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A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax
BACKGROUND: Primary spontaneous pneumothorax (PSP) or pulmonary cysts is one of the manifestations of Birt-Hogg-Dube syndrome (BHDS) that is caused by heterozygous mutations in FLCN gene. Most of the mutations are SNVs and small indels, and there are also approximately 10 % large intragenic deletion...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882857/ https://www.ncbi.nlm.nih.gov/pubmed/27229674 http://dx.doi.org/10.1186/s12931-016-0377-9 |
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author | Zhang, Xinxin Ma, Dehua Zou, Wei Ding, Yibing Zhu, Chengchu Min, Haiyan Zhang, Bin Wang, Wei Chen, Baofu Ye, Minhua Cai, Minghui Pan, Yanqing Cao, Lei Wan, Yueming Jin, Yu Gao, Qian Yi, Long |
author_facet | Zhang, Xinxin Ma, Dehua Zou, Wei Ding, Yibing Zhu, Chengchu Min, Haiyan Zhang, Bin Wang, Wei Chen, Baofu Ye, Minhua Cai, Minghui Pan, Yanqing Cao, Lei Wan, Yueming Jin, Yu Gao, Qian Yi, Long |
author_sort | Zhang, Xinxin |
collection | PubMed |
description | BACKGROUND: Primary spontaneous pneumothorax (PSP) or pulmonary cysts is one of the manifestations of Birt-Hogg-Dube syndrome (BHDS) that is caused by heterozygous mutations in FLCN gene. Most of the mutations are SNVs and small indels, and there are also approximately 10 % large intragenic deletions and duplications of the mutations. These molecular findings are generally obtained by disparate methods including Sanger sequencing and Multiple Ligation-dependent Probe Amplification in the clinical laboratory. In addition, as a genetically heterogeneous disorder, PSP may be caused by mutations in multiple genes include FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 genes. For differential diagnosis, these genes should also be screened which makes the diagnostic procedure more time-consuming and labor-intensive. METHODS: Forty PSP patients were divided into 2 groups. Nineteen patients with different pathogenic mutations of FLCN previously identified by conventional Sanger sequencing and MLPA were included in test group, 21 random PSP patients without any genetic screening were included in blinded sample group. 7 PSP genes including FLCN, FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 were designed and enriched by Haloplex system, sequenced on a Miseq platform and analyzed in the 40 patients to evaluate the performance of the targeted-NGS method. RESULTS: We demonstrated that the full spectrum of genes associated with pneumothorax including FLCN gene mutations can be identified simultaneously in multiplexed sequence data. Noteworthy, by our in-house copy number analysis of the sequence data, we could not only detect intragenic deletions, but also determine approximate deletion junctions simultaneously. CONCLUSIONS: NGS based Haloplex target enrichment technology is proved to be a rapid and cost-effective screening strategy for the comprehensive molecular diagnosis of BHDS in PSP patients, as it can replace Sanger sequencing and MLPA by simultaneously detecting exonic and intronic SNVs, small indels, large intragenic deletions and determining deletion junctions in PSP-related genes. |
format | Online Article Text |
id | pubmed-4882857 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-48828572016-05-28 A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax Zhang, Xinxin Ma, Dehua Zou, Wei Ding, Yibing Zhu, Chengchu Min, Haiyan Zhang, Bin Wang, Wei Chen, Baofu Ye, Minhua Cai, Minghui Pan, Yanqing Cao, Lei Wan, Yueming Jin, Yu Gao, Qian Yi, Long Respir Res Research BACKGROUND: Primary spontaneous pneumothorax (PSP) or pulmonary cysts is one of the manifestations of Birt-Hogg-Dube syndrome (BHDS) that is caused by heterozygous mutations in FLCN gene. Most of the mutations are SNVs and small indels, and there are also approximately 10 % large intragenic deletions and duplications of the mutations. These molecular findings are generally obtained by disparate methods including Sanger sequencing and Multiple Ligation-dependent Probe Amplification in the clinical laboratory. In addition, as a genetically heterogeneous disorder, PSP may be caused by mutations in multiple genes include FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 genes. For differential diagnosis, these genes should also be screened which makes the diagnostic procedure more time-consuming and labor-intensive. METHODS: Forty PSP patients were divided into 2 groups. Nineteen patients with different pathogenic mutations of FLCN previously identified by conventional Sanger sequencing and MLPA were included in test group, 21 random PSP patients without any genetic screening were included in blinded sample group. 7 PSP genes including FLCN, FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 were designed and enriched by Haloplex system, sequenced on a Miseq platform and analyzed in the 40 patients to evaluate the performance of the targeted-NGS method. RESULTS: We demonstrated that the full spectrum of genes associated with pneumothorax including FLCN gene mutations can be identified simultaneously in multiplexed sequence data. Noteworthy, by our in-house copy number analysis of the sequence data, we could not only detect intragenic deletions, but also determine approximate deletion junctions simultaneously. CONCLUSIONS: NGS based Haloplex target enrichment technology is proved to be a rapid and cost-effective screening strategy for the comprehensive molecular diagnosis of BHDS in PSP patients, as it can replace Sanger sequencing and MLPA by simultaneously detecting exonic and intronic SNVs, small indels, large intragenic deletions and determining deletion junctions in PSP-related genes. BioMed Central 2016-05-27 2016 /pmc/articles/PMC4882857/ /pubmed/27229674 http://dx.doi.org/10.1186/s12931-016-0377-9 Text en © Zhang et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Zhang, Xinxin Ma, Dehua Zou, Wei Ding, Yibing Zhu, Chengchu Min, Haiyan Zhang, Bin Wang, Wei Chen, Baofu Ye, Minhua Cai, Minghui Pan, Yanqing Cao, Lei Wan, Yueming Jin, Yu Gao, Qian Yi, Long A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax |
title | A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax |
title_full | A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax |
title_fullStr | A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax |
title_full_unstemmed | A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax |
title_short | A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax |
title_sort | rapid ngs strategy for comprehensive molecular diagnosis of birt-hogg-dubé syndrome in patients with primary spontaneous pneumothorax |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882857/ https://www.ncbi.nlm.nih.gov/pubmed/27229674 http://dx.doi.org/10.1186/s12931-016-0377-9 |
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