Objective:To investigate the diagnostic value of fluorescence quantitative method and G6PD/6PGD ratio method in glucose-6-phosphate dehydrogenase (G6PD) deficiency and the type of gene mutation.Methods:A total of 1 201 patients (711 males and 490 females) with suspected G6PD deficiency in Shanghai Children′s Hospital were collected from June 2018 to March 2021. Fluorescence quantification method, G6PD/6PGD ratio method and multicolor melting curve were used to detects enzyme activity, ratio and gene mutation type. Comparison of each index and evaluation of its diagnostic efficiency were performed.Results:Among 1 201 suspicious samples, 163 cases (135 males and 28 females) were finally diagnosed. 156 cases were diagnosed by fluorescence quantitative method with a detection rate of 95.71%, and 140 cases were diagnosed by G6PD/6PGD ratio method with a detection rate of 85.89%. enzymatic activity of G6PD and ratio of G6PD/6PGD in male were significantly lower than female, and the differences were statistically significant ( U=642.5, 734.5, P<0.001). 112 cases received G6PD gene mutation detection and 92 cases were diagnosed, 74 were hemizygous mutations, 1 were homozygous mutations, 15 were heterozygous mutations, and 2 were compound heterozygous mutations. Among 15 cases of heterozygous mutations, 11 cases were diagnosed by fluorescence quantitative method, the diagnosed rate was 73.33%, 4 cases were diagnosed by G6PD/6PGD ratio method, and the diagnosed rate was 26.67%. A total of 7 mutation sites were detected and the proportions were c.1388G>A (32.22%), c.1376G>T (30.00%), c.871G>A (13.33%), c.1024C>T (11.11%). c.95A>G (7.78%), c.487G>A (4.44%), c.392G>T (1.11%). The enzymatic activities of c.1376G>T and c.1024C>T, c.487G>A were statistically significant ( P<0.001,0.015); the G6PD/6PGD ratios of c.1024C>T and c.1388G>A, c.1376G>T were statistically significant ( P=0.017,0.002,0.011,0.013). Fluorescence quantitative method had sensitivity of 100%, specificity of 95.65%, and the area under the curve (AUC) is 0.972. The sensitivity of the G6PD/6PGD ratio method was 100%, the specificity was 94.57%, and the AUC was 0.979. The sensitivity of fluorescence quantitative method combined with G6PD/6PGD ratio was 96.7%, the specificity was 100%, and the AUC was 0.992. Conclusions:Compared with fluorescence quantification, the G6PD/6PGD ratio method might not be able to diagnose female heterozygotes effectively; The panel of G6PD fluorescence quantification and G6PD/6PGD ratio was helpful to reduce the missed diagnosis. Combined with gene mutation analysis, it could improve the diagnosis rate of G6PD deficiency in the children.

Objective:To analyze the difference and reliability of blood 17-hydroxyprogesterone (17-OHP), an indirect screening index for congenital adrenal hyperplasia (CAH), between preterm and full-term infants.Methods:In this retrospective cross-sectional study, a total of 210 285 newborns who underwent CAH screening at the Neonatal Screening Center of Shanghai Children′s Hospital from January 2019 to December 2022 were collected, including 14 312 premature infants and 195 973 full-term infants.The concentration of 17-OHP in dried blood spots on filter paper was determined by an automatic fluorescence analyzer.The distribution of 17-OHP levels in preterm and full-term infants and its statistical index were analyzed.The Kolmogorov-Smirnov test was used for normal distribution.The skewed distribution data was converted into approximately normal distribution using Box-Cox.Outliers were eliminated by the interquartile range method.The cumulative frequency distribution map was drawn by R language programming.The 99.5 th percentile value was used as the screening threshold and compared with the reference value given by the manufacturer or laboratory and with the reference change value (RCV). Results:According to the threshold provided by the laboratory, 26.76‰ of premature infants were tested positive in preliminary screening, and 4 were confirmed with an incidence of 1∶3 578, while 0.79‰ of full-term infants were tested positive in preliminary screening, and 11 were confirmed with an incidence of 1∶17 816.The thresholds for CAH screening established indirectly were 20.35 nmol/L in preterm infants and 10.78 nmol/L in full-term infants.The relative deviations between the indirect CAH screening thresholds and the manufacturer′s or laboratory′s CAH screening thresholds were higher than the RCV, respectively.According to the indirect CAH screening thresholds, the negative and positive coincidence rates of 65 samples in 13 batches from the Centers for Disease Control and Prevention interlaboratory quality assessment program in the United States reached 100%.A retrospective analysis of 210 285 neonates showed that 17-OHP concentration was higher than the screening threshold in all CAH-positive neonates.The application of this screening threshold reduced the false positive rate of preterm infants by 59.79%.Conclusions:It is feasible to establish the CAH screening thresholds for premature and full-term infants by an indirect method, which can improve the efficiency of screening and provide better diagnostic basis for clinical practice.

OBJECTIVE To establish the fingerprint of Gegenqinlian tablets and determine the content of eleven components(3’-hydroxy puerarin, puerarin, 3’-methoxy puerarin, daidzin, baicalin, palmatine hydrochloride, berberine hydrochloride, scutellarin, baicalein, ammonium glycyrrhizinate and wogonin) by quantitative analysis of multi-components by single-marker(QAMS).METHODS Ten batches of Gegenqinlian tablets were determined by HPLC and a common fingerprint was established. Baicalin was selected as internal reference. The relative correction factors of the component with 3’-hydroxy puerarin, puerarin, 3’-methoxy puerarin, daidzin, palmatine hydrochloride, berberine hydrochloride, scutellarin, baicalein, ammonium glycyrrhizinate and wogonin were calculated and their contents were calculated. The feasibility and scientificity of QAMS was evaluated by comparison on the results between the measured value and calculation value by external standard method and QAMS. The chromatographic separation was performed on ananalytical column of Waters Xbridge-C18(250 mm× 4.6 mm, 5 μm) with gradient elution, the mobile phase was acetonitrile 0.1% phosphoric acid aqueous solution, at a flow rate of 1.0 mL·min-1. The column temperature was 30 ℃ and the detection wavelength was 260 nm.RESULTS There were 20 peaks in 10 batches of Gegenqinlian tablets and 11 chemical constituents were identified. The similarity of 10 batches of Gegenqinlian tablets was >0.97. The linear range of 3’-hydroxy puerarin, puerarin, 3’-methoxy puerarin, daidzin, baicalin, palmatine hydrochloride, berberine hydrochloride, scutellarin, baicalein, ammonium glycyrrhizinate and wogonin were 0.056 6-2.830 2, 0.241 2-12.058 6, 0.128 0-6.401 0, 0.059 7-2.983 5, 0.242 7-12.134 9, 0.045 7-2.285 7, 0.192 8-9.641 0, 0.043 3-2.167 0, 0.018 0-0.900 2, 0.021 0-1.048 4, 0.011 5-0.575 4 μg (r2= 0.999 6-1.000) respectively. The average recovery were 100.23%, 102.01%, 101.66%, 102.73%, 100.17%, 98.45%, 98.41%, 100.95%, 101.85%, 97.97%, 100.09%(RSD=1.24%-2.57%, n=6) respectively. The relative correction factors of 3’-hydroxy puerarin, puerarin, 3’-methoxy puerarin, daidzin, palmatine hydrochloride, berberine hydrochloride, scutellarin, baicalein, ammonium glycyrrhizinate and wogonin were 0.860 4, 0.605 3, 0.850 9, 0.582 8, 0.557 1, 0.498 6, 0.767 2, 0.652 1, 2.608 1, 0.545 2 respectively. RAD between QAMS method and external standard method were 0.03%-2.12%. CONCLUSION The combination of QAMS and fingerprint can provide reference for the quantitative determination and quality control of Gegenqinlian tablets.

Objective:To explore the clinical, biochemical and genetic characteristics of three children with Isoleucine metabolic disorders due to variants of HSD17B10 and ACAT1 genes. Methods:Two children with 17β hydroxysteroid dehydrogenase 10 (HSD17B10) deficiency and a child with β-ketothiolase deficiency (BKD) diagnosed at Shanghai Children′s Hospital between 2014 and 2021 were selected as the study subjects. Clinical data of the children were collected. The children were subjected to blood acylcarnitine, urinary organic acid and genetic testing, and candidate variants were analyzed with bioinformatic tools.Results:The main symptoms of the three children had included epilepsy, developmental delay, hypotonia and acidosis. Their blood acylcarnitine methylcrotonyl carnitine (C5: 1), 3-hydroxyisovalerylcarnitine (C5-OH) and 3-hydroxybutylcarnitine (C4OH) were increased to various extents, and urine organic acids including methyl crotonylglycine and 2-methyl-3-hydroxybutyric acid were significantly increased. Child 1 and child 2 were respectively found to harbor a c. 347G>A (p.R116Q) variant and a c. 274G>A (p.A92T) variant of the HSD17B10 gene, and child 3 was found to harbor compound heterozygous variants of the ACAT1 gene, namely c. 547G>A (p.G183R) and a c. 331G>C (p.A111P). Among these, the c. 274G>A (p.A92T) and c. 331G>C (p.A111P) variants were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), they were respectively classified as variant of unknown significance (PP3_Strong+ PM2_supporting) and likely pathogenic (PM3+ PM2_Supporting+ PP3_Moderate+ PP4). Conclusion:Both the HSD17B10 deficiency and BKD can lead to Isoleucine metabolism disorders, which may be difficult to distinguish clinically. Genetic testing can further confirm the diagnosis. Discoveries of the HSD17B10: c. 274G>A (p.A92T) variant and the ACAT1: c. 331G>C (p.A111P) variant have enriched the mutational spectrum of the two diseases.