Molecular Karyotyping Research Articles

Karyotype evolution in the Pinaceae: implication with molecular phylogeny

The family Pinaceae is made up mostly of diploid species (2n = 24). Systematization of karyotype analysis was developed to make comparison of intra- and interspecific karyotypes among the Pinaceae more accurate and reliable. Considering all parameters, the genera Pseudotsuga and Pseudolarix have the "most derived" (or advanced) and asymmetric karyotypes in the Pinaceae, followed by Larix, Picea, Abies, and Cedrus. The genus Pinus was the "least derived" (or ancestral) of all the genera of the Pinaceae analyzed. Differences in karyotype formulae and asymmetry indices were found among species within the same genera, suggesting that structural changes may have contributed to the diversification of the genus. This review is a detailed analysis of comparative karyotyping based on similar parameters, including numeric data and cytogenetic information. Telomeric sequence repeats and rDNA distribution in the Pinaceae were surveyed. The role of transposition in rDNA chromosome distribution is analyzed. Cytogenetic implications of hybridization between related species are reported. Likewise, the relationships between molecular phylogenetic and karyotype evolution is discussed in light of several reports. Within many genera, chromosomal organization was conserved despite independent molecular divergence and adaptation through the evolutionary history of the species of the Pinaceae.

Duplication 16p13.3 and the CREBBP gene: Confirmation of the phenotype

The introduction of molecular karyotyping technologies into the diagnostic work-up of patients with congenital disorders permitted the identification and delineation of novel microdeletion and microduplication syndromes. Interstitial 16p13.3 duplication, encompassing the CREBBP gene, which is mutated or deleted in the Rubinstein–Taybi syndrome, have been proposed to cause a recognisable syndrome with variable intellectual disability, normal growth, mild facial dysmorphism, mild anomalies of the extremities, and occasional findings such as developmental defects of the heart, genitalia, palate or the eyes. We here report the phenotypic and genotypic delineation of 9 patients carrying a submicroscopic 16p13.3 duplication, including the smallest 16p13.3 duplication reported so far. Careful clinical assessment confirms the distinctive clinical phenotype and also defines frequent associated features : marked speech problems, frequent ocular region involvement with upslanting of the eyes, narrow palpebral fissures, ptosis and strabismus, frequent proximal implantation of thumbs, cleft palate/bifid uvula and inguinal hernia. It also confirms that CREBBP is the critical gene involved in the duplication 16p13.3 syndrome.

Spraguea lophii (Microsporidia) parasite of the teleost fish, Lophius piscatorius from Tunisian coasts: Evidence for an extensive chromosome length polymorphism

A microsporidian of the genus Spraguea was found parasitizing the nervous tissues of Lophius piscatorius collected from various localities in the Mediterranean coastal areas of Tunisia. The tissue localization, the infection focus aspect and sporal dimorphism are characteristics of Spraguea lophii species. Molecular data based on partial sequence of SSUrRNA encoding gene shows few nucleotide polymorphisms, compared to all described Spraguea isolates. Molecular karyotype obtained on pulsed field gel electrophoresis (1D-PFGE) shows a profile with 14 stained bands in the range of 230–880kbp and a genome size estimated to 6.700kbp. The rare cutter endonuclease MluI KARD 2-D-PFGE fingerprint shows an extensive chromosome length polymorphism, but the number of chromosome is unchanged and consists of 15 different molecules. The extensive chromosome length polymorphism is associated to a reduced number of genetic events.

Contribution of Rare Copy Number Variants to Isolated Human Malformations

BackgroundCongenital malformations are present in approximately 2–3% of liveborn babies and 20% of stillborn fetuses. The mechanisms underlying the majority of sporadic and isolated congenital malformations are poorly understood, although it is hypothesized that the accumulation of rare genetic, genomic and epigenetic variants converge to deregulate developmental networks.Methodology/Principal FindingsWe selected samples from 95 fetuses with congenital malformations not ascribed to a specific syndrome (68 with isolated malformations, 27 with multiple malformations). Karyotyping and Multiplex Ligation-dependent Probe Amplification (MLPA) discarded recurrent genomic and cytogenetic rearrangements. DNA extracted from the affected tissue (46%) or from lung or liver (54%) was analyzed by molecular karyotyping. Validations and inheritance were obtained by MLPA. We identified 22 rare copy number variants (CNV) [>100 kb, either absent (n = 7) or very uncommon (n = 15, <1/2,000) in the control population] in 20/95 fetuses with congenital malformations (21%), including 11 deletions and 11 duplications. One of the 9 tested rearrangements was de novo while the remaining were inherited from a healthy parent. The highest frequency was observed in fetuses with heart hypoplasia (8/17, 62.5%), with two events previously related with the phenotype. Double events hitting candidate genes were detected in two samples with brain malformations. Globally, the burden of deletions was significantly higher in fetuses with malformations compared to controls.Conclusions/SignificanceOur data reveal a significant contribution of rare deletion-type CNV, mostly inherited but also de novo, to human congenital malformations, especially heart hypoplasia, and reinforce the hypothesis of a multifactorial etiology in most cases.

Informatics-based molecular karyotyping of products of conception (POC) preserved in paraffin with maternal cell contamination (MCC) detection: report on 46 consecutive analyses

Chromosome testing on first trimester miscarriages is valuable in counseling patients regarding the cause of their miscarriage and has been shown to be cost effective in managing patients with recurrent pregnancy loss. However, traditional cytogenetics requires cell culture and can only be performed on fresh tissue. Here we report our initial experience with molecular karyotyping of paraffin POC using Single Nucleotide Polymorphism (SNP) microarray which determines number and parental origin of each chromosome.

Molecular karyotype analysis of single nucleotide polymorphism microarray for early spontaneous miscarriage after assisted reproductive technology

Molecular karyotype analysis of single nucleotide polymorphism microarray for early spontaneous miscarriage after assisted reproductive technology

Impact of abnormal embryonic molecular karyotype determined by single nucleotide polymorphism (SNP) microarray preimplantation genetic diagnosis (PGD) on embryo development and blastocyst formation

Impact of abnormal embryonic molecular karyotype determined by single nucleotide polymorphism (SNP) microarray preimplantation genetic diagnosis (PGD) on embryo development and blastocyst formation

Chromosomal polymorphism of Acremonium chrysogenum strains producing cephalosporin C

Using pulse electrophoresis in controlled homogenous electric field we conducted molecular karyotyping of highly-productive and laboratory strains of Acremonium chrysogenum generating antibiotic cephalosporin C (cefC). Differences in size of several chromosomes of highly active strain CB26/8 compared to the wild-type strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the highly active strain was not associated with alteration of localization and copy number ofcephalosporin C biosynthesis and transport genes. A cluster of "early" cefC biosynthesis genes is located on chromosome VI (4.4 Mb); a cluster of the "late genes", on chromosome II (2.3 Mb). Both clusters are presented as a single copy perA. chrysogenum genome in the wild-type and in CB26/8 producer strains. Based on comparative analysis of laboratory and industrial cefC producers, a karyotype scheme for A. chrysogenum strains of various origins was designed.

2p21 Deletions in hypotonia–cystinuria syndrome

The significant role of the SLC3A1 gene in the aetiology of cystinuria is meanwhile well established and more than 130 point mutations have been reported. With the reports on genomic deletions including at least both SLC3A1 and the neighboured PREPL gene the spectrum of cystinuria mutations and of clinical symptoms could recently be enlarged: patients homozygous for these deletions suffer from a general neonatal hypotonia and growth retardation in addition to cystinuria. The hypotonia in these hypotonia-cystinuria (HCS) patients has been attributed to the total loss of the PREPL protein. Here we report on the clinical course and molecular findings in a HCS patient compound heterozygote for a new deletion in 2p21 and a previously reported deletion, both identified by molecular karyotyping. The diagnostic workup in this patient illustrates the need for a careful clinical examination in context with powerful molecular genetic tools in patients with unusual phenotypes. The identification of unique genomic alterations and their interpretation serves as a prerequisite for the individual counselling of patients and their families. In diagnostic strategies to identify the molecular basis of both cystinuria and hypotonia 2p21 deletions should be considered as the molecular basis of the phenotype.

Familial Turner syndrome with an X;Y translocation mosaicism: Implications for genetic counseling

Spontaneous fertility is rare among patients with Turner syndrome and is most likely in women with mosaicism for a normal 46,XX cell line. We report an unusual case of familial Turner syndrome with mosaicism for a novel X;Y translocation involving Xp and Yp. The chromosomal analysis was carried out through cytogenetics and molecular karyotyping using a SNP array platform. The mother, a Turner syndrome woman, diagnosed in midchildhood because of short stature, was found to have a 45,X/46,X,der(X)t(X;Y)(p11.4;p11.2) karyotype, with a predominant 45,X cell line. Her parents decided against prophylactic gonadectomy, generally recommended at an early age when Y chromosome has been identified, because at age 13, she had spontaneous puberty and menarche. She reached a final height of 154 cm after treatment with growth hormone. At age 24, she became spontaneously pregnant. She had a mild aortic coarctation and close follow-up cardiac evaluation, including cardiac magnetic resonance imaging, had been performed during her pregnancy, which progressed uneventfully, except for intra-uterine growth retardation. Prenatal diagnosis revealed a female karyotype, with transmission of the maternal translocation with an unexpected different mosaic:47,X,der(X)t(X;Y)x2/46,X,der(X)t(X;Y) karyotype. This complex and unusual karyotype, including a mosaic partial trisomy X and a non-mosaic Xpter-Xp11.4 monosomy, results in transmission of Turner syndrome from mother to daughter. At birth, the girl had normal physical examination except for growth retardation. This family illustrates the complexity and difficulties, in term of patient counseling and management in Turner syndrome, in determining ovarian status, fertility planning, risks associated with pregnancies, particularly when mosaicism for Y material chromosome is identified.

Genomic chondrocyte culture profiling by array-CGH, interphase-FISH and RT-PCR

In vitro expansion is an important step to acquire sufficient cells in human tissue engineering technologies. The high number of chondrocytes needed for human articular cartilage implants requires in vitro expansion of the primary cells, bearing a theoretical risk of in vitro induced changes in the genomes. To gain more insights into this situation, model cultures were prepared and analyzed. 25 chondrocyte cell DNA samples from nine donors were analyzed by array comparative genomic hybridization (aCGH) on whole genome level and 28 chondrocyte cell samples from 16 individuals were analyzed by fluorescence in situ hybridization (FISH) on single cell level. The expanded cells were further characterized upon the chondrocytic mRNA phenotype by reverse-transciptase polymerase chain reaction (RT-PCR). The molecular karyotyping results revealed autosomal stability, but all male samples analyzed by aCGH displayed a variable loss of the Y-chromosome. These data were confirmed by FISH-experiments and suggest an age dependant effect toward the loss of the Y-chromosome in cultured chondrocytes. RT-PCR data for the mRNAs from collagen types I, II, and aggrecan and the pro-inflammatory cytokine interleukin-1ß (IL-1ß) did not reveal any correlation of transcriptional activity in cultures with Y-chromosome losses, nor were there statistically significant differences between cells from female and male donors. While cells of male origin may suffer from an age-related loss of the Y-chromosome, there was no indication of a functional impairment. The data suggest some caution toward applying proliferative steps when considering chondrocytes from elderly male patients for tissue engineering approaches.

Molecular Karyotyping as a Relevant Diagnostic Tool in Children with Growth Retardation with Silver-Russell Features

To determine the contribution of submicroscopic chromosomal imbalances to the etiology of Silver-Russell syndrome (SRS) and SRS-like phenotypes. We performed molecular karyotyping in 41 patients with SRS or SRS-like features without known chromosome 7 and 11 defects using the Affymetrix SNP Array 6.0 system (Affymetrix, High Wycombe, United Kingdom). In 8 patients, pathogenic copy number variations with sizes ranging from 672 kb to 9.158 Mb were identified. The deletions in 1q21, 15q26, 17p13, and 22q11 were associated with known microdeletion syndromes with overlapping features with SRS. The duplications in 22q13 and Xq25q27 represent unique novel copy number variations but have an obvious influence on the phenotype. In 5 additional patients, the pathogenetic relevance of the detected variants remained unclear. Pathogenic submicroscopic imbalances were detectable in a significant proportion of patients with short stature and features reminiscent of SRS. Therefore, molecular karyotyping should be implemented in routine diagnostics for growth-retarded patients with even slight dysmorphisms suggestive for SRS.

De novo triplication of the MAPT gene from the recurrent 17q21.31 microdeletion region in a patient with moderate intellectual disability and various minor anomalies

We report on a 16-year-old male patient with moderate intellectual disability, behavioral problems, and further anomalies such as facial dysmorphism, heart defect, and urogenital anomalies. By molecular karyotyping we identified the first de novo copy number gain to four copies on chromosome 17q21.31 including the MAPT gene but not the entire recurrent microdeletion/microduplication region. Recurrent microdeletions of this region including the MAPT and the CHRH1 genes have been shown to be a relatively frequent cause of intellectual disability, while only a few reciprocal duplications in patients with variable cognitive disorders have been published so far. A common inversion polymorphism in this region has been linked to a distinct H2 haplotype and seems to be associated with an increased risk for microdeletions and -duplications. Our patient and his father were both heterozygous for the H1/H2 haplotype, whereas the mother was homozygous for the H2 haplotype. In our patient the dosage gain apparently occurred on the paternal H1 allele and did not involve the H2 allele as in the previously published cases. This patient further delineates the genotypic and phenotypic variability associated with copy number variants from the 17q21.31 microdeletion region.

The clinical utility of molecular karyotyping using high-resolution array-comparative genomic hybridization

Clinical characteristics of patients are not always related to specific syndromes. Array-comparative genomic hybridization (aCGH) is used to detect submicroscopic copy number variants within the genome not visible by conventional karyotyping. The clinical application of aCGH has helped the genetic diagnosis of patients with unexplained developmental delay/intellectual disability, autism spectrum disorders, with or without multiple congenital anomalies. Since 2008, we have implemented aCGH with the 244K and 4×180K Agilent platform on 334 patients with various degrees of developmental delay/intellectual disability, seizures, autism spectrum disorders, multiple congenital anomalies and normal previous conventional karyotype. Many of the patients had also received a variety of other genetic tests (Fragile X syndrome, Rett syndrome, single FISH tests or metabolic screens), which were normal. Clinically significant submicroscopic imbalances with aCGH were detected in 84 (∼25.15%) patients. aCGH is proving to be a powerful tool for the identification of novel chromosomal syndromes, thus allowing accurate prognosis and phenotype–genotype correlations.

Further delineation of the phenotype of chromosome 14q13 deletions: (positional) involvement of FOXG1 appears the main determinant of phenotype severity, with no evidence for a holoprosencephaly locus

BackgroundDeletions including chromosome 14 band q13 have been linked to variable phenotypes. With current molecular methods the authors aim to elucidate a genotype–phenotype correlation by accurately determining the size and...

De novo microdeletions of chromosome 6q14.1-q14.3 and 6q12.1-q14.1 in two patients with intellectual disability - further delineation of the 6q14 microdeletion syndrome and review of the literature

Interstitial 6q deletions can cause a variable phenotype depending on the size and location of the deletion. 6q14 deletions have been associated with intellectual disability and a distinct pattern of minor anomalies, including upslanted palpebral fissures with epicanthal folds, a short nose with broad nasal tip, anteverted nares, long philtrum, and thin upper lip. In this study we describe two patients with overlapping 6q14 deletions presenting with developmental delay and characteristic dysmorphism. Molecular karyotyping using array CGH analysis revealed a de novo 8.9 Mb deletion at 6q14.1-q14.3 and a de novo 11.3 Mb deletion at 6q12.1-6q14.1, respectively. We provide a review of the clinical features of twelve other patients with 6q14 deletions detected by array CGH analysis. By assessing all reported data we could not identify a single common region of deletion. Possible candidate genes in 6q14 for intellectual disability might be FILIP1, MYO6, HTR1B, and SNX14.

Genome-wide arrays in routine diagnostics of hematological malignancies

Over the last three decades, cytogenetic analysis of malignancies has become an integral part of disease evaluation and prediction of prognosis or responsiveness to therapy. In most diagnostic laboratories, conventional karyotyping, in conjunction with targeted fluorescence in situ hybridization analysis, is routinely performed to detect recurrent aberrations with prognostic implications. However, the genetic complexity of cancer cells requires a sensitive genome-wide analysis, enabling the detection of small genomic changes in a mixed cell population, as well as of regions of homozygosity. The advent of comprehensive high-resolution genomic tools, such as molecular karyotyping using comparative genomic hybridization or single-nucleotide polymorphism microarrays, has overcome many of the limitations of traditional cytogenetic techniques and has been used to study complex genomic lesions in, for example, leukemia. The clinical impact of the genomic copy-number and copy-neutral alterations identified by microarray technologies is growing rapidly and genome-wide array analysis is evolving into a diagnostic tool, to better identify high-risk patients and predict patients' outcomes from their genomic profiles. Here, we review the added clinical value of an array-based genome-wide screen in leukemia, and discuss the technical challenges and an interpretation workflow in applying arrays in the acquired cytogenetic diagnostic setting.

Molecular Phylogeny, Laboratory Rearing, and Karyotype of the Bombycid Moth,Trilocha varians

This study describes the molecular phylogeny, laboratory rearing, and karyotype of a bombycid moth, Trilocha varians (F. Walker) (Lepidoptera: Bombycidae), which feeds on leaves of Ficus spp. (Rosales: Moraceae). The larvae of this species were collected in Taipei city, Taiwan, and the Ryukyu Archipelago (Ishigaki and Okinawa Islands, Japan). Molecular phylogenetic analyses revealed that T. varians belongs to the subfamily Bombycinae, thus showing a close relationship to the domesticated silkworm Bombyx mori (L.), a lepidopteran model insect. A laboratory method was developed for rearing T. varians and the time required for development from the embryo to adult was determined. From oviposition to adult emergence, the developmental zero was 10.47 °C and total effective temperature was 531.2 day—degrees, i.e., approximately 30 days for one generation when reared at 28 °C. The haploid of T. varians consisted of n = 26 chromosomes. In highly polyploid somatic nuclei, females showed a large heterochromatin body, indicating that the sex chromosome system in T. varians is WZ/ZZ (female/male). The results of the present study should facilitate the utilization of T. varians as a reference species for B. mori, thereby leading to a greater understanding of the ecology and evolution of bombycid moths.

Morphological structure of propagules and electrophoretic karyotype analysis of false smut Villosiclava virens in rice

The target pathogen Villosiclava virens (teleomorph: claviceps oryzae-sativae) was isolated from the infected rice, where it caused false smut. In our study, the forming processes of the chlamydospores, chlamydospore balls, conidiospores, and secondary conidiospores during the asexual reproduction were observed more precisely and in greater detail than previous descriptions. The microstructure of the infected rice kernel showed that the outer dense chlamydospores piled around the false smut balls grown on XBZ medium; moreover the sclerotia consisting of dense mycelium were found. The different morphology was observed across the different growing conditions. In addition, we observed the nuclear numbers of both the conidiospores and hyphae using 4',6-diamidino-2-phenylindole (DAPI) staining. Because the fungus has small chromosomes and the numbers were not previously known, we analyzed the electrophoretic karyotype using a pulsed field gel electrophoresis (PFGE) technique. The results showed that V. virens has at least 10 chromosomes ranging in size from 0.6 kb to 6 Mb. The V. virens genome size is estimated to be 23 Mb. Here, we report the morphological characteristics of the fungus and the process of asexual spores forming asexual propagules, along with the first analyze the molecular karyotype of V. virens. These results supply a foundation for further study of the pathogenicity and biology of this devastating pathogen.

Genetic counselling and ethical issues with chromosome microarray analysis in prenatal testing

Molecular karyotyping using chromosome microarray analysis (CMA) detects more pathogenic chromosomal anomalies than classical karyotyping, making CMA likely to become a first tier test for prenatal diagnosis. Detecting copy number variants of uncertain clinical significance raises ethical considerations. We consider the risk of harm to a woman or her fetus following the detection of a copy number variant of uncertain significance, whether it is ethically justifiable to withhold any test result information from a woman, what constitutes an 'informed choice' when women are offered CMA in pregnancy and whether clinicians are morally responsible for 'unnecessary' termination of pregnancy. Although we are cognisant of the distress associated with uncertain prenatal results, we argue in favour of the autonomy of women and their right to information from genome-wide CMA in order to make informed choices about their pregnancies. We propose that information material to a woman's decision-making process, including uncertain information, should not be withheld, and that it would be paternalistic for clinicians to try to take responsibility for women's decisions to terminate pregnancies. Non-directive pre-test and post-test genetic counselling is central to the delivery of these ethical objectives.