Integrated analysis reveals FLI1 regulates the tumor immune microenvironment via its cell-type-specific expression and transcriptional regulation of distinct target genes of immune cells in breast cancer.

BACKGROUND: Immunotherapy is a practical therapeutic approach in breast cancer (BRCA), and the role of FLI1 in immune regulation has gradually been unveiled. However, the specific role of FLI1 in BRCA was conflicted; thus, additional convincing evidence is needed. METHODS: We explored the upstream regulation of FLI1 expression via summary data-based Mendelian randomization (SMR) analysis and ncRNA network construction centering on FLI1 using BRCA genome-wide association study (GWAS) summary data with expression quantitative trait loci (eQTLs) and DNA methylation quantitative trait loci (mQTLs) from the blood and a series of in silico analyses, respectively. We illuminated the downstream function of FLI1 in immune regulation by integrating a series of analyses of single-cell RNA sequence data (scRNA-seq). RESULTS: We verified a causal pathway from FLI1 methylation to FLI1 gene expression to BRCA onset and demonstrated that FLI1 was downregulated in BRCA. FLI1, a transcription factor, served as myeloid and T cells' communication regulator by targeting immune-related ligands and receptor transcription in BRCA tissues. We constructed a ceRNA network centering on FLI1 that consisted of three LncRNAs (CKMT2-AS1, PSMA3-AS1, and DIO3OS) and a miRNA (hsa-miR-324-5p), and the expression of FLI1 was positively related to a series of immune-related markers, including immune cell infiltration, biomarkers of immune cells, and immune checkpoints. CONCLUSION: Low-methylation-induced or ncRNA-mediated downregulation of FLI1 is associated with poor prognosis, and FLI1 might regulate the tumor immune microenvironment via a cell-type-specific target genes manner in BRCA.

Novel compound heterozygous variants in ARL13B lead to Joubert syndrome.

Joubert syndrome (JBTS) is a systematic developmental disorder mainly characterized by a pathognomonic mid-hindbrain malformation. All known JBTS-associated genes encode proteins involved in the function of antenna-like cellular organelle, primary cilium, which plays essential roles in cellular signal transduction and development. Here, we identified four unreported variants in ARL13B in two patients with the classical features of JBTS. ARL13B is a member of the Ras GTPase family and functions in ciliogenesis and cilia-related signaling. The two missense variants in ARL13B harbored the substitutions of amino acids at evolutionarily conserved positions. Using model cell lines, we found that the accumulations of the missense variants in cilia were impaired and the variants showed attenuated functions in ciliogenesis or the trafficking of INPP5E. Overall, these findings expanded the ARL13B pathogenetic variant spectrum of JBTS.

The Association of Early Menopause with Increased Risk of Acute Myocardial Infarction: The INTERHEART China Study.

Background and Aim: Little is known about whether early menopause in Chinese ethnicity is associated with acute myocardial infarction (AMI). We aimed to determine whether self-reported early menopause (either surgical or natural menopause at an age <50 year) was associated with first AMI in Chinese women. Methods: The study population was from the INTERHEART China Study, part of the INTERHEART global study. INTERHEART global study was a standardized case-control study that was designed to evaluate the risk factors for first AMI among 52 countries. Data for demographic factors, education, income, and cardiovascular risk factors were obtained by structured questionnaires. A standard set of questions that inquired about menstrual history was included in the interview. Results: Of the 1,771 Chinese women, 1,563 (88.3%) reported either natural or surgical menopause. In univariate logistic regression model, women with early menopause had higher risk of AMI (odds ratio [OR]: 1.51; 95% confidence interval [CI]: 1.23-1.87). After controlling for age, birth control measures, type of menopause, and other traditional risk factors (including waist/hip ratio, lifestyle factors, history of hypertension and diabetes, psychosocial factors, and apolipoprotein B [ApoB]/A1 [ApoA1]), the risk for AMI remained (OR: 1.36; 95% CI: 1.03-1.79). The population attributable risk for AMI in women with early menopause at <50 years was 10.1% (95% CI: 4.0-20.0) compared with women who had menopause at ≥50 years. Conclusion: Early menopause is associated with increased risk of AMI in Chinese women, independent of other traditional coronary heart disease risk factors.

A novel 1.38-kb deletion combined with a single nucleotide variant in KIAA0586 as a cause of Joubert syndrome.

BACKGROUND: KIAA0586, also known as Talpid3, plays critical roles in primary cilia formation and hedgehog signaling in humans. Variants in KIAA0586 could cause some different ciliopathies, including Joubert syndrome (JBTS), which is a clinically and genetically heterogeneous group of autosomal recessive neurological disorders. METHODS AND RESULTS: A 9-month-old girl was diagnosed as JBTS by the "molar tooth sign" of the mid-brain and global developmental delay. By whole-exome sequencing, we identified a single nucleotide variant c.3303G > A and a 1.38-kb deletion in KIAA0586 in the proband. These two variants of KIAA0586 were consistent with the mode of autosomal recessive inheritance in the family, which was verified using Sanger sequencing. CONCLUSIONS: This finding of a compound heterozygote with a 1.38-kb deletion and c.3303G > A gave a precise genetic diagnosis for the patient, and the novel 1.38-kb deletion also expanded the pathogenic variation spectrum of JBTS caused by KIAA0586.

Clinical and Molecular Features of a Chinese Cohort With Syndromic and Nonsyndromic Retinal Dystrophies Related to the CEP290 Gene.

PURPOSE: To reveal the clinical and genetic features of 54 Chinese pedigrees with syndromic or nonsyndromic retinal dystrophies related to CEP290 and to explore the genotype-phenotype correlation. DESIGN: Retrospective cohort study. METHODS: Patients diagnosed with nonsyndromic inherited retinal dystrophy (IRD) or syndromic ciliopathy (SCP) were enrolled. We identified 61 patients from 54 families carrying biallelic pathogenic CEP290 variants using next-generation sequencing, Sanger sequencing, and co-segregation validation. Genotype-phenotype correlation was evaluated. RESULTS: This study included 37 IRD patients from 32 families and 24 patients with SCP from 22 pedigrees. Four retinal dystrophy phenotypes were confirmed: Leber congenital amaurosis (LCA, 46/61), early-onset severe retinal dystrophy (EOSRD, 4/61), retinitis pigmentosa (RP, 10/61), and cone-rod dystrophy (CORD, 1/61). The SCP phenotypes included Joubert syndrome (JS) (23/24) and Bardet-Biedl syndrome (BBS) (1/24). We detected 73 different CEP290 variants, of which 33 (45.2%) were not previously reported. Two novel copy number variations (CNVs) and 1 novel pathogenic synonymous change were identified. The most recurrent alterations in the IRD and SCP were p.Q123* (6/64, 9.4%) and p.I556Ffs*17 (10/44, 22.7%), respectively. IRD patients carried more stop-gain alleles (25/64, 39.1%), whereas SCP patients carried more frameshift alleles (23/44, 52.3%). CONCLUSIONS: LCA was the most common retinal dystrophy phenotype, and JS was the most prevalent syndrome in CEP290 patients; RP/CORD and BBS may be present in early adulthood. The hot spot variants and distribution of genotypes were distinct between IRD and SCP. Our study expands the CEP290 variant spectrum and enhances the current knowledge of CEP290 heterogeneity.

Machine-learning models utilizing CYP3A4*1G show improved prediction of hypoglycemic medication in Type 2 diabetes.

The effectiveness and side effects of Type 2 diabetes (T2D) medication are related to individual genetic background. SNPs CYP3A4 and CYP2C19 were introduced to machine-learning models to improve the performance of T2D medication prediction. Two multilabel classification models, ML-KNN and WRank-SVM, trained with clinical data and CYP3A4/CYP2C19 SNPs were evaluated. Prediction performance was evaluated with Hamming loss, one-error, coverage, ranking loss and average precision. The average precision of ML-KNN and WRank-SVM using clinical data was 92.74% and 92.9%, respectively. Combined with CYP2C19*2*3, the average precision dropped to 88.84% and 89.93%, respectively. While combined with CYP3A4*1G, the average precision was enhanced to 97.96% and 97.82%, respectively. Results suggest that CYP3A4*1G can improve the performance of ML-KNN and WRank-SVM models in predicting T2D medication performance.

About 10% of adults around the world are living with Type 2 Diabetes (T2D). Due to the huge number of patients and the complexity of individual makeup, it is a challenge for doctors to prescribe appropriate hypoglycemic drugs. To aid prescribing, machine-learning models were developed to predict medication schemes based on patients' demographic information and laboratory test results. These models treat prediction as a multilabel classification problem, with each class of medication as a label. This work was designed to determine whether the introduction of genetic information would improve prediction performance. The machine-learning models were trained using datasets with and without genetic information and their performance was compared. The performance of the machine-learning models was improved by incorporating the SNP CYP3A4*1G into the datasets. Thus, this work demonstrates a novel strategy to improve the prediction of T2D hypoglycemic medication performance and provides new ideas for how to support the T2D health system with machine-learning techniques.

Variant analysis of 92 Chinese Han families with hearing loss.

BACKGROUND: Hearing loss (HL) is the most frequent sensory deficit in humans, HL has strong genetic heterogeneity. The genetic diagnosis of HL is very important to aid treatment decisions and to provide prognostic information and genetic counseling for the patient's family. METHODS: We undertook pedigree analysis in 92 Chinese non-syndromic HL patients by targeted next-generation sequencing and Sanger sequencing. RESULTS: Among the 92 HL patients, 18 were assigned a molecular diagnosis with 33 different variants in 14 deafness genes. Eighteen of the variants in 12 deafness genes were novel. Variants in TMC1, CDH23, LOXHD1 and USH2A were each detected in two probands, and variants in POU3F4, OTOA, GPR98, GJB6, TRIOBP, SLC26A4, MYO15A, TNC, STRC and TMPRSS3 were each detected in one proband. CONCLUSION: Our findings expand the spectrum of deafness gene variation, which will inform genetic diagnosis of deafness and add to the theoretical basis for the prevention of deafness.

Novel CRISPR/Cas12a-based genetic diagnostic approach for SLC26A4 mutation-related hereditary hearing loss.

Hereditary hearing loss is a common defect of the auditory nervous system with high-incidence, seriously affecting the quality of life of the patients. The clinical manifestations of SLC26A4 mutation-related hearing loss are congenital sensorineural or mixed deafness. Sensitive and specific SLC26A4 mutation detection in the early clinical stage is key for the early indication of potential hearing loss in the lack of effective treatment. Using clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection technology, we designed a fast and sensitive detection system for SLC26A4 pathogenic mutations (c.919-2A > G, c.2168A > G and c.1229C > T). This recombinase-aided amplification-based detection system allows rapid target gene amplification and, in combination with the CRISPR-based nucleic acid testing (NAT) system, mutation site detection. Moreover, mismatches were introduced in CRISPR-derived RNA (crRNA) to increase signal differences between the wild-type genes and mutant genes. A total of 64 samples were examined using this approach and all results were verified using Sanger sequencing. The detection results were consistent with the polymerase chain reaction-Sanger sequencing results. Overall, this CRISPR-based NAT technology provides a sensitive and fast new approach for the detection of hereditary deafness and provides a crRNA optimization strategy for single-nucleotide polymorphism detection, which could be helpful for the clinical diagnosis of SLC26A4 mutation-related hereditary hearing loss.

Establishment of primary reference measurement procedures and reference materials for EGFR variant detection in non-small cell lung cancer.

Circulating tumor DNA (ctDNA)-based mutation detection is promising to change the clinical practice of genotype-directed therapy for cancer. A growing number of non-invasive tests for cancer screening and monitoring that involve the detection of ctDNA have been commercialized. Primary reference measurement procedures (PRMPs) and reference materials (RMs) are urgently needed to assess the non-invasive tests. In this study, a PRMP based on digital PCR (dPCR) and ctDNA RMs for quantification of the frequently occurring variant in epidermal growth factor receptor (EGFR L858R, T790M, and 19Del) in non-small cell lung cancer (NSCLC) were established. The candidate dPCR PRMP showed high specificity (false positive rate 0-0.003%), good repeatability (coefficient of variance (CV), 2-3% for 104 copies/reaction), and high interlaboratory reproducibility (3-10%). A good linearity (0.97 < slope < 1.03, R2 ≥ 0.9999) between the measured mutant (MU) value and prepared value was observed for all assays over the fractional abundance (FA) range, between 25% and 0.05%. The limit of quantification (LoQ) was determined to be 34 L858R, 23 T790M, and 34 19Del copies/reaction, corresponding to a FA of 0.2%. An inter-laboratory study of using the EGFR ctDNA RMs and dPCR assays demonstrated that the participating laboratories produced consistent concentrations of MU and wild-type (WT), as well as FA. This study demonstrates that dPCR can act as a potential PRMP for EGFR mutation for validation of NSCLC genotyping tests and ctDNA quantitative tests. The PRMP and RMs established here could improve interlaboratory repeatability and reproducibility, which supports rapid translation and application of non-invasive tests into clinical practice.

Disrupted intraflagellar transport due to IFT74 variants causes Joubert syndrome.

PURPOSE: Ciliopathies are a group of disorders caused by defects of the cilia. Joubert syndrome (JBTS) is a recessive and pleiotropic ciliopathy that causes cerebellar vermis hypoplasia and psychomotor delay. Although the intraflagellar transport (IFT) complex serves as a key module to maintain the ciliary structure and regulate ciliary signaling, the function of IFT in JBTS remains largely unknown. We aimed to explore the impact of IFT dysfunction in JBTS. METHODS: Exome sequencing was performed to screen for pathogenic variants in IFT genes in a JBTS cohort. Animal model and patient-derived fibroblasts were used to evaluate the pathogenic effects of the variants. RESULTS: We identified IFT74 as a JBTS-associated gene in three unrelated families. All the affected individuals carried truncated variants and shared one missense variant (p.Q179E) found only in East Asians. The expression of the human p.Q179E-IFT74 variant displayed compromised rescue effects in zebrafish ift74 morphants. Attenuated ciliogenesis; altered distribution of IFT proteins and ciliary membrane proteins, including ARL13B, INPP5E, and GPR161; and disrupted hedgehog signaling were observed in patient fibroblasts with IFT74 variants. CONCLUSION: IFT74 is identified as a JBTS-related gene. Cellular and biochemical mechanisms are also provided.

Identification of two novel pathogenic variants of PIBF1 by whole exome sequencing in a 2-year-old boy with Joubert syndrome.

BACKGROUND: Joubert syndrome (OMIM 213300) is an autosomal recessive disorder with gene heterogeneity. Causal genes and their variants have been identified by sequencing or other technologies for Joubert syndrome subtypes. CASE PRESENTATION: A two-year-old boy was diagnosed with Joubert syndrome by global development delay and molar tooth sign of mid-brain. Whole exome sequencing was performed to detect the causative gene variants in this individual, and the candidate pathogenic variants were verified by Sanger sequencing. We identified two pathogenic variants (NM_006346.2: c.1147delC and c.1054A > G) of PIBF1 in this Joubert syndrome individual, which is consistent with the mode of autosomal recessive inheritance. CONCLUSION: In this study, we identified two novel pathogenic variants in PIBF1 in a Joubert syndrome individual using whole exome sequencing, thereby expanding the PIBF1 pathogenic variant spectrum of Joubert syndrome.

Screening of PAH Common Mutations in Chinese Phenylketonuria Patients Using iPLEX MALDI-TOF MS.

Phenylketonuria (PKU) is caused by phenylalanine hydroxylase (PAH) gene variants. Previous research has identified some PAH mutation hotspots in Chinese patients with PKU. In this study, we introduce a novel MassArray panel for screening the 29 common PAH gene mutations in Chinese patients using iPLEX MALDI-TOF MS. 105 Patients with PKU and known PAH gene mutations were genotyped using this MassArray panel. All of the 29 mutations screened were detected, and MassArray panel results were consistent with those obtained by Sanger sequencing. Fifty patients newly diagnosed with PKU were recruited in the double-blind experiment. PAH gene variants were detected in these 50 patients using the MassArray panel, and the results were verified with Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) methods. Our results show that the mutation detection rate using the MassArray panel with 29 mutations is 74% (95% CI, 65-83%), and the clinical genetic diagnosis rate is 54% (95% CI, 40-68%). This panel can be used as a high throughput, low cost, and rapid method for screening and diagnosing PAH gene mutations. The establishment of this approach provides proof-of-concept for future large-scale PAH mutation carrier screening in areas with high rates of PKU.

Prevention and Control of Pathogens Based on Big-Data Mining and Visualization Analysis.

Morbidity and mortality caused by infectious diseases rank first among all human illnesses. Many pathogenic mechanisms remain unclear, while misuse of antibiotics has led to the emergence of drug-resistant strains. Infectious diseases spread rapidly and pathogens mutate quickly, posing new threats to human health. However, with the increasing use of high-throughput screening of pathogen genomes, research based on big data mining and visualization analysis has gradually become a hot topic for studies of infectious disease prevention and control. In this paper, the framework was performed on four infectious pathogens (Fusobacterium, Streptococcus, Neisseria, and Streptococcus salivarius) through five functions: 1) genome annotation, 2) phylogeny analysis based on core genome, 3) analysis of structure differences between genomes, 4) prediction of virulence genes/factors with their pathogenic mechanisms, and 5) prediction of resistance genes/factors with their signaling pathways. The experiments were carried out from three angles: phylogeny (macro perspective), structure differences of genomes (micro perspective), and virulence and drug-resistance characteristics (prediction perspective). Therefore, the framework can not only provide evidence to support the rapid identification of new or unknown pathogens and thus plays a role in the prevention and control of infectious diseases, but also help to recommend the most appropriate strains for clinical and scientific research. This paper presented a new genome information visualization analysis process framework based on big data mining technology with the accommodation of the depth and breadth of pathogens in molecular level research.

Whole exome sequencing reveals novel CEP104 mutations in a Chinese patient with Joubert syndrome.

BACKGROUND: Joubert syndrome (JS, OMIM: 213300) is a recessive developmental disorder characterized by cerebellar vermis hypoplasia and a distinctive mid-hindbrain malformation called the "molar tooth sign" on axial magnetic resonance imaging. To date, more than 35 ciliary genes have been identified as the causative genes of JS. METHODS: Whole exome sequencing was performed to detect the causative gene mutations in a Chinese patient with JS followed by Sanger sequencing. RT-PCR and Sanger sequencing were used to confirm the abnormal transcript of centrosomal protein 104 (CEP104, OMIM: 616690). RESULTS: We identified two novel heterozygous mutations of CEP104 in the proband, which were c.2364+1G>A and c.414delC (p.Asn138Lysfs*11) (GenBank: NM_014704.3) and consistent with the autosomal recessive inheritance mode. CONCLUSION: Our study reported the fourth case of JS patients with CEP104 mutations, which expands the mutation spectrum of CEP104 and elucidates the clinical heterogeneity of JS.

Evaluation of droplet digital PCR for non-invasive prenatal diagnosis of phenylketonuria.

This study was carried out to establish a non-invasive prenatal diagnosis method for phenylketonuria (PKU) based on droplet digital PCR (ddPCR) and to evaluate its accuracy by comparison with conventional invasive diagnostic methods. A total of 24 PKU pedigrees that required prenatal diagnosis were studied, in which the genetic mutations in the probands and parents were unambiguous. Prenatal diagnosis of sibling fetuses was performed using traditional invasive prenatal diagnostic methods as a standard. At the same time, cell-free DNA (cfDNA) was extracted from maternal plasma and the fetal genes contained within were typed and quantified using ddPCR method. Invasive prenatal diagnosis determined that 3 of the 24 fetuses were affected, 8 of them were normal, and 13 were heterozygous carriers of pathogenic mutations. Successful non-invasive prenatal diagnosis analysis of PAH gene mutations was performed for 8 of the families using ddPCR method. Non-invasive prenatal diagnosis results were consistent with the results of the invasive prenatal diagnoses and no false positive or false negative results were found. In conclusion, this study is the first to establish non-invasive prenatal diagnosis of PKU based on ddPCR. The method showed high sensitivity and specificity from cfDNA, indicating that ddPCR is a reliable non-invasive prenatal diagnosis tool for PKU diagnosis. Graphical abstract.

Mutation spectrum of PAH gene in phenylketonuria patients in Northwest China: identification of twenty novel variants.

This study was performed to analyze the mutational spectrum of the phenylalanine hydroxylase (PAH) gene in phenylketonuria (PKU) patients in Northwest China, to identify mutational hot spots, and to determine the correlation between variants and clinical phenotypes of PKU. A large cohort of 475 PKU families in Northwest China was enrolled to analyze PAH gene variants using Sanger sequencing, Multiplex ligation-dependent probe amplification (MLPA), and gap-PCR. Bioinformatics software was used to predict the pathogenicity of novel variants and analyze the correlations between PAH gene variants and phenotypes of PKU patients. A total of 895 variants were detected in the 950 alleles of 475 patients with PKU (detection rate: 94.21%), 20 of which were novel variants. Other 108, previously known variants, were also identified, with the three most frequent variants being p.Arg243Gln (14.00%), c.611A > G (5.58%), and p.Tyr356* (4.95%). Seven different large deletion/duplication variants were identified by the MLPA method, including the large deletion c.-4163_-406del3758 with high frequency. A correlation analysis between patient phenotype and gene variant frequency showed that p.Arg53His and p.Gln419Arg were correlated with mild hyperphenylalaninemia (MHP). In conclusion, the mutational spectrum underlying PKU in Northwest China was established for the first time. Functional analysis of 20 novel PAH gene variants enriched the PAH gene mutational spectrum. Correlation analysis between variants frequencies in compound heterozygous patients and phenotype severity is helpful for phenotypic prediction.

A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies.

Higher multiplexing in droplet digital PCR (ddPCR) can simplify the detection process of ddPCR-based non-invasive prenatal testing (NIPT) and improve its reliability, making it a practical approach in clinical practice. However, a high level of multiplex ddPCR-based NIPT has rarely been reported. In this study, we developed a multiplex ddPCR assay using universal locked nucleic acid (LNA) probes to reliably identify fetal aneuploidies. We first performed statistical analysis based on the Poisson distribution to evaluate the required number of target DNA molecules and the total number of droplets for a ddPCR assay. Next, we designed two sets of primers and probes to quantify cfDNA from chromosomes 21 and 18 and then determined the disease status of a sample. Finally, we evaluated our multiplex ddPCR assay with 60 clinical plasma samples. All of the 60 clinical samples were correctly identified. The accessibility and cost-effectiveness of our multiplex ddPCR-based NIPT make it a competitive prenatal testing method in clinical use.

Global Profiling of PknG Interactions Using a Human Proteome Microarray Reveals Novel Connections with CypA.

Mycobacterium tuberculosis (Mtb) serine/threonine kinase PknG plays an important role in the Mtb-host interaction by facilitating the survival of Mtb in macrophages. However, the human proteins with which the PknG interacts, and the underlying molecular mechanisms are still largely unknown. In this study, a HuProt array is been applied to globally identify the host proteins to which PknG binds. In this way, 125 interactors are discovered, including a cyclophilin protein, CypA. This interaction between PknG and CypA is validated both in vitro and in vivo, and functional studies show that PknG significantly reduces the protein levels of CypA through phosphorylation, which consequently inhibit the inflammatory response through downregulation of NF-κB and ERK1/2 pathways. Phenotypically, overexpression of PknG reduces cytokine levels and promotes the survival of Mycobacterium smegmatis (Msm) in macrophages. Overall, it is expected that the PknG interactors identified in this study will serve as a useful resource for further systematic studies of the roles that PknG plays in the Mtb-host interactions.