High accuracy mutation detection in leukemia on a selected panel of cancer genes.

With the advent of whole-genome and whole-exome sequencing, high-quality catalogs of recurrently mutated cancer genes are becoming available for many cancer types. Increasing access to sequencing technology, including bench-top sequencers, provide the opportunity to re-sequence a limited set of cancer genes across a patient cohort with limited processing time. Here, we re-sequenced a set of cancer genes in T-cell acute lymphoblastic leukemia (T-ALL) using Nimblegen sequence capture coupled with Roche/454 technology. First, we investigated how a maximal sensitivity and specificity of mutation detection can be achieved through a benchmark study. We tested nine combinations of different mapping and variant-calling methods, varied the variant calling parameters, and compared the predicted mutations with a large independent validation set obtained by capillary re-sequencing. We found that the combination of two mapping algorithms, namely BWA-SW and SSAHA2, coupled with the variant calling algorithm Atlas-SNP2 yields the highest sensitivity (95%) and the highest specificity (93%). Next, we applied this analysis pipeline to identify mutations in a set of 58 cancer genes, in a panel of 18 T-ALL cell lines and 15 T-ALL patient samples. We confirmed mutations in known T-ALL drivers, including PHF6, NF1, FBXW7, NOTCH1, KRAS, NRAS, PIK3CA, and PTEN. Interestingly, we also found mutations in several cancer genes that had not been linked to T-ALL before, including JAK3. Finally, we re-sequenced a small set of 39 candidate genes and identified recurrent mutations in TET1, SPRY3 and SPRY4. In conclusion, we established an optimized analysis pipeline for Roche/454 data that can be applied to accurately detect gene mutations in cancer, which led to the identification of several new candidate T-ALL driver mutations.

Analysis of human Pex19p's domain structure by pentapeptide scanning mutagenesis.

Pex19p, a primarily cytosolic protein, is essential for the biogenesis of numerous peroxisomal membrane proteins (PMPs); however, its precise function is unclear. Pex19p might function as a PMP-specific chaperone, a cycling PMP-receptor protein, a PMP membrane insertion factor, or an association/dissociation factor of membrane-associated protein complexes. Alternatively, Pex19p might act as a multifunctional peroxin and participate in a number of these activities. Here, we have employed transposon mutagenesis to generate a library of human pex19 alleles coding for Pex19p variants containing random in-frame pentapeptide insertions. A total of 87 different variants were characterized to identify functionally important regions. These studies revealed that Pex19p has a tripartite domain structure consisting of: (i) an amino-terminal domain that binds to Pex3p and is essential for docking at the peroxisome membrane; (ii) a central domain that competes with Pex5p and Pex13p for binding to Pex14p and may play a role in the assembly of PTS-receptor docking complexes; and (iii) a carboxy-terminal domain that interacts with multiple PMPs including Pex3p, Pex11pbeta, Pex12p, Pex13p, Pex16p, and Pex26p. Whether the latter interactions constitute the chaperone or transport functions (or both), remains to be determined. Finally, our observation that Pex19p contains two distinct binding sites for Pex3p suggests that the peroxin may bind PMPs in multiple places and for multiple purposes.

Potential role for Pex19p in assembly of PTS-receptor docking complexes.

Human Pex19p binds a broad spectrum of peroxisomal membrane proteins (PMPs). It has been proposed that this peroxin may: (i) act as a cycling PMP receptor protein, (ii) facilitate the insertion of newly synthesized PMPs into the peroxisomal membrane, or (iii) function as a chaperone to associate and/or dissociate complexes comprising integral PMPs already in the peroxisomal membrane. We previously demonstrated that human Pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences. Here we demonstrate that a mutant of Pex13p that fails to bind to Pex19p nevertheless targets to and integrates into the peroxisomal membrane. In addition, through in vitro biochemical analysis, we show that Pex19p competes with Pex5p and Pex13p for binding to Pex14p, supporting a role for this peroxin in regulating assembly/disassembly of membrane-associated protein complexes. To further examine the molecular mechanism underlying this competition, six evolutionarily conserved amino acids in the Pex5p/Pex13p/Pex19p binding domain of Pex14p were subjected to site-directed mutagenesis and the corresponding mutants functionally analyzed. Our results indicate that the physically overlapping binding sites of Pex14p for Pex5p, Pex13p, and Pex19p are functionally distinct, suggesting that competition occurs through induction of structural changes, rather than through direct competition. Importantly, we also found that amino acid substitutions resulting in a strongly reduced binding affinity for Pex13p affect the peroxisomal localization of Pex14p.