Untargeted metabolomic analysis of urine samples for diagnosis of inherited metabolic disorders.

Metabolomics has become an important tool for clinical research, especially for analyzing inherited metabolic disorders (IMDs). The purpose of this study was to explore the performance of metabolomics in diagnosing IMDs using an untargeted metabolomic approach. A total of 40 urine samples were collected: 20 samples from healthy children and 20 from pediatric patients, of whom 13 had confirmed IMDs and seven had suspected IMDs. Samples were analyzed by Orbitrap mass spectrometry in positive and negative mode alternately, coupled with ultra-high liquid chromatography. Raw data were processed using Compound Discovery 2.0 ™ and then exported for partial least squares discriminant analysis (PLS-DA) by SIMCA-P 14.1. After comparing with m/zCloud and chemSpider libraries, compounds with similarity above 80% were selected and normalized for subsequent relative quantification analysis. The uncommon compounds discovered were analyzed based on the Kyoto Encyclopedia of Genes and Genomes to explore their possible metabolic pathways. All IMDs patients were successfully distinguished from controls in the PLS-DA. Untargeted metabolomics revealed a broader metabolic spectrum in patients than what is observed using routine chromatographic methods for detecting IMDs. Higher levels of certain compounds were found in all 13 confirmed IMD patients and 5 of 7 suspected IMD patients. Several potential novel markers emerged after relative quantification. Untargeted metabolomics may be able to diagnose IMDs from urine and may deepen insights into the disease by revealing changes in various compounds such as amino acids, acylcarnitines, organic acids, and nucleosides. Such analyses may identify biomarkers to improve the study and treatment of IMDs.

Simultaneous quantification of fat-soluble vitamins A, 25-hydroxylvitamin D and vitamin E in plasma from children using liquid chromatography coupled to Orbitrap mass spectrometry.

The fat-soluble vitamins A, D, E and K are micronutrients essential for physiological activity, metabolism and growth. Accurate and sensitive analytical methods are needed to support growing research into fat-soluble vitamins and their impact on children's growth and health. Here we report the first method for simultaneous quantification of fat-soluble vitamins A (retinol), 25-hydroxylvitamin D2, 25-hydroxylvitamin D3, and vitamin E (α-tocopherol) using a Q-Exactive Orbitrap mass spectrometer in high-resolution, parallel reaction monitoring mode. This method can select desired ions with high efficiency, potentially making it superior to triple-quadrupole mass spectrometers that employ multiple reaction monitoring. The proposed method offers excellent accuracy, specificity, and sensitivity, as demonstrated with plasma samples from healthy children.

Improving quality management of newborn screening in southwest China.

OBJECTIVE: Newborn screening (NBS) programs benefit tens of millions of infants worldwide each year. However, the extremely large screening populations and number of laboratories involved pose great challenges to maintaining high screening quality. To achieve continuous quality improvement, we established a comprehensive quality management system (CQMS) in southwest China. METHODS: External quality assessment (EQA) and internal quality control were carried out for basic quality management. We used 16 quality indicators (QIs) to monitor the entire screening process, with external supervision from the China National Accreditation Service for Conformity Assessment. All retrospective data for quality assessment were collected consecutively from laboratory management and patient follow-up systems. RESULTS: From 2015 to 2019, satisfactory EQA performance was achieved, with an average score greater than 97 for each screening item. QI monitoring showed that NBS quality improved continuously. The rate of health education provision increased from 90.9% to 100% and the recall rate after a positive primary screening increased from 85.4% to 99.2%. The unsatisfactory specimen rate and rate of newborns lost to follow-up decreased to 0.38% and 0.08%, respectively. CONCLUSIONS: Implementing a CQMS and monitoring the whole screening process using QIs may yield continuous quality improvement of NBS.

Integrative identification of the pathogenic role of a novel G6PD missense mutation c.697G>C.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a hereditary disease caused by pathogenic mutations of G6PD. While most of the pathogenic variants of G6PD have been annotated, hemolysis of unknown etiology but analogous to that in G6PD deficiency persists, implying the existence of undocumented pathogenic variants. In our previous study, we reported four novel G6PD variants in China, for which the pathogenicity remains to be verified. METHODS: The variants were verified by exogenous expression in HEK-293 cells, and their functions were predicted by PolyPhen-2 and SIFT. The CRISPR/Cas9 system was exploited to edit the G6PD c.697G>C variant in HEK-293 cells and K562 cells. The expression of G6PD was detected by quantitative PCR (qPCR) and western blotting. The cell growth capacity was detected by the CCK-8 assay and crystal violet staining. The G6PD enzyme activity was reflected by the G6P/6PG ratio test. The apoptosis of cells was detected by Annexin V-APC/7-AAD staining. The secondary and crystallographic structures were denoted according to the literature and PyMOL software. The G6PD protein was purified from lysis of transformed Escherichia coli (E. coli) cell with Ni-charged Resin Column. The enzymatic activity was detected at different temperatures. RESULTS: The G6PD activity of exogenous G6PD c.697G>C in HEK-293 cells was significantly lower than that of wild type (WT) G6PD, a finding that was consistent with the observation in clinical samples. The functional predictions conducted by different algorithms indicated the damage role of the G6PD c.697G>C variant in its enzymatic activity. We recapitulated the G6PD c.697G>C variant both in HEK-293 cells and K562 cells by adapting the CRISPR/Cas9 strategy. Using distinct cell lines expressing the G6PD c.697G>C variant endogenously, we confirmed the deteriorative role of the G6PD c.697G>C variant in its enzymatic activity. Regarding the secondary and crystallographic structure, we found a mutated amino acid approaching the structural NADP+ binding site. Finally, we demonstrated the c.697G>C variant compromised the thermal stability of G6PD protein. CONCLUSIONS: Our data delineated the pathogenic role of G6PD c.697G>C variant for G6PD deficiency, implying the wide usage of CRISPR/Cas9 for genetic disease research.

Chinese expert brief consensus on newborn screening of inherited metabolic disorders during the novel coronavirus infection epidemic.

Novel coronavirus (2019-nCov) infection (COVID-19) rapidly spread across China and 25 countries in the worldwide, which infected not only adults but also children, even neonates. Each year, about 15 million newborns are delivered in China. Newborn screening (NBS) helps effectively prevent some mental retardation, premature death, and adverse outcomes in the early stage of baby, which could detect some inherited metabolic disorders (IMDs). During this COVID-19 epidemic, how to balance the risk of infected 2019-nCov and the risk of disability and teratogenesis of IMDs. Expert members of NBS extra quality assessment in National Clinical Center of Laboratory (NCCL) give a brief consensus for NBS of IMDs in the COVID-2019 epidemic, hoping that the brief consensus could be reference for NBS of IMDs in the other epidemic areas or periods all over the world.

Chinese newborn screening for the incidence of G6PD deficiency and variant of G6PD gene from 2013 to 2017.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common X-linked enzymopathies caused by G6PD gene variant. We aimed to provide the characteristics of G6PD deficiency and G6PD gene variant distribution in a large Chinese newborn screening population. We investigated the prevalence of G6PD in China from 2013 to 2017. Then, we examined G6PD activity and G6PD gene in representative Chinese birth cohort to explore the distribution of G6PD gene variant in 2016. We then performed multicolor melting curve analysis to classify G6PD gene variants in 10,357 neonates with activity-confirmed G6PD deficiency, and DNA Sanger sequencing for G6PD coding exons if hot site variants were not found. The screened population, organizations, and provinces of G6PD deficiency were increased from 2013 to 2017 in China. The top five frequency of G6PD gene variants were c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, and c.871G>A and varied in different provinces, with regional and ethnic features, and four pathogenic variant sites (c.152C>T, c.290A>T, c.697G>C, and c.1285A>G) were first reported. G6PD deficiency mainly occurs in South China, and the frequency of G6PD gene variant varies in different regions and ethnicities.

Severe clinical manifestation of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency associated with two novel mutations: a case report.

BACKGROUND: Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS) deficiency is an autosomal recessive inborn error of metabolism, which will give rise to failure of ketogenesis in liver during illness or fasting. It is a very rare disease with only a few patients reported worldwide, most of which had a good prognosis after proper therapies. CASE PRESENTATION: We report a 9-month-old boy with mHS deficiency presenting with unusually severe and persistent acidosis after diarrhea and reduced oral food intake. The metabolic acidosis persisted even after supplementation with sugar and alkaline solution. Blood purification and assisted respiration alleviated symptoms, but a second onset induced by respiratory infection several days later led to multiple organ failure and death. Urine organic acid analysis during the acute episode revealed a complex pattern of ketogenic dicarboxylic and 3-hydroxydicarboxylic aciduria with prominent elevation of glutaric acid and adipic acid, which seem to be specific to mHS deficiency. Plasma acylcarnitine analysis revealed elevated 3-hydroxybutyrylcarnitine and acetylcarnitine. This is the first report of elevated 3-hydroxybutyrylcarnitine in mHS deficiency. Whole exome sequencing revealed a novel compound heterozygous mutation in HMGCS2 (c.100C > T and c.1465delA). CONCLUSION: This severe case suggests the need for patients with mHS deficiency to avoid recurrent illness because it can induce severe metabolic crisis, possibly leading to death. Such patients may also require special treatment, such as blood purification. Urine organic acid profile during the acute episode may give a hint to the disease.

A novel tandem mass spectrometry method for first-line screening of mainly beta-thalassemia from dried blood spots.

Traditional methods for thalassemia screening are time-consuming and easily affected by cell hemolysis or hemoglobin degradation in stored blood samples. Tandem mass spectrometry (MS/MS) proved to be an effective technology for sickle cell disorders (SCD) screening. Here, we developed a novel MS/MS method for ß-thalassemia screening from dried blood spots (DBS). Stable isotopic-labeled peptides were used as internal standards for quantification and calculation of the α:ß-globin ratios. We used the α:ß-globin ratio cutoffs to differentiate between normal individuals and patients with thalassemia. About 781 patients and 300 normal individuals were analyzed. The α:ß-globin ratios showed significant difference between normal and ß-thalassemia patients (P<0.01), particularly when the disease was homozygous or double heterozygous with another α- or ß-thalassemia mutation. In the parallel study, all cases screened for suspected thalassemia from six hundred DBS samples by using this MS/MS method were successfully confirmed by genotyping. The intra-assay and inter-assay CVs of the ratios ranged from 2.4% to 3.9% and 4.7% to 7.1%, and there was no significant sample carryover or matrix effect for this MS/MS method. Combined with SCD screening, this MS/MS method could be used as a first-line screening assay for both structural and expression abnormalities of human hemoglobin. BIOLOGICAL SIGNIFICANCE: Traditional methods for thalassemia screening were depending on the structural integrity of tetramers and could be affected by hemolysis and degradation of whole blood samples, especially when stored. We used proteospecific peptides produced by the tryptic digestion of each globin to evaluate the production ratio between α- and ß-globin chains, which turned out to be quite stable even when stored for more than two months. Though most of the peptides were specific to α-globin or ß-globin, we only chose four most informative peptides and its stable isotopic-labeled peptides as internal standards for analysis, which could obtain a high accuracy. Currently, we are the first to address the application of MS/MS for thalassemia screening, when combined with SCD screening, this MS/MS method could be used as a first-line screening assay for both structural and expression abnormalities of human hemoglobin.

A novel method for quantification of human hemoglobin from dried blood spots by use of tandem mass spectrometry.

Quantification of human hemoglobin (Hb) is essential for diagnosis of anemia, especially for screening for thalassemia and sickle cell disease. The main methods currently used for quantification of Hb, including spectrophotometry, fluorimetry, and electrochemical assays, are all based on the structural integrity of Hb, which could be affected by hemolysis and degradation. When used for disease screening, whole blood specimens cannot meet requirements for sample collecting, transport, and storage. Here, we report a novel MS-MS method for quantification of Hb from dried blood spots (DBS) by use of a triple-quadrupole mass spectrometer. Proteospecific peptides from α-globin chains were selected after tryptic digestion. The precursor → product ion transitions of representative peptides were studied to identify the best choice with regard to sensitivity and chromatographic properties. For quantification, stable isotope-labeled peptides were used as internal standards. The concentration of Hb in each sample was obtained by calculation on the basis of established equations. The precision of the method was within 15 % and accuracy was in the range -7 to 13.0 %. Compared with routine clinical results obtained by use of the automated hematology analyzer (AHA) assay, the correlation, r (2), was >0.993. When used for determination of anemia levels the sensitivity of the assay was 95.7 % and specificity 96.5 %. Our new approach for quantification of the concentration of Hb from DBS is feasible, and precision is acceptable. The method could be used for determination of anemia levels when screening for hemoglobin disorders. Graphical Abstract Quantification of human hemoglobin from digested dried blood spot samples using tandem mass spectrometry.

Urine real-time polymerase chain reaction detection for children virus pneumonia with acute human cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus (HCMV) is an important pathogen of viral pneumonia in children. The diagnosis of acute HCMV infection is complicated and difficult. METHODS: Clinical and laboratory data of 6063 hospitalized children with respiratory infection and 509 with respiratory virus infection alone were retrospectively analyzed. Urine and respiratory specimens of 186 hospitalized children with pneumonia were also prospectively collected. Real-time polymerase chain reaction (PCR) and a chemiluminescent assay were used to detect HCMV DNA copy number, the pp65 gene, and HCMV IgM. RESULTS: The patients with respiratory virus infection alone and those with pulmonary HCMV infection (n = 422) were mostly children aged <6 months old (82.91%, 422/509). The accuracy of urine HCMV DNA (82.32%) was higher than that of HCMV IgM (67.78%), indicating that PCR of urine samples is suitable for determining pediatric acute pulmonary HCMV infection. There was no significant difference in detecting HCMV DNA or the pp65 gene between urinary and respiratory specimens (P > 0.05) in 186 pediatric pneumonia cases. The accuracy of the pp65 gene measured in urine for determining acute pulmonary HCMV infection was the highest (93.01%). CONCLUSIONS: Our study shows a novel method for investigating acute pulmonary HCMV infection in children by using real-time PCR and non-invasive samples. This study also highlights the superiority and potential use of the pp65 gene as an important target for the diagnosis of acute pulmonary HCMV infection.

Two novel mutations affecting the same splice site of PKD1 correlate with different phenotypes in ADPKD.

Genetic heterogeneity is the main factor for significant variation in the course of autosomal dominant polycystic kidney disease (ADPKD). PKD1 patients have more severe renal outcomes compared with PKD2 patients. Co-inheritance of a mutation in both genes is associated with more severe phenotypes than that found with either mutation alone. However, the genotype-phenotype relationship is far from clear in ADPKD. Here, we observed two novel mutations, PKD1:c.12444G > A and PKD1:c.12444 + 1G > A, which alter the same splice donor site of intron 45, correlate with different renal outcomes. To explain the phenomenon, we analyzed the genic and allelic background of the patients, as well as the genetic modifiers, DKK3 and HNF-1ß as suggested. Only PKD1 variants were found, which highlights the allelic influence of PKD1 gene to be the last candidate factor. Segregation analysis, online mutation prediction, and recurrence mutation searching were applied to sort the variants. However, none of variants was found to be damaging or associated with the disease except PKD1:c.12444G > A and PKD1:c.12444 + 1G > A. Cloning and sequencing of the mutated cDNA sequences had shown unexpected different splicing effects caused by the mutations. PKD1:c.12444 + 1G > A definitely destroyed the native splice site and created a novel donor site with truncating effect on PC1. In contrast, PKD1:c.12444G > A mainly weakened the site and decreased the expression of normal PC1. Since PC1 negatively regulates cell proliferation in the process of cyst formation and enlargement, our observation may explain this new genotype-phenotype correlation and help to improve genetic counseling and diagnosis of the disease.

Investigation of Mycoplasma pneumoniae infection in pediatric population from 12,025 cases with respiratory infection.

Although Mycoplasma pneumoniae (MP) is a major pathogen of primary atypical pneumonia in children, the clinical and laboratory characteristics of MP infection in large pediatric population are less reported. Here, we retrospectively analyzed 12,025 hospitalized children with respiratory infection by using serology and polymerase chain reaction (PCR) methods simultaneously. The results showed that 2433 (20.23%) children had MP infection, which mainly occurred in November to April. The presence of sore throat and pharyngitis was peculiar to MP infection. The positive percentage of MP-DNA was higher than that of MP-IgM in children aged <1 (P < 0.0001) and 1-3 years (P < 0.0001). Moreover, the positive rate of P1 gene, the key adhesion gene for MP infection, was higher in children with MP infection than in those with other pathogens (P < 0.0001). Our work provides the clinical information of children MP infection and highlights the superiority of PCR and potential usage of P1 as a diagnosis target for MP infection.