**Background:** Inborn errors of metabolism (IEM) are genetic disorders causing abnormalities in enzyme, receptor, transport protein, or membrane function, with a global birth rate of 50.9:100,000 live births. IEM often presents with nonspecific symptoms overlapping with sepsis, cerebral hemorrhage, and encephalitis, making early diagnosis challenging. Tandem mass spectrometry (MS/MS) can screen for dozens of IEMs simultaneously by analyzing amino acids, succinylacetone, free carnitine, and acylcarnitine from dried blood spots. This study aimed to evaluate MS/MS screening combined with next-generation sequencing for IEM detection in a high-risk area of China.
**Methods:** From July 1, 2019 to December 31, 2021, 16,207 newborns in Nanning, Guangxi Province, China underwent IEM screening via MS/MS. Inclusion criteria: newborns who had completed 72 hours after birth with at least 8 adequate feedings and complete medical history. Exclusion criteria: newborns undergoing emergency surgery or external blood transfusion; recent antibiotic or steroid use; maternal genetic metabolic disease, liver disease, or HELLP syndrome. Heel blood was collected on filter paper dried blood spots (diameter ≥8 mm, 3-4 spots) according to Technical Specification for Blood Spot Collection (WS 376.3-2013). MS/MS analysis used nonderivative multiple amino acid, carnitine and succinylacetone assay kits on a Waters TQD tandem mass spectrometry system. Newborns with abnormal results were recalled for rescreening. Those with two positive screens underwent blood biochemistry, repeat MS/MS, and genetic analysis via high-throughput panel sequencing of 130 IEM-related genes (NextSeq500, Illumina). Sanger sequencing was used for locus validation and parental verification. Quality control included low and high level controls with Z score QC charts (12s warning limit, 13s out-of-control limit) and regular external quality assessment.
**Key Results:** Of 16,207 newborns, 1,423 were positive on primary screening (8.78%). Among positives, 772 (54.25%) were male and 651 (45.75%) female; 378 (26.56%) were preterm (<37 weeks) and 1,045 (73.44%) full-term (≥37 weeks). Common abnormalities included decreased free carnitine (16.59%), decreased octenylcarnitine (12.86%), elevated free carnitine (12.30%), decreased arginine (8.08%), decreased citrulline (7.38%), elevated propionyl carnitine (7.73%), and elevated tyrosine (7.03%). Recall rate was 92.21% (1,311/1,423). Thirty-three cases were suspected IEM after rescreening, and 8 were confirmed by genetic testing, yielding a prevalence of 1:2,026. Confirmed cases included: 4 amino acid metabolic diseases (2 PKU, 1 citrin deficiency, 1 tyrosinemia type I), 2 organic acid metabolic diseases (1 methylmalonic acidemia [MMA], 1 glutaric aciduria type I [GA-I]), and 2 fatty acid oxidation disorders (1 carnitine palmitoyltransferase II [CPT II] deficiency, 1 primary carnitine deficiency [PCD]). Genetic analysis revealed: CPT II deficiency — compound heterozygous CPT2 variants (c.989dupT pathogenic, c.1393G>A of unclear significance); MMA — compound heterozygous MUT variants (c.729_730insTT pathogenic, c.424A>G of unclear significance); citrin deficiency — homozygous SLC25A13 c.852_855delTATG; HT-1 — homozygous FAH c.455G>A; PKU — homozygous PAH c.158G>A and c.728G>A; PCD — homozygous SLC22A5 c.1400C>G; GA-I — compound heterozygous GCDH variants (c.1109T>C, c.395G>A). At 2-year follow-up, all 8 children showed normal growth and development on Gesell Developmental Schedule assessment, with normal cranial MRI findings.
**Clinical Implications:** The IEM prevalence of 1:2,026 in Nanning is higher than the Chinese national average (approximately 1:5,834) and rates reported in Liuzhou (1:3,733), Beijing (1:3,666), Suzhou (1:3,163), Changsha (1:4,237), and Hong Kong (1:4,122), but lower than Xinxiang (1:1,617) and Jining (1:1,941). The high prevalence in Nanning may relate to the Zhuang minority population with historically high rates of consanguineous marriage. The study demonstrates that MS/MS screening combined with sequencing validation effectively identifies IEM cases and enables early intervention, preventing morbidity. The authors recommend: (1) establishing separate cut-off values for preterm versus full-term infants, as preterm infants comprised 26.56% of positive screens and have different metabolic profiles; (2) completing screening before intravenous nutritional supplementation or reviewing after stopping such treatment, as 12.30% of elevated free carnitine cases were associated with levocarnitine use in preterm infants; (3) ensuring adequate feeding before blood collection to avoid false positives from insufficient protein intake.