The ClinGen PTEN Expert Panel was organized by the ClinGen Hereditary Cancer Clinical Domain Working Group to assemble clinicians, researchers, and molecular diagnosticians with PTEN expertise to develop specifications to the 2015 ACMG/AMP Sequence Variant Interpretation Guidelines for PTEN variant interpretation. We describe finalized PTEN-specific variant classification criteria and outcomes from pilot testing of 42 variants with benign/likely benign (BEN/LBEN), pathogenic/likely pathogenic (PATH/LPATH), uncertain significance (VUS), and conflicting (CONF) ClinVar assertions. Utilizing these rules, classifications concordant with ClinVar assertions were achieved for 14/15 (93.3%) BEN/LBEN and 16/16 (100%) PATH/LPATH ClinVar consensus variants for an overall concordance of 96.8% (30/31). The variant where agreement was not reached was a synonymous variant near a splice donor with non-canonical sequence for which in silico models cannot predict the native site. Applying these rules to six VUS and five CONF variants, adding shared internal laboratory data enabled one VUS to be classified as LBEN and two CONF variants to be as classified as PATH and LPATH. This study highlights the benefit of gene-specific criteria and the value of sharing internal laboratory data for variant interpretation. Our PTEN-specific criteria and expertly reviewed assertions should prove helpful for laboratories and others curating PTEN variants.

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Despite significant structural diversity, present evidence suggests that EWS/ETS fusion proteins promote oncogenesis by transcriptionally modulating a common set of target genes. In order to identify these genes, microarray expression analyses were performed on NIH 3T3 polyclonal populations expressing one of three EWS/ETS fusion genes. The majority of these genes can be grouped into seven functional categories, including cellular metabolism and signal transduction. The biologic significance of these target genes was pursued. The effects of modulating genes involved in metabolism were assessed by flux studies and demonstrated shifts in glucose utilization and lactate production as a result of EWS/FLI1 expression. The proto-oncogene coding for serine/threonine kinase PIM3 was found to one of several genes encoding signal transduction proteins that were up-regulated by EWS/ETS fusions. PIM3 was found to be expressed in a panel of human Ewing's family tumor cell lines. Forced expression of PIM3 promoted anchorage-independent growth. Coexpression of a kinase-deficient PIM3 mutant attenuated EWS/FLI1-mediated NIH 3T3 tumorigenesis in immunodeficent mice.The EWS/ETS fusions comprise a group of structurally related oncoproteins that are found specifically in Ewing's family tumors (EFTs). The oncogenes coding for these oncoproteins are the result of chromosomal translocations that fuse a portion of the amino-terminal region of EWS to one of five members of the ETS family of transcription factors: FLI1, ERG, FEV, ETV1, and E1AF (for review, see reference 1). While ETS proteins have been extensively studied, the normal functions of EWS are still coming to light. EWS is part of a family of putative RNA binding proteins that also includes TLS and TAF II 68. A growing body of evidence suggests that members of this family (the TET family) may be involved in multiple levels of mRNA metabolism, from roles as transport chaperones to splicing factors (6,9,32,34,35). Interestingly, each of these three TET members is involved in tumor-associated chromosomal translocations associated with various human sarcomas, including desmoplastic small round cell tumor and clear cell sarcoma (3,11,17,21,25,26). In all cases, these translocations lead to the expression of chimeric proteins analogous to EWS/ETS fusions by incorporating DNA-binding domains (DBDs) from different transcription factors. Tumor histologies appear to correlate with the specific DBDs rather than the particular TET component in these fusions.All members of the ets-coded family of transcription factors contain a conserved 85-amino-acid domain that mediates sitespecific DNA binding at target gene promoters and enhancers (for review, see reference 16). Based on the primary amino acid sequences of their respective ets DBDs, the five EWS/ETS fusions can be partitioned into two groups. Group 1 contains EWS/FLI1, EWS/ERG, and EWS/FEV, and its members display up to 98% amino acid identity between their DBDs (FLI1 versus ERG). Similarly, in group 2, the ETV1 and E1AF DBDs are iden...

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder caused by mutations in ENG, ACVRL1 and SMAD4, which function in regulating the transforming growth factor beta and bone morphogenetic protein signaling pathways. Symptoms of HHT can be present in individuals who test negative for mutations in these three genes indicating other genes may be involved. In this study, we tested for mutations in two genes, RASA1 and GDF2, which were recently reported to be involved in vascular disorders. To determine whether RASA1 and GDF2 have phenotypic overlap with HHT and should be included in diagnostic testing, we developed a next-generation sequencing assay to detect mutations in 93 unrelated individuals who previously tested negative for mutations in ENG, ACVRL1 and SMAD4, but were clinically suspected to have HHT. Pathogenic mutations in RASA1 were identified in two samples (2.15%) and a variant of unknown significance in GDF2 was detected in one sample. All three individuals experienced epistaxis with dermal lesions described in medical records as telangiectases. These results indicate that the inclusion of RASA1 and GDF2 screening in individuals suspected to have HHT will increase the detection rate and aid clinicians in making an accurate diagnosis.

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