Xiaoming Wei

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.

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.

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.

Next-generation sequencing identifies a novel compound heterozygous mutation in MYO7A in a Chinese patient with Usher Syndrome 1B.

BACKGROUND Targeted enrichment and next-generation sequencing (NGS) have been employed for detection of genetic diseases. The purpose of this study was to validate the accuracy and sensitivity of our method for comprehensive mutation detection of hereditary hearing loss, and identify inherited mutations involved in human deafness accurately and economically. METHODS To make genetic diagnosis of hereditary hearing loss simple and timesaving, we designed a 0.60 MB array-based chip containing 69 nuclear genes and mitochondrial genome responsible for human deafness and conducted NGS toward ten patients with five known mutations and a Chinese family with hearing loss (never genetically investigated). RESULTS Ten patients with five known mutations were sequenced using next-generation sequencing to validate the sensitivity of the method. We identified four known mutations in two nuclear deafness causing genes (GJB2 and SLC26A4), one in mitochondrial DNA. We then performed this method to analyze the variants in a Chinese family with hearing loss and identified compound heterozygosity for two novel mutations in gene MYO7A. CONCLUSIONS The compound heterozygosity identified in gene MYO7A causes Usher Syndrome 1B with severe phenotypes. The results support that the combination of enrichment of targeted genes and next-generation sequencing is a valuable molecular diagnostic tool for hereditary deafness and suitable for clinical application.

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.Genet Med 16 8, 594–600.Genetics in Medicine (2013); 16 8, 594–600. doi:10.1038/gim.2013.197

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 patients father and one novel heterozygosis missense mutation (c.475G>C) was found in the patients 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.

A novel mutation of IDS gene in a Chinese patient with mucopolysaccharidosis II by next-generation sequencing.

BACKGROUND Mucopolysaccharidosis (MPS) is induced by the absence or malfunctioning of lysosomal enzymes. MPS I and MPS II are similar in phenotypes but they are different in genotypes, which are caused by the deficiencies of alpha-L-iduronidase gene (IDUA) and iduronate 2-sulfatase gene (IDS) respectively. In this work, a 5-year-old Chinese young male with manifestations of MPS in a family with unaffected parents was described. METHODS 12 kb of all the targeted exon sequences plus flanking sequences chromosomal DNA of IDS and IDUA genes from the proband and 20 other case-unrelated controls were captured and sequenced by using next-generation sequencing technology. RESULTS One single-nucleotide deletion variant (c.1270delG) resulting in frameshift and premature truncation of I2S enzyme was detected, out of 20 controls, only in the proband, and which was further verified by Sanger sequencing. The probands mother was also proved carrying c.1270delG by Sanger method but not for his father. CONCLUSIONS The novel variant (c.1270delG) is a candidate disease-causing mutation predicted to affect the normal structure and function of the enzyme. Target sequence capture and next-generation sequencing technology can be effective for the gene testing of MPS II disorder.

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 patients 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 for Clinical Diagnosis of 561 Mendelian Diseases

Background Targeted next-generation sequencing (NGS) is a cost-effective approach for rapid and accurate detection of genetic mutations in patients with suspected genetic disorders, which can facilitate effective diagnosis. Methodology/Principal Findings We designed a capture array to mainly capture all the coding sequence (CDS) of 2,181 genes associated with 561 Mendelian diseases and conducted NGS to detect mutations. The accuracy of NGS was 99.95%, which was obtained by comparing the genotypes of selected loci between our method and SNP Array in four samples from normal human adults. We also tested the stability of the method using a sample from normal human adults. The results showed that an average of 97.79% and 96.72% of single-nucleotide variants (SNVs) in the sample could be detected stably in a batch and different batches respectively. In addition, the method could detect various types of mutations. Some disease-causing mutations were detected in 69 clinical cases, including 62 SNVs, 14 insertions and deletions (Indels), 1 copy number variant (CNV), 1 microdeletion and 2 microduplications of chromosomes, of which 35 mutations were novel. Mutations were confirmed by Sanger sequencing or real-time polymerase chain reaction (PCR). Conclusions/Significance Results of the evaluation showed that targeted NGS enabled to detect disease-causing mutations with high accuracy, stability, speed and throughput. Thus, the technology can be used for the clinical diagnosis of 561 Mendelian diseases.