Showing papers on "Chromosome 22 published in 2013"

Single-cell Hi-C reveals cell-to-cell variability in chromosome structure

Abstract: Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns

Characterization of chromosome stability in diploid, polyploid and hybrid yeast cells.

Abstract: Chromosome instability is a key component of cancer progression and many heritable diseases. Understanding why some chromosomes are more unstable than others could provide insight into understanding genome integrity. Here we systematically investigate the spontaneous chromosome loss for all sixteen chromosomes in Saccharomyces cerevisiae in order to elucidate the mechanisms underlying chromosome instability. We observed that the stability of different chromosomes varied more than 100-fold. Consistent with previous studies on artificial chromosomes, chromosome loss frequency was negatively correlated to chromosome length in S. cerevisiae diploids, triploids and S. cerevisiae-S. bayanus hybrids. Chromosome III, an equivalent of sex chromosomes in budding yeast, was found to be the most unstable chromosome among all cases examined. Moreover, similar instability was observed in chromosome III of S. bayanus, a species that diverged from S. cerevisiae about 20 million years ago, suggesting that the instability is caused by a conserved mechanism. Chromosome III was found to have a highly relaxed spindle checkpoint response in the genome. Using a plasmid stability assay, we found that differences in the centromeric sequence may explain certain aspects of chromosome instability. Our results reveal that even under normal conditions, individual chromosomes in a genome are subject to different levels of pressure in chromosome loss (or gain).

High-throughput sequencing of a single chromosome: a moth W chromosome

Abstract: Y and W chromosomes have mostly been excluded from whole genome sequencing projects. Due to the high amount of repetitive sequences they are 'difficult' to assemble and therefore need special treatment in the form of, e.g. adapted assembly programs, a range of different libraries, and accurate maps, if possible. A minimum requirement for these approaches is pure template DNA. We therefore microdissected the W chromatin of highly polyploid cells from the flour moth, Ephestia kuehniella, and used Roche/454 and Sanger sequencing to generate 72.6 Mbp of DNA sequence. Nominal coverage was 4.3× of the 16.7 Mbp of W chromosomal DNA. We used these data to assess the genetic content of the W chromosome. This approach allowed us to determine constituent families of transposable elements, microsatellites, and recent insertion sites of mitochondrial DNA. However, no conventional protein-coding gene has yet been found. The sequence collection is a rich source for the definition of W-specific PCR markers and the reconstruction of W chromosome loci, as a step towards full reconstruction of the chromosome.

22q11 deletion syndrome: A review of the neuropsychiatric features and their neurobiological basis

Abstract: The 22q11.2 deletion syndrome (22q11DS) is caused by an autosomal dominant microdeletion of chromosome 22 at the long arm (q) 11.2 band. The 22q11DS is among the most clinically variable syndromes, with more than 180 features related with the deletion, and is associated with an increased risk of psychiatric disorders, accounting for up to 1%-2% of schizophrenia cases. In recent years, several genes located on chromosome 22q11 have been linked to schizophrenia, including those encoding catechol-O-methyltransferase and proline dehydrogenase, and the interaction between these and other candidate genes in the deleted region is an important area of research. It has been suggested that haploinsufficiency of some genes within the 22q11.2 region may contribute to the characteristic psychiatric phenotype and cognitive functioning of schizophrenia. Moreover, an extensive literature on neuroimaging shows reductions of the volumes of both gray and white matter, and these findings suggest that this reduction may be predictive of increased risk of prodromal psychotic symptoms in 22q11DS patients. Experimental and standardized cognitive assessments alongside neuroimaging may be important to identify one or more endophenotypes of schizophrenia, as well as a predictive prodrome that can be preventively treated during childhood and adolescence. In this review, we summarize recent data about the 22q11DS, in particular those addressing the neuropsychiatric and cognitive phenotypes associated with the deletion, underlining the recent advances in the studies about the genetic architecture of the syndrome.

Hippocampal Malrotation is Associated with Chromosome 22q11.2 Microdeletion

Abstract: Microdeletion at chromosome 22q11.2 is the most common pathogenic copy number variation (CNV) in humans, affecting 1/4000 live births1,2. Chromosome 22 has a region particularly susceptible to chromosomal rearrangements due to the presence of several areas of low copy repeats. Hemizygous deletions in this 22q11.2 region, most commonly involving 3Mb3,4 and encompassing more than 45 genes5, are responsible for 22q11.2 deletion syndrome (22q11DS). Patients with 22q11DS are clinically very complex with multisystem abnormalities. There are over 40 common clinical features6. Typical features include learning disabilities, subtle dysmorphic facies, velopharyngeal insufficiency, and congenital heart disease. Hypocalcemia due to parathyroid dysfunction are also very common6–8. Schizophrenia develops in up to 25% of patients9,10. Epilepsy is 7 times more common in patients with 22q11DS than the general population11,12. Non-hypocalcemic seizures in 22q11DS patients can have generalized or partial (i.e. focal) onset. According to Kao et al, partial onset seizures affect 5.5% of all patients with 22q11DS11. Known potential causes of partial onset seizures in this group of patients include rare case reports of polymicrogyria (PMG)13, periventricular nodular heterotopia (PNH)14, or hippocampal atrophy (HA)15. However, the prevalence of these abnormalities in the 22q11DS population is low, accounting for, at most, 25% of all cases of partial onset epilepsy11,12. Since there is a discrepancy between the prevalence of partial onset epilepsy and the prevalence of known causal structural abnormalities, the aim of this study was to evaluate a series of subjects with 22q11DS, using high resolution magnetic resonance imaging (MRI) and electroencephalography (EEG) to specifically search for additional findings to explain this difference.

Recurrent Chromosome 22 Deletions in Osteoblastoma Affect Inhibitors of the Wnt/Beta-Catenin Signaling Pathway

Abstract: Osteoblastoma is a bone forming tumor with histological features highly similar to osteoid osteoma; the discrimination between the tumor types is based on size and growth pattern. The vast majority of osteoblastomas are benign but there is a group of so-called aggressive osteoblastomas that can be diagnostically challenging at the histopathological level. The genetic aberrations required for osteoblastoma development are not known and no genetic difference between conventional and aggressive osteoblastoma has been reported. In order to identify recurrent genomic aberrations of importance for tumor development we applied cytogenetic and/or SNP array analyses on nine conventional and two aggressive osteoblastomas. The conventional osteoblastomas showed few or no acquired genetic aberrations while the aggressive tumors displayed heavily rearranged genomes. In one of the aggressive osteoblastomas, three neighboring regions in chromosome band 22q12 were homozygously deleted. Hemizygous deletions of these regions were found in two additional cases, one aggressive and one conventional. In total, 10 genes were recurrently and homozygously lost in osteoblastoma. Four of them are functionally involved in regulating osteogenesis and/or tumorigenesis. MN1 and NF2 have previously been implicated in the development of leukemia and solid tumors, and ZNRF3 and KREMEN1 are inhibitors of the Wnt/beta-catenin signaling pathway. In line with deletions of the latter two genes, high beta-catenin protein expression has previously been reported in osteoblastoma and aberrations affecting the Wnt/beta-catenin pathway have been found in other bone lesions, including osteoma and osteosarcoma.

Analysis of the clinico-hematological relevance of the breakpoint location within M-BCR in chronic myeloid leukemia.

Abstract: The Philadelphia chromosome (Ph) derives from the balanced translocation between chromosomes 9 and 22. This chromosomal translocation results in the fusion between the 5' part of the BCR gene, normally located on chromosome 22, and the 3' part of the ABL gene on chromosome 9 giving origin to a BCR-ABL fusion gene which is transcribed and then translated into a hybrid protein. In general, three breakpoint cluster regions in the BCR gene have been described: major (M-BCR), minor (m-BCR) and micro (μ-BCR). Three main variants of the BCR-ABL gene have been described depending on the length of the sequence of the BCR gene included that encode for the P190, P210, P230 proteins. Most patients (95 %) were found to have P210 protein that resulted from rearrangement in the M-BCR region in the BCR gene and thus gives rise to b2a2 or b3a2 variants. The incidence of one or other rearrangement in chronic myeloid leukemia (CML) patients varies in different reported series. These two variants are associated with distinct clinical types of human leukemias. In this study, we report the frequencies of M-BCR-ABL fusion transcripts in 44 CML patients and we review the data on the correlations between the type of M-BCR/ABL variant and the corresponding sex, age and biological features. Forty-four untreated chronic phase CML patients were studied. The type of BCR-ABL fusion transcripts was determined by reverse transcriptase polymerase chain reaction (RT-PCR). More than half of them showed b3a2 fusion transcript (64 %), while (36 %) showed b2a2 transcript. No patients coexpressed b3a2/b2a2. Correlation between biological data demonstrated that: (a) M-BCR rearrangements were not associated with the sex of the patients. (b) Patients with b3a2 rearrangements were older than patients with b2a2 transcripts. (c) M-BCR rearrangements were influenced neither by the white blood count (WBC) nor with hemoglobin levels. However, platelet level is more elevated in patients with b3a2 transcript (681.2/L vs. 207/L; P = 0.001). In conclusion, we observed significant correlations between age, platelet level and M-BCR-ABL transcript, these observations deserve further investigations.

Bernard-Soulier syndrome caused by a hemizygous GPIbβ mutation and 22q11.2 deletion.

Abstract: Background Bernard–Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by giant platelets, thrombocytopenia, and a prolonged bleeding time, which is caused by homozygous mutations in the GPIbα, GPIbβ, or GPIX genes. The 22q11.2 deletion syndrome (22q11.2DS) is caused by a microdeletion on chromosome 22, which includes the GPIbβ gene, and is characterized by abnormal development of the pharyngeal apparatus and heart. Thus, patients with 22q11.2DS are obligate carriers for BSS. Methods We evaluated two infants with BSS and performed the genetic analysis of the GPIbα, GPIbβ, or GPIX genes, and investigated the segregation of the mutation within the families. The status of the 22q11.2 deletion was examined by fluorescence in situ hybridization and single-nucleotide polymorphism array copy number analysis. Results DNA sequencing analysis revealed that the infants were compound heterozygous for a hemizygous mutation in the GPIbβ gene (p.Trp148X and p.Leu97Phe, respectively) and 22q11.2 deletion in the other chromosome. Both infants had the common 3Mb 22q11.2 deletion but did not show major phenotypic features of 22q11.2DS, such as developmental delay, cardiac defects, dysmorphic facial features, palatal anomalies, hypocalcemia, and immune deficiency. The 22q11.2DS would not have become clear if detailed molecular genetic analyses of BSS had not been performed. Conclusions Our cases illustrate that a suspicion of 22q11.2 deletion is warranted in pediatric BSS patients with a mutation in the GPIbβ gene, even without remarkable symptoms.

Genetic Basis of Y-Linked Hearing Impairment

Abstract: A single Mendelian trait has been mapped to the human Y chromosome: Y-linked hearing impairment. The molecular basis of this disorder is unknown. Here, we report the detailed characterization of the DFNY1 Y chromosome and its comparison with a closely related Y chromosome from an unaffected branch of the family. The DFNY1 chromosome carries a complex rearrangement, including duplication of several noncontiguous segments of the Y chromosome and insertion of ∼160 kb of DNA from chromosome 1, in the pericentric region of Yp. This segment of chromosome 1 is derived entirely from within a known hearing impairment locus, DFNA49. We suggest that a third copy of one or more genes from the shared segment of chromosome 1 might be responsible for the hearing-loss phenotype.

Microdissection and chromosome painting of the alien chromosome in an addition line of wheat--Thinopyrum intermedium.

Abstract: In this study, chromosome painting was developed and used to identify alien chromosomes in TAi-27, a wheat--Thinopyrum intermedium addition line, and the chromosomes of the three different genomes of Th. Intermedium. The smallest alien chromosome of TAi-27 was microdissected and its DNA amplified by DOP-PCR was used as a probe to hybridize with metaphase chromosomes of TAi-27 and Th. intermedium. Results showed that hybridization signals were observed in all regions of a pair of the smallest alien chromosomes and the pericentromeric area of another pair of alien chromosomes in TAi-27, indicating that the probe from microdissected chromosome is species specific. In Th. intermedium, 14 chromosomes had wide and strong hybridization signals distributed mainly on the pericentromere area and 9 chromosomes with narrow and weak signals on the pericentromere area. The remaining chromosomes displayed a very weak or no signal. Sequential FISH/GISH on Th. intermedium chromosomes using the DNAs of microdissected chromosome, Pseudoroegneria spicata (St genome) and pDbH12 (a J(s) genome specific probe) as the probes indicated that the microdissected chromosome belonged to the St genome, three genomes (J(s) , J and St) in Th. intermedium could be distinguished, in which there is no hybridization signal on J genome that is similar to the genome of Th. bessarabicum. Our results showed that the smallest alien chromosomes may represent a truncated chromosome and the repetitive sequence distribution might be similar in different chromosomes within the St genome. However, the repetitive sequence distributions are different within the J(s) genome, within a single chromosome, and among different genomes in Th. intermedium. Our results suggested that chromosome painting could be feasible in some plants and useful in detecting chromosome variation and repetitive sequence distribution in different genomes of polyploidy plants, which is helpful for understanding the evolution of different genomes in polyploid plants.

Association between mutation of the NF2 gene and monosomy 22 in menopausal women with sporadic meningiomas.

Abstract: Meningioma was the first solid tumor shown to contain a recurrent genetic alteration e.g. monosomy 22/del(22q), NF2 being the most relevant gene involved. Although monosomy 22/del(22q) is present in half of all meningiomas, and meningiomas frequently carry NF2 mutations, no study has been reported so far in which both alterations are simultaneously assessed and correlated with the features of the disease. Here, we analyzed the frequency of both copy number changes involving chromosome 22 and NF2 mutations in 20 sporadic meningiomas using high-density SNP-arrays, interphase-FISH and PCR techniques. Our results show a significant frequency of NF2 mutations (6/20 patients, 30%), most of which (5/6) had not been previously reported in sporadic meningiomas. NF2 mutations involved five different exons and led to a truncated protein (p.Leu163CysfsX46, p.Phe62LeufsX61, p.Asp281MetfsX15, p.Phe285LeufsX11, p.Gln389ArgfsX37) and an in frame deletion of Phe119. Interestingly, all NF2 mutated cases were menopausal women with monosomy 22 but not del(22q). These results confirm and extend on previous observations about the high frequency and heterogeneity of NF2 mutations in sporadic meningiomas and indicate they could be restricted to a well-defined cytogenetic and clinical subgroup of menopausal women. Further studies in large series of patients are required to confirm our observations.

Implications of COMT long-range interactions on the phenotypic variability of 22q11.2 deletion syndrome.

Abstract: 22q112 deletion syndrome (22q11DS) results from a hemizygous microdeletion on chromosome 22 and is characterized by extensive phenotypic variability Penetrance of signs, including congenital heart, craniofacial, and neurobehavioral abnormalities, varies widely and is not well correlated with genotype The three-dimensional structure of the genome may help explain some of this variability The physical interaction profile of a given gene locus with other genetic elements, such as enhancers and co-regulated genes, contributes to its regulation Thus, it is possible that regulatory interactions with elements outside the deletion region are disrupted in the disease state and modulate the resulting spectrum of symptoms COMT, a gene within the commonly deleted ~3 Mb region has been implicated as a contributor to the neurological features frequently found in 22q11DS patients We used this locus as bait in a 4C-seq experiment to investigate genome-wide interaction profiles in B lymphocyte and fibroblast cell l

CSF3R T618I co-occurs with mutations of splicing and epigenetic genes and with a new PIM3 truncated fusion gene in chronic neutrophilic leukemia

Abstract: Mutations in CSF3R have been recently defined as the common genetic event in patients with myeloid neoplasms, including the rare entity known as chronic neutrophilic leukemia (CNL),1, 2, 3 becoming a potentially useful biomarker for diagnosing and therapy target.4 CSF3R encodes the transmembrane receptor for granulocyte colony-stimulating factor (G-CSF; CSF3), which provides the proliferative and survival signal for granulocytes and also contributes to their differentiation and function.5 Although there are several studies on massive next-generation sequencing of myeloid disorders, not a single comprehensive study has been reported in CNL. Here, we used whole-exome sequencing (WES) and RNA sequencing (RNA-seq) to identify new candidate genes to the disease pathogenesis of an index CNL patient. A 66-year-old man was diagnosed with CNL, according to the 2008 World Health Organization (WHO) classification. At diagnosis, the patient presented peripheral blood leukocytosis (66 × 109/l), segmented neutrophils and band forms were 91.5% of the white blood cells counts (WBCs), immature granulocytes were <10% of WBCs and myeloblasts were <1%. The aspirate showed a hypercellular bone marrow (BM) with neutrophilic granulocytes increased in number and percentage and myeloblasts 0.5% of WBCs. No dysplastic features were observed in the myeloid lineages. His Zubrod Performance Status (ECOG) was 1. A GTG-banding chromosome analysis revealed a normal karyotype (46,XY[20]), and molecular biology studies were negative for BCR-ABL1 transcripts and JAK2 V617F mutation. The patient was treated with hydroxyurea but, unfortunately, died 7 months after the diagnosis due to an intensification of the disease. To improve our understanding of the genes involved in the pathogenesis of CNL, WES was performed on matched tumor and oral mucosa cell (germline) samples from the patient. Candidate somatic mutations were identified using RUbioSeq software.6 The bioinformatics analysis and the filtering steps to identify the coding variants are detailed in the Supplementary Material. In total, we found 1437 candidate variants; among them, 797 were somatic mutations (412 were intronic, intergenic, affecting non-conding-RNA or untranscribed regions and 385 were exonic). From the 385 exonic variants, we selected only those variants within coding regions that, after passing sequencing depth and quality filters, were, frameshift, stop gain/loss and non-synonymous amino acid changes predicted to produce a deleterious effect in the protein structure, resulting in 56 single-nucleototide variants (SNVs) and small insertions/deletions (indels). We selected 24 for further validation by Haloplex/Ion Torrent. In addition to the CSF3R p.Thr618Ile mutation, we validated mutations in U2AF1, TET2, LUC7L2 and ASXL1 (Figure 1a and Table 1). Figure 1 Molecular characterization of CNL. (a) In addition to the CSF3R T618I mutation, WES revealed mutations in U2AF1, TET2, LUC7L2 and ASXL1. LUC7L2 mutation was found in homozygosis; (b) LOH of 53.2 Mb in chromosome 7q including the locus of LUC7L2 ... Table 1 Description of validated mutations in the CNL patient The current study, first, confirms the observations by Maxon et al.1 and Pardanani et al.2 regarding the association between CNL and CSF3R mutations. Second, it presents a complete picture of the mutational profiling of CNL, certainly more complex than expected from these previous reports. In fact, we found and validated mutations affecting both splicing machinery and epigenetic genes. Kosmider et al.,3 very recently, showed that CSF3R somatic mutations can be identified in ∼4% of chronic myelomonocytic leukemias. These mutations, which affect distinct residues in CSF3R as compared with CNL, are frequently associated with mutations in the ASXL1 gene and have a poor prognostic impact on overall and acute myeloid leukemia (AML)-free survival. Together, these data indicate that CNL genome had a combination of few mutations with a pattern of cooperation with a strong biological relationship among genes and categories, similar to AML.7 Along this line of cooperating mutations on epigenetic genes, we also found in our CNL patient mutated copies of ASXL1 and TET2 genes. The variant allelic frequency of LUC7L2 mutation was found to be high (more than 95%). We previously described mutations of this gene in myelodysplastic syndrome (MDS).8 As this gene is located in the 7q region, a frequently deleted chromosomal region in myeloid leukemias,9 we decided to investigate whether a critical deletion or a loss of heterozygosity (LOH) affecting this genomic region was also present in the patient. To study this phenomenon, we interrogate our WES data for the LOH across the whole genome of the sample. Interestingly, we found an LOH of 53.2 Mb in chromosome 7q including the locus of the LUC7L2 gene. Allele frequencies of each SNP along chromosome 7 are shown in Figure 1b and Supplementary Figure 4. As no del(7q) was detected with metaphase cytogenetics, our study demonstrates for the first time mutations in LUC7L2 accompanied by a copy-neutral LOH (uniparental disomy) in 7q in a patient with an aggressive CNL phenotype. To further evaluate the biological consequences of this homozygous mutation, we explored LUC7L2 expression in the BM cells of the patient and in some myeloid leukemia cell lines (Figure 1c). By using real-time PCR, we observed a downregulation in the expression of LUC7L2 in the patient cells compared with normal granulocytes, as well as a general downregulation in myeloid leukemia cell lines. To determine the functional consequences of the mutations in LUC7L2 and U2AF1, genes involved in the splicing machinery, in the proper splicing process, we performed RNA-seq in our index patient as well as in CD34+ cells from a normal control BM. Although no clear genome-wide increase in intron retention was observed in the patient, as previously reported by us10 in some MDS cases, we found an altered pattern of splicing in the mRNA species transcribed from the RUNX1 gene. At the 3′ splice site of RUNX1 intron 5, the un-spliced reads were almost three times more frequent in the mutated patient than in the normal control (Supplementary Figure 1). Because of the large numbers of diverse mutations in the splicing machinery, larger studies will be needed to fully evaluate the impact of these mutations in splicing. In relation with the effects of an aberrant RNA splicing due to the presence of two mutations in the genes responsible for these processes, we used RNA-seq data to investigate the presence of aberrant fusion transcripts in our CNL patient. In fact, we identified a chimeric transcript involving the PIM3 and SCO2 genes (both were located on 22q13.33), which was the result of an intrachromosomal inversion of approximately 0.6 Mb in chromosome 22 (Figure 1d). The PIM3 oncogene belongs to the Ser/Thr protein kinase family and PIM subfamily. This gene is overexpressed in hematological and epithelial tumors and is associated with MYC co-expression. It has a role in the regulation of signal–transduction cascades, contributing to both cell proliferation and survival, and provides a selective advantage in tumorigenesis.11 Interestingly, the inhibition of PIM kinases by pim kinase inhibitors in Myc-induced lymphoma resulted in cell death.12 Functional studies are needed to elucidate the role of this fusion gene in leukemogenesis. Regarding the global expression profile of CNL, we obtained 2022 genes upregulated and 1884 genes downregulated in CNL compared with the control (FDR=0.05). Functional classification of genes differently expressed between CNL cells and CD34+, according to the Ingenuity Systems Analysis (Ingenuity Systems, www.ingenuity.com), revealed an enrichment of categories like cell signaling, cell death and survival, as well as gene expression (B-H P-value <0.05; Supplementary Figure 2). These findings reconfirmed the significant pathophysiology of such several synergetic unique genetic defects in the general CNL cohort, as well as in our index case. The oncogenic CSF3R mutations T618I strongly activate the JAK/STAT pathway and are sensitive to inhibitors of SRC family TNK2 and JAK kinases and may provide a new avenue for therapy.4 In contrast, pim kinase inhibition could be a viable treatment strategy in certain human leukemias that rely on the PIM3 kinase expression.12 In addition, epigenetic modifiers provide new targets for therapeutic intervention, and targeting these enzymatic activities is currently being explored from a therapeutic standpoint in several types of leukemia.13, 14 Although Pardanani et al.,2 considering 35 cases of clinically suspected CNL, did not found alteration in the survival on the basis of the presence or absence of CSF3R mutations, our reported patient had a rapid disease progression and died 7 months after diagnosis, which was probably explained by the profoundly aberrant landscape of gene mutations and rearrangements with functional effects on the biology of the tumor cells. In summary, our study provides, for the first time, a massive molecular and expression data, revealing a large amount of genomic alterations in CNL. In this complex scenario, a combination of new target therapies may be considered as reasonable options for the therapeutic management of this aggressive and rare subtype of leukemia.

Global Linkage Map Connects Meiotic Centromere Function to Chromosome Size in Budding Yeast

Abstract: Synthetic genetic array (SGA) analysis automates yeast genetics, enabling high-throughput construction of ordered arrays of double mutants. Quantitative colony sizes derived from SGA analysis can be used to measure cellular fitness and score for genetic interactions, such as synthetic lethality. Here we show that SGA colony sizes also can be used to obtain global maps of meiotic recombination because recombination frequency affects double-mutant formation for gene pairs located on the same chromosome and therefore influences the size of the resultant double-mutant colony. We obtained quantitative colony size data for ~1.2 million double mutants located on the same chromosome and constructed a genome-scale genetic linkage map at ~5 kb resolution. We found that our linkage map is reproducible and consistent with previous global studies of meiotic recombination. In particular, we confirmed that the total number of crossovers per chromosome tends to follow a simple linear model that depends on chromosome size. In addition, we observed a previously unappreciated relationship between the size of linkage regions surrounding each centromere and chromosome size, suggesting that crossovers tend to occur farther away from the centromere on larger chromosomes. The pericentric regions of larger chromosomes also appeared to load larger clusters of meiotic cohesin Rec8, and acquire fewer Spo11-catalyzed DNA double-strand breaks. Given that crossovers too near or too far from centromeres are detrimental to homolog disjunction and increase the incidence of aneuploidy, our data suggest that chromosome size may have a direct role in regulating the fidelity of chromosome segregation during meiosis.

Microdissection and painting of the Y chromosome in spinach (Spinacia oleracea)

Abstract: Spinach has long been used as a model for genetic and physiological studies of sex determination and expression Although trisomic analysis from a cross between diploid and triploid plants identified the XY chromosome as the largest chromosome, no direct evidence has been provided to support this at the molecular level In this study, the largest chromosomes of spinach from mitotic metaphase spreads were microdissected using glass needles Degenerate oligonucleotide primed polymerase chain reaction was used to amplify the dissected chromosomes The amplified products from the Y chromosome were identified using the male-specific marker T11A For the first time, the largest spinach chromosome was confirmed to be a sex chromosome at the molecular level PCR products from the isolated chromosomes were used in an in situ probe mixture for painting the Y chromosome The fluorescence signals were mainly distributed on all chromosomes and four pair of weaker punctate fluorescence signal sites were observed on the terminal region of two pair of autosomes These findings provide a foundation for the study of sex chromosome evolution in spinach

Co-existence of 9p deletion and Silver-Russell syndromes in a patient with maternally inherited cryptic complex chromosome rearrangement involving chromosomes 4, 9, and 11.

Abstract: We report a patient with a maternally inherited unbalanced complex chromosomal rearrangement (CCR) involving chromosomes 4, 9, and 11 detected by microarray comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). This patient presents with clinical features of 9p deletion syndrome and Silver-Russell syndrome (SRS). Chromosome analysis performed in 2000 showed what appeared to be a simple terminal deletion of chromosome 9p22.1. aCGH performed in 2010 revealed a 1.63 Mb duplication at 4q28.3, a 15.48 Mb deletion at 9p24.3p22.3, and a 1.95 Mb duplication at 11p15.5. FISH analysis revealed a derivative chromosome 9 resulting from an unbalanced translocation between chromosomes 9 and 11, a chromosome 4 fragment inserted near the breakpoint of the translocation. The 4q28.3 duplication does not contain any currently known genes. The 9p24.3p22.3 deletion region contains 36 OMIM genes including a 3.5 Mb critical region for the 9p-phenotype. The 11p15.5 duplication contains 49 OMIM genes including H19 and IGF2. Maternal aCGH was normal. However, maternal chromosomal and FISH analyses revealed an apparently balanced CCR involving chromosomes 4, 9, and 11. To the best of our knowledge, this is the first report of a patient with maternally inherited trans-duplication of the entire imprinting control region 1 (ICR1) among the 11p15.5 duplications reported in SRS patients. This report supports the hypothesis that the trans-duplication of the maternal copy of ICR1 alone is sufficient for the clinical manifestation of SRS and demonstrates the usefulness of combining aCGH with karyotyping and FISH for detecting cryptic genomic imbalances. © 2012 Wiley Periodicals, Inc.

Hominoid fission of chromosome 14/15 and the role of segmental duplications

Abstract: Human chromosomes 14 and 15 were generated by fission of an ancestral chromosome composed of chromosome 14 attached, head-to-tail, to chromosome 15 around 25 million years ago (Ventura et al. 2003). The rearrangement occurred in the hominoid ancestor and involved a cluster of olfactory receptor (OR) genes (Rudd et al. 2009). At the point of fission, the 15q telomere and the neocentromere of chromosome 14 were seeded (Ventura et al. 2003). The 15q subtelomeric region experienced numerous structural rearrangements, including interstitial deletions and transfers of material to and from other subtelomeric regions (Rudd et al. 2009). Low-copy repeats (LCR), also named segmental duplications (SDs) (Bailey et al. 2001; Cheung et al. 2001), mediate chromosomal rearrangements through non-allelic homologous recombination (NAHR) events (Hastings et al. 2009) and are enriched at pericentromeric and subterminal regions of the human genome (Bailey and Eichler 2006). LCR15 (low-copy repeats on human chromosome 15) loci have been widely studied (Pujana et al. 2001; Zody et al. 2006); those of primary focus are on 15q11–q13 because of their involvement in the Prader-Willi and Angelman syndromes (Pujana et al. 2002). In this work, we provide insights into and highlight the role of LCR in the evolution of human chromosomes 14 and 15. We assembled a 1.6-Mb contig of macaque BAC clones encompassing the 14/15 boundary and tracked evolutionary changes initiated by the fission in the human lineage. We detected duplications from the newly seeded subtelomeric region on chromosome 15 and pericentromeric region on chromosome 14 to other existing subtelomeric and pericentromeric regions, respectively. Further, we traced the dynamics of the formation of the chromosome 14 neocentromere and demonstrated the recruitment of new pericentromeric sequences mainly from the pericentromeric regions of ancestral chromosomes orthologous to human 2q14–qter and 10. We found a correlation between clusters of LCR15 and evolutionary “hotspot” regions. (Note: For simplicity, we used the orthologous numbering of great ape and human chromosomes as proposed for great ape genome sequencing projects [Supplemental Fig. S1; McConkey 2004].)

Combined fluorescent-chromogenic in situ hybridization for identification and laser microdissection of interphase chromosomes.

Abstract: Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis.

Deciphering the evolutionary origin of the RB1 imprint

Abstract: Previously we could show that the RB1 gene is imprinted. Skewed expression in favour of the maternal allele is due to a differentially methylated CpG-island within intron 2 of the RB1 gene. This CpG-island (CpG85) serves as a promoter for an alternative RB1 transcript and is part of a truncated processed pseudogene (PPP1R26P1), which is derived from the PPP1R26 gene (previously KIAA0649) located on chromosome 9. We could now narrow down the time interval of this retrotranspositional event by in silico analyses, which revealed that the ancestral gene PPP1R26 is present in all primates, whereas the pseudogene copy within the RB1 gene is only present in higher primates, which comprise Catarrhini (Old World Monkeys, Gibbons, Great Apes and Human) and Platyrrhini (New World Monkeys). Thus, the retrotransposition of PPP1R26 has occurred after the divergence of Strepsirrhini and higher primates, but before the split between Catarrhini and Platyrrhini. Although information for Tarsiidae as distant sister lineage to higher primates is lacking, the retrotransposition of PPP1R26 into the RB1 gene appears to coincide with the retrotranspositional explosion described by Ohshima et al., 2003. Moreover, the in silico analysis revealed that there are additional pseudogene copies on chromosome 22 in human and chimp, which must have been derived from independent retrotransposition events. Only the chimp and the marmoset have another copy on chromosome 8 and chromosome 4, respectively. For further examination of the evolutionary origin of the RB1 imprint we compared the methylation patterns of the ancestral gene PPP1R26 and its pseudogenes in different primates (human, chimp, rhesus, orangutan and marmoset). Methylation analysis by next generation bisulfite sequencing on the ROCHE/454 GS Junior showed that the pseudogene copy within the RB1 gene is differentially methylated in all primates studied. All other copies are fully methylated except the additional copy on chromosome 4 in the marmoset, which seemed to be differentially methylated. By using an informative SNP for the methylation analysis in 8 individuals from 4 different families we could show that the methylation pattern of the copy on chromosome 4 in the marmoset is not parent-of-origin-specific, but allele-specific. We conclude that the epigenetic fate of a PPP1R26 pseudogene after integration depends on the DNA sequence and selective forces at the integration site.

Genome organization and the role of centromeres in evolution of the erythroleukaemia cell line HEL

Abstract: Background and objectives: The human erythroleukaemia (HEL) cell line has a highly rearranged genome. We matched whole chromosome analysis with cytogenomic microarray data to build a detailed description of these rearrangements. Methodology: We used a combination of single nucleotide polymorphism array and multiple fluorescence in situ hybridization approaches, and compared our array data with publicly available data for different sublines of HEL. B allele frequencies revealed the fate of each homologue for most chromosomes. Results: At least two instances of the breakage–fusion–bridge cycle appear to have facilitated amplification of oncogenes and deletion of tumour suppressor genes. Because our study included centromere identification, we found that some abnormal chromosomes had centromeres that did not match the identity of the rest of the chromosome. Conclusions and implications: This study highlights the variety of complementary methods required to understand remodelling of the genome in cancer and uncover some of the mechanisms involved. We present evidence of centromere capture as a means of preserving broken chromosome segments. Testing for another highly repetitive DNA region, the nucleolus organizer region, helped identify the steps involved in chromosome 9 copy number aberrations. Increased use of techniques for identifying centromeres and other repetitive DNA regions will add to our understanding of genome remodelling and evolution. The pattern of chromosome 20 aberration in HEL supports an association of 20q11.21 original

Direct duplication of the Y chromosome with normal phenotype - incidental finding in two cases.

Abstract: Summary Structural rearrangement in the Y chromosome is closely involved in spermatogenesis. However, several Y chromosome variants may have no deleterious effects on male reproduction. Here, we report two cases of Y chromosomal duplication from incidental findings. Their FISH analysis revealed direct duplication of large segments of short and long arms of the Y chromosome. Nearly two intact Y chromosomes were carried in these two cases with normal phenotype.

Breakpoint characterization of the der(19)t(11;19)(q13;p13) in the ovarian cancer cell line SKOV-3

Abstract: About 20% of ovarian carcinomas show alterations of 19p13 and/or 19q13 in the form of added extra material whose origin often is from chromosome 11. Based on earlier spectral karyotype analysis of the ovarian cancer cell line SKOV-3, which shows an unbalanced translocation der(19)t(11;19), the aim of this study was to determine the precise breakpoints of that derivative chromosome. After rough delimitation of the breakpoints of microdissected derivative chromosomes by array analysis, we designed a matrix of primers spanning 11q13.2 and 19p13.2 detecting multiple amplicons on genomic and cDNA. Sequencing the amplicons, accurate localization of both breakpoints on both chromosomes was possible and we found that exon 14 of HOOK2 from chromosome 19 and exon 2 of ACTN3 from chromosome 11 were fused in the derivative chromosome. The breakpoint in the HOOK2 gene was in an intrinsic triplet of nucleic acids leading to a shift in the ACTN3 reading frame in the derivative chromosome. This frameshift alteration should give rise to an early stop codon causing a loss of function of ACTN3. Signals in two-dimensional Western blotting exactly match to calculated molecular mass and the isoelectric point of the fusion protein.

Mandibular Ewing sarcoma with chromosomal translocation t(21;22)(q22;q12).

Abstract: Ewing sarcoma (ES) is a primary bone malignant neoplasm and is the second most common primary malignancy of the bone found in childhood and adolescence after osteosarcoma. ES has an annual frequency in the population younger than 20 years of approximately 2.9 per million. ES occurs most frequently in the long bones of the extremities and pelvis and very rarely in the jaw. Recently, it was revealed that chromosomal translocation t(11;22)(q24;q12), which fuses the EWS gene on chromosome 22 and the FLI-1 gene on chromosome 11, occurs in most cases of ES. We report here a rare case of mandibular ES in a 10-year-old child with chromosomal translocation t(21;22)(q22;q12) in which the EWS gene is fused with the ERG gene on chromosome 21.

A comparative genetic and cytogenetic mapping of wheat chromosome 5B using introgression lines

Abstract: The genetic map of chromosome 5B has been constructed by using microsatellite (SSR) analysis of 381 plants from the F2 population produced by cross of the Chinese Spring (CS) and Renan cultivars. Initially, 180 SSR markers for the common wheat 5B chromosome have been used for analysis of these cultivars. The 32 markers able to detect polymorphism between these cultivars have been located on the genetic map of chromosome 5B. Cytogenetic mapping has involved a set of CS 5B chromosome deletion lines. Totally, 51 SSR markers have been located in ten regions (deletion bins) of this chromosome by SSR analysis of these deletion lines. Five genes--TaCBFIIIc-B10, Vrn--B1, Chi--B1, Skr, and Ph1--have been integrated into the cytogenetic map of chromosome 5B using the markers either specific of or tightly linked to the genes in question. Com- parison of the genetic and cytogenetic maps suggests that recombination is suppressed in the pericentromeric region of chromosome 5B, especially in the short arm segment. The 18 markers localized to deletion bins 5BL16-0.79-1.00 and 5BL8-0.66-0.79 have been used to analyze common wheat introgression lines L842, L5366-180, L73/00i, and L21-4, carrying fragments of alien genomes in the terminal region of 5B long arm. L5366-180 and L842 lines carry a fragment of the Triticum timopheevii 5GL chromosome, while L73/00i ? L21-4 lines, a fragment of the Aegilopsspeltoides 5SL chromosome. As has been shown, the translocated fragments in these four lines are of different lengths, allowing bin 5BL18-0.66-0.79 to be divided into three shorter regions. The utility of wheat introgression lines carrying alien translocations for increasing the resolution of cytogenetic mapping is discussed.

Parental Imbalances Involving Chromosomes 15q and 22q May Predispose to the Formation of De Novo Pathogenic Microdeletions and Microduplications in the Offspring

Abstract: Microarray-based comparative genomic hybridization (array-CGH) led to the discovery of genetic abnormalities among patients with complex phenotype and normal karyotype. Also several apparently normal individuals have been found to be carriers of cryptic imbalances, hence the importance to perform parental investigations after the identification of a deletion/duplication in a proband. Here, we report the molecular cytogenetic characterization of two individuals in which the microdeletions/duplications present in their parents could have predisposed and facilitated the formation of de novo pathogenic different copy number variations (CNVs). In family 1, a 4-year-old girl had a de novo pathogenic 10.5 Mb duplication at 15q21.2q22.2, while her mother showed a 2.262 Mb deletion at 15q13.2q13.3; in family 2, a 9-year-old boy had a de novo 1.417 Mb deletion at 22q11.21 and a second paternal deletion of 247 Kb at 22q11.23 on the same chromosome 22. Chromosome 22 at band q11.2 and chromosome 15 at band q11q13 are considered unstable regions. We could hypothesize that 15q13.2q13.3 and 22q11.21 deletions in the two respective parents might have increased the risk of rearrangements in their children. This study highlights the difficulty to make genetic counseling and predict the phenotypic consequences in these situations.

PCR and sequence analysis of barley chromosome 2H subjected to the gametocidal action of chromosome 2C

Abstract: Gametocidal (Gc) chromosomes induce various types of chromosomal mutations during gametogenesis in the chromosomes of common wheat and alien chromosomes added to common wheat. However, it is not yet known whether the Gc chromosome causes aberrations at the nucleotide level because mutations caused by Gc chromosomes have been studied only by cytological screening. In order to know whether the Gc chromosome induces point mutations, we conducted PCR analysis and sequencing with the progeny of a common wheat line that is disomic for barley chromosome 2H and monosomic for Gc chromosome 2C. We analyzed 18 2H-specific EST sequences using 81 progeny plants carrying a cytologically normal-appearing 2H chromosome and found no nucleotide changes in the analyzed 1,419 sequences (in total 647,075 bp). During this analysis, we found six plants for which some ESTs could not be PCR amplified, suggesting the presence of chromosomal mutations in these plants. The cytological and PCR analyses of the progeny of the six plants confirmed the occurrence of chromosomal mutations in the parental plants. These results suggested that the Gc chromosome mostly induced chromosomal aberrations, not nucleotide changes, and that the Gc-induced chromosomal mutations in the six plants occurred after fertilization.

Characterization of three small supernumerary marker chromosomes (sSMC) in humans

Abstract: In the present study, three prenatally detected small supernumerary marker chromosomes (sSMC) were identified by banding cytogenetics and characterized in detail by molecular cytogenetics. In one case an sSMC(10) leading to a pericentric partial trisomy and in two cases heterochromatic sSMC derived from chromosome 22 were characterized. Outcomes were reportedly normal for two of the three cases for whom this information was known.

Molecular alterations in meningiomas: association with clinical data.

Abstract: The aim of our study was to evaluate the frequency of deletions on chromosomes 1, 9, 10, 14, 18 and 22 in 75 benign and 15 atypical meningiomas and correlate them with clinical findings. Paired normal and tumor DNA samples were analyzed for loss of heterozygosity (LOH), using 24 microsatellite markers and PCR techniques. Statistical analysis showed that deletions on chromosomes 14 and 18 were significantly associated with tumor grade of meningiomas (p = 0.048 and p = 0.03, respectively). In addition, we found a marginally increased frequency of LOH on chromosome 9 in atypical meningiomas (p = 0.06). Interestingly, LOH on chromosome 14 was significantly associated with tumor size (p = 0.049), as the risk of developing a tumor of more than 4 cm in diameter was 6-times the risk of developing tumor with diameter below 4 cm. The most frequent genetic abnormality in meningiomas is 22 LOH, which seems to be confirmed by the present study in which high frequency of such abnormality was observed (67%). We found associations between chromosome 22 status and histological subtype. LOH on chromosome 22 was more frequent in fibrous meningiomas than in the meningothelial variant (p = 0.001). Besides that, we found a relationship between 22 LOH status and tumor localization: the frequency of LOH in skull base-localized tumors was significantly lower compared to parasagittal meningiomas (p = 0.0004). Our results indicated that allelic loss on chromosomes 9, 10, 14, 18 and 22 may be associated with meningioma pathogenesis and progression.