Comparative Genomic Hybridization Technique Research Articles

HGG-36. ASSESSMENT OF CHROMOSOMAL ABERRATIONS IN DIFFUSE ASTROCYTOMAS OF CHILDHOOD USING THE ARRAY COMPARATIVE GENOMIC HYBRIDIZATION (ACGH+SNP) TECHNIQUE – PRELIMINARY STUDY

Abstract BACKGROUND Comprehensive knowledge of the genomic changes underlying cancer is a key basis for diagnosis, prognosis and targeted therapies. With recent advances, the technique of comparative genomic hybridization (aCGH+SNP) facilitates genome-wide screening for genome copy number changes and rapid examination of numerical and structural chromosomal changes, as well as loss of heterozygosity at high resolution down to several tens of kilobases. METHODS The study included 12 patients aged 6 months to 15 years, operated on at the Department of Pediatric Neurosurgery in Krakow due to paediatric-type diffuse gliomas (according to WHO classification 5th Ed). Tumor tissue samples were collected from all included participants as remnants of routine examination. Pangenomic profiling of diffuse astrocytomas was performed with using SurePrint G3 Cancer CGH+SNP Kit 4x180K (Agilent, Santa Clara USA) at the Molecular Genetics Laboratory of the University Children’s Hospital in Krakow. Genomic DNA was extracted automatically from deep-frozen tumor tissue. Descriptive statistics were used to describe the basic characteristics of the data included in the study due to the small number of participants. RESULTS Of the 12 tumors examined by microarray, 5 patients had abnormalities in the form of diffuse high grade gliomas. In 1 patient, only numerical changes in chromosomes were observed, in 1 case only structural chromosomal abnormalities were detected. Combined numerical and structural aberrations were presented in 3 samples. Additionally, we detected 1 case of triploidy and 1 case of loss of heterozygosity. CONCLUSIONS Significant changes were observed in paediatric-type diffuse high-grade gliomas. Interestingly, we detected new undescribed alterations that were not only of a chromosomal nature, but also concerned individual genes. This may constitute the basis for further research aimed at establishing new diagnostic and prognostic markers, as well as effective therapy. A limitation of this study is the small patient sample, and further research is recommended.

First report on chromosomal abnormalities in Eastern Morocco: Identification of a new case of a de novo partial trisomy 13q using single-nucleotide polymorphism array

BackgroundChromosomal abnormalities are the main cause of birth defects, intellectual disability, and miscarriages. They contribute to significant human morbidity and infant mortality. Here we report for the first time the chromosomal abnormalities encountered in the population of Eastern Morocco. Furthermore, we describe a new case of a de novo partial trisomy 13q combined with a terminal deletion in an 11-day-old girl. MethodsFrom November 2015 to March 2022, 195 patients from the BRO Biobank who were clinically suspected of having chromosomal abnormalities were referred to the cytogenetics laboratory of the Genetics Unit of the Faculty of Medicine and Pharmacy of Oujda for cytogenetic study. Karyotyping analysis was performed on peripheral blood samples using standard R banding techniques. To identify single-nucleotide polymorphism (SNP) and copy number variants (CNVs), Illumina SNP array was used. ResultsAmong 195 studied cases, 32 (16.4 %) had abnormal karyotypes, of which 12 cases had numerical aberrations while 20 cases had structural aberrations. The most common numerical aberrations were Turner syndrome and Down syndrome followed by Edward, Patau, and Klinefelter syndromes. For structural aberrations, translocations were the most common, followed by derivative chromosomes, inversions, deletions, and an addition on chromosome 13 identified in an 11-day-old girl. To further characterize this addition, SNP array was carried out and revealed a 58.8-Mb duplication in region 13q14.3q34 associated with a 1-Mb deletion in region 13q34. Follow-up parental chromosomes analysis showed normal karyotypes for the parents, confirming that this partial trisomy 13q was de novo. Comparison of the phenotype associated with this novel duplication on chromosome 13q with those previously reported confirmed the considerable variability in the phenotype of the patients with partial trisomy 13q. ConclusionThis study provided the first report on chromosomal abnormalities in Eastern Morocco and it enriched the phenotype spectrum of partial trisomy 13q and further confirmed the genotype–phenotype correlations. Furthermore, these findings justify the need to set up microarray comparative genomic hybridization techniques in Morocco for better genetic diagnosis.

The Roles of the Fluorescent In Situ Hybridization (FISH) and Comparative Genomic Hybridization (CGH) Techniques in the Detection of the Breast Cancer

This paper aimed to understand and compare the two popular cytogenetic techniques of fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) in detecting breast cancer chromosomal abnormality. Several chromosomal anomalies play a role in the development of breast cancer, and the two above approaches play an important role in confirming fluorescence in situ hybridization in particular (FISH). However, comparative genomic hybridization has developed DNA copy number profiles for most of the publicly available breast cancer cell lines for the FISH methods rely on the fluorescent probes. Chromosomal profiles can be generated for the suspected chromosomal abnormality, copy number changes between the tumour and the DNA control can be compared, and the results can be registered. Today, modern cytogenetic tools such as fluorescence in situ hybridization (FISH) are more commonly used to detect any microdeletion that cannot be detected by conventional cytogenetic karyotypes that involve a high rate of cell division and good chromosomal morphology, which pose challenges for cytogeneticists, and a long period of testing and research. Usually, this is a problem for physicians, and there are still many drawbacks and disadvantages concerning the high benefits, such as false findings. Normal chromosome in situ hybridization requires the hybridization of a labelled DNA probe into denatured chromosomal DNA present in metaphase chromosomes in an air-dried microscope slide preparation. Metaphase spreads are used for traditional chromosome FISH (metaphase FISH). Positive and positive signs of hybridization also appear as a double spot, corresponding to the hybridized probe for both sister chromatids. A further extension of chromosome painting is comparative genomic hybridization (CCI-I). CCH involves simultaneous chromosome painting in two different colours using complete DNA from two similar sources as probes, which reveal variations concerning the benefit or loss of sub-chromosomal regions or even entire chromosomes.

Prenatal diagnosis of de novo small supernumerary marker chromosome 4q (4q11-q12): A case report

BackgroundSmall supernumerary marker chromosomes (sSMCs) are chromosomal fragments with abnormal structures found in patients with fertility problems and developmental delay. They may be detected in amniotic cell karyotypes. sSMCs are categorized as hereditary or de novo. Here, we describe a case of prenatal de novo 4q11q12 sSMC and its molecular cytogenetic features which had no apparent phenotypic abnormality.CaseThe fetus of a 36-yr-old pregnant woman was detected positive for Down's syndrome (trisomy 21) at the 16 wk of gestation. Quantitative fluorescent polymerase chain reaction technique was applied for the rapid detection of numerical aneuploidy of chromosomes X, Y, 13, 18, and 21 microsatellites. Array comparative genomic hybridization (array CGH) technique was also conducted following the karyotype analysis of amniotic cells. The karyotype analysis was also done for the parents. Quantitative fluorescent polymerase chain reaction result revealed a male fetus with a normal chromosomal pattern, while the amniocentesis karyotype analysis identified a male fetus with a marker chromosome (47, XY, +mar), and the sSMC were existing in 100% of amniocyte metaphase spreads. The parents' normal karyotypes indicated that the sSMC was de novo. Array CGH analysis revealed a 6.48-Mb duplication at 4q11q12. Eventually, the parents decided to terminate the pregnancy by legal abortion.ConclusionOur study highlights the importance of the application of array CGH in combination with karyotype analysis for rapid and precise prenatal diagnosis of partial aneuploidy region.

5. Further evidence of GABRA4 and TOP3B as autism susceptibility genes

Copy number variants (CNVs) are a known contributor to neurodevelopmental conditions such as autism spectrum disorder (ASD). Both array comparative genomic hybridization and Next-Generation sequencing techniques have led to increased detection of small CNVs and the identification of many candidate susceptibility genes for ASD. We report familial inheritance of two CNVs that include genes with known involvement in neurodevelopment. These CNVs are found in various combinations among four siblings with autism spectrum disorder, as well as in their phenotypically normal parents. We describe a 2.4 Mb duplication of 4p12 to 4p11 that includes GABRA4 and other GABA receptor genes, as well as a 246 kb deletion at 22q11.22 involving the TOP3B gene. The maternally inherited 4p duplication was detected in three siblings, two of whom also had the paternally inherited 22q11.22 deletion. The fourth sibling only had the 22q11.22 deletion. These CNVs have rarely been reported in the literature. Upon review, a single publication was found describing a similar 4p duplication in three generations of a family with neurodevelopmental and neuropsychiatric disorders, as well as in an unrelated patient with autism. TOP3B falls within the distal 22q11.22 deletion syndrome and has been associated with schizophrenia, neurodevelopmental disorders including epilepsy, and cardiac defects. The identification of this family contributes to the understanding of specific genetic contributors to neurodevelopmental disorders and an emerging phenotype associated with proximal 4p duplication.

Deletion of 15q26.1 region with absence epilepsy respond to valproic acid: A Literature Overview and A Case Report from Qatar

Chromosomal abnormalities involving deletions and duplications are known to cause severe developmental disorders, including mental retardation, dysmorphism, and seizures, in children. As the technique of array-based comparative genomic hybridization is being applied more frequently in the diagnostic evaluation of children with developmental disorders; novel pathologic chromosomal abnormalities are being identified in relation to various type of epilepsies in childhood. We report the case of a 4-year-old girl with a history of speech delay and communication disorder, mild dysmorphic features, and absence epilepsy with a de novo microdeletion 15q26.1. A much larger (5 Mb) but overlapping microdeletion has been previously reported in similar several cases with similar phenotype including intractable myoclonic and absence epilepsy, growth delay, and dysmorphic features. This leads us to propose that a potential candidate gene or genes within the deleted region involved in the pathogenesis of some forms of generalized intractable epilepsy, previously considered idiopathic should consider genetic study for childhood epilepsies especially if it was associated with underlying developmental delay in any particular aspect as speech delay in our case.

Frequent homozygous deletion of Cdkn2a/2b in tremolite-induced malignant mesothelioma in rats.

The onset of malignant mesothelioma (MM) is linked to exposure to asbestos fibers. Asbestos fibers are classified as serpentine (chrysotile) or amphibole, which includes the crocidolite, amosite, anthophyllite, tremolite, and actinolite types. Although few studies have been undertaken, anthophyllite has been shown to be associated with mesothelioma, and tremolite, a contaminant in talc and chrysotile, is a risk factor for carcinogenicity. Here, after characterizing the length and width of these fibers by scanning electron microscopy, we explored the cytotoxicity induced by tremolite and anthophyllite in cells from an immortalized human mesothelial cell line (MeT5A), murine macrophages (RAW264.7), and in a rat model. Tremolite and short anthophyllite fibers were phagocytosed and localized to vacuoles, whereas the long anthophyllite fibers were caught on the pseudopod of the MeT5A and Raw 264.7 cells, according to transmission electron microscopy. The results from a 2‐day time‐lapse study revealed that tremolite was engulfed and damaged the MeT5A and RAW264.7 cells, but anthophyllite was not cytotoxic to these cells. Intraperitoneal injection of tremolite in rats induced diffuse serosal thickening, whereas anthophyllite formed focal fibrosis and granulomas on peritoneal serosal surfaces. Furthermore, the loss of Cdkn2a/2b, which are the most frequently lost foci in human MM, were observed in 8 cases of rat MM (homozygous deletion [5/8] and loss of heterozygosity [3/8]) by array‐based comparative genomic hybridization techniques. These results indicate that tremolite initiates mesothelial injury and persistently frustrates phagocytes, causing subsequent peritoneal fibrosis and MM. The possible mechanisms of carcinogenicity based on fiber diameter/length are discussed.

Upper aerodigestive tract carcinoma: Development of a (epi)genomic predictive model for recurrence and metastasis.

Despite the increased molecular knowledge and the diagnostic and therapeutic improvements, the survival of patients with upper aerodigestive tract carcinoma remains poor. The identification of early diagnostic and prognostic biomarkers and the development of molecular models to distinguish patients that will recur and/or develop metastasis after treatment as well as to benefit with target therapies can be important to decrease mortality, improve survival rates and improve the quality of life of these patients. The current study analyzed 21 upper aerodigestive tract carcinomas through array comparative genomic hybridization and methylation-specific multiplex ligation-dependent probe amplification techniques. A number of chromosomal regions and genes were observed with copy number alterations and methylation. A predictive (epi)genomic model that comprises the 3p chromosomal region and WT1, VHL and THBS1 genes was built, highlighting a molecular signature with possible clinical use. The current study may aid in the development of a more individualized patient management and targeted drug design. The power of this genomic and epigenetic model to predict the recurrence and metastasis development should be evaluated and validated in future larger cohort study.

Identification of rare copy number variations reveals PJA2, APCS, SYNPO, and TAC1 as novel candidate genes in Autism Spectrum Disorders.

BackgroundThere is a strong evidence for genetic factors as the main causes of Autism Spectrum Disorders (ASD). To date, hundreds of genes have been identified either by copy number variations (CNVs) and/or single nucleotide variations. However, despite all the findings, the genetics of these disorders have not been totally explored.MethodsThus, the aim of our work was to identify rare CNVs and genes present in these regions in ASD children, using a high‐resolution comparative genomic hybridization technique and quantitative PCR (qPCR) approach.ResultsOur results have shown 60–70 chromosomal aberrations per patient. We have initially selected 66 CNVs that have been further assessed using qPCR. Finally, we have validated 22 CNVs including 11 deletions and 11 duplications. Ten CNVs are de novo, 11 are inherited and one of unknown origin of transmission. Among the CNVs detected, novel ASD candidate genes PJA2, SYNPO, APCS, and TAC1 have been identified in our group of Lebanese patients. In addition, previously described CNVs have been identified containing genes such as SHANK3, MBP, CHL1, and others.ConclusionOur study broadens the population spectrum of studied ASD patients and adds new candidates at the list of genes contributing to these disorders.

DNA copy number imbalances in primary cutaneous lymphomas.

Cutaneous lymphomas (CL) represent a clinically defined group of extranodal non-Hodgkin lymphomas harbouring heterogeneous and incompletely delineated molecular aberrations. Over the past decades, molecular studies have identified several chromosomal aberrations, but the interpretation of individual genomic studies can be challenging. With a comprehensive meta-analysis, we aim to delineate genomic alterations for different types of CL and propose a more accurate classification in line with their various pathogenicity. We searched PubMed and ISI Web of Knowledge for publications from 1996 to 2016 reporting the investigation of CL for genome-wide copy number alterations, by means of comparative genomic hybridization techniques and whole-genome sequencing and whole-exome sequencing. We then extracted and remapped the available copy number variation (CNV) data from these publications with the same pipeline and performed clustering and visualisation to aggregate samples of similar CNV profiles. For 449 samples from 22 publications, CNV data were accessible for sample based meta-analysis. Our findings illustrate structural and numerical chromosomal imbalance patterns. Most frequent CNAs were linked to oncogenes or tumour suppressor genes with important roles in the course of the disease. Summary profiles for genomic imbalances, generated from case-specific data, identified complex genomic imbalances, which could discriminate between different subtypes of CL and promise a more accurate classification. The collected data presented in this study are publicly available through the 'Progenetix' online repository.

High-throughput sequencing for the molecular diagnosis of Usher syndrome reveals 42 novel mutations and consolidates CEP250 as Usher-like disease causative

Usher syndrome is a rare disorder causing retinitis pigmentosa, together with sensorineural hearing loss. Due to the phenotypic and genetic heterogeneity of this disease, the best method to screen the causative mutations is by high-throughput sequencing. In this study, we tested a semiconductor chip based sequencing approach with 77 unrelated patients, as a molecular diagnosis routine. In addition, Multiplex Ligation-dependent Probe Amplification and microarray-based Comparative Genomic Hybridization techniques were applied to detect large rearrangements, and minigene assays were performed to confirm the mRNA processing aberrations caused by splice-site mutations. The designed panel included all the USH causative genes (MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, ADGRV1, WHRN and CLRN1) as well as four uncertainly associated genes (HARS, PDZD7, CEP250 and C2orf71). The outcome showed an overall mutation detection ratio of 82.8% and allowed the identification of 42 novel putatively pathogenic mutations. Furthermore, we detected two novel nonsense mutations in CEP250 in a patient with a disease mimicking Usher syndrome that associates visual impairment due to cone-rod dystrophy and progressive hearing loss. Therefore, this approach proved reliable results for the molecular diagnosis of the disease and also allowed the consolidation of the CEP250 gene as disease causative for an Usher-like phenotype.

The clinical, cytogenetics and molecular characterization of inverted duplication/deletion of chromosome 8p in a boy with mental and motor retardation: Genotype-phenotype correlation in a case report

BackgroundRearrangements that occur mainly through the non-allelic homologous recombination (NAHR) during maternal meiosis in short arms of chromosome 8 is relatively associated with various clinical spectrum. AimThe objective of this study was to report cytogenetics and molecular characterization of a mental and motor retarded boy with short arm of chromosome 8 rearrangements [invdupdel(8p)] in this current case report. Subjects and methods: We report an 11-year-old boy with scoliosis, intellectual disability, mental-motor retardation and characteristic facial features. Agenesis of corpus callosum was detected with brain Magnetic Resonance Imaging (MRI) analysis. Derivative chromosome 8 structure was identified after conventional cytogenetics – karyotype analysis, Multiplex Ligation-Dependent Probe Amplification (MLPA) and Microarray-based Comparative Genomic Hybridization (aCGH) techniques. Genotype-phenotype correlation in the current proband case will be discussed. ResultsCase was diagnosed as 46, XY, der (8), del (8) (p23.1) invdup (8) (p11.1-p23.1) by using advanced comparable techniques. Subtelomeric MLPA analysis showed deletion of FBXO25 gene which is located at 8p23.3 locus and FISH with subtelomeric probes for 8p shows also only deleted region. The microarray-CGH profilling showed 7,9 mb deletion for 8p23.1 and 31 mb duplication for 8p11.1 locuses. ConclusionResults from the current case emphasized that the cases with clinical manifestations of such disorders extremely need to be examined by combined comparable genetics techniques such as; karyotyping, FISH, MLPA and chromosomal microarray for the accurate phenotype – genotype correlation.

Large-scale copy number analysis reveals variations in genes not previously associated with malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumor that is often causally associated with asbestos exposure. Comparative genomic hybridization techniques and arrays demonstrated a complex set of copy number variations (CNVs) in the MPM-genome. These techniques however have a limited resolution, throughput and flexibility compared to next-generation sequencing platforms.In this study, the presence of CNVs in the MPM-genome was investigated using an MPM-cohort (N = 85) for which genomic microarray data are available through ‘The Cancer Genome Atlas’ (TCGA). To validate these results, the genomes of MPMs and matched normal samples (N = 21) were analyzed using low-pass whole genome sequencing on an ‘Illumina HiSeq’ platform. CNVs were detected using in-house developed analysis pipelines and frequencies of copy number loss and gain were calculated.In both datasets, losses on chromosomes 1, 3, 4, 6, 9, 13 and 22 and gains on chromosomes 1, 5, 7 and 17 were found in at least 25% and 15% of MPMs, respectively. Besides the well-known MPM-associated genes, CDKN2A, NF2 and BAP1, other interesting cancer-associated genes were listed as frequently involved in a copy number loss (e.g. EP300, SETD2 and PBRM1). Moreover, four cancer-associated genes showed a high frequency of copy number gain in both datasets (i.e. TERT, FCGR2B, CD79B and PRKAR1A). A statistically significant association between overall survival and the presence of copy number loss in the CDKN2A-containing region was observed in the TCGA-set.In conclusion, recurrent CNVs were detected in both datasets, occurring in regions harboring known MPM-associated genes and genes not previously linked to MPM.

Genome-wide copy number analysis reveals candidate gene loci that confer susceptibility to high-grade prostate cancer

BackgroundTwo key issues in prostate cancer (PCa) that demand attention currently are the need for a more precise and minimally invasive screening test owing to the inaccuracy of prostate-specific antigen and differential diagnosis to distinguish advanced vs. indolent cancers. This continues to pose a tremendous challenge in diagnosis and prognosis of PCa and could potentially lead to overdiagnosis and overtreatment complications. Copy number variations (CNVs) in the human genome have been linked to various carcinomas including PCa. Detection of these variants may improve clinical treatment as well as an understanding of the pathobiology underlying this complex disease. MethodsTo this end, we undertook a pilot genome-wide CNV analysis approach in 36 subjects (18 patients with high-grade PCa and 18 controls that were matched by age and ethnicity) in search of more accurate biomarkers that could potentially explain susceptibility toward high-grade PCa. We conducted this study using the array comparative genomic hybridization technique. Array results were validated in 92 independent samples (46 high-grade PCa, 23 benign prostatic hyperplasia, and 23 healthy controls) using polymerase chain reaction–based copy number counting method. ResultsA total of 314 CNV regions were found to be unique to PCa subjects in this cohort (P<0.05). A log2 ratio-based copy number analysis revealed 5 putative rare or novel CNV loci or both associated with susceptibility to PCa. The CNV gain regions were 1q21.3, 15q15, 7p12.1, and a novel CNV in PCa 12q23.1, harboring ARNT, THBS1, SLC5A8, and DDC genes that are crucial in the p53 and cancer pathways. A CNV loss and deletion event was observed at 8p11.21, which contains the SFRP1 gene from the Wnt signaling pathway. Cross-comparison analysis with genes associated to PCa revealed significant CNVs involved in biological processes that elicit cancer pathogenesis via cytokine production and endothelial cell proliferation. ConclusionIn conclusion, we postulated that the CNVs identified in this study could provide an insight into the development of advanced PCa.

Medical researchers unite for study on cancer intervention

Medical researchers unite for study on cancer intervention

Application of array-based comparative genomic hybridization technique in genetic analysis of patients with spontaneous abortion

To investigate the value of array-based comparative genomic hybridization (array-CGH) technique for the detection of chromosomal analysis of miscarried embryo, and to provide genetic counseling for couples with spontaneous abortion. Totally 382 patients who underwent miscarriage were enrolled in this study. All aborted tissues were analyzed with conventional cytogenetic karyotyping and array-CGH, respectively. Through genetic analysis, all of the 382 specimens were successfully analyzed by array-CGH (100.0%, 382/382), and the detection rate of chromosomal aberrations was 46.6% (178/382). However, conventional karyotype analysis was successfully performed in 281 cases (73.6%, 281/382), and 113 (40.2%, 113/281) were found with chromosomal aberrations. Of these 178 samples identified by array-CGH, 163 samples (91.6%, 163/178) were aneuploidy, 15 samples (8.4%, 15/178) were segmental deletion and (or) duplication cases. Four of 10 cases with small segmental deletion and duplication were validated to be transferred from their fathers or mathers who were carriers of submicroscopic reciprocal translocation. Of these 113 abnormal karyotypes founded by conventional karyotyping, 108 cases (95.6%, 108/113) were aneuploidy and 5 cases (4.4%, 5/113) had chromosome structural aberrations. Most array-CGH results were consistent with conventional karyotyping but with 3 cases of discrepancy, which included 2 cases of triploids, 1 case of low-level mosaicism that undetcted by array-CGH. Compared with conventional karyotyping, there is an increased detection rate of chromosomal abnormalities when array-CGH is used to analyse the products of conception, primarilly because of its sucess with nonviable tissues. It could be a first-line method to determine the reason of miscarrage with higher accuracy and sensitivity.

Genetic Basis of Posterior Urethral Valves Inheritance

To highlight genetic pattern of posterior urethral valves (PUVs), we performed a genetic study on 2 siblings affected. PUVs are the most common congenital cause of lower urinary tract obstruction and an important cause of renal failure in infants (50% progress to end-stage renal disease in 10 years). PUVs occur in 1 of 5000-8000 male infants, but real incidence is arduous to determine because of the wide spectrum of possible clinical presentation. A different recurrence rate is reported in African Americans and children with Down syndrome, although usually PUVs are not found in syndromic conditions but constitute an isolated disorder. Although most cases appear to be sporadic, some reports in literature suggest a partial genetic etiology. We report 2 brothers with PUVs. The children's mother was a healthy woman but had a history of urinary tract infections of unknown etiology. We investigated possible familial genetic anomalies using a DNA array comparative genomic hybridization technique. We identified 2 partial duplications in the short arm of chromosome 11 recurring in both children and mother. This finding, not previously reported to our knowledge, adds new data to support the hypothesis of the presence of a hereditary component in the occurrences of PUVs.

Low aneuploidy rate in early pregnancy loss abortuses from patients with polycystic ovary syndrome

A prospective cohort study was conducted to determine whether chromosome aneuploidy increases the risk of early spontaneous abortions in patients with polycystic ovary syndrome (PCOS). A total of 1461 patients who conceived after IVF and embryo transfer were followed; 100 patients who had experienced clinical spontaneous abortion were recruited, 32 with PCOS and 68 without PCOS. Before 2013, genetic analysis comprised conventional cultured villus chromosome karyotyping and a multiplex ligation-dependent probe amplification subtelomere assay combined with fluorescence in-situ hybridization; since 2013, array-based comparative genomic hybridization technique combined with chromosome karyotyping has been used. Age, BMI, pregnancy history, gestational age and total gonadotrophin dosage did not differ significantly between the PCOS and non-PCOS groups. In the PCOS group, 28.1% of abortuses demonstrated aneuploidy, which was significantly lower (P = 0.001) than in the non-PCOS group (72.1%). Further statistical analyses controlling for maternal age demonstrated that abortuses of women with PCOS were significantly less (P = 0.001) likely to have chromosome aneuploidy. Embryonic aneuploidy does not play a vital role in early spontaneous abortion in women with PCOS. Maternal factors resulting in endometrial disorders are more likely to be responsible for the increased risk of early spontaneous abortion in patients with PCOS.

Clinical Application of an Innovative Multiplex-Fluorescent-Labeled STRs Assay for Prader-Willi Syndrome and Angelman Syndrome.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurodevelopmental disorders caused by absence of paternally or maternally expressed imprinted genes on chr15q11.2-q13.3. Three mechanisms are known to be involved in the pathogenesis: microdeletions, uniparental disomy (UPD) and imprinting defects. Both disorders are difficult to be definitely diagnosed at early age if no available molecular cytogenetic tests. In this study, we identified 5 AS patients with the maternal deletion and 26 PWS patients with paternal deletion on chr15q11-q13 by using an innovative multiplex-fluorescent-labeled short tandem repeats (STRs) assay based on linkage analysis, and validated by the methylation-specific PCR and array comparative genomic hybridization techniques. More interesting, one of these PWS patients was confirmed as maternal uniparental isodisomy by the STR linkage analysis. The phenotypic and genotypic characteristics of these individuals were also presented. Our results indicate that the new linkage analysis is much faster and easier for large-scale screening deletion and uniparental disomy, thus providing a valuable method for early diagnosis of PWS/AS patients, which is critical for genetic diagnosis, management and improvement of prognosis.

Identification of Unbalanced Aberrations in the Genome of Equine Sarcoid Cells Using CGH Technique

Abstract Sarcoid is the most common skin cancer in horses. The etiology of the tumor is associated with BPV infection (BPV-1, -2, -13), which is an inducer of malignant transformation. The comparative genomic hybridization (CGH) technique identifying the unbalanced chromosome aberrations was used to analyze the genome of equine sarcoid cells and to diagnose the chromosome rearrangements involving large deletions or amplification. The results were based on the analysis of 100 metaphases and their karyograms as well as the diagram showing the average ratio of the intensity of the green to red fluorescence, using MetaSystems software (Isis). Based on a comparison of the fluorescence intensity ratios we found duplication in the subtelomeric regions of chromosome pairs 1, 4, 7, 8 and 23. Duplicated region of chromosome pair 1 also included the coding region of the rDNA. In the chromosome 23 next to the duplication occurring in the centromeric region of q arm (23q11) we also found the presence of deletions involving 23q18-23q19 region. For the chromosome pairs 25 to 31 and the X chromosome the software failed to generate CGH diagram, but on the individual karyograms we were able to observe fluorescence signals characteristic of duplication (red), in rDNA regions of chromosome pairs 28 and 31. The study showed that duplications of DNA present in the sarcoid cells are found mainly in the telomeric and rDNA regions. The presence of the duplication of telomeric regions is associated with increased activity of the telomerase enzyme, which is a hallmark of cancer cells, affecting the immortality of these cells. Accordingly, duplications of rDNA coding regions increase activity of nucleolar organizer region which is a tumor marker.