
==== Front
Mol Genet Genomic Med
Mol Genet Genomic Med
10.1002/(ISSN)2324-9269
MGG3
Molecular Genetics & Genomic Medicine
2324-9269
John Wiley and Sons Inc. Hoboken

10.1002/mgg3.70003
MGG370003
MGG3-2024-04-0405.R1
Original Article
Original Article
Incorporating Next‐Generation Sequencing as a Second‐Tier Test for Primary Carnitine Deficiency
Lin Yiming 1 linyiming0819@sina.com

Zheng Zhenzhu 1
Lin Weihua 2
Peng Weilin https://orcid.org/0000-0003-3921-6119
1 wellpeng@163.com

1 Department of Clinical Laboratory Quanzhou Maternity and Children's Hospital Quanzhou Fujian China
2 Neonatal Disease Screening Center Quanzhou Maternity and Children's Hospital Quanzhou Fujian China
* Correspondence:
Yiming Lin (linyiming0819@sina.com) | Weilin Peng (wellpeng@163.com)

09 9 2024
9 2024
12 9 10.1002/mgg3.v12.9 e7000321 7 2024
15 4 2024
13 8 2024
© 2024 The Author(s). Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

ABSTRACT

Background

Newborn screening (NBS) for primary carnitine deficiency (PCD) has poor performance. This study aimed to evaluate the feasibility of incorporating next‐generation sequencing (NGS) as a second‐tier PCD test.

Methods

Between March and December 2020, 60,070 newborns were screened for inherited metabolic disorders. Newborns with free carnitine (C0) levels below 8.5 μmol/L were selected for second‐tier genetic testing.

Results

In total, 130 (0.22%) newborns with low C0 levels underwent second‐tier genetic testing, 87 (66.92%) had positive genetic testing results, and 30 (23.08%) carried pathogenic variants of the SLC22A5 gene. Six newborns were diagnosed with PCD. The incidence of PCD was approximately 1 in 1:10,012 newborns. The PPV reached 20% after combining with second‐tier NGS. Of the eight variants identified in patients with PCD, the three most common variants were c.760C>T (p.Arg254*), c.51C>G (p.Phe17Leu), and c.1400C>G (p.Ser467Cys). The C0 levels of patients with PCD were significantly lower than those of PCD carriers (p = 0.0026) and PCD‐negative individuals (p = 0.0005).

Conclusions

Our results showed that the PPV reached 20% after combining with second‐tier NGS. The MS/MS‐based NBS and second‐tier NGS combination can effectively reduce the false‐positive rate and detect PCD in patients.

The biochemical and genetic information of 130 newborns suspected of PCD was elucidated. The combination of MS/MS‐based NBS and second‐tier NGS can effectively reduce the false positive rate and detect PCD patients. The PPV reached 20% after the combination of second‐tier NGS.

free carnitine
newborn screening
next‐generation sequencing
primary carnitine deficiency
second‐tier screening
Fujian Provincial Society of Laboratory Medicine and National (Fujian) Genetic Testing Technology Application Demonstration Center2023LHYC040 Quanzhou City Science and Technology Program of China2021C052R Joint Innovation Project of Huaqiao University2021YX003 source-schema-version-number2.0
cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:09.09.2024
Funding: This work was supported by grants from Fujian Provincial Society of Laboratory Medicine and National (Fujian) Genetic Testing Technology Application Demonstration Center (2023LHYC040), Quanzhou City Science and Technology Program of China (Grant No. 2021C052R), and Joint Innovation Project of Huaqiao University (Grant No. 2021YX003).
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pmcAbbreviations

C0 free carnitine

MS/MS tandem mass spectrometry

NBS newborn screening

NGS next‐generation sequencing

PCD primary carnitine deficiency

1 Introduction

Primary carnitine deficiency (PCD, OMIM #212140) is an inherited metabolic disease caused by biallelic mutations of the SLC22A5 gene (OMIM#603377) and is relatively common in southern China (Crefcoeur et al. 2022; Lin et al. 2021). Newborn screening (NBS) using tandem mass spectrometry (MS/MS) has been implemented worldwide. Newborns with low free carnitine (C0) levels can be flagged using MS/MS, enabling patients with PCD to be diagnosed and treated early (Crefcoeur et al. 2023). However, MS/MS‐based NBS for PCD shows poor performance because the C0 levels at birth are influenced by maternal C0 levels or other factors (Schiergens et al. 2021; Verbeeten et al. 2020; Wilson et al. 2019).

Second‐tier tests improved the performance of various inherited metabolic diseases (Chen et al. 2022; Peng et al. 2019; Luo et al. 2018). Therefore, the positive predictive value (PPV) can be increased by varying degrees. We have previously demonstrated that incorporating second‐tier genetic screening into NBS could increase PCD detection (Lin et al. 2021). However, a matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) assay could only identify 17 common SLC22A5 variants in the designed panel, and untargeted variants will be missed.

In recent years, with advances in next‐generation sequencing (NGS) technologies, many of these have been applied directly to NBS or second‐tier tests (Yang et al. 2023; Chen et al. 2023; Tong et al. 2022). Incorporating NGS‐based analysis into NBS has improved the early and accurate identification of newborns with inherited metabolic disorders; however, several challenges remain. NGS was used as a second‐tier molecular test for PCD in this study. Herein, we report the results of genetic screening to provide a reference for newborn genetic screening.

2 Materials and Methods

2.1 Editorial Policies and Ethical Considerations

This study was approved by the Ethics Committee of the Quanzhou Maternity and Children's Hospital (reference number: 2021‐IRB‐062). Written informed consent was obtained from the parents of all patients.

2.2 Study Cohort

A total of 60,070 newborns were screened for inherited metabolic disorders using MS/MS at Quanzhou Maternity and Children's Hospital from March 2020 to December 2020. Newborns with low C0 levels (C0 < 8.5 μmol/L, cut‐off value: 8.5–50 μmol/L) were recalled within 14 days, dried blood spot specimens were collected again for repeated MS/MS screening and second‐tier genetic testing.

2.3 Genetic Testing

Suspected PCD samples were subjected to targeted next‐generation sequencing (NGS) using a target‐sequencing panel including 264 genes associated with inherited disorders (Additional file 1: Table S1). Genomic DNA (gDNA) was extracted according to the manufacturer's instructions (MagPure Buffy Coat DNA Midi KF Kit). We used a BGI enzyme kit (Segmentase, BGI) to break the gDNA into 100–500 bp fragments, and then the 280–320 bp fragments were collected using magnetic beads. The collection added an “A” base at 3′ overhangs after repairing the ending, ensuring that the fragments could pare the “T” base with a special adapter, and built a single individual DNA library after LM‐PCR and purification. The library was enriched 16–24 h (47°C) by array hybridization (Roche NimbleGen, Madison, USA), followed by an elution and post‐capture amplification. After analyzing the products using an Agilent 2100 Bioanalyser and BMG to estimate the magnitude of enrichment, the qualified products were pooled and quantified according to different library quantities. Single strands of library products were prepared for circularization, and DNB was prepared. Finally, PE100 + 100 was used for sequencing using MGISEQ‐2000.

2.4 Data Analysis

After receiving the primary sequencing data, we performed the bioinformatics processing and data analysis. We used previously published filtering criteria to generate “clean reads” for further analysis (Wei et al. 2011). The “clean reads” (with a length of 90 bp), derived from targeted sequencing and filtering, were then aligned to the human genome reference (hg19) using the BWA (Burrows–Wheeler Aligner) Multi‐Vision software package. After alignment, the output files were used for sequencing coverage and depth analysis of the target region, single nucleotide variants (SNVs), and INDEL calling. We used GATK software to detect SNVs and indels. All SNVs and indels were filtered and estimated using multiple databases, including the NCBI dbSNP, HapMap, 1000 human genome datasets, and a database of 100 healthy Chinese adults. The reference sequence used in this study was based on the NCBI37/hg19 assembly of the human genome. NM_003060 was used as the reference sequence for SLC22A5.

2.5 Statistical Analyses

All the statistical analyses were performed using SPSS 26.0 (SPSS Corp., Armonk, NY, USA). Data were expressed as median (interquartile range), and non‐parametric tests were used for statistical comparisons. Differences between the groups were considered significant at *p < 0.05, **p < 0.01, and ***p < 0.001.

3 Results

3.1 NBS by MS/MS and Second‐Tier NGS

Among the 60,070 newborns, 130 (0.22%) with low C0 levels received second‐tier genetic testing, and 87 (66.92%) had positive genetic testing results (homozygote, compound heterozygote, or heterozygote). After removing genetic variants not associated with PCD, only 30 newborns (23.08%) harbored pathogenic variants in the SLC22A5 gene. Six newborns harboring biallelic pathogenic variants in the SLC22A5 gene were verified using Sanger sequencing and diagnosed with PCD; 24 newborns carrying one variant in the SLC22A5 gene were classified as PCD carriers, and the remaining 100 newborns with no variant in the SLC22A5 gene were deemed healthy individuals. The incidence of PCD was approximately 1 in 1:10,012 newborns (Tables 1 and 2). The PPV of traditional MS/MS screening was 4.62% (6/130). In comparison, only 30 newborns with positive genetic testing results needed recalling after combination with second‐tier NGS; thus, the PPV increased to 20% (6/30).

TABLE 1 Biochemical and genetic characteristics of six patients with primary carnitine deficiency (PCD).

Patient no.	Gender	C0	C0‐F1	Affected gene	Genotype	
1	Female	2.54	2.29	SLC22A5	c.695C>T (p.Thr232Met)	c.760C>T (p.Arg254*)	
2	Female	3.49	3.52	SLC22A5	c.51C>G (p.Phe17Leu)	c.760C>T (p.Arg254*)	
3	Female	3.25	2.78	SLC22A5	c.51C>G (p.Phe17Leu)	c.51C>G (p.Phe17Leu)	
4	Female	2.37	1.05	SLC22A5	c.338G>A (p.Cys113Tyr)	c.760C>T (p.Arg254*)	
5	Female	1.63	1.67	SLC22A5	c.760C>T (p.Arg254*)	c.760C>T (p.Arg254*)	
6	Male	7.65	5.04	SLC22A5	c.51C>G (p.Phe17Leu)	c.1400C>G (p.Ser467Cys)	
Note: C0, free carnitine detected at newborn screening; C0‐F1, C0 retested at recall stage, cutoff value: 8.5–50 μmol/L.

TABLE 2 Biochemical and genetic characteristics of PCD carriers.

No.	Gender	C0	C0‐F1	Affected gene	Genotype	
1	Male	7.3	19.57	SLC22A5	c.695C>T (p.Thr232Met)	
2	Female	7.41	14.63	SLC22A5	c.760C>T (p.Arg254*)	
3	Female	8.4	17.65	SLC22A5	c.760C>T (p.Arg254*)	
4	Female	8.26	17.28	SLC22A5	c.1400C>G (p.Ser467Cys)	
5	Male	8.4	20.34	SLC22A5	c.1400C>G (p.Ser467Cys)	
6	Female	6.99	15.36	SLC22A5	c.1400C>G (p.Ser467Cys)	
7	Male	6.87	22.75	SLC22A5	c.844C>T (p.Arg282*)	
8	Male	8.05	17.62	SLC22A5	c.1400C>G (p.Ser467Cys)	
9	Male	7.34	13.45	SLC22A5	c.760C>T (p.Arg254*)	
10	Female	6.81	18.14	SLC22A5	c.760C>T (p.Arg254*)	
11	Female	8.33	21.76	SLC22A5	c.51C>G (p.Phe17Leu)	
12	Female	7.9	17.1	SLC22A5	c.760C>T (p.Arg254*)	
13	Female	7.78	16.57	SLC22A5	c.760C>T (p.Arg254*)	
14	Female	8.41	12.62	SLC22A5	c.1252C>T (p.Gln418*)	
15	Female	6.99	19.22	SLC22A5	c.51C>G (p.Phe17Leu)	
16	Male	6.71	17.83	SLC22A5	c.51C>G (p.Phe17Leu)	
17	Female	5.12	15.94	SLC22A5	c.760C>T (p.Arg254*)	
18	Female	5.86	22.84	SLC22A5	c.51C>G (p.Phe17Leu)	
19	Male	5.18	22.26	SLC22A5	c.695C>T (p.Thr232Met)	
20	Female	7.86	15.48	SLC22A5	c.1400C>G (p.Ser467Cys)	
21	Female	8.17	19.38	SLC22A5	c.695C>T (p.Thr232Met)	
22	Female	5.81	18.3	SLC22A5	c.338G>A (p.Cys113Tyr)	
23	Female	5.71	16.06	SLC22A5	c.67_69delTTC (p.Phe23del)	
24	Female	7.6	19.31	SLC22A5	c.1400C>G (p.Ser467Cys)	
Note: C0, free carnitine detected at newborn screening; C0‐F1, C0 retested at recall stage, cutoff value: 8.5–50 μmol/L.

We identified eight variants in patients with PCD. The three most common variants were c.760C>T (p.Arg254*), c.51C>G (p.Phe17Leu), and c.1400C>G (p.Ser467Cys), with frequencies of 33.33% (12/36), 22.22% (8/36), and 19.44% (7/36), respectively (Table 3).

TABLE 3 The frequencies of SLC22A5 variants detected in PCD patients and carriers.

No.	Variants	Frequencies (%)	
1	c.760C>T (p.Arg254*)	33.33	
2	c.1400C>G (p.Ser467Cys)	19.44	
3	c.51C>G (p.Phe17Leu)	22.22	
4	c.695C>T (p.Thr232Met)	11.11	
5	c.338G>A (p.Cys113Tyr)	5.56	
6	c.844C>T (p.Arg282*)	2.78	
7	c.1252C>T (p.Gln418*)	2.78	
8	c.67_69delTTC (p.Phe23del)	2.78	
	In total	100.00	

3.2 Biochemical Features

As shown in Figure 1, the median values for the PCD, PCD carrier, and PCD‐negative groups were 2.90, 7.38, and 7.74, respectively. The C0 levels of patients with PCD were significantly lower than those of PCD carriers (p = 0.0026) and PCD‐negative individuals (p = 0.0005). C0 levels were not significantly different between PCD carriers and negative controls (p = 0.2246). All patients with PCD had low C0 levels on NBS and recall review; the mean C0 levels were 3.49 and 2.73, respectively. Comparatively, the mean C0 levels of PCD carriers at the NBS and recall review were 7.22 and 17.98, respectively.

FIGURE 1 Comparison of the C0 concentrations (μmol/L) in PCD patients, PCD carriers, and negatives. C0, Free carnitine; PCD, primary carnitine deficiency. Significant differences are indicated by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001) above the bracket connecting two groups.

4 Discussion

Although MS/MS‐based NBS for PCD can identify most patients with PCD, it also produces a large number of false‐positive results, which increases the financial burden and causes substantial anxiety for parents (Wilson et al. 2019). In addition, occasional false‐negative results render a small number of PCD patients unable to receive a timely diagnosis, which poses a great challenge to the existing NBS program (Lefevre et al. 2023). Our results showed that the PPV reached 20% after combining with second‐tier NGS, which was significantly higher than that of traditional MS/MS screening (4.62%). If second‐tier NGS were applied in clinical practice, 76.92% (100/130) of the false positives for PCD would eliminate the need for recall and unnecessary follow‐up.

Several studies have used genomic sequencing as a first‐tier screening test, markedly improving the detection capability of various genetic diseases (Yang et al. 2023; Chen et al. 2023; Luo et al. 2020). However, the cost is relatively high; it is difficult for most small and medium‐sized cities to conduct this project. Targeted sequencing of newborns with abnormal NBS results may be a cost‐effective screening strategy. In this study, 130 newborns with NBS results indicative of PCD were selected for targeted sequencing, which indicated that 76.92% (100/130) of newborns were healthy individuals, 18.46% (24/130) were PCD carriers, and 6 (4.62%) were patients with PCD.The combination of MS/MS‐based NBS and second‐tier NGS can effectively reduce the false‐positive rate and detect PCD in patients.

Most traditional NBS for PCD only provides biochemical and genetic information for patients (Yang et al. 2021; Lin et al. 2020; Zhou et al. 2019). Few studies have focused on the C0 levels in patients with PCD, carriers, or healthy individuals. This study presents biochemical and genetic information of newborns with different C0 levels. Initial NBS results showed that the C0 levels of patientswith PCD were significantly lower than those of PCD carriers and healthy individuals; however, there was no difference in C0 levels between PCD carriers and healthy individuals. The C0 levels of PCD duringthe recall review were lower than those in the NBS. In contrast, the C0 levels in PCD carriers increased significantly during the recall review.

The major limitation of this study was the limited sample size for genetic screening; however, our findings still have important reference values for newborn genetic screening. Second, carnitine transport activity analysis was unavailable to confirm the genetic testing results; the targeted NGS used in this study cannot detect disease‐causing pathogenic variants in regulatory regions (like c.‐149G>A variant in the 5′‐UTR) or deep introns, as well as duplications or large deletions. Third, only newborns with positive NBS results indicative of PCD were selected for targeted sequencing and a small percentage of PCD patients with normal C0 on NBS were ineligible for second‐tier testing. Finally, protocol optimization and cost‐effectiveness evaluations of second‐tier NGSwere not performed, necessitating further research.

5 Conclusion

In summary, the biochemical and genetic characteristics of 130 newborns with suspected PCD were elucidated. Our results showed that the PPV reached 20% after combination with second‐tier NGS. The combination of MS/MS‐based NBS and second‐tier NGS can effectively reduce the false‐positive rate and detect PCD in patients.

Author Contributions

Yiming Lin: Conceptualization, Methodology, Data acquisition, Data analysis, Data interpretation, Writing – Original draft. Zhenzhu Zheng: Data analysis. Weihua Lin: Data analysis. Weilin Peng: Writing – Review & Editing, Supervision.

Ethics Statement

This study was approved by the Ethical Committee of Quanzhou Maternity and Children's Hospital and was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from the parents of all infants for collection of samples and publication of medical data. Written informed consent was obtained from the parents of all infants for collection of samples and publication of medical data.

Consent

Consent was obtained from the parents of all patients for publication.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1.

Acknowledgements

We thank all the participants for their help and support. We would like to thank Editage (www.editage.cn) for English language editing.

Data Availability Statement

Data included in article/supplemental Material/referenced in article.
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