Clinical and genetic spectrum of Birt-Hogg-Dube syndrome patients in whom pneumothorax and/or multiple lung cysts are the presenting feature

Abstract

Background: Birt-Hogg-Dubé syndrome (BHDS) is an inherited autosomal genodermatosis characterised by fibrofolliculomas of the skin, renal tumours and multiple lung cysts. Genetic studies have disclosed that the clinical picture as well as responsible germline FLCN mutations are diverse.

Objectives: BHDS may be caused by a germline deletion which cannot be detected by a conventional genetic approach. Real-time quantitative polymerase chain reaction (qPCR) may be able to identify such a mutation and thus provide us with a more accurate clinical picture of BHDS.

Methods: This study analysed 36 patients with multiple lung cysts of undetermined causes. Denaturing high performance liquid chromatography (DHPLC) was applied for mutation screening. If no abnormality was detected by DHPLC, the amount of each FLCN exon in genome was quantified by qPCR.

Results: An FLCN germline mutation was found in 23 (63.9%) of the 36 patients by DHPLC and direct sequencing (13 unique small nucleotide alterations which included 11 novel mutations). A large genomic deletion was identified in two of the remaining 13 patients by qPCR (one patient with exon 14 deletion and one patient with a deletion encompassing exons 9 to 14). Mutations including genomic deletions were most frequently identified in the 3'-end of the FLCN gene including exons 12 and 13 (13/25=52.0%). The BHDS patients whose multiple cysts prompted the diagnosis in this study showed a very low incidence of skin and renal involvement.

Conclusions: BHDS is due to large deletions as well as small nucleotide alterations. Racial differences may occur between Japanese and patients of European decent in terms of FLCN mutations and clinical manifestations.

Figure 1

Analysis of the FLCN haploid copy number. FLCN copy number status (mean±SD) was determined by quantitative polymerase chain reaction (qPCR). The representative data from exon 8 to exon 14 of the FLCN gene, and the data of two reference genes, ZNF80 and GPR15, in two patients (JBR14 and JBR18) and normal genomic DNA (calibrator) are presented. JBR14 showed the gene dosage value close to 0.5 in 6 exons (exons 9 to 14), suggesting a large genomic deletion encompassing from exon 9 to exon 14. On the other hand, JBR18 showed the gene dosage value close to 0.5 only in exon 14, suggesting a genomic deletion of exon 14 and 3′-untranslated region. The gene dosage values of the remaining exons (exons 1 to 7) for JBR14 and JBR18 were close to 1.0 (data not shown).

Figure 2

Southern blot analysis of the FLCN gene. Genomic DNA from two patients (JBR14 and JBR18) and two normal controls (N1 and N2) was digested with either BamHI or EcoRI and Southern blotting was performed using ³²P-labelled hBHD cDNA probe (panel A). Size marker (Mr) was shown on the right. Distribution of 14 exons of the FLCN gene, restriction sites of BamHI and EcoRI, and expected length of fragment are schematically presented (panel B). The location of an undetermined restriction site which is involved in the generation of an aberrant band is indicated by a dotted arrow. Aberrant bands (a, b, c, d, and e) expected to be generated with a large genomic deletion (a thick shaded line) in JBR14 and JBR18 are depicted with the addition of a dotted line.

Figure 3

Comparison of FLCN germline mutation spectrum reported by the National Cancer Institute (NCI) group and the current study group. The upper half shows the result of NCI group while the lower half is the result of the current study (combined with the previously reported result and the present study). Symbols: red, single nucleotide substitution; blue, deletion and/or insertion; green, splice site mutation; the bars on the bottom, genomic deletion; a star, one patient.