Werner Emberger

Two transforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia.

Mutations leading to activation of the RAF-mitogen-activated protein kinase/extracellular signal-regulated (ERK) kinase (MEK)-ERK pathway are key events in the pathogenesis of human malignancies. In a screen of 82 acute myeloid leukemia (AML) samples, 45 (55%) showed activated ERK and thus were further analyzed for mutations in B-RAF and C-RAF. Two C-RAF germ-line mutations, S427G and I448V, were identified in patients with therapy-related AML in the absence of alterations in RAS and FLT3. Both exchanges were located within the kinase domain of C-RAF. In vitro and in vivo kinase assays revealed significantly increased activity for (S427G)C-RAF but not for (I448V)C-RAF. The involvement of the S427G C-RAF mutation in constitutive activation of ERK was further confirmed through demonstration of activating phosphorylations on C-RAF, MEK, and ERK in neoplastic cells, but not in nonneoplastic cells. Transformation and survival assays showed oncogenic and antiapoptotic properties for both mutations. Screening healthy individuals revealed a <1/400 frequency of these mutations and, in the case of I448V, inheritance was observed over three generations with another mutation carrier suffering from cancer. Taken together, these data are the first to relate C-RAF mutations to human malignancies. As both mutations are of germ-line origin, they might constitute a novel tumor-predisposing factor.

RT-PCR and FISH analysis of acute myeloid leukemia with t(8;16)(p11;p13) and chimeric MOZ and CBP transcripts: breakpoint cluster region and clinical implications.

The translocation t(8;16)(p11;p13) is associated with acute myeloid leukemia displaying monocytic differentiation (AML FAB M4/5) and fuses the MOZ (also named MYST3) gene (8p11) with the CBP (also named CREBBP) gene (16p13). Detection of the chimeric RNA fusions has proven difficult; only three studies have described successful amplification of the chimeric MOZ-CBP and CBP-MOZ fusions by reverse transcriptase-polymerase chain reaction (RT-PCR). We analyzed four cases of AML M4/5 with t(8;16)(p11;p13) by RT-PCR and fluorescence in situ hybridization (FISH) and characterized the reciprocal RNA fusions from three cases. We cloned both genomic translocation breakpoints from one case by long-range PCR and successfully applied RT-PCR to monitor minimal residual disease (MRD) between clinical complete remission and relapse. In three cases, the genomic breakpoints occurred in MOZ intron 16 and CBP intron 2. In one case, no fusion transcript was detected. The available data suggest clustering of t(8;16)(p11;p13) breakpoints in these introns leading to reciprocal in-frame MOZ exon 16/CBP exon 3 and in-frame CBP exon 2/MOZ exon 17 chimeric transcripts in the majority of cases. The described RT-PCR strategy may be valuable both for the routine detection of the t(8;16)(p11;p13) as well as for monitoring of MRD in this prognostically unfavorable patient group.

Assignment1 of the human GABAA receptor delta-subunit gene (GABRD) to chromosome band 1p36.3 distal to marker NIB1364 by radiation hybrid mapping

The gamma-aminobutyric acid type A (GABAA; Kuriyama et al., 1993) receptor represents a mechanism integral in functioning of the central nervous system and a locus for the action of many moodand emotion-altering agents such as benzodiazepines, barbiturates, steroids, and alcohol. Anxiety syndromes, sleep disorders, and convulsive disorders have been treated with therapeutic agents that enhance the action of gamma-aminobutyric acid (GABA) at the GABAA receptor or increase the concentration of GABA in nervous tissue (Rabow et al., 1995). GABAA receptors are heterooligomeric ligandgated ion channels that mediate the effect of the inhibitory neurotransmitter GABA which represents the main means of synaptic inhibition in neuronal tissue. The GABAA receptors consist of at least 15 different receptor subunits that can be classified into five subfamilies (alpha, beta, gamma, delta, rho). Sommer et al. isolated and characterized the murine gene for the GABAA receptor delta subunit (GABRD) by high-resolution mapping and DNA sequencing and they localized the human delta subunit gene to the short arm of chromosome 1. Materials and methods

Chronic myeloid leukemia with a rare variant Philadelphia translocation: t(9;10;22)(q34;q22;q11).

We report a 59-year-old, male, chronic myeloid leukemia patient with a rare variant Philadelphia (Ph) translocation t(9;10;22)(q34;q22;q11). Fluorescence in situ hybridization with whole chromosome paints was used to confirm the cytogenetic findings. With a BCR/ABL-specific probe, the known rearrangement on the derivative chromosome 22 was found. The prognostic implications as well as the relevance of the additional breakpoint region 10q22 are discussed.

Doublet-Mediated DNA Rearrangement—A Novel and Potentially Underestimated Mechanism for the Formation of Recurrent Pathogenic Deletions

Deletions and duplications of genomic DNA contribute to evolution, phenotypic diversity, and human disease. The underlying mechanisms are incompletely understood. We identified deletions of exon 10 of the SPAST gene in two unrelated families with hereditary spastic paraplegia. We excluded a founder event, but observed that the breakpoints map to identical repeat regions. These regions likely represent an intragenic “doublet,” that is, an enigmatic class of local duplications. The fusion sequences for both deletions are compatible with recombination‐based as well as with replication‐based mechanisms. Searching the literature, we identified a partial SLC24A4 deletion that involved two copies of another doublet, and was likely formed in an analogous way. Comparing the SPAST and the SLC24A4 doublets with doublets identified previously suggested that many additional doublets have a high potential for triggering rearrangements. Considering that doublets are still being formed in the human genome, and that they likely create high local instability, we suggest that a two‐step mechanism consisting of doublet generation and subsequent doublet‐mediated deletion/duplication may underlie certain copy‐number changes for which other mechanisms are currently assumed. Further studies are necessary to delineate the significance of the thus‐far understudied doublets for the formation of copy‐number variation.

Assignment1 of STAC to human chromosome band 3p22.3 between D3S3718 and D3S1611

Suzuki et al. (1996) recently isolated mouse and human cDNAs encoding the novel neuron-specific protein STAC. The protein has two functional domains, a cysteine-rich domain and an SH3 domain. Although the exact function of STAC is unknown so far, it is, because of it’s predominant expression in brain tissues, likely involved in a signal transduction pathway in neurons. Kawai et al. (1998) determined the chromosome location of the STAC gene in mouse to chromosome 9 and by radiation hybrid mapping in human to 3p24→p22. In another study we investigated a de novo complex chromosome rearrangement involving two breakpoints on 3p in a patient with spastic paraplegia (Emberger et al., in preparation). Since STAC is thought to contribute to several hereditary neurological diseases, we wanted to determine the location of this gene in order to evaluate its potential as a candidate gene for spastic paraplegia.