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

doi:10.1007/s11684-021-0846-5

Front. Med.

2022, Vol. 16

Issue (1) : 150-155 https://doi.org/10.1007/s11684-021-0846-5

RESEARCH ARTICLE

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

Xinyue Zhao¹, Keqiang Liu¹, Wenshuai Xu², Meng Xiao¹, Qianli Zhang¹, Jiaxing Song¹, Keqi Chen², Yaping Liu¹(

), Xinlun Tian²(

), Kai-Feng Xu², Xue Zhang¹

¹. McKusick-Zhang Center for Genetic Medicine, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China
². Department of Pulmonary and Critical Care Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China

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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

Corresponding Author(s): Yaping Liu,Xinlun Tian

Just Accepted Date: 12 July 2021 Online First Date: 26 July 2021 Issue Date: 28 March 2022

Cite this article:

Xinyue Zhao,Keqiang Liu,Wenshuai Xu, et al. 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[J]. Front. Med., 2022, 16(1): 150-155.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-021-0846-5
https://academic.hep.com.cn/fmd/EN/Y2022/V16/I1/150

Fig.1 Functional tests of splicing mutations via minigene assay. (A) A fragment of genomic DNA including exon 10, 414 nucleotide (nt) at the 3′ end of intron 9, and 402 nt at the 5′ end of intron 10 was amplified. Four types of target sequences encompassing two variants in intron 9 were designated as 7T+C (wild type), 5T+C, 7T+G, and 5T+G (carried by the patient). T_x stands for T₅ or T₇, and N stands for C or G nucleotide. (B) The amplicons were cloned into the pCAS2 vector, which has a PCMV promoter (orange) and two exons (exons A and B, brown and yellow, respectively). Two green boxes on the pCAS2 vector and target sequences represent two 15 bp fragments complementary to BamH1 ends, which help clone the target sequence into the pCAS2 vector. (C) Predicted resultant transcripts of the minigene: wild-type transcript, including exon A+exon 10+exon B, on the left; and mutant transcript, including exon A+exon B, skipping of exon 10, on the right.

Fig.2 CFTR mutations in the patient and his parents and their effect on splicing pattern. (A) Pedigree and genotype. Squares and circles symbolize males and females, respectively. No fill symbolizes unaffected individuals, and black fill symbolizes affected individuals. M1, mutation c.1210-3C>G; M2, mutation c.2909G>A (p.Gly970Asp); -, wild type. The arrow indicates the proband. (B) Sanger sequencing outcomes in the patient and his parents to verify their mutations. (C, D) Splicing patterns of plasmid: 7T+C, 5T+C, 7T+G, and 5T+G were shown by electrophoresis (C) and Sanger sequencing (D). Top band (band 1) is the wild-type transcript of exon A+exon 10+exon B; middle band (band 2) is the heterodimer of wild-type transcript and mutant transcript; bottom band (band 3) is the mutant transcript of exon A+exon B possessing a deletion of exon 10.

Fig.3 Dephosphorylated, ATP-bound human CFTR protein structure, with G970 colored yellow in the center. This view shows that G970 is a critical residue for chloride ion channel function.

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