Cystic fibrosis (CF) is a rare autosomal recessive inherited disease associated with impaired pulmonary function and a defective digestive system. With symptoms affecting multiple organs and a relatively high incidence rate, CF has become one of the most frequent lethal diseases for Caucasians[13]. Since CFTR was considered a CF-causative gene, more than 2000 mutations have been recognized, forming a defined CFTR mutation spectrum of Caucasian origin. In contrast, the morbidity of CF is low in China, given that there are considerably fewer reports of CF among large populations of Chinese. Atypical clinical syndromes and novel mutations unidentified previously in Caucasians also show an ethnicity-specific pattern in Chinese CF patients[9]. In this study, we aimed to identify potential pathogenic mutations in a CF patient of Chinese origin. By Sanger sequencing, we identified a low-frequency variant p.Gly970Asp (c.2909G>A, p.G970D), and a novel variant c.1210-3C>G in this patient.
p.Gly970Asp is located within the third cytoplasmic loop of the CFTR protein, and its impairment of chloride conductance may lead to a potentiator-sensitive gating defect as well as a partial trafficking defect without RNA splicing alteration[7,14]. In Amato’s report, p.Gly970Asp mutant plasmid-transfected HEK293 cells showed very low channel activity, demonstrating the mutation’s pathogenicity[14]. In addition, with an allele frequency of 9.8%, p.Gly970Asp is the most commonly seen hotspot mutation in CF patients of Chinese origin [9]. This may encourage the eligibility of pharmaco-gene treatment focusing on p.Gly970Asp in order to correct the CF phenotype in Chinese patients.
The second mutation in this patient, c.1210-3C>G, is adjacent to the splice acceptor site of CFTR exon 10, in cis with a poly-T tract of 5T, 3 bp upstream. Human splicing finder (http://www.umd.be/HSF3/) was chosen to predict the effect of c.1210-3C>G, as well as the complicated outcome of c.1210-3C>G combined with the poly-T tract of 5T, both of which were predicted to most likely affect splicing pattern. This result suggested that c.1210-3C>G might contribute to the pathogenicity in this patient, which prompted us to test its effect on the splicing pattern. Unfortunately, CFTR expression was too weak to be captured from peripheral blood leukocytes, and this study was restricted to a minigene assay in vitro because of the unavailability of the nasal epithelium from this patient.
CFTR exon 10 in-frame skipping mRNA exists in normal individuals with the poly-T tract of 7T, and among the 5T, 7T, and 9T forms of the poly-T tract, referring to the exon 10 splice acceptor site, the shorter the poly-T tract, the higher the amount of aberrant CFTR mRNA transcripts in the respiratory epithelium, as described in Chin-Shyan’s article[10]. However, the TG12T5 sequence may not be strong enough to be a disease-causing variant per se. (data shown on the CFTR2 website, https://www.cftr2.org/mutation/general/5T/). The level of human exon10+ transcripts in the bronchial epithelium of non-CF individuals can be as low as 8% of total CFTR transcripts[11]. This could be explained by the residual normal mRNA being abundant enough to exert CFTR function, as CF is an autosomal recessive disease but is not a haploinsufficiency. In contrast, c.1210-3C>G, a possibly pathogenic mutation carried by this patient, seems to arouse the CF phenotype alone, as the normal transcript can hardly be seen in the 7T+G and 5T+G plasmids, according to the results of the minigene assay. The 5T variant alone is not a definitive CF-causing variant, but it may act synergistically with c.1210-3C>G in cis to produce a null allele[15,16]. An antecedent case was reported in which the combined effect of TG12T5 and p.Arg117His in the same chromosome as p.Phe508del in trans contributed to the CF phenotype, which also hypothesized the combined effect of two mutations [16].
CFTR exon 10 (regarded as exon 9 in reference article for legacy name) encodes the first 21% of NBF1, a domain that is critical to CFTR function [17]. The defective protein encoded by a transcript without exon 10 cannot exit from the endoplasmic reticulum and traffic through the Golgi complex to the cell membrane. The resultant extremely decreased normal CFTR Cl- channel in the cell membrane may lead to the CF phenotype in our patient [11,18].