We have identified six intragenic deletions and one duplication in 47% (7/15) of unrelated BHDS families and characterized 5 of 7 breakpoints. Our data confirm that, in addition to protein-altering FLCN
frameshift, missense, nonsense and splice-site mutations, BHDS can be caused by large intragenic deletions as first described by Kunogi et al. (Kunogi et al., 2010
). Significantly, we have identified a “hot spot” in the exon 1/promoter region for FLCN
deletions and present here the first reported case of a large intragenic FLCN
repeats are associated with and may explain the majority of FLCN
deletions characterized in our cohort. Sixty percent (3/5) of the deletions identified (Families A, C and E) were flanked by Alu
repeats (SINEs), reported to be involved in Non-Allelic Homologous Recombination (NAHR) (Deininger et al., 1999
) in Alu
-mediated deletions implicated in other human cancers including VHL, breast cancer, Ewing’s sarcoma and HNPCC (Lehrman et al., 1986
; Mauillon et al., 1996
intragenic deletions in Families E and D involved more complex structural rearrangements. The inversion and subsequent intragenic deletion in Family E was the result of a recombination involving AluSg-
-flanked sequences, and another recombination event of unknown mechanism. The Family E deletion retained AluY
sequences at the 5′-deletion boundary but no definable repetitive elements at the 3′-deletion boundary; an AluJb
is located 982bp away. A similar mechanism has been described for the bleeding disorder Glanzmann Thrombasthenia (GT), where an Alu
-mediated inversion occurred followed by an Alu
-mediated deletion(Li et al., 1993
). The intragenic FLCN
deletion in Family D had no repetitive elements or homology to another DNA sequence on either side of the deletion boundaries. The closest repetitive elements were THE1B
located 436bp from the 5′-boundary, and AluJ,
located 1106bp from the 3′-deletion boundary. As has been suggested, (Stankiewicz et al., 2003
; Lee et al., 2006
) it is possible that repetitive elements near the gene could facilitate the deletion.
Low copy repeats (LCR) are DNA elements ranging from 1–200kb in size with >90% homology, which have been implicated in many chromosome 17 genetic rearrangements (Shchelochkov et al., 2010
). The density of these elements in the human genome ranges from 5–10%. However, their frequency in the proximal region of chromosome 17 is 23%, possibly accounting for the high rate of genetic rearrangements (Shchelochkov et al., 2010
). Stankiewicz et al. investigated the breakpoints of 18 genetic rearrangements in chromosome 17 and identified 9 patients (50% of their cohort) with LCRs at one breakpoint and non-LCR DNA sequences at the other boundary as identified in Family E (Stankiewicz et al., 2003
). Inoue et al. (2002)
also described 2 Pelizaeus-Merzbacher (PLP1
) families with deletions but no identifiable homologous sequences at the breakpoint boundaries. Possible mechanisms for the generation of these deletions include NAHR using only small segments of homology or Non-Homologous DNA End-Joining (NHEJ) where no homology is necessary (Lieber et al., 2003
). Another possibility is that novel repetitive elements could be involved in these rearrangements. These systems may not be mutually exclusive of each other.
We have identified the first intragenic duplication in the FLCN
gene. Although Alu
-mediated intragenic duplications have been well-described, (Schichman et al., 1994
; Yap et al., 2006
) no Alu
repeats or other repetitive elements were found at the breakpoint junction in BHDS Family G. We speculate that nonhomologous recombination or small homologous mechanisms are involved in the generation of the tandem duplication. Several examples in the literature, including two Duchenne muscular dystrophy families (Hu et al., 1991
) and a split hand–split foot malformation 3 (SHFM3) family (de Mollerat et al., 2003
) in which duplication events occurred at breakpoints without homologous sequences, further demonstrate that nonhomologous duplication events occur, although the mechanism remains poorly understood.
Our results demonstrate the importance of utilizing RQ-PCR, MLPA and/or aCGH as diagnostic methods in BHDS patients who are FLCN
mutation-negative by DNA sequencing, especially for the FLCN
promoter/exon1 region, where 67% of the deletions identified were located, and no point mutations had been reported (Toro et al., 2008
; Schmidt et al., 2005
). However, deletions and duplications involving exons did not account for all cases of BHDS in patients who were mutation-negative by sequencing (8/15 families, 53% of this cohort). Our RQ-PCR method will detect deletions and duplications within exonic regions of FLCN,
but will miss smaller deletions, duplications and other genetic rearrangements within introns (especially large introns 1, 3, 8 and 9) as well as epigenetic alterations and variations in the distal end of the 3′UTR.
The deletions and duplication we characterized most likely affect either the amount or function of the FLCN protein. Based on the marginal activity of the exon1/promoter deletion in the luciferase reporter assay, the exon 1 deletions in BHDS Families C-F would be predicted to dramatically reduce FLCN expression from the mutant allele suggesting that the commonly deleted region in these exon 1 deletion families contains the FLCN minimal promoter region.
The exon 2–5 deletion in BHDS Family A includes the initiation codon in exon 4, preventing normal translation. The duplication of exons 10 and 11 in BHDS Family G alters the reading frame of the transcribed mRNA resulting in a premature termination codon. The exon 7–14 deletion in BHDS Family B removes much of the coding region and the termination codon. If any protein were made from the encoded mRNA, its function would most likely be disrupted since FNIP1 and FNIP2, the FLCN-interacting proteins that also interact with AMPK, bind to the C-terminus of the FLCN protein (Baba et al., 2006
; Hasumi et al., 2008
; Takagi et al., 2008
). However, in most cases, the generation of a premature termination codon would result in degradation of the FLCN
mRNA by nonsense mediated decay (NMD) (Chang et al., 2007
Phenotypic findings in the BHDS patients with FLCN
deletions or duplication are very similar to those in the point mutation-positive patients (Schmidt et al., 2005
). Although major conclusions cannot be drawn from the small number of deletion and duplication-positive families (n=7), a few observations can be made. The most prominent findings are fibrofolliculomas, followed by lung cysts and kidney neoplasms. One patient had perifollicular fibromas (PFF), which have been described as part of the BHDS phenotype (Toro et al., 2008
). Notably, only one of 6 patients in the deletion-positive families developed kidney tumors. No significant difference was noted in the frequency of observed/reported phenotypic features between point mutation-positive patients and patients with a deletion or duplication. Additionally we report 6 of 15 patients with thyroid findings. A few BHDS cases have been described with thyroid adenomas or multinodular goiter, (De La Torre et al., 1999
; Drummond et al., 2002
) but the question of whether FLCN
plays a role in thyroid pathology, or BHD-associated thyroid findings reflect the high prevalence of thyroid disease in the general population (Rallison et al., 1991
), will await larger studies.
In conclusion this study confirms that large intragenic deletions in FLCN, in addition to sequence-altering germline mutations, are causative for BHDS, and reports the first large FLCN duplication in a BHDS patient. Large intragenic deletions and duplications of FLCN may account for at least 5% of cases of BHDS. Consequently RQ-PCR, MLPA and/or aCGH should be employed for clinical molecular diagnosis of BHDS in patients who are FLCN mutation-negative by DNA sequencing.