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This document summarizes a study on prenatal screening for copy number variants (CNVs) in the Duchenne muscular dystrophy (DMD) gene using non-invasive prenatal testing (NIPT) data. The study analyzed data from a large population of reproductive-age women in China and identified 33 different pathogenic/likely pathogenic maternal CNVs in the DMD gene. The study found that approximately 40% of offspring inherited true-positive maternal CNVs, and 1.63% of male offspring could potentially be prenatally diagnosed and managed using this method. The study provides important information for genetic counseling and DMD/BMD screening worldwide. [Extracted from the article]

A total of 15,267 pregnancies were tested by NIPT in this study. Grey zone (z score: 2.58 ∼ 4 and −4∼−2.58) was set for screening out aneuploidy 21, 18 and 13. Cases with z score located in the grey zone were retested starting from DNA extraction. The chi-squared test and/or the Fisher's exact test were used to compare variables. One hundred and eight screening-positive samples in the first run of NIPT were common trisomies 21 (N = 83), trisomies18 (N = 13) or trisomies 13 (N = 12), with PPVs of 87.18%, 76.92%, and 30% respectively. For the cases in the grey zone, most of them (67.15%, 184/274) were reported with Z score of Chromosome 21 in the grey zone and 176 were reclassified as negative by the second run of NIPT; while 3 cases reclassified as trisomy 21 and 1 case reclassified as trisomy 13 were finally confirmed by karyotyping analysis, with PPV 25% and 20% respectively. The grey zone and the second run of NIPT in this study showed that the grey zone and second run NIPT approach was able to accurately help categorise cases as negative and positive. Invasive diagnosis is recommended to prevent false negative aneuploidies for samples located in the special z score scope of 2.58–3 for two runs of NIPT. What is already known on this subject? Grey zone was widely used in NIPT. The performance of grey zone of clinical samples on Illumina HiSeq 2000 instrument has been reported, and the performance of grey zone on some mainstream sequencers with simulated samples has also been summarised. Reported treatments for samples located in the grey zone in NIPT usually included being classified into 'unclassified' or 'no call' followed by following up and/or karyotyping analysis. What do the results of this study add? This study investigated the performance of the grey zone on Ion Proton DA8600 with clinical samples; and it present an alternative treatment for samples in grey zone that reclassified them as negative or positive by the second run of sequencing. What are the implications of these findings for clinical practice and/or further research? Our own data for the performance of the grey zone in the cfDNA assay on the semiconductor sequencing platform might provide raw materials for other researchers' meta-analysis, cohort study, or other studies. Details of Z score distributions of chromosomes in grey zone, results of the second run of NIPT for samples in the grey zone, and false negative samples in the grey zone would help lab technicians better analyse the NIPT results and help doctors to improve genetic counselling. [ABSTRACT FROM AUTHOR]

Background: Whole-exome sequencing (WES) has been recommended as a first-tier clinical diagnostic test for individuals with neurodevelopmental disorders (NDDs). We aimed to identify the genetic causes of 17 children with developmental delay (DD) and/or intellectual disability (ID). Methods: WES and exome-based copy number variation (CNV) analysis were performed for 17 patients with unexplained DD/ID. Results: Single-nucleotide variant (SNV)/small insertion or deletion (Indel) analysis and exome-based CNV calling yielded an overall diagnostic rate of 58.8% (10/17), of which diagnostic SNVs/Indels accounted for 41.2% (7/17) and diagnostic CNVs accounted for 17.6% (3/17). Conclusion: Our findings expand the known mutation spectrum of genes related to DD/ID and indicate that exome-based CNV analysis could improve the diagnostic yield of patients with DD/ID. [ABSTRACT FROM AUTHOR]

Alternative lengthening of telomeres (ALT) is a homologous recombination-based telomere maintenance mechanism activated in 10–15% of human cancers. Although significant progress has been made, the key regulators of the ALT pathway and its role in cancer development remain elusive. Bioinformatics methods were used to predict novel telomere-associated proteins (TAPs) by analysis of large-scale ChIP-Seq data. Immunostaining and fluorescence in situ hybridization experiments were applied to detect the subcellular location of target genes and telomeres. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were used to examine the expression of targeting genes. Overall survival (OS) analyses were used to evaluate the relationship between gene expression and survival time; immunohistochemistry was used to detect the distribution of target genes in liver cancer tissues. We found that nuclear factor related to kappaB binding protein (NFRKB), a metazoan-specific subunit of the INO80 complex, can associate with telomeres in human ALT cells. Loss of NFRKB induces dysfunction of telomeres and less PML bodies in U2OS cells. In addition, NFRKB is low/moderately expressed in cytoplasm of normal hepatocytes but heavily accumulating in the nucleus of liver cancer cells. Finally, the high expression of NFRKB is associated with short OS time and poor prognosis. NFRKB is a TAP and protects telomeres from DNA damage in ALT cells. It is highly expressed in hepatocellular carcinoma (HCC) cells and predicts a poor prognosis. NFRKB may be a promising prognostic biomarker for the treatment of HCC in the future. [ABSTRACT FROM AUTHOR]

Objective: To explore the clinical effect of noninvasive prenatal screening (NIPS) for the women at advanced maternal age (AMA) and discuss the relationship between women's age and NIPS effect.Methods: Fourteen thousand thirty-five women at AMA who accepted NIPS from two prenatal diagnosis centers were recruited for this study. NIPS were checked by Illumina Next CN 500. All the AMA women received prenatal genetic counseling, selected prenatal diagnosis and different clinical treatments according to the results of NIPS.Results: A total of 114 cases (0.81%) got the NIPS-positive results of T21/T18/T13. One hundred four cases of them accepted prenatal diagnosis and 87 cases were proved as true positive. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 100, 99.88, 92.55 and 100%, respectively. Seventy-four women (0.53%) showed NIPS-positive results of sex chromosomal aneuploidies (SCAs). After informed consent, 46 women (62.2%) accepted fetus karyotype analysis. Nineteen cases were identified as true positive results, while 27 cases were false positive results. The PPV for SCAs in AMA women was 41.3%. The PPV of T21/T18/T13 in AMA women over 40 was 100%, while it was 81.91% for the women whose age was 35 ∼ 40 years old. There was also rising trend in PPV of fetal sex chromosome with the increased age (62.50 versus 36.84%).Conclusions: NIPS is a good choice for AMA pregnant women. It can not only achieve satisfactory clinical effect, but also greatly reduce invasive prenatal diagnosis. We will get better effect of NIPS by further managing AMA women stratified by their age. [ABSTRACT FROM AUTHOR]

Massively parallel sequencing (MPS) combined with bioinformatic analysis has been widely applied to detect fetal chromosomal aneuploidies such as trisomy 21, 18, 13 and sex chromosome aneuploidies (SCAs) by sequencing cell-free fetal DNA (cffDNA) from maternal plasma, so-called non-invasive prenatal testing (NIPT). However, many technical challenges, such as dependency on correct fetal sex prediction, large variations of chromosome Y measurement and high sensitivity to random reads mapping, may result in higher false negative rate (FNR) and false positive rate (FPR) in fetal sex prediction as well as in SCAs detection. Here, we developed an optimized method to improve the accuracy of the current method by filtering out randomly mapped reads in six specific regions of the Y chromosome. The method reduces the FNR and FPR of fetal sex prediction from nearly 1% to 0.01% and 0.06%, respectively and works robustly under conditions of low fetal DNA concentration (1%) in testing and simulation of 92 samples. The optimized method was further confirmed by large scale testing (1590 samples), suggesting that it is reliable and robust enough for clinical testing. [ABSTRACT FROM AUTHOR]

Spider venom comprises a mixture of compounds with diverse biological activities, which are used to capture prey and defend against predators. The peptide components bind a broad range of cellular targets with high affinity and selectivity, and appear to have remarkable structural diversity. Although spider venoms have been intensively investigated over the past few decades, venomic strategies to date have generally focused on high-abundance peptides. In addition, the lack of complete spider genomes or representative cDNA libraries has presented significant limitations for researchers interested in molecular diversity and understanding the genetic mechanisms of toxin evolution. In the present study, second-generation sequencing technologies, combined with proteomic analysis, were applied to determine the diverse peptide toxins in venom of the Chinese bird spider Ornithoctonus huwena. In total, 626 toxin precursor sequences were retrieved from transcriptomic data. All toxin precursors clustered into 16 gene superfamilies, which included six novel superfamilies and six novel cysteine patterns. A surprisingly high number of hypermutations and fragment insertions/deletions were detected, which accounted for the majority of toxin gene sequences with low-level expression. These mutations contribute to the formation of diverse cysteine patterns and highly variable isoforms. Furthermore, intraspecific venom variability, in combination with variable transcripts and peptide processing, contributes to the hypervariability of toxins in venoms, and associated rapid and adaptive evolution of toxins for prey capture and defense. [ABSTRACT FROM AUTHOR]

The hydrophobic nature of most membrane proteins severely complicates their extraction, proteolysis and identification. Although detergents can be used to enhance the solubility of the membrane proteins, it is often difficult for a detergent not only to have a strong ability to extract membrane proteins, but also to be compatible with the subsequent proteolysis and mass spectrometric analysis. In this study, we made evaluation on a novel application of sodium laurate (SL) to the shotgun analysis of membrane proteomes. SL was found not only to lyse the membranes and solubilize membrane proteins as efficiently as SDS, but also to be well compatible with trypsin and chymotrypsin. Furthermore, SL could be efficiently removed by phase transfer method from samples after acidification, thus ensuring not to interfere with the subsequent CapLC-MS/MS analysis of the proteolytic peptides of proteins. When SL was applied to assist the digestion and identification of a standard protein mixture containing bacteriorhodoposin and the proteins in rat liver plasma membrane-enriched fractions, it was found that, compared with other two representative enzyme- and MS-compatible detergents RapiGest SF (RGS) and sodium deoxycholate (SDC), SL exhibited obvious superiority in the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains. [ABSTRACT FROM AUTHOR]

Background: The detection limit of noninvasive prenatal testing (NIPT) by next generation sequencing for any given fetal copy number variants (CNV) can be influenced by several factors. In this study, we quantified the effects and predicted the value of parameters for CNVs detection by NIPT. Methods: Genomic DNA from patient's leucocytes with 3.16 Mb microdeletion in 22q11.21 was mixed with DNA from aborted fetal tissues without CNV at various concentrations by an enzyme digestion method. Abnormal DNA at 0% served as negative control. Sequencing of mixture samples (at 0%, 4%, 12%, and 20%) by Ion Proton Sequencer was performed at flow 500, with WISECONDOR as the pipeline in CNV‐calling and bin of 500, 750 and 1,000 kb for counting unique reads. The parameters were evaluated with Box–Behnken design. The region with Z score ≦−3 was marked as a potential microdeletion. Results: The equation of Z score depending on fetal fraction, unique read number and bin size was obtained by Box–Behnken design. The negative effect was quantified as the coefficient in the equation. The smallest values of these parameters were defined as 4 M unique read number, and 10.08% fetal DNA concentration at bin of 750 kb for detecting subchromosomal microdeletion of 3.16 Mb. Conclusion: The quantification of effect and value of parameters as well as the method used in this study can benefit the establishment of quality standards for CNVs detection and interpretation of CNVs detection results. [ABSTRACT FROM AUTHOR]

Latrodectus tredecimguttatus, commonly known as black widow spider, is well known for its dangerous bite. Although its venom has been characterized extensively, some fundamental questions about its molecular composition remain unanswered. The limited transcriptome and genome data available prevent further understanding of spider venom at the molecular level. In the present study, we combined next-generation sequencing and conventional DNA sequencing to construct a venom gland transcriptome of the spider L. tredecimguttatus, which resulted in the identification of 9,666 and 480 high-confidence proteins among 34,334 de novo sequences and 1,024 cDNA sequences, respectively, by assembly, translation, filtering, quantification and annotation. Extensive functional analyses of these proteins indicated that mRNAs involved in RNA transport and spliceosome, protein translation, processing and transport were highly enriched in the venom gland, which is consistent with the specific function of venom glands, namely the production of toxins. Furthermore, we identified 146 toxin-like proteins forming 12 families, including 6 new families in this spider in which α-LTX-Lt1a family2 is firstly identified as a subfamily of α-LTX-Lt1a family. The toxins were classified according to their bioactivities into five categories that functioned in a coordinate way. Few ion channels were expressed in venom gland cells, suggesting a possible mechanism of protection from the attack of their own toxins. The present study provides a gland transcriptome profile and extends our understanding of the toxinome of spiders and coordination mechanism for toxin production in protein expression quantity. [ABSTRACT FROM AUTHOR]

Spider venom comprises a mixture of compounds with diverse biological activities, which are used to capture prey and defend against predators. The peptide components bind a broad range of cellular targets with high affinity and selectivity, and appear to have remarkable structural diversity. Although spider venoms have been intensively investigated over the past few decades, venomic strategies to date have generally focused on high-abundance peptides. In addition, the lack of complete spider genomes or representative cDNA libraries has presented significant limitations for researchers interested in molecular diversity and understanding the genetic mechanisms of toxin evolution. In the present study, second-generation sequencing technologies, combined with proteomic analysis, were applied to determine the diverse peptide toxins in venom of the Chinese bird spider Ornithoctonus huwena. In total, 626 toxin precursor sequences were retrieved from transcriptomic data. All toxin precursors clustered into 16 gene superfamilies, which included six novel superfamilies and six novel cysteine patterns. A surprisingly high number of hypermutations and fragment insertions/deletions were detected, which accounted for the majority of toxin gene sequences with low-level expression. These mutations contribute to the formation of diverse cysteine patterns and highly variable isoforms. Furthermore, intraspecific venom variability, in combination with variable transcripts and peptide processing, contributes to the hypervariability of toxins in venoms, and associated rapid and adaptive evolution of toxins for prey capture and defense. [ABSTRACT FROM AUTHOR]

Genetic factors significantly influence the concentration of metabolites in adults. Nevertheless, the genetic influence on neonatal metabolites remains uncertain. To bridge this gap, we employed genotype imputation techniques on large-scale low-pass genome data obtained from non-invasive prenatal testing. Subsequently, we conducted association studies on a total of 75 metabolic components in neonates. The study identified 19 previously reported associations and 11 novel associations between single-nucleotide polymorphisms and metabolic components. These associations were initially found in the discovery cohort (8,744 participants) and subsequently confirmed in a replication cohort (19,041 participants). The average heritability of metabolic components was estimated to be 76.2%, with a range of 69%-78.8%. These findings offer valuable insights into the genetic architecture of neonatal metabolism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Understanding the mechanisms of uncontrolled proliferation in cancer cells provides valuable insights into tumor development and is benefit for discovering efficient methods in cancer treatment. In this study, we identified and quantified 2,057 phosphoproteins and 9,824 unique phosphosites in three liver cell lines with high (QGY, Hep3B) and low (L02) proliferative potentials and disclosed the wide variations in phosphorylation sites and levels among them. We found that the number of identified phosphoproteins and phosphosites in these cells were negatively correlated with their proliferative abilities. The function analysis suggested that the aberrant phosphorylation of SR proteins and activation of MAPK pathway might be two critical factors to promote cancer cell proliferation. Meanwhile, the phosphorylation status of mini-chromosome maintenance (MCM) and nuclear pore (NPC) complexes are significantly different between cell lines with high and low proliferative potentials. Furthermore, the phosphosites targeted by kinase families of CDK, STE and HIPK in the proteins coded by cancer driver genes showed distinct profiles between caner and normal cell lines. These results present key phosphorylation networks involving in abnormal proliferation of cancer cells and uncovered potential molecular markers for estimating the proliferation ability of liver cancer cells.

Background: To detect complex fetal subchromosomal abnormalities by noninvasive prenatal testing (NIPT)., Case Presentation: After routine prenatal serum screening, the plasma of high-risk pregnant women were tested via NIPT, and the NIPT results were further validated by fetal karyotype analysis and array-based comparative genomic hybridization (aCGH) through amniocentesis. In addition, the chromosome karyotypes of the parents were also analyzed. NIPT results indicated subchromosomal abnormalities in chromosomes 13 and 21; aCGH results showed 22 Mb and 16 Mb deletions in 13 q31.3 - q34 and 21q11.1 - q21.3, respectively; and the fetal karyotype was 45,XX, der(13),-21. The maternal karyotype 46,XX,inv(9)(p12q13),t(13;21)(q31.3;q21.3) was abnormal, while the paternal karyotype showed no obvious abnormality., Conclusion: In this study, we successfully detected complex deletions in chromosomes 13 and 21 in a fetus using NIPT, and NIPT can provide effective genetic information for the detection of fetal subchromosomal abnormalities.

It is well-known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structures and understanding protein functions. Although many efforts have been made in the field during the latest 2 decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we report an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in an Escherichia coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to the number of lysine in peptides, more reproducible and with higher MASCOT scores. Moreover, the introduction of the Single-Charge Ion Inclusion (SCII) method to alleviate the charge deficiency of small peptides allowed an additional 26% increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in E. coli in a triplicate experiments using 80 μg each. Our optimized method would benefit the deep screening of C-terminome and possibly help discover some novel C-terminal modifications. Data are available via ProteomeXchange with identifier PXD002409.

Identifying cancer driver genes and exploring their functions are essential and the most urgent need in basic cancer research. Developing efficient methods to differentiate between driver and passenger somatic mutations revealed from large-scale cancer genome sequencing data is critical to cancer driver gene discovery. Here, we compared distinct features of SNP with SNV data in detail and found that the weighted ratio of SNV to SNP (termed as WVPR) is an excellent indicator for cancer driver genes. The power of WVPR was validated by accurate predictions of known drivers. We ranked most of human genes by WVPR and did functional analyses on the list. The results demonstrate that driver genes are usually highly enriched in chromatin organization related genes/pathways. And some protein complexes, such as histone acetyltransferase, histone methyltransferase, telomerase, centrosome, sin3 and U12-type spliceosomal complexes, are hot spots of driver mutations. Furthermore, this study identified many new potential driver genes (e.g. NTRK3 and ZIC4) and pathways including oxidative phosphorylation pathway, which were not deemed by previous methods. Taken together, our study not only developed a method to identify cancer driver genes/pathways but also provided new insights into molecular mechanisms of cancer development.

Olfactory ensheathing cells (OECs) are a special type of glial cells that have characteristics of both astrocytes and Schwann cells. Evidence suggests that the regenerative capacity of OECs is induced by soluble, secreted factors that influence their microenvironment. These factors may regulate OECs self-renewal and/or induce their capacity to augment spinal cord regeneration. Profiling of plasma membrane and extracellular matrix through a high-throughput expression proteomics approach was undertaken to identify plasma membrane and extracellular matrix proteins of OECs under serum-free conditions. 1D-shotgun proteomics followed with gene ontology (GO) analysis was used to screen proteins from primary culture rat OECs. Four hundred and seventy nonredundant plasma membrane proteins and 168 extracellular matrix proteins were identified, the majority of which were never before reported to be produced by OECs. Furthermore, plasma membrane and extracellular proteins were classified based on their protein-protein interaction predicted by STRING quantitatively integrates interaction data. The proteomic profiling of the OECs plasma membrane proteins and their connection with the secretome in serum-free culture conditions provides new insights into the nature of their in vivo microenvironmental niche. Proteomic analysis for the discovery of clinical biomarkers of OECs mechanism warrants further study.

In-gel digestion is commonly used after proteins are resolved by polyacrylamide gel electrophoresis (SDS-PAGE, 2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the direct analysis of complex proteins. Here, we describe a strategy combining isolation of purified plasma membrane, efficient digestion of plasma membrane proteins in polyacrylamide gel, and high-sensitivity analysis by advanced mass spectrometry to create a new rapid and high-throughput method. The plasma membrane protein mixture is directly incorporated into a polyacrylamide gel matrix, After formation of the gel, proteins in the gel section are digested with trypsin, and the resulting peptides are subjected to reversed-phase, high-performance liquid chromatography followed by electrospray ion-trap tandem mass analysis. Using this optimized strategy, we have identified 883 rat liver membrane proteins, of which 490 had a gene ontology (GO) annotation indicating a cellular component, and 294 (60%) of the latter were known integral membrane proteins or membrane proteins. In total, 333 proteins are predicted by the TMHMM 2.0 algorithm to have transmembrane domains (TMDs) and 52% (175 of 333) proteins to contain 2-16 TMDs. The identified membrane proteins provide a broad representation of the rat plasma membrane proteome with little bias evident due to protein p I and molecular weight (MW). Also, membrane proteins with a high GRAVY score (grand average hydrophobicity score) were identified, and basic and acidic membrane proteins were evenly represented. This study not only offered an efficient and powerful method in shotgun proteomics for the identification of proteins of complex plasma membrane samples but also allowed in-depth study of liver membrane proteomes, such as of rat models of liver-related disease. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of rat liver plasma membrane in general.