Analysis of myelodysplastic syndromes with complex karyotypes by high-resolution comparative genomic hybridization and subtelomeric CGH array

Molecular cytogenetic techniques enabled us to clarify numerical and structural alterations previously detected by conventional cytogenetic techniques in 37 patients who had myelodysplastic syndromes with complex karyotypes. Using high-resolution comparative genomic hybridization (HR-CGH), we found the most recurrent alterations to be deletion of 5q (70%), 18q (35%), 7q (32%), 11q (30%), and 20q (24%), gain of 11q (35%) and 8q (24%), and trisomy of chromosome 8 (19%). Furthermore, in 35% of the patients, 20 amplifications were identified. These amplifications were shown by FISH to involve some genes previously described as amplified in hematological malignancies, such as ERBB2, MLL, and RUNX1. In addition, two other genes, BCL6 and BCL2, which are classically related to apoptosis and non-Hodgkin lymphoma, were shown for the first time to be involved in amplification. Genomic alterations involving different subtelomeric regions with losses in 4p16, 5p15.3, 6q27, 18p11.3, and 18q23 and gains in 1p36.3 and 19p13.3 were detected by HR-CGH. Array CGH analysis of the subtelomeric regions in some samples was able to confirm a number of these alterations and found some additional alterations not detected by conventional CGH.     

Statistical behavior of complex cancer karyotypes

Epithelial tumors commonly show complex and variable karyotypes that obscure the identification of general patterns of the karyotypic evolution. To overcome some of these problems, we previously systematically analyzed the accumulated cytogenetic data from individual tumor types by using various statistical means. In the present study, we compare previous results obtained for nine tumor types and perform several meta-analyses of data obtained from a number of epithelial tumors, including head and neck, kidney, bladder, breast, colorectal, ovarian, and lung cancer, as well as from malignant melanoma and Wilms tumor, with the specific aim of discovering common patterns of karyotypic evolution. We show that these tumors frequently develop through a hypo- or a hyperdiploid pathway and progress by an increasing number of alternative imbalances through at least two karyotypic phases, Phases I and II, and possibly through a third, Phase III. During Phase I, the karyotypes exhibited a power law distribution of both the number of changes per tumor and the frequency distribution at which bands were involved in breaks. At the transition from Phase I to Phase II/III, the observed power law distributions were lost, indicating a transition from an ordered and highly structured process to a disordered and chaotic pattern. The change in karyotypic orderliness at the transition from Phase I to Phase II/III was also shown by a drastic difference in karyotypic entropy.     

Unusual complex hyperdiploid karyotypes in myelodysplastic syndromes

Over an 18-year period, 10 myelodysplastic syndrome (MDS) patients with complex hyperdiploid karyotypes were identified. According to the FAB classification, the 10 patients were subclassified as three refractory anemias (RA), three refractory anemias with excess blasts (RAEB), two RAEB in transformation (RAEB-t), and two unclassified MDS. According to the WHO classification, the diagnoses were two RA, one refractory cytopenia with multilineage dysplasia, two RAEB-1, one RAEB-2, two unclassified MDS, and two acute myeloid leukemia. Six were secondary MDS. Four patients showed marked dyserythropoiesis; three of these were secondary MDS. The chromosome number ranged from 47 to 62, and clonal evolution or composite karyotypes were noted in 7 patients. Seven patients had at least one clone with >50 chromosomes. Recurrent defects included chromosome 5, 17, and 13 abnormalities. Notably, trisomy 8 and monosomy 7 were rare in that group of patients. Three of four patients with marked dyserythropoiesis shared abnormalities of both chromosomes 13 and 17.     

Array comparative genomic hybridization analysis in first-trimester spontaneous abortions with 'normal' karyotypes

Array comparative genomic hybridization (array CGH) analysis was conducted in chorionic villous samples from 20 first-trimester spontaneous abortions with G-banding normal chromosomes. A microarray, containing 2,173 BAC clones and covering the whole genome with a 1.5-Mb resolution, was constructed and used in the analysis. Two deletions were identified: a 1.4-Mb deletion at 3p26.2-p26.3 and a 13.7-Mb deletion at 13q32.3-qter. Reexamination of chromosome preparations from the sample with the 13.7-Mb deletion documented a mixture of cells with the 13q- chromosome and those with 46,XX chromosomes, the latter of which are likely to have been derived from contaminating decidual cells. This left the 1.4-Mb 3p deletion as the only instance with submicroscopic imbalance detected, giving a frequency of 1 in 19 (5%) G-banding normal abortions.     

Comparative analysis of trichomonad genome sizes and karyotypes

In parasitic protists, the genome sizes range from 2.9Mb in Encephalitozoon cuniculi to about 160Mb in Trichomonas vaginalis. The suprisingly large genome size of the former human parasite resulted from the expansion of various repetitive elements, specific gene families, and possibly from large-scale genome duplication. The reason for this phenomenon, as well as whether other trichomonad species have undergone a similar genome expansion, is not known. In this work we studied the genomes of nine selected species of the Trichomonadea group. We found that each species has a characteristic karyotype with a stable and haploid number of chromosomes. Relatively large genome sizes were found in all the tested species, although over a rather broad range (86-177Mb). The largest genomes were typically observed in the Trichomonas and Tritrichomonas genera (133-177Mb), while Tetratrichomonas gallinarum contains the smallest genome (86Mb). The genome size correlated with the cell volume, however, no relationship between genome size and the site of infection or trichomonad phagocytic ability was observed. The data presented here provide primary information towards selecting a trichomonad species for future large-scale sequencing to elucidate the evolution of unusual parabasalid genomes.     

Comparative genomic hybridization analysis of human oocytes and polar bodies

BACKGROUND: Classical cytogenetic methods and fluorescent in situ hybridization (FISH) have been employed for the analysis of chromosomal abnormalities in human oocytes. However, these methods are limited by the need to spread the sample on a microscope slide, a process that risks artefactual chromosome loss. Comparative genomic hybridization (CGH) is a DNA-based method that enables the investigation of the entire chromosome complement. We optimized and evaluated a CGH protocol for the chromosomal analysis of first polar bodies (PBs) and oocytes. The protocol was then employed to obtain a detailed picture of meiosis I errors in human oogenesis. METHODS: 107 MII oocyte-PB complexes were examined using whole genome amplification (WGA) and CGH. RESULTS: Data was obtained for 100 complexes, donated from 46 patients of average age 32.5 (range 18-42). 22 complexes from 15 patients were abnormal, giving an aneuploidy rate of 22%. CONCLUSIONS: The results presented in this study more than double the quantity of CGH data from female gametes currently available. Abnormalities caused by whole chromosome non-disjunction, unbalanced chromatid predivision and chromosome breakage were reliably identified using the CGH protocol. Analysis of the data revealed a preferential participation of chromosome X and the smaller autosomes in aneuploidy and provided further evidence for the existence of age-independent factors in female aneuploidy.     

Flow cytometric analysis of oncogene expression in human neoplasias

More than 40 oncogenes have been described and characterized. These oncogenes code for products intimately associated with cell proliferation. Their products include growth factors, growth factor receptors, transmembrane signaling molecules, second and third messengers, and nuclear proteins involved in gene regulation. Some oncogenes seem to be expressed in every malignancy, while others seem to be expressed only in certain malignancies. This expression pattern suggests some oncogenes may have tissue type specificity. Some oncogenes may also be expressed in only a fraction of the malignant cells, such as the proliferating cells. Flow cytometry has only recently been applied to the simultaneous measurement of oncoprotein levels and DNA content of malignant cells. As this application is developed, our understanding of oncogene expression in malignancy will be enhanced and its diagnostic importance will be realized.     

The role of CD24 in various human epithelial neoplasias

CD24 is a small, heavily glycosylated glycosylphosphatidylinositol-linked cell surface protein, which is expressed in hematologic malignancies and in a large variety of solid tumors. It appears to function as a ligand of P-selectin, an adhesion molecule present in activated platelets and endothelial cells. The authors aimed at evaluating CD24 protein expression in adenoma and adenocarcinoma of the stomach, colon, gallbladder, ovary, and breast to establish a correlation with clinicopathologic data. Staining was evaluated using four degrees of positivity (negative, weakly, moderately, strongly positive), and the staining patterns (membranous vs. intracytoplasmic) were analyzed for statistical analysis. The present study clearly demonstrates that CD24 is abundantly expressed in adenocarcinoma compared to adenoma of the colon and breast. Moreover, the positivity degree of CD24 expression increases with positive nodal status in advanced gastric carcinoma. Intracytoplasmic CD24 expression was found to be highly associated with adenocarcinoma of the colon, gallbladder, and ovary compared to the adenoma group of those organs, and with the positive nodal status compared to the negative nodal status of the colonic adenocarcinoma. We conclude that the degree of positivity and the staining pattern of CD24 constitute an important molecular marker for various epithelial neoplasms, which could help to define malignant transformation and to predict lymph node metastasis.     

Comparative genomic analyses of the human fungal pathogens Coccidioides and their relatives

While most Ascomycetes tend to associate principally with plants, the dimorphic fungi Coccidioides immitis and Coccidioides posadasii are primary pathogens of immunocompetent mammals, including humans. Infection results from environmental exposure to Coccidiodies, which is believed to grow as a soil saprophyte in arid deserts. To investigate hypotheses about the life history and evolution of Coccidioides, the genomes of several Onygenales, including C. immitis and C. posadasii; a close, nonpathogenic relative, Uncinocarpus reesii; and a more diverged pathogenic fungus, Histoplasma capsulatum, were sequenced and compared with those of 13 more distantly related Ascomycetes. This analysis identified increases and decreases in gene family size associated with a host/substrate shift from plants to animals in the Onygenales. In addition, comparison among Onygenales genomes revealed evolutionary changes in Coccidioides that may underlie its infectious phenotype, the identification of which may facilitate improved treatment and prevention of coccidioidomycosis. Overall, the results suggest that Coccidioides species are not soil saprophytes, but that they have evolved to remain associated with their dead animal hosts in soil, and that Coccidioides metabolism genes, membrane-related proteins, and putatively antigenic compounds have evolved in response to interaction with an animal host.Unlike most Ascomycete fungi, which live primarily as plant pathogens or plant saprobes, Coccidioides is capable of causing life-threatening disease in immunocompetent mammals, including humans. As the causal agent of coccidioidomycosis or “Valley fever,” Coccidioides infects at least 150,000 people annually, ∼40% of whom develop a pulmonary infection (Hector and Laniado-Laborin 2005). However, a chronic and disseminated form of coccidioidomycosis, for which existing treatments can be prolonged and difficult to tolerate, occurs in roughly 5% of patients (Galgiani et al. 2005). The virulent nature of this fungus and its potential for dispersal by airborne spores led to its listing as a U.S Health and Human Services Select Agent (Dixon 2001) and has fueled efforts to develop an effective vaccine and new treatments (Hung et al. 2002; Hector and Rutherford 2007).Coccidioides is an environmentally acquired, dimorphic pathogen. When not infecting a mammal, the fungus lives in the arid, alkaline New World deserts, where it is believed to grow as a filamentous soil saprophyte (Papagiannis 1967; Fisher et al. 2007). The filaments produce asexual spores (arthroconidia), which are inhaled to initiate infection. Once in the lungs, arthroconidia enlarge into spherules, documenting a morphological switch from polar to isotrophic growth. Spherules subsequently differentiate to produce internal spores (endospores) that are released upon spherule rupture. This latter morphology, endospore-containing spherules, is unique to Coccidioides among all known Ascomycota. Endospores are capable of disseminating in the host and re-initiating the spherulation cycle, but the host can sequester spherules in a granuloma to prevent disease dissemination. In the absence of a successful host response, chronic infection may persist for at least 12 yr (Hernandez et al. 1997), although human disease can progress to death in a much shorter period. Upon host death, the fungus reverts to filamentous growth and the production of arthroconidia.Coccidioides is composed of two closely related species, Coccidioides immitis and Coccidioides posadasii, and is a member of the Onygenales, an order characterized by many species that also tend to associate with animals. However, despite this shared characteristic, recently diverged fungal relatives of Coccidioides, such as Uncinocarpus, Gymnoascus, and Crysosporium species, are not known to cause disease (Untereiner et al. 2004). This observation has led to the parsimonious theory that Coccidioides recently acquired its pathogenic phenotype. Although a great deal is known about the clinical aspects of coccidioidomycosis and the biology of this fungus in laboratory mice, relatively little is known about the life history of Coccidioides between infections (Barker et al. 2007) or how it evolved the ability to cause disease in immunocompetent mammals. To address these questions, the genomes of C. immitis and C. posadasii, as well as their Onygenlean relatives Uncinocarpus reesii, a nonpathogenic fungus, and Histoplasma capsulatum, a mammalian pathogen, were sequenced and compared with those of 13 more distantly related Ascomycota, 12 of which associate with plants. Comparing the Coccidioides genome sequences across a range of evolutionary distances resolved various levels of genome evolution, including changes in gene family size, gene gain and loss, and the detection of positive natural selection, and provided an evolutionary context for observed differences between the taxa. Ultimately, the adoption of such a hierarchical comparative genomics approach reveals that myriad genomic changes are involved in shaping the evolution of phenotype and, in the case of Coccidioides, elucidates an understanding of how the fungus evolved to associate with animals.     

Analysis and rearrangement of human karyotypes by computer

A system useful for the collection and analysis of a large number of abnormal karyotypes and for studies on the relationship between cytogenetic abnormalities and clinical features of diseases is reported. The program is based on the disintegration and rearrangement of abnormal karyotypes recorded according to the 1978 International System for Human Cytogenetic Nomenclature (ISCN).     

Cross-species color banding in ten cases of myeloid malignancies with complex karyotypes

Cross-species color banding is a multiple-color fluorescence in situ hybridization (FISH) technique using probes developed from other animal species. Hybridization to human metaphases produces color banding patterns specific for each homologous chromosome pair. The technique has been evaluated in a complementary manner with G-banding and chromosome painting in a series of 10 myeloid malignancies with complex or unresolved karyotypes. Color banding detected the majority of chromosomal abnormalities, which had been identified by G-banding and in each case revealed chromosomal changes that G-banding had not identified. Painting was necessary to confirm these abnormalities due to the limitation of only seven colors in the color-banded karyotype. At the same time, painting fortuitously uncovered cryptic abnormalities in 6 of 10 cases that had not been detected by color banding. Insertions were visible by painting only. This study has demonstrated that in the analysis of complex karyotypes, the application of color banding revealed the involvement of the long arm of chromosome 3, indicating a poor risk, in two cases not identified by G-banding. Therefore, these techniques applied together have revealed cryptic chromosomal abnormalities with prognostic significance, which in some cases may have implications for patient management.     

Chromosomal aberrations as detected by array comparative genomic hybridization in early low-grade intraepithelial neoplasias of the breast

Stacher E, Boldt V, Leibl S, Halbwedl I, Popper H H, Ullmann R, Tavassoli F A & Moinfar F
(2011) Histopathology 59 , 549–555 Chromosomal aberrations as detected by array comparative genomic hybridization in early low‐grade intraepithelial neoplasias of the breast Aims: Low‐grade flat ductal intraepithelial neoplasia (DIN1a, flat epithelial atypia) is one of the earliest morphologically recognizable neoplastic lesions of the breast. Frequently, it occurs concomitantly with lobular intraepithelial neoplasia (LIN). We aimed to elucidate chromosomal aberrations in these early neoplastic breast lesions with the use of array comparative genomic hybridization analysis. Methods and results: Laser capture microdissection of 12 archival formalin‐fixed, paraffin‐embedded specimens harbouring foci of both DIN1a and LIN was performed. All analysed cases of DIN1a and LIN showed chromosomal gains and losses. The aberration encountered most often was loss of 16q, noted in seven DIN1a (70% of those successfully examined) and 10 LIN (91%) cases. The next most common alteration was a gain on 1q, noted in four DIN1a (40%) and seven LIN (64%) cases. Conclusions: The results show concurrent chromosomal aberrations of 1q gains and 16q losses in several cases with coexisting LIN and DIN1a. These aberrations are known to be common in low‐grade invasive (ductal and lobular) carcinomas as well as in more advanced (conventional) types of low‐grade ductal intraepithelial neoplasia (DIN) (low‐grade ductal carcinoma in situ). Our results raise the possibility of similar molecular‐genetic pathways in coexisting LIN and low‐grade flat DIN.     

Comparative genomic hybridization in ganglioneuroblastomas.

The ganglioneuroblastoma are rare lesions with widespread neuronal differentiation that have been classified as intermediate stages between neuroblastoma and ganglioneuroma. To identify overall chromosome aberrations in ganglioneuroblastoma, we performed comparative genomic hybridization. All of the five tumor samples were found to exhibit multiple gains involving different chromosomal regions. Chromosomal gains displayed by chromosomes and chromosome loci were 2p25 approximately pter (60%), 5p15.1 approximately p15.3 (60%), 7 (60%), 13q22 approximately q31 (60%), and 22 (60%), which were detected as minimal common regions in all five tumor samples. Chromosome 22 gain, which had not been reported in neuronal tumors before, and novel site 13q22 approximately q31 may be considered to play an important role in progression and differentiation of ganglioneuroblastoma.     

Global distribution of genomic diversity underscores rich complex history of continental human populations

Characterizing patterns of genetic variation within and among human populations is important for understanding human evolutionary history and for careful design of medical genetic studies. Here, we analyze patterns of variation across 443,434 single nucleotide polymorphisms (SNPs) genotyped in 3845 individuals from four continental regions. This unique resource allows us to illuminate patterns of diversity in previously under-studied populations at the genome-wide scale including Latin America, South Asia, and Southern Europe. Key insights afforded by our analysis include quantifying the degree of admixture in a large collection of individuals from Guadalajara, Mexico; identifying language and geography as key determinants of population structure within India; and elucidating a north–south gradient in haplotype diversity within Europe. We also present a novel method for identifying long-range tracts of homozygosity indicative of recent common ancestry. Application of our approach suggests great variation within and among populations in the extent of homozygosity, suggesting both demographic history (such as population bottlenecks) and recent ancestry events (such as consanguinity) play an important role in patterning variation in large modern human populations.Recent advances in sequencing and genotyping technology have transformed the study of human population genetics (Hinds et al. 2005; The International HapMap Consortium 2007). Analysis of dense genotype data has greatly expanded our understanding of the role natural selection has played in the recent evolution of our species (Voight et al. 2006; Sabeti et al. 2007; Williamson et al. 2007), the nature and causes of recombination rate variation (Myers et al. 2006; Coop et al. 2008), and the extent of structural variation within and among human genomes (Redon et al. 2006; Jakobsson et al. 2008; Kidd et al. 2008).Arguably, some of the most important insights have come from refining our views of human population structure and recent demographic history (The International HapMap Consortium 2005, 2007; Schaffner et al. 2005; Keinan et al. 2007; Jakobsson et al. 2008; Li et al. 2008). For example, the HapMap Project (The International HapMap Consortium 2005, 2007) has afforded unprecedented insight into fine-scale patterns of genotype and haplotype variation across more than 3.1 million single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from three major continental populations. Likewise, analysis of samples collected by the Human Genome Diversity Project (HGDP) (Jakobsson et al. 2008; Li et al. 2008) has elucidated patterns of diversity across ∼650,000 SNPs genotyped in nearly a 1000 individuals from 51 populations. One key feature of these projects is that they have focused on comparing geographically discontinuous populations with small-to-moderate sample sizes per group. Populations of known recent admixture were largely absent from these studies.In this paper, we analyze dense genotype data from 3845 individuals from the Population Reference Sample (POPRES; Nelson et al. 2008), with self-identified ancestry from four continental regions (Supplemental Table S1). The POPRES is comprised of samples from a number of studies, and includes individuals designated as healthy or with undisclosed disease status (Nelson et al. 2008). Individuals were generally sampled in urban locations, and were genotyped on the Affymetrix GeneChip Mapping Array 500K. Depending on the original study for which the samples were collected, further non-genetic data are often available, including self-reported ancestry up to, and including, grandparental information, and primary spoken language.The POPRES provides a complementary resource to both the HapMap and HGDP datasets, and presents an opportunity to further understand human genetic diversity. The POPRES has already been used to investigate fine-scale population structure in Europe (Novembre et al. 2008) and its implications on case-control association studies (Nelson et al. 2008). In this paper, we have investigated population structure, haplotype diversity, and patterns of homozygosity in the POPRES. Some of the key findings we have uncovered include evidence of historical South European admixture with the Mexican population, population stratification within South Asia, a gradient of haplotype diversity within Europe, and extended runs of homozygosity in almost all individuals examined. Together these analyses suggest the growing utility of large diverse samples of worldwide human populations.     

Comparative genomic hybridization reveals complex genetic changes in primary breast cancer tumors and their cell lines

DNA copy number changes were characterized by comparative genomic hybridization (CGH) in 18 breast cancer cell lines. In 5 of these, the results were comparable with those from the primary tumors of which the cell lines were established. All of the cell lines showed extensive DNA copy number changes, with a mean of 16.3 +/- 1.1 aberrations per sample (range 7-26). All of the cell lines had a gain at 8q22-qter. Other common gains of DNA sequences occurred at 1q31-32 (89%), 20q12-q13.2 (83%), 8q13 (72%), 3q26.1-qter (67%), 17q21-qter (67%) 5p14 (61%), 6p22 (56%), and 22pter-qter (50%). High-level amplifications were observed in all cell lines; the most frequent minimal common regions were 8q24.1 (89%), 20q12 (61%), 1q41 (39%), and 20p11.2 (28%). Losses were observed less frequently than gains and the minimal common regions of the most frequent losses were Xq11-q12 (56%), Xp11.2-pter (50%), 13q21 (50%), 8p12-pter (44%), 4p13-p14 (39%), 6q15-q22 (39%), and 18q11.2-qter (33%). Although the cell lines showed more DNA copy number changes than the primary tumors, all aberrations, except one found in a primary tumor, were always present in the corresponding cell line. High-level amplifications found both in primary tumors and cell lines were at 1q, 8q, 17q, and 20q. The DNA copy number changes detected in these cell lines can be valuable in investigation of tumor progression in vitro and for a more detailed mapping and isolation of genes implicated in breast cancer.