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1.  Interstitial Telomeric Sequences (ITS) and major rDNA mapping reveal insights into the karyotypical evolution of Neotropical leaf frogs species (Phyllomedusa, Hylidae, Anura) 
The combination of classical cytogenetics with molecular techniques represents a powerful approach for the comparative analysis of the genome, providing data for the systematic identification of chromosomal homologies among species and insights into patterns of chromosomal evolution within phylogenetically related groups. Here, we present cytogenetic data on four species of Neotropical treefrogs of the genus Phyllomedusa (P. vaillantii, P. tarsius, P. distincta, and P. bahiana), collected in Brazil and Ecuador, with the aim of contributing to the understanding of the chromosomal diversification of this genus.
With the exception of P. tarsius, which presented three telocentric pairs, all the species analyzed had conservative karyotypic features. Heterochromatic patterns in the genomes of these species revealed by C-banding and fluorochrome staining indicated the presence of a large number of non-centromeric blocks. Using the Ag-NOR method and FISH with an rDNA 28S probe, we detected NOR in the pericentromeric region of the short arm of pair 7 in P. vaillantii, pair 1 in P. tarsius, chromosomes 1 and 9 in P. distincta, and in chromosome 9 in P. bahiana, in addition to the presence of NOR in one homologue of chromosome pair 10 in some individuals of this species. As expected, the telomeric probe detected the terminal regions of the chromosomes of these four species, although it also detected Interstitial Telomeric Sequences (ITS) in some chromosomes of the P. vaillantii, P. distincta and P. bahiana karyotypes.
A number of conservative chromosomal structures permitted the recognition of karyotypic homologies. The data indicate that the presence of a NOR-bearing chromosome in pair 9 is the plesiomorphic condition in the P. burmeisteri group. The interspecific and intraspecific variation in the number and location of rDNA sites reflects the rapid rate of evolution of this character in Phyllomedusa. The ITS detected in this study does not appear to be a remnant of structural chromosome rearrangements. Telomeric repeats were frequently found in association with heterochromatin regions, primarily in the centromeres, which suggests that (TTAGGG)n repeats might be an important component of this heterochromatin. We propose that the ITSs originated independently during the chromosomal evolution of these species and may provide important insights into the role of these repeats in vertebrate karyotype diversification.
PMCID: PMC3975639  PMID: 24602295
Phyllomedusa; Karyotypes; rDNA; Interstitial telomeric sequences (ITS)
2.  The accuracy of chromosomal microarray testing for identification of embryonic mosaicism in human blastocysts 
Most previous studies of chromosomal mosaicism in IVF embryos were performed by fluorescence in situ hybridization (FISH) methods. While there are reports implicating chromosome aneuploidy in implantation failure following transfer and pregnancy loss by spontaneous miscarriage, the significance of mosaicism for the developmental potential of growing embryos is unknown. However, the low prevalence of chromosomal mosaicism in chorionic villus sampling and amniotic fluid specimens suggests the presence of selection against mosaic embryos for implantation and early pregnancy. The absence of evidence for selective allocation of abnormal cells to the trophectoderm (TE) of mosaic blastocysts permits these cells to be a good proxy for embryonic mosaicism detection by chromosomal microarrays (CMA). The purpose of this study was to establish the limits of detection and the prevalence of chromosome mosaicism in day 5/6 human embryos using CMA with TE biopsies.
From reconstitution experiments we established log2 ratio thresholds for mosaicism detection. These studies indicated that chromosomal mosaicism at levels as low as between 25-37% can be consistently identified. Follow-up studies by FISH on non-transferred abnormal embryos confirmed the diagnostic accuracy of CMA testing. The number of cells in a TE biopsy can influence mosaicism detection.
Chromosomal microarrays can detect mosaicism in TE biopsies when present at levels as low as between 25-37% and the prevalence of day 5/6 blastocysts which were mosaic and had no other abnormalities reached 15% among a cohort of 551 embryos examined. Validated protocols for establishing detection thresholds for mosaicism are important to reduce the likelihood of transferring abnormal embryos.
PMCID: PMC3944884  PMID: 24581286
3.  A parthenogenetic maternal and double paternal contribution to an ovotesticular disorder of sex development 
An ovotesticular disorder of sex development (OT-DSD) was rarely found in human. The mechanism causing such condition is poorly understood. We hereby reported a 11-year-old child with OT-DSD and a karyotype 46,XX/46,XY, a single maternal and double paternal genetic contribution to the patient.
Fluorescence in situ hybridization (FISH), blood grouping, HLA (human leukocyte antigen) haplotyping and a genome-wide scanning of lymphocytes with 398 short tandem repeat microsatellite markers were performed to investigate the origin of the cell lines concerned. ABO typing revealed that two populations of red cells were in the patient, which were group A and group B, both from paternal alleles. HLA haplotyping showed the patient had three haplotypes. Haplotype 1 was inherited from maternity, haplotype 2 and 3 were from paternity. The STR microsatellite analysis showed 25 of the 74 fully informative markers in both parents, three alleles were inherited: one of them was from mother, another two were from father. Seventeen of the thirty-eight paternal markers, the patient inherited two paternal alleles. For 121 informative maternal markers, the patient had a single maternal allele. There were two distinct alleles in locus DXS6810 and DXS1073 on X-chromosome, in which one was from the mother and the other from the father.
The patient was a single maternal and double paternal genetic, which was a type of a parthenogenetic division of a maternal haploid nucleus into two identical nuclei, followed by fertilization by two spermatozoa and fusion of the two zygotes into a single individual at the early embryonic stage. To the best of our knowledge, this is the oldest OT-DSD case of parthenogenetic chimerism. These data provide additional evidence that a parthenogenetic maternal and double paternal contribution causes 46,XX/46,XY OT-DSD.
PMCID: PMC3974030  PMID: 24581244
Ovotesticular disorder of sex development; Parthenogenetic chimera; Molecular genetics
4.  Anophthalmia, hearing loss, abnormal pituitary development and response to growth hormone therapy in three children with microdeletions of 14q22q23 
Microdeletions of 14q22q23 have been associated with eye abnormalities and pituitary defects. Other phenotypic features in deletion carriers including hearing loss and response to growth hormone therapy are less well recognized. We studied genotype and phenotype of three newly identified children with 14q22q23 deletions, two girls and one boy with bilateral anophthalmia, and compared them with previously published deletion patients and individuals with intragenic defects in genes residing in the region.
The three deletions were de novo and ranged in size between 5.8 and 8.9 Mb. All three children lacked one copy of the OTX2 gene and in one of them the deletion involved also the BMP4 gene. All three patients presented partial conductive hearing loss which tended to improve with age. Analysis of endocrine and growth phenotypes showed undetectable anterior pituitary, growth hormone deficiency and progressive growth retardation in all three patients. Growth hormone therapy led to partial catch-up growth in two of the three patients but just prevented further height loss in the third.
The pituitary hypoplasia, growth hormone deficiency and growth retardation associated with 14q22q23 microdeletions are very remarkable, and the latter appears to have an atypical response to growth hormone therapy in some of the cases.
PMCID: PMC3975945  PMID: 24581273
Anophthalmia; 14q22q23 microdeletion; OTX2; Hearing loss; Pituitary; Growth hormone therapy
5.  Monitoring of gas station attendants exposure to benzene, toluene, xylene (BTX) using three-color chromosome painting 
Chronic exposure of BTX (benzene, toluene, xylene) may lead to progressive degeneration of bone marrow, aplastic anemia and/or leukemia. In Brazil there is no self-service fuel in gas stations and attendants fill the fuel themselves. Due to this they are chronically exposed to high concentration of BTX. Occupational exposure to benzene has been associated with increased chromosomal aberrations in peripheral blood lymphocytes. Fluorescence in situ hybridization (FISH) using whole chromosome painting (wcp) probes allows the rapid detection of chromosomal aberration. In the present study three-color wcp probes for chromosomes 1, 2 and 4 were used for monitoring 60 gas station attendants.
Blood tests were done and interviews were conducted for each worker. For searching for possible associations between the clinical characteristics and the frequency of chromosomal aberrations the workers were divided into two groups (≤ 10 chromosomal abnormalities per 1,000 metaphases and > 10 chromosomal abnormalities per 1,000 metaphases).The studied workers had a low median age (36 year), albeit long period of BTX exposure (median was 16 years). Low prevalence of smoking and moderate consumption of alcoholic beverages were found in this population. The cytogenetic analysis showed 16.6% (10/60) of workers with a high frequency of chromosomal abnormalities (>10 chromosomal abnormalities per 1,000 metaphases). Translocations were the most frequently observed chromosome aberration. The statistical analysis revealed highly significant differences in skin color (p = 0.002) and a weak significant differences in gender (p = 0.052) distribution between the two groups.
16.6% of the studied population showed elevated frequencies of chromosomal abnormalities, which is highly likely to be correlated with their exposure to BTX during their work. Therefore, further studies are needed for better characterize the work associated damage of the genome in gas station workers. It is necessary to better understand the risks that these workers are exposed, so that we can be effective in preventing diseases and maintaining the health of these workers and possibly the offspring.
PMCID: PMC3974043  PMID: 24576355
Benzene; Toluene; Xylene; Monitoring; Cytogenetic; Painting; Chromosome
6.  Cytogenetic and molecular analyses of de novo translocation dic(9;13)(p11.2;p12) in an infertile male 
Whole arm t(9;13)(p11;p12) translocations are rare and have been described only a few times; all of the previously reported cases were familial.
We present here an infertile male carrier with a whole-arm reciprocal translocation dic(9;13)(p11.2;p12) revealed by GTG-, C-, and NOR-banding karyotypes with no mature sperm cells in his ejaculate. FISH and genome-wide 400 K CGH microarray (Agilent) analyses demonstrated a balanced chromosome complement and further characterised the abnormality as a dicentric chromosome (9;13): dic(9;13)(pter→p11.2::p12→qter),neo(9)(pter→p12→neo→p11.2). An analysis of the patient’s ejaculated cells identified immature germ cells at different phases of spermatogenesis but no mature spermatozoa. Most (82.5%) of the germ cells were recognised as spermatocytes at stage I, and the cell nuclei were most frequently found in pachytene I (41.8%). We have also undertaken FISH analysis and documented an increased rate of aneuploidy of chromosomes 15, 18, X and Y in the peripheral blood leukocytes of our patient. To study the aneuploidy risk in leukocytes, we have additionally included 9 patients with non-obstructive azoospermia with normal karyotypes.
We propose that the azoospermia observed in the patient with the dic(9;13)(p11.2;p12) translocation was most likely a consequence of a very high proportion (90%) of association between XY bivalents and quadrivalent formations in prophase I.
PMCID: PMC3944724  PMID: 24559467
De novo whole arm dic(9;13)(p11.2;p12) translocation; Meiotic chromosomes; Aneuploidy rates in leukocytes; Male infertility
7.  Reviewer acknowledgement 2014 
Contributing reviewers
The Editors of Molecular Cytogenetics would like to thank all our reviewers who have contributed to the journal in volume 6 (2013).
PMCID: PMC3924429  PMID: 24529454
8.  PAX5 fusion genes in t(7;9)(q11.2;p13) leukemia: a case report and review of the literature 
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by recurrent genetic alterations including chromosomal translocations. The transcription factor PAX5, which is pivotal for B-cell commitment and maintenance, is affected by rearrangements, which lead to the expression of in-frame fusion genes in about 2.5% of the cases.
Using conventional cytogenetics, fluorescence in situ hybridization (FISH), and molecular methods, an additional case with a der(9)t(7;9)(q11.23;p13) resulting in the expression of a PAX5-ELN fusion gene was identified. Furthermore, a general review of leukemia harboring a t(7;9)(q11.2;p13) or der(9)t(7;9)(q11.2;p13), which occurs more often in children than in adults and shows a remarkably high male preponderance, is given. These cytogenetically highly similar translocations lead to the expression of one of three different in frame PAX5-fusions, namely with AUTS2 (7q11.22), ELN (7q11.23), or POM121 (7q11.23), which constitute the only currently known cluster of PAX5 partner genes.
Our report underlines the recurrent involvement of PAX5 in different fusion genes resulting either from t(7;9)(q11.2;p13) or der(9)t(7;9)(q11.2;p13), which cannot be distinguished cytogenetically and whose discrimination requires molecular analysis.
PMCID: PMC3937052  PMID: 24507461
B-cell precursor acute lymphoblastic leukemia; t(7;9)(q11.2;p13); der(9)t(7;9)(q11.2;p13); PAX5-fusions; ELN; AUTS2; POM121
9.  Establishment of a molecular cytogenetic analysis for native tumor tissue of meningiomas-suitable for clinical application 
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.
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.
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.
PMCID: PMC3937053  PMID: 24499596
Meningioma; Chromosomes; Cell culture; Fluorescence in situ hybridization
10.  MECP2 duplication phenotype in symptomatic females: report of three further cases 
Xq28 duplications, including MECP2 (methyl CpG-binding protein 2; OMIM 300005), have been identified in approximately 140 male patients presenting with hypotonia, severe developmental delay/intellectual disability, limited or absent speech and ambulation, and recurrent respiratory infections. Female patients with Xq28 duplication have been rarely reported and are usually asymptomatic. Altogether, only fifteen symptomatic females with Xq28 duplications including MECP2 have been reported so far: six of them had interstitial duplications while the remaining had a duplication due to an unbalanced X;autosome translocation. Some of these females present with unspecific mild to moderate intellectual disability whereas a more complex phenotype is reported for females with unbalanced X;autosome translocations.
Here we report on the clinical features of three other adolescent to adult female patients with Xq28 interstitial duplications of variable size, all including MECP2 gene.
Mild to moderate cognitive impairment together with learning difficulties and speech delay were evident in each of our patients. Moreover, early inadequate behavioral patterns followed by persistent difficulties in the social and communication domains, as well as the occurrence of mild psychiatric disturbances, are common features of these three patients.
PMCID: PMC3922903  PMID: 24472397
MECP2; Xq28 duplication; X chromosome inactivation
11.  Induction of polyploidy by nuclear fusion mechanism upon decreased expression of the nuclear envelope protein LAP2β in the human osteosarcoma cell line U2OS 
Polyploidy has been recognized for many years as an important hallmark of cancer cells. Polyploid cells can arise through cell fusion, endoreplication and abortive cell cycle. The inner nuclear membrane protein LAP2β plays key roles in nuclear envelope breakdown and reassembly during mitosis, initiation of replication and transcriptional repression. Here we studied the function of LAP2β in the maintenance of cell ploidy state, a role which has not yet been assigned to this protein.
By knocking down the expression of LAP2β, using both viral and non-viral RNAi approaches in osteosarcoma derived U2OS cells, we detected enlarged nuclear size, nearly doubling of DNA content and chromosomal duplications, as analyzed by fluorescent in situ hybridization and spectral karyotyping methodologies. Spectral karyotyping analyses revealed that near-hexaploid karyotypes of LAP2β knocked down cells consisted of not only seven duplicated chromosomal markers, as could be anticipated by genome duplication mechanism, but also of four single chromosomal markers. Furthermore, spectral karyotyping analysis revealed that both of two near-triploid U2OS sub-clones contained the seven markers that were duplicated in LAP2β knocked down cells, whereas the four single chromosomal markers were detected only in one of them. Gene expression profiling of LAP2β knocked down cells revealed that up to a third of the genes exhibiting significant changes in their expression are involved in cancer progression.
Our results suggest that nuclear fusion mechanism underlies the polyploidization induction upon LAP2β reduced expression. Our study implies on a novel role of LAP2β in the maintenance of cell ploidy status. LAP2β depleted U2OS cells can serve as a model to investigate polyploidy and aneuploidy formation by nuclear fusion mechanism and its involvement in cancerogenesis.
PMCID: PMC3926685  PMID: 24472424
LAP2; Cell fusion; Nuclear fusion; Polyploidy; Nuclear envelope; Osteosarcoma; SKY; U2OS
12.  De novo duplication of chromosome 16p in a female infant with signs of neonatal hemochromatosis 
Reported cases of “pure” duplication of the entire short arm of chromosome 16 (16p) are rare, with only 7 patients described in the literature. We report on a female infant with de novo 16p duplication localized to the short arm of chromosome 6, detected by chromosomal analysis and characterized by array CGH and fluorescence in situ hybridization. This baby girl presented with clinical symptoms characteristic of patients with duplications of the short arm of chromosome 16: psychomotor retardation, constitutional growth delay and specific dysmorphic features, including proximally placed hypoplastic thumbs. In addition, she exhibited evidence of neonatal hemochromatosis as shown by direct hyperbilirubinemia, iron overload and elevated liver enzyme levels. To our knowledge, this is the first report of signs of neonatal hemochromatosis in a patient with 16p duplication.
PMCID: PMC3905920  PMID: 24456940
16p duplication; 16p dup; 16p whole arm duplication; Chromosome 16; Duplication of short arm of chromosome 16; Neonatal hemochromatosis; Iron overload; Neonatal liver failure
13.  Differences and homologies of chromosomal alterations within and between breast cancer cell lines: a clustering analysis 
The MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism.
A range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines.
Our study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines.
The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level.
PMCID: PMC3914704  PMID: 24456987
Cytogenetic; Chromosomal abnormalities; Breast cancer cell lines; Hierarchical cluster
14.  Best diagnostic approach for the genetic evaluation of fetuses after intrauterine death in first, second or third trimester: QF-PCR, karyotyping and/or genome wide SNP array analysis 
The aim of this study was to evaluate the best diagnostic approach for the genetic analysis of samples from first, second and third trimester intrauterine fetal deaths (IUFDs). We examined a total of 417 IUFD samples from fetuses with and without congenital anomalies. On 414 samples, karyotyping (N = 46) and/or rapid aneuploidy testing by QF-PCR (N = 371) was performed). One hundred sixty eight samples with a normal test result were subsequently tested by genome wide Single Nucleotide Polymorphism (SNP) array analysis. Three samples were only analyzed by array.
In 50 (12.0%) samples an aneuploidy was detected by QF-PCR and/or karyotyping, representing 47.1% of first, 13.2% of second and 3.4% of third trimester pregnancies. Karyotyping and QF-PCR failed in 4 (8.7%) and 7 (1.9%) samples, respectively, concerning mostly contaminated amniotic fluid samples from third trimester pregnancies.
Clinically relevant aberrations were identified in 4.2% (all fetuses with malformations) of the 168 samples tested by SNP array. Inherited copy number variants (CNVs) were detected in 5.4% and 8.9% showed CNVs of unknown clinical relevance as parental inheritance could not be studied yet. In a sample from a fetus suspect for Meckel-Grüber syndrome, the genotype information from the SNP array revealed various stretches of homozygosity, including one stretch encompassing the CEP290 gene. Subsequent CEP290 mutation analysis revealed a homozygous, pathogenic mutation in this gene.
Based on our experience we recommend QF-PCR as the first-line test in IUFD samples of first and second trimester pregnancies to exclude aneuploidy before performing array analysis. The chance to detect aneuploidy in third trimester pregnancies is relatively low and therefore array analysis can be performed as a first-tier test. A tissue sample, instead of amniotic fluid, is preferred because of a higher success rate in testing.
We emphasize the need for analysis of parental samples whenever a rare, unique CNV is detected to allow for better interpretation of such findings and to improve future pregnancy management. Furthermore, we illustrate the strength of SNP arrays for genotype analysis, even though we realize it is crucial to have detailed phenotypic information to make optimal use of the genotype data in finding candidate recessive genes that may be related to the fetal phenotype.
PMCID: PMC3906897  PMID: 24428858
Intrauterine fetal death; IUFD; DNA; QF-PCR; Karyotyping; SNP array
15.  In memoriam of Anna D Polityko (17.12.1959 — 20.04.2013) 
PMCID: PMC3895789  PMID: 24423091
16.  An evaluation of SOX2 and hTERC gene amplifications as screening markers in oral and oropharyngeal squamous cell carcinomas 
Oral and oropharyngeal squamous cell carcinomas (OSCC) are among the most common cancers. The poor survival rate among oral cancer patients can be attributed to several factors, one of them being lack of early detection. A key approach to this problem would be to detect potentially malignant lesion at their early stage. Using the FISH technique, oral brush cytology slides can be an easy and rapid screening approach for malignant cell detection. The present study was designed to detect hTERC and SOX2 amplifications in OSSC exfoliative tumor cells and evaluate whether those two gene amplifications might serve as a supportive biomarker in early detection and diagnosis of oral and oropharyngeal SCC.
Brush biopsies were collected from exophytic and exulcerated oral and oropharyngeal lesions of the oral cavity of 71 patients and 22 healthy controls. FISH techniques using a TERC-specific DNA probe and a SOX2 DNA specific probe both combined with a centromere 3-specific control probe was performed on the cytology slides. A 100 squamous epithelial cell nuclei of the smears per slide were analysed. As abnormal FISH pattern were considered amplified and polyploid patterns.
From 71 brush biopsies of oropharynx and other locations in oral cavity analysed by FISH 49 were considered to be abnormal (69%). The over representation of polyploidy and/or TERC/SOX2 amplification in tumour samples was statistically significant when compared to controls (p = 0.01).
SOX2 and TERC gene amplifications are common in all squamous cell carcinomas and their detection in early stages could be crucial for early detection and more accurate prognosis. Our study strongly suggests that early detection by FISH on cytobrushed samples could be a possible non-invasive screening method even before a tissue biopsy is performed.
PMCID: PMC3900261  PMID: 24410919
SOX and hTERC gene amplifications; Brush biopsy; Oral; Oropharyngeal squamous cell carcinoma
17.  Cytogenomic mapping and bioinformatic mining reveal interacting brain expressed genes for intellectual disability 
Microarray analysis has been used as the first-tier genetic testing to detect chromosomal imbalances and copy number variants (CNVs) for pediatric patients with intellectual and developmental disabilities (ID/DD). To further investigate the candidate genes and underlying dosage-sensitive mechanisms related to ID, cytogenomic mapping of critical regions and bioinformatic mining of candidate brain-expressed genes (BEGs) and their functional interactions were performed. Critical regions of chromosomal imbalances and pathogenic CNVs were mapped by subtracting known benign CNVs from the Databases of Genomic Variants (DGV) and extracting smallest overlap regions with cases from DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER). BEGs from these critical regions were revealed by functional annotation using Database for Annotation, Visualization, and Integrated Discovery (DAVID) and by tissue expression pattern from Uniprot. Cross-region interrelations and functional networks of the BEGs were analyzed using Gene Relationships Across Implicated Loci (GRAIL) and Ingenuity Pathway Analysis (IPA).
Of the 1,354 patients analyzed by oligonucleotide array comparative genomic hybridization (aCGH), pathogenic abnormalities were detected in 176 patients including genomic disorders in 66 patients (37.5%), subtelomeric rearrangements in 45 patients (25.6%), interstitial imbalances in 33 patients (18.8%), chromosomal structural rearrangements in 17 patients (9.7%) and aneuploidies in 15 patients (8.5%). Subtractive and extractive mapping defined 82 disjointed critical regions from the detected abnormalities. A total of 461 BEGs was generated from 73 disjointed critical regions. Enrichment of central nervous system specific genes in these regions was noted. The number of BEGs increased with the size of the regions. A list of 108 candidate BEGs with significant cross region interrelation was identified by GRAIL and five significant gene networks involving cell cycle, cell-to-cell signaling, cellular assembly, cell morphology, and gene expression regulations were denoted by IPA.
These results characterized ID related cross-region interrelations and multiple networks of candidate BEGs from the detected genomic imbalances. Further experimental study of these BEGs and their interactions will lead to a better understanding of dosage-sensitive mechanisms and modifying effects of human mental development.
PMCID: PMC3905969  PMID: 24410907
Intellectual disability; Critical regions; Brain expressed genes; Cross-region gene interrelation; Functional network
18.  Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling 
Characteristic genomic abnormalities in patients with B cell chronic lymphocytic leukemia (CLL) have been shown to provide important prognostic information. Fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA), currently used in clinical diagnostics of CLL, are targeted tests aimed at specific genomic loci. Microarray-based genomic profiling is a new high-resolution tool that enables genome-wide analyses. The aim of this study was to compare two recently launched genomic microarray platforms, i.e., the CytoScan HD Array (Affymetrix) and the HumanOmniExpress Array (Illumina), with FISH and MLPA to ascertain whether these latter tests can be replaced by either one of the microarray platforms in a clinical diagnostic setting.
Microarray-based genomic profiling and FISH were performed in all 28 CLL patients. For an unbiased comparison of the performance of both microarray platforms 9 patients were evaluated on both platforms, resulting in the identification of exactly identical genomic aberrations. To evaluate the detection limit of the microarray platforms we included 7 patients in which the genomic abnormalities were present in a relatively low percentage of the cells (range 5-28%) as previously determined by FISH. We found that both microarray platforms allowed the detection of copy number abnormalities present in as few as 16% of the cells. In addition, we found that microarray-based genomic profiling allowed the identification of genomic abnormalities that could not be detected by FISH and/or MLPA, including a focal TP53 loss and copy neutral losses of heterozygosity of chromosome 17p.
From our results we conclude that although the microarray platforms exhibit a somewhat lower limit of detection compared to FISH, they still allow the detection of copy number abnormalities present in as few as 16% of the cells. By applying similar interpretation criteria, the results obtained from both platforms were comparable. In addition, we conclude that both microarray platforms allow the identification of additional potential prognostic relevant abnormalities such as focal TP53 deletions and copy neutral losses of heterozygosity of chromosome 17p, which would have remained undetected by FISH or MLPA. The prognostic relevance of these novel genomic alterations requires further evaluation in prospective clinical trials.
PMCID: PMC3905918  PMID: 24401281
Chronic lymphocytic leukemia; Microarray-based genomic profiling; FISH; MLPA
19.  Homozygous deletion of TNFRSF4, TP73, PPAP2B and DPYD at 1p and PDCD5 at 19q identified by multiplex ligation-dependent probe amplification (MLPA) analysis in pediatric anaplastic glioma with questionable oligodendroglial component 
Pediatric oligodendrogliomas are rare and appear to show a different molecular profile from adult tumors. Some gliomas display allelic losses at 1p/19q in pediatric patients, although less frequently than in adult patients, but this is rare in tumors with an oligodendroglial component. The molecular basis of this genomic abnormality is unknown in pediatric gliomas, but it represents a relatively common finding in pediatric oligodendroglioma-like neoplasms with leptomeningeal dissemination.
Multiplex ligation-dependent probe amplification (MLPA) analysis using SALSA P088-B1 for the analysis of the 1p/19q allelic constitution in a pediatric anaplastic (oligodendro)-glioma showed homozygous co-deletion for markers: TNFRSF4 (located at 1p36.33), TP73 (1p36.32), PPAP2B (1pter-p22.1), DPYD (1p21.3), and PDCD5 (19q13.12), and hemizygous deletion of BAX (19q13.3-q13.4). No sequence changes for R132 and R172 of the IDH1/2 genes were identified.
The molecular findings in this pediatric anaplastic glioma do not allow for a clearly definitive pathological diagnosis. However, the findings provide data on a number of 1p/19q genomic regions that, because of homozygotic deletion, might be the location of genes that are important for the development and clinical evolution of some malignant gliomas in children.
PMCID: PMC3905963  PMID: 24387276
Pediatric anaplastic glioma; Oligodendroglioma; Homozygous deletion 1p/19q; MLPA
20.  Body maps on the human genome 
Chromosomes have territories, or preferred locales, in the cell nucleus. When these sites are taken into account, some large-scale structure of the human genome emerges.
The synoptic picture is that genes highly expressed in particular topologically compact tissues are not randomly distributed on the genome. Rather, such tissue-specific genes tend to map somatotopically onto the complete chromosome set. They seem to form a “genome homunculus”: a multi-dimensional, genome-wide body representation extending across chromosome territories of the entire spermcell nucleus. The antero-posterior axis of the body significantly corresponds to the head-tail axis of the nucleus, and the dorso-ventral body axis to the central-peripheral nucleus axis.
This large-scale genomic structure includes thousands of genes. One rationale for a homuncular genome structure would be to minimize connection costs in genetic networks. Somatotopic maps in cerebral cortex have been reported for over a century.
PMCID: PMC3905923  PMID: 24354739
Somatotopic map; Homunculus; Tissue-specific gene; Chromosome territory; Connection optimization
21.  Molecular cytogenetic analysis of Xq critical regions in premature ovarian failure 
One of the frequent reasons for unsuccessful conception is premature ovarian failure/primary ovarian insufficiency (POF/POI) that is defined as the loss of functional follicles below the age of 40 years. Among the genetic causes the most common one involves the X chromosome, as in Turner syndrome, partial X deletion and X-autosome translocations. Here we report a case of a 27-year-old female patient referred to genetic counselling because of premature ovarian failure. The aim of this case study to perform molecular genetic and cytogenetic analyses in order to identify the exact genetic background of the pathogenic phenotype.
For premature ovarian failure disease diagnostics we performed the Fragile mental retardation 1 gene analysis using Southern blot technique and Repeat Primed PCR in order to identify the relationship between the Fragile mental retardation 1 gene premutation status and the premature ovarion failure disease. At this early onset, the premature ovarian failure affected patient we detected one normal allele of Fragile mental retardation 1 gene and we couldn’t verify the methylated allele, therefore we performed the cytogenetic analyses using G-banding and fluorescent in situ hybridization methods and a high resolution molecular cytogenetic method, the array comparative genomic hybridization technique. For this patient applying the G-banding, we identified a large deletion on the X chromosome at the critical region (ChrX q21.31-q28) which is associated with the premature ovarian failure phenotype. In order to detect the exact breakpoints, we used a special cytogenetic array ISCA plus CGH array and we verified a 67.355 Mb size loss at the critical region which include total 795 genes.
We conclude for this case study that the karyotyping is definitely helpful in the evaluation of premature ovarian failure patients, to identify the non submicroscopic chromosomal rearrangement, and using the array CGH technique we can contribute to the most efficient detection and mapping of exact deletion breakpoints of the deleted Xq region.
PMCID: PMC3914679  PMID: 24359613
Sterility; Premature premature ovarian failure (POF); Primary ovarian insufficiency (POI); FMR1 gene analysis; Array–comparative genomic hybridization (aCGH); X chromosome deletion; Repeat primed PCR; G-banding; Deletion breakpoint; Turner syndrome
22.  Karyotypic analysis and FISH mapping of microsatellite motifs reveal highly differentiated XX/XY sex chromosomes in the pink-tailed worm-lizard (Aprasia parapulchella, Pygopodidae, Squamata) 
The infraorder Gekkota is intriguing because it contains multiple chromosomal and environmental sex determination systems that vary even among closely related taxa. Here, we compare male and females karyotypes of the pink-tailed worm-lizard (Aprasia parapulchella), a small legless lizard belonging to the endemic Australian family Pygopodidae.
We applied comparative genomic hybridization to reveal an XX/XY sex chromosome system in which the Y chromosome is highly differentiated from the X in both gross morphology and DNA sequence. In addition, FISH mapping has revealed that two microsatellite repeat motifs, (AGAT)n and (AC)n, have been amplified multiple times on the Y chromosome.
XY karyotypes are found in other pygopodids (Delma inornata and Lialis burtonis), suggesting that the common ancestor of Pygopodidae also had XY sex chromosomes. However, the morphology and size of the Y chromosomes are different among the three species, suggesting that the processes underlying the evolution of sex chromosomes in the Pygopodidae involved chromosome rearrangements and accumulation and amplification of repeats.
PMCID: PMC3905675  PMID: 24344753
Reptile; Sex chromosome differentiation; Y chromosome degeneration
23.  Polyploidy in myelofibrosis: analysis by cytogenetic and SNP array indicates association with advancing disease 
Myelofibrosis occurs as primary myelofibrosis or as a late occurrence in the evolution of essential thrombocythaemia and polycythaemia vera. It is the rarest of the three classic myeloproliferative neoplasms (MPN). Polyploidy has only rarely been reported in MPN despite the prominent involvement of abnormal megakaryocytes. The use of peripheral blood samples containing increased numbers of haematopoietic progenitors has improved the output from cytogenetic studies in myelofibrosis and together with the use of single nucleotide polymorphism arrays (SNPa) has contributed to an improved knowledge regarding the diverse genetic landscape of this rare disease.
Cytogenetic studies performed on a consecutive cohort of 42 patients with primary or post ET/PV myelofibrosis showed an abnormal karyotype in 24 cases and of these, nine showed a polyploid clone. Six of the nine cases showed a tetraploid (4n) subclone, whereas three showed mixed polyploid subclones with both tetraploid and octoploid (4n/8n) cell lines. The abnormal clone evolved from a near diploid karyotype at the initial investigation to a tetraploid karyotype in follow-up cytogenetic analysis in four cases. In total, six of the nine polyploid cases showed gain of 1q material. The remaining three cases showed polyploid metaphases, but with no detectable structural karyotypic rearrangements. Three of the nine cases showed chromosome abnormalities of 6p, either at diagnosis or later acquired. SNPa analysis on eight polyploid cases showed additional changes not previously recognised by karyotype analysis alone, including recurring changes involving 9p, 14q, 17q and 22q. Except for gain of 1q, SNPa findings from the polyploid group compared to eight non-polyploid cases with myelofibrosis found no significant differences in the type of abnormality detected.
The study showed the use of peripheral blood samples to be suitable for standard karyotyping evaluation and DNA based studies. The overall profile of abnormalities found were comparable with that of post-MPN acute myeloid leukaemia or secondary myelodysplastic syndrome and cases in the polyploidy group were associated with features of high risk disease. The above represents the first documented series of polyploid karyotypes in myelofibrosis and shows a high representation of gain of 1q.
PMCID: PMC3906908  PMID: 24341401
Gain of 1q; Myelofibrosis; Tetraploidy; Polyploidy; SNP array
24.  First detailed reconstruction of the karyotype of Trachypithecus cristatus (Mammalia: Cercopithecidae) 
The chromosomal homologies of human (Homo sapiens = HSA) and silvered leaf monkey (Trachypithecus cristatus = TCR) have been previously studied by classical chromosome staining and by fluorescence in situ hybridization (FISH) applying chromosome-specific DNA probes of all human chromosomes in the 1980s and 1990s, respectively.
However, as the resolution of these techniques is limited we used multicolor banding (MCB) at an ~250-band level, and other selected human DNA probes to establish a detailed chromosomal map of TCR. Therefore it was possible to precisely determine evolutionary conserved breakpoints, orientation of segments and distribution of specific regions in TCR compared to HSA. Overall, 69 evolutionary conserved breakpoints including chromosomal segments, which failed to be resolved in previous reports, were exactly identified and characterized.
This work also represents the first molecular cytogenetic one characterizing a multiple sex chromosome system with a male karyotype 44,XY1Y2. The obtained results are compared to other available data for old world monkeys and drawbacks in hominoid evolution are discussed.
PMCID: PMC3914712  PMID: 24341374
Evolutionary conserved breakpoints; Multicolor banding; Old world monkeys; XY1Y2 sex system
25.  Will we cure cancer by sequencing thousands of genomes? 
The promise to understand cancer and develop efficacious therapies by sequencing thousands of cancers has not occurred. Mutations in specific genes termed oncogenes and tumor suppressor genes are extremely heterogeneous amongst the same type of cancer as well as between cancers. They provide little selective advantage to the cancer and in functional tests have yet to be shown to be sufficient for transformation. Here I discuss the karyotyptic theory of cancer and ask if it is time for a new approach to understanding and ultimately treating cancer.
PMCID: PMC3906905  PMID: 24330806
Cancer genome; Cancer; Karyotype; Oncogenes; Tumor suppressor genes

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