Novel mutation c.1210-3C>G in cis with a poly-T tract of 5T affects CFTR mRNA splicing in a Chinese patient with cystic fibrosis

Abstract

Cystic fibrosis (CF) is a rare autosomal recessive disease with only one pathogenic gene cystic fibrosis transmembrane conductance regulator (CFTR). To identify the potential pathogenic mutations in a Chinese patient with CF, we conducted Sanger sequencing on the genomic DNA of the patient and his parents and detected all 27 coding exons of CFTR and their flanking intronic regions. The patient is a compound heterozygote of c.2909G>A, p.Gly970Asp in exon 18 and c.1210-3C>G in cis with a poly-T of 5T (T5) sequence, 3 bp upstream in intron 9. The splicing effect of c.1210-3C>G was verified via minigene assay in vitro, indicating that wild-type plasmid containing c.1210-3C together with T7 sequence produced a normal transcript and partial exon 10-skipping-transcript, whereas mutant plasmid containing c.1210-3G in cis with T5 sequence caused almost all mRNA to skip exon 10. Overall, c.1210-3C>G, the newly identified pathogenic mutation in our patient, in combination with T5 sequence in cis, affects the CFTR gene splicing and produces nearly no normal transcript in vitro. Moreover, this patient carries a p.Gly970Asp mutation, thus confirming the high-frequency of this mutation in Chinese patients with CF.

Keywords

cystic fibrosis / CFTR / splicing mutation / minigene

References

[1] Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, .Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245(4922): 1066–1073

[2] Dorwart M, Thibodeau P, Thomas P. Cystic fibrosis: recent structural insights. J Cyst Fibros 2004; 3(Suppl 2): 91–94

[3] Marshall B, Elbert A, Petren K, Grimes M, Fink A, Myers V, Sewall A. Cystic Fibrosis Foundation Patient Registry 2013 Annual Data Report to the Center Directors. Cystic Fibrosis Foundation 2014

[4] Rosenstein BJ, Cutting GR. The diagnosis of cystic fibrosis: a consensus statement. Cystic Fibrosis Foundation Consensus Panel. J Pediatr 1998; 132(4): 589–595

[5] Sahami A, Alibakhshi R, Ghadiri K, Sadeghi H. Mutation analysis of exons 10 and 17a of CFTR gene in patients with cystic fibrosis in Kermanshah Province, Western Iran. J Reprod Infertil 2014; 15(1): 49–56

[6] Moskowitz SM, Chmiel JF, Sternen DL, Cheng E, Gibson RL, Marshall SG, Cutting GR. Clinical practice and genetic counseling for cystic fibrosis and CFTR-related disorders. Genet Med 2008; 10(12): 851–868

[7] Tian X, Liu Y, Yang J, Wang H, Liu T, Xu W, Li X, Zhu Y, Xu KF, Zhang X. p.G970D is the most frequent CFTR mutation in Chinese patients with cystic fibrosis. Hum Genome Var 2016; 3(1): 15063

[8] Liu Y, Wang L, Tian X, Xu KF, Xu W, Li X, Yue C, Zhang P, Xiao Y, Zhang X. Characterization of gene mutations and phenotypes of cystic fibrosis in Chinese patients. Respirology 2015; 20(2): 312–318

[9] Guo X, Liu K, Liu Y, Situ Y, Tian X, Xu KF, Zhang X. Clinical and genetic characteristics of cystic fibrosis in Chinese patients: a systemic review of reported cases. Orphanet J Rare Dis 2018; 13(1): 224

[10] Chu CS, Trapnell BC, Curristin S, Cutting GR, Crystal RG. Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nat Genet 1993; 3(2): 151–156

[11] Delaney SJ, Rich DP, Thomson SA, Hargrave MR, Lovelock PK, Welsh MJ, Wainwright BJ. Cystic fibrosis transmembrane conductance regulator splice variants are not conserved and fail to produce chloride channels. Nat Genet 1993; 4(4): 426–430

[12] Gaildrat P, Killian A, Martins A, Tournier I, Frébourg T, Tosi M. Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants. Methods Mol Biol 2010; 653: 249–257

[13] Salinas DB, Azen C, Young S, Keens TG, Kharrazi M, Parad RB. Phenotypes of California CF newborn screen-positive children with CFTR 5T allele by TG repeat length. Genet Test Mol Biomarkers 2016; 20(9): 496–503

[14] Huang Q, Ding W, Wei MX. Comparative analysis of common CFTR polymorphisms poly-T, TG-repeats and M470V in a healthy Chinese population. World J Gastroenterol 2008; 14(12): 1925–1930

[15] Amato F, Scudieri P, Musante I, Tomati V, Caci E, Comegna M, Maietta S, Manzoni F, Di Lullo AM, De Wachter E, Vanderhelst E, Terlizzi V, Braggion C, Castaldo G, Galietta LJV. Two CFTR mutations within codon 970 differently impact on the chloride channel functionality. Hum Mutat 2019; 40(6): 742–748

[16] Kiesewetter S, Macek M Jr, Davis C, Curristin SM, Chu CS, Graham C, Shrimpton AE, Cashman SM, Tsui LC, Mickle J, Amos J, Highsmith WE, Shuber A, Witt DR, Crystal RG, Cutting GR. A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet 1993; 5(3): 274–278

[17] de Nooijer RA, Nobel JM, Arets HG, Bot AG, van Berkhout FT, de Rijke YB, de Jonge HR, Bronsveld I. Assessment of CFTR function in homozygous R117H-7T subjects. J Cyst Fibros 2011; 10(5): 326–332

[18] Kimura S, Okabayashi Y, Inushima K, Yutsudo Y, Kasuga M. Polymorphism of cystic fibrosis gene in Japanese patients with chronic pancreatitis. Dig Dis Sci 2000; 45(10): 2007–2012

[19] Gregory RJ, Rich DP, Cheng SH, Souza DW, Paul S, Manavalan P, Anderson MP, Welsh MJ, Smith AE. Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2. Mol Cell Biol 1991; 11(8): 3886–3893

Acknowledgements

We thank the patient and his parents, as well as other participants in this study. The study was supported by the National Key Research and Development Program of China (No. 2016YFC0901502 to Kai-Feng Xu; No. 2016YFC0905100 to Xue Zhang; No. 2017YFC1001201 to Yaping Liu), the National Natural Science Foundation of China (NSFC) (Nos. 81788101 and 81230015 to Xue Zhang; No. 31271345 to Yaping Liu), and the CAMS Initiative for Medical Sciences (CIFMS) (No. 2016-I2M-1-002 to Xue Zhang and Yaping Liu; No. 2018-I2M-1-003 to Xinlun Tian; No. 2017-I2M-2-001 to Kai-Feng Xu).

Compliance with ethics guidelines

Xinyue Zhao, Keqiang Liu, Wenshuai Xu, Meng Xiao, Qianli Zhang, Jiaxing Song, Keqi Chen, Yaping Liu, Xinlun Tian, Kai-Feng Xu, and Xue Zhang declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 2000 and were approved by the Institutional Review Board committee at PUMCH. Informed consent was obtained from the patient for being included in the study.

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