Understanding the phenotype and genotype features in a specific population helps to make correct diagnosis for CF patients. Numerous CF studies conducted in Caucasians has successfully characterized the clinical features and CFTR mutation spectrum of Caucasian CF patients. While, limited knowledge of the spectrum of CF phenotype and genotype in Chinese is available. Recently, a systematic review preliminarily summarized the clinical and genetic characteristics of 71 reported Chinese CF patients. [1] The authors also reported a significant under-recognition and diagnosis delay of CF in China, with the median age at onset of 1 y and median age at diagnosis of 10 y. [1] The present study collected 20 CF patients, of which the median age at onset and diagnosis were 9.3 y and 19 y, respectively. Most of the patients were firstly diagnosed with CF by sweat chloride tests or genetic screening. The reason for the much larger age at onset and diagnosis in this cohort might be due to selection bias, because all these patients were recruited from the Department of Pulmonary and Critical Care Medicine, a clinic mainly for adults in PUMCH, while, children with suspected CF often visit pediatricians. Despite that, a similar significant diagnosis delay of about 10 years was also observed in this study. Out of 20 patients, 15 (15/20, 75%) had experienced misdiagnosis, including bronchiectasis, diffusive pan-bronchiolitis, pulmonary tuberculosis and pneumonia.
As to the clinical manifestation, respiratory symptom was most commonly observed in our cohort, consistent with the results of previous studies performed in CF patients of Chinese origin. [1, 3, 5] Interestingly, about 30% patients in this study presented with ABPA, which was far more than that reported in Caucasians (7–9%). [8] Similarly, several recent studies [2, 3, 5] also reported higher rate of ABPA in Chinese patients, ranging from 37.5–57.1%. Furthermore, our recent review demonstrated that out of all 71 reported Chinese CF patients, 15 had developed ABPA, reaching a high rate of 21.1%. [1] Therefore, from the data in these independent studies, we would conclude that Chinese CF patients might be more prone to developing ABPA than Caucasians.
In addition, a lower frequency (4/20, 20%) of PI was found in this cohort, diagnosed by positive Sudan Ⅲ staining and/or fatty diarrhea. In contrast, the same frequency was about 85% in Caucasians as reported in the 2017 Cystic Fibrosis Foundation Patient Registry Annual Data Report (available at https://www.cff.org/Research/Researcher-Resources/Patient-Registry/). Similar results can be found in several recent studies conducted in Chinese population. [1–3, 5] PI and PS status in CF are predisposed by the genotype at the CFTR locus. [14, 15] The most common mutation p.F508del detected in Caucasians and other mutations severely impairing CFTR function were reported to be associated with PI. [14] Thus, the substantial different CFTR mutation spectrum from Caucasians (see below) might be responsible, at least partially, for the lower frequency of PI observed in Chinese.
CBAVD is a one of the comorbidities commonly seen in Caucasian male CF patients. [16] As reported previously, [1] only one case was adult male who suffered from CBAVD. In this study, 3 of the 5 male patients received semen tests and/or urological ultrasound examination, and all turned out to have CBAVD. Thus, all the 4 Chinese male patients received CBAVD examination, including 3 reported in this cohort, showed manifestation of CBAVD. In other words, it seems like that CBAVD is also frequently presented in male CF patients of Chinese origin. Further studies with larger sample size or long-term follow-up data may make us more confident to draw the conclusion.
Three decades have passed away since the cystic fibrosis gene was firstly identified. [17] Extensive efforts have been directed towards the research of CFTR and this disease. Over 2000 mutations have been detected in CF patients across the world as recorded in the Cystic Fibrosis Mutation Database (http://www.genet.sickkids.on.ca). The mutation spectrum has been well established in Caucasians. A distinct CFTR mutation spectrum in Chinese has been suggested in previous studies. [1, 2] In the present study, we detected 22 distinct mutations, with p.G970D as the most common mutation counting for 31.6% (12/38 alleles) of CFTR alleles. Mutations detected more than once also included two other variants, p.Q98R and c.1766 + 5G > T. The three mutations were all among the five most common mutations observed in Chinese. [1] In contrast, the most frequent mutation in Caucasians, p.F508del, was not observed; only one mutation, p.R553X detected in patient 11, was in the screening panel recommended by the American College of Medical Genetics, which consists of the 23 most common mutations in Caucasians. [18] Thus, the present study further confirmed the ethnic-specific mutation spectrum in patients of Chinese origin.
Five novel mutations were identified in this study. The variants p.Y109D and p.R1066S both were novel missense amino acid changes at the same positions as reported pathogenic missense mutations, p.Y109N and p. R1066C, respectively. This is considered moderate evidence supporting their pathogenicity but cannot be assumed to be pathogenic. [19] The potential pathogenicity of the rest 2 novel variants, p.I203F and p.D572E, was unknown. Further functional study for these missense mutations are warranted to determine their effects on CFTR function. The deletion of CFTR exons 2–3 found in patient 9 was the fourth gross rearrangement reported in CF patients of Chinese origin. △E2-3 is very like to be a pathogenic mutation, because it was predicted to result in a pre-mature termination codon and no functional CFTR protein. A similar deletion of 21 Kb also removing exons 2–3 was found to be a frequent and severe CF mutation in Central and Eastern Europe, [20] supporting the pathogenicity. It is noteworthy that the other three gross deletion were also detected by our group. [2–4] There might be an underestimated detection rate of rearrangement in CFTR in Chinese patients, for which overlook and the inaccessibility of MLPA analysis should be responsible. Further emphasis should be placed on the necessity of MLPA analysis in routine CFTR mutation screening in Chinese patients, especially for those with only one or no mutations identified via direct sequencing of CFTR exons.
In our cohort, substantial differences in pulmonary function and radiological findings in the lung (Table 1 and Fig. 3b) were observed in the siblings 6 − 1 and 6 − 2, who carried the same homozygous p.G970D. It has been well known that the severity of CF lung disease varies dramatically even in patients with identical CFTR genotypes, in which genetic factors play an important role. [21–23] A review collected recent efforts aiming at identifying non-CFTR modifier genes for lung disease severity in cystic fibrosis. [21] Genotyping of reported modifier loci showed that patient 6 − 1 carried several risk alleles in the TGFB1, MUC4/MUC20 and HLA Ⅱ genes but patient 6 − 2 did not; the rest loci turned out to be the same for the siblings (Table S1). The TGFB1 codon 10 CC genotype (rs1800470) detected in patient 6 − 1 was reported to be related to severe lung function with an odds ratio of about 2.2. [10] This genotype has been associated with elevated TFGβ1 expression level, which could be responsible for the severe lung function due to increased inflammation. [24] In the same gene, the protective C-T-C haplotype [11] was seen in patient 6 − 1 with very mild pulmonary manifestations. TGFβ1 has multiple functions including regulating immune responses and inflammation process, and it has been associated with other lung diseases like chronic obstructive pulmonary disease and asthma. [25, 26] The MUC4/MUC20 genes encode proteins located on ciliated airway mucosal surface, which are involved in mucus secretion and mucociliary clearance.[27] As to the risk alleles in HLA Ⅱ, several recent studies reported the association of SNPs in HLA Ⅱ or HLA class Ⅱ pathway with severe lung disease in CF patients, using genome-wide association study or gene expression approaches [12, 13, 28]. However, what we should notice is that studies about these reported modifier genes often yielded conflicting results, due to small sample size and the lack of replications. For example, the purported modifier gene TGFB1 mentioned above failed to reach the genome-wide significance in another GWAS study. [13] Furthermore, regardless of the reliability of the associations, these genes can only explain limited proportion of the variability of lung disease. Finally, environmental factors like secondhand smoke exposure may also contributed to the variability of pulmonary phenotype observed in the patients.
There were some limitations in our research. First, all patients were recruited from a single center, Department of Pulmonary and Critical Care Medicine, PUMCH. Some of the patients came to our center due to suspected ABPA. So, the higher rate of ABPA in Chinese CF patients found in the present study should be carefully used because of potential selection bias. Second, due to cultural reasons, patients often refuse semen examination, making it difficult to screen CBAVD in Chinese CF patients. Studies with more patients accepting CBAVD screening are warranted to confirm the high frequency of CBAVD observed in this study.