Analysis of gene variation and long-term follow-up in children with phenylalanine hydroxylase deficiency diagnosed by newborn screening. / æ–°ç”Ÿå„¿ç­›æŸ¥ç¡®è¯Šè‹¯ä¸™æ°¨é ¸ç¾ŸåŒ–é ¶ç¼ºä¹ç—‡æ‚£è€ åŸºå› å˜å¼‚åŠé•¿æœŸéšè®¿åˆ†æž.

OBJECTIVES: To retrospectively analyze the variation and characteristics of phenylalanine hydroxylase (PAH) gene, and to observe the long-term treatment effect and follow-up of newborns with PAH deficiency. METHODS: Clinical data, treatment and follow-up results of 198 patients with PAH deficiency diagnosed by newborn screening in Jinan from 1996 to 2021 were collected. The genetic analysis of 55 patients with PAH deficiency diagnosed by newborn screening in Jinan and 213 patients referred from the surrounding areas of Jinan were summarized. Gene variations were checked by a customized Panel gene detection method. Blood phenylalanine-concentration and physical development indicators including height and weight were regularly monitored. Intellectual development was assessed using a neuropsychological development scale for patients aged 0-6 years and academic performance, and brain injury in patients was assessed using brain magnetic resonance imaging. RESULTS: c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. The genotype of c.158G>A variation is compound heterozygous variation, with mainly a mild hyperpheny-lalaninemia. 168 patients with PAH deficiency who were followed-up regularly had normal physical development without dwarfism or malnutrition. Among the 33 preschool patients who underwent mental development assessment, 2 were mentally retarded and the initial treatment age was older than 6 months. Nine patients with an average age of (17.13±2.42) years completed brain magnetic resonance imaging, one case was normal, and 8 cases were abnormal. There were patchy or patchy hyperintense foci near the bilateral lateral ventricles on T2WI, and the intellectual development was normal. Compared with the other eight patients, the blood phenylalanine concentration of the normal child was better and stably controlled within the ideal range. CONCLUSIONS: c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. After standardized treatment, most patients with PAH deficiency diagnosed by screening can obtain normal growth and intellectual development in adolescence, but there are different degrees of organic lesions in the cerebral white matter.

Cut-off values of neonatal lysosomal storage disease-related enzymes detected by tandem mass spectrometry.

OBJECTIVE: To establish cut-off values of lysosomal storage disease (LSD)-related enzymes by tandem mass spectrometry. METHODS: A total of 26 689 newborns and 7 clinically confirmed LSD children underwent screening for LSDs (glycogen storage disease typeⅡ, Fabry disease, mucopolysaccharidosis type Ⅰ, Krabbe disease, Niemann-Pick disease A/B and Gaucher disease). The activities of LSD-related enzymes were detected by tandem mass spectrometry. The 20% of the median enzyme activity of each batch of acid ß-glucocerebrosidase, acid sphingomyelinase, ß-galactocerebroside, α- L-iduronidase and acid α-glucosidase, and the 30% of the median enzyme activity of α-galactosidase were taken as cut-off values of corresponding enzymes. The genetic diagnosis was performed in neonates whose enzyme activity was lower than 70% of the cut-off value. RESULTS: The enzyme activities of 7 clinically confirmed cases were all lower than the cut-off values. Among 26 689 newborns, 142 cases (0.53%) were suspected positive for LSDs, including 25 cases of ß-galactocerebroside deficiency, 1 case of α- L-iduronidase deficiency, 19 cases of α-galactosidase deficiency, and 97 cases of acid α-glucosidase deficiency. Eight infants were genetically diagnosed with LSDs, including 3 cases of glycogen storage disease type Ⅱ, 3 cases of Krabbe disease, and 2 cases of Fabry disease, with a positive predictive value of about 5.6%. Cut-off values ​​of the 6 LSD enzyme activities all showed a downward trend from March to August, and an upward trend from September to December. There was a statistically significant difference in LSD enzyme activity among different months ( P<0.05). CONCLUSION: The established cut-off values of LSD-related enzyme activities detected by tandem mass spectrometry can be used for screening LSDs in neonates, and the enzyme activity would be affected by temperature and humidity.

Establishment of Cutoff Values for Newborn Screening of Six Lysosomal Storage Disorders by Tandem Mass Spectrometry.

Objective: Lysosomal storage disorders (LSDs) are becoming increasingly important in newborn screening, and tandem mass spectrometry (MS/MS) is widely used in newborn screening for LSDs through measurement of enzymatic activities in dried blood spots (DBSs). Overall, the determination of the cutoff value is important in such screening, and different laboratories have different methods of determining this value; most do not use a fixed cutoff value but rather calculate the corresponding batch cutoff value based on each batch of experimental data. In this study, we used MS/MS to screen for LSDs and sought to find an appropriate method to establish the cutoff value for LSD screening. Methods: A total of 38,945 samples from newborn blood tablets collected from various maternity hospitals in six cities in Shandong province, including Jinan, Dezhou, Heze, Linyi, Weifang, and Zibo, were tested using a Waters Xevo TQD tandem mass spectrometer; the experimental data were analyzed with MassLynx V4.1. The laboratory used 30% of the median GLA enzyme activity and 20% of the median ABG, ASM, GALC, IDUA, and GAA enzyme activities in every test as the cutoff values for that batch of experiments. Results: There were 254 suspicious positives in the initial screening test, including one case of Gaucher disease, one of Niemann-Pick disease, 47 of Krabbe disease, four of MPS-I, 21 of Fabry disease, and 180 of Pompe disease. After genetic screening, 11 children were diagnosed, including three with Pompe disease, three with Fabry disease, and five with Krabbe disease. In addition, the enzyme activity cutoff value of this experiment showed seasonal variation, which was initially believed to be related to the ambient temperature, such as the effect of ambient temperature on the human body or the temperature when the blood tablets dried naturally. Conclusion: Overall, MS/MS can be used in LSD screening, and using different cutoff values in each batch of experiments is feasible. The ambient temperature might be a reason why the enzyme activity cutoff value has seasonal variation. More samples are needed to develop a method of determining cutoff values in laboratories.