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1.  Interstitial deletion at 11q14.2-11q22.1 may cause severe learning difficulties, mental retardation and mild heart defects in 13-year old male 
Interstitial deletions of the long arm of chromosome 11 are rare, and they could be assumed as non-recurrent chromosomal rearrangements due to high variability of the size and the breakpoints of the deleted region. The exact region of the deletion was difficult to be determined before the use of molecular cytogenetic techniques such as array comparative genomic hybridization (aCGH). Here, a 13-year old boy with severe learning difficulties, mental retardation and mild heart defects is described. Conventional G-band karyotyping was performed and it is found that the patient is a carrier of a de novo interstitial deletion on the long arm of chromosome 11, involving 11q14 and 11q22 breakpoints. Further investigation, using aCGH, specified the deleted region to 11q14.2-11q22.1. There was a difficulty in correlating the genotype with the phenotype of the patient due to lack of similar cases in literature. More studies should be done in order to understand the genetic background that underlies the phenotypic differences observed in similar cases.
PMCID: PMC4574176  PMID: 26388939
2.  A chromosomal rearrangement in a child with severe speech and language disorder separates FOXP2 from a functional enhancer 
Mutations of FOXP2 in 7q31 cause a rare disorder involving speech apraxia, accompanied by expressive and receptive language impairments. A recent report described a child with speech and language deficits, and a genomic rearrangement affecting chromosomes 7 and 11. One breakpoint mapped to 7q31 and, although outside its coding region, was hypothesised to disrupt FOXP2 expression. We identified an element 2 kb downstream of this breakpoint with epigenetic characteristics of an enhancer. We show that this element drives reporter gene expression in human cell-lines. Thus, displacement of this element by translocation may disturb gene expression, contributing to the observed language phenotype.
PMCID: PMC4546047  PMID: 26300977
3.  Disomy 21 in spermatozoa and the paternal origin of trisomy 21 Down syndrome 
Trisomy 21 Down syndrome is the most common genetic cause for congenital malformations and intellectual disability. It is well known that in the outstanding majority of cases the extra chromosome 21 originates from the mother but only in less than 10 % from the father. The mechanism underlying this striking difference in parental origin of Trisomy 21 Down syndrome is still unknown. However, it seems likely that the main reason is a much higher stringency in the elimination of any trisomy 21 cells during fetal testicular than ovarian development. We have here focussed attention on the paternal gametic output, i.e. the incidence of disomy 21 in spermatozoa.
We have used fluorescence in situ hybridisation (FISH) to determine the copy number of chromosome 21 in spermatozoa from 11 men with normal spermiograms. Due to the well-known risk of false positive and false negative signals using a single FISH probe, we have applied two chromosome 21q probes, and we have added a chromosome 18-specific probe to allow differentiation between disomy 21 and diploidy. Analysing a total number of 2000 spermatozoa per case, we documented an average incidence of disomy 21 at 0.13 %, with a range of 0.00-0.25 % and a SD of 0.08. There was no indication of diploidy in this cohort of 22,000 sperm.
Numerous previous studies on the incidence of disomy 21 in sperm have been published, using FISH. As far as we are aware, none of these have applied more than a single chromosome 21-specific probe. Accepting our mean of 0.13 % of disomy 21, and providing there is no selective fertilisation capability of disomy 21 sperm in relation to the normal, we conclude that around 1 in 800 conceptions is expected to be trisomic for chromosome 21 of paternal origin. Bearing in mind that the maternal origin likely is at least 10 times more common, we tentatively propose that around 1 in 80 oocytes in the maternal ovarian reserve may be disomy 21. One reason for this discrepancy may be a more stringent selection against aberrant chromosome numbers during spermatogenesis than oogenesis. Further work is required to determine the relevant stages of spermatogenesis at which such a selection may take place.
PMCID: PMC4545544  PMID: 26300975
Trisomy 21; Down syndrome; Spermatozoa; Chromosome copy number; Fluorescence in situ hybridisation (FISH); Disomy 21; Paternal origin
4.  Double inv(3)(q21q26.2) in acute myeloid leukemia is resulted from an acquired copy neutral loss of heterozygosity of chromosome 3q and associated with disease progression 
Acute myeloid leukemia (AML) with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) is a distinct clinicopathologic entity with a poor prognosis. However, double inv(3)(q21q26.2) is extremely rare in AML. We report here 3 cases analyzed by oligonucleotide microarray comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP). Clinicopathologic, cytogenetic and molecular findings were correlated with clinical outcome to better understand the entity.
The study group included one man and two women at 56–74 years of age. The AML arose from myelodysplastic syndrome in one patient and from chronic myelomonocytic leukemia in another patient. Monosomy 7 was found as additional cytogenetic finding in one patient. One patient had a single inv(3) in the initial clone and acquired double inv(3) as part of clonal evolution. EVI1 (MECOM) rearrangement was confirmed using metaphase/interphase fluorescence in situ hybridization (FISH). Microarray (aCGH + SNP) data analysis revealed that the double inv(3) was a result of acquiring copy neutral loss of heterozygosity of chromosome 3q: arr[hg19] 3q13.21q29(10,344,387–197,802,470)x2 hmz, spanning ~ 94.3 Mb in size. Mutational profiling showed a PTPN11 mutation at a low level (~10 %) in one patient and wild type FLT3 and RAS in all patients. No patients achieved cytogenetic remission and all died with an overall survival (OS) of 23, 12 and 5 months, respectively.
Double inv(3) is a result of acquired copy neutral loss of heterozygosity, a somatic repair event occurring as a part of mitotic recombination of the partial chromosome 3q. The double inv(3) in AML patients is highly associated with a rapid disease progression.
PMCID: PMC4545786  PMID: 26300976
5.  De novo microduplication of CHL1 in a patient with non-syndromic developmental phenotypes 
The CHL1 gene codes for a member of the L1 family of neural cell adhesion molecules. It is highly expressed in the central and peripheral nervous system playing an important role in the building and functioning on the brain. CHL1 proteins are also involved in axonal migration, synaptic formation and plasticity. In mice, functional studies showed that the haploinsufficiency of Chl1 gene in the developing brain results in cognitive deficits suggesting that the CHL1 gene at 3p26.3 is a candidate for an autosomal form of intellectual disability. Furthermore, in humans deletions of CHL1 have been described in patients with neurodevelopmental delay characterized by learning and language difficulties, seizures. Less is known about the potential effect of CHL1 overexpression, and microduplications of CHL1 have been rarely identified.
Case presentation
In this report, we describe a male patient with a phenotype characterized by developmental delay, symptoms of hyperactivity, short attention span and speech delay. In addition, minor facial dysmorphic features have been observed. Chromosomal microarray analysis revealed a rare de novo 0.85 Mb microduplication on the short arm (p26.3) of chromosome 3, encompassing a single gene, CHL1. To the best of our knowledge, duplication of chromosome 3p26.3, including only the CHL1 gene, has been described in only one intellectually disabled girl with epilepsy. The duplication described here is the smallest reported so far. In addition, this is the first report describing a patient in which the CHL1 duplication is a de novo event.
The clinical and molecular findings reported here are useful to provide further evidence that CHL1 is a dosage sensitive gene suggesting that not only the deletion but also its duplication can cause non-syndromic neurodevelopmental phenotypes.
PMCID: PMC4537544  PMID: 26279679
6.  Reversing chromatin accessibility differences that distinguish homologous mitotic metaphase chromosomes 
Chromatin-modifying reagents that alter histone associating proteins, DNA conformation or its sequence are well established strategies for studying chromatin structure in interphase (G1, S, G2). Little is known about how these compounds act during metaphase. We assessed the effects of these reagents at genomic loci that show reproducible, non-random differences in accessibility to chromatin that distinguish homologous targets by single copy DNA probe fluorescence in situ hybridization (scFISH). By super-resolution 3-D structured illumination microscopy (3D-SIM) and other criteria, the differences correspond to ‘differential accessibility’ (DA) to these chromosomal regions. At these chromosomal loci, DA of the same homologous chromosome is stable and epigenetic hallmarks of less accessible interphase chromatin are present.
To understand the basis for DA, we investigate the impact of epigenetic modifiers on these allelic differences in chromatin accessibility between metaphase homologs in lymphoblastoid cell lines. Allelic differences in metaphase chromosome accessibility represent a stable chromatin mark on mitotic metaphase chromosomes. Inhibition of the topoisomerase IIα-DNA cleavage complex reversed DA. Inter-homolog probe fluorescence intensity ratios between chromosomes treated with ICRF-193 were significantly lower than untreated controls. 3D-SIM demonstrated that differences in hybridized probe volume and depth between allelic targets were equalized by this treatment. By contrast, DA was impervious to chromosome decondensation treatments targeting histone modifying enzymes, cytosine methylation, as well as in cells with regulatory defects in chromatid cohesion. These data altogether suggest that DA is a reflection of allelic differences in metaphase chromosome compaction, dictated by the localized catenation state of the chromosome, rather than by other epigenetic marks.
Inhibition of the topoisomerase IIα-DNA cleavage complex mitigated DA by decreasing DNA superhelicity and axial metaphase chromosome condensation. This has potential implications for the mechanism of preservation of cellular phenotypes that enables the same chromatin structure to be correctly reestablished in progeny cells of the same tissue or individual.
Electronic supplementary material
The online version of this article (doi:10.1186/s13039-015-0159-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4535684  PMID: 26273322
Chromosome decondensation; Metaphase chromosome structure; Super-resolution microscopy; DNA Topoisomerases Type II; Fluorescence In Situ Hybridization; Epigenetics
7.  Aneusomy detection with Karyolite-Bac on Beads® is a cost-efficient and high throughput strategy in the molecular analyses of the early pregnancy conception losses 
Approximately 10–15 % of all clinically recognized pregnancies end in miscarriage, the majority of them occur during the first trimester, underlying the cause of the loss. Genetic analysis of fetal tissues has the potential to provide valuable information and is highly recommended in some cases. Around 3–4 years ago, the gold standard for the analysis was the GTG Kayrotype, is well known that around 50 % of the tissue samples received failed to grow in culture. Different molecular techniques are used to improve the quality and the specificity of the study, intending to circumvent the limits of the Karyotype.
Karyolite-BoBs™ (KL-BoB™) assay is a recent bead-based suspension, low density array technology with consistent results, probed that is an efficient molecular method to detect aneusomies in early pregnancy losses. Fifty samples from abortions were analyzed in order to probe and give more information about the methodology and analyze if KL-BoBs™ is a good and cost-efficient strategy. We detected 32 % of chromosomal abnormalities, in some of the cases more than one aberration was identified, the array CGH validate the observations.
This molecular strategy is a cost-effective sensitive tool in the early pregnancy loss study.
PMCID: PMC4534128  PMID: 26269714
8.  Observations of the genomic landscape beyond 1p19q deletions and EGFR amplification in glioma 
With recent advancements in molecular techniques, the opportunities to gather whole genome information have increased, even in degraded samples such as FFPE tissues. As a result, a broader view of the genomic landscape of solid tumors may be explored. Whole genome copy number and loss of heterozygosity patterns can advance our understanding of mechanisms and complexity of various tumors.
Genome-wide alterations involving copy number changes and loss of heterozygosity were identified in 17 glioma samples with positive FISH results for 1p19q co-deletions (n = 9) or EGFR amplification (n = 8). Gliomas positive for 1p19q co-deletions did not have other frequently recurrent genomic alterations. Additional copy-number alterations were observed in individual cases, and consisted primarily of large-scale changes, including gains or losses of entire chromosomes. The genomic architecture of EGFR amplified gliomas was much more complex, with a high number of gains and losses across the genome. Recurrent alterations in EGFR amplified gliomas were both focal, such as CDKN2A homozygous deletions, and large, such as chromosome 10 loss.
Microarray enabled a broader picture of the genomic alterations occurring in glioma cases. Gliomas with 1p19q co-deletion had a relatively quiet genome, apart from the selected co-deletion. Additional alterations in isolated cases, involved primarily larger aberrations. On the other hand, EGFR amplified cases tended to be more complex and have specific abnormalities associated with the EGFR amplification. Furthermore, 1p19q co-deletions and EGFR amplification associated copy number changes appeared to often be mutually exclusive.
PMCID: PMC4528708  PMID: 26257825
Microarray; OncoScan; Glioma; Genomic landscape; Copy number change; FFPE array
9.  Molecular cytogenetic studies characterizing a novel complex karyotype with an uncommon 5q22 deletion in childhood acute myeloid leukemia 
Deletions in the long arm of chromosome 5 or loss of the whole chromosome are rare in childhood Acute Myeloid Leukemia (AML) patients. It is also unknown if the wide variety of breakpoints have diverging implications in the patient’s outcome. Despite -5/5q- abnormalities have usually been described as a poor prognostic feature, however, the low frequency of -5/5q- in pediatric AML patients limits a full knowledge about this cytogenetic and clinical category, which is an intriguing factor for further research and new findings. Here, we report an AML child showing an uncommon deletion in 5q associated with 2 new abnormalities involving chromosome 2 within a complex karyotype well-characterized by several molecular cytogenetic approaches. Our work stimulates upcoming studies with more detailed descriptions about 5q abnormalities to better define its role in the stratification risk of such cytogenetic subgroup in childhood AML.
PMCID: PMC4528810  PMID: 26257826
Childhood AML; Uncommon deletion 5q22; Complex karyotype
10.  Chromosome number and ploidy level of balm (Melissa officinalis) 
Lemon balm (Melissa officinalis L.) is of increasing importance resulting in rising growth area. Improved knowledge on the genome structure, number of chromosomes in connection with the taxonomical structure of balm is indispensable for improved new varieties.
A collection of 40 balm accessions (M. officinalis) was characterized by flow cytometry and FISH (18/25S and 5S rDNA) to determine the chromosome number and ploidy level. Three different types were found: diploid genotypes with 2n = 2× = 32 chromosomes; tetraploid 2n = 4× = 64 chromosomes and triploid 2n = 3× = 48 chromosomes. A haploid base number of × = 16 chromosomes is likely. First time described triploid accessions are sterile but cytologically and morphologically stable for many years. Triploids express better winter hardiness and regeneration after harvesting cuts as well as bigger leaves and internodes.
A basic chromosome number of x = 16 is reported for the first time for the species M. officinalis.
PMCID: PMC4545860  PMID: 26300974
11.  Deletion 9p23 to 9p11.1 as sole additional abnormality in a Philadelphia positive chronic myeloid leukemia in blast crisis: a rare event 
Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence of a derivative chromosome 22 [der(22)] commonly called Philadelphia chromosome (Ph). The Ph chromosome is a product of the reciprocal translocation t(9;22)(q34.1;q11.2). Additional genetic changes occur in less than 10 % of CML cases at the time of diagnosis and other genetic changes are seen in 60–80 % of the cases in advanced disease. Even though deletions in chromosome 9 are not rare findings in advanced phase-CML, del(9)(p23p11.1) as sole additional abnormality detected by fluorescence in situ hybridization (FISH) technique, to our knowledge has not been described in the literature.
A complete cytogenetic and molecular cytogenetic analysis, molecular biology method (reverse transcription polymerase chain reaction (RT-PCR)), and immunophenotype confirmed to be a CML case in blast crisis (BC). It revealed del(9)(p23p11.1) as sole abnormality detected by FISH technique besides Ph chromosome, which leads to monoallely of tumor suppressor gene CDKN2A (cyclin-dependent kinase inhibitor 2A) before Imatinib mesylate (IM) treatment.
The patient did not demonstrate a good response to IM treatment. The underlying mechanisms and prognostic implications of these cytogenetic abnormalities are discussed.
PMCID: PMC4523925  PMID: 26244056
Chronic myeloid leukemia; Philadelphia chromosome; del(9)(p24p12); CDKN2A gene; Prognostic factors
12.  Cytogenetic and molecular characterization of a recombinant X chromosome in a family with a severe neurologic phenotype and macular degeneration 
Duplications of MECP2 gene in males cause a syndrome characterized by distinctive clinical features, including severe to profound mental retardation, infantile hypotonia, mild dysmorphic features, poor speech development, autistic features, seizures, progressive spasticity and recurrent infections. Patients with complex chromosome rearrangements, leading to Xq28 duplication, share most of the clinical features of individuals with tandem duplications, in particular neurologic problems, suggesting a major pathogenetic role of MECP2 overexpression.
We performed cytogenetic and molecular cytogenetic studies in a previously described family with affected males showing congenital ataxia, late-onset progressive myoclonic encephalopathy and selective macular degeneration. Microsatellite, FISH and array-CGH analyses identified a recombinant X chromosome with a deletion of the PAR1 region, encompassing SHOX, replaced by a duplicated segment of the Xq28 terminal portion, including MECP2.
Our report describes the identification of the actual genetic cause underlying a severe syndrome that previous preliminary analyses erroneously associated to a terminal Xp22.33 region. In the present family as well as in previously reported patients with similar rearrangements, the observed neurologic phenotype is ascribable to MECP2 duplication, with an undefined contribution of the other involved genes. Maculopathy, presented by affected males reported here, could be a novel clinical feature associated to Xq28 disomy due to recombinant X chromosomes, but at present the underlying pathogenetic mechanism is unknown and this potential clinical correlation should be confirmed through the collection of additional patients.
PMCID: PMC4522089
MECP2 duplication; Xq28 disomy; Recombinant X chromosome; Macular degeneration
13.  A recurrent deletion on chromosome 2q13 is associated with developmental delay and mild facial dysmorphisms 
We report two unrelated patients with overlapping chromosome 2q13 deletions (patient 1 in chr2:111415137-113194067 bp and patient 2 in chr2:110980342-113007823 bp, hg 19). Patient 1 presents with developmental delay, microcephaly and mild dysmorphic facial features, and patient 2 with autism spectrum disorder, borderline cognitive abilities, deficits in attention and executive functions and mild dysmorphic facial features. The mother and maternal grandmother of patient 1 were healthy carriers of the deletion. Previously, 2q13 deletions were reported in 27 patients, and the interpretation of its clinical significance varied. Our findings support that the 2q13 deletion is associated with a developmental delay syndrome manifesting with variable expressivity and reduced penetrance which poses a challenge for genetic counselling as well as the clinical recognition of 2q13 deletion patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s13039-015-0157-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4521466
2q13 deletion; Developmental delay; Facial dysmorphism; aCGH; BCL2L11; FBLN7; TMEM87B
14.  Comparative cytogenetics in the genus Hoplias (Characiformes, Erythrinidae) highlights contrasting karyotype evolution among congeneric species 
The Erythrinidae fish family contains three genera, Hoplias, Erythrinus and Hoplerythrinus widely distributed in Neotropical region. Remarkably, species from this family are characterized by an extensive karyotype diversity, with 2n ranging from 39 to 54 chromosomes and the occurrence of single and/or multiple sex chromosome systems in some species. However, inside the Hoplias genus, while H. malabaricus was subject of many studies, the cytogenetics of other congeneric species remains poorly explored. In this study, we have investigated chromosomal characteristics of four Hoplias species, namely H. lacerdae, H. brasiliensis, H. intermedius and H. aimara. We used conventional staining techniques (C-banding, Ag-impregnation and CMA3 -fluorescence) as well as fluorescence in situ hybridization (FISH) with minor and major rDNA and microsatellite DNAs as probes in order to analyze the karyotype evolution within the genus.
All species showed invariably 2n = 50 chromosomes and practically identical karyotypes dominated only by meta- and submetacentric chromosomes, the absence of heteromorphic sex chromosomes, similar pattern of C-positive heterochromatin blocks and homologous Ag-NOR-bearing pairs. The cytogenetic mapping of five repetitive DNA sequences revealed some particular interspecific differences between them. However, the examined chromosomal characteristics indicate that their speciation was not associated with major changes in their karyotypes.
Such conserved karyotypes contrasts with the extensive karyotype diversity that has been observed in other Erythrinidae species, particularly in the congeneric species H. malabaricus. Nevertheless, what forces drive such particularly different modes of karyotype evolution among closely related species? Different life styles, population structure and inner chromosomal characteristics related to similar cases in other vertebrate groups can also account for the contrasting modes of karyotype evolution in Hoplias genus.
PMCID: PMC4518567
Trahiras; Fish cytogenetics; FISH; Repetitive DNA; Chromosome change and speciation
15.  Construction of BAC contig maps of homoeologous chromosomes A12 and D12 of Gossypium hirsutum L. acc. TM-1 
The Gossypium hirsutum homoeologous chromosome 12 encodes important genes that contribute to fiber fuzz, lethality, gland development and male sterility. In this study a physical map of the cotton TM-1 chromosome 12 was constructed. A number of large-insert cotton genome libraries are available, and genome-wide physical mapping using large insert segments combined with bacterial cloning is a thriving area of genome research. However, sequencing of the cotton genome is difficult due to sequence repeats and homoeologous regions. In order to effectively distinguish the homologous segments, a new method for adjusting the parameters of the FPC software was applied for contig map construction.
All available markers on chromosomes A12 and D12 were used to screen the TM-1 BAC library by PCR. A total of 775 clones (387 for A12, 388 for D12) were obtained using Hind III fingerprinting and used for construction of the contig map. Seven pairs of SSR markers located on A12 and D12 were chosen for contig analysis. Following optimization of the tolerance (10) and cutoff (1e-12) parameters, combining all clones from A12 and D12 produced two separate contigs.
The BAC contig map of chromosomes A12 and D12 was constructed and FPC software parameters were optimized for analysis. The resulting approach is a powerful platform for genome-wide and evolutionary research on cotton.
PMCID: PMC4517413  PMID: 26221184
Contig; BAC; Fingerprint; Homoeologous chromosome; Cotton; Genomics
16.  Periventricular heterotopia and white matter abnormalities in a girl with mosaic ring chromosome 6 
Ring chromosome 6 is a rare chromosome abnormality that arises typically de novo. The phenotypes can be highly variable, ranging from almost normal to severe malformations and neurological defects. We report a case of a 3-year-old girl with mosaic ring chromosome 6 who presented with being small for gestational age and intellectual disability, and whose brain MRI later revealed periventricular heterotopia and white matter abnormalities. Mosaicism was identified in peripheral blood cells examined by standard G-bands, mos 46,XX,r(6)(p25q27)[67]/45,XX,-6[25]/46,XX,dic r(6:6)(p25q27:p25q27)[6]/47,XX,r(6)(p25q27) × 2[2]. Using array-comparative genomic hybridization, we identified terminal deletion of 6q27 (1.5 Mb) and no deletion on 6p. To our knowledge, this is the first report of periventricular heterotopia and white matter abnormalities manifested in a patient with ring chromosome 6. These central nervous system malformations are further discussed in relation to molecular genetics.
PMCID: PMC4514952  PMID: 26213576
Ring chromosome 6; Brain MRI; Periventricular heterotopia; White matter abnormality; Array-comparative genomic hybridization; Small for gestational age
17.  Trisomy Xp and partial tetrasomy Xq resulting from gain of a rearranged X chromosome in a female fetus: pathogenic or not? 
Cytogenetic analysis of chorionic villous sampling revealed a mosaic karyotype with gain of a rearranged X chromosome. Microarray and additional studies indicated that the rearranged X carried an inverted duplication, a deletion and a satellited Xqter. Gain of this rearranged X was confirmed by follow-up amniocentesis and postnatal cord blood sample. A full-term infant girl was delivered and showed normal physical findings at both birth and 21-month follow-up examinations. Late replication studies demonstrated that the rearranged X was inactivated in all abnormal cells analyzed. Skewed X-inactivation may suppress the potentially deleterious effects of genomic imbalance; however, gain of X chromosomes, particularly rearranged X chromosomes, often presents challenges for prenatal genetic counseling. The gradation of clinical phenotype severity generally correlates with the number of additional X chromosomes. However, the X chromosome regions responsible for the abnormal phenotypes are poorly understood. This case will further elucidate the phenotypic effects of X inactivation and X chromosome abnormalities.
PMCID: PMC4513972  PMID: 26213575
Prenatal diagnosis; Sex chromosome abnormality; Array CGH; X inactivation; High risk pregnancy; Genetic counseling; Chorionic villus sampling; Amniocentesis
18.  Prader-Willi syndrome - type 1 deletion, a consequence of an unbalanced translocation of chromosomes 13 and 15, easily to be mixed up with a Robertsonian translocation 
Prader-Willi syndrome, due to microdeletion of proximal 15q, is a well-known cause of syndromic obesity.
Case characteristics
A couple with history of repeated first trimester abortions had a son with balanced Robertsonian translocation of chromosomes 13 and 15 according to cytogenetic banding technique.
Chromosomal analysis for the couple was performed. A balanced translocation involving BP1-BP3 region of proximal 15q was observed in the father.
Investigations of the parents is mandatory when a structural rearrangement is detected in a dysmorphic child.
PMCID: PMC4510909  PMID: 26203302
Structural rearrangement; sSMC; Deletion; Prader-Willi syndrome; Unbalanced translocation; FISH
19.  Complex X chromosome rearrangement associated with multiorgan autoimmunity 
Turner syndrome, a congenital condition that affects 1/2,500 births, results from absence or structural alteration of the second sex chromosome. Turner syndrome is usually associated with short stature, gonadal dysgenesis and variable dysmorphic features.
The classical 45,X karyotype accounts approximately for half of all patients, the remainder exhibit mosaicism or structural abnormalities of the X chromosome. However, complex intra-X chromosomal rearrangements involving more than three breakpoints are extremely rare.
We present a unique case of a novel complex X chromosome rearrangement in a young female patient presenting successively a wide range of autoimmune diseases including insulin dependent diabetes mellitus, Hashimoto’s thyroiditis, celiac disease, anaemia perniciosa, possible inner ear disease and severe hair loss. For the genetic evaluation, conventional cytogenetic analysis and FISH with different X specific probes were initially performed. The complexity of these results and the variety of autoimmune problems of the patient prompted us to identify the exact composition and breakpoints of the rearranged X as well as methylation status of the X chromosomes. The high resolution array-CGH (assembly GRCh37/hg19) detected single copy for the whole chromosome X short arm. Two different sized segments of Xq arm were present in three copies: one large size of 80,3 Mb from Xq11.1 to Xq27.3 region and another smaller (11,1 Mb) from Xq27.3 to Xq28 region. An 1,6 Mb Xq27.3 region of the long arm was present in two copies. Southern blot analysis identified a skewed X inactivation with ≈ 70:30 % ratios of methylated/unmethylated fragments. The G-band and FISH patterns of the rearranged X suggested the aspect of a restructured i(Xq) chromosome which was shattered and fortuitously repaired. The X-STR genotype analysis of the family detected that the patient inherited intact maternal X chromosome and a rearranged paternal X chromosome.
The multiple Xq breakages and fusions as well as inverted duplication would have been expected to cause a severe Turner phenotype. However, the patient lacks many of the classic somatic features of Turner syndrome, instead she presented multiorgan autoimmune diseases.
The clinical data of the presented patient suggest that fragmentation of the i(Xq) chromosome elevates the risk of autoimmune diseases.
PMCID: PMC4506572  PMID: 26191082
Complex X chromosome rearrangement; Autoimmune disease; Turner syndrome; Type 1 diabetes mellitus; X inactivation disorder
20.  Short stature and primary ovarian insufficiency possibly due to chromosomal position effect in a balanced X;1 translocation 
Primary ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea), a decrease in the initial primordial follicle number, high follicle-stimulating hormone (FSH) levels and hypoestrogenism. Although the etiology of a majority of POI cases is not yet identified, several data suggest that POI has a strong genetic component. Conventional cytogenetic and molecular analyses have identified regions of the X chromosome that are associated with ovarian function, as well as POI candidate genes, such as FMR1 and DIAPH2.
Here we describe a 10.5-year-old girl presenting with high FSH and luteinizing hormone (LH) levels, pathologic GH stimulation arginine and clonidine tests, short stature, pterygium, ovarian dysgenesis, hirsutism and POI.
Cytogenetic analysis demonstrated a balanced reciprocal translocation between the q arms of chromosomes X and 1, with breakpoints falling in Xq21 and 1q41 bands. Molecular studies did not unravel any chromosome microdeletion/microduplication, and no XIST-mediated inactivation was found on the derivative chromosome 1. Interestingly, through immunofluorescence assays, we found that part of the Xq21q22 trait, translocated to chromosome 1q41, was late replicating and therefore possibly inactivated in 30 % metaphases both in lymphocytes and skin fibroblasts, in addition to a skewed 100 % inactivation of the normal X chromosome. These findings suggest that a dysregulation of gene expression might occur in this region. Two genes mapping to the Xq translocated region, namely DIAPH2 and FMR1, were found overexpressed if compared with controls.
We report a case in which gonadal dysgenesis and POI are associated with over-expression of DIAPH2 gene and of FMR1 gene in wild type form. We hypothesize that this over-expression is possibly due to a phenomenon known as “chromosomal position effect”, which accounts for gene expression variations depending on their localization within the nucleus. For the same effect a double mosaic inactivation of genes mapping to the Xq21-q22 region, demonstrated by immunofluorescence assays, may be the cause of a functional Xq partial monosomy leading to most Turner traits of the proband’s phenotype.
PMCID: PMC4501070  PMID: 26175800
DIAPH2; FMR1; Primary ovarian insufficiency; Chromosomal position effect; X chromosome translocation; Turner syndrome; X;autosome translocation
21.  Preimplantation genetic screening of blastocysts by multiplex qPCR followed by fresh embryo transfer: validation and verification 
Aneuploidy is an important etiology of implantation failure and quantitative real-time polymerase chain reaction (qPCR) seems a promising preimplantation genetic screening (PGS) technology to detect aneuploidies. This verification study aimed at verifying the impact on reproductive outcomes in in vitro fertilization (IVF) cycles using fresh embryo transfer (FET) in which the embryos were selected by blastocyst biopsy with qPCR-based PGS in our settings.
A total of 13 infertile couples with more than once failed in vitro fertilization were enrolled during July to October of 2014. PGS was conducted by qPCR with selectively amplified markers to detect common aneuploidies (chromosomes 13, 18, 21, X, and Y). The design of the qPCR molecular markers adopted the locked nucleic acid (LNA) strategy. The blastocyst biopsy was performed on Day 5/6 and the PGS was done on the same day, which enabled FET. A total of 72 blastocysts were biopsied. Successful diagnoses were established in all embryos and the rate of successful diagnosis was 100 %. The aneuploidy rate was 38.9 % (28/72). 28 embryos were transferred. The clinical pregnancy rate was 61.5 % (8/13) per cycle. Early first trimester abortion was encountered in 1 and the ongoing pregnancy rate was 53.8 % (7/13) per cycle.
This study verified the favorable outcome of adopting PGS with qPCR + FET in our own setting. Expanding the repertoire of aneuploidies being investigated (from a limited set to all 24 chromosomes) is underway and a randomized study by comparing qPCR and other PGS technologies is warranted.
Electronic supplementary material
The online version of this article (doi:10.1186/s13039-015-0140-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4495615  PMID: 26157486
Aneuploidy; Blastocyst; Fresh embryo transfer; PGS; qPCR
22.  PAX5-KIAA1549L: a novel fusion gene in a case of pediatric B-cell precursor acute lymphoblastic leukemia 
In B-cell precursor acute lymphoblastic leukemia (BCP-ALL) PAX5, a transcription factor pivotal for B-cell commitment and maintenance, is frequently affected by genetic alterations. In 2-3 % of the cases PAX5 rearrangements result in the expression of oncogenic fusion genes. The encoded chimeric proteins consist of the N-terminal PAX5 DNA-binding paired domain, which is fused to the C-terminal domains of a remarkable heterogeneous group of partner proteins.
Employing fluorescence in situ hybridization and molecular methods PAX5-KIAA1549L was identified as novel fusion gene in a case of pediatric BCP-ALL.
Our report underlines the high diversity of PAX5 fusion partners in BCP-ALL and we describe the second involvement of KIAA1549L in a genetic rearrangement in acute leukemia.
Electronic supplementary material
The online version of this article (doi:10.1186/s13039-015-0138-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4495688  PMID: 26157485
B-cell precursor acute lymphoblastic leukemia; PAX5 fusion; KIAA1549L
23.  Chromosome territory repositioning induced by PHA-activation of lymphocytes: A 2D and 3D appraisal 
Genomes and by extension chromosome territories (CTs) in a variety of organisms exhibit nonrandom organization within interphase nuclei. CTs are susceptible to movement upon induction by a variety of stimuli, including: cell differentiation, growth factors, genotoxic agents, proliferating status, and stimulants that induce novel transcription profiles. These findings suggest nuclear architecture can undergo reorganization, providing support for a functional significance of CT organization. The effect of the initiation of transcription on global scale chromatin architecture has been underexplored. This study investigates the organization of all 24 human chromosomes in lymphocytes from two individuals in resting and phytohaemagglutinin activated lymphocytes using 2D and 3D approaches.
The radial organization of CTs in lymphocytes in both resting and activated lymphocytes follows a gene-density pattern. However, CT organization in activated nuclei appears less constrained exhibiting a more random organization. We report differences in the spatial relationship between homologous and heterologous CTs in activated nuclei. In addition, a reproducible radial hierarchy of CTs was identified and evidence of a CT repositioning was observed in activated nuclei using both 2D and 3D approaches.
Alterations between resting and activated lymphocytes could be adaptation of CTs to the new transcription profile and possibly the formation of new neighborhoods of interest or interaction of CTs with nuclear landmarks. The increased distances between homologous and heterologous CTs in activated lymphocytes could be a reflection of a defensive mechanism to reduce potential interaction to prevent any structural chromosome abnormalities (e.g. translocations) as a result of DNA damage that increases during lymphocyte activation.
PMCID: PMC4490598  PMID: 26146516
Genome organization; Chromosome territories; Phytohaemagglutinin; Repositioning
24.  Assessment of copy number variations in the brain genome of schizophrenia patients 
Cytogenomic mutations and chromosomal abnormality are implicated in the neuropathology of several brain diseases. Cell heterogeneity of brain tissues makes their detection and validation difficult, however. In the present study, we analyzed gene dosage alterations in brain DNA of schizophrenia patients and compared those with the copy number variations (CNVs) identified in schizophrenia patients as well as with those in Asian lymphocyte DNA and attempted to obtain hints at the pathological contribution of cytogenomic instability to schizophrenia.
Brain DNA was extracted from postmortem striatum of schizophrenia patients and control subjects (n = 48 each) and subjected to the direct two color microarray analysis that limits technical data variations. Disease-associated biases of relative DNA doses were statistically analyzed with Bonferroni’s compensation on the premise of brain cell mosaicism. We found that the relative gene dosage of 85 regions significantly varied among a million of probe sites. In the candidate CNV regions, 26 regions had no overlaps with the common CNVs found in Asian populations and included the genes (i.e., ANTXRL, CHST9, DNM3, NDST3, SDK1, STRC, SKY) that are associated with schizophrenia and/or other psychiatric diseases. The majority of these candidate CNVs exhibited high statistical probabilities but their signal differences in gene dosage were less than 1.5-fold. For test evaluation, we rather selected the 10 candidate CNV regions that exhibited higher aberration scores or larger global effects and were thus confirmable by PCR. Quantitative PCR verified the loss of gene dosage at two loci (1p36.21 and 1p13.3) and confirmed the global variation of the copy number distributions at two loci (11p15.4 and 13q21.1), both indicating the utility of the present strategy. These test loci, however, exhibited the same somatic CNV patterns in the other brain region.
The present study lists the candidate regions potentially representing cytogenomic CNVs in the brain of schizophrenia patients, although the significant but modest alterations in their brain genome doses largely remain to be characterized further.
Electronic supplementary material
The online version of this article (doi:10.1186/s13039-015-0144-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4487564  PMID: 26136833
CNV; Caudate; Genome instability; Schizophrenia; Somatic mutation
25.  High rates of submicroscopic aberrations in karyotypically normal acute lymphoblastic leukemia 
Acute lymphoblastic leukemia (ALL) is not a single uniform disease. It consists of several subgroups with different cytogenetic and molecular genetic aberrations, clinical presentations and outcomes. Banding cytogenetics plays a pivotal role in the detection of recurrent chromosomal rearrangements and is the starting point of genetic analysis in ALL, still. Nowadays, molecular (cyto)genetic tools provide substantially to identify previously non-detectable, so-called cryptic chromosomal aberrations in ALL. However, ALL according to banding cytogenetics with normal karyotype - in short cytogenetically normal ALL (CN-ALL) - represent up to ~50 % of all new diagnosed ALL cases. The overall goal of this study was to identify and characterize the rate of cryptic alterations in CN-ALL and to rule out if one single routine approach may be sufficient to detect most of the cryptic alterations present.
Sixty-one ALL patients with CN-ALL were introduced in this study. All of them underwent high resolution fluorescence in situ hybridization (FISH) analysis. Also DNA could be extracted from 34 ALL samples. These DNA-samples were studied using a commercially available MLPA (multiplex ligation-dependent probe amplification) probe set directed against 37 loci in hematological malignancies and/or array-comparative genomic hybridization (aCGH). Chromosomal aberrations were detected in 21 of 61 samples (~34 %) applying FISH approaches: structural abnormalities were present in 15 cases and even numerical ones were identified in 6 cases. Applying molecular approaches copy number alterations (CNAs) were detected in 27/34 samples. Overall, 126 CNAs were identified and only 34 of them were detectable by MLPA (~27 %). Loss of CNs was identified in ~80 % while gain of CNs was present in ~20 % of the 126 CNAs. A maximum of 13 aberrations was detected per case; however, only one aberration per case was found in 8 of all in detail studied 34 cases. Of special interest among the detected CNAs are the following new findings: del(15)(q26.1q26.1) including CHD2 gene was found in 20 % of the studied ALL cases, dup(18)(q21.2q21.2) with the DCC gene was present in 9 % of the cases, and the CDK6 gene in 7q21.2 was deleted in 12 % of the here in detail studied ALL cases.
In conclusion, high resolution molecular cytogenetic tools and molecular approaches like MLPA and aCGH need to be combined in a cost-efficient way, to identify disease and progression causing alterations in ALL, as majority of them are cryptic in banding cytogenetic analyses.
Electronic supplementary material
The online version of this article (doi:10.1186/s13039-015-0153-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4486437  PMID: 26136832
Multitude multicolor banding (mMCB); Acute lymphoblastic leukemia (ALL); Cryptic rearrangements; Fluorescence in situ hybridization (FISH); Multiplex ligation-dependent probe amplification (MLPA); Array-comparative genomic hybridization (aCGH)

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