Characterization of phenylalanine hydroxylase gene variants and analysis of genotype-phenotype correlation in patients with phenylalanine hydroxylase deficiency from Fujian Province, Southeastern China.

BACKGROUND: Phenylalanine hydroxylase deficiency (PAHD) is the most prevalent inherited disorder of amino acid metabolism in China. Its complex phenotype includes many variants and genotypes among different populations. METHODS AND RESULTS: In this study, we analyzed the phenylalanine hydroxylase gene (PAH) variants in a cohort of 93 PAHD patients from Fujian Province. We also assessed genotype and phenotype correlation in patients with PAHD. A total of 44 different pathogenic variants were identified, including five novel variants. The three most prevalent variants among all patents were c.158G > A, p.(Arg53His) (18.03%), c.721C > T, p.(Arg241Cys) (14.75%), and c.728G > A, p.(Arg243Gln) (7.65%). The frequency of the c.158G > A, p.(Arg53His) variant was highest in patients with mild hyperphenylalaninemia, whereas the frequency of the c.1197A > T, p.(Val399 =) and c.331C > T, p.(Arg111Ter) variants was highest in patients with classic phenylketonuria. The most abundant genotypes observed in PAHD patients were c.[158G > A];[728G > A], c.[158G > A];[442-1G > A], and c.[158G > A];[721C > T]. Comparing allelic phenotype to genotypic phenotype values yielded fairly accurate predictions of phenotype, with an overall consistency rate was 85.71% for PAHD patients. CONCLUSIONS: Our study identified a PAH variant spectrum in PAHD patients from Fujian Province, Southeastern China. Quantitative correlation analysis between genotype and phenotype severity is helpful for genetic counseling and management.

[Analysis of GCDH gene mutations in 3 patients from Fujian area with glutaric academia type I].

OBJECTIVE: To explore clinical features and mutation types in patients from Fujian area with glutaric academia type I(GA I). METHODS: Serum acylcarnitine and urine organic acid of 3 patients were determined with tandem mass spectrometry and gas chromatographic mass spectrometry. The patients also underwent magnetic resonance imaging analysis for the cranial region. Genomic DNA was extracted from peripheral blood samples, and the 12 exons and flanking regions of the GCDH gene were amplified with PCR and subjected to direct DNA sequencing. One hundred healthy newborns were used as controls. RESULTS: Mutations of the GCDH gene were identified in all of the 3 patients. Two patients have carried compound heterozygous mutations including c.1244-2A>C and c.1147C>T(p.R383C), c.406G>T(p.G136C) and c.1169G>A(p.G390E), respectively. One has carried homozygous c.1244-2A>C mutation. The same mutations were not detected among the 100 healthy newborns. Only one patient received early intervention and did not develop the disease. The other two had irreversible damagesto their intelligence. CONCLUSION: c.1169G>A(p.G390E) is likely pathogenic mutations for GA I patients from Fujianarea. Early screening of neonatal metabolic diseases is crucial for such patients.

[Analysis of CYP21A2 gene mutations among patients with classical steroid 21-hydroxylase deficiency].

OBJECTIVE: To assess the frequencies of CYP21A2 gene mutations among patients from Fujian area with classical 21-hydroxylase deficiency. METHODS: For 19 probands from different families affected with classical steroid 21-hydroxylase deficiency and 74 family members, mutations of the CYP21A2 gene were analyzed with combined nested polymerase chain reaction, Sanger sequencing and multiplex ligation-dependent probe amplification. Time resolved fluorescence immunoassay was performed to determine the level of 17-hydroxyprogesterone (17-OHP) in all family members. Clinical data and laboratory results of the probands and their family members were analyzed. RESULTS: Eleven mutations were identified among the 38 alleles from the 19 probands. 92.1% (35/38) of the mutant CYP21A2 alleles were due to recombination between CYP21A2 and CYP21A1P. Gene conversion and deletions were identified in 84.2% (32/38) and 7.9% (3/38) of the alleles, respectively. IVS2-13A/C>G and chimeras were the most common mutations, which respectively accounted for 34.2% (13/38) and 18.4% (7/38) of all mutant alleles. Among these, IVS2+1G>A and Q318X+356W were first reported in China. 74.3% (55/74) of the family members were carriers of heterozygous mutations. However, no significant difference was found in the 17-OHP levels between carriers and non-carriers (P>0.05). CONCLUSION: There seems to be a specific spectrum of CYP21A2 gene mutations in Fujian area, where IVS2-13A/C>G and chimeras are the most common mutations.

Newborn Screening of 6 Lysosomal Storage Disorders by Tandem Mass Spectrometry.

This study was designed to screen 6 lysosomal storage diseases (LSDs) in neonates using tandem mass spectrometry (MS/MS), and establish cutoff values for these LSDs with 3000 dried blood spots (DBS) samples. Cutoff values for α-L-iduronidase (IDUA), α-galactosidase (GLA), acid beta glucosidase (ABG), ß-galactocerebrosidase (GALC), acid sphingomyelinase (ASM), and acid alpha glucosidase (GAA) were as follows: GLA, > 2.06 µmol/L·h; ABG, > 1.78 µmol/L·h; ASM, > 0.99 µmol/L·h; IDUA, > 1.33 µmol/L·h; GALC, > 0.84 µmol/L·h; and GAA, > 2.06 µmol/L·h. There were 30 positives in initial MS/MS screening test, and 15 samples were still positive with repeat testing. Their parents/guardians were recontacted and DBS samples were collected again for test. Only 1 child showed abnormal GAA enzyme activity after recontacting process, and was diagnosed with Pompe disease after genetic screening. Eventually, cutoff values of 6 specific enzyme activities were established and MS/MS is effective for early LSDs screening.

Development and internal validation of a multivariable prediction model for 6-year risk of stroke: a cohort study in middle-aged and elderly Chinese population.

OBJECTIVE: To develop and internally validate a prediction model for 6-year risk of stroke and its primary subtypes in middle-aged and elderly Chinese population. DESIGN: This is a retrospective cohort study from a prospectively collected database. PARTICIPANTS: We included a total 3124 adults aged 45-80 years, free of stroke or myocardial infarction at baseline in the 2009-2015 cohort of China Health and Nutrition Survey. PRIMARY AND SECONDARY OUTCOME MEASURES: The outcome of the prediction model was stroke. Investigated predictors were: age, gender, body mass index (BMI), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), hypertension (HBP), drinking status, smoking status, diabetes and site. Stepwise multiple Cox regression was applied to identify independent predictors. A nomogram was constructed to predict 6-year risk of stroke based on the multiple analysis results. Bootstraps with 1000 resamples were applied to both C-index and calibration curve. RESULT: The overall incidence of overall stroke was 2.98%. Age, gender, HBP and TC were found as significant risk predictors for overall stroke; age, gender, HBP and LDL-C were found as significant risk predictors for ischaemic stroke; age, gender, HBP, BMI and HDL-C were found as significant risk predictors for haemorrhagic stroke. The nomogram was constructed using significant variables included in the model, with a C-index of 0.74 (95% CI: 0.72 to 0.76), 0.74 (95% CI: 0.71 to 0.77), and 0.81 (95% CI: 0.78 to 0.84) for overall stroke, ischaemic stroke, and haemorrhagic stroke model, respectively. The calibration curves demonstrated the good agreements between predicted and observed 6-year risk probability. CONCLUSION: Our nomogram could be convenient, easy to use and effective prognoses for predicting 6-year risk of stroke in middle-aged and elderly Chinese population.

Gene diagnosis and pedigree analysis of two Han ethnicity families with propionic acidemia in Fujian.

ABSTRACT: Propionic acidemia is associated with pathogenic variants in PCCA or PCCB gene. We investigated the potential pathogenic variants in PCCA or PCCB genes in Fujian Han population.Two probands and their families of Han ethnicity containing two generations were subject to newborn screening using tandem mass spectrometry, followed by diagnosis using urine gas chromatography mass spectrometry. Sanger sequencing was used to identify potential mutations in PCCA and PCCB genes.Compound heterozygous variants were identified in PCCB gene in two siblings of the first family, the youngest girl showed a novel missense variant c.1381G>C (p.Ala461Pro) in exon 13 and a heterozygous missense variant c.1301C>T (p.Ala434Val) in exon 13, which were inherited respectively from their parents. The oldest boy is a carrier with a novel missense variant c.1381G>C (p.Ala461Pro) in exon 13 which were inherited from his father. In the second family, c.1535G>A homozygous mutations were identified in the baby girl, which were inherited respectively from their parents. In silico analysis, several different types of bioinformatic software were utilized, which predicted that the novel variant c.1381G>C in PCCB gene was damaged. According to ACMG principle, the missense variant c.1381G>C (p.Ala461Pro) in exon 13 was a Variant of Undetermined Significance (VUS).One novel missense variant and two missense variants in PCCB gene were identified in the study. The novel variant of PCCB gene identified VUS was identified for the first time in the Chinese population, which enriched the mutational spectrum of PCCB gene.

Gene spectrum and clinical traits of 10 patients with primary carnitine deficiency.

BACKGROUND: Rare studies focused on the tandem mass spectrometry (MS/MS) findings for the primary carnitine deficiency (PCD) in the neonates in China mainland. In this study, we aim to analyze the gene mutation spectrum of PCD in Fujian Province in China mainland. METHODS: Primary carnitine deficiency (PCD) samples used in this study were selected from 95,453 cases underwent neonatal screening between May 2015 and February 2020. SLC22A5 gene sequencing was performed on the neonates and their parents with C0 level of less than 8.8 µmol/L. RESULTS: Ten patients (male: 7; female: 3) were finally included in this study. Among these patients, nine were neonates, and one was maternal decline of C0 of less than 8.8 µmol/L. The maternal case showed two types of mutations of SLC22A5 including c.760C>T(p.R254*) and c.1400C>G(p.S467C). The other nine neonates showed compound mutations involving nine types in 18 sites, among which two mutations [i.e., c.37G>T(p.E13*) and c.694A>G(p.T232A)] were novel that had never been reported before. Bioinformatic analysis indicated that c.37G>T(p.E13*) was a pathogenic mutation, while the c.694A>G (p.T232A) was considered to be likely pathogenic. CONCLUSION: MS/MS screening on PCD contributed to the early diagnosis and screening. In addition, SLC22A5 gene mutation analysis contributed to the PCD screening.

A verification of the application of the non-derivatized mass spectrometry method in newborns screening of metabolic disorders.

It is required that the clinical screening of metabolic disorders in newborns meet International Organization for Standardization 15189-2012 approval. The new tandem mass spectrometry (MS/MS) based screening system and its companion reagent should be independently authenticated before their implementation in clinical diagnosis laboratories.Linearity, stability, accuracy, and precision evaluations were carried out to verify the performance of the Waters ACQUITY TQD MS/MS system with the NeoBase non-derivatized MS/MS PerkinElmer kit for detecting amino acids and acylcarnitine in newborns with metabolic disorders.Statistically, the correlation coefficient (R) of 0.9982 to 0.9999 indicates good linearity. The measurements at the beginning and end of the reagent storage procedure were taken for stability verification. No significant difference was detected between the 2 periods. The amino acid exhibited a degree of bias in the range of 0% to 14.17%, with acylcarnitine's being was in the range of 0% to 14.84%; they consequently passed the quality assessment requirements for clinical laboratories of the China National Centre. The amino acids' within-run, between-run, and day-to-day run precision were 1.19% to 7.68%, 1.63% to 5.01%, and 4.77% to 12.48%, respectively, while the total imprecision was 5.55% to 13.33%. Acylcarnitine's within-run, between-run, and day-to-day run precision was 1.2% to 8.43%, 0.19% to 9.60%, and 2.33% to 10.74%, respectively, while it's total imprecision was 6.57% to 13.99%. The manufacturer declared that the total imprecision of the tests, using Multiple Reaction Monitoring, should be less than or equal to 25% of the coefficient of variation for the kit's high and low-quality control levels.The performance of the non-derivatized MS/MS screening system in detecting the amino acids and acylcarnitines passed the test's requirements. It was maintained in accordance with the routine clinical chemical detection system.

Pretreatment of apramycin wastewater by catalytic wet air oxidation.

The pretreatment technology of wet air oxidation (WAO) and coagulation and acidic hydrolysis for apramycin wastewater was investigated in this paper. The COD, apramycin, NH4+ concentration, and the ratio of BOD5/COD were analyzed, and the color and odor of the effluent were observed. WAO of apramycin wastewater, without catalyst and with RuO2/Al2O3 and RuO2-CeO2/Al2O3 catalysts, was carried out at degradation temperature of 200 degrees C and the total pressure of 4 MPa in a 1 L batch reactor. The result showed that the apramycin removals were respectively 50.2% and 55.0%, COD removals were 40.0% and 46.0%, and the ratio of BOD5/COD was increased to 0.49 and 0.54 with RuO2/Al2O3 and RuO2-CeO2/Al2O3 catalysts in catylytic wet air oxidation (CWAO) after the reaction of 150 min. With the pretreatment of coagulation and acidic hydrolysis, COD and apramycin removals were slight decreased, and the ratio of BOD5/COD was increased to 0.45, and the effluents was not suitable to biological treatment. The color and odor of the wastewater were effectively controlled and the reaction time was obviously shortened with WAO. HO2 may promote organic compounds oxidized in WAO of the apramycin wastewater. The addition of CeO2 could promote the activity and stability of RuO2/Al2O3 in WAO of apramycin wastewater.

[Risk factors of 125 cases of neonatal congenital hypothyroidism during perinatal period].

OBJECTIVE: To understand the relationship between perinatal factors and congenital hypothyroidism (CH) and provide scientific evidence for the prevention of CH. METHODS: A case-control study was conducted among 125 neonates with CH (case group) and 375 neonates without CH (control group) in Fujian Neonatal Screening Center from January in 2012 to December in 2013. Univariate and multivariate logistic regression analysis were performed to identify the risk factors to CH during perinatal period. RESULTS: Univariate logistic regression analysis indicated that compared with control group, gestational hypertension, gestational diabetes mellitus, gestational thyroid disease and older age of mother were the risk factors to CH, the difference was statistically significant (P < 0.05) and the risk of CH was higher in female babies, preterm babies, post-term babies low birth weight babies, macrosomia, twins, babies with birth defects and infection in cases group than those in control group, the difference was statistically significant (P < 0.05). Multivariate logistic analysis showed that older age of mother (OR = 2.518, 95% CI: 1.186-5.347), gestational diabetes mellitus (OR = 1.904, 95% CI: 1.190-3.045), gestational hypothyroidism or hyperthyroidism (OR = 12.883 and 30.797, 95% CI: 2.055-80.751 and 3.309-286.594), preterm birth (OR = 4.238, 95% CI: 1.269-14.155), and post-term birth (OR = 12.799, 95% CI: 1.257-130.327), low birth weight (OR = 3.505, 95% CI: 1.059-11.601), macrosomia (OR = 3.733, 95% CI: 1.415-9.851), twin or multiparous delivery (OR = 5.493, 95% CI: 1.701-17.735), birth defects (OR = 3.665, 95% CI: 1.604-8.371) and fetal distress (OR = 3.130, 95% CI: 1.317-7.440) were the high risk factors to CH (P < 0.05). CONCLUSION: CH was correlated with mother's age, gestational diabetes, gestational thyroid disease as well as neonate's birth weight and gestational age, foetus number, fetal distress and other complicated birth defects at certain degree. More attention should be paid to perinatal care to reduce risk factors and the incidence of CH.

Expression of a Ligninase by a New Hybrid Baculovirus with Expanded Host Range.

We have generated and purified a new recombinant baculovirus with expanded host range. AcNPV DNA and BamHI-digested BmNPV DNA were co-transfected into Spodoptera frugiperda SF21 cells. Progeny viruses were used to infect BmN cells, which are normally resistant to AcNPV infection, in order to screen for recombinant viruses with cross-infectivity. A recombinant virus was isolated by plaque purification and analyzed. This isolate was able to infect, replicate and produce polyhedrin in both the SF21 and BmN cells. DNA restriction endonuclease analysis showed that it was a hybrid (HyNPV) of AcNPV and BmNPV. Co-transfection of this HyNPV virus with a transfer vector containing a ligninase H8 gene insert into SF21 cells yielded a recombinant baculovirus expressing the ligninase. When this recombinant baculovirus was used to infect either SF21 or BmN cells, ligninase proteins could be found in the lysed cells as well as in the cell culture media. We have thus demonstrated that this hybrid virus can be utilized in the generation of recombinant baculovirus expressing foreign proteins in two host cells which normally can not be infected by AcNPV nor BmNPV.