Barnacle: detecting and characterizing tandem duplications and fusions in transcriptome assemblies.

BACKGROUND: Chimeric transcripts, including partial and internal tandem duplications (PTDs, ITDs) and gene fusions, are important in the detection, prognosis, and treatment of human cancers. RESULTS: We describe Barnacle, a production-grade analysis tool that detects such chimeras in de novo assemblies of RNA-seq data, and supports prioritizing them for review and validation by reporting the relative coverage of co-occurring chimeric and wild-type transcripts. We demonstrate applications in large-scale disease studies, by identifying PTDs in MLL, ITDs in FLT3, and reciprocal fusions between PML and RARA, in two deeply sequenced acute myeloid leukemia (AML) RNA-seq datasets. CONCLUSIONS: Our analyses of real and simulated data sets show that, with appropriate filter settings, Barnacle makes highly specific predictions for three types of chimeric transcripts that are important in a range of cancers: PTDs, ITDs, and fusions. High specificity makes manual review and validation efficient, which is necessary in large-scale disease studies. Characterizing an extended range of chimera types will help generate insights into progression, treatment, and outcomes for complex diseases.

Correlation of clinical response and response duration with miR-145 induction by lenalidomide in CD34(+) cells from patients with del(5q) myelodysplastic syndrome.

We examined whether lenalidomide exposure up-regulates miRNAs and mRNAs, previously shown to play a role in the disease phenotype of del(5q) myelodysplastic syndrome, in pre-treatment CD34(+) marrow cells. We hypothesized that increased expression would predict for clinical response. Changes in miR-143, miR-145, miR-146a, miR-146b, miR-378, miR-584, SPARC and RPS14 were examined in del(5q) (n=10) and non-del(5q) (n=18) myelodysplastic syndrome patient samples. Significantly increased expression of miR-143 (1.8-fold and 1.5-fold in del(5q) and non-del(5q), respectively), and miR-145 (1.9-fold and 1.6-fold in del(5q) and non-del(5q), respectively) was observed. In the del(5q) myelodysplastic syndrome cohort, transfusion independence correlated with a 1.3-fold or more increase in miR-145 expression and response over 12 months correlated with a 1.5-fold or more increase. Knockdown of miR-143 and miR-145 in cord blood CD34(+) cells resulted in increased erythroid progenitor activity. Lenalidomide selectively abrogated progenitor activity in cells depleted of miR-143 and miR-145 supporting a key role for miR-143/145 in the sensitivity to lenalidomide of del(5q) myelodysplastic syndrome patients.

High-resolution whole genome tiling path array CGH analysis of CD34+ cells from patients with low-risk myelodysplastic syndromes reveals cryptic copy number alterations and predicts overall and leukemia-free survival.

Myelodysplastic syndromes (MDSs) pose an important diagnostic and treatment challenge because of the genetic heterogeneity and poorly understood biology of the disease. To investigate initiating genomic alterations and the potential prognostic significance of cryptic genomic changes in low-risk MDS, we performed whole genome tiling path array comparative genomic hybridization (aCGH) on CD34(+) cells from 44 patients with an International Prognostic Scoring System score less than or equal to 1.0. Clonal copy number differences were detected in cells from 36 of 44 patients. In contrast, cells from only 16 of the 44 patients displayed karyotypic abnormalities. Although most patients had normal karyotype, aCGH identified 21 recurring copy number alterations. Examples of frequent cryptic alterations included gains at 11q24.2-qter, 17q11.2, and 17q12 and losses at 2q33.1-q33.2, 5q13.1-q13.2, and 10q21.3. Maintenance of genomic integrity defined as less than 3 Mb total disruption of the genome correlated with better overall survival (P = .002) and was less frequently associated with transformation to acute myeloid leukemia (P = .033). This study suggests a potential role for the use of aCGH in the clinical workup of MDS patients.

Heterogeneity of breast cancer stem cells as evidenced with Notch-dependent and Notch-independent populations.

Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors, but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue, we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct, which inhibits signaling through all Notch receptors, and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease, but not a complete abrogation, of these cells in dnMAML-expressing cells. Interestingly, when assessed in secondary assays in vitro or in vivo, there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool, which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population.

Copy number alterations at polymorphic loci may be acquired somatically in patients with myelodysplastic syndromes.

Loss of genomic integrity is thought to be one of the underlying causes of myelodysplastic syndromes (MDS). However, it is unclear whether changes in copy number at loci that are common sites of copy number polymorphisms play a pathogenic role. Here we show that copy number changes in the MDS clone that occur at polymorphic loci are frequently somatic alterations rather than constitutional variants, and the extent of copy number changes at polymorphic loci is increased in CD34(+) cells of MDS patients compared to age-matched controls. This study suggests a potential pathophysiological role for copy number alterations at polymorphic loci in patients with MDS, and highlights the need for somatic control tissues for each patient studied in high-resolution genome-wide investigations.

Array comparative genomic hybridization of peripheral blood granulocytes of patients with myelodysplastic syndrome detects karyotypic abnormalities.

The diagnosis of myelodysplastic syndromes (MDSs) relies largely on morphologic and karyotypic abnormalities, present in about 50% of patients with MDS. Array-based genomic platforms have identified copy number alterations in 50% to 70% of bone marrow samples of patients with MDS with a normal karyotype, suggesting a diagnostic role for these platforms. We investigated whether blood granulocytes harbor the same copy number alterations as the marrow of affected patients. Of 11 patients, 4 had cytogenetic abnormalities shown by conventional karyotyping involving chromosomes 5, 8, 11, 20, and X, and these changes were seen in the granulocytes of all 4 patients by using array comparative genomic hybridization (aCGH). Cryptic alterations were identified at a significantly higher level in marrow CD34+ cells compared with granulocytes (P < .0001). These data suggest that aCGH analysis of circulating granulocytes may be useful in detecting gross karyotypic alterations in patients with MDS when marrow examination has failed or not been done.

Identification of miR-145 and miR-146a as mediators of the 5q- syndrome phenotype.

5q- syndrome is a subtype of myelodysplastic syndrome characterized by severe anemia and variable neutropenia but normal or high platelet counts with dysplastic megakaryocytes. We examined expression of microRNAs (miRNAs) encoded on chromosome 5q as a possible cause of haploinsufficiency. We show that deletion of chromosome 5q correlates with loss of two miRNAs that are abundant in hematopoietic stem/progenitor cells (HSPCs), miR-145 and miR-146a, and we identify Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) and tumor necrosis factor receptor-associated factor-6 (TRAF6) as respective targets of these miRNAs. TIRAP is known to lie upstream of TRAF6 in innate immune signaling. Knockdown of miR-145 and miR-146a together or enforced expression of TRAF6 in mouse HSPCs resulted in thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice transplanted with TRAF6-expressing marrow progressed either to marrow failure or acute myeloid leukemia. Thus, inappropriate activation of innate immune signals in HSPCs phenocopies several clinical features of 5q- syndrome.

Directional migration in the Hindu castes: inferences from mitochondrial, autosomal and Y-chromosomal data.

Genetic, ethnographic, and historical evidence suggests that the Hindu castes have been highly endogamous for several thousand years and that, when movement between castes does occur, it typically consists of females joining castes of higher social status. However, little is known about migration rates in these populations or the extent to which migration occurs between caste groups of low, middle, and high social status. To investigate these aspects of migration, we analyzed the largest collection of genetic markers collected to date in Hindu caste populations. These data included 45 newly typed autosomal short tandem repeat polymorphisms (STRPs), 411 bp of mitochondrial DNA sequence, and 43 Y-chromosomal single-nucleotide polymorphisms that were assayed in more than 200 individuals of known caste status sampled in Andrah Pradesh, in South India. Application of recently developed likelihood-based analyses to this dataset enabled us to obtain genetically derived estimates of intercaste migration rates. STRPs indicated migration rates of 1-2% per generation between high-, middle-, and low-status caste groups. We also found support for the hypothesis that rates of gene flow differ between maternally and paternally inherited genes. Migration rates were substantially higher in maternally than in paternally inherited markers. In addition, while prevailing patterns of migration involved movement between castes of similar rank, paternally inherited markers in the low-status castes were most likely to move into high-status castes. Our findings support earlier evidence that the caste system has been a significant, long-term source of population structuring in South Indian Hindu populations, and that patterns of migration differ between males and females.

Mutations in genes encoding fast-twitch contractile proteins cause distal arthrogryposis syndromes.

The distal arthrogryposes (DAs) are a group of disorders characterized by multiple congenital contractures of the limbs. We previously mapped a locus for DA type 2B (DA2B), the most common of the DAs, to chromosome 11. We now report that DA2B is caused by mutations in TNNI2 that are predicted to disrupt the carboxy-terminal domain of an isoform of troponin I (TnI) specific to the troponin-tropomyosin (Tc-Tm) complex of fast-twitch myofibers. Because the DAs are genetically heterogeneous, we sought additional candidate genes by examining modifiers of mutant Drosophila isoforms of TnI. One of these modifiers, Tm2, encodes tropomyosin, another component of the Tc-Tm complex. A human homologue of Tm2, TPM2, encodes beta-tropomyosin and maps to the critical interval of DA type 1 (DA1). We discovered that DA1 is caused by substitution of a highly conserved amino acid residue in beta-tropomyosin. These findings suggest that DAs, in general, may be caused by mutations in genes encoding proteins of the contractile apparatus specific to fast-twitch myofibers. This provides a new opportunity to directly study the etiology and pathogenesis of multiple-congenital-contracture syndromes.

Expressivity of Holt-Oram syndrome is not predicted by TBX5 genotype.

Mutations in TBX5, a T-box-containing transcription factor, cause cardiac and limb malformations in individuals with Holt-Oram syndrome (HOS). Mutations that result in haploinsufficiency of TBX5 are purported to cause cardiac and limb defects of similar severity, whereas missense mutations, depending on their location in the T box, are thought to cause either more severe heart or more severe limb abnormalities. These inferences are, however, based on the analysis of a relatively small number of independent cases of HOS. To better understand the relationship between mutations in TBX5 and the variable expressivity of HOS, we screened the coding and noncoding regions of TBX5 and SALL4 for mutations in 55 probands with HOS. Seventeen mutations, including six missense mutations in TBX5 and two mutations in SALL4, were found in 19 kindreds with HOS. Fewer than 50% of individuals with nonsense or frameshift mutations in TBX5 had heart and limb defects of similar severity, and only 2 of 20 individuals had heart or limb malformations of the severity predicted by the location of their mutations in the T box. These results suggest that neither the type of mutation in TBX5 nor the location of a mutation in the T box is predictive of the expressivity of malformations in individuals with HOS.