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a-CGH partial profiles of chromosome 7 in patient 1. A) Patient's 100% DNA. B) Synthetic mosaicism at 10% level, C) 8%, D) 7%.
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Background
The results of cytogenetic investigations on unbalanced chromosome anomalies, both constitutional and acquired, were largely improved by comparative genomic hybridization on microarray (a-CGH), but in mosaicism the ability of a-CGH to reliably detect imbalances is not yet well established. This problem of sensitivity is even more relevan...
Contexts in source publication
Context 1
... were scanned using Agilent's microarray scanner G2565BA and features were extracted by Agilent's Feature Extrac- tion 9.5.1 software. The a-CGH profiles of patients were extrapolated by the Agilent's Genomic Workbench soft- ware (5.0.14), and are shown in Figures 1, 2 [17]. So, in total, seven different unbalanced regions of different size were present in the DNA of the three patients. ...Context 2
... no case 5% and 6% mosaicism was clearly discernible. The profiles of the chromosome 7, 4, and 9 of mosaicism at levels 7, 8, and 10% in patients 1 and 2 are illustrated in Figures 1, 2, 3 (panels B, C, D): the analysis system was able to detect these three imbalances at 8 and 10% level, and not at 7%. ...Context 3
... patients 1 and 2 we were able to get easily evidence of the abnormal cell population at 10 and 8% levels of mosaicism (Figures 1, 2, 3), because we had already defined the presence of the imbalance. In our material the mosaicism was built up artificially, but in general in patients in which the imbalance may be suspected from the results of a-CGH at levels comparable to those here presented, the tool to reach a conclusion is FISH with informative probes, possibly on interphase nuclei, which will be able to confirm or deny the presence of the acquired mosaicism. ...Similar publications
ACTB and ACTG1 mutations have recently been reported to cause Baraitser-Winter syndrome (BRWS) - a rare condition characterized by ptosis, colobomata, neuronal migration disorder, distinct facial anomalies and intellectual disability. One of the patients carrying an ACTB mutation was previously diagnosed with Fryns-Aftimos syndrome (FAS), which is...
Citations
... The finding that array CGH detected no aneuploidy is not inconsistent. In fact, the detection threshold limit is around 8% as previously shown [52]. ...
Background SMC1A is a subunit of the cohesin complex that participates in many DNA-and chromosome-related biological processes. Previous studies have established that SMC1A is involved in cancer development and in particular , is overexpressed in chromosomally unstable human colorectal cancer (CRC). This study aimed to investigate whether SMC1A could serve as a therapeutic target for CRC. Methods At first, we studied the effects of either SMC1A overexpression or knockdown in vitro. Next, the outcome of SMC1A knocking down (alone or in combination with bevacizumab, a monoclonal antibody against vascular endothelial growth factor) was analyzed in vivo. Results We found that SMC1A knockdown affects cell proliferation and reduces the ability to grow in anchorage-independent manner. Next, we demonstrated that the silencing of SMC1A and the combo treatment were effective in increasing overall survival in a xenograft mouse model. Functional analyses indicated that both treatments lead to atypical mitotic figures and gene expression dysregulation. Differentially expressed genes were implicated in several pathways including gene transcription regulation, cellular proliferation, and other transformation-associated processes. Conclusions These results indicate that SMC1A silencing, in combination with bevacizumab, can represent a promising therapeutic strategy for human CRC.
... However, conventional karyotyping is a highly laborious and time-consuming method, with a limited resolution of 5-10 Mb and hampered by maternal cell contamination and culture failure [38]. Recently, we witnessed the spread of a wide variety of complementary molecular techniques available for the analysis of product-ofconception analyses, including QF-PCR, MLPA, FISH, and chromosomal microarray (CMA) [39,40]. ...
It is well known that first-trimester miscarriages are associated with chromosome abnormalities, with numerical chromosome abnormalities being the ones most commonly detected. Conventional karyotyping is still considered the gold standard in the analysis of products of conception, despite the extended use of molecular genetic techniques. However, conventional karyotyping is a laborious and time-consuming method, with a limited resolution of 5–10 Mb and hampered by maternal cell contamination and culture failure. The aim of our study was to assess the type and frequency of chromosomal abnormalities detected by conventional karyotyping in specimens of sporadic first-trimester miscarriages in a Romanian cohort, using QF-PCR to exclude maternal cell contamination. Long-term cultures were established and standard protocols were applied for cell harvesting, slide preparation, and GTG banding. All samples with 46,XX karyotype were tested for maternal cell contamination by QF-PCR, comparing multiple microsatellite markers in maternal blood with cell culture and tissue samples. Out of the initial 311 specimens collected from patients with sporadic first-trimester miscarriages, a total of 230 samples were successfully analyzed after the exclusion of 81 specimens based on unsuitable sampling, culture failure, or QF-PCR-proven maternal cell contamination. Chromosome abnormalities were detected in 135 cases (58.7%), with the most common type being single autosomal trisomy (71/135—52.6%), followed by monosomy (monosomy X being the only one detected, 24/135—17.8%), and polyploidy (23/135—17.0%). The subgroup analysis based on maternal age showed a statistically significant higher rate of single trisomy for women aged 35 years or older (40.3%) compared to the young maternal age group (26.1%) (p = 0.029). In conclusion, the combination of conventional karyotyping and QF-PCR can lead to an increased chromosome abnormality detection rate in first-trimester miscarriages. Our study provides reliable information for the genetic counseling of patients with first-trimester miscarriages, and further large-scale studies using different genetic techniques are required.
... On the other hand, it is a cost-effective, methodologically simple screening method for larger cytogenetic alterations and can detect numerical and structural aneuploidies as well as transpositions, deletions, duplications, and even inversions of chromosomal arms or larger chromosomal fragments (being the only convenient method to detect balanced genomic rearrangements) [51]. Comparative Genomic Hybridization array, in turn, enables high-resolution detection of submicroscopic unbalanced genomic alterations [52]. Nonetheless, the frequent use of this technique is limited by its higher costs compared to conventional cytogenetic techniques. ...
Human pluripotent stem cells (hPSCs) have generated unprecedented interest in the scientific community, given their potential applications in regenerative medicine, disease modeling, toxicology and drug screening. However, hPSCs are prone to acquire genomic alterations in vitro, mainly due to suboptimal culture conditions and inappropriate routines to monitor genome integrity. This poses a challenge to both the safety of clinical applications and the reliability of basic and translational hPSC research. In this study, we aim to investigate if the implementation of a Quality Management System (QMS) such as ISO9001:2015 to ensure reproducible and standardized cell culture conditions and genomic screening strategies can decrease the prevalence of genomic alterations affecting hPSCs used for research applications. To this aim, we performed a retrospective analysis of G-banding karyotype and Comparative Genomic Hybridization array (aCGH) data generated by our group over a 5-year span of different hESC and hiPSC cultures. This work demonstrates that application of a QMS to standardize cell culture conditions and genomic monitoring routines leads to a striking improvement of genomic stability in hPSCs cultured in vitro, as evidenced by a reduced probability of potentially pathogenic chromosomal aberrations and subchromosomal genomic alterations. These results support the need to implement QMS in academic laboratories performing hPSC research.
... Additionally, SNP arrays are also able to detect copy neutral losses, gains and mosaics 13,132,133 . Both CGH and SNP microarrays are capable of identifying mosaic aberrations present in 4-8% (SNPs) to 10% (CGH) of the cells [134][135][136] . ...
Mosaicism denotes an individual who has at least two populations of cells with distinct genotypes that are derived from a single fertilized egg. Genetic variation among the cell lines can involve whole chromosomes, structural or copy-number variants, small or single-nucleotide variants, or epigenetic variants. The mutational events that underlie mosaic variants occur during mitotic cell divisions after fertilization and zygote formation. The initiating mutational event can occur in any types of cell at any time in development, leading to enormous variation in the distribution and phenotypic effect of mosaicism. A number of classification proposals have been put forward to classify genetic mosaicism into categories based on the location, pattern, and mechanisms of the disease. We here propose a new classification of genetic mosaicism that considers the affected tissue, the pattern and distribution of the mosaicism, the pathogenicity of the variant, the direction of the change (benign to pathogenic vs. pathogenic to benign), and the postzygotic mutational mechanism. The accurate and comprehensive categorization and subtyping of mosaicisms is important and has potential clinical utility to define the natural history of these disorders, tailor follow-up frequency and interventions, estimate recurrence risks, and guide therapeutic decisions.
... 13,132,133 Both CGH and SNP microarrays are capable of identifying mosaic aberrations present in 4-8% (SNPs) to 10% (CGH) of the cells. [134][135][136] Mosaic structural variations are seen in about 1:200 to 1:400 children investigated for clinical diagnostic testing (Fig. 1, Fig. S5). 137 ...
Mosaicism denotes an individual who has at least two populations of cells with distinct genotypes that are derived from a single fertilized egg. Genetic variation among the cell lines can involve whole chromosomes, structural or copy-number variants, small or single nucleotide variants, or epigenetic variants. The mutational events that underlie mosaic variants occur during mitotic cell divisions after fertilization and zygote formation. The initiating mutational event can occur in any types of cell at any time in development, leading to enormous variation in the distribution and phenotypic effect of mosaicism. A number of classification proposals have been put forward to classify genetic mosaicism into categories based on the location, pattern, and mechanisms of the disease. We here propose a new classification of genetic mosaicism that considers the affected tissue, the pattern and distribution of the mosaicism, the pathogenicity of the variant, the direction of the change (benign to pathogenic vs. pathogenic to benign), and the postzygotic mutational mechanism. The accurate and comprehensive categorization and subtyping of mosaicisms is important and has potential clinical utility to define the natural history of these disorders, tailor follow-up frequency and interventions, estimate recurrence risks, and guide therapeutic decisions.
... Features were extracted using Agilent Feature extraction software version 12.0.2.2. As expected, amplified DNAs produce high levels of background/noise, so molecular karyotype and percentage of mosaicism were reconstructed by the use of Agilent's Genomic Workbench software version 7.0.4.0, with parameters and algorithms set as recommended in our previous studies [69][70][71] with the only difference of two parameters: centralization = off and Fuzzy zero = off. This was chosen due to the nature itself of the experiment in which a low number of chromosomal changes and variable percentage of mosaics are expected; the centralization algorithm is used only in the case of an expected high number of chromosome imbalances and the fuzzy zero algorithm only in the case of an expected high percentage of mosaicism. ...
The growing trend for women to postpone childbearing has resulted in a dramatic increase in the incidence of aneuploid pregnancies. Despite the importance to human reproductive health, the events precipitating female age-related meiotic errors are poorly understood. To gain new insight into the molecular basis of age-related chromosome missegregation in human oocytes, we combined the transcriptome profiles of twenty single oocytes (derived from females divided into two groups according to age <35 and ≥35 years) with their chromosome status obtained by array comparative genomic hybridization (aCGH). Furthermore, we compared the transcription profile of the single oocyte with the surrounding cumulus cells (CCs). RNA-seq data showed differences in gene expression between young and old oocytes. Dysregulated genes play a role in important biological processes such as gene transcription regulation, cytoskeleton organization, pathways related to RNA maturation and translation. The comparison of the transcription profile of the oocyte and the corresponding CCs highlighted the differential expression of genes belonging to the G protein-coupled receptor superfamily. Finally, we detected the loss of a X chromosome in two oocytes derived from women belonging to the ≥35 years age group. These aneuploidies may be caused by the detriment of REEP4, an endoplasmic reticulum protein, in women aged ≥35 years. Here we gained new insight into the complex regulatory circuit between the oocyte and the surrounding CCs and uncovered a new putative molecular basis of age-related chromosome missegregation in human oocytes.
... The threshold for genomic deletion is x = −1; the threshold for genomic gain is x = +0 58. Notably, in a mosaic scenario, the threshold is between -1 and 0 for deletions and between 0 and +0.58 for duplications [20]. Amplifications and homozygous deletions are considered with threshold >+2 and <-1, respectively. ...
Glioblastoma (GBM) is the most malignant human brain tumour, characterized by rapid progression, invasion, intense angiogenesis, high genomic instability, and resistance to therapies. Despite countless experimental researches for new therapeutic strategies and promising clinical trials, the prognosis remains extremely poor, with a mean survival of less than 14 months. GBM aggressive behaviour is due to a subpopulation of tumourigenic stem-like cells, GBM stem cells (GSCs), which hierarchically drive onset, proliferation, and tumour recurrence. The morbidity and mortality of this disease strongly encourage exploring genetic characteristics of GSCs. Here, using array-CGH platform, we investigated genetic and genomic aberration profiles of GBM parent tumour ( n=10 ) and their primarily derived GSCs. Statistical analysis was performed by using R software and complex heatmap and corrplot packages. Pearson correlation and K -means algorithm were exploited to compare genetic alterations and to group similar genetic profiles in matched pairs of GBM and derived GSCs. We identified, in both GBM and matched GSCs, recurrent copy number alterations, as chromosome 7 polysomy, chromosome 10 monosomy, and chromosome 9p21deletions, which are typical features of primary GBM, essential for gliomagenesis. These observations suggest a condition of strong genomic instability both in GBM as GSCs. Our findings showed the robust similarity between GBM mass and GSCs (Pearson corr.≥0.65 ) but also highlighted a marked variability among different patients. Indeed, the heatmap reporting Gain/Loss State for 21022 coding/noncoding genes demonstrated high interpatient divergence. Furthermore, K -means algorithm identified an impairment of pathways related to the development and progression of cancer, such as angiogenesis, as well as pathways related to the immune system regulation, such as T cell activation. Our data confirmed the preservation of the genomic landscape from tumour tissue to GSCs, supporting the relevance of this cellular model to test in vitro new target therapies for GBM.
... In mosaic samples, there will be proportion of normal cells and tumor cells resulting in a fractional of total CN in nondiploid regions. Previous studies have theorized the presence of mosaicism by assessing log2 ratios or allele specific copy number and predicate the differences as potential indicators of clonal heterogeneity [25][26][27]. In a study on clonal evolution in hematology malignancies by combining comparative genomic hybridization and single nucleotide polymorphism array on 16 patients, manual peak reassignment analysis enabled the detection of heterogeneity in 10 patients [27]. ...
... Secondly, the presence of low tumor cells admixed with normal cells reduces the ability to detect tumor-related changes. However, Valli et al. reported as low as 8% of abnormal cells in mosaic samples [26]. Array CGH is unable to identify tetraploidy and other ploidy levels [29] [30] but a combined array CGH+SNP platform is capable in revealing poplyploidy. ...
Acute myeloid leukemia (AML) is an aggressive disease characterized by the overproduction of immature myeloid cells that accumulate in blood and bone marrow. Integration of genetic findings and clinicopathological information is crucial in establishing the diagnosis, prognosis and determining the therapeutic approach in the management of AML patients. In recent years, the AML classification has evolved from morphology to cytogenetics/molecular genetics-based findings, which is essential in the detection of chromosomal abnormalities and has provided the framework for the diagnosis and risk-stratification in AML. Moreover, with advances in molecular karyotyping such as comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) arrays, various limitations of conventional diagnostic approaches have been overcome. Hence, this review focuses on the insights into molecular karyotyping using CGH and SNP arrays which enable the identification of copy number variations (CNVs) at a higher resolution and facilitate the detection of copy neutral loss of heterozygosity (CN-LOH) otherwise undetectable by conventional cytogenetics. Technical hindrances of these methods (e.g. regions of losses, gains, or "undulating waves") are also discussed in the context of AML.
... Analysis was performed with Agilent Genomic Workbench 7.0 using the ADM2 algorithm (threshold 6, five consecutive probes and 10 kb size needed for a call). The 'fuzzy zero' long range correction was used, because removing it may help detect mosaicism (Valli et al., 2011), but also allows artefacts due to GCrelated DNA isolation bias to be called (Nacheva et al., 2017). Data were mapped to hg19. ...
The α-synuclein protein, encoded by SNCA, has a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Although usually sporadic, Parkinson's disease can result from inherited copy number variants in SNCA and other genes. We have hypothesized a role of somatic SNCA mutations, leading to mosaicism, in sporadic synucleinopathies. The evidence for mosaicism in healthy and diseased brain is increasing rapidly, with somatic copy number gains of APP reported in Alzheimer's brain. Here we demonstrate somatic SNCA copy number gains in synucleinopathies (Parkinson's disease and multiple system atrophy), focusing on substantia nigra. We selected sporadic cases with relatively young onset or short disease duration, and first excluded high level copy number variant mosaicism by DNA analysis using digital PCR for SNCA, and/or customized array comparative genomic hybridization. To detect low level SNCA copy number variant mosaicism, we used fluorescent in situ hybridization with oligonucleotide custom-designed probes for SNCA, validated on brain and fibroblasts with known copy number variants. We determined SNCA copy number in nigral dopaminergic neurons and other cells in frozen nigra sections from 40 cases with Parkinson's disease and five with multiple system atrophy, and 25 controls, in a blinded fashion. Parkinson's disease cases were significantly more likely than controls to have any SNCA gains in dopaminergic neurons (P = 0.0036), and overall (P = 0.0052). The average proportion of dopaminergic neurons with gains in each nigra was significantly higher in Parkinson's disease than controls (0.78% versus 0.45%; P = 0.017). There was a negative correlation between the proportion of dopaminergic neurons with gains and onset age in Parkinson's disease (P = 0.013), but not with disease duration, or age of death in cases or controls. Cases with tremor at onset were less likely to have gains (P = 0.035). All multiple system atrophy cases had gains, and the highest levels in dopaminergic neurons were in two of these cases (2.76%, 2.48%). We performed selective validation with different probes after dye swapping. All three control probes used showed minimal or no gains (≤0.1% in dopaminergic neurons). We also found occasional SNCA gains in frontal neurons of cases with Parkinson's disease, and the putamen of one multiple system atrophy case. We present evidence of somatic SNCA gains in brain, more commonly in nigral dopaminergic neurons of Parkinson's disease than controls, negatively correlated with onset age, and possibly commonest in some multiple system atrophy cases. Somatic SNCA gains may be a risk factor for sporadic synucleinopathies, or a result of the disease process.
... Another potential limitation of the platform refers to the mosaicism conditions. As previously reported, the aCGH technology allows detecting lowgrade as well as submicroscopic mosaics (<10%) [85,86]. Anyway, the possibility to reach a correct identification of a low-represented mosaic depends on several factors (i.e., the DNA quality, the algorithm used for analysis, the platform design itself). ...
Background:
Neurological disorders are a highly heterogeneous group of pathological conditions that affect both the peripheral and the central nervous system. These pathologies are characterized by a complex and multifactorial etiology involving numerous environmental agents and genetic susceptibility factors. For this reason, the investigation of their pathogenetic basis by means of traditional methodological approaches is rather arduous. High-throughput genotyping technologies, including the microarray-based comparative genomic hybridization (aCGH), are currently replacing classical detection methods, providing powerful molecular tools to identify genomic unbalanced structural rearrangements and explore their role in the pathogenesis of many complex human diseases.
Methods:
In this report, we comprehensively describe the design method, the procedures, validation, and implementation of an exon-centric customized aCGH (NeuroArray 1.0), tailored to detect both single and multi-exon deletions or duplications in a large set of multi- and monogenic neurological diseases. This focused platform enables a targeted measurement of structural imbalances across the human genome, targeting the clinically relevant genes at exon-level resolution.
Conclusion:
An increasing use of the NeuroArray platform may offer new insights in investigating potential overlapping gene signatures among neurological conditions and defining genotype-phenotype relationships.
