Identification of recurring tumor-specific somatic mutations in acute myeloid leukemia by transcriptome sequencing.

Genetic lesions are crucial for cancer initiation. Recently, whole genome sequencing, using next generation technology, was used as a systematic approach to identify mutations in genomes of various types of tumors including melanoma, lung and breast cancer, as well as acute myeloid leukemia (AML). Here, we identify tumor-specific somatic mutations by sequencing transcriptionally active genes. Mutations were detected by comparing the transcriptome sequence of an AML sample with the corresponding remission sample. Using this approach, we found five non-synonymous mutations specific to the tumor sample. They include a nonsense mutation affecting the RUNX1 gene, which is a known mutational target in AML, and a missense mutation in the putative tumor suppressor gene TLE4, which encodes a RUNX1 interacting protein. Another missense mutation was identified in SHKBP1, which acts downstream of FLT3, a receptor tyrosine kinase mutated in about 30% of AML cases. The frequency of mutations in TLE4 and SHKBP1 in 95 cytogenetically normal AML patients was 2%. Our study demonstrates that whole transcriptome sequencing leads to the rapid detection of recurring point mutations in the coding regions of genes relevant to malignant transformation.

Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23.

BACKGROUND: The gene for the renal phosphate wasting disorder autosomal-dominant hypophosphatemic rickets (ADHR) is FGF23, which encodes a secreted protein related to the fibroblast growth factors (FGFs). We previously detected missense mutations R176Q, R179W, and R179Q in FGF23 from ADHR kindreds. The mutations replace R residues within a subtilisin-like proprotein convertase (SPC) cleavage site 176RHTR-179 (RXXR motif). The goal of these studies was to determine if the ADHR mutations lead to protease resistance of FGF-23. METHODS: The ADHR mutations were introduced into human FGF-23 cDNA clones with or without an N-terminal FLAG tag by site-directed mutagenesis and were transiently transfected into HEK293 cells. Protein expression was determined by Western analyses. RESULTS: Antibodies directed toward the C-terminal portion of FGF-23 revealed that the native FGF-23 protein resolved as 32 kD and 12 kD species in HEK293 conditioned media; however, the three mutated proteins were detected only as the 32 kD band. An N-terminal FLAG-tagged native FGF-23 resolved as two bands of 36 kD and 26 kD when detected with a FLAG antibody, whereas the R176Q mutant resolved primarily as the 36 kD protein species. Cleavage of FGF-23 was not enhanced by extracellular incubation of FGF-23 with HEK293 cells. Native and mutant FGF-23s bound heparin. CONCLUSIONS: FGF-23 proteins containing the ADHR mutations are secreted, and produce polypeptides less sensitive to protease cleavage than wild-type FGF-23. Therefore, the ADHR mutations may protect FGF-23 from proteolysis, thereby potentially elevating circulating concentrations of FGF-23 and leading to phosphate wasting in ADHR patients.

The autosomal dominant hypophosphatemic rickets (ADHR) gene is a secreted polypeptide overexpressed by tumors that cause phosphate wasting.

The gene mutated in autosomal dominant hypophosphatemic rickets (ADHR), a phosphate wasting disorder, has been identified as FGF-23, a protein that shares sequence homology with fibroblast growth factors (FGFs). Patients with ADHR display many of the clinical and laboratory characteristics that are observed in patients with oncogenic hypophosphatemic osteomalacia (OHO), a disorder thought to arise by the secretion of a phosphate wasting factor from different mesenchymal tumors. In the present studies, we therefore investigated whether FGF-23 is a secreted factor and whether it is abundantly expressed in OHO tumors. After transient transfection of OK-E, COS-7, and HEK293 cells with the plasmid encoding full-length FGF-23, all three cell lines efficiently secreted two protein species into the medium that were approximately 32 and 12 kDa upon SDS-PAGE and subsequent Western blot analysis using an affinity-purified polyclonal antibody to FGF-23. Furthermore, Northern blot analysis using total RNA from five different OHO tumors revealed extremely high levels of FGF-23 mRNA, and Western blot analysis of extracts from a sixth tumor detected the 32 kDa FGF-23 protein species. In summary, FGF-23, the gene mutated in ADHR, is a secreted protein and its mRNA is abundantly expressed by several different OHO tumors. Our findings indicate that FGF-23 may be a candidate phosphate wasting factor, previously designated "phosphatonin".