TMPLi (TMP=2,2,6,6-tetramethylpiperidide) reacts with CuI salts in the presence of Et2O to give the dimers [{(TMP)2Cu(X)Li2(OEt2)}2] (X=CN, halide). In contrast, the use of DMPLi (DMP=cis-2,6-dimethylpiperidide) gives an unprecedented structural motif; [{(DMP)2CuLi(OEt2)}2LiX] (X=halide). This formulation suggests a hitherto unexplored route to the in situ formation of Gilman-type bases that are of proven reactivity in directed ortho cupration.

Insertional translocations (IT) are rare structural rearrangements. Offspring of IT balanced carriers are at high risk to have either pure partial trisomy or monosomy for the inserted segment as manifested by "pure" phenotypes. We describe an IT between chromosomes 3 and 13 segregating in a three-generation pedigree. Short tandem repeat (STR) segregation analysis and array-comparative genomic hybridization were used to define the IT as a 25.1 Mb segment spanning 13q21.2-q31.1. The phenotype of pure monosomy included deafness, duodenal stenosis, developmental and growth delay, vertebral anomalies, and facial dysmorphisms; the trisomy was manifested by only minor dysmorphisms. As the AUNA1 deafness locus on 13q14-21 overlaps the IT in the PCDH9 (protocadherin-9) gene region, PCDH9 was investigated as a candidate gene for deafness in both families. Genotyping of STRs and single nucleotide polymorphisms defined the AUNA1 breakpoint as 35 kb 5' to PCDH9, with a 2.4 Mb area of overlap with the IT. DNA sequencing of coding regions in the AUNA1 family and in the retained homologue chromosome in the monosomic patient revealed no mutations. We conclude that AUNA1 deafness does not share a common etiology with deafness associated with monosomy 13q21.2-q31.3; deafness may result from monosomy of PCHD9 or another gene in the IT, as has been demonstrated in contiguous gene deletion syndromes. Precise characterization of the breakpoints of the translocated region is useful to identify which genes may be contributing to the phenotype, either through haploinsufficiency or extra dosage effects, in order to define genotype-phenotype correlations.

Whole genome sequencing technologies recently identified novel recurrent mutations in childhood medulloblastomas (MB), including missense mutations in the DEAD box RNA helicase DDX3X. In the present study, we used complementary genomic approaches to illuminate normal DDX3X function and the impact of cancer-associated mutations. We used CLIPseq to identify the RNA targets of endogenous DDX3X. This analysis revealed that DDX3X primarily binds mature mRNAs and, further, that the distribution of DDX3X binding sites in targets spans the full length of these mRNAs, with enrichment in the coding regions, suggesting a role for DDX3X in translation elongation. Consistent with this observation, we detected a fraction of wild type DDX3X in polysomes by sedimentation experiments. In contrast, MB-associated DDX3X mutants appear to associate poorly with polysomes, but induce spontaneous stress granule (SG) formation in cells, suggesting a role of mutant DDX3X in triggering translation silencing. In fact, the RNA binding capabilities of DDX3X are still preserved in the context of cancer-associated mutations further supporting a role of mutant DDX3X in repressing translation of specific RNAs by retaining them within SGs. Interestingly, gene ontology analyses find mRNAs encoding translation factors themselves to be highly enriched among DDX3X RNA targets, suggesting that perturbations of DDX3X functions could have far-reaching impact on cellular translation. Indeed, knocking down DDX3X with siRNA or overexpressing a mutant led to a significant impairment in global translation as measured by S35-methionine metabolic labeling. Furthermore, assessment of whole-genome protein translation by ribosomal profiling analyses in cells overexpressing cancer-associated mutant DDX3X confirmed a decreased translation rate of direct DDX3X mRNA targets in particular, but also numerous other mRNAs. Finally, we have generated an allelic series of transgenic fruit flies expressing wild type or mutant versions of human DDX3X or its Drosophila ortholog Belle, and have captured mutation-dependent phenotypes when these transgenes are expressed in specific tissues. Together, our data show that DDX3X regulates the translation of a key subset of mRNAs and thereby indirectly influences cellular translation globally, and that this function is perturbed by cancer-associated mutations. This abstract is also presented as poster C48. Citation Format: Yasmine A. Valentin-Vega, Matthew Parker, Michael Rusch, Demelza Smeeth, Nam Chul Kim, Jinghui Zhang, Richard J. Gilbertson, Joseph P. Taylor. Medulloblastoma-associated mutations in the DEAD box RNA helicase DDX3X impair protein translation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR11.

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