Expanded newborn screening for inborn errors of metabolism by tandem mass spectrometry in newborns from Xinxiang city in China.

Abstract Background Tandem mass spectrometry is a powerful technology available in China over the last 15 years. The development of tandem mass spectrometry had made it possible to rapidly screen newborns for inborn errors of metabolism. The aim of this study was to determine the birth incidence of inborn errors of metabolism through expanded screening of newborns by tandem mass spectrometry in Xinxiang area. Methods Dried blood spots from 50 112 newborns were assessed for inborn errors of metabolism by tandem mass spectrometry. The diagnoses were confirmed based on the clinical features, conventional laboratory tests, and the organic acid levels tested in urine by gas chromatography‐mass spectrometry. Results The study findings revealed that 31 newborns were diagnosed with inborn errors of metabolism. The total incidence rate of inborn errors of metabolism was 1/1617, and these included 16 cases of amino acid disorders (51.6%), nine cases of organic acid disorders (29.0%), and 6 (19.4%) cases of fatty acid beta‐oxidation disorders. Conclusions The screening for the incidence of inborn errors of metabolism in Xinxiang area showed that the rate was higher than previously reported. This study provides valuable data which may be useful in facilitating improvements in the expansion of screening to enable early diagnosis and treatment of inborn errors of metabolism before the onset of symptoms.


| INTRODUC TI ON
Inborn errors of metabolism (IEM) are a large group of complex and rare genetic diseases caused by a severe deficiency in enzymes or carrier proteins function. 1 Although individual metabolic diseases are relatively rare, IEM collectively presents a significant healthcare burden. Early diagnosis is important to timely treatment, thereby preventing IEM related mortality and morbidity.
Newborn screening for IEM is a vital and efficient approach for early diagnosis of serious inherited disorders in apparently healthy newborns and pre-symptomatic treatment to improve their survival.
Over the last few decades, tandem mass spectrometry (MS/MS) was introduced as a powerful technology used in newborn screening. In Mainland China, MS/MS newborn screening started in 2004 and since then, the coordination of screening services has continued to expand. 2 Analyses of amino acids and acylcarnitine in blood have led to simultaneous detection of three groups of IEM which include amino acid disorders, organic acid disorders, and fatty acid beta-oxidation disorders.

| Amino acids and acylcarnitine analysis
An API3200 triple quadrupole mass spectrometer (AB SCIEX LLC) equipped with an electrospray ionization (ESI) interface operated in positive ionization mode was used for the mass spectrometric detection. A multiple reaction monitoring mode was operated to detect specific precursor and product ions. The ESI parameters were curtain gas, 25 psi; collision gas, 6 psi; lonspray voltage, 5500 V; temperature, 450°C; ion source gas1, 45 psi; ion source gas2, 55 psi; interface heater, on. The mass spectrum parameters for analytes are shown in Table S1.
The NeoBase kit (Perkin-Elmer Life Sciences) was used as the detection reagent for blood amino acids and acylcarnitine. The detection platforms included a UFLC XR liquid chromatography (Shimadzu Corp) and an API3200 mass spectrometer (AB SCIEX LLC). Prior to the testing, blood spot samples (3 mm) were punched out from the dry blood spots containing approximately 3.2 μL of whole blood. The dry blood samples spot specimens were placed each on a single well of a 96-well microtiter plate to which 100 µL of the extracting solution containing the internal standard of amino acids and acylcarnitine was added. The plates were placed on a shaker at 45°C for 45 minutes, 75 μL of the extracted solution transferred to a V-bottomed plate and covered with aluminium foil. The samples were incubated at room temperature for 1 hour and then placed in the autosampler for detection.

| Organic acid analysis
GCMS-QP2010 (Shimadzu Corp) was used for the detection of urinary organic acids. The test kits included urease, heptadecanoic acid, tetracosane, tropinic acid, hydroxylamine hydrochloride, anhydrous sodium sulfate, sodium hydroxide, hydrochloric acid, and ethyl acetate (Sigma). BSTFA with 10% TMCS was provided by Pierce Chemical. The specific method used was as described in the article. 5

| Confirmatory studies
In addition to the amino acids and acylcarnitine's levels, diagnosis of the disorder was confirmed through clinical features, conventional laboratory tests, and organic acid levels in urine as tested by gas chromatography-mass spectrometry. 6

| Quality control program
Monitoring of the quality of the laboratory performance for the newborn screening was achieved by participating in an External Quality Assessment Programs of National Center for Clinical Laboratories.
The age of blood collected from the newborns ranged from 3 to 42 days with an average age of (5.0 ± 3.2) days.

| Newborn screening
Among the 50 112 screened newborns, 1149 (2.29%) newborns were suspected to be positive for inborn errors of metabolism during the first screening. Of these, 1102 newborns were successfully recalled for re-testing (95.9%). Confirmatory tests showed that 31 patients had IEMs with a positive predictive value (PPV) of 2.8%.
The overall incidence of IEMs was 1/1617 while that of amino acid disorders, organic acid disorders, and fatty acid beta-oxidation disorders was 1/3132, 1/5568, and 1/8352, respectively. Amino acid disorders accounted for nearly 50% (16 cases) of the total number of patients while organic acid disorders accounted for 29.0% (nine cases) and fatty acid beta-oxidation disorders accounted for 19.4% (six cases; Table 1).

| D ISCUSS I ON
In China, the incidence of IEMs greatly varies across various regions. The overall incidence for this study was approximately 1:1617 which was higher than those reported in other parts of China. Table 3 shows the incidences of IEMs reported in other regions of China. 2,4,7-10 Guo et al 9 reported the incidence of IEMs in Jining, Shandong Province, China as 1/1178, which was close to but slightly higher than the incidence of IEMs reported in this study for Xinxiang.
The incidence of IEMs detected varies greatly in different countries. For example, the incidence of IEM reported in Korea was 1/2000, 11 1/3159 in Singapore, 12 1 /3600 in India, 13 1/2400 in Germany, 14 1/2396 in Italy, 15 and 1/4300 in America. 16 The highest reported incidence of IEM was in the Arab nations (Saudi Arabia 1/1381, Lebanon 1/1482, Egyptian 1/1286). [17][18][19] The wide variation in the incidence is caused by many factors such as the environment, ethnicity, detection criteria for IEMs and the sample source. In the Arab region, the high incidence of IEMs has been explained by the high rates of consanguineous marriages. 20

| Amino acid disorders
Amino acid disorders accounted for 51.6% of the reported cases of IEMs with HPA being the most common type. HPA incidence varies widely in different human populations. In this study, the incidence was 1/3341 which was significantly higher than that reported in other areas of China as well as in other countries. Kun Zhong et al 21 reported that the incidence of HPA was 1/12 189 in China. Huang et al 8 reported that the incidence of HPA was 1/18 487 in Zhejiang Province.
In Germany, the incidence of HPA was reported as 1/4500. 14

| Organic acid disorders
Organic acidemia accounted for 29.0% of all the cases of IEMs in this study. The most common organic acidemias were MMA (eight cases), followed by IVA (1 case). This study results showed that the incidence of MMA was approximately 1/6264. Tu 22  Xinxiang city hence an indication that the incidence of MMA was relatively high in Xinxiang and neighboring areas.

| Fatty acid beta-oxidation disorders
Fatty acid beta-oxidation disorders have been reported not to be common within the Asian population. 26 These study findings revealed that the incidence of fatty acid beta-oxidation disorders was 1/8352 and Other types of fatty acid beta-oxidation disorders are rare in the Chinese population. However, blood sampling was not performed under strict fasting conditions for most of the children; therefore, an underestimation of the incidence of fatty acid beta-oxidation disorders could have occurred.

| CON CLUS IONS
These study findings revealed that the incidence of IEMs in Xinxiang area was higher compared to reports from other regions within China.
Expanded newborn screening by MS/MS method is important for early diagnosis of IEM. The findings from this study can be used to facilitate improvements in the implementation of expanded screening for inborn errors of metabolism in newborns for early diagnosis and treatment.

ACK N OWLED G M ENTS
We thank the patients and their families for their cooperation.

CO N FLI C T O F I NTE R E S T S
The authors declare that there are no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article.