Torbjörn Säll

Coping with complexity: multivariate analysis of tumor karyotypes

Human cancers are characterized by chromosomal aberrations, and an increasing number of specific balanced rearrangements have been found among malignant hematologic disorders. Most solid tumors, however, exhibit a much more complex cytogenetic pattern. Although these chromosome changes show a nonrandom distribution, tumor-specific aberrations are uncommon, and the solid tumors often contain a large number of abnormalities and also display extensive cytogenetic variability. The high level of karyotypic complexity has made a systematic characterization of the chromosomal patterns difficult. In order to better understand the biological relevance of highly abnormal karyotypes in tumor cell populations, novel statistical strategies are needed. We have developed and adapted several methods that may be useful for the evaluation of general patterns of karyotypic complexity, including distribution analysis of cytogenetic imbalances, temporal analysis for time of occurrence of aberrations, and principal component analysis for reconstructing karyotypic pathways. By applying these methods on the chromosomal changes presently known, distinct subgroups have been identified among breast, kidney, bladder, colon, and brain tumors.

Competition as a source of errors in RAPD analysis.

We have used artificial 1∶1 DNA mixtures of all pairwise combinations of four doubled haploid Brassica napus lines to test the ability of RAPDs to function as reliable dominant genetic markers. In situations where a specific RAPD band is present in one homozygous line but absent in the other, the band is expected in the artificial heterozygote, i.e. in the 1∶1 DNA mixture. In 84 of all 613 heterozygous situations analysed, the expected band failed to amplify in the RAPD reaction. Thus, RAPD markers will lead to an erroneous genetic interpretation in 14% of all cases. In contrast, the formation of non-parental heteroduplex bands was found at a frequency of only 0.2%. Analysis of 1∶ 1 mixtures using (1) a different set of optimized reaction conditions and (2) a material with low genomic complexity (Bacillus cereus) gave identical results. Serial dilutions of one genome into another, in steps of 10%, showed that all of the polymorphic bands decreased in intensity as a linear function of their respective proportion in the mixture. In dilutions with water no differences in band intensity were detected. Thus, competition occurs in the amplification of all RAPD fragments and is a major source of genotyping errors in RAPD analysis.

Evaluation of RFLP and RAPD markers in a comparison of Brassica napus breeding lines

RFLP and RAPD markers were evaluated and compared for their ability to determine genetic relationships in a set of three B. napus breeding lines. Using a total of 50 RFLP and 92 RAPD markers, the relatedness between the lines was determined. In total, the RFLP and the RAPD analysis revealed more than 500 and 400 bands, respectively. The relative frequencies of loci with allele differences were estimated from the band data. The RFLP and RAPD marker sets detected very similar relationships among the three lines, consistent with known pedigree data. Bootstrap analyses showed that the use of approximately 30 probes or primers would have been sufficient to achieve these relationships. This indicates that RAPD markers have the same resolving power as RFLP markers when used on exactly the same set of B. napus genotypes. Since RAPD markers are easier and quicker to use, these markers may be preferred in applications where the relationships between closely-related breeding lines are of interest. The use of RAPD markers in fingerprinting applications may, however, not be warranted, and this is discussed in relation to the reliability of RAPD markers.

Identification of cytogenetic subgroups and karyotypic pathways of clonal evolution in follicular lymphomas

Follicular lymphoma (FL) is characterized by the activation of BCL2 through t(14;18)(q32;q21). Additional acquired mutations are necessary to generate a fully malignant clonal proliferation. Many of these secondary genetic alterations are visible in the clonal karyotype; however, the sequence by which they arise and their influence on clinical behavior have not been determined. The ability to address these issues has been hampered by the lack of computational methods to manipulate complex chromosomal data in a sufficiently large cohort of cases. In the present investigation, we analyzed secondary karyotypic alterations in 336 cases of FL with t(14;18) to identify the most common regions of recurrent chromosomal gain or loss. This revealed 29 recurrent changes present in more than 5% of the tumors. Each tumor karyotype was then assessed for the presence or absence of each of these 29 specific changes. By statistical means, we show that the chromosomal changes arise in an apparent temporal order, with distinct early and late changes. We identify, by principal‐components analysis, four possible cytogenetic pathways that characterize the early stages of clonal evolution, which converge to a common route at later stages. We show that FLs with t(14;18) may be classified into cytogenetic subgroups determined by the presence or absence of 6q−, +7, or der(18)t(14;18). Correlation with clinical outcomes in a subset of cases with clinical data revealed del(17p) and +12 to be correlated with an adverse clinical outcome. The clinical implications of these pathways of clonal evolution need to be examined on a prospective basis in a large cohort of FLs.

Dissecting karyotypic patterns in malignant melanomas: Temporal clustering of losses and gains in melanoma karyotypic evolution.

Malignant melanomas can be divided into two major subtypes, involving either the skin or eye melanomas. Both tumor forms exhibit highly complex karyotypes with nonrandom recurrent chromosomal imbalances. Loss of chromosome 3, the short arm of chromosome 1, and gain of 8q have been suggested to be associated with eye melanomas, whereas gain of 6p and loss of 6q have been more often seen in skin melanomas. Imbalances implicated in tumor progression include among others, −10 and +7. In spite of the abundance of cytogenetic information, with more than 300 published karyotypes, very little is known about the mode of karyotypic evolution or of the presence of possible cytogenetic pathways. In our investigation, we have used 362 melanoma karyotypes, including both the skin and eye subtypes, to identify the most frequently occurring imbalances. Tumor cases were then classified with respect to the presence or absence of these imbalances and statistically analyzed in order to assess the order of appearance of chromosomal imbalances, the presence of karyotypic pathways, as well as possible cytogenetic subtypes. We show that the melanomas develop through one mode of karyotypic evolution, common to both low and high complexity karyotypes, and we establish the temporal order by which the different imbalances occur. By applying several statistical methods, we show that at least two cytogenetic pathways of clonal evolution exist in malignant melanomas, one initiated with −3 and one with +6p, and that these pathways operate in both skin and eye melanomas.

Multivariate analyses of genomic imbalances in solid tumors reveal distinct and converging pathways of karyotypic evolution

A total of 3,016 malignant solid tumors (kidney, colorectal, breast, head and neck, ovarian, and lung carcinomas, neuroglial tumors, malignant melanoma, and testicular germ cell tumors) were selected for statistical analyses regarding karyotypic evolution. Genomic imbalances, i.e., net gains and losses, present in more than 5% of each tumor type were identified. Individual tumors were then classified with respect to absence or presence of these imbalances. To analyze for possible patterns of correlated imbalances, principal component analyses (PCA) were performed. Furthermore, algorithms were developed to analyze the temporal order of the imbalances, as well as the possible selection for early or late appearance in the karyotypic evolution. By analyzing the temporal order of imbalances common to many tumor types, a general order for nine of these emerged, namely, +7, −3p, −6q, −1p, −8p, −17p, −9p, −18, and −22. The distributions of the number of imbalances per case revealed a geometrical distribution, ranging from one to nine imbalances per tumor, in the majority of the tumor types. In tumor types in which cases with a high number of imbalances per case were frequent, notably head and neck, ovarian, and lung carcinomas, the overall distributions were bimodal, indicating the presence of two modes of chromosome evolution. By combining data from the PCA with the temporal analyses, it was possible to identify karyotypic pathways. It was found that the majority of the tumor types displayed more than one cytogenetic route, but, as the karyotypic evolution continued, these converged to a common pathway.

Phylogenetic analysis of the genus Hordeum using repetitive DNA sequences

A set of six cloned barley (Hordeum vulgare) repetitive DNA sequences was used for the analysis of phylogenetic relationships among 31 species (46 taxa) of the genus Hordeum, using molecular hybridization techniques. in situ hybridization experiments showed dispersed organization of the sequences over all chromosomes of H. vulgare and the wild barley species H. bulbosum, H. marinum and H. murinum. Southern blot hybridization revealed different levels of polymorphism among barley species and the RFLP data were used to generate a phylogenetic tree for the genus Hordeum. Our data are in a good agreement with the classification system which suggests the division of the genus into four major groups, containing the genomes I, X, Y, and H. However, our investigation also supports previous molecular studies of barley species where the unique position of H. bulbosum has been pointed out. In our experiments, H. bulbosum generally had hybridization patterns different from those of H. vulgare, although both carry the I genome. Based on our results we present a hypothesis concerning the possible origin and phylogeny of the polyploid barley species H. secalinum, H. depressum and the H. brachyantherum complex.

Yeast ''Make-Accumulate-Consume'' Life Strategy Evolved as a Multi-Step Process That Predates the Whole Genome Duplication

When fruits ripen, microbial communities start a fierce competition for the freely available fruit sugars. Three yeast lineages, including baker’s yeast Saccharomyces cerevisiae, have independently developed the metabolic activity to convert simple sugars into ethanol even under fully aerobic conditions. This fermentation capacity, named Crabtree effect, reduces the cell-biomass production but provides in nature a tool to out-compete other microorganisms. Here, we analyzed over forty Saccharomycetaceae yeasts, covering over 200 million years of the evolutionary history, for their carbon metabolism. The experiments were done under strictly controlled and uniform conditions, which has not been done before. We show that the origin of Crabtree effect in Saccharomycetaceae predates the whole genome duplication and became a settled metabolic trait after the split of the S. cerevisiae and Kluyveromyces lineages, and coincided with the origin of modern fruit bearing plants. Our results suggest that ethanol fermentation evolved progressively, involving several successive molecular events that have gradually remodeled the yeast carbon metabolism. While some of the final evolutionary events, like gene duplications of glucose transporters and glycolytic enzymes, have been deduced, the earliest molecular events initiating Crabtree effect are still to be determined.

Chiasma and recombination data in plants: are they compatible?

It has long been considered that the number of chiasmata formed during meiosis corresponds to the number of crossovers indicated by the genetic map. However, recent investigations in plants show an unexpected discrepancy in the results obtained when calculating the total number of crossover events per meiosis by these two methods. Is this discrepancy due to methodological difficulties? Or is there something fundamentally wrong with our understanding of crossovers and chiasmata?

Linkage disequilibrium mapping of the bolting gene in sea beet using AFLP markers.

The possibility of using linkage disequilibrium mapping in natural plant populations was assessed. In studying linkage disequilibrium among 137 mapped AFLP markers in four populations of sea beet (Beta vulgaris ssp. maritima (L.) Arcang.) it was shown that tightly linked loci could be detected by screening for associations. It was hypothesized that the short distances spanned by linkage disequilibrium enable markers that are very tightly linked to a target gene to be identified. The hypothesis was tested by whole-genome screening of AFLP markers for association with the gene for the annual growth habit, the B gene, in a sample of 106 sea beets. Despite the dominant nature of AFLP, two markers showing significant linkage disequilibrium with the B gene were detected. The results indicate the potential use of linkage disequilibrium for gene mapping in natural plant populations.