Germline BHD-mutation spectrum and phenotype analysis of a large cohort of families with Birt-Hogg-Dubé syndrome

Abstract

Birt-Hogg-Dubé syndrome (BHD), a genodermatosis characterized by multiple hamartomas of the hair follicle (fibrofolliculoma), predisposes individuals to an increased risk of developing renal neoplasms and spontaneous pneumothorax. Previously, we localized the BHD locus (also known as FLCN) to chromosome 17p11.2 by linkage analysis and subsequently identified germline mutations in a novel gene in probands from eight of the nine families with BHD in our screening panel. Affected members of five of the families inherited an insertion/deletion of a cytosine in a C8 tract in exon 11. This mutation was also identified by exon 11 screening in probands from 22 of 52 additional families with BHD and therefore represents a hypermutable "hotspot" for mutation in BHD. Here, we screened the remaining 30 families from this large BHD cohort by direct sequence analysis and identified germline BHD mutations in 84% (51/61) of all families with BHD recruited to our study. Mutations were located along the entire length of the coding region, including 16 insertion/deletion, 3 nonsense, and 3 splice-site mutations. The majority of BHD mutations were predicted to truncate the BHD protein, folliculin. Among patients with a mutation in the exon 11 hotspot, significantly fewer renal tumors were observed in patients with the C-deletion than those with the C-insertion mutation. Coding-sequence mutations were not found, however, in probands from two large families with BHD whose affected members shared their family's BHD-affected haplotype. Of the 53 families with BHD whose members inherited either a germline mutation or the affected haplotype, 24 (45%) had at least one member with renal neoplasms. Three families classified with familial renal oncocytoma were identified with BHD mutations, which represents the first disease gene associated with this rare form of renal neoplasm. This study expands the BHD-mutation spectrum and evaluates genotype-phenotype correlations among families with BHD.

Figure 1

BHD mutations in affected families. Sequencing chromatograms of genomic DNA of patients with BHD are shown on the left (the arrow indicates position of nucleotide variation), and family pedigrees are shown on the right. Quadrants within pedigree symbols indicate presence of FFs (blue), lung cysts/pneumothorax (yellow), and/or renal tumor (red) in each family member. WT=wild-type BHD sequence; ND=not determined; diagonal line=deceased. A, Mutation in a splice-donor site in intron 9 (IVS9+2T→G) is predicted to cause exon skipping. B and C, Mutations at c.2034C→T and c.1844C→G encode premature termination codons predicted to truncate the BHD protein. D, A 2-bp deletion, c.1126delCA, will cause a shift in the reading frame and lead to a premature stop codon downstream, truncating the BHD protein.

Figure 2

BHD exon-intron structure showing the location of germline mutations identified in 51 families with BHD. Frameshift (red) and nonsense (blue) mutations, predicted to prematurely truncate the BHD protein, were identified along the entire length of the coding region (exons 4–14). Splice-site (yellow) mutations predicted to cause exon skipping were identified in introns 4 and 9. The insertion or deletion of a cytosine in a C₈ tract in exon 11 was identified in 27 of 51 families with BHD mutations.

Figure 3

Frequency of BHD phenotypic manifestations in patients with the C-insertion mutation versus the C-deletion mutation in the C₈ tract of exon 11. Patients with the c.1733delC mutation developed renal tumors at a significantly lower frequency than did patients with the c.1733insC mutation (P=.03). Hatched bar indicates c.1733insC mutation carriers (n=64); blackened bar indicates c.1733delC mutation carriers (n=27); unblackened bar indicates all BHD-mutation/haplotype carriers (n=219), included for comparison. Renal tumors were identified from medical histories, surgical reports, and abdominal CT scans. Spontaneous pneumothorax episodes were determined by patient history and thoracic CT scans. Lung cysts were identified by thoracic CT scans. Skin biopsies were evaluated by a dermatologist for FF-positive histology.