Targeted next-generation sequencing as a comprehensive test for Mendelian diseases: a cohort diagnostic study

Scientific Reports, Aug 2018

With the development of next generation sequencing, more and more common inherited diseases have been reported. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of disease causing mutations. In this study, we introduced a new single-step method for the genetic analysis of patients and carriers in real clinical settings. All kinds of disease causing mutations can be detected at the same time in patients with Mendelian diseases or carriers. First, we evaluated this technology using YH cell line DNA and 9 samples with known mutations. Accuracy and stability of 99.80% and 99.58% were achieved respectively. Then, a total of 303 patients were tested using our targeted NGS approaches, 50.17% of which were found to have deleterious mutations and molecular confirmation of the clinical diagnosis. We identified 219 disease causing mutations, 43.84% (96/219) of which has never been reported before. Additionally, we developed a new deleteriousness prediction method for nonsynonymous SNVs, and an automating annotation and diagnosis system for Mendelian diseases, thus greatly assisting and enhancing Mendelian diseases diagnosis and helping to make a precise diagnosis for patients with Mendelian diseases.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://www.nature.com/articles/s41598-018-30151-z.pdf

Targeted next-generation sequencing as a comprehensive test for Mendelian diseases: a cohort diagnostic study

Abstract With the development of next generation sequencing, more and more common inherited diseases have been reported. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of disease causing mutations. In this study, we introduced a new single-step method for the genetic analysis of patients and carriers in real clinical settings. All kinds of disease causing mutations can be detected at the same time in patients with Mendelian diseases or carriers. First, we evaluated this technology using YH cell line DNA and 9 samples with known mutations. Accuracy and stability of 99.80% and 99.58% were achieved respectively. Then, a total of 303 patients were tested using our targeted NGS approaches, 50.17% of which were found to have deleterious mutations and molecular confirmation of the clinical diagnosis. We identified 219 disease causing mutations, 43.84% (96/219) of which has never been reported before. Additionally, we developed a new deleteriousness prediction method for nonsynonymous SNVs, and an automating annotation and diagnosis system for Mendelian diseases, thus greatly assisting and enhancing Mendelian diseases diagnosis and helping to make a precise diagnosis for patients with Mendelian diseases. Introduction Mendelian diseases are a series of diseases following the principles of Mendelian inheritance. So far, more than 8,000 Mendelian inheritance diseases have been included in Online Mendelian Inheritance in Man (the OMIM database, www.omim.org). Many methods have been performed for the diagnosis of Mendelian diseases in clinical practice, for example, Sanger sequencing of known disease causing genes. As is known, Sanger sequencing is widely considered as the gold standard for sequencing, however, there are some drawbacks in this technology: laborious, expensive, and time consuming. With the rapid development of next generation sequencing (NGS) technology, all kinds of diseasing causing mutations, such as SNVs (single nucleotide variants), Indel (small insertion/deletion) and CNVs (copy number variants), can be detected at the same time in patients with Mendelian diseases or carriers1. It has been proved that NGS based technology is a powerful tool for the detection of pathogenic mutations, especially in patients with monogenic disorders2. Researchers performed NGS to analyze tens to thousands of genes simultaneously in a single assay1,3,4. This makes NGS more suitable in the diagnosis of Mendelian diseases, and highly improved the diagnostic yield5. Comprehensive characterization of genetic diseases using NGS technology has become an option for researchers and clinicians. However, annotation and interpretation of NGS data is tedious and time-consuming, and require highly specific expertise in this filed. Our new method introduced here can help clinical scientists to efficiently integrate all their data more quickly and form a whole picture of the detected variants. WGS (whole genome sequencing) and WES (Whole exome sequencing) have been applied in the diagnostic of Mendelian genetic disorders or the screening of carriers6,7,8,9. However, it is still a major challenge to detect few disease causing mutations in the vast potential variants in human genome10. Targeted NGS is recognized as a cost effective method for the diagnostic of Mendelian genetic disorders1,3,4. Targeted NGS with high throughput and lower cost can target disease related regions in human genome more efficiently, and detect variants more sensitively. This technology has been widely used in the screening of mutations and effective diagnosis of genetic diseases in clinical setting11,12,13,14. What is more, targeted NGS targets a set of genes related to specific disease phenotypes, which could result in a higher sequencing coverage. Variant calling is more accurate for region of interest when performing targeted NGS15. In this study, we designed a chip (array based) containing 4,689 nuclear genes related to Mendelian diseases, and tested its performance in detecting clinical relevant mutations using HiSeq platform. Then, we applied this panel to 303 clinical cases, and identified disease causing mutations in 152 patients. What is more, we developed a new deleteriousness prediction method for nonsynonymous SNVs, and an automating annotation and diagnosis system for Mendelian diseases. It can greatly assist and enhance Mendelian diseases diagnosis and help to make a precise diagnosis for the patients. Results Evaluation of accuracy and stability Before applying this technology to real clinical patients, we evaluated the accuracy and stability of targeted NGS as reported before1. To assess the accuracy of this technology, we compared the SNPs of a YH cell line sample (C1) detected using targeted NGS with the genotyping results detected using Illumina’s Human Zhonghua-8 Bead Chips (SNP Array). The analysis of C1 using SNP Array was carried out blindly. In C1, there are a to (...truncated)


This is a preview of a remote PDF: https://www.nature.com/articles/s41598-018-30151-z.pdf

Yan Sun, Jianfen Man, Yang Wan, Gao Pan, Lique Du, Long Li, Yun Yang, Liru Qiu, Qing Gao, Handong Dan, Liangwei Mao, Zhengyu Cheng, Chen Fan, Jing Yu, Mufei Lin, Karsten Kristiansen, Yin Shen, Xiaoming Wei. Targeted next-generation sequencing as a comprehensive test for Mendelian diseases: a cohort diagnostic study, Scientific Reports, 2018, Issue: 8, DOI: 10.1038/s41598-018-30151-z