Results of a Pilot External Quality Assessment Scheme for Genetic Testing of Newborns with Spinal Muscular Atrophy.

BACKGROUND: This study aims to evaluate the ability of laboratories to perform spinal muscular atrophy (SMA) genetic testing in newborns based on dried blood spot (DBS) samples, and to provide reference data and advance preparation for establishing the pilot external quality assessment (EQA) scheme for SMA genetic testing of newborns in China. METHODS: The pilot EQA scheme contents and evaluation principles of this project were designed by National Center for Clinical Laboratories (NCCL), National Health Commission. Two surveys were carried out in 2022, and 5 batches of blood spots were submitted to the participating laboratory each time. All participating laboratories conducted testing upon receiving samples, and test results were submitted to NCCL within the specified date. RESULTS: The return rates were 75.0% (21/28) and 95.2% (20/21) in the first and second surveys, respectively. The total return rate of the two examinations was 83.7% (41/49). Nineteen laboratories (19/21, 90.5%) had a full score passing on the first survey, while in the second survey twenty laboratories (20/20, 100%) scored full. CONCLUSIONS: This pilot EQA survey provides a preliminary understanding of the capability of SMA genetic testing for newborns across laboratories in China. A few laboratories had technical or operational problems in testing. It is, therefore, of importance to strengthen laboratory management and to improve testing capacity for the establishment of a national EQA scheme for newborn SMA genetic testing.

Discordant performances of non-invasive prenatal testing for foetal trisomy 21 screening in subgroups of pregnancies.

BACKGROUND: Non-invasive prenatal testing (NIPT) has been widely adopted as an approach for foetal aneuploidy screening. This study was to evaluate the performance of NIPT for foetal T21 detection in subgroups of pregnancies and the correlation between Z-score and discordant positive predictive values (PPVs). METHODS: We retrospectively reviewed the NIPT results among 22361 pregnancies undergoing combined second-trimester screening (cSTS) previously. Sixty-four cases with positive NIPT results for foetal T21 were validated by invasive prenatal diagnosis. RESULTS: In pregnancies with cSTS-T21 low-, intermediate-, and high-risk, the PPVs at NIPT were 14.3%, 64.3%, and 86.4%, respectively. Mean Z-scores of positive NIPT cases with cSTS-T21 high- and intermediate-risk were comparable, while were higher than that of cases with pre-test low-risk. Furthermore, PPVs for positive NIPT cases at 3 < Z < 5, 5 ≤ Z < 9, and Z ≥ 9 were 16.7%, 63.2%, and 100.0%, respectively. CONCLUSIONS: This study suggested that Z-score value of positive cases might be associated with discordant PPVs for T21 screening in subgroups of pregnancies.

Non-invasive prenatal testing has been offered as a primary screening option to high-risk or general pregnancy. However, the accuracy of non-invasive prenatal testing in patients with various pre-test risks remained unveiled. The current study revealed that the true positive probability for foetal trisomy 21 screening in pregnancies with prior high-risk was higher than that in pregnancies of intermediate-risk, and both of them were much higher than that of those with pre-test low-risk. The average of Z-score for chromosome 21 of positive non-invasive prenatal testing case in high-risk group was comparable with that of intermediate-risk group, while was higher than that of low-risk group. There was also an upward trend for the true positive probability of foetal trisomy 21 screening with the increase of Z-score. Our study revealed that pre-test risk and Z-score for chromosome 21 were helpful for accurately interpreting the reliability of positive results for foetal trisomy 21.

Healthy Plasma Exosomes Exert Potential Neuroprotective Effects against Methylmalonic Acid-Induced Hippocampal Neuron Injury.

Exosomes have shown good potential for alleviating neurological deficits and delaying memory deterioration, but the neuroprotective effects of exosomes remain unknown. Methylmalonic acidemia is a metabolic disorder characterized by the accumulation of methylmalonic acid (MMA) in various tissues that inhibits neuronal survival and function, leading to accelerated neurological deterioration. Effective therapies to mitigate these symptoms are lacking. The purpose of this study was to explore the neuroprotective effects of plasma exosomes on cells and a mouse model of MMA-induced injury. We evaluated the ability of plasma exosomes to reduce the neuronal apoptosis, cross the blood-brain barrier, and affect various parameters related to neuronal function. MMA promoted cell apoptosis, disrupted the metabolic balance, and altered the expression of B-cell lymphoma-2 (Bcl-2), Bcl2-associated X (Bax), and synaptophysin-1 (Syp-1), and these changes may be involved in MMA-induced neuronal apoptosis. Additionally, plasma exosomes normalized learning and memory and protected against MMA-induced neuronal apoptosis. Our findings indicate that neurological deficits are linked to the pathogenesis of methylmalonic acidemia, and healthy plasma exosomes may exert neuroprotective and therapeutic effects by altering the expression of exosomal microRNAs, facilitating neuronal functional recovery in the context of this inherited metabolic disease. Intravenous plasma-derived exosome treatment may be a novel clinical therapeutic strategy for methylmalonic acidemia.

Hepatic protein phosphatase 1 regulatory subunit 3G alleviates obesity and liver steatosis by regulating the gut microbiota and bile acid metabolism.

Intestinal dysbiosis and disrupted bile acid (BA) homeostasis are associated with obesity, but the precise mechanisms remain insufficiently explored. Hepatic protein phosphatase 1 regulatory subunit 3G (PPP1R3G) plays a pivotal role in regulating glycolipid metabolism; nevertheless, its obesity-combatting potency remains unclear. In this study, a substantial reduction was observed in serum PPP1R3G levels in high-body mass index (BMI) and high-fat diet (HFD)-exposed mice, establishing a positive correlation between PPP1R3G and non-12α-hydroxylated (non-12-OH) BA content. Additionally, hepatocyte-specific overexpression of Ppp1r3g (PPP1R3G HOE) mitigated HFD-induced obesity as evidenced by reduced weight, fat mass, and an improved serum lipid profile; hepatic steatosis alleviation was confirmed by normalized liver enzymes and histology. PPP1R3G HOE considerably impacted systemic BA homeostasis, which notably increased the non-12-OH BAs ratio, particularly lithocholic acid (LCA). 16S ribosomal DNA (16S rDNA) sequencing assay indicated that PPP1R3G HOE reversed HFD-induced gut dysbiosis by reducing the Firmicutes/Bacteroidetes ratio and Lactobacillus population, and elevating the relative abundance of Blautia, which exhibited a positive correlation with serum LCA levels. A fecal microbiome transplantation test confirmed that the anti-obesity effect of hepatic PPP1R3G was gut microbiota-dependent. Mechanistically, PPP1R3G HOE markedly suppressed hepatic cholesterol 7α-hydroxylase (CYP7A1) and sterol-12α-hydroxylase (CYP8B1), and concurrently upregulated oxysterol 7-α hydroxylase and G protein-coupled BA receptor 5 (TGR5) expression under HFD conditions. Furthermore, LCA administration significantly mitigated the HFD-induced obesity phenotype and elevated non-12-OH BA levels. These findings emphasize the significance of hepatic PPP1R3G in ameliorating diet-induced adiposity and hepatic steatosis through the gut microbiota-BA axis, which may serve as potential therapeutic targets for obesity-related disorders.

Targeted exome sequencing strategy (NeoEXOME) for Chinese newborns using a pilot study with 3423 neonates.

BACKGROUND: Newborn screening (NBS) aims to detect congenital anomalies, and next-generation sequencing (NGS) has shown promise in this aspect. However, the NBS strategy for monogenic inherited diseases in China remains insufficient. METHODS: We developed a NeoEXOME panel comprising 601 genes that are relevant to the Chinese population found through extensive research on available databases. An interpretation system to grade the results into positive (high-risk, moderate-risk, and low-risk genotypes), negative, and carrier according to the American College of Medical Genetics (ACMG) guidelines was also developed. We validated the panel to evaluate its efficacy by using data from the "1000 Genomes Project" and conducted a pilot multicenter study involving 3423 neonates. RESULTS: The NGS positive rate in the 1000 Genomes Project was 7.6% (23/301), whereas the rate was 12.0% in the multicenter study, including 3249 recruited neonates. Notably, in 200 neonates, positive per conventional NBS, 58.5% (69/118) showed results consistent with NGS. In the remaining 3049 neonates showing negative results in conventional NBS, 271 (8.9%) were positive per NGS, and nine of them were clinically diagnosed with diseases in the follow-up. CONCLUSION: We successfully designed a NeoEXOME panel for targeted sequencing of monogenic inherited diseases in NBS. The panel demonstrated high performance in the Chinese population, particularly for the early detection of diseases with no biochemical markers.

Decreased PPP1R3G in pre-eclampsia impairs human trophoblast invasion and migration via Akt/MMP-9 signaling pathway.

Pre-eclampsia (PE) is a severe pregnancy complication characterized by impaired trophoblast invasion and spiral artery remodeling and can have serious consequences for both mother and child. Protein phosphatase 1 regulatory subunit 3G (PPP1R3G) is involved in numerous tumor-related biological processes. However, the biological action and underlying mechanisms of PPP1R3G in PE progression remain unclear. We used western blotting and immunohistochemistry to investigate PPP1R3G expression in gestational age-matched pre-eclamptic and normal placental tissues. After lentivirus transfection, wound-healing, Transwell, cell-counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and TdT mediateddUTP Nick End Labeling (TUNEL) assays were used to assess trophoblast migration, invasion, proliferation, and apoptosis, respectively. The relative expression levels of PPP1R3G and the proteins involved in the Akt signaling pathway were determined using western blotting. The results showed that PPP1R3G levels were significantly lower in the placental tissues and GSE74341 microarray of the PE group than those of the healthy control group. We also found that neonatal weight and Apgar score were lower at birth, and peak systolic blood pressure and diastolic blood pressure were higher in the PE group than in the non-PE group. In addition, PPP1R3G knockdown decreased p-Akt/Akt expression and inhibited migration, invasion, and proliferation in HTR-8/SVneo trophoblasts but had no discernible effect on cell apoptosis. Furthermore, PPP1R3G positively regulated matrix metallopeptidase 9 (MMP-9), which was downregulated in placental tissues of pregnant women with PE. These results provided the first evidence that the reduced levels of PPP1R3G might contribute to PE by suppressing the invasion and migration of trophoblasts and targeting the Akt/MMP-9 signaling pathway.

Development of a fast and robust liquid chromatography-mass spectrometry-based metabolomics analysis method for neonatal dried blood spots.

Dried blood spot (DBS) samples have been widely used in many fields including newborn screening, with the advantages in transportation, storage and non-invasiveness. The DBS metabolomics research of neonatal congenital diseases will greatly expand the understanding of the disease. In this study, we developed a liquid chromatography-mass spectrometry-based method for neonatal metabolomics analysis of DBS. The influences of blood volume and chromatographic effects on the filter paper on metabolite levels were studied. The levels of 11.11 % metabolites were different between 75 µL and 35 µL of blood volumes used for DBS preparation. Chromatographic effects on the filter paper occurred in DBS prepared with 75 µL whole blood and 6.67 % metabolites had different MS responses when central disks were compared with outer disks. The DBS storage stability study showed that compared with - 80 °C storage, storing at 4 °C for 1 year had obvious influences on more than half metabolites. Storing at 4 °C and - 20 °C for short term (< 14 days) and - 20 °C for longer term (1 year) had less influences on amino acids, acyl-carnitines and sphingomyelins, but greater influences on partial phospholipids. Method validation showed that this method has a good repeatability, intra-day and inter-day precision and linearity. Finally, this method was applied to investigate metabolic disruptions of congenital hypothyroidism (CH), metabolic changes of CH newborns were mainly involved in amino acid metabolism and lipid metabolism.

Clinical utilization of chromosomal microarray analysis for the genetic analysis in subgroups of pregnancy loss.

OBJECTIVE: The underlying etiologies of pregnancy loss are heterogeneous and in many cases unexplained. This study was to explore the genetic causes of early and late pregnancy loss using chromosomal microarray analysis (CMA). METHODS: A cohort of 222 specimens of conceptions underwent genetic analysis using Affymetrix CytoScan 750 K arrays, which includes both SNP markers and copy number markers. RESULTS: Of the 222-products of conception (POC), the overall detection rate for clinical significantly chromosomal anomalies was 40.54%, including 53 autosomal aneuploidy (23.87%), 16 sex chromosome aneuploidy (7.21%), 5 mutiple aneuploidy (2.25%), 4 triploidy (1.80%), and 12 pathogenic copy number variants (pCNVs) (5.41%). In addition, variants of uncertain significance and loss of heterozygosity were detected in 9 samples and 2 samples, respectively. The detection rates for total chromosomal abnormalities, autosomal aneuploidy, sex chromosome aneuploidy, multiple aneuploidy, and triploidy in specimens of early pregnancy loss was higher than that of late pregnancy loss, while had lower detection rate of pCNVs. Moreover, the detection rate in POC of mothers younger than 35 years was lower than that of advanced maternal age. The detection rate was 40.57% in POC of mothers with adverse pregnancy histories, in which was comparable with that of mothers without adverse pregnancy histories. CONCLUSIONS: CMA yielded a superior detection rate in early pregnancy loss than that of late pregnancy loss. Moreover, the incidence of chromosome abnormality in cases with advanced maternal age was higher than that of cases with younger maternal age, while adverse pregnancy history seemed not to be the factors affecting the detection rate for chromosomal abnormality in pregnancy loss.

Quantification of Differential Metabolites in Dried Blood Spots Using Second-Tier Testing for SCADD/IBDD Disorders Based on Large-Scale Newborn Screening in a Chinese Population.

Background: Although newborn screening (NBS) for metabolic defects using the marker butyl carnitine (C4) combined with the C4-to-acetylcarnitine ratio is adequate, the incorporation of novel parameters may improve differential testing for these disorders without compromising sensitivity. Methods: Analytical and clinical performance was evaluated by MS/MS using 237 initially positive neonatal samples between March 2019 and March 2020 at the Newborn Screening Center of Xuzhou Maternity and Child Health Care Hospital. Additionally, second-tier testing by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with the quantification of ethylmalonate (EMA) or isobutyryl-glycine (IBG) in dried blood spots (DBSs) was performed to reduce the false-positive rate. Results: We reviewed initial MS/MS data for DBSs from 469,730 neonates, and a second-tier test was performed using 237 samples that exceeded the C4 concentration cutoff value. Eleven variants of the ACADS gene were identified, with c.1031A>G (p.E344G) being the most common. Fifteen ACAD8 mutations were identified in seven patients, and Swiss modeling and amino acid conservation analyses were conducted for the novel variants. Based on a retrospective analysis of EMA and IBG, the application of second-tier tests before the release of neonatal screening results reduced referrals by over 91.89% and improved the positive predictive value (PPV) for short-chain acyl-CoA dehydrogenase deficiency/isobutyryl-CoA dehydrogenase deficiency (SCADD/IBDD) screening. Conclusion: A screening algorithm including EMA/IBG improves target differential testing for NBS and may eliminate unnecessary referrals while maintaining 100% sensitivity. Second-tier screening using UPLC-MS/MS as a rapid and convenient supplemental DNA sequencing method may be beneficial for differential detection.

Identification and analyzes of DUOX2 mutations in two familial congenital hypothyroidism cases.

BACKGROUND: Mutations in DUOX2 are the frequent cause of congenital hypothyroidism (CH), a common neonatal metabolic disorder characterized by great phenotypic variability. CH can be traditionally subclassified into two subtypes: thyroid dysgenesis (TD) and thyroid dyshormonogenesis. The objectives of this study were to analyze the genetic data of two familial CH cases, to elucidate the pathogenesis from the perspective of genetics and to review and summarize the previous findings. METHODS: Targeted regions sequencing (TRS) technology covering all exons and intron-exon boundaries of 35 known and potential CH-related candidate target genes in combination with Sanger sequencing were performed to identify the likely pathogenic mutations of the six patients with familial CH. RESULTS: In family 1, two DUOX2 missense mutations, namely, c.1060C>T/p.R354W in exon 10 and c.3200C>T/p.S1067L in exon 25, were found. Patient 1 (P1), P2 and P3 were transient CH (TCH) patients with eutopic thyroid glands of normal size and function. In family 2, only the mutation c.3200C>T/p.S1067L was identified. P4, P5, and P6 were diagnosed with permanent CH (PCH), which requires lifelong levothyroxine (L-T4) treatment. Furthermore, both P4 and P5 harbored properly located thyroid glands, whereas P6 had a mildly reduced gland. P1, P3, P6, and other family members carrying monoallelic or biallelic DUOX2 mutations showed no obvious abnormal clinical symptoms or signs, while P2, P4, and P5 showed umbilical hernias. CONCLUSIONS: The present study suggests that the phenotypic features resulting from DUOX2 mutations vary greatly. The p.R354W and p.S1067L alterations or the combination of the two alterations in DUOX2 are probably only predisposing to CH and DUOX2 may be involved in the morphogenesis of the human thyroid gland. Simultaneously, the compensation of DUOX1 for the loss of DUOX2, undetectable pathogenic mutations, the effects of environmental factors, epigenetic mechanisms and the involvement of multiple genes cannot be excluded in the explanation of these genetic results.