Comprehensive Molecular Genetic Diagnostics of Birt-Hogg-Dube Syndrome in a Russian Patient with Renal Cancer and Lung Cysts: A Case Report.

We report a case of Birt-Hogg-Dube syndrome (BHDS), a rare hereditary syndrome, the main visible sign of which is the development of multiple skin fibrofolliculomas. In our case, there was a manifestation of BHDS consisting in the absence of fibrofolliculomas and presence of other characteristic features of this syndrome: lung cysts and renal cancer. The 26-year-old woman was admitted to a clinic for diagnosis and treatment of a neoplasm of the left kidney and had a history of renal cell cancer (RCC) of the right kidney and spontaneous pneumothorax. Multiple tumors of the left kidney and lung cysts were observed upon clinical and laboratory testing. Tumors of the left kidney were resected and diagnosed by a pathologist as chromophobe RCC. Sequencing of FLCN exons 4-14 from blood DNA revealed the heterozygous germline nonsense mutation c.1429C>T (p.R477*), confirming the diagnosis of BHDS. Several somatic variants were detected by tumor DNA sequencing using the Comprehensive Cancer Panel and Ion S5 platform. Medical-genetic counseling was conducted, and follow-up management was outlined. To our knowledge, this case report is the first comprehensive clinical and genetic examination of a patient with BHDS in Russia. The p.R477* mutation has been described by other authors in patients with fibrofolliculomas and lung cysts, but not in those with RCC, while RCC was the first manifestation of BHDS in our case. The case report may help geneticists, oncologists, and other specialists to better understand the clinical and genetic heterogeneity of BHDS in various populations.

Novel tools for unbiased DNA differential methylation screening.

DNA differential methylation screening approaches may be hypothesis driven (preselection of the loci to screen) or unbiased (screening precedes mapping of differentially methylated loci). The latter allow for the identification of sequences demonstrating 'nonclassical' methylation behavior in cancer and, thus, widen our concept of tumor biology. Extensive employment of unbiased screening methods is hampered by the troublesome procedures involved in physically mapping the identified differentially methylated DNA fragments. In this special report, we describe a positive experience of optimizing two screening methods, methylation-sensitive arbitrarily primed PCR and amplification of intermethylated sites, with a special focus on simplification, or even exclusion, of sequencing procedures. With our modifications, unbiased screening of DNA differential methylation acquires an easy-to-use workflow, including sophisticated experimental design, high-resolution analysis and simple genomic mapping of the fragments of interest.

Methylation of the BIN1 gene promoter CpG island associated with breast and prostate cancer.

BACKGROUND: Loss of BIN1 tumor suppressor expression is abundant in human cancer and its frequency exceeds that of genetic alterations, suggesting the role of epigenetic regulators (DNA methylation). BIN1 re-expression in the DU145 prostate cancer cell line after 5-aza-2'-deoxycytidine treatment was recently reported but no methylation of the BIN1 promoter CpG island was found in DU145. METHODS: Methylation-sensitive arbitrarily-primed PCR was used to detect genomic loci abnormally methylated in breast cancer. BIN1 CpG island fragment was identified among the differentially methylated loci as a result of direct sequencing of the methylation-sensitive arbitrarily-primed PCR product and subsequent BLAST alliance. BIN1 CpG island cancer related methylation in breast and prostate cancers was confirmed by bisulphite sequencing and its methylation frequency was evaluated by methylation sensitive PCR. Loss of heterozygosity analysis of the BIN1 region was performed with two introgenic and one closely adjacent extragenic microsatellite markers.BIN1 expression was evaluated by real-time RT-PCR. RESULTS: We have identified a 3'-part of BIN1 promoter CpG island among the genomic loci abnormally methylated in breast cancer. The fragment proved to be methylated in 18/99 (18%) and 4/46 (9%) breast and prostate tumors, correspondingly, as well as in MCF7 and T47D breast cancer cell lines, but was never methylated in normal tissues and lymphocytes as well as in DU145 and LNCaP prostate cancer cell lines. The 5'-part of the CpG island revealed no methylation in all samples tested. BIN1 expression losses were detected in MCF7 and T47D cells and were characteristic of primary breast tumors (10/13; 77%), while loss of heterozygosity was a rare event in tissue samples (2/22 informative cases; 9%) and was ruled out for MCF7. CONCLUSION: BIN1 promoter CpG island is composed of two parts differing drastically in the methylation patterns in cancer. This appears to be a common feature of cancer related genes and demands further functional significance exploration. Although we have found no evidence of the functional role of such a non-core methylation in BIN1 expression regulation, our data do not altogether rule this possibility out.