Preamble

Exploratory Study of Newborn Screening for Multiple Inherited Metabolic Diseases in Hainan Province

Abstract

Objective: To investigate the screening results, disease spectrum, and genetic characteristics of inherited metabolic diseases (IMDs) among newborns in Hainan Province, providing a scientific basis for the prevention and control of birth defects in the region.

Methods: A retrospective analysis was conducted on the screening data of newborns born in Hainan Province from [Year] to [Year]. Dried blood spot samples were collected and analyzed using tandem mass spectrometry (MS/MS) to detect levels of amino acids, acylcarnitines, and succinylacetone. Positive cases were followed up for clinical diagnosis, biochemical verification, and genetic testing.

Results: Among the screened population, a total of [Number] cases of IMDs were confirmed. The overall incidence rate was 1 in [Number]. The disease spectrum primarily included organic acidemias, fatty acid oxidation disorders, and amino acid metabolic disorders. Specifically, [Disease Name 1] and [Disease Name 2] were the most prevalent. Genetic analysis revealed several common mutations specific to the Hainan population, including [Mutation 1] and [Mutation 2].

Conclusion: Tandem mass spectrometry is an effective tool for the early detection of IMDs in newborns. The disease spectrum in Hainan Province exhibits regional characteristics. Early screening, diagnosis, and intervention are crucial for improving the prognosis of affected children and reducing the burden of birth defects.

Introduction

Inherited metabolic diseases (IMDs) are a large group of genetic disorders caused by defects in enzymes, receptors, or transport proteins involved in metabolic pathways. Although individual IMDs are rare, their cumulative incidence is significant. Without timely intervention, these diseases often lead to irreversible neurological damage, physical disability, or even death.

In recent years, the application of tandem mass spectrometry (MS/MS) has revolutionized newborn screening (NBS) by allowing the simultaneous detection of dozens of metabolic markers from a single dried blood spot. Hainan Province, characterized by its unique geographical location and ethnic diversity (including a significant Li ethnic minority population), may present a distinct genetic landscape for IMDs compared to mainland China. This study aims to analyze the results of MS/MS-based newborn screening in Hainan to better understand the local epidemiology and genetic architecture of these disorders.

Materials and Methods

1.1 Study Population

The study included all newborns who underwent MS/MS screening at the Hain

1.570311 海南省海口市，海南省妇女儿童医学中心，检验科

Neonatal Screening Center, Hainan Women and Children's Medical Center, Haikou, Hainan Province

背景

The prevalence of neonatal inherited metabolic diseases in China exceeds 0.5%. This study represents the first comprehensive screening for these conditions across the entire province of Hainan. Our findings contribute significantly to the prevention and control of birth defects in Hainan Province and provide essential laboratory diagnostic tools for identifying inherited metabolic disorders.

The objective of this study is to investigate the incidence of various neonatal inherited metabolic diseases in Hainan Province, thereby providing a reliable methodology and reference framework for the prevention and control of regional birth defects.

方法

The study utilized dried blood spot (DBS) samples collected from the heels of newborns born between February and December 2024 at various midwifery medical institutions in Hainan Province. Screening was conducted using tandem mass spectrometry (MS/MS), and the reference ranges for relevant detection indicators were calculated using the percentile method. For suspected positive samples, recall and diagnostic validation were performed using gas chromatography-mass spectrometry (GC-MS) and genetic sequencing methods.

结果

A total of 84,184 newborn heel prick dried blood spot samples were collected. Using the percentile method on 29,676 of these samples, the reference ranges for newborns in Hainan Province were established for 14 amino acids, 36 carnitines, 1 ketone, 2 adenosines, and 4 lysophosphatidylcholines.

Thirty-eight cases of various neonatal inherited metabolic diseases (IMDs) were diagnosed, representing an overall incidence rate of 1/2,215 (38/84,184). Twelve distinct types of diseases were confirmed: Phenylketonuria (PKU), Hypermethioninemia (HMet), Maple Syrup Urine Disease (MSUD), Citrin Deficiency (CD), Primary Carnitine Deficiency (PCD), Short-Chain Acyl-CoA Dehydrogenase Deficiency (SCADD), Carnitine Palmitoyltransferase I Deficiency (CPT I), Carnitine-Acylcarnitine Translocase Deficiency (CACT), 3-Methylcrotonyl-CoA Carboxylase Deficiency (MCCD), Glutaric Acidemia Type I (GA-I), Glutaric Acidemia Type II (GA-II), and 2-Methylbutyryl-CoA Dehydrogenase Deficiency (SBCADD).

The incidence rates for individual conditions varied significantly, ranging from 1/84,184 to 1/6,475. The three most frequently detected diseases were PCD (13 cases), SCADD (6 cases), and CD (5 cases). Genetic analysis identified 42 mutation sites across 12 different genes.

结论

This study establishes, for the first time, the normal reference ranges for screening multiple inherited metabolic disorders (IMDs) in newborns within Hainan Province. These ranges provide essential diagnostic indicators for subsequent screening programs. Following the implementation of comprehensive free screening, the study identified a relatively high incidence of various IMDs in Hainan, characterized by complex and diverse genetic profiles. Among these, fatty acid oxidation disorders—specifically primary carnitine deficiency (PCD), short-chain acyl-CoA dehydrogenase deficiency (SCADD), and carnitine palmitoyltransferase II deficiency (CPT II)—were found to be the most prevalent. These findings underscore the critical importance of neonatal screening for multiple IMDs in the prevention and control of birth defects in Hainan Province.

Keywords: Neonatal screening; Inherited metabolic disorders; Dried blood spots; Incidence; Newborn disease screening; Hainan Province

[CLC Number] R 722
[Document Code] A

Exploratory Study on Screening of Multiple Inherited Metabolic Disorders in Newborns in Hainan Province

Peizhen Zhang, Zhendong Li, Haizhu Yan
Hainan Women and Children’s Medical Center, Haikou 570311, China

Background

The prevalence of neonatal inherited metabolic disorders exceeds 0.5%. This study represents the first comprehensive population-wide screening for these conditions in Hainan Province, contributing to the prevention and control of birth defects in the region and establishing diagnostic laboratory capabilities for inherited metabolic diseases.

Objective To investigate the incidence of multiple inherited metabolic disorders (IMDs) among newborns in Hainan Province and provide reliable methods and data to support birth defect prevention and control.

Methods

This study used dried blood spot (DBS) samples collected from heel pricks of newborns born between February and December 2024 across all obstetric healthcare facilities in Hainan Province. Screening was performed using tandem mass spectrometry, and reference ranges for relevant biomarkers were established using the percentile method. Suspected cases were recalled for confirmatory

Chinese General Practice diagnosis via gas chromatography-mass spectrometry (GC-MS) and gene sequencing.

Results

A total of 84 184 DBS samples from newborns were collected. Using the percentile method, reference ranges for 14 amino acids, 36 acylcarnitines, 1 ketone, 2 adenosines, and 4 lysophosphatidylcholines were established in 29 676 samples, providing the first normative reference data for Hainan Province. Thirty-eight cases of IMDs were diagnosed, yielding an incidence rate of 1/2 215 (38/84 184). Twelve distinct diseases were confirmed, including phenylketonuria (PKU), hypermethioninemia (HMet), multiple sulfatase deficiency (MUSD), citrin deficiency (CD), primary carnitine deficiency (PCD), short-chain acyl-CoA dehydrogenase deficiency (SCADD), carnitine palmitoyltransferase Ⅰ deficiency (CPTⅠ), carnitine-acylcarnitine translocase deficiency (CACT), 3-methylcrotonyl-CoA carboxylase deficiency (MCCD), glutaric acidemia type Ⅰ (GA-Ⅰ), glutaric acidemia type Ⅱ (GA- Ⅱ), and short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD), with incidence rates ranging widely from 1/84 184 to 1/6 475. PCD (13 cases), SCADD (6 cases), and CD (5 cases) ranked as the three most prevalent disorders. 42 mutation sites across 12 genes were identified.

Conclusion

This study establishes the first reference ranges for newborn IMD screening in Hainan Province, providing critical reference criteria for subsequent screening programs. Following the implementation of a comprehensive free screening initiative, the data revealed a relatively high incidence of IMDs in Hainan newborns, with notable genetic complexity and diversity. Disorders of fatty acid metabolism-PCD, SCADD, and CD-were the most frequently detected. These findings underscore the importance of expanded newborn IMD screening in Hainan Province for the prevention and control of birth defects.

Key words: Neonatal screening; Inherited metabolic disorders; Dried blood spots; Incidence rate; Newborn screening; Hainan Province

Neonatal inherited metabolic disorders (IEM), also known as inborn errors of metabolism, comprise a large category of diseases characterized by abnormal metabolic biochemical markers. As single-gene disorders, they pose a serious threat to the quality of the birth population. The vast majority of IEMs follow recessive inheritance patterns, while a minority are characterized by dominant, X-linked, or mitochondrial inheritance. These disorders encompass various metabolic abnormalities, including disturbances in amino acid metabolism, organic acid metabolism, the urea cycle, fatty acid oxidation, and carbohydrate metabolism. Although the incidence of any single IEM is typically less than 1 in 10,000—leading them to be classified as rare diseases—their cumulative incidence is high, with some reports suggesting a neonatal prevalence exceeding 0.5%. Consequently, implementing multi-disease IEM screening within neonatal populations is a critical task for birth defect prevention worldwide. Currently, tandem mass spectrometry (MS/MS) for multi-IEM screening has been implemented in several countries \cite{4-5}. According to Article 3 of the "Administrative Measures for Neonatal Disease Screening" (formerly Ministry of Health Order No. 64) in China, health administrative departments at the provincial, autonomous region, and municipal levels may increase the number of screened neonatal diseases based on local medical resources, public demand, and disease incidence rates. Although Hainan Province initiated MS/MS screening as early as 2016, the volume of screened IEM samples remained below one-tenth of the province's birth population as of January 2024. In recent years, the number of detected neonatal IEM cases has steadily increased (for instance, the incidence rate in ethnic minority areas of Hainan reached 19/37,143), drawing significant attention from the Hainan Provincial Government. Following the issuance of the "Notice on the Implementation Plan for the 2024 Hainan Provincial Free Neonatal Multi-Inherited Metabolic Disease Screening Project" (Qiong Wei Fu You Han [2024]) by the Hainan Provincial Health Commission, province-wide free screening was realized for the first time under the guiding principle of "screening all who should be screened."

The authors have compiled and analyzed the 2024 screening data to provide a comprehensive overview of the incidence of various inherited metabolic disorders among newborns in Hainan Province. This report aims to provide a reliable methodology and reference basis for the prevention and control of birth defects in the region. Notably, the recorded incidence rate of IEM among newborns in Haikou City was 88/185,660.

1.1 研究对象

With the implementation of the "Notice," this study focused on newborns born in various midwifery institutions in Hainan Province between February 1, 2024, and December 31, 2024.

Adhering to the principle of informed consent and voluntary participation, the "Hainan Provincial Neonatal Genetic Metabolic Disease Screening Informed Consent Form" was signed for each participant. Following the requirements of the Expert Consensus on the Collection, Delivery, and Preservation of Filter Paper Blood Spots for Neonatal Disease Screening, heel blood was collected from newborns at 48 hours of age to prepare dried blood spot (DBS) samples. These samples were used to implement free tandem mass spectrometry (MS/MS) screening for Inborn Errors of Metabolism (IEM). A total of 84,184 neonatal DBS samples were collected. This study was approved by the Medical Ethics Committee of the Hainan Women and Children’s Medical Center [Approval No.: HNWCMC Ethics Review 2024 No. (55)].

Neonatal screening DBS samples were prepared for testing in accordance with the Expert Consensus on the Collection, Delivery, and Preservation of Filter Paper Blood Spots for Neonatal Disease Screening. According to the Technical Specifications for Blood Spot Collection for Neonatal Disease Screening, the screening cards must be fully completed. Each card required four blood spots with a diameter of $\ge 8$ mm, ensuring uniform penetration on both sides, no overlapping blood drops, and natural air-drying.

Experimental Instruments and Reagents: 2081 Panthera-Puncher™ 9 puncher (PerkinElmer, Finland; punch diameter 3 mm), QSight 225MD tandem mass spectrometer (Jiangsu PerkinElmer Fenghua Bio-engineering Co., Ltd.), Agilent 7890B-5977A gas chromatograph-mass spectrometer (Agilent Technologies, USA), BS-2000M biochemical analyzer (Mindray, China), MicBio-IV incubator shaker (Abenson, China), XW-80A vortex mixer (Haimen Kylin-Bell Lab Instruments, China), KQ-100 ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), and ABI 3730xl sequencer (Applied Biosystems, USA).

Chinese General Practice: Reagent kits for the determination of non-derivatized amino acids, carnitine, adenosine, lysophosphatidylcholine, and succinylacetone (Jiangsu PerkinElmer Fenghua Bio-engineering Co., Ltd.; tandem mass spectrometry method, hereinafter referred to as the NeoBase™ 2 kit), urinary organic acid kits (Biosan), creatinine determination kits (Beijing Beijian Xinchuangyuan Biotechnology Co., Ltd.), QIAamp Whole Blood DNA Extraction Kit (Qiagen, Germany), NEBNext DNA Library Prep Kit for Illumina (Beijing MyGenostics Co., Ltd.), and Whole Exome Capture Kit (Beijing MyGenostics Co., Ltd.).

Sample Testing and Clinical Interpretation

1.4.1 串联质谱技术新生儿疾病筛查与临床判读

The NeoBase™2 kit was utilized for tandem mass spectrometry (MS/MS) detection of 14 amino acids, 36 acylcarnitines, 1 ketone, 2 adenosines, and 4 lysophosphatidylcholines in newborn dried blood spot samples. From February to May 2024, the manufacturer-provided reference ranges were initially employed. After accumulating 29,676 valid screening data points, the laboratory established its own specific screening reference ranges using the percentile method. These laboratory-derived reference ranges were subsequently implemented from June to December 2024.

The data generated from the tandem mass spectrometry screening were interpreted and analyzed by clinical physicians, who then issued relevant clinical recommendations. These findings were uploaded to the "Hainan Provincial Maternal and Child Health System Platform" for the online publication of screening reports. Furthermore, follow-up diagnostic verification, including recall and re-examination, was conducted for newborns suspected of having inherited errors of metabolism (IEM).

1.4.2 尿有机酸分析与临床判读

Clinicians recommend that neonates suspected of having Inborn Errors of Metabolism (IEM) undergo urinary organic acid analysis using Gas Chromatography-Mass Spectrometry (GC-MS). The resulting data should be analyzed and reported by clinicians to ensure accurate diagnosis and clinical correlation.

1.4.3 基因测序与临床判读

Peripheral whole blood samples (2 mL each) were collected from neonates suspected of having inherited errors of metabolism (IEM) and their parents. Genomic DNA (3 $\mu$L) was extracted and fragmented via ultrasonication for library construction. Whole-exome capture was performed using a commercial capture kit targeting all human exons, followed by paired-end sequencing on a next-generation sequencing (NGS) platform. Genetic reports were issued following a rigorous review of the sequencing data by physicians at the Molecular Diagnosis Center.

Clinical diagnoses for the neonates were established by clinical specialists at the Hainan Provincial Neonatal Screening Center. These diagnoses were based on a comprehensive evaluation of tandem mass spectrometry (MS/MS) data, urinary organic acid analysis, and genetic testing reports.

Cut-off values were calculated and statistical analyses were performed using SPSS 13.0 software. Following the recommendations of the Expert Consensus on Neonatal Screening Technology using Tandem Mass Spectrometry and the Expert Consensus on Methods for Establishing Cut-off Values for Neonatal Inherited Metabolic Disease Screening, the percentile method was employed. The reference population consisted of healthy full-term neonates, excluding factors known to affect metabolite levels such as low birth weight, prematurity, and post-term birth. Based on the reported incidence of specific diseases and the clinical significance of each biomarker, preliminary reference intervals were determined using the 1st and 99th percentiles ($P_{1.0}$ and $P_{99.0}$), the 0.5th and 99.5th percentiles ($P_{0.5}$ and $P_{99.5}$), or the 0.1st and 99.9th percentiles ($P_{0.1}$ and $P_{99.9}$). These values were rounded to define the upper and lower limits of the initial reference ranges, which are scheduled for recalibration once the cumulative dataset exceeds 100,000 cases. The newly calculated reference ranges were implemented for clinical use only after consultation with and confirmation by clinical physicians and formal documentation. Categorical data are presented as relative numbers, and a $P$-value of less than 0.05 was considered statistically significant.

2.1 海南省新生儿多种

The incidence rate of various inherited errors of metabolism (IEM) among newborns in Hainan Province was found to be 1/2,215. Additionally, the normal reference ranges for 14 amino acids, 36 carnitines, 1 ketone, 2 adenosines, 4 lysophosphatidylcholines, and their respective ratios were established for the neonatal population in Hainan. Between February and May 2024, using the original manufacturer's kit reference ranges, a total of 29,676 valid data entries were accumulated. During this period, 984 cases screened positive, resulting in an initial screening positive rate of 3.29%. Of these, 748 cases were recalled, yielding a recall rate of 76.02%, and 11 cases were ultimately diagnosed with IEM. Based on these data, the percentile method was employed to calculate new reference ranges for each indicator specifically for newborns in Hainan Province. Subsequently, 54,302 samples were collected from June to December 2024:

Among these samples, 1,879 cases screened positive, representing an initial screening positive rate of 3.40%. A total of 1,716 cases were recalled, achieving a recall rate of 91.33%, and 27 cases were ultimately diagnosed with IEM, as shown in [TABLE:1] and Table 2.

Comparisons between the initial screening positive rates using the kit's ranges and the newly calculated ranges showed no statistically significant differences ($\chi^2 = 0.712, P = 0.399$). However, the positive predictive value (PPV) of the newly calculated reference range (1.57%) was higher than that of the kit's reference range (1.47%), as detailed in [TABLE:2].

Distribution of Neonatal IEM Diseases in Hainan Province

In this study, 38 cases of IEM were diagnosed among newborns in Hainan Province. Fatty acid oxidation disorders were the most prevalent, followed by organic acidemias ([TABLE:3]). The specific breakdown included: (1) 4 cases of amino acid metabolic disorders: 2 cases of phenylketonuria (PKU), 1 case of maple syrup urine disease (MSUD), and 1 case of hypermethioninemia (HMET); and 5 cases of urea cycle disorders. (2) 5 cases of Citrin deficiency (CD). (3) 7 cases of organic acid metabolic disorders: 2 cases of 3-methylcrotonyl-CoA carboxylase deficiency (MCCD), 2 cases of glutaric acidemia type I (GA-I), and 3 cases of 2-methylbutyryl-glycinuria (SBCADD). (4) 22 cases of fatty acid oxidation disorders: 13 cases of primary carnitine deficiency (PCD), 6 cases of short-chain acyl-CoA dehydrogenase deficiency (SCADD), 1 case of carnitine palmitoyltransferase I deficiency (CPT I), 1 case of carnitine-acylcarnitine translocase deficiency (CACT), and 1 case of glutaric acidemia type II (GA-II). Among the aforementioned conditions, PCD, CD, and SBCADD were the most common single-disease types of IEM in Hainan Province.

There were no statistically significant differences in the IEM diagnostic rates ($\chi^2 = 1.643, P = 0.200$) or other primary screening metrics when comparing the two periods.

3 讨论

Since the implementation of free newborn screening for multiple inherited metabolic diseases in Hainan Province, the project has expanded rapidly, achieving a 100% coverage rate and a 99.63% participation rate. In accordance with the requirements of technical expert consensus, a total of 29,676 valid data entries were collected between February and May 2024. These data were used to calculate and establish the first reference ranges in Hainan Province for various newborn indicators, including amino acids, carnitine, succinylacetone, adenosine, and lecithin.

A comparison was conducted between the number of initial screening positives and confirmed diagnostic cases using two different reference ranges. Statistical analysis revealed no significant differences between the two, demonstrating that the application of these reference ranges for screening is scientifically sound. However, by utilizing the laboratory-specific reference ranges established in this study alongside the established expert consensus on methodology, the screening process can be further refined.

Results and Analysis

Percentile Positioning Method for Metabolic Markers

The application of the percentile positioning method provides a standardized reference range for various acylcarnitine species within the study population. This method is critical for identifying metabolic deviations in clinical practice. The following data details the observed ranges for specific long-chain and medium-chain acylcarnitines, categorized by their respective carbon chain lengths and degrees of unsaturation.

[TABLE:1]

The distribution of these metabolites demonstrates specific concentration intervals. For instance, the reference range for decanoylcarnitine (C10:1) was established at 0.01~0.11 and 0.02~0.1 across the observed cohorts. Similarly, dodecenoylcarnitine (C12:1) showed a consistent range of 0.01~0.16.

As the carbon chain length increases, the observed variance in concentration also shifts. Tetradecenoylcarnitine (C14:1) values ranged from 0.02~0.23 to 0.02~0.21, while tetradecadienoylcarnitine (C14:2) remained relatively low and stable at 0.01~0.04. Hexadecenoylcarnitine (C16:1) exhibited a broader distribution, ranging from 0.03~0.44 to 0.02~0.36, with its hydroxylated form (C16:1OH) maintaining a narrower range of 0.01~0.09.

The most significant concentrations were observed in the C18 series. Oleoylcarnitine (C18:1) showed the highest concentration levels, with a percentile range spanning 0.55~2.62 and 0.37~2.72. Linoleylcarnitine (C18:2) followed with a range of 0.06~0.65 and 0.06~0.6. These percentile-based benchmarks serve as a vital diagnostic tool in general practice for evaluating fatty acid oxidation and metabolic health.

[TABLE:1] (Continued)

Analyte Percentile Localization Method (Range 1) Percentile Localization Method (Range 2) C18:1OH 0.01~0.05 0.01~0.04 C18:2OH 0~0.04 0~0.03 C20:0-LPC 0.05~0.68 0.07~0.95 C22:0-LPC 0.02~0.45 0.07~0.56 C24:0-LPC 0.12~1.04 0.13~0.89 C26:0-LPC 0.05~0.79 0.08~0.59

Note:
ALA = Alanine; ARG = Arginine; ASA = Argininosuccinic acid; CIT = Citrulline; GLN = Glutamine; GLU = Glutamic acid; GLY = Glycine; LEU = Leucine; C0 = Free carnitine; C2 = Acetylcarnitine; C3 = Propionylcarnitine; C3DC+C4OH = Malonylcarnitine + 3-hydroxybutyrylcarnitine; C4 = Butyrylcarnitine; C4DC+C5OH = Methylmalonylcarnitine + 3-hydroxyvalerylcarnitine; C5 = Isovalerylcarnitine; C5:1 = Tiglylcarnitine; C5DC+C6OH = Glutarylcarnitine + 3-hydroxyhexanoylcarnitine; C6 = Hexanoylcarnitine; C6DC = Adipoylcarnitine; C8 = Octanoylcarnitine; C8:1 = Octenoylcarnitine; C10 = Decanoylcarnitine; C10:1 = Decenoylcarnitine; C10:2 = Decadienoylcarnitine; C12 = Lauroylcarnitine.

The following abbreviations and corresponding chemical names are used throughout this study:

• C16:1: Palmitoleoylcarnitine
• C16OH: 3-hydroxypalmitoylcarnitine
• C16:1OH: 3-hydroxypalmitoleoylcarnitine
• C18: Octadecanoylcarnitine (Stearoylcarnitine)
• C18:1: Octadecenoylcarnitine (Oleoylcarnitine)
• C18:2: Octadecadienoylcarnitine (Linoleoylcarnitine)
• C18OH: 3-hydroxyoctadecanoylcarnitine
• C18:1OH: 3-hydroxyoctadecenoylcarnitine
• C18:2OH: 3-hydroxyoctadecadienoylcarnitine
• C20: Eicosanoylcarnitine (Arachidoylcarnitine)
• C22: Docosanoylcarnitine (Behenoylcarnitine)
• C24: Tetracosanoylcarnitine (Lignoceroylcarnitine)
• C26: Hexacosanoylcarnitine (Cerotoylcarnitine)
• C20:0-LPC: Eicosanoyl lysophosphatidylcholine
• C22:0-LPC: Docosanoyl lysophosphatidylcholine
• C24:0-LPC: Tetracosanoyl lysophosphatidylcholine
• C26:0-LPC: Hexacosanoyl lysophosphatidylcholine

Screening Status of Inherited Metabolic Diseases (IEM) in Newborns in Hainan Province (February–December 2024)

[TABLE:1]

The screening results for Inherited Metabolic Diseases (IEM) in Hainan Province from February to December 2024 are summarized below. The data includes the total number of screenings, initial positive cases, recall rates, and definitive diagnoses, alongside specific biochemical and genetic findings for confirmed cases.

Screening and Diagnostic Metrics

During the specified period, the screening program monitored several key performance indicators:
- Total Screened: [Number of cases]
- Initial Positives: [Number of cases]
- Initial Positive Rate: [%]
- Recalled: [Number of cases]
- Recall Rate: [%]
- Confirmed Diagnoses: [Number of cases]
- Positive Predictive Value (PPV): [%]

Clinical and Laboratory Findings for Confirmed Cases

The following cases were identified through Tandem Mass Spectrometry (MS/MS) and confirmed via Gas Chromatography-Mass Spectrometry (GC-MS) and genetic analysis:

1. Phenylketonuria (PKU)

Two cases of PKU were identified with the following biochemical and genetic profiles:
- Case 1: MS/MS results showed elevated Phenylalanine (PKU/PHE) at $329.18 \text{ \mu mol/L} \uparrow$ and a PHE/TYR ratio of $6.35 \uparrow$. Genetic analysis revealed compound heterozygous mutations: $c.1315+6T>A$ and $c.122C>T$.
- Case 2: MS/MS results showed PHE at $225.14 \text{ \mu mol/L} \uparrow$ and a PHE/TYR ratio of $3.03 \uparrow$. Genetic testing identified compound heterozygous mutations: $c.478C>T$ and $c.320A>G$.

2. Hypermethioninemia

One case presented with elevated Methionine (MET) levels at $56 \text{ \mu mol/L} \uparrow$ and a MET/PHE ratio of $1.41 \uparrow$. Genetic analysis confirmed a heterozygous mutation in the $MAT1A$ gene ($c.7

Case Report: Identification of BCKDHB Mutations

Genetic Analysis Results

In this clinical case, genetic testing identified two distinct mutations in the BCKDHB gene, which encodes the E1 beta subunit of the branched-chain alpha-keto acid dehydrogenase complex. The patient carries a compound heterozygous genotype consisting of the following variants:

1. c.658C>T (p.Arg220*): This is a nonsense mutation occurring at position 658 of the coding sequence, where a cytosine is replaced by a thymine. This substitution results in the conversion of an Arginine codon to a premature stop codon at position 220 of the protein sequence, likely leading to a truncated protein or nonsense-mediated decay of the mRNA.

2. c.920dup (p.Tyr308*): This variant is a duplication mutation at position 920 of the cDNA. The insertion of an extra base causes a frameshift starting at Tyrosine-308, which subsequently results in a premature termination of the polypeptide chain.

Clinical Significance

Mutations in the BCKDHB gene are primarily associated with Maple Syrup Urine Disease (MSUD) Type 1B. MSUD is an autosomal recessive metabolic disorder characterized by the body's inability to properly process certain amino acids (leucine, isoleucine, and valine).

The presence of these two heterozygous mutations in a compound state typically impairs the enzymatic activity of the branched-chain alpha-keto acid dehydrogenase complex. This enzymatic deficiency leads to the accumulation of branched-chain amino acids and their corresponding toxic keto acids in the blood and urine, which can result in neurological damage and the characteristic "maple syrup" odor of bodily fluids if not managed through dietary intervention.

The biochemical analysis and genetic testing results are summarized as follows:

The patient exhibited significantly elevated levels of several metabolic markers, including a VAL/PHE ratio of 11.70, 2-K-ICA at 11447.6, CIT at 22.82, 3-OHPLA at 86.32, and 4-OHPPA at 132.86.

Genetic sequencing identified a homozygous mutation in the SLC25A13 gene (c.852_855del). This genotype is associated with markedly increased biochemical indicators, specifically a CIT level of 374.54 and a 5-OHPLA level of 39.68.

Clinical Findings and Genetic Analysis

The biochemical analysis revealed a significant elevation in 6-4-OHPPA (4-hydroxyphenyllactate), with a measured value of 42.0 ↑. Genetic testing identified two variants in the SLC25A13 gene: the c.615+5G>A splice-site mutation and the c.852_855del (p.Met285fs) frameshift mutation, both in a heterozygous state. These findings are consistent with a diagnosis related to citrin deficiency.

The following biochemical markers and genetic findings indicate significant metabolic elevations and specific genetic variants:

MET: 79.32 ↑; CIT: 36.78 ↑; 4-OHPLA: 499.16 ↑; 5-4-OHPPA: [Value not provided].

A specific mutation was identified: SLC25A13 c.852_855del. The associated biochemical profile shows CIT: 23.82 ↑; 4-OHPLA: 499.16 ↑; and 5-4-OHPPA: 397.86 ↑.

Furthermore, a homozygous mutation of SLC25A13 c.852_855del was recorded, presenting with the following metabolic levels: CIT: 34.98 ↑; 4-OHPLA: 3127.28 ↑; and 5-4-OHPPA: [Value not provided].

1060.40 ↑ SLC25A13 c.852_855del (Heterozygous) c.615+5G>A

(Continued from Table 3) MS/MS Results ($\mu mol/L$): $C0: 5.31 \downarrow$; $C16: 12.4 \uparrow$; $C18: 2.51 \uparrow$; $C18:1: 5.35 \uparrow$. GA-II: $C5DC: 0.6 \uparrow$; $C8: 0.65 \uparrow$.

The preliminary screening positive rate and incidence rate calculated using laboratory-derived reference ranges were slightly higher than those obtained using the original kit's reference ranges. Furthermore, the positive predictive value (PPV) using laboratory-derived ranges was higher than that of the kit's ranges. This not only provides a diagnostic standard tailored to the population of Hainan Province for subsequent screenings but also reduces the occurrence of missed diagnoses, thereby facilitating the screening, prevention, and control of various inherited metabolic diseases (IMDs) in the region.

Due to the late issuance of the "Notice on the Implementation Plan for the 2024 Hainan Provincial Free Screening Project for Multiple Neonatal Inherited Metabolic Diseases," samples from February were screened retrospectively. Consequently, the recall rate for the period from February to May was only 76.02%. As screening operations became more systematic, the recall rate gradually improved, reaching 91.33% for the period from June to December.

Through comprehensive screening and diagnostic efforts conducted across the province from February to December 2024, we have, for the first time, systematically...

MCCC2 c.464G>A (Heterozygous) + c.1538T>C (Heterozygous)

The systematic screening results reveal that the incidence of inborn errors of metabolism (IEM) among newborns in Hainan Province is 1/2,215 (38/84,184). This figure is consistent with the overall incidence rates reported internationally, which range from approximately 1/2,555 to 1/784 \cite{13-14}. Notably, this rate is higher than the national average in China, which stands at 1/2,585. These findings underscore the necessity of implementing comprehensive free screening programs for multiple genetic metabolic diseases in Hainan Province.

According to Table 3, the three most prevalent specific IEM disorders in Hainan newborns are Primary Carnitine Deficiency (PCD), Citrin Deficiency (CD), and Short-Chain Acyl-CoA Dehydrogenase Deficiency (SBCAD). The incidence of PCD in Hainan is 1/6,476, which exceeds the threshold of 1/10,000. While this is lower than the 1/3,740 incidence rate previously reported in ethnic minority areas of Hainan, further analysis indicates that the PCD incidence among Li newborns in this study (5/11,499) is comparable to that of Han newborns.

[TABLE:3]

The diagnostic data, including Gas Chromatography-Mass Spectrometry (GC-MS) results and genetic analysis, are summarized below for confirmed cases:

Primary Carnitine Deficiency (PCD)

The biochemical hallmark for these cases was a significant decrease in free carnitine ($C0$). Genetic testing identified various mutations in the $SLC22A5$ gene:
- $C0: 6.09 \downarrow$; $SLC22A5$ c.51C>G heterozygous + c.92C>G
- $C0: 3.16 \downarrow$; $SLC22A5$ c.51C>G homozygous
- $C0: 7.88 \downarrow$; $SLC22A5$ c.694A>G heterozygous + c.1064C>T heterozygous
- $C0: 5.96 \downarrow$; $SLC22A5$ c.51C>G heterozygous + c.760C>T heterozygous
- $C0: 6.23 \downarrow$; $SLC22A5$ c.51C>G heterozygous + c.415G>A heterozygous
- $C0: 2.80 \downarrow$; $SLC22A5$ c.51C>G heterozygous + c.760C>T heterozygous
- $C0: 4.09 \downarrow$; $SLC22A5$ c.51C>G heterozygous + c.760C>T heterozygous
- $C0: 6.16 \downarrow$; $SLC22A5$ c.51C>G heterozygous + c.797C>T heterozygous
- $C0: 1.99 \downarrow$; $SLC22A5$ c.839C>T heterozygous + c.1064C>T
- $C0: 5.01 \downarrow$; $SLC22A5$ c.51C>G heterozygous + c.1400C>G heterozygous
- $C0: 6.79 \downarrow$; $SLC22A5$ c.51C>G heterozygous + c.1400C>G heterozygous
- $C0: 3.15 \downarrow$; $SLC22A5$ c.51C>G heterozygous + c.338G>A heterozygous

Short-Chain Acyl-CoA Dehydrogenase Deficiency (SCADD)

Cases presented with elevated butyrylcarnitine ($C4$) and $C4/C3$ ratios, often accompanied by increased ethylmalonic acid (EMA) in urine. Mutations were identified in the $ACADS$ gene:
- $C4: 0.69 \uparrow, C4/C3: 0.44 \uparrow, EMA: 36.09 \uparrow$; $ACADS$ c.322G>A heterozygous + c.625G>A heterozygous
- $C4: 0.60 \uparrow, C4/C3: 0.40 \uparrow$; $ACADS$ c.431C>T heterozygous + c.625G>A heterozygous
- $C4: 0.68 \uparrow, C4/C3: 13.60 \uparrow$; $ACADS$ c.322G>A heterozygous + c.625G>A heterozygous
- $C4: 0.49 \uparrow, C4/C3: 0.44 \uparrow$; $ACADS$ c.322G>A heterozygous + c.625G>A heterozygous
- $C4: 0.60 \uparrow, C4/C3: 0.34 \uparrow, EMA: 69 \uparrow$;

DecA: 83.29 SLC25A20 c.199-10T>G heterozygous + c.326+1G>A heterozygous

The biochemical and genetic analysis revealed several significant findings related to metabolic disorders. The results are summarized as follows:

Metabolic and Genetic Findings

The patient exhibited elevated levels of decanoylcarnitine (C10: 1.32 $\uparrow$) and dodecanoylcarnitine (C12: 1.14 $\uparrow$). Genetic testing identified compound heterozygous mutations in the ETFDH gene: c.250G>A and c.353G>T. These findings are consistent with a diagnosis related to electron transfer flavoprotein dehydrogenase deficiency.

Furthermore, hydroxyvalerylcarnitine levels were significantly elevated (C5OH: 0.88 $\uparrow$). Genetic analysis of the MCCC2 gene revealed compound heterozygous mutations: c.914A>G and a splice-site mutation c.903+1G>A.

Additional metabolic markers showed a marked increase in C5OH at 3.49 $\uparrow$, with a C5OH/C3 ratio of 0.03 $\uparrow$. Urinary organic acid analysis further supported these findings, showing significantly elevated levels of 3-methylcrotonylglycine (3MCG: 91.57 $\uparrow$) and 3-hydroxyisovaleric acid (3-OH-IVA). These results are highly indicative of 3-methylcrotonyl-CoA carboxylase deficiency.

Clinical and Genetic Profiles of Identified Metabolic Disorders

The diagnostic results for Glutaric Acidemia Type I (GA-I) and Short-Chain L-3-Hydroxyacyl-CoA Dehydrogenase Deficiency (SBCADD) are summarized below, including biochemical markers and genetic variants.

Glutaric Acidemia Type I (GA-I)

In the first case of GA-I, biochemical analysis revealed significantly elevated levels of glutarylcarnitine (C5DC) at 2.28 and a C5DC/C8 ratio of 45.60. Urinary organic acid analysis showed a marked increase in glutaric acid (GA) at 5010.17 and 2-hydroxyglutaric acid (2-OHGA) at 42.27. Genetic testing identified compound heterozygous mutations: c.1063C>T and c.271+1G>A.

In the second case of GA-I, C5DC was elevated to 2.90 with a C5DC/C8 ratio of 96.67. Urinary GA was measured at 1667.44. Genetic analysis confirmed compound heterozygosity for the c.881G>A and c.1063C>T variants.

2-Methylbutyryl-CoA Dehydrogenase Deficiency (SBCADD)

Three cases of SBCADD were identified through elevations in 2-methylbutyrylcarnitine (C5) and specific urinary metabolites.

1. The first subject presented with a C5 level of 1.15 and a C5/C3 ratio of 1.42. Urinary 2-methylbutyrylglycine (2-MBT-GLY) was 70.93. Genetic testing of the ACADSB gene revealed compound heterozygous mutations: c.275C>G and c.746del.
2. The second subject showed a C5 level of 0.42 and a C5/C3 ratio of 0.62, with a 2-MBT-GLY level of 105.01. Genetic analysis identified compound heterozygous variants c.746del and c.655G>A in the ACADSB gene.
3. The third subject exhibited a C5 level of 1.14 and a C5/C3 ratio of 1.21, with a 2-MBT-GLY level of 18.04.

Chinese General Practice ($P < 0.011$), which may suggest that the Li ethnic group is a population predisposed to Primary Carnitine Deficiency (PCD). This disparity is likely associated with the genetic founder effect resulting from long-term geographical isolation and the practice of endogamy within the Li population.

Previous studies have demonstrated significant ethnic variations in the incidence of PCD, which is consistent with the established patterns of genetic disease transmission. For instance, the incidence of PCD in the Egyptian population is reported at 1/250,000, whereas the incidence among Faroese children is significantly higher at 8/70,732 ($\chi^2 = 6.477, P = 0.011$). This study further confirms such ethnic disparities.

Furthermore, nine distinct mutations of the $SLC22A5$ gene were identified in this cohort. Among these, $c.51C>G$ was found to be a hot-spot mutation. This finding differs from the most common $SLC22A5$ gene variant typically observed in the broader Chinese population ($c.1400C>G$), as well as the prevalent mutations found in the Faroese population (1/300).

[19], which also differs from the variant observed in the Quanzhou region of Fujian Province (c.760C>T).

[20], however, it is close to the frequency observed in the Guangzhou region (c.51C>G), which is adjacent to Hainan Province.

According to PolyPhen-2 predictions, although c.51C>G (p.Phe17Leu) is a synonymous mutation, it may lead to the loss of carnitine transporter function by affecting mRNA splicing stability. This "regional mutation hotspot" suggests that Hainan Province may serve as a critical node for research into Inborn Errors of Metabolism (IEM) gene variants in East Asia. In particular, attention should be paid to the potential impact of tropical climates on genetic selection—for instance, whether genes related to fatty acid metabolism (such as $ACADS$) are more prone to accumulating functional mutations due to adaptive evolution in high-temperature environments.

In cases of Short-Chain Acyl-CoA Dehydrogenase Deficiency (SCADD), the identified mutation hotspots in the $ACADS$ gene were c.322G>A and c.655G>A. This distribution differs significantly from the hotspot mutation sites reported in the Lianyungang region (c.1031A>G and c.1130C>T). Furthermore, the c.852_855del mutation in the $SLC25A13$ gene accounted for 80% (4/5) of Citrin deficiency (CD) cases, which is consistent with the prevalence of the c.851_854del variant commonly observed in domestic populations.

and the hotspot mutation site (c.1031A > G) in the Hefei region.

Although these variants involve only a few bases, they can lead to more severe protein truncation. Whether such "Hainan-specific" variants result from gene-environment interactions warrants further in-depth functional experimental analysis. Notably, two Citrin Deficiency (CD) cases in this diagnostic cohort presented initial screening values for citrulline (Cit) within both the kit’s reference range (5.85–28.99 $\mu$mol/L) and the reference range calculated in this study (4.9–27.0 $\mu$mol/L), leading to initial negative clinical interpretations. These two patients were subsequently admitted to our hospital’s neonatal department due to the onset of cholestasis. Follow-up tandem mass spectrometry (MS/MS) was performed twice; the final tests revealed elevated Cit concentrations (64.87 $\mu$mol/L and 95.50 $\mu$mol/L), and genetic testing confirmed homozygous c.852_855del mutations in both cases. Literature reviews indicate that only approximately 40% of neonatal intrahepatic cholestasis caused by Citrin deficiency (NICCD) is detectable via MS/MS screening during the neonatal period \cite{25-26}. This is attributed to the relatively low total amino acid levels in these infants, which results in absolute values of citrulline, phenylalanine, tyrosine, and methionine that remain below the detection thresholds of MS/MS.

This study confirms that the implementation of free universal Inborn Errors of Metabolism (IEM) screening increased the participation rate from 6.85% (5,934/86,585) in 2023 to 100% in 2024. Furthermore, the detection rate of confirmed cases increased nearly 40-fold (3/38) compared to the self-pay period, indicating that the birth defect prevention system in Hainan Province is becoming increasingly robust. Simultaneously, we have mapped the genetic and metabolic profiles of neonatal IEMs in Hainan, revealing the unique characteristics of IEMs in this tropical region.

Author Contributions

Zhao Zhendong proposed the research concept and designed the study protocol and objectives. Zhao Peizhen and Xu Haizhu were responsible for conducting the experiments, implementing the research process, selecting subjects, and performing index testing and detection. Zhao Peizhen and Xu Haizhu were responsible for data collection, acquisition, cleaning, and statistical analysis. Zhao Peizhen drafted the initial manuscript. Xu Haizhu was responsible for the final revision and holds overall responsibility for the paper.

The authors declare no conflicts of interest.

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