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Birt–Hogg–Dubé syndrome (BHDS) is a rare autosomal dominant disorder characterized by fibrofolliculomas, renal cancer, multiple pulmonary cysts, and spontaneous pneumothoraces [1]; however, its diagnosis requires satisfaction of one major or two minor diagnostic criteria as suggested by the European Birt-Hogg-Dubé consortium [1], and it predominantly results from mutations in the FLCN gene on chromosome 17p11.2, encoding folliculin, a tumor suppressor protein [2].
The estimated global prevalence of BHDS is 1.86 (95% confidence interval, 1.16–3.00) per million, with an unknown prevalence in Asia. BHDS can manifest at any age, typically at 40 [3]. Differences in incidence between the sexes remain debatable [4], as do genotype-phenotype correlations [3, 5]. BHDS exhibits phenotypic variability, with 80% of skin lesions [3, 6, 7], 80% of pulmonary cysts [3], 24–35% of pneumothorax [3, 8, 9], and 19–40% of renal cancer [3, 7, 10, 11] in Caucasian patients. Asian patients show slight differences, with fewer instances of skin lesions and renal cancer and more pulmonary manifestations, including pulmonary cysts [6, 8, 10, 12, 13]. Few BHDS cases have been reported in Korea, with clinical manifestations similar to those reported in previous Asian studies [5, 9]. This study aims to elucidate the clinical manifestations and genetic profiles of Korean patients with BHDS.
This study included nine patients from five families with pathogenic FLCN variants. Whole exome sequencing (WES) and multiple ligation-dependent probe amplification (MLPA) were performed on five probands, along with Sanger sequencing of the family members to confirm family-specific variants. The data included demographics, clinical traits, and test results from electronic medical records. Informed consent was obtained from the patients; the study adhered to the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Seoul National University Hospital (H-2204-144-1319).
Peripheral blood from patients, stored in EDTA disodium salt tubes, was subjected to DNA extraction using a Gentra Puregene Blood kit (Qiagen, Hilden, Germany). DNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). After sonication of the DNA using a Covaris system (Covaris, Inc., Woburn, MA, USA), target enrichment was performed using SureSelect Human All Exon software (version 5; Agilent, Santa Clara, CA, USA). Paired-end sequencing after exome capture was performed using an Illumina HiSeq 2000 or 2500 platform (Illumina, San Diego, CA, USA). DNA sequence analysis was performed using Next-GENe software (v2.4.0.1) (SoftGenetics, State College, PA, USA), and the sequences were aligned with the GRCh37/hg19 human reference genome. NM_144997.5 served as the reference sequence for the FLCN gene. Benign variants were filtered using Genome Aggregation and Korean Reference Genome Databases. In silico prediction tools such as SIFT, Mutation Taster, and Poly-Phen2 were employed. The Human Gene Mutation and ClinVar databases were referenced. The pathogenicity of each variant was determined as per the American College of Medical Genetics and Association for Molecular Pathology (ACMG/AMP) guidelines [14] and the latest guidelines by the ClinGen Sequence Variant Interpretation Working Group [15-17]. Variants were visualized using ProteinPaint (https://proteinpaint.stjude.org).
SALSA MLPA Probemix P256-C1 FLCN (MRC-Holland, Amsterdam, The Netherlands) was used to screen for large deletions or duplications of the FLCN gene according to the manufacturer’s instructions. Deletions and duplications were indicated by normalized peak ratios <0.8 and >1.2, respectively. We selected four nearby SNPs within a 2.5 Mb range of FLCN, including two downstream (rs56926918, rs56153623) and two upstream SNPs (rs2072652, rs2075659). The FLCN haplotype was reconstructed using PHASE 2.1.1 with 56 unrelated normal controls and four patients.
Among the nine patients with BHDS, five were from unrelated families. At the time of diagnosis, the mean age of the nine patients was 49.2±18.2 years, and 8/9 (89%) patients were females (Table 1). In our study, we identified four different pathogenic FLCN variants in nine patients from five families (Table 2). Splice site variants were observed in 3/5 (60%) patients. A nonsense and a frameshift variant were observed in each of the 1/5 (20%) families. All four variants were classified as pathogenic according to the ACMG/AMP [14] and ClinGen guidelines [15-17]. These pathogenic variants were predominantly localized to the distal FLCN, spanning exons 11 and 14 and introns 11 and 13. Among the BHDS families meeting clinical diagnostic criteria [1] (3.I.2 and 4), all of them (100%) had pathogenic FLCN variants.
The FLCN c.1539-2A>G variant, previously unreported in Korean patients with BHDS, was discovered in two unrelated families (1 and 3.I.2). Nine haplotypes were reconstructed (Supplementary Table 1), and haplotype H1 was observed in both unrelated families with the FLCN c.1539-2A>G variant (Supplementary Table 2), suggesting a common genetic background between these two families. MLPA analysis showed no large deletions or insertions, including a mutational hotspot [11]. The genotype-phenotype correlation was not significant, consistent with previous findings [6].
Bilateral multiple lung cysts were observed in 6/7 (86%) patients (mean age 52.2±12.0) from five unrelated families (Supplementary Fig. 1). Pneumothorax occurred in 2/9 (22%) patients, with one experiencing recurrent episodes starting five years before diagnosis. Pulmonary function tests were conducted in 4/9 unrelated patients, showing mild restrictive patterns in 1/4 (25%) and mildly decreased diffusion capacity in 2/3 (67%) (Supplementary Table 3). Further, 3/8 (38%) patients from the same family had multiple neck fibrofolliculomas, raising concerns regarding selection bias and necessitating histological confirmation. Renal cancer was observed in 2/9 (22%) patients, one with bilateral chromophobe renal cell carcinoma and the other with multilocular cystic renal neoplasm of low malignant potential. Renal cysts were observed in 4/5 (80%) patients. Malignant tumors, including breast cancer and papillary thyroid carcinoma, were observed in 2/9 (22%) unrelated patients. Benign tumors were observed in 6/9 (67%) patients and arterial hypertension in 3/9 (33%). Family history included pneumothorax in 3/9 (33%) and lung cancer in 1/9 (11%) patients, and five patients from one family had a family history of pathogenic FLCN variants. In family 3 with the c.1539-2A>G variant, the proband showed all pulmonary, renal, and skin manifestations. In contrast, the other family members showed varying clinical patterns. As BHDS is typically described in the 40s [3], patient 3.III.3 may not fully present the disease symptoms (Fig. 1).
This study investigated nine Korean patients with BHDS, with a mean diagnosis age of 49.2±18.2 years, consistent with previous Korean studies [5, 9]. The rates of pulmonary cysts (86%), renal cancer (22%), and skin lesions (38%) were similar to those reported in earlier studies on Korean patients with BHDS [9, 18]. However, the rate of pneumothorax (22%) was lower [5, 9, 18]. This pattern was comparable to that reported in previous studies on Asian patients [8, 13]. Compared to Caucasians, Korean patients with BHDS exhibited similar rates for pulmonary cysts [3], pneumothorax [3, 7, 8], and renal cancer [3, 7, 10, 11]; however, they had lower rates for skin lesions [3, 6, 7]. Recurrent pneumothorax, typically higher (75–80%) in Caucasians [4] and Koreans [9], was 50% in this study, emphasizing the need for larger-scale research due to the small population. The discrepant skin lesion rates in Asian patients [9, 12, 13] may be related to low biopsy rates [13], necessitating further research. The common renal cancers in Korean patients with BHDS are chromophobe renal cell carcinoma and hybrid oncocytic/chromophobe tumors [9]. Patients with BHDS have a sevenfold higher risk of renal cancer [3] and a 19.2% incidence of renal cysts [10]. Further, 4/5 (80%) of unrelated patients in this study had renal cysts, necessitating continuous monitoring. Breast cancer or papillary thyroid carcinoma was observed in 2/9 (22%) unrelated patients, underlining the importance of early screening.
Our study identified FLCN variants mainly in the exon and intron regions of FLCN, predominantly including splice site variants (60%), among nine patients from five families. This warrants further investigation into genotype-phenotype correlations. Similar to previous research [3, 7], a high variant detection rate of 100% was observed in clinical BHDS families that met the set criteria [1]; however, larger studies are needed for validation due to the limited number of included cases.
In conclusion, our study describes the genetic and clinical heterogeneity of BHDS in Korean patients and contributes to a better understanding of their comprehensive profiles.
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