Inherited metabolic disorders (IMDs), also called inborn errors of metabolism, are a type of disease associated with abnormal accumulation of metabolites and deficiency of essential substances caused by metabolic pathway defects (Ferreira and van Karnebeek, 2019). To date, more than 3,000 types of IMDs have been identified (Mak et al., 2013), most of which have no clinical presentation at the early onset, which makes them difficult for clinicians to diagnose. Once abnormalities occur, they lead to intellectual disability, developmental delay, and even death. Therefore, early screening, diagnosis, and treatment of neonatal IMDs are of great importance for the prevention of birth defects (Martínez-Morillo et al., 2016). In 1990, tandem mass spectrometry (MS/MS) was expanded to the field of neonatal disease screening for the first time. Qualitative and quantitative analyses are performed by detecting the mass-to-charge ratio of substances in samples for screening. Dozens of metabolites can be analyzed simultaneously in 2 min, and more than 40 genetic metabolic diseases can be detected, including metabolic disorders of amino acids, organic acids, and fatty acids. At present, the use of MS/MS in screening of IMDs has been widely carried out in developed countries and regions, and the coverage rate of screening has also increased annually (Wilcken and Wiley, 2008). However, the incidence of IMDs detected by MS/MS varies greatly among different countries and regions. A pilot study of over seven million newborns in China between 2016 and 2017 revealed that the incidence of IMDs detected by MS/MS was 38.69 per 100,000 births (1:2,585) (Deng et al., 2021). However, data from the present study differed from what was reported by Deng et al. The disease spectrum and genetic background of neonates in the Changsha area that a city with a population of more than ten million, were elucidated for the first time in this study. The Neonatal Disease Screening Center of Changsha Hospital for Maternal & Child Health Care launched the screening of neonatal IMDs by MS/MS in 2014. It is the first institution in Hunan Province to carry out the screening of IMDs using MS/MS, and currently performs the largest amount of screening in the Hunan Province. The findings of this study are of great significance as a reference for the whole Hunan region.

The incidence rate of IMDs in Changsha varies greatly across countries. The incidence rate of IMDs in California, United States, is 1:3,367 (Frazier et al., 2006), 1:2,960 in Spain (la Marca et al., 2008), 1:2,396 in Portugal (Vilarinho et al., 2010), and 1:2,855 in Australia (Kasper et al., 2010). In Asian countries, the incidence rate of IMDs in Korea is 1:13,205; 1:2,916 in Malaysia; 1:3,159 in Singapore; and 1:8,557 in Japan (Yoon et al., 2005; Lim et al., 2014; Yunus et al., 2016; Shibata et al., 2018). The incidence rates of IMDs vary in different regions of China. For example, the incidence rate of IMDs in Jining is 1:1,178 (Guo et al., 2018), which is higher than that in Changsha, but only seven kinds of diseases were found. Yiming reported that the overall incidence rate in Quanzhou was 1:2,804 (Lin et al., 2019); however, a study showed that the incidence rate of IMDs in Zhejiang is 1:5,626 (Huang et al., 2011). In this study, we found that the overall incidence rate of IMDs detected by MS/MS in Changsha was 1:4,237, which is dissimilar to the incidence rates reported in Jining, Quanzhou, and Zhejiang.

There were five kinds of AAMDs in Changsha, with HPA and CD having the two highest incidence rates. HPA is a group of the most common AAMDs due to the deficiency of phenylalanine hydroxylase or tetrahydrobiopterin, resulting in elevated phenylalanine. The biochemical pathway of phenylalanine metabolism was illustrated in Figure 3. The incidence rate of HPA is reportedly between 1:2,600 and 1:143,000 (Mitchell et al., 2011). It has been previously reported that the incidence rate of HPA in China is 1:11,572 (Zhan et al., 2009), and the incidence rate of HPA is significantly high in the north. The incidence rate of HPA in Changsha is 1:16,714, higher than that in Zhejiang (1:20,445), and lower than that in Jining (1:4,391), a northern city in China (Guo et al., 2018), which is consistent with previous reports. c.158G > A is the most common hotspot mutation of PAH (OMIM* 612,349), which accounting for 12.5% of mutations in HPA (Table 10). 17 kinds of mutations were found and the total mutations associated with HPA that have accounted for 20.34% of all mutations in our patients with IMDs. Citrullinemia is divided into CIT-Ⅰ and citrullinemia type Ⅱ (CD), both of which are autosomal recessive disorders of the urea cycle. Citrullinemia type Ⅱ is due to mutations in the SLC25A13 gene (OMIM* 603,859) (localized at 7q21.3), which encodes Citrin, causing impaired urea cycling and NADH transport and associated metabolic disturbances, The urea cycle and associated pathways were shown in Figure 3. The overall incidence rate of CD is 1:60,170 in Changsha, which is consistent with previous reports (Wang et al., 2019). According to a previous report, IVS16ins3kb, c.852_855delTATG are hotspot mutation sites in Chinese patients with CD (Lin et al., 2016). The results of this study are consistent with this report, and these two mutations accounted for 66.67% of the total mutations in SLC25A13 (Table 10). In one case of CD, the concentration of citrulline at initial screening was normal, but CD was confirmed by genetic testing, suggesting that early detection is of great importance for diagnosis, but not all patients with CD had an increase in citrulline (Lin et al., 2020). Two cases of neonatal CIT-I and H-MET were found, with an incidence rate of 1:150,425. The incidence rate of TYR-Ⅲ was the lowest, with an incidence rate of 1:300,849.

We found that five types of OAMDs in Changsha, with a incidence rate of 1:100,283.3-MCCD, 2-MBDD, and PA were the most common OAMDs. This is different from other reports in other areas of China, where methylmalonic acidemia was reported as the most common organic acid disorder in Zhejiang and Shandong. The incidence rate of methylmalonic acidemia in China is 1:3,960–1:26,000 (Zhou et al., 2018), but no cases have been reported in the Changsha area. Our screening data showed 4,923 newborns were suspected positive, and 286 of them were related to elevated C3 acylcarnitine and its ratio. After second test and the analyzation of urine by gas chromatography-mass spectrometry, there were still 16 indicated positive, and then suspected patients were further diagnosed by gene mutation detection, but only two cases of PA were confirmed. Methylmalonic acid, methylcitric acid and homocysteine are widely known biomarkers of genetic conditions leading isolated or combined MMA and HCY, or PA. However, elevations of methylmalonic acid, methylcitric acid and homocysteine can also be the result of secondary alterations, such as secondary vitamin B12 deficiency (Pajares et al., 2021). Therefore the second-tier test of methylmalonic acid, methylcitric acid and homocysteine is necessary to differentially diagnose the secondary vitamin B12 deficiency when we screening for MMA and HCY.

The deficiency of 3-methylcrotonyl-CoA carboxylase leads to disruption of the leucine metabolic pathway and accumulation of 3-hydroxyisovalerylcarnitine, 3-hydroxycrotonoylglycine, 3-hydroxyisovaleric acid and other metabolities, the metabolic pathways associated with 3-MCCD and other OAMDs were illustrated in Figure 3. 3-MCCD is classified into type I and type Ⅱ, caused by MCCC1 (OMIM* 609010) gene and MCCC2 (OMIM* 609014) gene, respectively. To date, at least 66 MCCC1 and 83 MCCC2 mutations have been reported (Yang et al., 2015). But there is no case with MCCC1 gene mutation in our screening results of 300,849 newborns. The current data show that about 90% of patients with 2-MBDD are asymptomatic (Porta et al., 2019). And the conclusion is corroborated in our findings, the conditions of three cases with 2-MBDD we found were normal during the follow-up. A study reported that the incidence rate of IVA was approximately 1:50,000 (Yoon et al., 2005), and in our study, two patients were found in approximately 300,000 neonates, with an incidence rate of 1:150,425. The least common organic acid disorder was GA-I, with an incidence rate of 1:300,849.

In Australia, Germany, and the United States, the overall incidence rate of FAODs is nearly 1:9,300; however, they are uncommon in Asia (Lindner et al., 2010; Shibata et al., 2018). It was also reported that in China, FAODs are very unusual, accounting for only 13% of the total IMDs (Han et al., 2015). However, research on the incidence of IMDs in the Chinese mainland population indicated that FAODs had a high incidence of 9.16 per 100,000 births (Deng et al., 2021). Our findings were consistent with these results. FAODs have the highest incidence rate of IMDs in Changsha, with an overall incidence rate of 1:9,705. PCD is not only the most common fatty acid oxidation disorder, but also the most common IMD in this study, and its incidence rate even exceeded that of HPA, reaching 1:13,675, which is consistent with the findings from the Quanzhou area (Lin et al., 2019). PCD is an autosomal recessive disorder of FOADs, due to mutations in SLC22A5 gene (OMIM* 603,377), which encodes the carnitine transporter protein, the biochemical metabolic pathway was shown in Figure 3. Carnitine transporter protein has a high affinity for carnitine in the cell membrane. In our research, c.1400C > G, c.51C > G, and c.760C > T were the hotspot mutation sites of SLC22A5, with the mutation frequencies of 24.32, 21.62, and 16.22% (Table 10), respectively, which is consistent with the literature (Guo et al., 2018). 15 kinds of mutations were found and the overall mutation numbers associated with PCD are 37, which accounting for 31.36% in total mutations of IMDs in Changsha (Table 10), that are the most mutations discovered in our research. However, our research is different from Suzhou, which found nine kinds of mutation associated with PCD, and PCD is not the most common disorder in Suzhou either (Wang et al., 2019). SCADD is the second most common fatty acid oxidation disorder, with an incidence rate of 1:42,978. C.1031A > G in ACADS (OMIM* 606,885) gene is the most common mutation, accounting for 23.08% in all mutations associated with SCAD found in Changsha. The newborn screening programs in Australia (Wilcken et al., 2009) and Massachusetts (Waisbren et al., 2008) have followed SCADD infants detected on the basis of an elevated C4 acylcarnitine in newborn blood spots and have found that most of the infants grow and develop normally. And in those that do not, clinical disease can often be attributed to another cause. These data, which suggest that SCADD does not cause disease in the great majority of cases. However some researches reveal that short chain fatty acids are very important for maintaining mucosal dynamic balance, their deficiency may affect the pathogenesis of a diverse range of diseases, from allergies and asthma to cancers, autoimmune diseases, metabolic diseases, and neurological diseases (Tan et al., 2014; van der Hee and Wells, 2021). It is important to pay close attention to the level of short chain fatty acids.

In summary, we report the incidence, disease spectrum, and genetic characteristics of neonatal IMDs for the first time in Changsha. FAODs had the highest incidence rate of IMDs in Changsha, which is contrary to previous reports. This may be due to variation in the different regions and populations. The most common IMDs were PCD, HPA and SCADD. Finally, the screening of neonatal IMDs by MS/MS in Changsha is conducive to early diagnosis and intervention. And our findings show newborn screening by MS/MS may miss part of the IMDs, the performance of our newborn screen program needs to be improved, and the combination of MS/MS with gene screening is necessary in future.