A Novel FLCN Intragenic Deletion Identified by NGS in a BHDS Family and Literature Review

Abstract

Birt-Hogg-Dubé syndrome (BHDS, MIM #135150), caused by germline mutations of FLCN gene, is a rare autosomal dominant inherited disorder characterized by skin fibrofolliculomas, renal cancer, pulmonary cysts and spontaneous pneumothorax. The syndrome is considered to be under-diagnosed due to variable and atypical manifestations. Herein we present a BHDS family. Targeted next generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) revealed a novel FLCN intragenic deletion spanning exons 10-14 in four members including the proband with pulmonary cysts and spontaneous pneumothorax, one member with suspicious skin lesions and a few pulmonary cysts, as well as two asymptomatic family members. In addition, a linkage analysis further demonstrated one member with pulmonary bullae to be a BHDS-ruled-out case, whose bullae presented more likely as an aspect of paraseptal emphysema. Furthermore, the targeted NGS and MLPA data including our previous and present findings were reviewed and analyzed to compare the advantages and disadvantages of the two methods, and a brief review of the relevant literature is included. Considering the capability of the targeted NGS method to detect large intragenic deletions as well as determining deletion junctions, and the occasional false positives of MLPA, we highly recommend targeted NGS to be used for clinical molecular diagnosis in suspected BHDS patients.

Keywords: BHD syndrome; FLCN gene; MLPA; large intragenic deletion; targeted NGS.

FIGURE 1

Pedigree and clinical pictures of the family. (A) Pedigree and haplotype analysis. Numbers shown below each individual indicate the genotypes of the microsatellite markers in chromosomal order. The black bars represent the disease-associated haplotype. (B–D) Representative CT images of II-2 (B), I-1 (C), and II-1 (D); red frame: cyst; red arrow: bullae. (E) Several dome-shaped, skin-colored papules were seen on the face of II-1.

FIGURE 2

Identification of FLCN intragenic deletions and breakpoint analysis in II-2. (A) Representation of the normalize depth ratio of 20-bp intervals in FLCN gene detected by NGS method. The red dotted lines represent the average ratio of the deletion area. (B) MLPA analysis showed heterozygous exonic deletions (red arrows) in II-2 compared with the control individual. The characters and numbers below the probes indicate exons and control probes. (C) PCR products of junction fragments were separated by 2% agarose gel electrophoresis. The numbers above the lanes corresponded to the subjects of the Pedigree in Figure 1A. (D) Bidirectional sequencing of the junction region. The sequences of microhomology between the up and downstream breakpoints were marked in red; boxed nucleotides indicate the deletion and an arrow indicate the location of the deletion.

FIGURE 3

A comparative analysis of NGS and MLPA and a review of previously reported FLCN intragenic deletions. (A) Box-plot (median, box: 5–95th percentile, whiskers) representation of haploid copy number values for FLCN deleted exons (NGS-D and MLPA-D) and normal controls (NGS-N and MLPA-N) by NGS and MLPA, respectively. The red dotted line represents the cut-off value; exons with values <0.7 were scored as deleted, while >0.7 as normal status. (B) Genomic structure of the FLCN gene, showing the locations and sizes of previously reported large intragenic deletions found in BHD patients; black line: deletions previously reported. Red line: deletion found in this study. The lines with solid circles or arrowheads at both ends indicate the deletions with breakpoints determined or undetermined, respectively. (C) Small indels can show peak heights of heterozygous exonic deletions (normalized ratio <0.7) compared with the control individuals (black arrow) in MLPA analysis. DNA changes/FLCN probe sequences are presented in black frames, respectively. LPO (Left Probe Oligo) is the 5′ half of the probe; RPO (Right Probe Oligo) is the 3′ half of the probe. Red underlined characters indicate deleted bases. The characters and numbers below the probes indicate exons and control probes. The number below each MLPA result indicate sample number in our private sample database.