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Targeted Next-Generation Sequencing Effectively Analyzed the Cystic Fibrosis Transmembrane Conductance Regulator Gene in Pancreatitis

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Abstract

Background

The cystic fibrosis transmembrane conductance regulator (CFTR) gene, responsible for the development of cystic fibrosis, is known as a pancreatitis susceptibility gene. Direct DNA sequencing of PCR-amplified CFTR gene segments is a first-line method to detect unknown mutations, but it is a tedious and labor-intensive endeavor given the large size of the gene (27 exons, 1,480 amino acids). Next-generation sequencing (NGS) is becoming standardized, reducing the cost of DNA sequencing, and enabling the generation of millions of reads per run. We here report a comprehensive analysis of CFTR variants in Japanese patients with chronic pancreatitis using NGS coupling with target capture.

Methods

Exon sequences of the CFTR gene from 193 patients with chronic pancreatitis (121 idiopathic, 46 alcoholic, 17 hereditary, and nine familial) were captured by HaloPlex target enrichment technology, followed by NGS.

Results

The sequencing data covered 91.6 % of the coding regions of the CFTR gene by ≥20 reads with a mean read depth of 449. We could identify 12 non-synonymous variants including three novel ones [c.A1231G (p.K411E), c.1753G>T (p.E585X) and c.2869delC (p.L957fs)] and seven synonymous variants including three novel ones in the exonic regions. The frequencies of the c.4056G>C (p.Q1352H) and the c.3468G>T (p.L1156F) variants were higher in patients with chronic pancreatitis than those in controls.

Conclusions

Target sequence capture combined with NGS is an effective method for the analysis of pancreatitis susceptibility genes.

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Abbreviations

bp:

Base pair

CF:

Cystic fibrosis

CFTR:

Cystic fibrosis transmembrane conductance regulator

CP:

Chronic pancreatitis

ERCP:

Endoscopic retrograde cholangiopancreatography

NGS:

Next-generation sequencing

PCR:

Polymerase chain reaction

RD:

Related disorder

SIFT:

Sorting Intolerant From Tolerant

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Acknowledgments

The authors are grateful to Ms. Yoko Tateda for the excellent technical assistance. This work was supported in part by the Pancreas Research Foundation of Japan (to E. Nakano), the HIROMI Medical Research Foundation (to A. Masamune), the Mother and Child Health Foundation (to A. Masamune), the Smoking Research Foundation (to A. Masamune), and by the Ministry of Health, Labour, and Welfare of Japan.

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None.

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Authors and Affiliations

  1. Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan

    Eriko Nakano, Atsushi Masamune, Kiyoshi Kume, Shin Hamada & Tooru Shimosegawa

  2. Department of Medical Genetics, Tohoku University Graduate School of Medicine, Sendai, 980-8574, Japan

    Tetsuya Niihori, Yoko Aoki & Yoichi Matsubara

  3. National Research Institute for Child Health and Development, Tokyo, 157-8535, Japan

    Yoichi Matsubara

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  1. Eriko Nakano
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  2. Atsushi Masamune
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  3. Tetsuya Niihori
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  4. Kiyoshi Kume
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  5. Shin Hamada
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Correspondence to Atsushi Masamune.

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Nakano, E., Masamune, A., Niihori, T. et al. Targeted Next-Generation Sequencing Effectively Analyzed the Cystic Fibrosis Transmembrane Conductance Regulator Gene in Pancreatitis. Dig Dis Sci 60, 1297–1307 (2015). https://doi.org/10.1007/s10620-014-3476-9

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  • DOI: https://doi.org/10.1007/s10620-014-3476-9

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