Inherited metabolic diseases (IMDs) constitute a large group of disorders in which a single gene defect results in a metabolic block, leading to biochemical abnormalities that may manifest at birth and/or later in life. IMDs are classified as single-gene genetic diseases, most of which follow an autosomal recessive pattern of inheritance. The majority of children with IMDs present with non-specific clinical manifestations at birth and may remain undiagnosed unless neonatal screening is conducted. Without prompt diagnosis and treatment, IMDs can impair the function of multiple organs and systems, cause progressive and irreversible damage to the nervous system, and lead to developmental delay or even death. These outcomes impose a considerable burden on affected families and society. Early diagnosis and timely intervention for neonates identified with IMDs through screening can prevent severe clinical consequences, such as mild to severe irreversible intellectual disability, lifelong disability, physical handicaps, coma, and early death (1).

Newborn screening (NBS) for IMDs represents a highly successful public-health initiative aimed at detecting life-threatening or long-term health conditions to reduce morbidity and mortality (2). Tandem mass spectrometry (MS/MS) serves as a cornerstone technology in NBS programs, enabling the rapid detection of numerous metabolites in dried blood spots (DBSs). The simultaneous quantification of amino acids and acylcarnitines allows identification of approximately 40–50 IMDs within a few days after birth (3). Expanded NBS using MS/MS has been widely adopted worldwide due to its advantages, including rapid and convenient analysis, significantly increased detection rates of IMDs (4), early diagnosis, prevention of premature death, and cost-effectiveness (5, 6).

With the advent of target capture and next-generation sequencing (NGS), it has become possible to sequence large panels of disease-related genes simultaneously, making NGS the preferred approach for identifying the genetic etiology in newborns with abnormal MS/MS screening results for IMDs. Since 2018, Huai'an City in China has implemented newborn screening for IMDs using tandem mass spectrometry, achieving a screening rate exceeding 98%. This study aimed to determine the incidence, disease spectrum, and gene spectrum of IMDs among newborns in Huai'an City, China.

There are many types of IMDs, and the total number of identified disorders is estimated to exceed several thousand. Although the incidence of any single IMD is generally low, ranging from several per 10,000 to several per 100,000 live births, the cumulative incidence of all IMDs is considerably higher. Since the early 1990s, MS/MS has been widely implemented in neonatal screening programs for IMDs due to its relatively low cost, high sensitivity (99%), and high specificity (>99.8%) (7). The majority of IMDs detected through MS/MS are disorders of organic acid, amino acid, or fatty acid metabolism. Reported IMD types and their incidences vary substantially among countries and regions, reflecting differences in screening panels, population genetics, and healthcare systems.

As summarized in Table 4, expanded newborn screening programs worldwide demonstrate diverse detection rates. For example, approximately 40 types of IMDs are screened in Italy, with an incidence of about 1 in 6,041 (4). In Spain, 13 IMDs are screened with an incidence of approximately 1 in 2,577 (8). Germany, Japan, and Korea screen for 24, 18, and 24 IMDs, respectively, with corresponding incidences of 1/2,200, 1/8,557, and 1/13,205 (9). In China, 38 IMDs are screened by MS/MS across multiple neonatal screening centers, with an overall incidence of about 1 in 1,410 (10). In the present study, in total 161,966 newborns were screened in Huai'an, and 57 IMD cases were confirmed by NGS, corresponding to an incidence of 1/2,842. The three most common IMDs were phenylalanine hydroxylase deficiency (1/8,098), PCD (1/23,138), and MMA (1/32,393), consistent with previously reported data from other regions of China (10). All 57 affected children exhibited biochemical abnormalities of varying degrees that were successfully detected by MS/MS, confirming its reliability and critical role in early IMD screening and diagnosis.

Hyperphenylalaninemia is the most prevalent amino acid metabolism disorder, resulting from a deficiency of either phenylalanine hydroxylase (PAH) or its cofactor, tetrahydrobiopterin (BH4). The deficiency leads to elevated phenylalanine concentrations in the blood, which can cause neurotoxicity and irreversible brain damage if left untreated. To date, more than 800 PAH gene variants have been identified in patients with PAH deficiency (11), with several recurrent variants reported in distinct populations. The most frequent variants include c.168+5G>C in Western Iranians (12), IVS10-11G>A in Iranians (13, 14) and Spaniards (15), c.1238G>C in Japanese (16), c.1162G>A in Brazilians (17), c.728G>A in Chinese (11, 18, 19), c.1068C>A and c.728G>A in South Koreans (20), c.1222C>T in Australians (21), and c.782G>A in Syrians (22).

In the Huai'an cohort, all 20 patients diagnosed with PAH deficiency harbored variants in the PAH gene. The detected variant included c.728G>A, c.611A>G, c.721C>T, c.598dup, c.1068C>A, c.331C>T, c.509+1G>A, c.1045T>G, c.699C>G, c.1301C>A, c.194T>C, c.158G>A, c.929C>T, c.301G>A, c.1256A>G, c.460T>C, c.827T>A, and c.1031G>A. Consistent with findings from other Chinese populations, the most frequent variant in this study was c.728G>A, accounting for 22.5% of alleles, followed by c.721C>T (17.5%) and c.611A>G (12.5%).

PCD was the second most common IMD detected in this study. PCD is caused by defects in the carnitine transporter protein encoded by the SLC22A5 gene, leading to impaired fatty acid β-oxidation within cells. The reported incidence of PCD in China varies regionally, ranging from 1/26,777 to 1/8,938 (18, 23, 24), while the incidence in Huai'an was 1/23,138. More than 110 SLC22A5variants have been identified to date, with distinct recurrent variants observed across different ethnic groups (25). For example, c.844T>C is common among Caucasian patients (26–28), while c.1400C>Gis the predominant hotspot variant in Southeast Asian populations (29–31). In Chinese populations, recurrent variants are relatively consistent across regions. The c.1400C>G variant is the most frequent, with a relative frequency of 50%, accounting for 80% of PCD cases in Suzhou (22). Chen et al. reported c.760C>T (32.9%) and c.1400C>G (21.1%) as the most common variants (32). Similarly, in the Huai'an cohort, c.1400C>G was the predominant SLC22A5 variant, with a relative frequency of 64.29%, aligning with findings from most other studies.

MMA was identified as the third most prevalent IMD and the most common organic acid metabolic disorder among the Huai'an population. MMA can be classified according to the specific defect into two categories: cobalamin (cbl) metabolic defects and methylmalonyl-CoA mutase deficiency (MUT type). It is a genetically and phenotypically heterogeneous disorder characterized by diverse clinical manifestations and significant morbidity. Currently, isolated MMA is attributed to variants in five genes—MMADHC, MUT, MCEE, MMAA, and MMAB. Among Chinese populations, MUT gene variants are the most frequently observed pathogenic type, with more than 400 related variant sites cataloged in ClinVar. The cblC type of MMA is the most common form associated with concurrent homocysteinemia. Reported incidences of MMA are approximately 1/50,000 in Japan (33) and 1/250,000 in Germany (34). However, in mainland China, the incidence ranges from 1/3,920 to 1/26,000 (18, 35). The incidence of MMA in Huai'an, approximately 1/32,393, is evidently higher than that in Japan and Germany but lower than in Shandong, Henan, Beijing, Shanghai, and Suzhou.

In this study, five patients were diagnosed with MMA, and all were classified as the cblC type. These patients carried six MMACHC gene variants: c.609G>A, c.567dup, c.482G>A, c.599G>A, c.80A>G, and an exon 1 deletion. These variants accounted for 28.6% of the total detected genetic sites, a distribution consistent with findings from most Chinese studies (36–38). All children diagnosed with MMA in the present cohort exclusively exhibited the combined type, and no variants in other related genes were identified. Given the limited sample size, the precision of variant frequency representation in the affected population may be constrained. Nevertheless, the identification of recurrent MMACHC gene variants—c.609G>A, c.567dup, and c.482G>A—highlights their importance as potential hotspots for subsequent MMA gene screening, prenatal diagnosis, and genetic counseling.

SCADD is an autosomal recessive disorder of fatty acid oxidation caused by defects in the ACADS gene, leading to the accumulation of butyrylcarnitine (C4) and ethylmalonic acid in urine. The ACADS gene is located on chromosome 12q24.31, spans approximately 13 kb, contains 10 exons, and encodes a 412-amino-acid enzyme. In this study, three cases of SCADD were identified with ACADS gene variants at four sites: c.1055C>T, c.881C>T, c.1130C>T, and c.164C>T. Among these, c.1055C>T and c.1130C>T exhibited the highest variant frequencies, each accounting for 33.33% of the detected sites. According to the American College of Medical Genetics and Genomics (ACMG) variant classification standards, c.881C>T (p.Ala294Val) was deemed clinically insignificant. Variants c.511C>T and c.625G>A are prevalent in Euramerican and Jewish populations (39), whereas c.1031A>G, c.164C>T, and c.323G>A have been reported in Japan (40). Long-term follow-up studies of SCADD cases detected through newborn screening have shown most patients remain asymptomatic, underlying a rationale that SCADD should not be routinely screened in newborns (41, 42). SCADD has been labeled a “biochemical phenotype” rather than a clinically significant monogenic disorder (43). This remains a controversial issue regarding the clinical significance of SCADD and whether it should be included in newborn screening panels.

Isobutyryl-CoA dehydrogenase deficiency (IBDD) is a rare autosomal recessive metabolic disorder that results from defects in the ACAD8gene, which encodes an enzyme responsible for the catabolism of the essential amino acid valine. Disruption of this metabolic pathway leads to the accumulation of isobutyrylglycine, which is excreted in urine and detectable through organic acid analysis. Accordingly, the condition is often referred to as isobutyrylglycinuria. The ACAD8gene is located on chromosome 11q25, comprises 11 exons, and encodes a member of the acyl-CoA dehydrogenase family. In this study, three cases of isobutyrylglycinuria were diagnosed, all associated with ACAD8 gene variants. Five variants— c.1000C>T, c.286G>A, c.526G>A, c.236G>A, and c.568-3C>G—as well as one structural variant (g.UNK1134123466 deletion) were identified. Based on ACMG classification criteria, c.526G>A, c.236G>A, and c.568-3C>G were categorized as variants of uncertain clinical significance. Reports from both Chinese and international studies imply that most IBDD patients are asymptomatic, making it difficult to establish clear genotype-phenotype correlations.

The etiology of hyperhomocysteinemia (HHCY) is multifactorial and can be categorized into congenital and acquired causes. Congenital factors primarily involve genetic variants in key enzymes such as thioether-β-synthase, methionine synthase reductase, methylenetetrahydrofolate reductase (MTHFR), and methionine synthase. These genetic defects lead to deficiencies in cofactors required for homocysteine (HCY) metabolism, resulting in HCY accumulation and subsequent development of HHCY. In the present study, one case of homocysteinemia was identified, in which a homozygous variant of the MTHFR gene (c.1316T>C) was detected. This case was initially identified through MS/MS screening, which revealed methionine levels below the diagnostic threshold, and was subsequently confirmed through molecular diagnosis using NGS. These findings indicate that HHCY can be detected by MS/MS-based newborn screening, acknowledging that screening for low methionine without screening for elevated HCY wouldn't effectively screen for all genetic etiologies of HHCY.

Citrullinemia type I (CTLN1), also referred to as argininosuccinate synthase (ASS) deficiency, is caused by pathogenic variants in the ASS1gene, a key enzyme in the urea cycle. The disorder is characterized by hyperammonemia and elevated citrulline levels and follows an autosomal recessive inheritance pattern (44). The ASS1 gene is located on chromosome 9q34.11, spans approximately 56 kb, comprises 16 exons, and encodes a 412-amino-acid protein. Reported common variants include c.1085G>T and c.970G>A in Turkish patients, and c.1088G>A in German patients (45, 46). In East Asian populations, c.421-2A>G is the most frequently observed variant, whereas c.970G>A predominates in Korean patients (47, 48). Additionally, c.794G>A is the most common variant reported among Japanese CTLN1 patients (49). In the present study, two cases of CTLN1 were diagnosed, and four heterozygous ASS1 gene variants were detected: c.349G>A, c.805G>A, c.919C>T, and c.1069C>G.

Maple syrup urine disease (MSUD) is an autosomal recessive disorder affecting the catabolism of branched-chain amino acids. The condition derives its name from the characteristic sweet, “maple syrup–like” odor of the urine. MSUD results from a functional defect in the branched-chain α-keto acid dehydrogenase complex. The primary pathogenic genes implicated in this disorder include BCKDHA (34%), BCKDHB (29%), DLD, and DBT. In this study, we identified an MSUD case in which two heterozygous variants of the BCKDHB gene were detected: c.93_103dup and c.673_675del. According to the ACMG variant classification guidelines, the c.673_675del (p.Leu226del) variant is classified as having uncertain clinical significance, warranting further functional validation. It shows how NGS as a standalone first tier screen would be insufficient as VUS clinical correlation is only feasible because of the MS/MS NBS result.

3-Methylcrotonyl-CoA carboxylase deficiency (3-MCCD) is an autosomal recessive inborn error of leucine metabolism that exhibits considerable phenotypic variability. Most affected individuals remain asymptomatic unless secondary systemic carnitine deficiency develops. However, severe cases may present with leukodystrophy, developmental delay, hypoglycemia, metabolic acidosis, failure to thrive, lactic acidosis, and hyperammonemia (50). Deficiency of 3-MCCC, caused by pathogenic variants in either MCCC1 or MCCC2, leads to elevated levels of 3-hydroxyisovalerylcarnitine in the blood. The enzyme comprises α and β subunits encoded by the MCCC1 and MCCC2 genes, respectively. Previous studies have shown that MCCC2variants are more frequently reported worldwide, whereas MCCC1variants are predominant in Chinese populations (51). In the present study, one patient with 3-MCCD was diagnosed, carrying a homozygous MCCC2 gene variant (c.728G>A).

This study reports, for the first time, the results of newborn screening for IMDs and their epidemiological characteristics in Huai'an over the past 7 years. Among 161,966 newborns screened using MS/MS, 57 children were diagnosed with 17 different types of IMDs, yielding an overall incidence rate of 1 in 2,842. The three most prevalent disorders were phenylalanine hydroxylase deficiency, PCD, and MMA, findings that were consistent with the reported incidence patterns of these diseases in other regions of China.

Of the total 77 genetic variants identified, 49.35% (38/77) were classified as pathogenic, 32.47% (25/77) as likely pathogenic, and 18.18% (14/77) as variants of uncertain clinical significance, the latter requiring further functional validation. Several recurrent variants were identified across 10 IMDs, including PAH gene variants (c.728G>A, c.611A>G, and c.721C>T) for phenylketonuria; PAH gene variants (c.158G>A, c.721C>T, and c.728G>A) for mild hyperphenylalaninemia; SLC22A5gene variant (c.1400C>G) for PCD; MMACHC gene variants (c.609G>A, c.567dup, and c.482G>A) for MMA; ACADS gene variants (c.1055C>T and c.1130C>T) for SCADD; and ACADSB gene variant (c.923G>A) for SBCADD. All these recurrent variants have been previously reported as pathogenic or likely pathogenic, except for the PAH gene variant c.158G>A.

In conclusion, the majority of the IMD patients identified through expanded newborn screening in Huai'an carried pathogenic or potentially pathogenic variants. These findings underscore the clinical value of combining MS/MS-based screening with NGS for the accurate diagnosis and genetic confirmation of IMDs in newborns.