Showing papers on "Chromosome 22 published in 2014"

Origin and Evolution of B Chromosomes in the Cichlid Fish Astatotilapia latifasciata Based on Integrated Genomic Analyses

Abstract: Approximately 15% of eukaryotes contain supernumerary B chromosomes. When present, B chromosomes frequently represent as much as 5% of the genome. Despite thousands of reports describing the distribution of supernumeraries in various taxa, a comprehensive theory for the origin, maintenance, and evolution of B chromosomes has not emerged. Here, we sequence the complete genomes of individual cichlid fish (Astatotilapia latifasciata )w ith and without B chromosomes, as well as microdissected B chromosomes, to identify DNA sequences on the B. B sequences were further analyzed through quantitative polymerase chain reaction and in situ hybridization. We find that the B chromosome contains thousands of sequences duplicated from essentiall ye very chromosome in the ancestral karyotype. Although most genes on the B chromosome are fragmented, a few are largely intact, and we detect evidence that at least three of them are transcriptionally active. We propose a model in which the B chromosome originated early in the evolutionary history of Lake Victoria cichlids from as mall fragment of one autosome. DNA sequences originating from several autosomes, including protein-coding genes and transposable elements, subsequently inserted into this proto-B. We propose that intact B chromosome genes involved with microtubule organization, kinetochore structure, recombination and progression through the cell cycle may play a role in driving the transmission of the B chromosome. Furthermore, our work suggests that karyotyping is an essential step prior to genome sequencing to avoid problems in genome assembly and analytical biases created by the presence of high copy number sequences on the B chromosome.

Decreased DGCR8 expression and miRNA dysregulation in individuals with 22q11.2 deletion syndrome

Abstract: Deletion of the 1.5-3 Mb region of chromosome 22 at locus 11.2 gives rise to the chromosome 22q11.2 deletion syndrome (22q11DS), also known as DiGeorge and Velocardiofacial Syndromes. It is the most common micro-deletion disorder in humans and one of the most common multiple malformation syndromes. The syndrome is characterized by a broad phenotype, whose characterization has expanded considerably within the last decade and includes many associated findings such as craniofacial anomalies (40%), conotruncal defects of the heart (CHD; 70-80%), hypocalcemia (20-60%), and a range of neurocognitive anomalies with high risk of schizophrenia, all with a broad phenotypic variability. These phenotypic features are believed to be the result of a change in the copy number or dosage of the genes located in the deleted region. Despite this relatively clear genetic etiology, very little is known about which genes modulate phenotypic variations in humans or if they are due to combinatorial effects of reduced dosage of multiple genes acting in concert. Here, we report on decreased expression levels of genes within the deletion region of chromosome 22, including DGCR8, in peripheral leukocytes derived from individuals with 22q11DS compared to healthy controls. Furthermore, we found dysregulated miRNA expression in individuals with 22q11DS, including miR-150, miR-194 and miR-185. We postulate this to be related to DGCR8 haploinsufficiency as DGCR8 regulates miRNA biogenesis. Importantly we demonstrate that the level of some miRNAs correlates with brain measures, CHD and thyroid abnormalities, suggesting that the dysregulated miRNAs may contribute to these phenotypes and/or represent relevant blood biomarkers of the disease in individuals with 22q11DS.

Interstitial 22q13 deletions not involving SHANK3 gene: a new contiguous gene syndrome.

Abstract: Phelan–McDermid syndrome (22q13.3 deletion syndrome) is a contiguous gene disorder resulting from the deletion of the distal long arm of chromosome 22. SHANK3, a gene within the minimal critical region, is a candidate gene for the major neurological features of this syndrome. We report clinical and molecular data from a study of nine patients with overlapping interstitial deletions in 22q13 not involving SHANK3. All of these deletions overlap with the largest, but not with the smallest deletion associated with Phelan–McDermid syndrome. The deletion sizes and breakpoints varied considerably among our patients, with the largest deletion spanning 6.9 Mb and the smallest deletion spanning 2.7 Mb. Eight out of nine patients had a de novo deletion, while in one patient the origin of deletion was unknown. These patients shared clinical features common to Phelan–McDermid syndrome: developmental delay (11/12), speech delay (11/12), hypotonia (9/12), and feeding difficulties (7/12). Moreover, the majority of patients (8/12) exhibited macrocephaly. In the minimal deleted region, we identified two candidate genes, SULT4A1 and PARVB (associated with the PTEN pathway), which could be associated in our cohort with neurological features and macrocephaly/hypotonia, respectively. This study suggests that the haploinsufficiency of genes in the 22q13 region beside SHANK3 contributes to cognitive and speech development, and that these genes are involved in the phenotype associated with the larger Phelan–McDermid syndrome 22q13 deletions. Moreover, because the deletions in our patients do not involve the SHANK3 gene, we posit the existence of a new contiguous gene syndrome proximal to the smallest terminal deletions in the 22q13 region. © 2014 Wiley Periodicals, Inc.

The Two Cis-Acting Sites, parS1 and oriC1 , Contribute to the Longitudinal Organisation of Vibrio cholerae Chromosome I

Abstract: The segregation of bacterial chromosomes follows a precise choreography of spatial organisation. It is initiated by the bipolar migration of the sister copies of the replication origin (ori). Most bacterial chromosomes contain a partition system (Par) with parS sites in close proximity to ori that contribute to the active mobilisation of the ori region towards the old pole. This is thought to result in a longitudinal chromosomal arrangement within the cell. In this study, we followed the duplication frequency and the cellular position of 19 Vibrio cholerae genome loci as a function of cell length. The genome of V. cholerae is divided between two chromosomes, chromosome I and II, which both contain a Par system. The ori region of chromosome I (oriI) is tethered to the old pole, whereas the ori region of chromosome II is found at midcell. Nevertheless, we found that both chromosomes adopted a longitudinal organisation. Chromosome I extended over the entire cell while chromosome II extended over the younger cell half. We further demonstrate that displacing parS sites away from the oriI region rotates the bulk of chromosome I. The only exception was the region where replication terminates, which still localised to the septum. However, the longitudinal arrangement of chromosome I persisted in Par mutants and, as was reported earlier, the ori region still localised towards the old pole. Finally, we show that the Par-independent longitudinal organisation and oriI polarity were perturbed by the introduction of a second origin. Taken together, these results suggest that the Par system is the major contributor to the longitudinal organisation of chromosome I but that the replication program also influences the arrangement of bacterial chromosomes.

Single-copy gene-based chromosome painting in cucumber and its application for chromosome rearrangement analysis in Cucumis.

Abstract: Chromosome painting based on fluorescence in situ hybridization (FISH) has played an important role in chromosome identification and research into chromosome rearrangements, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. In the present work, a chromosome painting method for single-copy gene pools in Cucumis sativus was successfully developed. Gene probes with sizes above 2 kb were detected consistently. A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome-specific gene probes. This single-copy gene-based chromosome painting (ScgCP) technique was performed by PCR amplification, purification, pooling, labeling and hybridization onto chromosome spreads. Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes. Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C. sativus. Three mis-aligned regions on chromosome 4 were identified by the painting patterns. A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross-species FISH in the Cucumis genus. Distinct painting patterns of this region were observed in C. sativus, C. melo and C. metuliferus species. A comparative chromosome map of this region was constructed between cucumber and melon. With increasing sequence resources, this ScgCP technique may be applied on any other sequenced species for chromosome painting research.

Whole exome sequencing reveals that the majority of schwannomatosis cases remain unexplained after excluding SMARCB1 and LZTR1 germline variants.

Abstract: familial and ~10 % of sporadic schwannomatosis patients [4, 10, 12]. recently, another schwannomatosis-predisposing gene within the 22q candidate region has been identified. piotrowski et al. [9] reported germline loss-of-function mutations in LZTR1 in 80 % of familial and sporadic schwannomatosis cases with combined chr22 loss and somatic NF2 mutation (MIM #615670). LZTr1 functions as an adaptor of the cullin 3-containing e3 ubiquitin ligase complex and has recently been implicated in glioblastoma multiforme development [3], indicating a broader role in tumorigenesis. We therefore sought to further explore the prevalence of LZTR1 mutations in a cohort of 23 sporadic schwannomatosis patients. eight tumor-blood DNA pairs from de novo schwannomatosis patients were subjected to whole exome schwannomatosis (MIM #162091) is characterized by the development of multiple schwannomas without vestibular nerve involvement (which is a characteristic of neurofibromatosis type 2—NF2—MIM #101000). About 10 % of patients have a family history of the disease, with the remaining 90 % presumed to be sporadic [7]. Germline mutations of NF2 are not observed [8], although schwannomas frequently harbor somatic NF2 mutations and often display chromosome 22 loss [6]. rare cases with somatic mosaicism for NF2 alterations have also been described [5]. Germline mutations in SMARCB1 (INI1), located proximal to NF2 on chromosome 22, are found in ~30–60 % of

De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder

Abstract: BACKGROUND: Autism spectrum disorders (ASDs) are common and have a strong genetic basis, yet the cause of ?70-80% ASDs remains unknown. By clinical cytogenetic testing, we identified a family in which two brothers had ASD, mild intellectual disability and a chromosome 22 pericentric inversion, not detected in either parent, indicating de novo mutation with parental germinal mosaicism. We hypothesised that the rearrangement was causative of their ASD and localised the chromosome 22 breakpoints. METHODS: The rearrangement was characterised using fluorescence in situ hybridisation, Southern blotting, inverse PCR and dideoxy-sequencing. Open reading frames and intron/exon boundaries of the two physically disrupted genes identified, TCF20 and TNRC6B, were sequenced in 342 families (260 multiplex and 82 simplex) ascertained by the International Molecular Genetic Study of Autism Consortium (IMGSAC). RESULTS: IMGSAC family screening identified a de novo missense mutation of TCF20 in a single case and significant association of a different missense mutation of TCF20 with ASD in three further families. Through exome sequencing in another project, we independently identified a de novo frameshifting mutation of TCF20 in a woman with ASD and moderate intellectual disability. We did not identify a significant association of TNRC6B mutations with ASD. CONCLUSIONS: TCF20 encodes a transcriptional coregulator (also termed SPBP) that is structurally and functionally related to RAI1, the critical dosage-sensitive protein implicated in the behavioural phenotypes of the Smith-Magenis and Potocki-Lupski 17p11.2 deletion/duplication syndromes, in which ASD is frequently diagnosed. This study provides the first evidence that mutations in TCF20 are also associated with ASD.

Novel X-Linked Genes Revealed by Quantitative Polymerase Chain Reaction in the Green Anole, Anolis carolinensis

Abstract: The green anole, Anolis carolinensis (ACA), is the model reptile for a vast array of biological disciplines. It was the first nonavian reptile to have its genome fully sequenced. During the genome project, the XX/XY system of sex chromosomes homologous to chicken chromosome 15 (GGA15) was revealed, and 106 X-linked genes were identified. We selected 38 genes located on eight scaffolds in ACA and having orthologs located on GGA15, then tested their linkage to ACA X chromosome by using comparative quantitative fluorescent real-time polymerase chain reaction applied to male and female genomic DNA. All tested genes appeared to be X-specific and not present on the Y chromosome. Assuming that all genes located on these scaffolds should be localized to the ACA X chromosome, we more than doubled the number of known X-linked genes in ACA, from 106 to 250. While demonstrating that the gene content of chromosome X in ACA and GGA15 is largely conserved, we nevertheless showed that numerous interchromosomal rearrangements had occurred since the splitting of the chicken and anole evolutionary lineages. The presence of many ACA X-specific genes localized to distinct contigs indicates that the ACA Y chromosome should be highly degenerated, having lost a large amount of its original gene content during evolution. The identification of novel genes linked to the X chromosome and absent on the Y chromosome in the model lizard species contributes to ongoing research as to the evolution of sex determination in reptiles and provides important information for future comparative and functional genomics.

The Sister Chromatid Cohesion Pathway Suppresses Multiple Chromosome Gain and Chromosome Amplification

Abstract: Gain or loss of chromosomes resulting in aneuploidy can be important factors in cancer and adaptive evolution. Although chromosome gain is a frequent event in eukaryotes, there is limited information on its genetic control. Here we measured the rates of chromosome gain in wild-type yeast and sister chromatid cohesion (SCC) compromised strains. SCC tethers the newly replicated chromatids until anaphase via the cohesin complex. Chromosome gain was measured by selecting and characterizing copper-resistant colonies that emerged due to increased copies of the metallothionein gene CUP1. Although all defective SCC diploid strains exhibited increased rates of chromosome gain, there were 15-fold differences between them. Of all mutants examined, a hypomorphic mutation at the cohesin complex caused the highest rate of chromosome gain while disruption of WPL1, an important regulator of SCC and chromosome condensation, resulted in the smallest increase in chromosome gain. In addition to defects in SCC, yeast cell type contributed significantly to chromosome gain, with the greatest rates observed for homozygous mating-type diploids, followed by heterozygous mating type, and smallest in haploids. In fact, wpl1-deficient haploids did not show any difference in chromosome gain rates compared to wild-type haploids. Genomic analysis of copper-resistant colonies revealed that the “driver” chromosome for which selection was applied could be amplified to over five copies per diploid cell. In addition, an increase in the expected driver chromosome was often accompanied by a gain of a small number of other chromosomes. We suggest that while chromosome gain due to SCC malfunction can have negative effects through gene imbalance, it could also facilitate opportunities for adaptive changes. In multicellular organisms, both factors could lead to somatic diseases including cancer.

Pseudoautosomal region 1 length polymorphism in the human population.

Abstract: The human sex chromosomes differ in sequence, except for the pseudoautosomal regions (PAR) at the terminus of the short and the long arms, denoted as PAR1 and PAR2. The boundary between PAR1 and the unique X and Y sequences was established during the divergence of the great apes. During a copy number variation screen, we noted a paternally inherited chromosome X duplication in 15 independent families. Subsequent genomic analysis demonstrated that an insertional translocation of X chromosomal sequence into theMa Y chromosome generates an extended PAR. The insertion is generated by non-allelic homologous recombination between a 548 bp LTR6B repeat within the Y chromosome PAR1 and a second LTR6B repeat located 105 kb from the PAR boundary on the X chromosome. The identification of the reciprocal deletion on the X chromosome in one family and the occurrence of the variant in different chromosome Y haplogroups demonstrate this is a recurrent genomic rearrangement in the human population. This finding represents a novel mechanism shaping sex chromosomal evolution.

SMARCB1 involvement in the development of leiomyoma in a patient with schwannomatosis.

Abstract: Germline SMARCB1 mutations predispose in schwannomatosis patients to the development of multiple benign schwannomas and, in some cases, meningiomas. Here, we report on a 34-year-old female patient who developed multiple schwannomas at various locations and in addition a leiomyoma of the cervix uteri. She carried a c.362+1G>A mutation that inactivates the donor splice site of exon 3. This mutation caused the schwannomatosis phenotype in this patient and was also demonstrated to be present in her affected mother. The leiomyoma displayed the genetic features that are characteristic for germline SMARCB1 mutation-associated tumors. The mutant allele retained in the tumor, whereas the wild-type allele was lost by loss of heterozygosity. Furthermore, the loss of heterozygosity involved net loss of chromosome 22. An NF2 mutation was not found. However, quantitative polymerase chain reaction suggested that both NF2 copies were lost in the tumor. Immunostaining with a SMARCB1 antibody revealed the mosaic expression pattern that is typical for schwannomatosis-associated tumors. To our knowledge, this is the first reported case of leiomyoma associated with a germline SMARCB1 mutation. As such, it widens the spectrum of benign tumors associated with a germline SMARCB1 mutation.

Functional annotation of proteome encoded by human chromosome 22.

Abstract: As part of the chromosome-centric human proteome project (C-HPP) initiative, we report our progress on the annotation of chromosome 22. Chromosome 22, spanning 51 million base pairs, was the first chromosome to be sequenced. Gene dosage alterations on this chromosome have been shown to be associated with a number of congenital anomalies. In addition, several rare but aggressive tumors have been associated with this chromosome. A number of important gene families including immunoglobulin lambda locus, Crystallin beta family, and APOBEC gene family are located on this chromosome. On the basis of proteomic profiling of 30 histologically normal tissues and cells using high-resolution mass spectrometry, we show protein evidence of 367 genes on chromosome 22. Importantly, this includes 47 proteins, which are currently annotated as "missing" proteins. We also confirmed the translation start sites of 120 chromosome 22-encoded proteins. Employing a comprehensive proteogenomics analysis pipeline, we provide evidence of novel coding regions on this chromosome which include upstream ORFs and novel exons in addition to correcting existing gene structures. We describe tissue-wise expression of the proteins and the distribution of gene families on this chromosome. These data have been deposited to ProteomeXchange with the identifier PXD000561.

Mapping the deletion endpoints in individuals with 22q11.2 Deletion Syndrome by droplet digital PCR

Abstract: Chromosome 22q11.2 deletion syndrome (22q11DS) is the most common human microdeletion syndrome and is associated with many cognitive, neurological and psychiatric disorders. The majority of individuals have a 3 Mb deletion while others have a nested 1.5 Mb deletion, but rare atypical deletions have also been described. To date, a study using droplet digital PCR (ddPCR) has not been conducted to systematically map the chromosomal breakpoints in individuals with 22q11DS, which would provide important genotypic insight into the various phenotypes observed in this syndrome. This study uses ddPCR to assess copy number (CN) changes within the chromosome 22q11 deletion region and allows the mapping of the deletion endpoints. We used eight TaqMan assays interspersed throughout the deleted region of 22q11.2 to characterize the deleted region of chromosome 22 in 80 individuals known to have 22q11DS by FISH. Ten EvaGreen assays were used for finer mapping of the six identified individuals with 22q11DS atypical deletions and covering different regions of chromosome 22. ddPCR provided non-ambiguous CN measurements across the region, confirmed the presence of the deletion in the individuals screened, and led to the identification of five differently sized and located deletions. The majority of the participants (n = 74) had the large 3 Mb deletions, whereas three had the smaller 1.5 Mb deletions, and the remaining three had an interstitial deletion of different size. The lower cost, rapid execution and high reliability and specificity provided by ddPCR for CN measurements in the 22q11 region constitutes a significant improvement over the variable CN values generated by other technologies. The ability of the ddPCR approach, to provide a high resolution mapping of deletion endpoints may result in the identification of genes that are haplo-insufficient and play a role in the pathogenesis of 22q11DS. Finally, this methodology can be applied to the characterization of other microdeletions throughout the genome.

Unraveling the chromosome 17 patterns of FISH in interphase nuclei: an in-depth analysis of the HER2 amplicon and chromosome 17 centromere by karyotyping, FISH and M-FISH in breast cancer cells

Abstract: In diagnostic pathology, HER2 status is determined in interphase nuclei by fluorescence in situ hybridization (FISH) with probes for the HER2 gene and for the chromosome 17 centromere (CEP17). The latter probe is used as a surrogate for chromosome 17 copies, however chromosome 17 (Chr17) is frequently rearranged. The frequency and type of specific structural Chr17 alterations in breast cancer have been studied by using comparative genomic hybridization and spectral karyotyping, but not fully detailed. Actually, balanced chromosome rearrangements (e.g. translocations or inversions) and low frequency mosaicisms are assessable on metaphases using G-banding karyotype and multicolor FISH (M-FISH) only. We sought to elucidate the CEP17 and HER2 FISH patterns of interphase nuclei by evaluating Chr17 rearrangements in metaphases of 9 breast cancer cell lines and a primary culture from a triple negative breast carcinoma by using G-banding, FISH and M-FISH. Thirty-nine rearranged chromosomes containing a portion of Chr17 were observed. Chromosomes 8 and 11 were the most frequent partners of Chr17 translocations. The lowest frequency of Chr17 abnormalities was observed in the HER2-negative cell lines, while the highest was observed in the HER2-positive SKBR3 cells. The MDA-MB231 triple negative cell line was the sole to show only non-altered copies of Chr17, while the SKBR3, MDA-MB361 and JIMT-1 HER2-positive cells carried no normal Chr17 copies. True polysomy was observed in MDA-MB231 as the only Chr17 alteration. In BT474 cells polysomy was associated to Chr17 structural alterations. By comparing M-FISH and FISH data, in 8 out of 39 rearranged chromosomes only CEP17 signals were detectable, whereas in 14 rearranged chromosomes HER2 and STARD3 genes were present without CEP17 signals. HER2 and STARD3 always co-localized on the same chromosomes and were always co-amplified, whereas TOP2A also mapped to different derivatives and was co-amplified with HER2 and STARD3 on SKBR3 cells only. The high frequency of complex Chr17 abnormalities suggests that the interpretation of FISH results on interphase nuclei using a dual probe assay to assess gene amplification should be performed “with caution”, given that CEP17 signals are not always indicative of normal unaltered or rearranged copies of Chr17.

Vibrio chromosome-specific families

Abstract: We have compared chromosome-specific genes in a set of 18 finished Vibrio genomes, and, in addition, also calculated the pan- and core-genomes from a data set of more than 250 draft Vibrio genome sequences. These genomes come from 9 known species and 2 unknown species. Within the finished chromosomes, we find a core set of 1269 encoded protein families for chromosome 1, and a core of 252 encoded protein families for chromosome 2. Many of these core proteins are also found in the draft genomes (although which chromosome they are located on is unknown.) Of the chromosome specific core protein families, 1169 and 153 are uniquely found in chromosomes 1 and 2, respectively. Gene ontology (GO) terms for each of the protein families were determined, and the different sets for each chromosome were compared. A total of 363 different `Molecular Function` GO categories were found for chromosome 1 specific protein families, and these include several broad activities: pyridoxine 5' phosphate synthetase, glucosylceramidase, heme transport, DNA ligase, amino acid binding, and ribosomal components; in contrast, chromosome 2 specific protein families have only 66 Molecular Function GO terms and include many membrane-associated activities, such as ion channels, transmembrane transporters, and electron transport chain proteins. Thus, it appears that whilst there are many 'housekeeping systems' encoded in chromosome 1, there are far fewer core functions found in chromosome 2. However, the presence of many membrane-associated encoded proteins in chromosome 2 is surprising.

'Escaping' the X chromosome leads to increased gene expression in the male germline of Drosophila melanogaster.

Abstract: Genomic analyses of Drosophila species suggest that the X chromosome presents an unfavourable environment for the expression of genes in the male germline. A previous study in D. melanogaster used a reporter gene driven by a testis-specific promoter to show that expression was greatly reduced when the gene was inserted onto the X chromosome as compared with the autosomes. However, a limitation of this study was that only the expression regulated by a single, autosomal-derived promoter was investigated. To test for an increase in expression associated with ‘escaping' the X chromosome, we analysed reporter gene expression driven by the promoters of three X-linked, testis-expressed genes (CG10920, CG12681 and CG1314) that were inserted randomly throughout the D. melanogaster genome. In all cases, insertions on the autosomes showed significantly higher expression than those on the X chromosome. Thus, even genes whose regulation has adapted to the X-chromosomal environment show increased male germline expression when relocated to an autosome. Our results provide direct experimental evidence for the suppression of X-linked gene expression in the Drosophila male germline that is independent of gene dose.

A novel mouse model for Down syndrome that harbor a single copy of human artificial chromosome (HAC) carrying a limited number of genes from human chromosome 21.

Abstract: Down syndrome (DS), also known as Trisomy 21, is the most common chromosome aneuploidy in live-born children and displays a complicated symptom. To date, several kinds of mouse models have been generated to understand the molecular pathology of DS, yet the gene dosage effects and gene(s)-phenotype(s) correlation are not well understood. In this study, we established a novel method to generate a partial trisomy mice using the mouse ES cells that harbor a single copy of human artificial chromosome (HAC), into which a small human DNA segment containing human chromosome 21 genes cloned in a bacterial artificial chromosome (BAC) was recombined. The produced mice were found to maintain the HAC carrying human genes as a mini-chromosome, hence termed as a Trans-Mini-Chromosomal (TMC) mouse, and HAC was transmitted for more than twenty generations independent from endogenous mouse chromosomes. The three human transgenes including cystathionine β-synthase, U2 auxiliary factor and crystalline alpha A were expressed in several mouse tissues with various expression levels relative to mouse endogenous genes. The novel system is applicable to any of human and/or mouse BAC clones. Thus, the TMC mouse carrying a HAC with a limited number of genes would provide a novel tool for studying gene dosage effects involved in the DS molecular pathogenesis and the gene(s)-phenotype(s) correlation.

Submicroscopic deletion of 5q involving tumor suppressor genes (CTNNA1, HSPA9) and copy neutral loss of heterozygosity associated with TET2 and EZH2 mutations in a case of MDS with normal chromosome and FISH results

Abstract: Advances in genome-wide molecular cytogenetics allow identification of novel submicroscopic DNA copy number alterations (aCNAs) and copy-neutral loss of heterozygosity (cnLOH) resulting in homozygosity for known gene mutations in myeloid neoplasms. We describe the use of an oligo-SNP array for genomic profiling of aCNA and cnLOH, together with sequence analysis of recurrently mutated genes, in a patient with myelodysplastic syndrome (MDS) presenting with normal karyotype and FISH results. Oligo-SNP array analysis revealed a hemizygous deletion of 896 kb at chromosome 5q31.2, representing the smallest 5q deletion reported to date. The deletion involved multiple genes, including two tumor suppressor candidate genes (CTNNA1 and HSPA9) that are associated with MDS/AML. The SNP-array study also detected 3 segments of somatic cnLOH: one involved the entire long arm of chromosome 4; the second involved the distal half of the long arm of chromosome 7, and the third encompassed the entire chromosome 22 (UPD 22). Sequence analysis revealed mutations in TET2 (4q), EZH2 (7q), ASXL1 (20q11.21), and RUNX1 (21q22.3). Coincidently, TET2 and EZH2 were located at segments of cnLOH resulting in their homozygosity. Loss of heterozygosity affecting these two chromosomes and mutations in TET2 and EZH2 are indicative of a myelodysplastic syndrome with a poor prognosis. Deletion of the tumor suppressor genes CTNNA1 and HSPA9 is also likely to contribute to a poor prognosis. Furthermore, the original cnLOHs in multiple chromosomes and additional cnLOH 14q in the follow-up study suggest genetic evolution of the disease and poor prognosis. This study attests to the fact that some patients with a myelodysplastic syndrome who exhibit a normal karyotype may have underlying genetic abnormalities detectable by chromosomal microarray and/or targeted mutation analyses.

Prenatal diagnosis and molecular cytogenetic characterization of chromosome 22q11.2 deletion syndrome associated with congenital heart defects.

Abstract: Objective To report prenatal diagnosis of 22q11.2 deletion syndrome in a pregnancy with congenital heart defects in the fetus. Case report A 26-year-old, primigravid woman was referred for counseling at 24 weeks of gestation because of abnormal ultrasound findings of fetal congenital heart defects. The Level II ultrasound revealed a singleton fetus with heart defects including overriding aorta, small pulmonary artery, and ventricular septal defect. Cordocentesis was performed. The DNA extracted from the cord blood was analyzed by multiplex ligation-dependent amplification (MLPA). The MLPA showed deletion in the DiGeorge syndrome (DGS) critical region of chromosome 22 low copy number repeat (LCR) 22-A∼C. Conventional cytogenetic analysis revealed a normal male karyotype. Repeated amniocentesis and cordocentesis were performed. Whole-genome array comparative genomic hybridization (aCGH) on cord blood was performed. aCGH detected a 3.07-Mb deletion at 22q11.21. Conventional cytogenetic analysis of cultured amniocytes revealed a karyotype 46,XY. Metaphase fluorescence in situ hybridization (FISH) analysis on cultured amniocytes confirmed an interstitial 22q11.2 deletion. Conclusion Prenatal ultrasound findings of congenital heart defects indicate that the fetuses are at increased risk for chromosome abnormalities. Studies for 22q11.2 deletion syndrome should be considered adjunct to conventional karyotyping. Although FISH has become a standard procedure for diagnosis of 22q11.2 deletion syndrome, MLPA can potentially diagnose a broader spectrum of abnormalities, and aCGH analysis has the advantage of refining the 22q11.2 deletion breakpoints and detecting uncharacterized chromosome rearrangements or genomic imbalances.

Recent Selection on the Y-to-Dot Translocation in Drosophila pseudoobscura

Abstract: The Drosophila pseudoobscura dot chromosome acquired genes from the ancestral Drosophila Y chromosome in a Y-to-dot translocation event that occurred between 12.7 and 20.8 Ma. The formerly Y-linked genes mostly retained their testis-specific expression but shrank drastically in size, mostly through intron reduction, since becoming part of the dot chromosome in this species. We investigated the impact of this translocation on the evolution of the both the Y-to-dot translocated region and the original segments of the dot chromosome in D. pseudoobscura. Our survey of polymorphism and divergence across the chromosome reveals a reduction in variation, a deletion polymorphism segregating at high frequency, and a shift in the frequency spectra, all consistent with a history of recent selective sweeps in the Y-to-dot translocated region but not on the rest of the dot chromosome. We do find evidence for recombination primarily as gene conversion on the dot chromosome; however, predicted recombination events are restricted to the part of the dot chromosome outside the translocation. It therefore appears that recombination has resulted in a degree of decoupling between the ancestral Y region and the conserved region of the dot chromosome.

Establishment of a molecular cytogenetic analysis for native tumor tissue of meningiomas-suitable for clinical application

Abstract: Background: Meningiomas are mostly benign tumors which arise from the meninges. They are among the cytogenetically best-studied solid tumors, mostly displaying a normal karyotype or, as a typical primary aberration, monosomy of chromosome 22. Further secondary chromosomal aberrations, especially the deletion of chromosome 1p, are correlated with increasing biological aggressiveness up to malignancy. These data are derived from the cytogenetical characterization of 661 meningiomas, from which the genetic progression score (GPS) has been developed. Due to the high expenditure of time and the expert knowledge for the cytogenetical characterization, the aim of this work was to establish an equally reliable yet more rapid clinical diagnosis based on fluorescence in situ hybridization (FISH) on meningiomas. Thus a comparison between the native tumor tissue and the primary culture of the same tumor was done in order to determine the most efficient method for a molecular cytogenetic characterization. The diagnostic procedure has to deliver fast and robust results, since they must enable the attending physician to plan the appropriate follow-up regimens for the patients. All in all, preparations of native tumor tissue as well as preparations of cell culture of 22 meningiomas were tested with FISH for aberrations concerning the prognostically relevant chromosome regions 1p and 9p, and the chromosomes 10, 14, 18 and 22 in comparison with the particular karyotypes revealed by conventional karyotyping using G-banding. Results: The FISH examinations between native and cultured cells showed an accordance of 93.4%. The comparison of FISH data and karyotyping presented accordance to the greatest possible extent concerning the chromosomes 14, 18 and 22, but to detect the progression associated losses of 1p and 9p FISH is the most sensitive method. Conclusions: The raised data reveal that both methods can be used for a significant analysis of chromosome aberrations on meningiomas. As a result of that the complex primary culture could also be avoided. Therefore a clinical diagnosis based on FISH on meningiomas is at hand for the assignment of patients to a suitable follow-up regimen.

Linking chromosome duplication and segregation via sister chromatid cohesion.

Abstract: DNA replication during S phase generates two identical copies of each chromosome Each chromosome is destined for a daughter cell, but each daughter must receive one and only one copy of each chromosome To ensure accurate chromosome segregation, eukaryotic cells are equipped with a mechanism to pair the chromosomes during chromosome duplication and hold the pairs until a bi-oriented mitotic spindle is formed and the pairs are pulled apart This mechanism is known as sister chromatid cohesion, and its actions span the entire cell cycle During G1, before DNA is copied during S phase, proteins termed cohesins are loaded onto DNA Paired chromosomes are held together through G2 phase, and finally the cohesins are dismantled during mitosis The processes governing sister chromatid cohesion ensure that newly replicated sisters are held together from the moment they are generated to the metaphase–anaphase transition, when sisters separate

Distinct nuclear orientation patterns for mouse chromosome 11 in normal B lymphocytes

Abstract: Characterizing the nuclear orientation of chromosomes in the three-dimensional (3D) nucleus by multicolor banding (mBANDing) is a new approach towards understanding nuclear organization of chromosome territories. An mBANDing paint is composed of multiple overlapping subchromosomal probes that represent different regions of a single chromosome. In this study, we used it for the analysis of chromosome orientation in 3D interphase nuclei. We determined whether the nuclear orientation of the two chromosome 11 homologs was random or preferential, and if it was conserved between diploid mouse Pre B lymphocytes of BALB/c origin and primary B lymphocytes of congenic [T38HxBALB/c]N wild-type mice. The chromosome orientation was assessed visually and through a semi-automated quantitative analysis of the radial and angular orientation patterns observed in both B cell types. Our data indicate that there are different preferential patterns of chromosome 11 orientation, which are not significantly different between both mouse cell types (p > 0.05). In the most common case for both cell types, both copies of chromosome 11 were oriented in parallel with the nuclear border. The second most common pattern in both types of B lymphocytes was with one homolog of chromosome 11 positioned with its telomeric end towards the nuclear center and with its centromeric end towards the periphery, while the other chromosome 11 was found parallel with the nuclear border. In addition to these two most common orientations present in approximately 50% of nuclei from each cell type, other orientations were observed at lower frequencies. We conclude that there are probabilistic, non-random orientation patterns for mouse chromosome 11 in the mouse B lymphocytes we investigated (p < 0.0001).

Reduced rDNA Copy Number Does Not Affect “Competitive” Chromosome Pairing in XYY Males of Drosophila melanogaster

Abstract: The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a “competitive” situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments.

Oocyte morphology on day 0 correlates with aneuploidy as detected by polar body biopsy and FISH.

Abstract: For better selection of oocytes and embryos, preimplantation genetic screening (PGS) was introduced. As from the beginning of IVF, morphology was used as selection criteria; we investigated the combination of both. If there was a correlation between phenotype and genotype, invasive PGS might be replaced. Therefore, 104 cycles with PGS were done by biopsy of the first polar body and FISH with five chromosomes. Morphology of the oocyte was recorded digitally and noted for 12 categories in 4–13 values; evaluation of the chromosomes was noted for five chromosomes in five values. Morphology and genetics were correlated to each other. Correlations between morphology and genetics for day 0 were found: oocytes with an irregular or dark zona are less probable to have a normal chromosome 13 (80 vs. 53 %, p = 0.001). A medium amount of detritus in the perivitelline space makes it more probable to have a normal chromosome 18 (94 vs. 78 %, p = 0.001). A halo in the cytoplasm makes it less probable to be euploid for chromosome 22 (56 vs. 75 %, p = 0.018). For day 1, pattern “1, 2, 3 and fine” in the pronuclei makes it more probable to be euploid for chromosome 22 (78 vs. 63 %, p = 0.002). There are correlations between the oocyte genome and its morphology also on day 0. These correlations are not sufficient to replace PGS.

c.G2114A MYH9 mutation (DFNA17) causes non-syndromic autosomal dominant hearing loss in a Brazilian family

Abstract: We studied a family presenting 10 individuals affected by autosomal dominant deafness in all frequencies and three individuals affected by high frequency hearing loss. Genomic scanning using the 50k Affymetrix microarray technology yielded a Lod Score of 2.1 in chromosome 14 and a Lod Score of 1.9 in chromosome 22. Mapping refinement using microsatellites placed the chromosome 14 candidate region between markers D14S288 and D14S276 (8.85 cM) and the chromosome 22 near marker D22S283. Exome sequencing identified two candidate variants to explain hearing loss in chromosome 14 [PTGDR - c.G894A:p.R298R and PTGER2 - c.T247G:p.C83G], and one in chromosome 22 [MYH9, c.G2114A:p.R705H]. Pedigree segregation analysis allowed exclusion of the PTGDR and PTGER2 variants as the cause of deafness. However, the MYH9 variant segregated with the phenotype in all affected members, except the three individuals with different phenotype. This gene has been previously described as mutated in autosomal dominant hereditary hearing loss and corresponds to DFNA17. The mutation identified in our study is the same described in the prior report. Thus, although linkage studies suggested a candidate gene in chromosome 14, we concluded that the mutation in chromosome 22 better explains the hearing loss phenotype in the Brazilian family.