The phenotypes associated with BHD – fibrofolliculomas, pulmonary cysts with an increased risk of pneumothorax, and an increased risk of renal cell carcinoma (RCC) – show variable penetrance between and within families, making diagnosis difficult when only one symptom is present. Studies have found that up to 10% of patient cohorts diagnosed with Primary Spontaneous Pneumothorax (PSP) have folliculin (FLCN) mutations (Ren et al., 2008, Johannesma et al., 2014).
Patients suspected of having BHD undergo genetic testing via DNA sequencing to detect FLCN mutations. However, for a minority patients with clinical BHD no mutation can be detected using these techniques. Large intragenic deletions and duplications have been identified in such patients (Kunogi et al., 2010, Sempau et al., 2010, Benhammou et al., 2011). New work from Ding et al., (2015) has identified and mapped three large intragenic deletions in the FLCN genes of 40 patients from nine Chinese families with a family history of PSP and lung cysts.
Ding et al., (2015) analysed 12 families in total including five families identified in Ren et al., (2008) that were found not to have small mutations detectable by DNA sequencing. The families showed reduced fibrofolliculoma penetrance and no RCC. The families were identified through a proband admitted after a spontaneous pneumothorax, so this selection bias might make the reduction in fibrofolliculomas and RCC less significant. Larger comparative studies are required to determine if there are significant difference in phenotype penetrance in different ethnic groups.
Ding et al., used Multiplex Ligation-Dependent Probe Amplification (MLPA) and breakpoint analysis to identify a large deletion across exons 9-14 (c.872-492_1740+1763del) in five families, a deletion across exon 14 (c.1539-536_1740+1701del) in two families and a large deletion across exons 1-3 (c.-504-1303_-25+845del) in two families. The FLCN start codon is in exon 4 therefore deletion of exons 1-3 would disrupt the promotor reducing expression (Benhammou et al., 2011). The other deletions would prematurely truncate FLCN and as several identified functions rely on the C-terminal (Baba et al., 2006) these mutations would be pathogenic.
A 5.5Mb disease haplotype was identified around the exon 9-14 deletion suggesting a founder mutation. In addition all five families shared a point mutation in exon 5. This deletion was estimated to have occurred approximately 16 generations ago. Haplotypes around the exon 14 and exon 1-3 deletions were also found, however estimating the age was not possible from two families.
To date one large intragenic duplication and 11 large intragenic deletions (Figure 1) have been identified in 20 BHD families and 2 sporadic patients. Two of the deletions identified by Ding et al., have previously been reported (Kunogi et al., 2010) but not mapped in detail. The three deletions mapped in this work and several of the other deletions have Alu repeat sequences (black arrows in Figure 1) on either side. Interestingly the FLCN gene has a high density of Alu repeats compared to the genome in general and this increases the risk of homologous recombination resulting in deletions and duplications.
This work supports the conclusion that a percentage of PSP patients are actually undiagnosed BHD patients, and that the 10% previously suggested could be an underestimation if only DNA sequencing is used to detect FLCN mutations. MLPA analysis would therefore be an important and useful additional DNA testing tool for detecting mutations in suspected BHD patients.
Figure 1: Intragenic deletions and duplications identified in FLCN
Previously identified deletions and duplications are shown in blue and the three mutations found in Ding et al., (2015) are shown in red. Alu repeats are represented by black arrows. Taken from Ding et al., 2015, American Journal of Medical Genetics Part A 21 MAR 2015 DOI: 10.1002/ajmg.a.36979
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