BHD syndrome is caused by small nucleotide alterations in the FLCN gene. A total of 132 different mutations have been identified, which are described in the Folliculin Sequence Variation Database. In a large study of 102 BHD syndrome families, only 86% had FLCN mutations identifiable by DNA sequencing (Toro et al., 2008). Although the remaining 14% were clinically characterised as having BHD syndrome, no FLCN mutations could be identified.
A recent paper by Benhammou et al. (2011) goes some way to explaining this lack of FLCN mutations. The authors used real-time quantitative PCR (RQ-PCR), multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridization (aCGH) to analyse 23 individuals from 15 unrelated families clinically confirmed as having BHD syndrome, but with no previous identifiable FLCN mutation. These techniques can detect deletions and duplications which are not identifiable by DNA sequencing.
The authors found intragenic FLCN deletions in six families and a FLCN duplication in one family. This is the first report of a FLCN duplication mutation. Of the six FLCN deletion mutations identified, four were found in the non-coding exon 1, suggesting a deletion ‘hot-spot’. This region was found to contain the FLCN promoter. A luciferase reporter assay confirmed that deletion of exon 1 dramatically reduces expression of the gene. The other two FLCN deletions were found to span exons 2-5 and exons 7-14. The duplication involves exons 10 and 11 and introduces a premature stop codon.
The Benhammou group at NIH also looked for genotype-phenotype correlations between the small nucleotide alterations and the large deletions/duplication. However, no difference was observed in the manifestations present or the age of BHD syndrome diagnosis, suggesting no genotype-phenotype correlation.
Although this study identified FLCN mutations in patients who were mutation-negative by DNA sequencing, 8/15 of families still had no identifiable FLCN mutation. RQ-PCR, MLPA and aCGH will detect deletions and duplications within exonic regions of FLCN but will miss mutations within introns as well as other epigenetic alterations. RNA transcript analysis would be necessary to identify mutations within introns. This study shows the importance of using alterative techniques to diagnose BHD syndrome when no FLCN mutations are detected by DNA sequencing.
- Benhammou, J., Vocke, C., Santani, A., Schmidt, L., Baba, M., Seyama, K., Wu, X., Korolevich, S., Nathanson, K., Stolle, C., & Linehan, W. (2011). Identification of intragenic deletions and duplication in the FLCN gene in Birt-Hogg-Dubé syndrome Genes, Chromosomes and Cancer DOI: 10.1002/gcc.20872
- Toro, J., Wei, M., Glenn, G., Weinreich, M., Toure, O., Vocke, C., Turner, M., Choyke, P., Merino, M., Pinto, P., Steinberg, S., Schmidt, L., & Linehan, W. (2008). BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dube syndrome: a new series of 50 families and a review of published reports Journal of Medical Genetics, 45 (6), 321-331 DOI: 10.1136/jmg.2007.054304