IDENTIFICATION OF THE GENE RESPONSIBLE FOR THE BIRT-HOGG-DUBÉ SYNDROME AND STUDIES OF BHD GENE EXPRESSION BY IN SITU HYBRIDIZATION

Abstract

The Birt-Hogg-Dubè (BHD) syndrome phenotype involves the development of fibrofolliculomas, spontaneous pneumothorax, and an increased risk for development of renal tumors. In order to localize the genetic locus responsible for BHD, a genome wide scan using 185 microsatellite markers was conducted using a large family with 40 affected members. Linkage analysis in 8 additional BHD families narrowed the linked region to 4 cM on chromosome 17p11.2. A physical map was created in silico using available genome browsers to identify candidate genes for mutation analysis in a panel of BHD patients. Simultaneously, 20 new microsatellite markers were designed in the linked region to analyze additional BHD families and linkage analysis identified new recombinants, which narrowed the linked region to 700kb between markers CA109 and D17S2196. After screening 39 candidate genes from the region of non combination, germline mutations were identified in 8 of 9 BHD families in a pair of uncharacterized overlapping mRNAs which detected a single transcript in multiple tissues by Northern blot analysis. Five different mutations were identified in 9 BHD families. Four of the 5 mutations resulted in a frameshift to protein truncation after a number of missense amino acids. The first mutation was a deletion of 2 nucleotides and the insertion of one nucleotide in exon 7 (1087delAGinsC). The second is a 28bp duplication in exon 9 (nt 1378-1405 duplication). Five families had an insertion or deletion of a C at exon 11 nt 1733. In addition 14 of 53 additional families tested had the same 1733insC mutation and 8 of 53 families had the 1733deI0 mutation. The fifth mutation was an amino acid substitution to a stop codon (C1844G). Extensive BLAST searches have not shown homology to any known gene and not identified key functional domains, suggesting that the BHD gene product, folliculin, is a novel protein. To gain information about BHD expression in kidney, lung and skin in situ hybridization was performed using an antisense fluorescent riboprobe. BHD mRNA expression was seen in kidney distal tubules, the inner and outer root sheaths of the hair follicle, keratinocytes surrounding the sebaceous glands, the spinous layer of the epidermis, stromal cells and type 1 pneumocytes in the lung. A tissue microarray was also used to identify other tissues that express the BHD mRNA including samples from the brain, tonsils, spleen, prostate, breast, pancreas and parotid. The acinar cells of the pancreas and parotid, as well as the epithelial ducts of the breast and prostate, strongly expressed BHD mRNA. Strong expression was seen in neurons of the cerebrum, Purkinje cells in the cerebellum, macrophage, and lymphocytes in the tonsils and spleen.