Background
Birt-Hogg-Dubé syndrome (BHDS) is an autosomal dominant inherited disorder caused by germline mutations in the
folliculin (
FLCN) gene [
1], which encodes a tumor suppressor protein. BHDS phenotype is characterized by fibrofolliculomas on the face, neck and upper torso, lung cysts, spontaneous pneumothorax, and increased risk for renal tumors. The symptoms of skin, pulmonary, and renal appear independently with high association, and BHDS patients can present with a single phenotype or a combination of any of them [
2]. This combination contributes to the great variability in clinical features of BHD syndrome, making clinical diagnosis generally difficult. Clinical and genetic studies about BHDS were mainly conducted among Caucasian population in the US and Europe [
3‐
5], which help to characterize the phenotype and mutation spectrum. A recent epidemiologic study with a large cohort in Japan found different phenotypes and
FLCN mutational hotspots (c.1347_1353dupCCACCCT and c.1533_1536delGATG) in Japanese BHDS patients [
6]. While, BHDS studies were rarely reported in China and most of them were case reports. Our previous study [
7] reported a much lower prevalence of skin lesions (11.1%, 3/27) and a different
FLCN mutation spectrum in Chinese BHDS patients compared with Caucasians. Further studies are warranted to better understand the clinical and genetic characteristics of Chinese BHDS patients.
Since
FLCN was identified as the causative gene of BHDS in 2002 [
1], over 200 mutations have been reported in BHDS patients according to the Human Genome Mutation Database (HGMD) [
8]. A mutation hotspot of 1-bp deletion or duplication (c.1285delC/dupC) within the polyC (8) tract in exon 11 has been detected as the most common mutation in several different countries or races [
3,
4,
6,
7]. Interestingly, the majority of reported
FLCN mutations are protein-truncating mutations resulting in complete loss of function of the gene product [
9], including frameshifts, nonsenses, canonical +/− 1 or 2 splice site mutations and single- or multi-exon deletions. By comparison, missense and in-frame mutations are less frequently observed in BHDS patients comprising only 12.6% (26/206) of all
FLCN mutations in HGMD. In vitro experimental evidence has shown that most of the
FLCN missense/in-frame mutations impaired folliculin tumor suppressor function by disrupting the stability of the protein [
10]. However, emerging evidence shows that quite a portion of presumed missense mutations in other genes abolish protein function actually by disrupting the normal mRNA splicing pattern [
11‐
13]. Whether this mechanism is involved in the pathogenicity of
FLCN missense and in-frame mutations remains to be elucidated.
In the present study, we described the clinical and genetic features of a cohort of 39 Chinese BHDS patients from 31 distinct families, and systematically evaluated the effect of all FLCN missense and in-frame mutations reported in HGMD database on mRNA splicing pattern.
Discussion
FLCN was first identified as the causative gene for BHDS in 2002 [
1], making genetic testing an effective diagnosis method for this disease. While clinical and genetic studies of BHDS are rare in Asian population, especially in Chinese. In the present study, we reported a large Chinese BHDS cohort with 39 patients from 31 unrelated families diagnosed by genetic testing, including 37 female and 2 male patients. Much more female patients were observed in our patients, which is likely due to a selection bias because Peking Union Medical College Hospital (PUMCH) is the largest lymphagioleiomyomatosis referral center in China. The same selection bias has been reported in a Japanese study [
16].
Respiratory system was the most frequently affected system in these BHDS patient as we reported before [
7], with frequencies of almost 100 and 58.3% for lung cysts and pneumothorax respectively. Similarly, a recent epidemiologic study of Japanese BHDS patients reported that all except one family had lung cysts and 73.7% presented with episodes of pneumothorax in their cohort [
6]. By comparison, lower prevalence of lung cysts (70–85%) was reported in Caucasian BHDS patients and only about one-third patients reported a history of spontaneous pneumothorax [
4,
5,
17,
18]. Thus, it seems that pulmonary manifestations are more frequently observed in Chinese BHDS patients. What is worth mentioning is that the Caucasian patients from literature were all recruited through referrals from department of dermatology or urology. While, most patients in this study came to our clinic because of cysts in the lung. So, selection bias should be responsible, at least partially, for the different frequencies of pulmonary manifestations between Chinese and Caucasian BHDS.
BHDS affected individuals are reported to have a higher risk to develop renal tumors with a prevalence of 12–34% in Caucasian population [
3‐
5,
18,
19] and about 34.8% (40/115) in Japanese patients over the age of 40 [
6] . The most common histologic type of renal tumor in BHDS patients are chromophobe renal cell carcinomas and hybrid oncocytic neoplasms with features of chromophobe renal cell carcinoma and oncocytoma [
20]. While, among 30 patients with available data in our cohort, there were 8 patients with unilateral or bilateral renal cysts and 1 with renal hamartoma, but no renal malignancy. Similar findings have been reported in our previous studies, in which no malignant renal tumor was diagnosed in 27 probands, except for 1 with hamartomas and 4 with renal cysts [
7]. Despite potential selection and non-response bias in these two studies, malignant renal tumor was rarely detected in these Chinese
FLCN mutation carriers, which strongly suggests a lower prevalence of renal malignancy in Chinses BHDS patients.
More interestingly, about half of the patients (47.2%, 17/36) in this cohort were found to have cutaneous lesions, much more than that we reported before (11.1%, 3/27) [
7]. Relatively small sample size and more clinical awareness in China might be responsible for the inconsistency. A similar frequency (48.7%, 76/156) of skin lesions was documented in Japanese BHDS patients [
6]. In contrast, cutaneous findings were reported to be the most common symptoms among Caucasian patients with a percentage of around 90% [
4,
5,
17]. So, there was much less skin manifestation observed in Chinese BHDS individuals compared with Caucasians. Collectively, this study showed that Chinese BHDS patients have different clinical features from Caucasians, with more pulmonary manifestations but fewer skin lesions and renal malignancy.
In our previous study, 14 out of 20 variants found in a Chinese BHDS cohort were novel [
7], indicating a different mutation spectrum from that of Caucasians. And in the present study, as a follow-up study, more than half (52.4%, 11/21) of the 21 detected different variants have never been reported. Collectively, these two studies demonstrated the variability of
FLCN mutation spectrum between Chinese and other races. A previously reported mutational hot spot, c.1285dupC/delC, is also the most frequent mutation observed in these two studies, with a frequency (28.3%, 15/53) comparable to that of Japanese patients (28.3%, 34/120) [
6]. However, the other two mutational hot spots c.1347_1353dupCCACCCT (p.Val452ProfsX6) and c.1533_1536delGATG (p.Trp511X) reported in Japanese were not observed in our cohort. The only one mutation observed in more than 5 patients is c.1285dupC. We compared the pulmonary, cutaneous and renal manifestations between c.1285dupC carriers and other patients. No association was observed between this mutation with any clinical features in our cohort.
Gross intragenic rearrangements of
FLCN were less frequently reported. To our knowledge, the Exon 1 deletion identified in this study is the second gross deletion reported in Chinese BHDS patients. Unlike the first reported △Exon 8 mutation [
7], this deletion is located in the 5′-untranslated region. Long-range PCR and bidirectional sequencing revealed a 3.6 Kb deletion encompassing the entire exon 1 flanked by the repeat elements
AluSx in intron 1 and
AluSq in the upstream region of
FLCN. Benhammou et al. [
21] reported several BHDS families carrying deletions involving exon 1 and found that the genomic sequences of this region contain a significantly higher number of
Alu elements than the rest of the entire
FLCN gene, which in turn might be responsible for the deletions due to unequal crossover mediated by these
Alu elements. In vitro luciferase reporter assay indicated that this region contains the putative
FLCN promoter [
21], deletion of which will significantly reduce the expression of
FLCN, supporting the pathogenicity of this △Exon 1 mutation.
The synonymous variant, c.1287C > T (p.His429=), found in patient 1 was classified as VUS and was observed in gnomAD with the highest frequency of about 1e− 4 in Ashkenazi Jewish. Besides, the clinical significance of this variant was evaluated as likely benign in ClinVar by multiple submitters with no conflicts. Moreover, minigene assay showed that this variant did not affect mRNA splicing. Therefore, this variant might be not responsible for the phenotypes in patient 1. Further investigations including phenotypic validation and application of other mutation detection tool are needed.
As a putative tumor suppressor, the vast majority of reported
FLCN mutations are truncating mutations including nonsenses, frameshifts, splice site mutations and large deletions/duplications, which will result in truncated protein or absent of mRNA by nonsense-mediated decay. By contrast, variants causing amino acid substitution or in-frame insertion/deletion are much infrequently reported. The
FLCN mutation database established by Lim and colleagues [
9] reported that missense mutations only count for 8.6% (6/70) of all
FLCN mutations reported at that time. In a large Japanese BHDS cohort, missense/in-frame mutations counted for 9.2% of all patients with germline mutation. In addition, only 1 missense (c.1067 T > C, p.Leu356Pro) [
7] and 1 in-frame deletion (c.469_471delTTC, p.Phe157del identified in this study) were observed in our cohort of 53 Chinese patients. Unlike truncating mutations, the pathogenicity of missense/in-frame mutations are relatively difficult to determine, which requires co-segregation of the variants with disease status in relatively large kindred and solid functional evidence. Previously, researchers transiently expressed folliculin in wild-type form or mutant form carrying specific missenses/in-frame deletions in FTC-133 cell line, and found that most of these non-truncating mutations significantly disrupted the stability of folliculin protein [
10]. However, by directly inserting the full-length
FLCN cDNA into expression vector, this strategy did not rule out the possibility that these mutations might cause loss of gene function actually by affecting the mRNA splicing process [
11‐
13].
To investigate the potential effects of
FLCN non-truncating mutations on mRNA processing, we analyzed the splicing patterns of all 21 reported non-truncating mutations (15 missenses and 6 in-frame deletions) as well as the c.1287C > T variant (predicted as p.His429=), in an in vitro system based on the splicing reporter minigene [
22]. All wild-type minigenes produced expected chimeric transcripts, while the c.249 + 1G > A variant, used as a positive control, produced a same aberrant transcript as shown in vivo. To a certain degree, it reflected the reliability of this splicing system. However, none of 21 non-truncating mutations changed the splicing pattern of minigenes. Moreover, normally spliced transcripts bearing the corresponding substitutions or deletions were observed by direct sequencing. These results denied the hypothesis that
FLCN missense/in-frame mutations cause BHDS phenotypes through affecting mRNA splicing. And, with this observation, the hypothesis of disrupted folliculin stability seems more reasonable. However, we should also note that the in vitro minigene assay may not fully represent the splicing regulatory machinery involved in BHDS affected tissues.
There are some limitations in our study. Firstly, data missing is a major problem, especially for renal and cutaneous examinations. Data about kidney examination in 9/39 patients are not available, which make it difficult to correctly define the prevalence of renal impairments. Because skin lesions like fibrofolliculomas are generally neither painful nor pruritic, most patients were reluctant to accept invasive biopsy. Thus, only one patient was diagnosed with typical skin lesions by histological confirmation. Secondly, most of the patients were recruited from a single center, Department of Pulmonary and Critical Care Medicine, PUMCH, which is a referral center for rare pulmonary diseases in China. Patient were likely to be referred to our hospital when they have lung cysts on radiological scans. So, the finding of 100% lung cysts in this cohort should be used carefully, due to obvious selection bias. However, in these genetically confirmed BHDS patients, cutaneous involvements and renal tumors were much less frequently detected than in those reported in Caucasians. Given that skin, pulmonary, and renal symptoms appear independently in BHDS, our data even incomplete still support that Chinese BHDS patients have fewer skin lesions and kidney tumors.
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