Tandem Mass Spectrometry Screening Research Articles

Clinical characteristics and genetic analysis of six children with carnitine palmitoyltransferase 2 deficiency.

To investigate the clinical characteristic and genetic variants of children with carnitine palmitoyltransferase 2 (CPT2) deficiency. The clinical and genetic data of 6 children with CPT2 deficiency were retrospectively analyzed. The blood acylcarnitines and genetic variants were detected with tandem mass spectrometry and whole-exon gene sequencing, respectively. There were 4 males and 2 females with a mean age of 32 months (15 d-9 years) at diagnosis. One case was asymptomatic and with normal laboratory test results, 2 had delayed onset, and 3 were of infantile type. Three cases were diagnosed at neonatal screening, and 3 cases presented with clinical manifestations of fever, muscle weakness, and increased muscle enzymes. Five children presented with decreased free carnitine and elevated levels of palmitoyl and octadecenoyl carnitines. CPT2 gene variants were detected at 8 loci in 6 children (4 harboring biallelic mutations and 2 harboring single locus mutations), including 3 known variants (p.R631C, p.T589M, and p.D255G) and 5 newly reported variants (p.F352L, p.R498L, p.F434S, p.A515P, and c.153-2A>G). It was predicted by PolyPhen2 and SIFT software that c.153-2A>G and p.F352L were suspected pathogenic variants, while p.R498L, p.F434S and p.A515P were variants of unknown clinical significance. The clinical phenotypes of CPT2 deficiency are diverse. An early diagnosis can be facilitated by neonatal blood tandem mass spectrometry screening and genetic testing, and most patients have good prognosis after a timely diagnosis and treatment.

Analysis of Imprecision for Internal Quality Control of Newborn Screening by Tandem Mass Spectrometry in China, 2015 - 2021.

This study aimed to assess the performance of the newborn screening laboratories in China through retrospective analysis of the coefficient of variation (CV) of the internal quality control (IQC) data in the national tandem mass spectrometry screening for inherited metabolic disorders in newborns. From 2015 to 2021, the IQC data of amino acid and acylcarnitine test were collected twice each year. CVmonthly in-control was calculated by excluding outliers for the current month and its discrete distribution and changes in trend were comprehensively evaluated for both normal and high concentration levels. The proportion of laboratories meeting both 1/3 and 1/4 quality criteria of the total error allowable (TEa), based on the CVmonthly in-control for each testing item, was calculated. The analysis of CVmonthly in-control for the two concentration levels for the amino acids and acylcarnitine parameters showed that CVmonthly in-control for the normal concentration levels were more discrete before 2018, while CVmonthly in-control for the high concentration levels were less discrete than the normal concentration levels, but there were relatively more outliers. More than 80% of laboratories were able to meet the 1/3 TEa standard for each test at the high concentration level, while the pass rate for the 1/4 TEa standard was significantly lower than 80% (except for C2). According to the current status of testing in China, it is recommended to use 1/3TEa as the imprecision level standard; for laboratories with relatively high precision, the 1/4TEa standard can be used.

Analysis of clinical phenotypes and MMACHC gene variants in 65 children with Methylmalonic acidemia and homocysteinemia

To carry out Sanger sequencing for MMACHC gene variants among 65 Chinese pedigrees affected with combined methylmalonic aciduria and homocysteinemia, and summarize their genetic and clinical characteristics and prognosis. Clinical characteristics of the 65 children identified with Methylmalonic acidemia and homocysteinemia at the Children's Hospital Affiliated to Zhengzhou University (Zhengzhou Children's Hospital) from April 2017 to April 2022 were selected as the study subjects. Potential variants of the MMACHC gene were detected by direct sequencing of the PCR products. The median age of the 65 children was 3 months (14 days to 17 years old). These included 28 cases (43.08%) from neonatal screening, 11 cases (16.92%) with a history of jaundice, and 9 cases (13.85%) with various degrees of anemia. The main clinical symptoms included development delay, slow growth, epilepsy, hydrocephalus, lethargy, feeding difficulty, regression or decline in motor ability, recurrent respiratory infections, anemia, jaundice, respiratory and heart failures, hydrocephalus, limb weakness, and hypertension. Blood and urine tandem mass spectrometry screening has revealed increase of methylmalonic acid, propionyl carnitine, propionyl carnitine/acetylcarnitine ratio, and propionyl carnitine/free carnitine ratio to various extents, and blood homocysteine was increased in all patients. The detection rate of genetic variants was 98.46% (128/130), and in total 22 types of MMACHC gene variants were detected. The most common ones have included c.609G>A (W203X) (58/128), c.658-660del (K220del) (19/128), and c.80A>G (Q27A) (16/128). Two novel variants have been identified, namely c.565C>T (p.R189C) and c.624_ 625delTG (p.A208Afs), which were respectively predicted as likely pathogenic (PM2_Supporting+PM3+PP2+PP3) and pathogenic (PVS1+PM2_Supporting+PM3+PP2) based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). Exon 4 had the highest frequency for the detection. Identification of MMACHC gene variants has confirmed the diagnosis in the children, among which the c.609G>A variant has the highest frequency. Discovery of the new variants has enriched the mutational spectrum of the MMACHC gene.

Evidence and Recommendation for Guanidinoacetate Methyltransferase Deficiency Newborn Screening.

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive disorder of creatine biosynthesis due to pathogenic variants in the GAMT gene that lead to cerebral creatine deficiency and neurotoxic levels of guanidinoacetate. Untreated, GAMT deficiency is associated with hypotonia, significant intellectual disability, limited speech development, recurrent seizures, behavior problems, and involuntary movements. The birth prevalence of GAMT deficiency is likely between 0.5 and 2 per million live births. On the basis of small case series and sibling data, presymptomatic treatment with oral supplements of creatine, ornithine, and sodium benzoate, and a protein-restricted diet to reduce arginine intake, appear to substantially improve health and developmental outcomes. Without newborn screening, diagnosis typically happens after the development of significant impairment, when treatment has limited utility. GAMT deficiency newborn screening can be incorporated into the tandem-mass spectrometry screening that is already routinely used for newborn screening, with about 1 per 100 000 newborns screening positive. After a positive screen, diagnosis is established by finding an elevated guanidinoacetate concentration and low creatine concentration in the blood. Although GAMT deficiency is significantly more rare than other conditions included in newborn screening, the feasibility of screening, the low number of positive results, the relative ease of diagnosis, and the expected benefit of presymptomatic dietary therapy led to a recommendation from the Advisory Committee on Heritable Disorders in Newborns and Children to the Secretary of Health and Human Services that GAMT deficiency be added to the Recommended Uniform Screening Panel. This recommendation was accepted in January 2023.

Clinical and Gene Analysis of Fatty Acid Oxidation Disorders Found in Neonatal Tandem Mass Spectrometry Screening.

To investigate the clinical and gene mutation characteristics of fatty acid oxidative metabolic diseases found in neonatal screening. A retrospective analysis was performed on 29,948 neonatal blood tandem mass spectrometry screening samples from January 2018 to December 2021 in our neonatal screening centre. For screening positive, recall review is still suspected of fatty acid oxidation metabolic disorders in children as soon as possible to improve the genetic metabolic disease-related gene detection package to confirm the diagnosis. All diagnosed children were followed up to the deadline. Among 29,948 neonates screened by tandem mass spectrometry, 14 cases of primary carnitine deficiency, six cases of short-chain acyl coenzyme A dehydrogenase deficiency, two cases of carnitine palmitoyltransferase-I deficiency and one case of multiple acyl coenzyme A dehydrogenase deficiency were recalled. Except for two cases of multiple acyl coenzyme A dehydrogenase deficiency that exhibited [manifestations], the other 21 cases were diagnosed pre-symptomatically. Eight mutations of SLC22A5 gene were detected, including c.51C>G, c.403G>A, c.506G>A, c.1400C>G, c.1085C>T, c.706C>T, c.1540G>C and c.338G>A. Compound heterozygous mutation of CPT1A gene c.2201T>C, c.1318G>A, c.2246G>A, c.2125G>A and ETFA gene c.365G>A and c.699_701delGTT were detected, and new mutation sites were found. Neonatal tandem mass spectrometry screening is an effective method for identifying fatty acid oxidative metabolic diseases, but it should be combined with urine gas chromatography-mass spectrometry and gene sequencing technology. Our findings enrich the gene mutation profile of fatty acid oxidative metabolic disease and provide evidence for genetic counselling and prenatal diagnosis in families.

UHPLC-MS/MS phytochemical screening, polyphenolic content and antioxidant potential of Diplorhynchus condylocarpon (Müll.Arg.) Pichon (Apocynaceae), a medicinal plant

Diplorhynchus condylocarpon (Müll.Arg.) Pichon (Apocynaceae) leaves, stem bark, and roots are commonly used as popular folk medicine in Zambia and other African countries. This work aimed to find out the broad categories of phytocompounds present in the plant, screen the polyphenols, and assess the total polyphenolic content and the antioxidant activity of methanolic extracts of leaf, stem bark, and roots. Maceration was used to extract secondary metabolites for preliminary phytochemical screening. QuEChERS (quick, easy, cheap, effective, rugged, and safe), based technique was used to recover polyphenols for liquid chromatography with tandem mass spectrometry (LC-MS/MS) screening. Standard chemical tests were used for preliminary phytochemical screening. Total phenolic content (TPC) was evaluated using the Folin-Ciocalteu (FC) method at 765 nm. Total flavonoid content (TFC) was evaluated by the aluminum chloride colorimetric assay at 510 nm. Antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method at 517 nm. The preliminary phytochemical screening revealed the presence of antioxidants, polyphenols, and other valuable phytocompounds such as alkaloids, saponins, and terpenoids. The LCMS/MS polyphenolic screening revealed the presence of mainly polyphenolic metabolites. TPC values were higher in the leaf (171.099±0.515 mg GAE/g) and root (88.8093±3.156 mg GAE/g) than in the stem bark (71.061±0.515 mg GAE/g) extracts. Further, the leaf extract contained 129.402±0.407, the root contained 64.672±0.813, and the stem bark had 37.643±0.704 mg QE/g total flavonoids. The leaf extract exhibited in a dose-dependent manner higher DPPH radical scavenging activities (34.250, 50.625, 66.562, 81.406, and 97.344%), followed by the root (16.406, 31.406, 50.781, 65.625, and 82.188%), and the stem bark extract only had the least DPP quenching activities (15.469, 27.969, 40.469, 52.500, and 68.590%) for the concentration of 20, 40, 60, 80 and 100 µg/mL respectively. Further, the results from the DPPH assay show that the leaf extract had the best IC50 value of 19.973 µg/mL, followed by the root extract with 60.867 µg/mL, and the stem bark extract with 107.150 µg/mL. These antioxidant activities support the traditionally claimed therapeutic benefits of D. condylocarpon leaves, stem bark, and roots in the treatment of oxidative stress-related disorders such as diabetes mellitus, chronic wounds, and diarrhea. This study may lead to the production of new herbal medicines using D. condylocarpon.

Molecular genetic screening of full-term small for gestational age

ObjectiveTo examine the clinical application of genomic screening in newborns small for gestational age (SGA), hoping to provide an efficient technique for early discovery of neonatal diseases, which is necessary to elevate survival rates and the quality of life in infants.MethodsTotally 93 full-term SGA newborns were assessed. Dried blood spot (DBS) samples were obtained at 72 h after birth, and tandem mass spectrometry (TMS) and Angel Care genomic screening (GS, using Targeted next generation sequencing) were carried out.ResultsAll 93 subjects were examined by Angel Care GS and TMS. No children showing inborn errors of metabolism (IEM) were detected by TMS, while 2 pediatric cases (2.15%, 2/93) were confirmed as thyroid dyshormonogenesis 6 (TDH6) by Angel Care GS. Additionally, 45 pediatric cases (48.4%) had one or more variants conferring a carrier status for recessive childhood-onset disorders, with 31 genes and 42 variants associated with 26 diseases. The top three gene-related diseases with carrier status were autosomal recessive deafness (DFNB), abnormal thyroid hormone and Krabbe disease.ConclusionsSGA is tightly associated with genetic variation. Molecular Genetic Screening allows early detection of congenital hypothyroidism and may be a potent genomic sequencing technique for screening newborns.

Cost-effectiveness analysis of newborn screening by tandem mass spectrometry in Shenzhen, China: value and affordability of new screening technology

BackgroundNewborn screening (NBS) can prevent inborn errors of metabolism (IEMs), which may cause long-term disability and even death in newborns. However, in China, tandem mass spectrometry (MS/MS) screening has just started. This study aimed to assess the cost-effectiveness of NBS using MS/MS in Shenzhen under the nationally recommended program, as well as evaluate the value and affordability of introducing this new screening technology.MethodsA Markov model was built to estimate the cost and quality-adjusted life-years (QALYs) of different screening programs. We compared PKU screening using traditional immunofluorescence (IF) with the other 11 IEMs not screened and all 12 IEMs screened using MS/MS, and the programs detecting different numbers of IEMs chosen from the national recommended program were also compared. A sensitivity analysis and budget impact analysis (BIA) were performed.ResultsThe incremental cost-effectiveness ratio (ICER) of detecting all 12 IEMs in the national program is 277,823 RMB per QALY, below three times per capita GDP in Shenzhen. MS/MS screening in Shenzhen can be cost-effective only if at least three diseases (PKU, PCD and MMA) are covered and when the screening program covers five diseases (PKU, PCD, MMA, MSUD, IVA), the ICER closely approaches its critical threshold. The BIA indicated the implementation cost of the national program to be around 490 million RMB over 10 years and showed no difference in budget between programs detecting different numbers of IEMs.ConclusionsWe conclude that the newborn screening using MS/MS in Shenzhen is cost-effective, and the budget affordable for the Shenzhen government. Two concepts for selecting the IEMs to be detected are also presented. One is to choose the most cost-effective screening programs detecting highest number of IEMs to achieve a minimal ICER. The other considers the curability and affordability of the disease as the basis of healthcare decisions to screen suitable IEMs, achieving an ICER under the threshold and close to the minimum value.

Toxicity of heroin laced with synthetic opioid β-U10 and etizolam

Illicit heroin can be substituted or adulterated with New Psychoactive Substances (NPS) including highly potent fentanyl-type substances or other opioid-like compounds to counteract low-purity supply. The addition of unknown NPS to the heroin supply poses an additional risk of overdose and death. In 2020, the Emerging Drug Network of Australia VIC (EDNAV) study was funded to monitor drug related harm in individuals attending emergency departments (ED) at major hospitals within Victoria. In September 2020, a previously unreported NPS, β-U10 (2-napthyl-U-47700), was detected in discarded drug paraphernalia samples collected during a pilot study of an early warning system for illicit drug use in Melbourne, Australia. Subsequently, blood samples of EDNAV project individuals with positive heroin detections were analysed for β-U10, in addition to NPS and traditional illicit drugs. After liquid-liquid extraction, blood samples underwent extensive toxicological analysis via targeted liquid-chromatography tandem mass spectrometry (LC-MS/MS) and untargeted screening via high-resolution quadrupole time-of-flight mass spectrometry (LC-QTOF). Confirmation of β-U10 concentrations were measured via LC-MS/MS using certified reference material. In total, seven cases were found to contain synthetic opioid β-U10. Six samples were collected in October 2020, with the remaining case detected more than a year later in December 2021. Measured β-U10 concentrations ranged from 0.01 to 22 ng/mL (median 3 ng/mL). All cases also contained methylamphetamine and etizolam. Morphine was detected in all cases along with 6-acetylmorphine in three of these cases. Other drugs included diphenhydramine, diazepam, doxylamine, gamma-hydroxybutyrate, MDMA, methadone, and mitragynine. The average age was 37 and ranged from 20 to 52 years of age. All individuals presented to ED with varying levels of sedation (Glascow Coma Scale 3–14, median 9) and recovered with supportive care (median stay 6.8 hours). Two cases showed a positive response to naloxone. The Poisons Severity Score for one case was 3, with 4 cases assessed as level 2. The pharmacological activity of β-U10 is largely unknown. β-U10 was first published by the Upjohn Company in the 1970's and is a structural analogue of the widely reported NPS U-47700, which has approximately seven times greater affinity to the μ-opioid receptor than morphine (Cheney. Journal of Medicinal Chemistry 1985;28:1853–1864). U-47700 has been utilised recreationally for its sedative properties in addition to positive effect on mood. The co-ingestion of other sedatives and respiratory depressants, including opioids and the NPS benzodiazepine etizolam in each of these reported cases precludes definitive conclusions regarding the clinical effect of β-U10. Additional studies that examined heroin supply at the time found the addition of β-U10 was associated with a decrease in heroin purity. The detection of β-U10 in one case 12 months following the previous detection may indicate the drug re-supply of the adulterated mixture in Australia. The detection of β-U10 has also informed toxicological investigations into heroin-related deaths that occurred during the same time-period. The consumption of adulterated illicit substances by unsuspecting individuals with unknown compounds such as β-U10, particularly in conjunction with other opioids and CNS depressants such as etizolam, is an alarming trend and poses a significant public health risk.

Are We Ready for Newborn Genetic Screening? A Cross-Sectional Survey of Healthcare Professionals in Southeast China.

ObjectivesTo understand the knowledge, attitude, willingness, and ability of healthcare professionals working in newborn screening (NBS) centers regarding newborn genetic screening (nGS).MethodsThe questionnaire consisted of four sections with 27 questions and the data were collected by the WJX platform. All participants accessed the questionnaire by scanning a specific QR code with their mobile phones. Two researchers independently completed the summary and analysis.ResultsA total of 258 valid questionnaires were collected from 43 NBS centers in six provinces of southeast China. In total, 209 (81.01%) participants were interested in nGS, and almost all participants (97.67%) thought that nGS was necessary in China. About 89.53% of participants thought that it could be used to effectively expand the diseases that could be screened, but 72.87% also worried about the inability to provide genetic counseling. About 55.34% suggested that nGS and tandem mass spectrometry (TMS) screening could be applied in a unite screening mode. The higher the institution and personal education levels, the higher the interest healthcare professionals displayed toward nGS. However, they also showed greater concern about the inability to provide genetic counseling and ethical issues. If a center had engaged in TMS screening, its staff would have been more likely to believe that nGS had great advantages. In addition, most participants had ethical concerns, such as “the psychological burden caused by carrying information regarding adult morbidity risk.”ConclusionMost participants were interested and considered nGS necessary. The inability to provide genetic counseling may be the primary impediment to clinical practice. Three important influencing factors were level of education, institution level, and engagement in TMS screening.

Molecular Genetic Screening of Neonatal Intensive Care Units: Hyperbilirubinemia as an Example.

ObjectiveTo explore the clinical value of newborn genomic screening (nGS) for neonatal intensive care units (NICU) infants (taking neonatal hyperbilirubinemia as an example).MethodsDried blood spots (DBSs) were collected after 72 hours of birth. The tandem mass spectrometry (TMS) screening and Angel Care genomic screening (GS, based on Targeted next-generation sequencing) were performed at the same time.ResultsNinety-six hyperbilirubinemia newborns were enrolled in this study and none was identified with inborn errors of metabolism (IEM) by TMS, while 6 infants (6.25%, 6/96) were suspected to have a genetic disorder by Angel Care, including 2 cases of glucose-6-phosphate dehydrogenase deficiency (G6PD), and 1 case of maple syrup urine disease type 1B (MSUD1B), autosomal recessive deafness 1A (DFNB1A), Leber hereditary optic neuropathy (LHON), thyroid dyshormonogenesis 6 (TDH6) each. In addition, 44 infants (45.8%) were detected having at least one variant which conferred a carrier status for a recessive childhood-onset disorder. A total of 33 out of 60 variants (55.0%) reported for carrier status were pathogenic (P), 24 (40.0%) were likely pathogenic (LP), and 3 variants were variant of uncertain significance (VUS). Top six common genes of carrier status were GJB2, DUOX2, PRODH, ATP7B, SLC12A3, SLC26A4. Two newborns showed abnormalities in elementary screening of TMS, but were confirmed as false positive after recall. Their results of Angel Care did not found abnormality.ConclusionUsing neonatal hyperbilirubinemia as an example, genome sequencing screening can find more evidence of genetic variation in NICU newborns, and “Angel Care” is an effective method.

Neonatal sudden death caused by a novel heterozygous mutation in SLC25A20 gene: A case report and brief literature review

Carnitine-acylcarnitine translocase deficiency (CACTD) is a rare and life-threatening autosomal recessive disorder of fatty acid β-oxidation (FAO). Most patients with CACTD develop severe metabolic decompensation which deteriorates progressively and rapidly, causing death in infancy or childhood. As CACTD in some patients is asymptomatic or only with some nonspecific symptoms, the diagnosis is easy to be ignored, resulting in sudden death, which often triggers medical disputes. Herein, we report a case of neonatal sudden death with CACTD. The neonate showed a series of severe metabolic crisis, deteriorated rapidly and eventually died 3 days after delivery. Tandem mass spectrometry (MS-MS) screening of dry blood spots before death showed that the level of long-chain acylcarnitines, especially C12-C18 acylcarnitine, was increased significantly, and therefore a diagnosis of inherited metabolic disease (IMD) was suspected. Autopsy and histopathological results demonstrated that there were diffuse vacuoles in the heart and liver of the deceased. Mutation analysis revealed that the patient was a compound heterozygote with c.199–10 T > G and a novel c.1A > T mutation in the SLC25A20 gene. Pathological changes such as heart failure, arrhythmia and cardiac arrest related to mitochondrial FAO disorders are the direct cause of death, while gene mutation is the underlying cause of death.

Neonatal genetic metabolic disease entities ought to be included in tandem mass spectrometry screening in China

Objective To systematically retrieve and sort out the information on neonatal genetic metabolic disease entities included in tandem mass spectrometry (MS/MS) screening in some countries and to provide a reference for entity expansion in the screening on neonatal genetic metabolic diseases with MS/MS in China. Methods With literature analysis and expert consultation, we assessed the priority order of the neonatal genetic metabolic diseases to be included in screening with MS/MS using a comprehensive score. Results Totally 53 neonatal genetic metabolic diseases were identified with the practicability of to be screened with MS/MS; of which, 23, 16, and 14 were aminoacidopathies, organic acidemias and fatty amino acid metabolic diseases, respectively. The diseases with top ten comprehensive scores in descending order are isovaleric acidemia, glutaric acidemia type Ⅰ, medium-chain acyl-CoA dehydrogenase deficiency, maple syrup urine disease, homocystinuria, propionic acidemia, methyl malonic acidemia, phenylketonuria, citrullinemia type Ⅰ, and very long-chain acyl-CoA dehydrogenase deficiency. Conclusion Based on the study results, we recommend that the ten neonatal genetic metabolic diseases with higher comprehensive scores ought to be included in MS/MS screening and the subsequent disease entities for the screening could be adjusted timely according to screening detection outcomes and the incidences of neonatal genetic metabolic diseases.

Comparing ELISA and LC–MS-MS: A Simple, Targeted Postmortem Blood Screen

A comprehensive screening method that is specific, accurate and customizable is necessary in any forensic toxicology laboratory. Most laboratories utilize some form of immunoassay testing as it is reliable and sensitive with minimal sample preparation and is relatively inexpensive to simultaneously screen for multiple classes of drugs with different chemical properties. However, accessibility to more specific technology and instrumentation such as mass spectrometry has increased and therefore using immunoassay as the screening method of choice may be revisited. A screening method for 42 drugs in postmortem blood was developed and validated following the Organization of Scientific Area Committees for Forensic Science guidelines for toxicology method validation. The method was developed using minimal sample preparation of postmortem blood consisting only of a protein precipitation. Only two internal standards were used, which greatly reduces the cost of implementing this method. Limit of detection, interference studies, processed sample stability and ion suppression/enhancement were examined. Additionally, over 100 case samples were analyzed by both the current enzyme-linked immunosorbent assay (ELISA) testing procedure and the proposed liquid chromatography-tandem mass spectrometry (LC-MS-MS) screening method. The comparison determined that the LC-MS-MS method performed as well as or better than the ELISA in nearly all cases. The ability to add additional target drugs increases the laboratory's scope of analysis as well. This method is ideal for forensic laboratories wishing to improve screening while working within budget constraints.

Tandem Mass Spectrometry Screening for Inborn Errors of Metabolism in Newborns and High-Risk Infants in Southern China: Disease Spectrum and Genetic Characteristics in a Chinese Population

Inborn errors of metabolism (IEMs) often causing progressive and irreversible neurological damage, physical and intellectual development lag or even death, and serious harm to the family and society. The screening of neonatal IEMs by tandem mass spectrometry (MS/MS) is an effective method for early diagnosis and presymptomatic treatment to prevent severe permanent sequelae and death. A total of 111,986 healthy newborns and 7,461 hospitalized high-risk infants were screened for IEMs using MS/MS to understand the characteristics of IEMs and related gene mutations in newborns and high-risk infants in Liuzhou. Positive samples were analyzed by Sanger sequencing or next-generation sequencing. The results showed that the incidence of IEMs in newborns in the Liuzhou area was 1/3,733, and the incidence of IEMs in high-risk infants was 1/393. Primary carnitine deficiency (1/9,332), phenylketonuria (1/18,664), and isovaleric acidemia (1/37,329) ranked the highest in neonates, while citrullinemia type II ranked the highest in high-risk infants (1/1,865). Further, 56 mutations of 17 IEMs-related genes were found in 49 diagnosed children. Among these, HPD c.941T > C, CBS c.1465C > T, ACADS c.337G > A, c.1195C > T, ETFA c.737G > T, MMACHC 1076bp deletion, PCCB c.132-134delGACinsAT, IVD c.548C > T, c.757A > G, GCDH c.1060G > T, and HMGCL c.501C > G were all unreported variants. Some related hotspot mutations were found, including SLC22A5 c.51C > G, PAH c.1223G > A, IVD c.1208A > G, ACADS c.625G > A, and GCDH c.532G > A. These results show that the overall incidence of IEMs in the Liuzhou area is high. Hence, the scope of IEMs screening and publicity and education should be expanded for a clear diagnosis in the early stage of the disease.

Herbal Medicine Must Be Treated with Care—A Case Report on Aconitine

Pathologists usually only request a screening for natural toxic substances if plant material has been observed during autopsy or if there exists a hint in the police investigation file. This situation is aggravated by the fact that most toxins are not covered by typical immunoassays and gas chromatography–mass spectrometry (GC–MS) profiling systems. In addition, only a few forensic toxicological libraries based on liquid chromatography coupled to high-resolution tandem mass spectrometry (LC–HRMS/MS) exist. In the following case, femoral blood and urine were applied to systematic toxicological analysis (STA). However, the concentrations determined in blood did not lead to death. Consequently, a liquid chromatography high-resolution tandem mass spectrometry (LC–HRMS/MS) screening approach was applied. Aconitine was quantitated in all specimens taken during autopsy and urine and bile fluid screened for aconitine metabolites. Aconitine, jesaconitine, hypaconitine, and mesaconitine were found in the root piece collected from the duodenum. Apart from aconitine, no other alkaloids were detected in the urine or in the femoral blood sample. The highest concentrations of aconitine were found in gastric content (55.2 μg/mL), bile fluid (11.7 μg/mL), and liver (9.14 μg/g), and least in femoral blood (0.15 μg/mL) and cerebrospinal fluid (0.07 μg/mL). The liver/peripheral blood ratio amounted to 61 L/kg and indicated that aconitine undergoes postmortem redistribution. During our metabolism investigation, we found 3-dehydrogen-aconitine in the urine and bile fluid sample and N-deethyl-aconitine only in the bile fluid sample. If the routine GC–MS screening approach does not come up with a toxin, then LC–HRMS/MS profiling could represent the method of choice. In this case aconitine was identified. The concentrations determined were compared to those reported in literature and clearly indicate that the deceased died due to an aconitine overdose.

Establishment of an auxiliary diagnosis system of newborn screening for inherited metabolic diseases based on artificial intelligence technology and a clinical trial

Objective: To establish a disease risk prediction model for the newborn screening system of inherited metabolic diseases by artificial intelligence technology. Methods: This was a retrospectively study. Newborn screening data (n=5 907 547) from February 2010 to May 2019 from 31 hospitals in China and verified data (n=3 028) from 34 hospitals of the same period were collected to establish the artificial intelligence model for the prediction of inherited metabolic diseases in neonates. The validity of the artificial intelligence disease risk prediction model was verified by 360 814 newborns' screening data from January 2018 to September 2018 through a single-blind experiment. The effectiveness of the artificial intelligence disease risk prediction model was verified by comparing the detection rate of clinically confirmed cases, the positive rate of initial screening and the positive predictive value between the clinicians and the artificial intelligence prediction model of inherited metabolic diseases. Results: A total of 3 665 697 newborns' screening data were collected including 3 019 cases' positive data to establish the 16 artificial intelligence models for 32 inherited metabolic diseases. The single-blind experiment (n=360 814) showed that 45 clinically diagnosed infants were detected by both artificial intelligence model and clinicians. A total of 2 684 cases were positive in tandem mass spectrometry screening and 1 694 cases were with high risk in artificial intelligence prediction model of inherited metabolic diseases, with the positive rates of tandem 0.74% (2 684/360 814)and 0.46% (1 694/360 814), respectively. Compared to clinicians, the positive rate of newborns was reduced by 36.89% (990/2 684) after the application of the artificial intelligence model, and the positive predictive values of clinicians and artificial intelligence prediction model of inherited metabolic diseases were 1.68% (45/2 684) and 2.66% (45/1 694) respectively. Conclusion: An accurate, fast, and the lower false positive rate auxiliary diagnosis system for neonatal inherited metabolic diseases by artificial intelligence technology has been established, which may have an important clinical value.

A pilot study of expanded newborn screening for 573 genes related to severe inherited disorders in China: results from 1,127 newborns.

BackgroundNewborn screening (NBS) in China is mainly aimed at detecting biochemical levels of metabolites in the blood, which may generate false-positive/negative results. Current biochemical NBS includes tandem mass spectrometry (MS/MS) screening for metabolites as well as phenylalanine (Phe), thyroid-stimulating hormone (TSH), 17-α-hydroxyprogesterone (17-OHP), and glucose-6-phosphate dehydrogenase (G6PD) test. This study intended to explore whether next-generation sequencing (NGS) for dried blood spots combining with biochemical screening could improve the current screening efficiency and to investigate the carrier frequencies of mutations in causative genes related to amino acid metabolism, organic acid metabolism, and fatty acid oxidation in this cohort.MethodsWe designed a panel of 573 genes related to severe inherited disorders and performed NGS in 1,127 individuals who had undergone biochemical NBS. The NGS screening results of neonates were used to compare with the biochemical results.ResultsNGS screening results revealed that all the four newborns with abnormal G6PD values carried hemizygous G6PD mutations, which were consistent with the decreased G6PD enzymatic activity. The NGS results revealed an individual with compound heterozygous mutations of SLC22A5, who was biochemically negative in 2016. The MS/MS screening results in 2019 showed free carnitine deficiency, which was consistent with the genetic findings. The top five genes with the highest carrier frequencies of mutations in these newborns were PAH (1:56, 1.79%), ETFDH (1:81, 1.23%), MMACHC (1:87, 1.15%), SLC25A13 (1:102, 0.98%), and GCDH (1:125, 0.80%).ConclusionsOur study highlighted that combining NGS screening with biochemical screening could improve the current NBS efficiency. This is the first study to investigate carrier frequencies of mutations in 77 genes causing inherited metabolic diseases (IMDs) in China.

A Comprehensive HPLC-MS-MS Screening Method for 77 New Psychoactive Substances, 24 Classic Drugs and 18 Related Metabolites in Blood, Urine and Oral Fluid.

To date, more than 800 molecules are classified as New Psychoactive Substances (NPS), and it is reported that this number increases every year. Whereas several cases of polydrug consumption that led to acute intoxication and death are reported, a lack of effective analytical screening method to detect NPS and classical drug of abuse in human matrices affects the prompt identification of the probable cause of intoxication in emergency department of hospitals. In this concern, a fast, simple and comprehensive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) screening method to detect and quantify 77 NPS, 24 classic drugs and 18 related metabolites has been successfully developed and validated in blood, urine and oral fluid. A small volume (100 µL) of whole blood samples spiked with internal standard deuterated mixture was added to 70 µL of M3® buffer, and after precipitation of blood proteins, the supernatant was evaporated to dryness and reconstituted in 1 mL of mobile phase. Same volume (100 µL) of urine and oral fluid samples spiked with internal standard deuterated mix were only diluted with 500 µL of M3® reagent. One microliter of samples of each matrix was injected into HPLC-MS-MS equipment. The run time lasted 10 min with a gradient mobile phase. Mass spectrometric analysis was performed in positive ion multiple reaction monitoring mode. The method was linear for all analytes under investigation with a determination coefficient always better than 0.99. The calibration range for blood and oral fluid was from limits of quantification (LOQs) to 200 ng/mL, whereas that for urine was LOQs to 1000 ng/mL. Recovery and matrix effect were always higher than 80%, whereas intra-assay and inter-assay precision were always better than 19% and accuracy was always within 19% of target in every matrix. Applicability of the method was verified by analysis of samples from real cases.

Exploring the Potential of Antibiotic Production From Rare Actinobacteria by Whole-Genome Sequencing and Guided MS/MS Analysis.

Actinobacteria are well recognized for their production of structurally diverse bioactive secondary metabolites, but the rare actinobacterial genera have been underexploited for such potential. To search for new sources of active compounds, an experiment combining genomic analysis and tandem mass spectrometry (MS/MS) screening was designed to isolate and characterize actinobacterial strains from a mangrove environment in Macau. Fourteen actinobacterial strains were isolated from the collected samples. Partial 16S sequences indicated that they were from six genera, including Brevibacterium, Curtobacterium, Kineococcus, Micromonospora, Mycobacterium, and Streptomyces. The isolate sp.01 showing 99.28% sequence similarity with a reference rare actinobacterial species Micromonospora aurantiaca ATCC 27029T was selected for whole genome sequencing. Organization of its gene clusters for secondary metabolite biosynthesis revealed 21 clusters encoded to antibiotic production, which is higher than other Micromonospora species. Of the genome-predicted antibiotics, kanamycin was found through guided MS/MS analysis producible by the M. aurantiaca strain for the first time. The present study highlighted that genomic analysis combined with MS/MS screening is a promising method to discover potential of antibiotic production from rare actinobacteria.