[Feasibility of non-invasive prenatal testing for the detection of fetal chromosomal copy number variants].

OBJECTIVE: To explore the feasibility of non-invasive prenatal testing (NIPT) for detecting fetal chromosomal copy number variants (CNV). METHODS: A retrospective analysis was carried out on NIPT positive samples in Suzhou Municipal Hospital from January 1, 2019 to December 31, 2021. The effect of NIPT on fetal CNV detection was assessed by comparison with the results of karyotype analysis and/or chromosomal microarray analysis (CMA). RESULTS: Among the 525 NIPT positive samples, 146 were CNV cases, of which 84 were further verified by karyotyping and/or CMA, 29 (34.5%) were true positive. Among them, 12 cases were pathogenic variants, 2 cases were likely pathogenic variants and 15 cases were variants of uncertain significance. CONCLUSION: NIPT could detect CNV with high accuracy, and to combine CNV detection and chromosomal aneuploidy detection has great significance to improve the prenatal and postnatal care.

ZBTB40 is a telomere-associated protein and protects telomeres in human ALT cells.

Alternative lengthening of telomeres (ALTs) mechanism is activated in some somatic, germ cells, and human cancer cells. However, the key regulators and mechanisms of the ALT pathway remain elusive. Here we demonstrated that ZBTB40 is a novel telomere-associated protein and binds to telomeric dsDNA through its N-terminal BTB (BR-C, ttk and bab) or POZ (Pox virus and Zinc finger) domain in ALT cells. Notably, the knockout or knockdown of ZBTB40 resulted in the telomere dysfunction-induced foci and telomere lengthening in the ALT cells. The results also show that ZBTB40 is associated with ALT-associated promyelocytic leukemia nuclear bodies, and the loss of ZBTB40 induces the accumulation of the ALT-associated promyelocytic leukemia nuclear bodies in U2OS cells. Taken together, our results implicate that ZBTB40 is a key player of telomere protection and telomere lengthening regulation in human ALT cells.

The performance of grey zone in common foetal aneuploidy screening by semiconductor sequencing.

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. IMPACT STATEMENTWhat 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.

Genetic Analysis of Children With Unexplained Developmental Delay and/or Intellectual Disability by Whole-Exome Sequencing.

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.

Nuclear Factor Related to KappaB Binding Protein (NFRKB) Is a Telomere-Associated Protein and Involved in Liver Cancer Development.

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.

Integrated genomic analysis reveals regulatory pathways and dynamic landscapes of the tRNA transcriptome.

tRNAs and tRNA-derived RNA fragments (tRFs) play various roles in many cellular processes outside of protein synthesis. However, comprehensive investigations of tRNA/tRF regulation are rare. In this study, we used new algorithms to extensively analyze the publicly available data from 1332 ChIP-Seq and 42 small-RNA-Seq experiments in human cell lines and tissues to investigate the transcriptional and posttranscriptional regulatory mechanisms of tRNAs. We found that histone acetylation, cAMP, and pluripotency pathways play important roles in the regulation of the tRNA gene transcription in a cell-specific manner. Analysis of RNA-Seq data identified 950 high-confidence tRFs, and the results suggested that tRNA pools are dramatically distinct across the samples in terms of expression profiles and tRF composition. The mismatch analysis identified new potential modification sites and specific modification patterns in tRNA families. The results also show that RNA library preparation technologies have a considerable impact on tRNA profiling and need to be optimized in the future.

BCREval: a computational method to estimate the bisulfite conversion ratio in WGBS.

BACKGROUND: Whole genome bisulfite sequencing (WGBS) also known as BS-seq has been widely used to measure the methylation of whole genome at single-base resolution. One of the key steps in the assay is converting unmethylated cytosines into thymines (BS conversion). Incomplete conversion of unmethylated cytosines can introduce false positive methylation call. Developing a quick method to evaluate bisulfite conversion ratio (BCR) is benefit for both quality control and data analysis of WGBS. RESULTS: Here we provide a computational method named "BCREval" to estimate the unconverted rate (UCR) by using telomeric repetitive DNA as native spike-in control. We tested the method by using public WGBS data and found that it is very stable and most of BS conversion assays can achieve> 99.5% efficiency. The non-CpG DNA methylation at telomere fits a binomial model and may result from a random process with very low possibility (the ratio < 0.4%). And the comparison between BCREval and Bismark (Krueger and Andrews, Bioinformatics 27:1571-1572, 2011), a widely used BCR evaluator, suggests that our algorithm is much faster and more efficient than the latter. CONCLUSION: Our method is a simple but robust method to QC and speculates BCR for WGBS experiments to make sure it achieves acceptable level. It is faster and more efficient than current tools and can be easily integrated into presented WGBS pipelines.

Sequencing shorter cfDNA fragments improves the fetal DNA fraction in noninvasive prenatal testing.

BACKGROUND: Sequencing cell-free DNA in maternal plasma is an effective noninvasive prenatal testing technique that has been used in fetal aneuploidy screening worldwide. However, its clinical application is limited by the low fetal fraction (<4%) of cell-free DNA in many singleton pregnancies, which usually results in screen failures or no calls. In addition, dizygotic twin contributions of cell-free DNA into the maternal circulation can vary by 2-fold, complicating the quantitative diagnosis of fetal aneuploidy. OBJECTIVE: We performed semiconductor sequencing of shorter fragments (107-145 bp) of circulating cell-free DNA to improve the fetal DNA fraction at lower uniquely mapped reads (1-8.5 MB) to reduce the probability of no calls. STUDY DESIGN: We identified 2903 plasma samples from pregnant women, including 86 dizygotic twin pregnancy, that were collected at a single prenatal diagnostic center between October 2015 and July 2018. Size-selection noninvasive prenatal testing for fetal aneuploidy was applied to 2817 plasma samples (1409 male and 1408 female fetuses) and 86 dizygotic twins using noninvasive prenatal testing with and without size selection. Shorter fragment size was the key factor affecting fetal fraction in multivariable linear regression models as well as to validate the accuracy of the size selection for noninvasive prenatal testing. RESULTS: Analysis of 1409 male fetuses by multivariable linear regression showed that maternal age, body mass index, number of pregnancies, average cell-free DNA size, maternal plasma cell-free DNA concentration, library concentration, and multiple gestation were negatively correlated with fetal fraction. Conversely, gestational age and uniquely mapped reads were positively correlated with fetal fraction. Compared with ≤120 bp cell-free DNA fragments, mean fetal fraction differences were -3.57% (95% confidence interval, -5.95% to -1.19%), for 121-130 bp, -9.52% (95% confidence interval, -11.89% to -7.14%) for 131-140 bp, and -14.47% (95% confidence interval, -18.37% to -10.58%) for ≥141 bp (Ptrend < .0001). These results were statistically significant after multivariable adjustments in models for fetal fraction. Meanwhile, results from 86 dizygotic twins showed that the size selection increased the fetal fraction by ∼3.2-fold, with 98.8% of samples reaching a fetal fraction >10%. Improved detection accuracy was also achieved. CONCLUSION: Sequencing shorter cell-free DNA fragments is a reasonable strategy to reduce the probability of no calls results because of low fetal fraction and should be recommended to pregnant subjects.

Effect quantification and value prediction of factors in noninvasive detection for specific fetal copy number variants by semiconductor sequencing.

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.

Clinical evaluation of NIPS for women at advanced maternal age: a multicenter retrospective study.

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.

Enrichment of the fetal fraction in non-invasive prenatal screening reduces maternal background interference.

Measurement of cell-free fetal DNA (cffDNA) is an indispensable process for non-invasive prenatal screening (NIPS). According to recent studies, cffDNA in maternal plasma can be enriched for various lengths of fragments, and a sufficient amount of cffDNA can effectively eliminate background interference on the part of maternal DNA. Therefore, we developed a simple and effective separation method, improved NIPS (iNIPS), that enriches the fetal fraction and improves the accuracy of NIPS for fetal aneuploid detection. We adopted a novel strategy to achieve enrichment of 125-135 bp cell-free DNA (cfDNA) by e-gel electrophoresis. To evaluate clinical performance, we compared NIPS and iNIPS results from 2153 retrospective clinical samples. Of the 22 samples with NIPS results of "no call", 17 samples were reclassified as "unaffected" (9 cases of chr13, 5 cases of chr18, and 3 cases of chr21); 2 samples remained classified as "no call" (1 case of chr18 and 1 case of chr21); and 3 samples were identified as T21 by iNIPS. The average increase in abundance of cfDNA fragments of 125-135 bp was 2.5 times, and the average decrease in maternal background interference was 1.3 times. On this basis, the detection of fetal aneuploidy was highly improved with the fetal fraction as low as 2%; iNIPS achieved 100% sensitivity and 99.90% specificity in retrospective samples.

Quantitative Phosphoproteomic Analysis Reveals Key Mechanisms of Cellular Proliferation in Liver Cancer Cells.

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.

An Optimized Method for Accurate Fetal Sex Prediction and Sex Chromosome Aneuploidy Detection in Non-Invasive Prenatal Testing.

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.

Rac1-PAK2 pathway is essential for zebrafish heart regeneration.

P-21 activated kinases, or PAKs, are serine-threonine kinases that play important roles in diverse heart functions include heart development, cardiovascular development and function in a range of models; however, the mechanisms by which PAKs mediate heart regeneration are unknown. Here, we demonstrate that PAK2 and PAK4 expression is induced in cardiomyocytes and vessels, respectively, following zebrafish heart injury. Inhibition of PAK2 and PAK4 using a specific small molecule inhibitor impedes cardiomyocyte proliferation/dedifferentiation and cardiovascular regeneration, respectively. Cdc42 is specifically expressed in the ventricle and may function upstream of PAK2 but not PAK4 under normal conditions and that cardiomyocyte proliferentation during heart regeneration relies on Rac1-mediated activation of Pak2. Our results indicate that PAKs play a key role in heart regeneration.

Single Nucleotide Polymorphisms and Osteoarthritis: An Overview and a Meta-Analysis.

Osteoarthritis (OA) is a complex disorder characterized by degenerative articular cartilage and is largely attributed to genetic risk factors. Single nucleotide polymorphisms (SNPs) are common DNA variants that have shown promising and efficiency, compared with positional cloning, to map candidate genes of complex diseases, including OA. In this study, we aim to provide an overview of multiple SNPs from a number of genes that have recently been linked to OA susceptibility. We also performed a comprehensive meta-analysis to evaluate the association of SNP rs7639618 of double von Willebrand factor A domains (DVWA) gene with OA susceptibility. A systematic search of studies on the association of SNPs with susceptibility to OA was conducted in PubMed and Google scholar. Studies subjected to meta-analysis include human and case-control studies that met the Hardy-Weinberg equilibrium model and provide sufficient data to calculate an odds ratio (OR). A total of 9500 OA cases and 9365 controls in 7 case-control studies relating to SNP rs7639618 were included in this study and the ORs with 95% confidence intervals (CIs) were calculated. Over 50 SNPs from different genes have been shown to be associated with either hip (23), or knee (20), or both (13) OA. The ORs of these SNPs for OA and the subtypes are not consistent. As to SNP rs7639618 of DVWA, increased knee OA risk was observed in all genetic models analyzed. Specifically, people from Asian with G-allele showed significantly increased risk of knee OA (A versus G: OR = 1.28, 95% CI 1.13-1.46; AA versus GG: OR = 1.60, 95% CI 1.25-2.05; GA versus GG: OR = 1.31, 95% CI 1.18-1.44; AA versus GA+GG: OR = 1.34, 95% CI 1.12-1.61; AA+GA versus GG: OR = 1.40, 95% CI 1.19-1.64), but not in Caucasians or with hip OA. Our results suggest that multiple SNPs play different roles in the pathogenesis of OA and its subtypes; SNP rs7639618 of DVWA gene is associated with a significantly increased risk of knee OA in Asians. Given the limited sample size, further studies are needed to evaluate this observation.

Detection of complex deletions in chromosomes 13 and 21 in a fetus by noninvasive prenatal testing.

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.

Systematic Optimization of C-Terminal Amine-Based Isotope Labeling of Substrates Approach for Deep Screening of C-Terminome.

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.

Structural and Functional Diversity of Peptide Toxins from Tarantula Haplopelma hainanum (Ornithoctonus hainana) Venom Revealed by Transcriptomic, Peptidomic, and Patch Clamp Approaches.

Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum. The genes expressed in the venom glands were identified, and the bioactivity of their protein products was analyzed using the patch clamp technique. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity.