Novel germline mutations in FLCN gene identified in two Chinese patients with Birt–Hogg–Dubé syndrome

Birt–Hogg–Dubé (BHD) syndrome, a hereditary renal cancer syndrome caused by mutations in the folliculin (FLCN) gene, is characterized by the presence of fibrofolliculomas, pulmonary cysts, spontaneous pneumothorax, and renal cell carcinoma (RCC). Few BHD syndrome cases have been reported in Asian countries, and cutaneous presentations are relatively rare in Asian patients. Asian BHD patients may be misdiagnosed due to their atypical manifestations. Here, we report two Chinese BHD patients with novel FLCN mutations (c.946-947delAG in exon 9 and c.770-772delCCT in exon 7). Both of them had RCC and spontaneous pneumothorax without fibrofolliculomas. In patients with RCC and pulmonary cysts but without cutaneous lesions, screening for mutations in the FLCN gene should be performed, especially for those with a family history of RCC or pulmonary cysts (pneumothorax).


Background
Birt-Hogg-Dubé syndrome (BHD, OMIM #135150) is a rare autosomal dominant hereditary disease first described by Birt et al. in 1977 [1]. This syndrome is characterized by the presence of fibrofolliculomas (FFs), pulmonary cysts, spontaneous pneumothorax, and renal cell carcinoma (RCC) [2]. Germline mutations in the folliculin (FLCN) gene mapped to chromosome 17p11.2 were identified in BHD patients in 2002 [3]. FLCN mutations have also been detected in isolated familial spontaneous pneumothorax and familial clear cell carcinoma [4]. FLCN is considered a tumor suppressor gene and interacts with the mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways. The spectrum of FLCN gene mutations has been outlined in several reports and has been summarized in a database (http://www.lovd.nl/flcn) [5]; however, the genotype-phenotype associations between the FLCN gene and BHD syndrome are not well known.

Most BHD cases have been identified in Caucasians.
Only several reports of compiled BHD cases in the literature have been from Asia, and most of them have been from Japan [6][7][8]. Many of the BHD patients from Asia have not had all symptoms (FFs, pulmonary cysts, spontaneous pneumothorax, and RCC) [8]. The most life-threatening manifestation of BHD syndrome is renal cancer, which is present in 27%-41% of patients [2,9,10]. RCCs in BHD patients can be multiple or bilateral and include various histopathologic types, such as hybrid oncocytic tumor, chromophobe RCC, clear-cell RCC, oncocytoma, and papillary RCC [10,11].
Here, we report two Chinese BHD patients with two novel germline FLCN mutations. Analysis of these patients was approved by the Medical Ethics Committee of Peking University First Hospital. Written informed consent was obtained from the patients and their families. pneumothorax (Fig. 1c). No apparent cutaneous lesions were found by careful inspection and palpation of the skin. The patient had a history of spontaneous pneumothorax at the age of 30. His son also had a history of spontaneous pneumothorax at the age of 17. Open bilateral and partial nephrectomies were performed. Histopathologic examination revealed that the tumor in the left kidney was chromophobe RCC, nuclear grade G2 (partial G1), and 1.7 cm × 1.5 cm × 1.5 cm in size (Fig. 1d); the tumor in the right kidney was clear-cell RCC, nuclear grade G2, and 5.0 cm × 4.7 cm × 4.5 cm in size (Fig. 1e).
Genomic DNA was extracted from blood samples collected from the patient and his son. Mutation examination revealed that the proband and his son carried a deletion of two bases (AG) at nucleotides c.946-947 in exon 9 (c.946-947delAG) of the FLCN gene (Fig. 1f ), which caused a frameshift mutation starting at the 316th amino acid (p.316fs388x). No RCC was detected by abdominal CT scan on the son.

Case 2
A 37-year-old man presented with an asymptomatic right renal mass detected by ultrasonography and CT (Fig. 2a). No apparent cutaneous lesions were found by careful inspection and palpation of the skin. The patient had a history of spontaneous pneumothorax on both sides and underwent pulmonary bullectomy on the right side at the age of 30. His father had a history of recurrent spontaneous pneumothorax starting at the age of 28 and died of stoke at the age of 63. His uncle had a history of recurrent spontaneous pneumothorax starting at the age of 24 and died of an accident at the age of 56. Open partial nephrectomy was performed on the patient. Histopathologic examination revealed that the tumor was chromophobe RCC, nuclear grade G2, and 2.2 cm × 2.0 cm × 2.0 cm in size (Fig. 2b).
Genomic DNA was extracted from blood samples collected from the patient and his family members. Mutation examination revealed that the proband carried a deletion of three bases (CCT) at nucleotides c.770-772 in exon 7 (c.770-772delCCT) (Fig. 2c), resulting in deletion of the 257th serine residue (p.257delS). No FLCN mutations were found in any other members of the patient's family.

Discussion
Most reported cases of BHD have been from Western countries, and reports of cases from Asia are rare, likely due to the lack of awareness and atypical manifestations of this disease in Asian patients. Identification of BHD patients is usually based on dermatologic signs. However, Kunogi et al. [6] found that among 30 Japanese BHD patients, only 6 (20.0%) had cutaneous lesions, 1 (3.3%) was histologically diagnosed with FFs, and 29 (96.7%)  [7] reported that 13 (28.8%) patients had FFs, 40 (88.9%) had pulmonary cysts, and 9 (20.0%) had RCC among 45 BHD patients from 19 Japanese families. In addition, Murakami et al. [8] compiled 62 BHD cases from Asia and found that FFs were detected in 17 (27.4%), pulmonary cysts in 49 (79.0%), and RCC in 11 (17.7%). In comparison, Toro et al. [9] found that among 51 BHD families in the United States, 46 (90.2%) had FFs, 45 (88.2%) had pulmonary cysts, and 25 (49.0%) had renal tumors. Kluger et al. [12] reported 22 patients from ten unrelated families with BHD in France; 18 (81.8%) patients had FFs, 16 (72.7%) had pulmonary cysts or a history of pneumothorax, and 10 (45.5%) had renal tumors. Therefore, the incidence of FFs may be lower among Asian BHD patients compared with the higher incidence of 80%-90% reported among patients from the United States and Europe, whereas the pulmonary cyst incidences are similar between patients from Asian and Western countries. FLCN mutation screening is recommended for patients with RCC and pulmonary cysts without cutaneous lesions, especially for those with a clear family history of RCC or pulmonary cysts/pneumothorax.
Over 100 unique mutations in the coding region of FLCN have been identified. Most of these mutations are frameshift, nonsense, insertion/deletion, or splice site mutations, resulting in truncation and inactivation of the encoded protein folliculin [15]. However, no clear genotype-phenotype associations between FLCN mutation type or location and skin, lung, or renal manifestations have been identified to date; only a significantly lower frequency of renal neoplasia has been reported in patients with a deleted cytosine in the exon 11 mutational "hot spot" compared with the patients with an inserted cytosine at this location [15,16]. A definite diagnosis of BHD can only be made when a pathogenic germline FLCN mutation is detected. Detection of an FLCN gene mutation not only confirms the diagnosis in suspected patients but also enables the diagnosis of at-risk family members with this disease. Once RCC is detected in BHD patients, it can be closely monitored as long as the dominant lesion is less than 3 cm in diameter. Surgical resection is recommended when the solid lesion or solid portion of a mixed lesion exceeds the threshold diameter of 3 cm [10]. For patients with bilateral renal tumors measuring greater than 3 cm, staged surgeries in two separate settings are recommended [11]. The tumor growth rate and location are additional factors that should be considered [2,11]. Recently, a kidney-specific knockout model was developed by disrupting mouse FLCN in proximal tubules [17]. This model provides a valuable tool for further study of FLCN-deficient renal tumorigenesis and for tests of new drugs (such as mTOR inhibitors) for the treatment of BHD-associated renal tumors.
Here, we report two Chinese patients with RCC and spontaneous pneumothorax who were diagnosed with