Prospective prenatal cell-free DNA screening for genetic conditions of heterogenous etiologies.

Prenatal cell-free DNA (cfDNA) screening uses extracellular fetal DNA circulating in the peripheral blood of pregnant women to detect prevalent fetal chromosomal anomalies. However, numerous severe conditions with underlying single-gene defects are not included in current prenatal cfDNA screening. In this prospective, multicenter and observational study, pregnant women at elevated risk for fetal genetic conditions were enrolled for a cfDNA screening test based on coordinative allele-aware target enrichment sequencing. This test encompasses the following three of the most frequent pathogenic genetic variations: aneuploidies, microdeletions and monogenic variants. The cfDNA screening results were compared to invasive prenatal or postnatal diagnostic test results for 1,090 qualified participants. The comprehensive cfDNA screening detected a genetic alteration in 135 pregnancies with 98.5% sensitivity and 99.3% specificity relative to standard diagnostics. Of 876 fetuses with suspected structural anomalies on ultrasound examination, comprehensive cfDNA screening identified 55 (56.1%) aneuploidies, 6 (6.1%) microdeletions and 37 (37.8%) single-gene pathogenic variants. The inclusion of targeted monogenic conditions alongside chromosomal aberrations led to a 60.7% increase (from 61 to 98) in the detection rate. Overall, these data provide preliminary evidence that a comprehensive cfDNA screening test can accurately identify fetal pathogenic variants at both the chromosome and single-gene levels in high-risk pregnancies through a noninvasive approach, which has the potential to improve prenatal evaluation of fetal risks for severe genetic conditions arising from heterogenous molecular etiologies. ClinicalTrials.gov registration: ChiCTR2100045739 .

Genetic deconvolution of fetal and maternal cell-free DNA in maternal plasma enables next-generation non-invasive prenatal screening.

Current non-invasive prenatal screening (NIPS) analyzes circulating fetal cell-free DNA (cfDNA) in maternal peripheral blood for selected aneuploidies or microdeletion/duplication syndromes. Many genetic disorders are refractory to NIPS largely because the maternal genetic material constitutes most of the total cfDNA present in the maternal plasma, which hinders the detection of fetus-specific genetic variants. Here, we developed an innovative sequencing method, termed coordinative allele-aware target enrichment sequencing (COATE-seq), followed by multidimensional genomic analyses of sequencing read depth, allelic fraction, and linked single nucleotide polymorphisms, to accurately separate the fetal genome from the maternal background. Analytical confounders including multiple gestations, maternal copy number variations, and absence of heterozygosity were successfully recognized and precluded for fetal variant analyses. In addition, fetus-specific genomic characteristics, including the cfDNA fragment length, meiotic error origins, meiotic recombination, and recombination breakpoints were identified which reinforced the fetal variant assessment. In 1129 qualified pregnancies tested, 54 fetal aneuploidies, 8 microdeletions/microduplications, and 8 monogenic variants were detected with 100% sensitivity and 99.3% specificity. Using the comprehensive cfDNA genomic analysis tools developed, we found that 60.3% of aneuploidy samples had aberrant meiotic recombination providing important insights into the mechanism underlying meiotic nondisjunctions. Altogether, we show that the genetic deconvolution of the fetal and maternal cfDNA enables thorough and accurate delineation of fetal genome which paves the way for the next-generation prenatal screening of essentially all types of human genetic disorders.

PGK1 contributes to tumorigenesis and sorafenib resistance of renal clear cell carcinoma via activating CXCR4/ERK signaling pathway and accelerating glycolysis.

Phosphoglycerate kinase 1 (PGK1) has complicated and multiple functions in cancer occurrence, tumor progression and drug resistance. Sorafenib is the first-line treatment targeted drug for patients with kidney renal clear cell carcinoma (KIRC) as a tyrosine kinase inhibitor, but sorafenib resistance is extremely common to retard therapy efficiency. So far, it is unclear whether and how PGK1 is involved in the pathogenesis and sorafenib resistance of KIRC. Herein, the molecular mechanisms of PGK1-mediated KIRC progression and sorafenib resistance have been explored by comprehensively integrative studies using biochemical approaches, mass spectrometry (MS) identification, microarray assay, nude mouse xenograft model and bioinformatics analysis. We have confirmed PGK1 is specifically upregulated in KIRC based on the transcriptome data generated by our own gene chip experiment, proteomics identification and the bioinformatics analysis for five online transcriptome datasets, and PGK1 upregulation in tumor tissues and serum is indicative with poor prognosis of KIRC patients. In the KIRC tissues, a high expression of PGK1 is often accompanied with an increase of glycolysis-related enzymes and CXCR4. PGK1 exhibits pro-tumorigenic properties in vitro and in a xenograft tumor model by accelerating glycolysis and inducing CXCR4-mediated phosphorylation of AKT and ERK. Moreover, PGK1 promotes sorafenib resistance via increasing CXCR4-mediated ERK phosphorylation. In conclusion, PGK1-invovled metabolic reprogramming and activation of CXCR4/ERK signaling pathway contributes to tumor growth and sorafenib resistance of KIRC.

Novel compound heterozygous frameshift variants in WDR81 associated with congenital hydrocephalus 3 with brain anomalies: First Chinese prenatal case confirms WDR81 involvement.

BACKGROUND: Congenital hydrocephalus-3 with brain anomalies (HYC3, MIM 617967) is a rare form of congenital hydrocephalus characterized by severe hydrocephalus and cerebellar abnormalities, the onset of the disease occurs in utero even resulting in fetal death. A very limited spectrum of WDR81 pathogenic variants had been reported in three unrelated families with HYC3. This study aims at presenting novel compound heterozygous frameshift variants in WDR81 in a Chinese fetus. METHODS: Whole-exome sequencing (WES) was performed for a fetus with multiple congenital anomalies including sever hydrocephalus, cleft lip and palate, hydrops fetalis, hepatomegaly, and cerebellar hypoplasia. Sanger sequencing was performed to confirm the origin of the variants subsequently. Variants classification was based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. RESULTS: Two novel heterozygous variants c.146_147insG (p.Thr52fs) and c.673delC (p.Leu225fs) in WDR81 were identified. Sanger sequencing revealed that the c.146_147insG mutation was maternal origin and the c.673delC mutation was paternal origin. Both variants were pathogenic according to the ACMG/AMP guidelines. CONCLUSION: The present study expands the mutation spectrum of WDR81 and help further define the genotype-phenotype correlations of HYC3. WDR81-related HYC3 were highly clinical heterogeneity. We suggested that fetal hydrocephalus with extracerebral manifestations may be suggestive of WDR81 deficiency and WES is effective for achieving a conclusive diagnosis for disorder.

CNV profiles of Chinese pediatric patients with developmental disorders.

PURPOSE: To examine the overall genomic copy-number variant (CNV) landscape of Chinese pediatric patients with developmental disorders. METHODS: De-identified chromosomal microarray (CMA) data from 10,026 pediatric patients with developmental disorders were collected for re-evaluating the pathogenic CNV (pCNV) yields of different medical conditions and for comparing the frequency and phenotypic variability of genomic disorders between the Chinese and Western patient populations. RESULTS: The overall yield of pCNVs in the Chinese pediatric patient cohort was 21.37%, with variable yields for different disorders. Yields of pCNVs were positively associated with phenotypic complexity and intellectual disability/developmental delay (ID/DD) comorbidity for most disorders. The genomic burden and pCNV yield in neurodevelopmental disorders supported a female protective effect. However, the stratification analysis revealed that it was seen only in nonsyndromic ID/DD, not in nonsyndromic autism spectrum disorders or seizure. Furthermore, 15 known genomic disorders showed significantly different frequencies in Chinese and Western patient cohorts, and profiles of referred clinical features for 15 known genomic disorders were also significantly different in the two cohorts. CONCLUSION: We defined the pCNV yields and profiles of the Chinese pediatric patients with different medical conditions and uncovered differences in the frequency and phenotypic diversity of genomic disorders between Chinese and Western patients.

Clinical and Genetic Analysis of CHD7 Expands the Genotype and Phenotype of CHARGE Syndrome.

CHARGE syndrome is a life-threatening disease caused by mutations of chromodomain helicase DNA-binding protein 7 gene (CHD7). The disease is characterized by a pattern of congenital anomalies that involve multiple organs. In this study, five patients were diagnosed as CHARGE syndrome with CHD7 mutations by whole exome sequencing. Although the clinical phenotypes of probands are highly variable and typical symptoms such as coloboma and choanal atresia are not commonly manifested in this cohort, they all presented congenital heart defects. Of note, dyspnea is the most prominent symptom in all five neonatal patients, suggesting that dyspnea might be a phenotypic clue of CHARGE syndrome.

Novel compound heterozygous pathogenic variants in ASCC1 in a Chinese patient with spinal muscular atrophy with congenital bone fractures 2 : Evidence supporting a "Definitive" gene-disease relationship.

BACKGROUND: A very limited spectrum of ASCC1 pathogenic variants had been reported in six (mostly consanguineous) families with spinal muscular atrophy with congenital bone fractures 2 [OMIM #616867] since 2016. METHODS: A proband from a non-consanguineous Chinese family presented with neonatal severe hypotonia, respiratory distress, muscle weakness, and atrophy, as well as congenital bone fractures was performed by exome sequencing. RESULTS: A compound heterozygosity of a nonsense (c.932C>G,p.Ser311Ter) and an exon 5 deletion in ASCC1 segregating with phenotypes was detected, both variants are novel and pathogenic. Since ASCC1 is a relatively new disease gene, we performed the gene curation by ClinGen SOP. The existing evidence is sufficient to support a "Definitive" level of disease-gene relationship. CONCLUSION: This case report expended the mutation spectrum of ASCC1 and support the notion that this novel disease also occurs in outbreed populations and this is a rare disease but may still be underdiagnosed due to its perinatal lethal outcomes.

Loss of hnRNPA2B1 inhibits malignant capability and promotes apoptosis via down-regulating Lin28B expression in ovarian cancer.

Ovarian cancer has the highest mortality rate among all gynecological cancers with its pathogenic mechanisms largely unknown. Here, we uncovered that ovarian cancer tissues exhibit higher heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) expression than normal ovarian epithelium tissues. Increased hnRNPA2B1 level matches along with poor prognosis of ovarian cancer patients. Importantly, hnRNPA2B1 inhibition hampers growth, reduces mobility of ovarian cancer cells in vitro and hinders xenograft tumor formation in vivo. Transcriptome profiling analysis reveals that hnRNPA2B1 dictates the expression of various important genes involved in tumorigenesis and Lin-28 Homolog B (Lin28B) is down-regulated upon hnRNPA2B1 loss. hnRNPA2B1 regulates expression of Lin28B via binding to Lin28B mRNA and enhancing its stability. Furthermore, knockdown of Lin28B reduces proliferation and mobility of ovarian cancer cells and impairs tumorigenesis in vivo, whereas Lin28B overexpression promotes xenograft tumor formation. Finally, re-expression of Lin28B in hnRNPA2B1 knockdown cells results in rescued phenotypes. Collectively, our results demonstrate that hnRNPA2B1 facilitates the malignant phenotype of ovarian cancer through activating Lin28B expression.

Prenatal diagnosis of cri-du-chat syndrome by SNP array: report of twelve cases and review of the literature.

BACKGROUND: Cri-du-chat syndrome (CdCS; OMIM#123450) is a classic contiguous gene syndrome caused by chromosome 5p terminal deletion (5p-), which characterized by a high-pitched cat-like cry, developmental delay, severe psychomotor, mental retardation, and dysmorphic features in infancy. Prenatal diagnosis of CdCS is difficult due to the non-specific ultrasound features. And reports using array analysis are rare. This study presented the first retrospective analysis of prenatal series of CdCS fetuses diagnosed by single nucleotide polymorphism (SNP) array in China. CASE PRESENTATION: A total of 35,233 pregnant women were enrolled from Jan 2014 to April 2019 in our center, there are twelve 5p- cases with abnormal sonographic signs revealed by SNP array, giving an incidence of 0.034% (12/35,233). Clinical information and molecular basis included: maternal demographics, indications for invasive testing, sonographic findings and SNP array results. Among all the 5p- cases revealed, nine cases were diagnosed by both karyotyping and SNP array, three cases were detected only by SNP array. Half of our cases (6/12) had an isolated 5p terminal deletion, which sizes ranged from 9.0 Mb to 30 Mb. The other half of cases (6/12) characterized by unbalanced translocation, with sex ratio 7:5 (female: male), when combine the clinical features observed from this study and available literature, the most frequent anomaly observed in prenatal ultrasound examination of CdCS was cerebral abnormalities, accounted for 44.4% (16/36) of the existing cases. Features that are less consistent included: choroid plexus cyst (13.8%, 5/36), single umbilical artery (13.3%, 4/30), ventricular septal defect (11.1%, 4/36), hydrops fetalis (8.3%, 3/36), ascites (8.3%, 3/36), increased NT/NF (8.3%, 3/36), absent/severely hypoplastic nasal bone (5.5%, 2/36), in order. CONCLUSION: Prenatal findings such as cerebral abnormalities, absent/hypoplastic nasal bone, hydrops fetalis, ascites or encephalocele may act as suggestive signs of CdCS or other microdeletion/duplication syndromes. Combining typical karyotyping with chromosomal microarray analysis (CMA) is a definitive method for a precise diagnosis of CdCS and provides more accurate results in order to offer genetic counseling to families which need to deal with cryptic aberrations.

Using a Large Margin Context-Aware Convolutional Neural Network to Automatically Extract Disease-Disease Association from Literature: Comparative Analytic Study.

BACKGROUND: Research on disease-disease association (DDA), like comorbidity and complication, provides important insights into disease treatment and drug discovery, and a large body of the literature has been published in the field. However, using current search tools, it is not easy for researchers to retrieve information on the latest DDA findings. First, comorbidity and complication keywords pull up large numbers of PubMed studies. Second, disease is not highlighted in search results. Finally, DDA is not identified, as currently no disease-disease association extraction (DDAE) dataset or tools are available. OBJECTIVE: As there are no available DDAE datasets or tools, this study aimed to develop (1) a DDAE dataset and (2) a neural network model for extracting DDA from the literature. METHODS: In this study, we formulated DDAE as a supervised machine learning classification problem. To develop the system, we first built a DDAE dataset. We then employed two machine learning models, support vector machine and convolutional neural network, to extract DDA. Furthermore, we evaluated the effect of using the output layer as features of the support vector machine-based model. Finally, we implemented large margin context-aware convolutional neural network architecture to integrate context features and convolutional neural networks through the large margin function. RESULTS: Our DDAE dataset consisted of 521 PubMed abstracts. Experiment results showed that the support vector machine-based approach achieved an F1 measure of 80.32%, which is higher than the convolutional neural network-based approach (73.32%). Using the output layer of convolutional neural network as a feature for the support vector machine does not further improve the performance of support vector machine. However, our large margin context-aware-convolutional neural network achieved the highest F1 measure of 84.18% and demonstrated that combining the hinge loss function of support vector machine with a convolutional neural network into a single neural network architecture outperforms other approaches. CONCLUSIONS: To facilitate the development of text-mining research for DDAE, we developed the first publicly available DDAE dataset consisting of disease mentions, Medical Subject Heading IDs, and relation annotations. We developed different conventional machine learning models and neural network architectures and evaluated their effects on our DDAE dataset. To further improve DDAE performance, we propose an large margin context-aware-convolutional neural network model for DDAE that outperforms other approaches.

PGRMC1 Is a Novel Potential Tumor Biomarker of Human Renal Cell Carcinoma Based on Quantitative Proteomic and Integrative Biological Assessments.

Progesterone receptor membrane component 1 (PGRMC1) is widely observed with an elevated level in multiple human cancers. However, the roles of PGRMC1 in renal cancer are not clear and merit further study. In this report, we made a systematic, integrative biological assessment for PGRMC1 in renal cell carcinoma (RCC) by a quantitative proteomic identification, immunohistochemical detection, and its clinic pathologic significance analysis. We found that PGRMC1 abundance is increased by 3.91-fold in RCC tissues compared with its autologous para-cancerous tissues by a quantitative proteome identification. To validate the proteomic result with more confidence, 135 clinic RCC tissues were recruited to measure PGRMC1 abundance by immunohistochemical staining, and 63.7% RCC samples (n = 86) showed a higher abundance of PGRMC1 than the noncancerous counterparts. And the elevated PGRMC1 level was related to the tumor malignancy degree and overall survival of RCC patients. Meanwhile the average serum PGRMC1 concentration for RCC patients (n = 18) was significantly increased by 1.67 fold compared with healthy persons. Moreover an exogenous elevated abundance of PGRMC1 by plasmid transfections significantly enhanced cell proliferation of renal cancer cells in vitro. Our findings demonstrate PGRMC1, which promotes RCC progression phenotypes in vitro and in vivo, is a novel potential biomarker and therapeutic target for RCC.

Downregulation of ARHGDIA contributes to human glioma progression through activation of Rho GTPase signaling pathway.

The protein ARHGDIA has been found to play distinct roles in cancer progression for several tumors. However, it remains elusive whether and how ARHGDIA plays functions in human glioma. In this study, we discovered that ARHGDIA is much downregulated in human glioma; meanwhile, its expression negatively correlates with glioma malignancy and positively relates to prognosis of glioma patients. It has independent predictive value of ARHGDIA expression level for overall survival of human glioma patients. Glioma patients with ARHGDIA-positive expression have a longer overall survival time than ARHGDIA-negative patients. Knockdown of ARHGDIA promotes cell proliferation, cell cycle progression, and cell migration due to the activation of Rho GTPases (Rac1, Cdc42, and RhoA) and Akt phosphorylation, whereas overexpression of ARHGDIA suppresses cell growth, cell cycle progression, and cell migration. ARHGDIA is a potential prognostic marker and therapeutic target for human glioma.

The Overexpression of IQGAP1 and ß-Catenin Is Associated with Tumor Progression in Hepatocellular Carcinoma In Vitro and In Vivo.

The IQ-domain GTPase-activating protein 1 (IQGAP1) is a multifunctional scaffold protein, which interacts with diverse proteins to regulate cell adhesion and cell migration. The abnormal expression of IQGAP1 widely exists in many cancers, but biological roles of IQGAP1 cooperation with its interacting proteins to involve in tumorigenesis remain to clarify. In this study, we have found that IQGAP1 interacts with ß-catenin and regulates ß-catenin expression in hepatocellular carcinoma (HCC) cells. The expression levels of IQGAP1 and ß-catenin and their associations have a positive correlation with cell metastasis ability in several HCC cell lines. The up-regulation of IQGAP1 and ß-catenin improves cell proliferation and migration ability of HCC cells, whereas the knockdown of IQGAP1 by small interfering RNA can decrease ß-catenin expression, which results in the reduction of cell proliferation and migration ability in vitro. In addition, a significantly higher expression of IQGAP1 and ß-catenin also usually exists in human HCC tissues, especially their overexpression is clinicopathologically associated with tumor malignancy. Generally the overexpression and interactions of IQGAP1 and ß-catenin contribute to HCC progression by promoting cell proliferation and migration.

MicroRNA-26a regulates glucose metabolism by direct targeting PDHX in colorectal cancer cells.

BACKGROUND: Reprogramming energy metabolism has been an emerging hallmark of cancer cells. MicroRNAs play important roles in glucose metabolism. METHODS: The targets of microRNA-26a (miR-26a) were predicted by bioinformatics tools. The efficacy of miR-26a binding the 3'-untranslated region (UTR) of pyruvate dehydrogenase protein X component (PDHX) mRNA was evaluated using a dual-luciferase reporter assay. The PDHX expression at the mRNA and protein level in several colon cancer cell lines was quantified with real-time PCR and Western blot analysis respectively. The effects of miR-26a on glucose metabolism were determined by detecting the content of glucose consumption, production of lactate, pyruvate, and acetyl-coenzyme A. RESULTS: The expression of miR-26a is inversely associated with the level of its targeting protein PDHX in several colon cancer cell lines with different malignancy potentials. MiR-26a inhibits PDHX expression by direct targeting the 3'-UTR of PDHX mRNA. The glucose consumption and lactate concentration were both greatly increased in colon cancer cells than the normal colon mucosal epithelia under physiological conditions. The overexpression of miR-26a in HCT116 cells efficiently improved the accumulation of pyruvate and decreased the production of acetyl coenzyme A. Meanwhile the inhibition of miR-26a expression induced inverse biological effects. CONCLUSIONS: MiR-26a regulates glucose metabolism of colorectal cancer cells by direct targeting the PDHX, which inhibits the conversion of pyruvate to acetyl coenzyme A in the citric acid cycle.