Clinical characteristics and genetic analysis of familial spontaneous pneumothorax in China


 Familial spontaneous pneumothorax (FSP) is a hereditary disease, and Birt-Hogg-Dubé (BHD) syndrome is its main cause. FSP is an autosomal dominant genetic disease related to folliculin (FLCN). The goal of this study was to investigate the clinical characteristics and possible causes of FSP in China compared with those of primary spontaneous pneumothorax (PSP). We reviewed the detailed clinical data of 8 FSP patients in a family and analyzed the clinical characteristics of FSP combined with literatures. The FLCN gene of these 8 FSP patients was sequenced by the next-generation sequencing technology (NGS). The results showed that the clinical features of FSP were significantly different from those of PSP: the incidence of pneumothorax in women is significantly higher, and the age of pneumothorax is later, most of them are two generations, which may be related to heredity. And the effect of surgical treatment is the best. Novel nonsense mutation was found in the 8 patients, and the 8 patients had the same mutation, thus demonstrating the diversity of mutation spots along the gene. Therefore, the FLCN gene screening and early surgical intervention is recommended for FSP patients.


Introduction
Primary spontaneous pneumothorax (PSP, OMIM 173600) is de ned as the spontaneous occurrence of pneumothorax in patients without clinically obvious potential lung disease. There are tens of thousands of patients with PSP every year [1][2][3]. Spontaneous pneumothorax is usually not considered a hereditary disease, but about 11.5%-16.2% of patients with spontaneous pneumothorax had a positive family

Materials And Methods
One patient with spontaneous pneumothorax was treated in our department in April 2018. By inquiring about the medical history, we found that 8 patients in their family had suffered from spontaneous pneumothorax and were hospitalized nearby. General information, diagnosis and treatment of these 8 patients were obtained through the proband and their families.
Because FSP is rare in clinic, in order to further analyze its clinical characteristics, we searched all the literatures about FSP before 2018 in China National Knowledge Infrastructure (CNKI) and Wanfang Data. The keywords of "familial spontaneous pneumothorax" were used to search and summarize literatures (excluding cases with incomplete clinical data), We obtained 23 related literatures, 75 families, plus this case, a total of 76 families, 265 patients with FSP. The clinical characteristics of FSP patients were analyzed combined with literature.
We used the target region probe capture technology and next-generation sequencing technology (NGS) based on illumina sequencing platform to detect FLCN gene in 8 patients of this family (details are available upon request). The results were compared in a large population database, and the previously unreported mutations were de ned as new mutations.

Results
The clinical information of 8 patients with pneumothorax are listed in Table 1. We also made a simple family pedigree for this family (the spouse is omitted) as shown in Fig. 1.  Sequence analysis of the FLCN gene revealed a novel nonsense mutation (c.558G A) in exon 6, and 8 patients in the family harbored the same mutation, thus demonstrating the diversity of mutation spots along the gene. (Fig. 2) Discussion PSP sporadic cases are common among young people, most of whom are 20-30 years old, and the incidence rate is 9/ 100,000 [7]. There are more men than women, with a ratio of 4 Ehlers Danlos syndrome, α 1-antitrypsin de ciency and BHD syndrome [11]. We found that a large proportion of reported FSP cases are closely related to FLCN gene mutations in BHD syndrome 12-15 .
In this study, novel nonsense mutation of FLCN gene was found in the proband and other patients in the family. Further, we found that Amino acid 186 of FLCN gene was mutated from tryptophan to terminator. Premature terminator may lead to nonsense mediated mRNA degradation (NMD), resulting in loss of protein expression. The mutation has not been found in large population databases (including gnomad, exac, 1000genome, etc.), suggesting that it is a rare mutation. Whether there are FLCN mutations in other members of the family needs further genetic testing to determine.
The expression of FLCN mRNA plays a role in the transformation of surfactant in phagocytosis and is expressed in alveolar macrophages, it is speculated that FLCN protein may have endocytosis or phagocytosis [16]. It is speculated that when FLCN gene mutation occurs, a large number of in ammatory factors are secreted by macrophages and broblasts to induce in ammation, which eventually leads to the destruction of elastic bers in the lung, which may also be the cause of pneumothorax. Therefore, it is speculated that when FLCN gene is mutated, a large number of in ammatory factors are secreted by macrophages and broblasts, which induce in ammation, and nally cause the destruction of elastic bers in the lung and lead to pneumothorax, which may also be the cause of pneumothorax. Whether it belongs to BHD syndrome requires further genetic testing to make a de nite diagnosis.
Combined with literature review, we found that the surgical treatment of FSP is the best, with a recurrence rate of only 4%, followed by medical conservative treatment with a recurrence rate of 21.62%, while the recurrence rates of thoracic puncture and closed thoracic drainage are higher, which are 48.39% and 38.89%, respectively. However, the recurrence rate of PSP sporadic patients undergoing non-surgical treatment, including follow-up observation, puncture aspiration and closed thoracic drainage, is about 30% [17]. The difference of recurrence rate may be due to the fact that FSP patients often complicated with multiple pulmonary bullae, and the number and size of pulmonary bullae in these patients will develop with the extension of time, and eventually rupture and cause pneumothorax; Therefore, if the imaging data of FSP patients clearly have pulmonary bullae, surgical treatment is recommended; sometimes when patients have a large number of diffuse pulmonary vesicles, the operation is di cult to achieve the intended result and there is still a chance of pneumothorax recurrence. Mechanical or chemical pleurodesis is used in the operation to promote pleural cavity adhesion. According to Japanese reports, bioabsorbable pleura coverings have successfully protected patients from intractable pneumothorax 18-19 . This method enables patients to safely receive video-assisted thoracoscopic (VATS) treatment after several years of other lung diseases; However, there is still a lack of effective measures and follow-up data collection to support the treatment of pneumothorax with pleural covering technology, which needs to be veri ed by a large number of cases.
Although the study of FLCN gene mutation in FSP is more concerned than before, the development of pulmonary cysts in BHD syndrome is still unknown, we need to further study the relationship between these diseases. If BHD syndrome is diagnosed, the risk of renal tumor will be increased by 7 times, and the risk of pneumothorax will be increased by 50 times [20][21]. FSP patients may be more likely to develop kidney tumors. It is recommended that these patients should be regularly screened for renal ultrasound or nuclear magnetic resonance imaging.
This study has some limitations. First of all, we only tested the gene of the patients in the family. We need to further verify whether other members of the family have the same gene mutation. Secondly, we only made a retrospective analysis of this study, and the conclusion may be biased. Therefore, we need to conduct a large number of prospective studies to verify our conclusions.

Conclusion
In conclusion, this study shows that BHD syndrome is one of the most common causes of FSP. Patients with FSP should be recommended for mutation screening for the FLCN gene to facilitate early diagnosis and surgical intervention. Further studies are warranted to better understand the nature of this disease. Simpli ed family pedigree (spouses are omitted due to space limitation). The index number of generations is listed on the left using Roman numerals. The index number of certain individuals in each generation is listed on the lower using Arabic numerals. The proband is indicated by an arrow with a "P" on the lower left. Patients with pneumothorax are highlighted in black.