GENOMICS 9, 446-460 (1991)

cDNA Sequence and Genomic Structure of EWZB, a Lying within an lntron of the Neurofibromatosis Type 1 Gene

RICHARD M. CAWTHON,* LONE B. ANDmsm,t ARTHUR M. BUCHBERG,$ GANGFENG Xu,* PETER O’CONNELL, * DAVID VISKOCHIL, * ROBERT B. WEISS, * MARGARET R. WALL&x, t DOUGLAS A. MARcHuK,t MELANIE CULVER,* JEFFREY STEVENS,* NANCY A. JENKINS,* NEAL G. COPELAND,+ FRANCIS S. CoLms,t AND RAY WHITE* *Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84732; tHoward Hughes Medical institute and Departments of Internal Medicine and Human Gene06 University of Michigan, Ann Arbor, Michigan 48709; and SABL-Basic Research Program, NC/-Frederick Cancer Research and Development Center, Frederick, Maryland 2 1702

Received August6, 1990

nant disorder occurring at a frequency of 1 in 3000 The gene responsible for neurofibromatosis type 1 (Crowe et al., 1956). NFl is a clinical diagnosis de- (NFl), one of the more common inherited human disorders, fined by criteria agreed upon in a consensus confer- was identified recently, and segments of it were cloned. ence at the National Institutes of Health (Stumpf et Two translocation breakpoints that interrupt the NFl al., 1987). The most common physical signs of this gene in NFl patients flank a 60-kb segment of DNA that condition are multiple subcutaneous nodules (neuro- contains the EVIZA locus (previously reported as the EV12 fibromas) and hyperpigmented patches of skin (cafe- locus), the human homolog of a mouse gene, Evi-2A, impli- au-lait spots). Patients with NFl are at significantly cated in retrovirus-induced murine myeloid tumors. increased risk for development of malignancies (e.g., EVI2A lies within an intron of the NFl gene and is tran- see Riccardi and Eichner, 1986), including malignant scribed from telomere toward centromere, opposite to the schwannoma, pheochromocytoma, and rhabdomyo- direction of transcription of the NFl gene. Here we de- sarcoma. scribe a second locus, EVI2B, also located between the two NFl translocation breakpoints. Full-length cDNAs from Recently, the NFl gene was identified and seg- the EVZZB locus detect a 2.1-kb transcript in bone ments of it were cloned (Cawthon et al., 1990a; Visko- marrow, peripheral blood mononuclear cells, and fibro- chil et al., 1990a; Wallace et al., 1990a). Two balanced blasts. Sequencing studies predict an EVI2B of 448 translocations involving band 17q11.2 amino acids that is proline-rich and contains an N-terminal have been found in two unrelated NFl patients signal peptide, an extracellular domain with four potential (Schmidt et al., 1987; Ledbetter et al., 1989; Menon et glycosylation sites, a single hydrophobic transmembrane al., 1989); the breakpoints on lie ap- domain, and a cytoplasmic hydrophilic domain. At the level proximately 60 kb apart (O’Connell et al., 1990). An of genomic DNA the EVI2B locus lies within the same in- RNA PCR-based assay indicated that both transloca- tron of the NFl gene as EVI2A and contains a 57-bp B’exon tions prevent transcription of the NFl gene (Wallace that is noncoding, an 8-kb intron, and a 2078-bp 3’ exon et al., 1990a) on the affected chromosome 17, and hy- that includes the entire open reading frame. EVI2B is tran- bridization studies showed that NFl cDNAs are in- scribed in the same direction as EVI2A; its 5’exon lies only terrupted by both the t( 17;22) break (Viskochil et al., 4 kb downstream from the 3’ exon of the EVI2A locus. In 1990a) and the more centromeric t(1;17) break (Xu et the mouse the 5’ exon of the homologous gene, Evi-2B, lies al., 1990). We have previously reported the mapping approximately 2.8 kb from the 3’ end of Evi-2A, in the of the human homolog of a mouse gene implicated in midst of a cluster of viral integration sites identified in ret- leukemogenesis, hi-2A (ecotropic viral i