ABSTRACT
Introduction: Cell-free DNA (cfDNA) methylation is an important molecular biomarker, which provides information about the regulation of gene expression in the tissue of origin. There is an inverse correlation between SOST gene methylation and expression levels. Methods: We analyzed SOST promoter methylation in cfDNA from serum, and compared it with DNA from blood and bone cells from patients undergoing hip replacement surgery. We also measured cfDNA methylation in 28 osteoporotic patients at baseline and after 6 months of antiosteoporotic therapy (alendronate, teriparatide, or denosumab). Results: SOST gene promoter methylation levels in serum cfDNA were very similar to those of bone-derived DNA (79% ± 12% and 82% ± 7%, respectively), but lower than methylation levels in blood cell DNA (87% ± 10%). Furthermore, there was a positive correlation between an individual's SOST DNA methylation values in serum and bone. No differences in either serum sclerostin levels or SOST methylation were found after 6-months of therapy with antiosteoporotic drugs. Conclusions: Our results suggest that serum cfDNA does not originate from blood cells, but rather from bone. However, since we did not confirm changes in this marker after therapy with bone-active drugs, further studies examining the correlation between bone changes of SOST expression and SOST methylation in cfDNA are needed to confirm its potential role as a bone biomarker.
Subject(s)
Adaptor Proteins, Signal Transducing , Arthroplasty, Replacement, Hip , Cell-Free Nucleic Acids/metabolism , DNA Methylation , Osteoporosis/blood , Promoter Regions, Genetic , Aged , Aged, 80 and over , Biomarkers/metabolism , Female , Humans , Male , Middle AgedABSTRACT
Neurofibromatosis type 1 (NF1) is a hereditary disorder caused by mutations in the NF1 gene. Detecting mutation in NF1 is hindered by the gene's large size, the lack of mutation hotspots, the presence of pseudogenes, and the wide variety of possible lesions. We developed a method for detecting germline mutations by combining an original RNA-based cDNA-PCR mutation detection method and denaturing high-performance liquid chromatography (DHPLC) with multiplex ligation-dependent probe amplification (MLPA). The protocol was validated in a cohort of 56 blood samples from NF1 patients who fulfilled NIH diagnostic criteria, identifying the germline mutation in 53 cases (95% sensitivity). The efficiency and reliability of this approach facilitated detection of different types of mutations, including single-base substitutions, deletions or insertions of one to several nucleotides, microdeletions, and changes in intragenic copy number. Because mutational screening for minor lesions was performed using cDNA and the characterization of mutated alleles was performed at both the RNA and genomic DNA level, the analysis provided insight into the nature of the different mutations and their effect on NF1 mRNA splicing. After validation, we implemented the protocol as a routine test. Here we present the overall unbiased spectrum of NF1 mutations identified in 93 patients in a cohort of 105. The results indicate that this protocol is a powerful new tool for the molecular diagnosis of NF1.
Subject(s)
Chromatography, High Pressure Liquid/methods , DNA Mutational Analysis/methods , Genes, Neurofibromatosis 1 , Germ-Line Mutation , Neurofibromatosis 1/diagnosis , Neurofibromatosis 1/genetics , Polymerase Chain Reaction/methods , DNA Copy Number Variations , Humans , Molecular Diagnostic Techniques , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
BACKGROUND: Neurofibromatosis type 1 is a common autosomal dominant disorder with full penetrance and variable expression. The condition predisposes individuals to the development of malignant nervous system tumours, most frequently Malignant Peripheral Nerve Sheath Tumours (MPNSTs). Previous studies indicate that genetic factors other than mutations in NF1 may be responsible for the condition's variable expression. CASE REPORT: Here we present data from a pair of monozygotic twins affected by Neurofibromatosis type 1 resulting from a de novo mutation. Both twins developed a left sciatic plexiform neurofibroma that evolved into MPNST at a similar age and they also developed pulmonary metastasis at the same age. Other concordant traits between the twins were: macrocephaly, psychomotor delay, café-au-lait spots, cutaneous neurofibromas, retroperitoneal, pleural and paraspinal neurofibromas. The main discordant features observed were tibial pseudoarthrosis, pectus carinatum, osteoporosis and thymus hyperplasia. CONCLUSIONS: This is the first report of monozygotic twins with Neurofibromatosis type 1 that develop MPNSTs, the localization and chronological evolution of which, and its metastasis, is concordant in both twins. These cases suggest that the events involved in the transformation of benign plexiform neurofibromas to MPNSTs in Neurofibromatosis type 1, follow a spatiotemporally programme that is influenced by heritable factors other than NF1 mutations.
