CircPRDM5 inhibits the proliferation, migration, invasion, and glucose metabolism of gastric cancer cells by reducing GCNT4 expression in a miR-485-3p-dependent manner.

Circular RNA (circRNA) plays a key part in the pathological process of gastric cancer (GC). The study is organized to analyze the function of circPRDM5 in GC cell tumor properties. Expression levels of circPRDM5, miR-485-3p, glucosaminyl (N-acetyl) transferase 4 (GCNT4), ki67, E-cadherin, N-cadherin, and hexokinase 2 (HK2) were analyzed by quantitative real-time polymerase chain reaction (PCR), Western blotting or immunohistochemistry assay. Cell proliferation was assessed by cell colony formation assay and 5-ethynyl-2'-deoxyuridine assay. Cell migration and invasion were investigated by transwell assay. Glycolysis was evaluated by the Seahorse XF Glycolysis Stress Test Kit. Dual-luciferase reporter assay and RNA pull-down assay were performed to identify the associations among circPRDM5, miR-485-3p, and GCNT4. Xenograft mouse model assay was conducted to determine the effects of circPRDM5 on tumor formation in vivo. CircPRDM5 and GCNT4 expression were downregulated, while miR-485-3p expression was upregulated in GC tissues and cells when compared with paracancerous tissues or human gastric epithelial cells. CircPRDM5 overexpression inhibited proliferation, migration, invasion, and glucose metabolism of GC cells; however, circPRDM5 depletion had the opposite effects. CircPRDM5 repressed tumor properties of GC cells in vivo. MiR-485-3p restoration relieved circPRDM5-induced effects in GC cells. GCNT4 overexpression remitted the promoting effects of miR-485-3p mimics on GC cell malignancy. CircPRDM5 acted as a sponge for miR-485-3p, and GCNT4 was identified as a target gene of miR-485-3p. Moreover, circPRDM5 regulated GCNT4 expression by interacting with miR-485-3p.CircPRDM5 acted as a miR-485-3p sponge to inhibit GC progression by increasing GCNT4 expression, proving a potential target for GC therapy.

Copy number variants landscape of multiple cancers and clinical applications based on NGS gene panel.

BACKGROUND: The rapid adoption of next-generation sequencing in clinical oncology has enabled detection of molecular biomarkers which are shared between multiple tumour types. Intra-tumour heterogeneity is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the tumour-related copy number variants (CNVs), as key regulators of cancer origination, development, and progression, across various types of cancers are poorly understood. METHODS: We performed pan-cancer CNV analysis of cancer-related genes in 15 types of cancers including 1438 cancerous patients by next-generation sequencing using a commercially available pan-cancer panel (Onco PanScan™). Downstream bioinformatics analysis was performed in order to detect CNVs, cluster analysis of the found CNVs, and comparison of the frequency of gained CNVs between different types of cancers. LASSO analysis was used for identification of the most important CNVs. RESULTS: We also identified 523 CNVs among which 16 CNVs were common while 22 CNVs were caner-specific CNVs. Meanwhile, FAM58A was most commonly found in all studied cancers in this study and significant differences were found in FAM58A between female and male patients (p = .001). Common CNVs, such as FOXA1, NFKBIA, HEY1, MECOM, CHD7, AGO2, were mutated in 6.79%, 8.45%, 7.51%, 6.43%, 7.59%, 8.16% of tumours, while most of these mutations have proven roles in positive regulation of transcription from RNA polymerase II promoter. 11 features including sex, DIS3, EPHB1, ERBB2, FLT1, HCK, KEAP1, MYD88, PARP3, TBX3, and TOP2A were found as the key features for classification of cancers using CNVs. CONCLUSION: The 16 common CNVs between cancers can be used to identify the target of pan-cancer drug design and targeted therapies. Additionally, 22 caner-specific CNVs can be used as unique diagnostic markers for each cancer type.

Comprehensive bioinformatics analysis of functional molecules in colorectal cancer.

Background: Colorectal cancer (CRC) is the 3rd most common cancer and the 2nd leading cause of cancer-related death. Numerous studies have found that aberrations in cellular molecules play an important role in the development of tumors. Studying and determining the interactions between these molecules can contribute to the diagnosis, treatment, and prognosis of tumors. Methods: The GSE151021, GSE156720, and GSE156719 data sets were analyzed to screen the differentially expressed messenger RNAs (DEmRNAs), long non-coding RNAs (DElncRNAs), and microRNAs (DEmiRNAs) in CRC. Database for Annotation, Visualization and Integrated Discovery (DAVID) and the Search Tool for the Retrieval of Interacting Genes/Proteins software were used to examine gene enrichment and the hub genes. Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and UALCAN was used to verify the expression of the hub genes. To analyze the overall survival (OS) of the hub genes, Kaplan-Meier plotter (KM plotter) was performed. Finally, the miRCancer database, TargetScan, and GSE156719 were used to identify the targets of the identified miRNAs. To predict the lncRNA-miRNA interactions, we used DIANA-LncBase v2 and GSE156720. Finally, the visualization protein­protein interaction (PPI), competitive endogenous RNA (ceRNA) network was constructed using Cytoscape v3.1. Results: By analyzing GSE151021 and GSE156720, 23 upregulated mRNAs and 10 downregulated mRNAs were identified as sharing the differentially expressed genes (DEGs) between CRC and adjacent tissues. Furthermore, nucleolar protein 14 (NOP14), the sonic hedgehog (SHH) signaling molecule, phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), the BCL2 apoptosis regulator (BCL2), and zinc finger E-box binding homeobox 2 (ZEB2) were considered hub genes. The constructed lncRNA-miRNA-mRNA network revealed 7 intersecting miRNAs (4 upregulated and 3 downregulated), 79 lncRNAs (40 upregulated and 39 downregulated), and 5 mRNAs (3 upregulated and 2 downregulated). Finally, we determined that the dysregulation of lncRNAs, such as HCG16, CASC9, SNHG16, HAND2-AS1, and NR2F1-AS1, secluded altered the expression of several miRNAs, such as hsa-miR-193a-5p, hsa-miR-485-5p, hsa-miR-17-5p, and hsa-miR-92a-3p, and affected the occurrence and development of CRC. Conclusions: We identified a series of DElncRNAs, DEmRNAs, and DEmiRNAs in CRC that might be considered potential biomarkers in understanding the complex molecular pathways leading to CRC development.

Serum metabolomic and lipidomic profiling identifies diagnostic biomarkers for seropositive and seronegative rheumatoid arthritis patients.

BACKGROUND: Diagnosing seronegative rheumatoid arthritis (RA) can be challenging due to complex diagnostic criteria. We sought to discover diagnostic biomarkers for seronegative RA cases by studying metabolomic and lipidomic changes in RA patient serum. METHODS: We performed comprehensive metabolomic and lipidomic profiling in serum of 225 RA patients and 100 normal controls. These samples were divided into a discovery set (n = 243) and a validation set (n = 82). A machine-learning-based multivariate classification model was constructed using distinctive metabolites and lipids signals. RESULTS: Twenty-six metabolites and lipids were identified from the discovery cohort to construct a RA diagnosis model. The model was subsequently tested on a validation set and achieved accuracy of 90.2%, with sensitivity of 89.7% and specificity of 90.6%. Both seropositive and seronegative patients were identified using this model. A co-occurrence network using serum omics profiles was built and parsed into six modules, showing significant association between the inflammation and immune activity markers and aberrant metabolism of energy metabolism, lipids metabolism and amino acid metabolism. Acyl carnitines (20:3), aspartyl-phenylalanine, pipecolic acid, phosphatidylethanolamine PE (18:1) and lysophosphatidylethanolamine LPE (20:3) were positively correlated with the RA disease activity, while histidine and phosphatidic acid PA (28:0) were negatively correlated with the RA disease activity. CONCLUSIONS: A panel of 26 serum markers were selected from omics profiles to build a machine-learning-based prediction model that could aid in diagnosing seronegative RA patients. Potential markers were also identified in stratifying RA cases based on disease activity.

A Chinese family with Noonan syndrome caused by a heterozygous variant in LZTR1: a case report and literature review.

BACKGROUND: Noonan syndrome is an inherited disease involving multiple systems. More than 15 related genes have been discovered, among which LZTR1 was discovered recently. However, the pathogenesis and inheritance pattern of LZTR1 in Noonan syndrome have not yet been elucidated. CASE PRESENTATION: We herein describe a family with LZTR1-related Noonan syndrome. In our study, the proband, sister, mother, maternal aunt and grandmother and female cousin showed the typical or atypical features of Noonan syndrome. Only 3 patients underwent the whole-exome sequencing analysis and results showed that the proband as well as her sister inherited the same heterozygous LZTR1 variant (c.1149 + 1G > T) from their affected mother. Moreover, the proband accompanied by growth hormone deficiency without other associated variants. CONCLUSION: In a Chinese family with Noonan syndrome, we find that the c.1149 + 1G > T variant in LZTR1 gene shows a different autosomal dominant inheritance from previous reports, which changes our understanding of its inheritance and improves our understanding of Noonan syndrome.

Analysis of a Chinese Pedigree With Familial Chylomicronemia Syndrome Reveals Two Novel LPL Mutations by Whole-Exome Sequencing.

Familial chylomicronemia syndrome (FCS) is a rare monogenic autosomal recessive disease caused by loss-of-function mutations in genes involved in chylomicron breakdown through hydrolysis of triglycerides into free fatty acids. Patients are often diagnosed in early childhood with extremely high triglyceride levels and symptoms including abdominal pain, eruptive cutaneous xanthomata, hepatosplenomegaly, and significant cognitive, psychological, and social impairment. The most serious medical condition suffered by FCS patients is recurrent acute pancreatitis. Lipoprotein lipase (LPL) gene mutation accounts for majority of the known pathogenic mutations. Early diagnosis and strict low-fat diet are critical for successful management of the triglyceride concentration to lower the risk of pancreatitis. The true prevalence of FCS in China is unknown and here we report a Chinese female preterm neonate presented with an extremely high triglyceride level of 22.11 mmol/L on day 13 after birth. Clinical and laboratory workup including whole-exome sequencing revealed two novel compound heterozygous LPL mutations (c.406G > C and c.829G > C) that are co-segregated with her non-consanguineous parents, consistent with autosomal recessive inheritance. A diagnosis of FCS based on clinical, biochemical, and genetic ground was made to guide her management.

CPVA: a web-based metabolomic tool for chromatographic peak visualization and annotation.

MOTIVATION: Liquid chromatography-mass spectrometry-based non-targeted metabolomics is routinely performed to qualitatively and quantitatively analyze a tremendous amount of metabolite signals in complex biological samples. However, false-positive peaks in the datasets are commonly detected as metabolite signals by using many popular software, resulting in non-reliable measurement. RESULTS: To reduce false-positive calling, we developed an interactive web tool, termed CPVA, for visualization and accurate annotation of the detected peaks in non-targeted metabolomics data. We used a chromatogram-centric strategy to unfold the characteristics of chromatographic peaks through visualization of peak morphology metrics, with additional functions to annotate adducts, isotopes and contaminants. CPVA is a free, user-friendly tool to help users to identify peak background noises and contaminants, resulting in decrease of false-positive or redundant peak calling, thereby improving the data quality of non-targeted metabolomics studies. AVAILABILITY AND IMPLEMENTATION: The CPVA is freely available at http://cpva.eastus.cloudapp.azure.com. Source code and installation instructions are available on GitHub: https://github.com/13479776/cpva. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Isolation and whole genome sequencing of fetal cells from maternal blood towards the ultimate non-invasive prenatal testing.

OBJECTIVE: The purpose of this study were to develop a methodology of isolating fetal cells from maternal blood and use deep sequence demonstrating the promise for complete and accurate genetic screening compared to other non-invasive prenatal testing. METHODS: Here in this study, we developed a double negative selection (DNS) procedure to unbiasedly enrich fetal cells. After validated by short tandem repeat (STR), the isolated circulating fetal cells (CFCs) were subjected to deep whole genome sequencing analysis. RESULTS: Our DNS protocol significantly increasing the purity of the mimic fetal cells from 1 in 1 million nucleated cells in whole blood to 1:8 to 1:30 (12.5%-3.33%) after 2 steps of enrichment. Isolated single fetal cell obtained a coverage rate (86.8%) and allelic dropout rate (24.90%) comparative to the reported results of human cell line. Several disease-associated variants were identified in the whole genome sequencing data of isolated CFCs and further confirmed in the sequencing data of unamplified gDNA. CONCLUSION: In conclusion, the robustness of DNS and STR to collect CFCs from peripheral maternal blood for the first time coupled with deep sequencing technique demonstrates the possibility of comprehensive non-invasive prenatal testing of genetic disorders using isolated CFCs.

Syntool: A Novel Region-Based Intolerance Score to Single Nucleotide Substitution for Synonymous Mutations Predictions Based on 123,136 Individuals.

BACKGROUND: Synonymous mutation is the single nucleotide change that does not cause an amino acid change but can affect the rate and efficiency of translation. So recent increase in our knowledge has revealed a substantial contribution of synonymous mutations to human disease risk and other complex traits. Nevertheless, there are still rarely synonymous mutation prediction methods. METHODS: Nonsynonymous and synonymous coding SNPs show similar likelihood and effect size of human disease association. Here we defined synonymous and missense variation as single nucleotide substitution variation. And then we evaluated the intolerance of genic transcripts to single nucleotide substitution variation based on gnomAD 123136 individuals. After regressing all variations on common variations, we defined residuals of regression model as every genomics region intolerance scores. RESULTS: We constructed a total of 24799 nonoverlapped region-based intolerance score by their intolerance to single nucleotide substitution variation (Syntool). The results show that Syntool score can discriminate synonymous disease causing mutations in Human Gene Mutation Database (HGMD Professional) and ClinVar database much better than others. Taken together, this study provides a novel prediction system for synonymous mutations, called Syntool, which could be helpful in identifying candidate synonymous disease causing mutations.

Clinical, biochemical and molecular analysis of two infants with familial chylomicronemia syndrome.

Familial chylomicronemia syndrome (FCS) is a rare autosomal recessive disease due mainly to inherited deficiencies in the proteins or enzymes involved in the clearance of triglycerides from circulation. It usually happens in late childhood and adolescence, which can have serious consequences if misdiagnosed or untreated. In the present study, we investigated two Chinese male babies (A and B), 30d and 48d in age, respectively, who have milky plasma. Clinical, biochemical, and radiological assessments were performed, while samples from the patients were referred for molecular diagnosis, including genetic testing and subsequent analysis of related genes. The fasting serum lipids of the two patients showed extreme lipid abnormalities. Through a low-lipid formula diet including skimmed milk and dietary advice, their plasma lipid levels were significantly lower and more stable at the time of hospital discharge. The genetic testing revealed compound heterozygote mutations in the lipoprotein lipase (LPL) gene for patient A and two known compound heterozygote LPL gene mutations for the patient B. FCS is the most dramatic example of severe hypertriglyceridemia. Early diagnosis and timely dietary intervention is very important for affected children.

A comprehensive genetic diagnosis of Chinese muscular dystrophy and congenital myopathy patients by targeted next-generation sequencing.

Muscular dystrophies and congenital myopathies are a large group of heterogeneous inherited muscle disorders. The spectrum of muscular dystrophies and congenital myopathies extends to more than 50 diseases today, even excluding the common forms Duchenne Muscular Dystrophy, Myotonic Dystrophy and Facioscapulohumeral Dystrophy. Unfortunately, even by critical clinical evaluation and muscle pathology, diagnosis is still difficult. To potentially remediate this difficulty, we applied a microarray-based targeted next-generation sequencing (NGS) technology to diagnose these patients. There were 55 consecutive unrelated patients who underwent the test, 36 of which (65%) were found to have a causative mutation. Our result shows the accuracy and efficiency of next-generation sequencing in clinical circumstances and reflects the features and relative distribution of inherited myopathies in the Chinese population.

Integration of targeted sequencing and NIPT into clinical practice in a Chinese family with maple syrup urine disease.

PURPOSE: This article demonstrates a prominent noninvasive prenatal approach to assist the clinical diagnosis of a single-gene disorder disease, maple syrup urine disease, using targeted sequencing knowledge from the affected family. METHODS: The method reported here combines novel mutant discovery in known genes by targeted massively parallel sequencing with noninvasive prenatal testing. RESULTS: By applying this new strategy, we successfully revealed novel mutations in the gene BCKDHA (Ex2_4dup and c.392A>G) in this Chinese family and developed a prenatal haplotype-assisted approach to noninvasively detect the genotype of the fetus (transmitted from both parents). CONCLUSION: This is the first report of integration of targeted sequencing and noninvasive prenatal testing into clinical practice. Our study has demonstrated that this massively parallel sequencing-based strategy can potentially be used for single-gene disorder diagnosis in the future.

A novel mutation of the SLC25A13 gene in a Chinese patient with citrin deficiency detected by target next-generation sequencing.

Type II citrullinaemia, also known as citrin deficiency, is an autosomal recessive metabolic disorder, which is caused by pathogenic mutations in the SLC25A13 gene on chromosome 7q21.3. One of the clinical manifestations of type II citrullinaemia is neonatal intrahepatic cholestatic hepatitis caused by citrin deficiency (NICCD, OMIM# 605814). In this study, a 5-month-old female Chinese neonate diagnosed with type II citrullinaemia was examined. The diagnosis was based on biochemical and clinical findings, including organic acid profiling using a gas chromatography mass spectrometry (GC/MS), and the patient's parents were unaffected. Approximately 14 kb of the exon sequences of the SLC25A13 and two relative genes (ASS1 and FAH) from the proband and 100 case-unrelated controls were captured by array-based capture method followed by high-throughput next-generation sequencing. Two single-nucleotide mutations were detected in the proband, including the previous reported c.1177+1G>A mutation and a novel c.754 G>A mutation in the SLC25A13 gene. Sanger sequence results showed that the patient was a compound heterozygote for the two mutations. The novel mutation (c.754 G>A), which is predicted to affect the normal structure and function of citrin, is a candidate pathogenic mutation. Target sequence capture combined with high-throughput next-generation sequencing technologies is proven to be an effective method for molecular genetic testing of type II citrullinaemia.

Targeted next-generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD: a multi-population diagnostic study.

Duchenne and Becker muscular dystrophies (DMD/BMD) are the most commonly inherited neuromuscular disease. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of the dystrophin gene and complex causative mutation spectrum. Such traditional methods as multiplex ligation-dependent probe amplification plus Sanger sequencing require multiple steps to fulfill the diagnosis of DMD/BMD. Here, we introduce a new single-step method for the genetic analysis of DMD patients and female carriers in real clinical settings and demonstrate the validation of its accuracy. A total of 89 patients, 18 female carriers and 245 non-DMD patients were evaluated using our targeted NGS approaches. Compared with traditional methods, our new method yielded 99.99% specificity and 98.96% sensitivity for copy number variations detection and 100% accuracy for the identification of single-nucleotide variation mutations. Additionally, this method is able to detect partial deletions/duplications, thus offering precise personal DMD gene information for gene therapy. We detected novel partial deletions of exons in nine samples for which the breakpoints were located within exonic regions. The results proved that our new method is suitable for routine clinical practice, with shorter turnaround time, higher accuracy, and better insight into comprehensive genetic information (detailed breakpoints) for ensuing gene therapy.

A novel missense mutation of FGFR3 in a Chinese female and her fetus with Hypochondroplasia by next-generation sequencing.

BACKGROUND: Hypochondroplasia (HCH) is a mild, autosomal dominant human skeletal dysplasias characterized by short extremities, short stature and lumbar lordosis. There are three other kinds of dwarfism (Pseudoachondroplasia, Achondroplasia and Thanatophoric Syndromes) with similar clinical features, which makes it difficult to give a precise diagnosis. Molecular genetic analysis of related genes should be employed. METHODS: In this study, we reported a Chinese family diagnosed as a type of skeletal dysplasia based on clinical and radiologic findings. To make an accurate diagnosis quickly and economically, we performed microarray-based next-generation sequencing (NGS) to detect the variants in the disease-related genes (FGFR3 and COMP). RESULTS: The mother presents short limbed stature, short iliac bones, short femoral necks, short stubby tibia and mildly increased fibular length and genu varum. Her fetus demonstrated abnormally short femur at 23 and 28week's gestation by ultrasound scan, and was highly suspected with dwarfism. Eventually, a novel missense mutation (c.1024G>T) in FGFR3 was identified by next-generation sequencing. The substitution is found in both the mother and her fetus. The mutation was further confirmed by Sanger sequencing. CONCLUSIONS: This is the first report of missense mutation identified in the IgIII domain of the FGFR3 gene using NGS. Our results extended the mutational spectrum of FGFR3 and proved that applications of NGS and bioinformatics are effective methods for skeletal dysplasia diagnosis in clinical practices.

Genetic diagnosis of autosomal dominant polycystic kidney disease by targeted capture and next-generation sequencing: utility and limitations.

Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by genetic and allelic heterogeneity, large multi-exon genes, and duplication sequences of PKD1. Recently, targeted resequencing by pooling long-range polymerase chain reaction (LR-PCR) amplicons has been used in the identification of mutations in ADPKD. Despite its high sensitivity, specificity and accuracy, LR-PCR is still complicated. We performed whole-exome sequencing on two unrelated typical Chinese ADPKD probands and evaluated the effectiveness of this approach compared with Sanger sequencing. Meanwhile, we performed targeted gene and next-generation sequencing (targeted DNA-HiSeq) on 8 individuals (1 patient from one family, 5 patients and 2 normal individuals from another family). Both whole-exome sequencing and targeted DNA-HiSeq confirmed c.11364delC (p.H3788QfsX37) within the unduplicated region of PKD1 in one proband; in the other family, targeted DNA-HiSeq identified a small insertion, c.401_402insG (p.V134VfsX79), in PKD2. These methods do not overcome the screening complexity of homology. However, the true positives of variants confirmed by targeted gene and next-generation sequencing were 69.4%, 50% and 100% without a false positive in the whole coding region and the duplicated and unduplicated regions, which indicated that the screening accuracy of PKD1 and PKD2 can be largely improved by using a greater sequencing depth and elaborate design of the capture probe.

Analysis of a Chinese pedigree with Zellweger syndrome reveals a novel PEX1 mutation by next-generation sequencing.

BACKGROUND: Autosomal recessive Zellweger spectrum disorder (ZSD), the main subgroup of the peroxisome biogenesis disorders (PBDs), can be caused by mutations in any of the 13 PEX genes. Zellweger syndrome (ZS) is the most common and severe phenotype in the heterogeneous ZSD. For the large number genes involved, it is difficult to make a precise genetic diagnosis by traditional methods at a time. A combination of enrichment of targeted genes and next-generation sequencing (NGS) would result in both high efficiency and low cost for targeted sequencing of genes of interest. METHODS: To identify potential mutations in a Chinese family associated with Zellweger syndrome, 1930kb of all the targeted region of PEX genes were captured and sequenced using NGS. We also performed Sanger sequencing to validate the NGS results. RESULTS: Here, we reported a Chinese patient diagnosed as a severe classic type of PBD based on a clinical investigation. We then performed microarray-based NGS to detect the variants in PEX genes of the whole family. One reported heterozygosis mutation (c.782_783delAA) was identified in the patient's father and one novel heterozygosis missense mutation (c.475G>C) was found in the patient's mother, the patient inherited both mutations. CONCLUSIONS: The results proved that the application of target sequence capture using chip and high-throughput NGS is a valuable tool for the molecular diagnosis of peroxisome biogenesis disorders. The accuracy, high-throughput and speed of the method make it suitable for clinical application.

Detection of truncated dystrophin lacking the C-terminal domain in a Chinese pedigree by next-generation sequencing.

Dystrophin (DMD) gene is the largest gene containing 79 exons involving various mutation types and regions, and targeted next-generation sequencing (NGS) was employed in detecting DMD gene mutation in the present study. A literature-annotated disease nonsense mutation (c.10141C>T, NM_004006.1) in exon 70 that has been reported as Duchenne Muscular Dystrophy (DMD)-causing mutation was found in our two patients, the proband and his cousin. In the present study two main methods were used, the next-generation sequencing and the classic Sanger sequencing. The exon capture followed by HiSeq2000 sequencing was specifically used in this study. Combined applications of the next-generation sequencing platform and bioinformatics are proved to be effective methods for DMD diagnosis.

Identification of sequence variants in genetic disease-causing genes using targeted next-generation sequencing.

BACKGROUND: Identification of gene variants plays an important role in research on and diagnosis of genetic diseases. A combination of enrichment of targeted genes and next-generation sequencing (targeted DNA-HiSeq) results in both high efficiency and low cost for targeted sequencing of genes of interest. METHODOLOGY/PRINCIPAL FINDINGS: To identify mutations associated with genetic diseases, we designed an array-based gene chip to capture all of the exons of 193 genes involved in 103 genetic diseases. To evaluate this technology, we selected 7 samples from seven patients with six different genetic diseases resulting from six disease-causing genes and 100 samples from normal human adults as controls. The data obtained showed that on average, 99.14% of 3,382 exons with more than 30-fold coverage were successfully detected using Targeted DNA-HiSeq technology, and we found six known variants in four disease-causing genes and two novel mutations in two other disease-causing genes (the STS gene for XLI and the FBN1 gene for MFS) as well as one exon deletion mutation in the DMD gene. These results were confirmed in their entirety using either the Sanger sequencing method or real-time PCR. CONCLUSIONS/SIGNIFICANCE: Targeted DNA-HiSeq combines next-generation sequencing with the capture of sequences from a relevant subset of high-interest genes. This method was tested by capturing sequences from a DNA library through hybridization to oligonucleotide probes specific for genetic disorder-related genes and was found to show high selectivity, improve the detection of mutations, enabling the discovery of novel variants, and provide additional indel data. Thus, targeted DNA-HiSeq can be used to analyze the gene variant profiles of monogenic diseases with high sensitivity, fidelity, throughput and speed.