A total of 76 patients from 50 unrelated families were diagnosed as BHDS in our Rare Lung Disease Clinic from January 1, 2017 to August 31, 2022. All patients with BHDS were of Han ethnic group. Seventy patients were confirmed by genetic testing and the remaining six by clinical diagnosis. Among them, forty-seven were female and the ratio of male to female was 1:1.6. The average age of diagnosis was 44 ± 14 years (range, 18–76 years). Eight (11%) patients had a smoking history.

DNA samples from these 50 unrelated families were analyzed, of which unique pathogenic FLCN variants were identified in 62 patients from 45 families (90%) by Sanger sequencing (38 families) and NGS strategy (7 families), and three patients (12.2, 21.2 and 44.2) did not undergo genetic testing since their first-degree relatives carried germline FLCN mutations (Additional file 1: Table S1). The mutation spectrum revealed 20 different mutations, including seven nonsense, seven frameshift, three splice site, two missense, and one in-frame mutations (Fig. 1). A known mutational “hot spot”, c.1285dupC in exon 11, was detected in 9 patients from 7 families (14%) (Additional file 2: Fig. S1B). In addition, four heterozygous novel mutations were identified: c.761 T > C detected in three families, c.599 T > C in one family, c.1381_1382insA in one and c.1283_1284insA in one, respectively. The remaining 5 FLCN sequence-mutation negative families (10%) were detected to have a large intragenic deletion encompassing exons 1–3, of which 8 patients from 5 families were diagnosed by MLPA analysis (F13、F14、F15、F34、F40) (Fig. 2A). In addition, 3 patients (34.2, 34.3 and 34.4) in F34 were clinically diagnosed since they exhibited at least one of BHDS-related manifestations and the proband (34.1) was an exons 1–3 deletion carrier. The 1.8 kb PCR products of the junction fragment was separated by agarose gel electrophoresis, whereas the 9.3 kb wildtype sequence was too long to be amplified (Fig. 2A). Analysis of the sequencing results demonstrated one breakpoint to be located near chr 17: 17,134,286 and another breakpoint near chr 17: 17,141,828 with the estimated deletion size of 7,543 bp in length. Further analysis illustrated the involvement of AluSz and AluSc located on the up and downstream breakpoint junctions, respectively (Fig. 2B and C). A 23 bp stretch of microhomology in both Alu repeats suggested microhomology-mediated genomic recombination as a possible mechanism underlying in the deletion.

Point mutations at FLCN found in our cohort are listed. White boxes represent exons and yellow boxes represent exons with pathogenic point mutations in FLCN. The number of patients for each mutation is given in parentheses. *One patient in each of the three mutations did not undergo genetic testing, but the mutations were identified in their first-degree relatives

The results of identification of FLCN intragenic deletion in BHDS patients. A The electrophoretogram by MLPA on the left shows that the patient (F14-3) has a loss of heterozygosity in exon 1, 2 and 3 (in red); PCR products of junction fragments were separated by 2% agarose gel electrophoresis on the right. The characters and numbers above the lanes corresponded to the subjects (P1-P4) and controls (C1-C2). B and C The electrophoretogram and DNA sequence analysis of breakpoint junction show the 7,543 bp deletion mediated by the microhomology (blue bold bases in C) in Alu repeats (Alu SZ and Alu SC indicated by blue rectangular box in Fig. 2B and blue bases in C) span the exon 1 to 3 (E1-E3) of FLCN gene. The black arrows indicate the position of the primer for amplification of breakpoint junction in B

Interestingly, all 5 families harboring exons 1–3 deletion came from the same region, Feidong County, located near the center of Anhui Province; no kinship was identified among these families. Since nearly all BHDS patients in Anhui Province (including Feidong County) are evaluated and managed at our hospital, based on the most recent demographic data for Anhui Province at the end of 2021, the prevalence of BHDS in Anhui Province (population 61.13 million [14]) was estimated to be 1.24 cases per million. In Feidong County (population 1.09 million [15]), the carrier frequency of exons 1–3 deletion was 10.09 cases per million, which is 8.1 times higher prevalence of BHDS compared with that in Anhui population overall. The markedly increased frequency of exons 1–3 deletion carriers in a local region shows a clustered phenomenon of this variant in the FLCN gene.

Of 76 affected individuals, 11 cases were carriers of exons 1–3 deletion, while the other 65 harbored point mutations. The demographic characteristics of these subjects are summarized in Table 1. The average age at diagnosis for BHDS in intragenic deletion carriers was 47 ± 12 years (range, 19–73 years); 8 patients (73%) were females and only 1 had a history of smoking. There was no significant difference between the patients with exons 1–3 deletion and those with point mutations in age at diagnosis, gender distribution, and smoking history.

All 11 patients with large deletion had multiple bilateral pulmonary cysts, predominantly distributed in the lower lung fields (Fig. 3A). The cyst morphology varied greatly, mainly of round, oval or irregular shapes. All patients had pulmonary cysts > 20 mm, while the largest cysts in four (36%) were > 50 mm with the maximum size at 74 mm. For total number of cysts, 9 patients (82%) had more than 20 pulmonary cysts, 1 (9%) had 10–20 cysts; the remaining patient (9%) with fewer than 10 cysts were a 19-year-old female who was treated by bullectomy after suffering an episode of pneumothorax.

Pulmonary, kidney and skin involvements in BHDS patients with exons 1–3 deletion. A Chest CT showing multiple cysts distributed in the lower lungs (red arrows) and pneumothorax on the right. B Abdominal CT showing multiple bilateral kidney tumors with irregular edges (white arrows). C Hematoxylin and eosin staining of skin biopsy showing histologic features consistent with fibrofolliculoma (× 200)

Excluding 3 individuals without detailed CT data, multiple cysts were observed in 60 patients (97%) carrying point mutations including a 30-year-old male with only left-sided cysts. The largest cysts measured 20 to 50 mm in 31 patients (52%), and measured > 50 mm in 17 patients (28%). Sixty-three percent (38/60) of patients had more than 20 lung cysts and 25% (15/60) had 10–20 cysts. Although no significant difference in the sizes pulmonary cysts when comparing the two groups (exons 1–3 deletion versus point mutations), there was a tendency for the number of cysts in carriers of exons 1–3 deletion to be higher compared to those with point mutations (82% vs. 63%, p value 0.312).

Among 11 patients with intragenic deletion, 91% (10/11) experienced at least one episode of pneumothorax, with the mean age at the first episode of 36 ± 11 years (range, 19–58 years). The average number of pneumothorax was 2 ± 3 (range, 1–11). A female patient suffered the initial left pneumothorax at age 24 years and six episodes of ipsilateral recurrences, four episodes of contralateral pneumothoraces respectively in the following years despite three bullectomies and three chest tube drainage. A 43-year-old female has not developed pneumothorax yet, but her chest CT demonstrated more than 20 cysts with the largest cyst measuring 47 mm and located adjacent to the pleura.

58% (38/65) of sequence mutation-positive patients had experienced pneumothorax with an average number of 2 ± 2 episodes (range, 1–7). The mean age at onset of pneumothorax was 37 ± 12 years (range, 18–62 years). Mutation c.1285dupC was associated with a 33% risk (3/9) of pneumothorax. Significant difference was noted in pneumothorax risk between individuals with exons 1–3 deletion and those with point mutations (91% vs. 58%, p value 0.047). The risk of pneumothorax for subjects with exons 1–3 deletion was 2.8-times higher compared to those with c.1285dupC mutation (91% vs. 33%, p value 0.017). In addition, the relationship between history of pneumothorax and related factors mentioned above (smoking history, parameters related to pulmonary cysts, exons 1–3 deletion) was further examined by logistic regression analysis, which showed that only exons 1–3 deletion was associated with the presence of pneumothorax in BHDS (p value 0.028, odds ratio 10.000, 95% CI 1.280–78.117). However, there was no significant difference in the age at onset (p value 0.753) and the number of pneumothorax episodes between exons 1–3 deletion carriers and those with point mutation (p value 0.752).

Renal tumors were detected in 2 of 11 patients with exons 1–3 deletion (18%) (Fig. 3B). One patient underwent a partial nephrectomy for clear cell renal cell carcinoma at the age of 38 years. Due to poor performance status, another 73-year-old female was clinically diagnosed as having bilateral multifocal renal tumors by PET-CT. This patient underwent three renal artery embolization and died of advanced renal cancer 17 months later. Two patients with point mutations (4%) were found to have renal tumors as we previously described [6]. Large intragenic deletion carriers tended to be more likely to develop renal tumors compared to those with point mutations (18% vs. 4%, p value 0.126).

Eighty-nine percent (8/9) of carriers with exons 1–3 deletion manifested multiple pale or normal skin-colored papules on the face, neck, or upper trunk, of whom one underwent a biopsy and was confirmed to have fibrofolliculoma (Fig. 3C). Cutaneous involvement was seen in 78% (47/60) of point mutations carriers. Fibrofolliculomas were confirmed in 3 patients and trichodiscoma in 1. The prevalence of skin involvement in patients with large deletion and point mutations appeared to be similar (p value 0.674).