Genetic insight into Birt-Hogg-Dubé syndrome in Indian patients reveals novel mutations at FLCN

Abstract

Background: Birt-Hogg-Dubé syndrome (BHDS) is a rare monogenic condition mostly associated with germline mutations at FLCN. It is characterized by either one or more manifestations of primary spontaneous pneumothorax (PSP), skin fibrofolliculomas and renal carcinoma (chromophobe). Here, we comprehensively studied the mutational background of 31 clinically diagnosed BHDS patients and their 74 asymptomatic related members from 15 Indian families.

Results: Targeted amplicon next-generation sequencing (NGS) and Sanger sequencing of FLCN in patients and asymptomatic members revealed a total of 76 variants. Among these variants, six different types of pathogenic FLCN mutations were detected in 26 patients and some asymptomatic family members. Two of the variants were novel mutations: an 11-nucleotide deletion (c.1150_1160delGTCCAGTCAGC) and a splice acceptor mutation (c.1301-1G > A). Two variants were Clinvar reported pathogenic mutations: a stop-gain (c.634C > T) and a 4-nucleotide duplication (c.1329_1332dupAGCC). Two known variants were: hotspot deletion (c.1285delC) and a splice donor mutation (c.1300 + 1G > A). FLCN mutations could not be detected in patients and asymptomatic members from 5 families. All these mutations greatly affected the protein stability and FLCN-FNIP2 interaction as observed by molecular docking method. Family-based association study inferred pathogenic FLCN mutations are significantly associated with BHDS.

Conclusion: Six pathogenic FLCN mutations were detected in patients from 10 families out of 15 families in the cohort. Therefore, genetic screening is necessary to validate the clinical diagnosis. The pathogenic mutations at FLCN affects the protein-protein interaction, which plays key roles in various metabolic pathways. Since, pathogenic mutations could not be detected in exonic regions of FLCN in 5 families, whole genome sequencing is necessary to detect all mutations at FLCN and/or any undescribed gene/s that may also be implicated in BHDS.

Keywords: BHDS; FLCN mutations; Family-based association; Indian patients; Molecular docking; Primary spontaneous pneumothorax.

Fig. 1

Pathogenic mutations at FLCN found in patients and asymptomatic members along with their phenotypes. Light blue boxes represent exons and green boxes are exons with pathogenic mutations in FLCN. Most pathogenic mutations were found between exons 10–13. Circles indicate females, squares males. Family numbers are denoted in grey boxes with patients and asymptomatic members harboring pathogenic FLCN mutations. Patient phenotypes are also indicated in red, blue and green colors in individual symbols, and asymptomatic carriers are denoted with a pink dot

Fig. 2

FLCN exonic mutations and their effect on protein structure and interacting proteins. Green: N-terminal/Longin of FLCN, Brown: C-terminal of FLCN. Structures of homology modelled monomers of FLCN protein—(i) wild-type FLCN with amino-acid residues which were affected by mutations; (ii) stop-gain mutant FLCN affecting chain termination after Glu211 (i.e., p.Gln212*); (iii) indel (11-nucleotide) mutant FLCN affecting chain termination after Leu383 (i.e., p.Val384Phe*2); (iv) indel (single-nucleotide) mutant FLCN, altering the frame from His429 to Thr429, and subsequently creating a stop-codon after Leu435 in the model (i.e., p.His429Thr*39); (v) four-nucleotide duplicate mutant FLCN, altering the frame and terminating the chain after His442 in the model (i.e., p.Ala445Ser*11)

Fig. 3

Normalized ∆Ct (dCT) values of patients, asymptomatic and unrelated healthy controls obtained from FLCN Taqman copy number assays for exons 4, 8 and 13. For calculation of p-values, 2^−∆ct values from patients and asymptomatic members were compared with unrelated controls. Patients and unrelated controls (p-value: 0.019), and asymptomatic members and unrelated controls (p-value: 0.008) had a significant difference in copy number only for exon 8. Non-significant p-values have not been shown in the figures