The study was approved by the Institutional Review Board of Yonsei University Health System (IRB, 42017-0127). Korean neonates or infants who were admitted to the NICU at Severance Children's Hospital (Seoul, Korea) from May 2017 to September 2017 were included in the study after obtaining written informed consent from at least one biological parent/guardian. None of the babies was selected for metabolic disorders in line with the population-based NBS program.

A TNGS panel for neonatal diseases was designed and validated in a previous study.12 A targeted gene enrichment method was used to construct libraries for subsequent determination of sequences using an NGS method with HiSeq2000 (Illumina, San Diego, CA, USA). In this study, the probe set was designed to capture 1.45 Mb, covering the exons and 25 nucleotides at the flanking introns for 198 targeted genes (Supplementary Table 1, only online). Probe-library hybridization followed by capture of target genes was performed according to the manufacturer's instructions.

A total of 48 samples were registered for the TNGS panel, which when run together using the developed panel of a 1-Mb region with a 1-Gb output would likely cover ≤×500 reads. We used blood samples collected on filter paper, and DNA was isolated from dried blood spots with QIAamp DNA Mini Kits (Qiagen, Valencia, CA, USA). The DNA was enzyme-fragmented with NEBNext dsDNA Fragmentase® (New England Biolabs, Ipswich, MA, USA), which was used to construct sequence libraries, according to the protocol provided by Cellemix (Seoul, Korea). For library enrichment, we used a custom probe set synthesized by Cellemix (Seoul). The subsequent capturing procedure was performed with the MYbaits® kit (Arbor Biosciences, Ann Arbor, MI, USA), and sequencing was performed on a HiSeq 2000 system (Illumina).

BaseSpace (Illumina) was used for data processing, alignment, variant calling, and annotations. The average coverage of target bases was 99.8%, with 98.1% of the regions of interest having at least 10X coverage and 95.4% having at least 20% coverage (Supplementary Table 2, only online). The sequenced reads were mapped to the human reference genome (UCSC hg19) with Burrows-Wheeler Aligner (BWA-0.7.7-isis-1.0.0), and variants were identified with the Genome Analysis toolkit (GATK version 1.6.23-gf0210b3). After filtering out variants using an internal database, variants with a minor allele frequency of less than 1% in either the 1000 Genomes Project or Exome Aggregation Consortium database were assessed further. The criteria for classification of variants were based on the principle recommended by the American College of Medical Genetics and Genomics (ACMG) standards.13

In Korea, NBS is mandated by law, and initial NBS samples were collected based on conventional processes within 7 days with TNGS samples consecutively. For newborns weighing less than 2000 g or those starting TPN, repeated screening tests for 28 days after birth or before discharge were required. Abnormal NBS was conducted once more and considered presumptively positive. Additional biochemical tests were then performed based on ACMG ACT sheets (https://www.acmg.net), and medical records were reviewed.

To evaluate the usefulness of the TNGS panel in NBS for neonates showing high numbers of FPRs, we tested 48 clinical samples for newborn diseases associated with 198 genes (Supplementary Table 1, only online). The individual participants in the NICU were not selected for metabolic diseases in advance and included both full-term and preterm infants. In this study, there were 18 (38%) preterm infants and 27 (56%) newborns with a birth weight of less than 2000 g (Table 1). The average birth weight was 1998 g (range: 610–3790 g).

In our hospital, additional NBS tests are routinely performed to obtain more reliable results when babies have had 48 h of oral feeding after TPN is discontinued. Among the infants in the NICU that underwent additional NBS, those born with low birth weight (<2000 g) and preterm babies were assessed at later ages (days since birth) than full-term neonates to minimize the FPRs (Table 1).

The clinical characteristics of all newborns included in this pilot study are summarized in Supplementary Table 3 (only online). A total of 25 variants were identified in 19 patients (Table 2). In accordance with ACMG guidelines, 13 variants classified as “likely pathogenic” or “pathogenic” were detected in 11 (22.9%) of the neonates. In Korea, an extended NBS test using MS/MS was performed to detect approximately 54 metabolic diseases in a conventional NBS program. However, we identified pathogenic or likely pathogenic variants reported in six genes, including those associated with metabolic diseases not covered in the NBS: DHCR7 (Smith-Lemli-Opitz syndrome), PCBD1 (BH4-deficient hyperphenylalaninemia), GAA (glycogen storage disease II), ALDOB (fructose intolerance, hereditary), ATP7B (Wilson disease), and GBA (Gaucher disease). Two pathogenic GAA variants were identified in patient D17; however, those alleles were found in the same strand in the raw data and were negative in the enzyme analysis, suggesting a diagnosis as a carrier of Pompe disease. Of the known pathogenic mutations of the CYP21A2 gene, p. Gln319Ter is one of the most common variants related to congenital adrenal hyperplasia in Koreans, and the heterozygote mutation was identified in two unrelated patients in this study.14 One of the patients, D30, a female with ambiguous genitalia, showed normal 17-OHP levels in the NBS screening test, and the electrolyte balance was also normal. The other newborn, D43, was a male who underwent a colostomy operation due to a congenital imperforate anus.

Three of the newborns (D01, D03, D04) showed positive results in a comprehensive NBS. In the case of D01, elevation of C4 using MS/MS resulted in suspicion of an isobutyl-CoA dehydrogenase (IBDH) deficiency and short-chain acyl-CoA dehydrogenase deficiency. According to the ACMG algorithm, ethylmalonic encephalopathy (associated with the ETHE1 gene) is one of the diseases included in the differential diagnosis NBS.15 As a result, two mutations in ACAD8 were identified in patient D01, and the patient was ultimately diagnosed with IBDH deficiency associated with a novel compound heterozygous variant. For patients D03 and D04, increases in C5-OH acylcarnitine (3-OH isovalerylcarnitine) were reported to result in the same suspected disease of 3-methylcrotonyl-CoA carboxylase deficiency inherited in an autosomal recessive pattern. However, in the former patient, a heterozygote mutation (NM_020166.3: c.1391A>C; p. His464Pro) was identified in the MCCC1 gene, which is reported to be benign. In the latter case, D04, one unknown variant of the MCCC1 gene was identified and classified as a variant of unknown significance. In addition, biotinidase deficiency (associated with the BTD gene), which is one of the diseases causing elevated C5-OH, is not included in the Korean screening program at present, but could be evaluated through the TNGS panel. However, in patient D04, considering the limitations of not finding additional variants, such as deletion or duplication, it is necessary to confirm the clinical characteristics of the patient, and additional biochemical tests may be needed. Ultimately, the abnormal NBS tests were considered presumptively false-positive in these two asymptomatic patients based on assessment with the TNGS panel.