activity screening for lysosomal storage disorders, especially in Asian regions such as Japan, Taiwan, and China (Table 4)^{6, 7, 10, 28–35}.

Table 4

FRP and PPV in NBGS + Enzyme activity detection group
	NBGS			Enzyme activity screening
Disease	Screened newborns	FPR	PPV	Region	Screened newborns	FPR	PPV	Reference
Krabbe	22,687	0%	100%	Shanghai of China	50,108	0.046%	28.13%	⁹
				New York of USA	550,000	0.004%	8.00%	²⁴
GSD II	22,687	0.0132%	25%	Shanghai of China	50,108	0.584%	1.01%	⁹
				Taiwan of China	191,786	0.44%	1.83%	²⁵
				Taiwan of China	402,281	1.03%	0.62%	²⁶
				Taiwan of China	473,738	0.47%	1.25%	²⁷
				Taiwan of China	132,538	0.82%	0.37%	²⁸
				Japan	297,387	0.033%	7.34%	²⁹
				Japan	103,204	0.215%	1.33%	³⁰
				North East Italy	44,411	0.007%	25.00%	²²
				Missouri of USA	43,702	0.023%	44.44%	⁷
				Hungary	40,024	0.14%	14.06%	³¹
				Austria	34,736	0.003%	80.00%	³²
NPD-A/B	22,687	0%	100%	Shanghai of China	50,108	0.002%	83.33%	⁹
				Austria	34,736	0.003%	0%	³²
				Hungary	40,024	0.007%	40.00%	³¹

FPR, false positive rate = false positives / (false positives + true negatives); PPV, positive predictive value = true positives / (true positives + false positives).

Furthermore, when the FRP and PPV were compared between the genomic screening and enzyme activity screening methods for Krabbe, GSD II, and NPD in the NBGS + enzyme activity detection group, it was observed that the genomic screening FRP for LSDs was significantly lower than that of the enzyme activity detection (P < 0.05), whereas the PPV was significantly greater than that of the enzyme activity detection method (P < 0.05) (Table 5). This finding indicates that, compared with relying solely on lysosomal enzyme activity screening, incorporating NBGS has a more proactive role in detecting LSDs.

Table 5

FRP and PPV in NBGS + Enzyme activity detection group
	Disease	Krabbe	GSD II	NPD-A/B
NBGS	Negative	290	290	290
	Carrier	220	153	117
	True positive	8	1	1
	False positive	0	3	1
	FPR	0%	0.67%	0.24%
	PPV	100.00%	25.00%	50.00%
Enzyme activity detection	Negative	495	431	403
	True positive	6	1	1
	False positive	7	11	3
	NBGS Negative	2	2	2
	NBGS Carrier	5	9	1
	False negative	2	0	0
	FPR	1.37%	2.48%	0.74%
	FPR (exclude NBGS carrier)	0.009%	0.009%	0.009%
	PPV	46.15%	8.33%	25.00%
	PPV (exclude NBGS carrier)	75.00%	33.33%	33.33%
P-value	FPR1 (NBGS vs Enzyme activity detection)	< 0.05
	FPR2 (NBGS vs Enzyme activity detection excluded NBGS carrier)	NS
	PPV1 (NBGS vs Enzyme activity detection)	< 0.05
	PPV2 (NBGS Negative vs Enzyme activity detection excluded NBGS carrier)	NS

FPR, false positive rate = false positives / (false positives + true negatives); PPV, positive predictive value = true positives / (true positives + false positives).

6. Understanding factors influencing lysosomal enzyme activity through the NBGS

Among carriers of LSDs, approximately 50% exhibit enzyme activity within the 10–20th percentile of negative samples, whereas 70–85% fall within the 50th percentile (Table S5). This finding indicates that lysosomal enzyme activity in carriers of pathogenic genes for LSDs often decreases to half of the levels observed in the general population, with approximately half of the carriers displaying even lower enzyme activity, which may increase the likelihood of false-positive results in lysosomal activity testing for carriers.

Moreover, carriers with multiple genes that are pathogenic for LSDs show a reduction in the corresponding enzyme activities of each gene (Fig. 3A). When carriers have both pathogenic genes for LSDs and genes for other conditions, such as those identified in traditional newborn screening or diseases lacking biochemical testing methods, lysosomal enzyme activity remains unaffected, which indicates that carrier pathogenic variants for other diseases may not necessarily impact the detection of lysosomal enzyme activity (Figure S3). Testing lysosomal enzyme activity in positive cases from NBGS for several common newborn genetic disorders revealed a decrease in IDUA enzyme activity in infants with hypothyroidism, indicating a greater likelihood of false positives in IDUA enzyme activity for hypothyroid infants (Fig. 3B).

To explore the significance and clinical application of NBGS for LSD screening, we conducted prospective NBGS on 22,687 newborns and selected 900 newborns for lysosomal enzyme activity testing to compare the two screening methods preliminarily. To our knowledge, this study is the first to focus on the role of NBGS in LSD screening and compare it with current enzymatic screening methods, aiming to evaluate the potential benefits of applying NBGS to LSD screening.

Our prospective cohort study revealed that among the 22,687 newborns screened with NBGS, 30 were positive, with an initial screening positivity rate of 0.13%. Four cases were excluded after familial validation, resulting in a false positive rate of 0.0177%, which is much lower than the reported false positive rate for enzyme activity screening³⁶. Fifteen cases were confirmed, with a positive predictive value (PPV) of approximately 50%. The overall prevalence is 1/1,512, much higher than the prevalence estimated on the basis of enzyme activity screening statistics^36–38, indicating that NBGS-based neonatal LSD screening can identify more potentially affected infants before the onset of symptoms. Additionally, approximately one potential LSD patient per 1,512 newborns may benefit from timely disease tracking management and intervention through the NBGS. Furthermore, by comparing the targeted capture-based NGS technology used in NBGS with the enzymatic detection technology used in LSD screening in some regions, we found that genomic screening, compared with enzymatic screening, can not only reduce the FPR but also significantly increase the PPV (Table 4 and Table 5), indicating that genomic screening can effectively improve the detection rate of LSDs and play a more proactive role in LSD screening.

The prevalence of LSDs reported in our study may be greater than that previously reported in clinically symptomatic populations³⁸. This difference arises because earlier statistics primarily included patients who had already developed symptoms and sought medical attention, potentially overlooking individuals who are asymptomatic, misdiagnosed, or have mild symptoms and did not seek medical care. In contrast, the NBGS can detect presymptomatic patients who have not yet exhibited obvious clinical symptoms. A recent study on newborn screening for LSDs on the basis of lysosomal enzyme activity in the Shanghai region of China reported a prevalence similar to that reported in our study⁹.

LSDs encompass a wide range of diseases, with up to 70 types³⁸. Currently, LSDs clinically screened through enzyme activity mainly include four to six types, such as Fabry disease, GSD II, NPD, Gaucher disease, mucopolysaccharidosis type I, and Krabbe disease. We propose a screening approach that uses genomic testing as first-tier screening supplemented by enzymatic testing. The conditions included are not all LSDs but 15 specific LSDs selected after comprehensive evaluation according to the W&J criteria¹⁸, expanding the scope of screened diseases compared with enzyme activity screening. Each of these 15 LSDs has a clear diagnostic method, primarily combining enzymatic and genetic testing, which allows for presymptomatic diagnosis. Furthermore, all 15 LSDs have treatment options available (Table S1), including symptomatic and supportive therapy, small molecule therapy, substrate reduction therapy, enzyme replacement therapy, and gene therapy^{38, 39}. Individuals diagnosed with LSDs through genomic screening and enzyme activity detection may not yet exhibit clinical symptoms, often presenting with subclinical phenotypes. In this study, these presymptomatic individuals are termed "potential patients" and undergo regular follow-ups, similar to health management for high-risk populations. We closely monitor their health status more intensively than that of normal individuals, allowing us to detect and treat early clinical symptoms promptly, leading to better treatment outcomes. A consensus on the long-term follow-up of newborns who test positive for Krabbe disease serves as a reference for guidelines on other LSDs⁴⁰, underscoring the necessity of early detection and ongoing follow-up. Without early screening, irreversible organ damage often occurs when symptoms prompt medical attention, adversely affecting treatment outcomes. Thus, genomic screening for LSDs ensures comprehensive health management for subclinical patients, promoting healthier and more secure lives.

In this study, by comparing the FPR and PPV of NBGS and enzymatic detection in the same population, we found that the interference of carriers is more significant in enzymatic detection methods. However, when carrier samples were excluded, no significant differences were found in the FPR and PPV of NBGS compared with enzyme activity detection (Table 5 and Table S5), suggesting that lysosomal disease carriers are prone to false positives in enzyme activity detection, leading to a decrease in PPV. In addition to carriers, enzyme activity screening may also be affected by other factors, such as interference from pseudo-defective alleles (No. 26 in Table 3) or other diseases (Fig. 3). Genomic screening, starting from a genetic perspective, can effectively overcome the limitations of enzyme activity-based LSD screening by avoiding interference from carriers and other external factors.

The onset of clinical symptoms varies across different LSDs and even among patients with the same LSD due to genotypic differences, with some cases being mild or late-onset and not appearing in the neonatal period (Table S6). NBGS for LSDs with varying onset times not only allows close monitoring of potential patients but also provides insights into family health and aids in genetic counseling. For example, for X-linked diseases such as Fabry disease, NBGS can trace affected family members after identifying potential patients and enabling timely detection and intervention. For autosomal recessive disorders, NBGS can reveal carrier status in family members, helping to assess genetic risk and optimize genetic counseling⁴¹.

By analyzing carrier data from our study, we found that in LSDs, diseases that can be screened through enzyme activity screening constitute the majority. However, there are still more than ten other LSDs, with carriers accounting for approximately 1/77 of the population (Table S2), suggesting a prevalence of approximately 1/23,716. This means that NBGS may identify one LSD patient per approximately 20,000 newborns who cannot be detected through enzyme activity screening alone. Early detection of these LSD patients not only facilitates timely intervention but also provides valuable clinical samples for the development of targeted drugs, further promoting the treatment of rare LSDs.

Previous studies have discussed the advantages of targeted capture-based NGS technology over techniques such as whole-genome sequencing and whole-exome sequencing, citing its lower cost, relatively easy operation, shorter reporting cycle, and easier interpretation^{12, 42}, making it the most suitable technology for newborn screening at present. However, with further optimization of the techniques and continuous reduction in costs, whole-genome sequencing may become a better alternative in the future. On the basis of existing screening methods, more attention should be given to improving disease management after screening, standardizing the follow-up process for potential patients to facilitate timely and effective intervention, and improving the quality of life of affected infants. Additionally, considering that many LSDs may be asymptomatic in the neonatal period, in future work, if we can establish connections between the genotypes, enzyme activities, disease severity, and onset time of these diseases through long-term follow-up, we can use genotype and enzyme activity to assess disease severity and approximate onset time, which will further improve the screening, diagnosis, and treatment processes of LSDs.

This study has four key limitations. First, its sample size and geographical scope are restricted, providing only a partial understanding of lysosomal disease epidemiology. Future research should consider broader, multicenter newborn screening to increase data reliability. Second, although our screening encompasses all coding regions of lysosomal diseases, only variants categorized as pathogenic or likely pathogenic according to the ACMG are reported. This simplifies interpretation for clinicians and reduces anxiety for newborn families but increases the risk of false negatives. Third, some lysosomal diseases are not suitable for NBGS because of homologous sequences and other reasons. Future screenings could benefit from integrating diverse methodological approaches to increase efficiency. Finally, newborns identified as positive may not exhibit symptoms, hindering our understanding of their condition. However, early detection combined with regular follow-up and increased family awareness of the disease can aid in timely diagnosis and treatment.

This study revealed that NBGS is more efficient than traditional enzymatic detection methods for the screening of LSDs and can significantly increase the detection rate of LSDs, especially in Asian populations. Early detection of these LSD patients is essential for providing healthcare and genetic counseling, and it allows newborns and their families to benefit from timely treatment and monitoring. Our study provides a foundation for the clinical application of NBGS in LSDs screening and lays the groundwork for further research in this field.

LSDs Lysosomal storage disorders

NBGS Newborn genomic screening

tNBS Traditional newborn screening

ABG Acid-β-glucocerebrosidase

ASM Acid-sphingomyelinase

GAA Acid-α-glucosidase

GALC β-galactocerebrosidase

GLA α-galactosidase A

IDUA α-L-iduronidase

GSD II Glycogen storage disease type II

NPD Niemann‒Pick disease

MPS II Mucopolysaccharidosis type II

FPRs False positive rates

PPVs Positive predictive values

Ethics approval and consent to participate

This study was reviewed and approved by the Ethics Committee of Nanjing Maternity and Child Health Care Hospital affiliated with Nanjing Medical University (No. 2021KY-071). Written informed consent was obtained from all the participants.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32100680).

Authors’ contributions

Zheng-Feng Xu and Tao Jiang designed the research and made the final version of the manuscript; Xin Wang and Yun Sun analyzed the data and wrote the manuscript with contributions from all the authors; Xian-Wei Guan, Yan-Yun Wang and Dong-Yang Hong collected the samples; Zhi-Lei Zhang, Ya-Hong Li and Pei-Ying Yang were involved in the test. All the authors approved the final manuscript.

Acknowledgments

We thank all individuals and families for participating in newborn screening and genomic screening. We are thankful to the newborn screening team from BGI Co. Ltd. for the technical support of genomic screening.

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Effect of Newborn Genomic Screening for Lysosomal Storage Disorders: A Randomized Clinical Trial in China

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Method

Study population and data collection

Heel blood collection

NBGS based on targeted capture-based NGS

Bioinformatics analysis

Sanger sequencing

Lysosomal enzyme activity detection

Statistical analysis

Results

1. Study population

2. Screening status for LSDs genes

6. Understanding factors influencing lysosomal enzyme activity through the NBGS

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1