Rearrangement Breakpoints Research Articles

Breakpoint analysis of balanced chromosome rearrangements by next-generation paired-end sequencing

Characterisation of breakpoints in disease-associated balanced chromosome rearrangements (DBCRs), which disrupt or inactivate specific genes, has facilitated the molecular elucidation of a wide variety of genetic disorders. However, conventional methods for mapping chromosome breakpoints, such as in situ hybridisation with fluorescent dye-labelled bacterial artificial chromosome clones (BAC-FISH), are laborious, time consuming and often with insufficient resolution to unequivocally identify the disrupted gene. By combining DNA array hybridisation with chromosome sorting, the efficiency of breakpoint mapping has dramatically improved. However, this can only be applied when the physical properties of the derivative chromosomes allow them to be flow sorted. To characterise the breakpoints in all types of balanced chromosome rearrangements more efficiently and more accurately, we performed massively parallel sequencing using Illumina 1G analyser and ABI SOLiD systems to generate short sequencing reads from both ends of DNA fragments. We applied this method to four different DBCRs, including two reciprocal translocations and two inversions. By identifying read pairs spanning the breakpoints, we were able to map the breakpoints to a region of a few hundred base pairs that could be confirmed by subsequent PCR amplification and Sanger sequencing of the junction fragments. Our results show the feasibility of paired-end sequencing of systematic breakpoint mapping and gene finding in patients with disease-associated chromosome rearrangements.

Base-Pair Resolution of Somatic and Germline-Derived Genome Rearrangement Breakpoints in Follicular Lymphoma.

Abstract 439 Introduction:Follicular lymphoma (FL) is the most common indolent lymphoid malignancy in North America with approximately 20,000 new cases of this incurable cancer diagnosed each year. In approximately 85% of patients, FL is associated with the reciprocal translocation t(14;18)(q32;q21), which results in a fusion between IGH and BCL2 genes and consequent over-expression of the anti-apoptotic protein BCL2. This translocation likely represents an initiating event for FL, requiring additional mutational events for the onset of clinical disease. To investigate the relationship between genome rearrangements and FL we identified rearrangement locations in the genome followed by detailed, fine-structure analysis of the rearrangements to ascertain their effects on genes and other features of biological interest. Patients and Methods:We used a whole-genome bacterial artificial chromosome (BAC) fingerprint-based approach, termed Fingerprint Profiling (FPP, Krzywinski, M. et al. 2007), to detect genome rearrangements relative to the reference human genome in neoplastic B cells purified from 24 FL patient biopsies. Analysis of 2,640,707 BAC fingerprints revealed 721 candidate genomic rearrangements. To validate these observations and provide base-pair resolution of the rearrangement breakpoints we performed paired-end massively parallel sequencing, on the Illumina Genome Analyzer II platform, of the breakpoint-containing regions captured in the BAC clones. Sequence reads were assembled into contigs using our in-house de novo assembly algorithm ABySS (Assembly By Short Sequences, Simpson, J. et al. 2009) then aligned to the reference human genome. Following manual annotation of the breakpoint junctions PCR primers were designed to assay patient tumour and matched constitutional DNA and thus determine whether the observed genome rearrangements were somatic (acquired) or germline in origin. Results:727 BACs with apparent large-scale genome rearrangements, representing 354 distinct genome rearrangements across 20 patients, were sequenced in 95 pools, generating 72 Gbp of sequence. The 354 distinct events include 163 deletions, 71 inversions, 27 insertions, 83 translocations and 10 duplications, ranging in size from 3 kb to 67 Mb. PCR assays for 194 of the distinct events have been performed thus far identifying 80 distinct somatic and 114 germline-derived structural variations at base-pair resolution. Of the somatic events 5 are present in two or more of the 20 patients analyzed including a 720 kb inversion of 3q27.3 that results in expression of a BCL6-ST6GAL1 fusion transcript. Identification at base-pair resolution of breakpoint sequences enabled a detailed study of breakpoint and fusion mechanisms. We classified breakpoint junctions into 4 groups; those with microhomology (48%), those with sequence additions (28%), those with blunt fusions (20%) and those with flanking low copy repeats (4%). We were particularly interested in establishing the origin of the observed nucleotide sequence additions in 97 breakpoint junctions. The sequence additions ranged in size from a single nucleotide to 454 bp. In one case we have unambiguously mapped a 53 bp sequence, lying within one of the 3q27.3 inversion breakpoints, to chromosome 5q12.3. This finding is consistent with the recently proposed fork stalling and template switching (FoSTeS) DNA replication-based mechanism and thus represents a novel mechanism in FL lymphomagenesis. Conclusions:We have successfully employed high-throughput clone fingerprinting and sequencing to identify numerous novel somatic and germline genome rearrangements from FL primary tumour samples. Furthermore, base-pair resolution of rearrangement breakpoints provides mechanistic insights. With the complete inventory of somatic and germline events in hand we will be able to propose recurrent structurally altered genes in FL patients for validation in independent datasets and improve our understanding of FL biology. Pathway analyses to identify emerging themes from somatic mutations are also being performed. The PCR assays we have developed will also be of utility in identifying germline predisposition alleles in larger FL patient cohorts. Disclosures:No relevant conflicts of interest to declare.

Searching for Genes for Cleft Lip and/or Palate Based on Breakpoint Analysis of a Balanced Translocation t(9;17)(q32;q12)

Identification of the breakpoints of disease-associated chromosome rearrangements can provide informative clues to a positional cloning approach for genes responsible for inherited diseases. Recently, we found a three-generation Japanese family segregating balanced chromosome translocation t(9;17)(q32;q12). One of the subjects had cleft lip and palate. We examined whether regions near the breakpoint could be associated with cleft lip and/or palate. We determined the breakpoints involved in the translocation by fluorescence in situ hybridization analysis and subsequent long-range polymerase chain reaction. In order to study the role of these disrupted regions in nonsyndromic cleft lip and/or palate, we performed mutation analysis and a haplotype-based transmission disequilibrium test using tagging single-nucleotide polymorphisms in the flanking regions of the breakpoints in white and Filipino nonsyndromic cleft lip and/or palate populations. Sequence analysis demonstrated that two genes, SLC31A1 (solute carrier family 31 member 1) on chromosome 9 and CCL2 (chemokine ligand 2) on chromosome 17, were rearranged with the breaks occurring within their introns. It is interesting that SLC31A1 lies closed to BSPRY (B-box and SPRY domain), which is a candidate for involvement with cleft lip and/or palate. Some of the variants in BSPRY and CCL2 showed significant p values in the cleft lip and/or palate population compared with the control population. There was also statistically significant evidence of transmission distortion for haplotypes on both chromosomes 9 and 17. The data support previous reports that genes on chromosomal regions of 9q and 17q play an important role in facial development.

Analysis of fine-scale mammalian evolutionary breakpoints provides new insight into their relation to genome organisation

BackgroundThe Intergenic Breakage Model, which is the current model of structural genome evolution, considers that evolutionary rearrangement breakages happen with a uniform propensity along the genome but are selected against in genes, their regulatory regions and in-between. However, a growing body of evidence shows that there exists regions along mammalian genomes that present a high susceptibility to breakage. We reconsidered this question taking advantage of a recently published methodology for the precise detection of rearrangement breakpoints based on pairwise genome comparisons.ResultsWe applied this methodology between the genome of human and those of five sequenced eutherian mammals which allowed us to delineate evolutionary breakpoint regions along the human genome with a finer resolution (median size 26.6 kb) than obtained before. We investigated the distribution of these breakpoints with respect to genome organisation into domains of different activity. In agreement with the Intergenic Breakage Model, we observed that breakpoints are under-represented in genes. Surprisingly however, the density of breakpoints in small intergenes (1 per Mb) appears significantly higher than in gene deserts (0.1 per Mb).More generally, we found a heterogeneous distribution of breakpoints that follows the organisation of the genome into isochores (breakpoints are more frequent in GC-rich regions). We then discuss the hypothesis that regions with an enhanced susceptibility to breakage correspond to regions of high transcriptional activity and replication initiation.ConclusionWe propose a model to describe the heterogeneous distribution of evolutionary breakpoints along human chromosomes that combines natural selection and a mutational bias linked to local open chromatin state.

Ocular manifestations in Wolf-Hirschhorn syndrome

Wolf-Hirschhorn syndrome (WHS) multiple congenital anomalies/mental retardation is caused by partial deletion of the short arm of chromosome 4 and can be considered a contiguous gene syndrome, characterized by typical facial appearance, mental retardation, growth delay, and seizures. We investigated the ocular defects in a population of 10 patients with WHS and analyzed the relationship between ocular findings and the extent of deletion on chromosome 4. The ocular abnormalities found included hypertelorism, strabismus, refractive errors, epicanthal folds, proptosis, downslanting palpebral fissures, microphthalmos, microcornea, iris coloboma, optic nerve coloboma, ocular cyst, ptosis, glaucoma, and nystagmus. Different breakpoints of the chromosomal rearrangement were observed in individual patients, ranging from 4p15.1 to 4p16.3, and the size of chromosomal deletion ranged from 2.6 to 26 million base pairs. Congenital glaucoma and colobomatous ocular cysts have rarely been described in WHS patients that were previously reported. In all cases exhibiting strabismus, an exodeviation was present. Comparing genotype with ocular phenotype, a relationship between the size of deletion and the severity of the ocular involvement was observed in all cases but one.

Reconstructing the History of Yeast Genomes

Reconstructing the History of Yeast Genomes

High-resolution mapping of heterochromatin redistribution in a Drosophila position-effect variegation model.

BackgroundPosition-effect variegation (PEV) is the stochastic transcriptional silencing of a gene positioned adjacent to heterochromatin. white-mottled X-chromosomal inversions in Drosophila are classic PEV models that show variegation of the eye color gene white due to its relocation next to pericentric heterochromatin. It has been suggested that in these models the spreading of heterochromatin across the rearrangement breakpoint causes the silencing of white. However, the extent of this spreading and the precise pattern of heterochromatin redistribution have remained unclear. To obtain insight into the mechanism of PEV, we constructed high-resolution binding maps of Heterochromatin Protein 1 (HP1) on white-mottled chromosomes.ResultsWe find that HP1 invades euchromatin across the inversion breakpoints over ~175 kb and ~30 kb, causing de novo association of HP1 with 20 genes. However, HP1 binding levels in these regions show substantial local variation, and white is the most strongly bound gene. Remarkably, white is also the only gene that is detectably repressed by heterochromatin. Furthermore, we find that HP1 binding to the invaded region is particularly sensitive to the dosage of the histone methyltransferase Su(var)3-9, indicating that the de novo formed heterochromatin is less stable than naturally occurring constitutive heterochromatin.ConclusionOur molecular maps demonstrate that heterochromatin can invade a normally euchromatic region, yet the strength of HP1 binding and effects on gene expression are highly dependent on local context. Our data suggest that the white gene has an unusual intrinsic affinity for heterochromatin, which may cause this gene to be more sensitive to PEV than most other genes.

Rearrangements of the MLL Gene Are Influenced by DNA Secondary Structure, Potentially Mediated by Topoisomerase II Binding

Rearrangements of the MLL gene are found in approximately 75–80% of both infant acute leukemia (IAL) and therapy related myeloid leukemia (tAML), the latter linked to prior exposure to Topoisomerase II inhibitors. In order to study the mechanism whereby such rearrangements are induced, a fine mapping screen was performed using Inverse PCR to analyze a MLL location at the intron 11/exon 12 border that is known to be a hot spot for the induction of DNA breaks and rearrangements. Human lymphoblastoid TK6 cells were exposed to the pro-apoptotic agent anti-CD95 and estrogen as both the apoptotic process and birth control formulations have been associated with the development of MLL-linked leukemia. The latter association was determined as part of an epidemiological analysis of IAL. In addition, blood samples from patients receiving Topoisomerase II inhibitor therapy, subsequent to a diagnosis of breast cancer or lymphoma, were examined in the same manner as such drugs are strongly associated with the etiology of tAML. From an initial screen of cells treated in-vitro, 13/27 of all rearrangement break points (translocations/insertions) were located within a 3 bp tract at the 5′ edge of a 10 bp palindrome that defined a potential 101 bp stem-loop with its 3′ palindrome partner. Analysis of blood samples taken up to a year after treatment initiation from lymphoma and breast cancer patients, showed a similar distribution of translocations with 10/20 events restricted to the same location on the 5′ of the palindrome. Further examination of the putative stem-loop structure showed a high stringency Topoisomerase II consensus sequence binding site at the geometric midpoint of the proposed stem-loop. In order to link the rearrangements observed with DNA cleavage events, TK6 cells exposed to anti-CD95 antibody were also screened for DNA breaks within the same region of MLL. Here, LM-PCR products spanning the region of interest were extracted from DNA gels and the location of cleavage determined by cloning and sequencing. From four to twenty four hours after anti-CD95 exposure, analysis of the breaks induced showed 24/37 (65%) were located at the base of the proposed stem loop associated with the DNA palindrome. 14/37 (38%) of breaks were found within a 40 bp tract at the 5′ side and 10/37 (27%) within a 20 bp tract at the 3′ side of the palindrome base. In each case, the hot spots for cleavage identified by LM-PCR included the palindromic sequences. Thus, unlike the 5′ restriction of MLL rearrangements, DNA fragmentation occurred at both sides of the proposed stem loop base, indirectly supporting the creation of such a structure in-vivo that is subject to local attack at the stem base. Palindrome association may be driven by the bending Topoisomerase II exerts when bound at the midpoint between each palindrome, such that each half of the proposed palindrome is brought into physical contact. This effect would be accentuated by poisoning of Topoisomerase II, subsequent to binding of drugs such as etoposide that stabilize the cleavable complex. We propose a model for a sub-group of MLL rearrangements that utilize Topoisomerase II mediated stabilization, either natively or subsequent to chemical poisoning, to assist in the creation of stable DNA structures that are permissive for DNA fragmentation. Further, the conversion of DNA double strand breaks at these sites to detectable rearrangements may be influenced by 5′ to 3′ DNA processing functions, such as transcription or replication.

Mapping of 5q35 chromosomal rearrangements within a genomically unstable region

Background:Recent molecular studies of breakpoints of recurrent chromosome rearrangements revealed the role of genomic architecture in their formation. In particular, segmental duplications representing blocks of >1 kb with >90% sequence...

Detailed phenotype–genotype study in five patients with chromosome 6q16 deletion: narrowing the critical region for Prader–Willi-like phenotype

Most patients with an interstitial deletion of 6q16 have Prader-Willi-like phenotype, featuring obesity, hypotonia, short hands and feet, and developmental delay. In all reported studies, the chromosome rearrangement was detected by karyotype analysis, which provides an overview of the entire genome but has limited resolution. Here we describe a detailed clinical presentation of five patients, two of whom were previously reported, with overlapping interstitial 6q16 deletions and Prader-Willi-like phenotype. Our patients share the following main features with previously reported cases: global developmental delay, hypotonia, obesity, hyperphagia, and eye/vision anomalies. All rearrangement breakpoints have been accurately defined through array-CGH at about 100 Kb resolution. We were able to narrow the shortest region of deletion overlap for the presumed gene(s) involved in the Prader-Willi-like syndrome to 4.1 Mb located at 6q16.1q16.2. Our results support the evidence that haploinsufficiency of the SIM1 gene is responsible for obesity in these patients. A possible involvement of the GRIK2 gene in autistic-like behaviour, of POPDC3 in heart development, and of MCHR2 in the control of feeding behaviour and energy metabolism is also hypothesized.

FOXG1 Is Responsible for the Congenital Variant of Rett Syndrome

Rett syndrome is a severe neurodevelopmental disease caused by mutations in the X-linked gene encoding for the methyl-CpG-binding protein MeCP2. Here, we report the identification of FOXG1-truncating mutations in two patients affected by the congenital variant of Rett syndrome. FOXG1 encodes a brain-specific transcriptional repressor that is essential for early development of the telencephalon. Molecular analysis revealed that Foxg1 might also share common molecular mechanisms with MeCP2 during neuronal development, exhibiting partially overlapping expression domain in postnatal cortex and neuronal subnuclear localization.

Precise detection of rearrangement breakpoints in mammalian chromosomes

BackgroundGenomes undergo large structural changes that alter their organisation. The chromosomal regions affected by these rearrangements are called breakpoints, while those which have not been rearranged are called synteny blocks. We developed a method to precisely delimit rearrangement breakpoints on a genome by comparison with the genome of a related species. Contrary to current methods which search for synteny blocks and simply return what remains in the genome as breakpoints, we propose to go further and to investigate the breakpoints themselves in order to refine them.ResultsGiven some reliable and non overlapping synteny blocks, the core of the method consists in refining the regions that are not contained in them. By aligning each breakpoint sequence against its specific orthologous sequences in the other species, we can look for weak similarities inside the breakpoint, thus extending the synteny blocks and narrowing the breakpoints. The identification of the narrowed breakpoints relies on a segmentation algorithm and is statistically assessed. Since this method requires as input synteny blocks with some properties which, though they appear natural, are not verified by current methods for detecting such blocks, we further give a formal definition and provide an algorithm to compute them.The whole method is applied to delimit breakpoints on the human genome when compared to the mouse and dog genomes. Among the 355 human-mouse and 240 human-dog breakpoints, 168 and 146 respectively span less than 50 Kb. We compared the resulting breakpoints with some publicly available ones and show that we achieve a better resolution. Furthermore, we suggest that breakpoints are rarely reduced to a point, and instead consist in often large regions that can be distinguished from the sequences around in terms of segmental duplications, similarity with related species, and transposable elements.ConclusionOur method leads to smaller breakpoints than already published ones and allows for a better description of their internal structure. In the majority of cases, our refined regions of breakpoint exhibit specific biological properties (no similarity, presence of segmental duplications and of transposable elements). We hope that this new result may provide some insight into the mechanism and evolutionary properties of chromosomal rearrangements.

Distribution of induced chromosome rearrangement breakpoints along the chromosome length and the problem of intercalary heterochromatin in Drosophila melanogaster

Historically, the term "intercalary heterochromatin" was based on the finding that induced chromosome rearrangements occur at a higher frequency in the corresponding regions. The available molecular genetic data and, in particular, the results of the Drosophila Genome Project made it possible to decide between two possible explanations of the preferential location of chromosome rearrangement breakpoints in intercalary heterochromatin regions. Namely, a higher frequency of radiation-induced rearrangements in these regions correlates with the DNA content and probably lacks an association with the features of chromatin organization.

A de novo apparently balanced translocation [46,XY,t(2;9)(p13;p24)] interrupting RAB11FIP5 identifies a potential candidate gene for autism spectrum disorder

Autism spectrum disorder (ASD) is a severe developmental disorder of the central nervous system characterized by impairments in social interaction, communication, and range of interests and behaviors. The syndrome's prevalence is estimated to be as high as 1 in 150 American children yet its etiology remains largely unknown. Examination of observed cytogenetic variants in individuals with ASD may identify genes involved in its pathogenesis. As part of a multidisciplinary study, an apparently balanced de novo translocation between chromosomes 2 and 9 [46,XY,t(2;9)(p13;p24)] was identified in a subject with pervasive developmental disorder not otherwise specified (PDD-NOS), and no distinctive dysmorphic features. Molecular characterization of the rearrangement revealed direct interruption of the RAB11 family interacting protein 5 (RAB11FIP5) gene. RAB11FIP5 is a Rab effector involved in protein trafficking from apical recycling endosomes to the apical plasma membrane. It is ubiquitously expressed and reported to contribute to both neurotransmitter release and neurotransmitter uptake at the synaptic junction. Detailed analysis of the rearrangement breakpoints suggests that the reciprocal translocation may have formed secondary to incorrect repair of double strand breaks (DSBs) by nonhomologous end-joining (NHEJ).

A small trip in the untranquil world of genomes: A survey on the detection and analysis of genome rearrangement breakpoints

Genomes are dynamic molecules that are constantly undergoing mutations and rearrangements. The latter are large scale changes in a genome organisation that participate in the evolutionary and speciation process, but may also be involved in inherited diseases and in cancer. They have since long been studied by the biologists whereas computational biologists have more recently only been attracted to the topic. One of the (exciting) objectives for studying rearrangements is to understand the underlying molecular mechanisms of evolution. One possible line of investigation is to analyse, at the sequence level, the regions which have undergone a rearrangement, assuming we are able to very precisely locate them. This paper presents a survey of the different methods that have been developed to identify such regions, in particular the approaches that are based on the alignment of genomes. The main purpose of the paper is then to investigate what is currently known about the characteristics of the regions where a rearrangement took place, and about the mechanism(s) which have led to such large scale changes.

Large-insert genome analysis technology detects structural variation in Pseudomonas aeruginosa clinical strains from cystic fibrosis patients

Large-insert genome analysis (LIGAN) is a broadly applicable, high-throughput technology designed to characterize genome-scale structural variation. Fosmid paired-end sequences and DNA fingerprints from a query genome are compared to a reference sequence using the Genomic Variation Analysis (GenVal) suite of software tools to pinpoint locations of insertions, deletions, and rearrangements. Fosmids spanning regions that contain new structural variants can then be sequenced. Clonal pairs of Pseudomonas aeruginosa isolates from four cystic fibrosis patients were used to validate the LIGAN technology. Approximately 1.5 Mb of inserted sequences were identified, including 743 kb containing 615 ORFs that are absent from published P. aeruginosa genomes. Six rearrangement breakpoints and 220 kb of deleted sequences were also identified. Our study expands the “genome universe” of P. aeruginosa and validates a technology that complements emerging, short-read sequencing methods that are better suited to characterizing single-nucleotide polymorphisms than structural variation.

Evaluation of Paired-End Sequencing Strategies for Detection of Genome Rearrangements in Cancer

Paired-end sequencing is emerging as a key technique for assessing genome rearrangements and structural variation on a genome-wide scale. This technique is particularly useful for detecting copy-neutral rearrangements, such as inversions and translocations, which are common in cancer and can produce novel fusion genes. We address the question of how much sequencing is required to detect rearrangement breakpoints and to localize them precisely using both theoretical models and simulation. We derive a formula for the probability that a fusion gene exists in a cancer genome given a collection of paired-end sequences from this genome. We use this formula to compute fusion gene probabilities in several breast cancer samples, and we find that we are able to accurately predict fusion genes in these samples with a relatively small number of fragments of large size. We further demonstrate how the ability to detect fusion genes depends on the distribution of gene lengths, and we evaluate how different parameters of a sequencing strategy impact breakpoint detection, breakpoint localization, and fusion gene detection, even in the presence of errors that suggest false rearrangements. These results will be useful in calibrating future cancer sequencing efforts, particularly large-scale studies of many cancer genomes that are enabled by next-generation sequencing technologies.

FISH Mapping of De Novo Apparently Balanced Chromosome Rearrangements Identifies Characteristics Associated with Phenotypic Abnormality

We report fluorescence in situ hybridization (FISH) mapping of 152, mostly de novo, apparently balanced chromosomal rearrangement (ABCR) breakpoints in 76 individuals, 30 of whom had no obvious phenotypic abnormality (control group) and 46 of whom had an associated disease (case group). The aim of this study was to identify breakpoint characteristics that could discriminate between these groups and which might be of predictive value in de novo ABCR (DN-ABCR) cases detected antenatally. We found no difference in the proportion of breakpoints that interrupted a gene, although in three cases, direct interruption or deletion of known autosomal-dominant or X-linked recessive Mendelian disease genes was diagnostic. The only significant predictor of phenotypic abnormality in the group as a whole was the localization of one or both breakpoints to an R-positive (G-negative) band with estimated predictive values of 0.69 (95% CL 0.54-0.81) and 0.90 (95% CL 0.60-0.98), respectively. R-positive bands are known to contain more genes and have a higher guanine-cytosine (GC) content than do G-positive (R-negative) bands; however, whether a gene was interrupted by the breakpoint or the GC content in the 200 kB around the breakpoint had no discriminant ability. Our results suggest that the large-scale genomic context of the breakpoint has prognostic utility and that the pathological mechanism of mapping to an R-band cannot be accounted for by direct gene inactivation.

Rearrangement and duplication as main forces in chloroplast genome evolution in Pines

The entire nucleotide sequence of the chloroplast genome (117,190bp) of Korean pine (Pinus koraiensis) was determined and compared with the already published genome of Japanese black pine. As in the case of Japanese black pine, it retains 475 bp inverted repeats containing 3' portion of psbA gene but lacking rRNA genes. The translated amino acid sequences of respective potential coding genes showed 77% to 100% sequence similarity to those of corresponding genes of P. thunbergii. When compared with Japanese black pine, the order of the genes is fairly conserved except for the psaM gene and ndh pseudo genes. Thirteen indels that are larger than 100 bp were identified from the alignment of the two pine genomes. Except for the region containing ndh pseudo genes and that between psaI and accD, all the large indels occur near the potential rearrangement breakpoints involving several tRNA genes and their neighboring sequences as repeat units. Multiple rearrangement events appeared to be responsible for the loss of a copy of psaM gene and rps4 and ycf12 pseudo genes found in Japanese black pine. In addition, the further proceeded truncation of ndhK pseudo gene in Korean pine and ndhB pseudo gene in Japanese black pine indicate that these genes are functionally redundant in pines. Surprisingly, the loss of the gene and pseudo genes are conserved among haploxylon pines and not in diploxylon pines, suggesting that they occurred simultaneously at the time of divergence or within a short period of time after divergence of the two pine subgenera and that since then little change has occurred. There also appeared many indels that are less than 50 bps. Many of these indels appeared to be the tandem duplication products of the adjacent sequences. These tandem repeats are also very well conserved among haploxylon and among diploxylon pines.

Genome-wide mapping and characterization of hypomethylated sites in human tissues and breast cancer cell lines

We have developed a method for mapping unmethylated sites in the human genome based on the resistance of TspRI-digested ends to ExoIII nuclease degradation. Digestion with TspRI and methylation-sensitive restriction endonuclease HpaII, followed by ExoIII and single-strand DNA nuclease allowed removal of DNA fragments containing unmethylated HpaII sites. We then used array comparative genomic hybridization (CGH) to map the sequences depleted by these procedures in human genomes derived from five human tissues, a primary breast tumor, and two breast tumor cell lines. Analysis of methylation patterns of the normal tissue genomes indicates that the hypomethylated sites are enriched in the 5' end of widely expressed genes, including promoter, first exon, and first intron. In contrast, genomes of the MCF-7 and MDA-MB-231 cell lines show extensive hypomethylation in the intragenic and intergenic regions whereas the primary tumor exhibits a pattern between those of the normal tissue and the cell lines. A striking characteristic of tumor cell lines is the presence of megabase-sized hypomethylated zones. These hypomethylated zones are associated with large genes, fragile sites, evolutionary breakpoints, chromosomal rearrangement breakpoints, tumor suppressor genes, and with regions containing tissue-specific gene clusters or with gene-poor regions containing novel tissue-specific genes. Correlation with microarray analysis shows that genes with a hypomethylated sequence 2 kb up- or downstream of the transcription start site are highly expressed, whereas genes with extensive intragenic and 3' untranslated region (UTR) hypomethylation are silenced. The method described herein can be used for large-scale screening of changes in the methylation pattern in the genome of interest.