Chromosome Fluorescence In Situ Hybridization Research Articles

Molecular cytogenetic characterization of 9 populations of four species in the genus Polygonatum (Asparagaceae).

To characterize the chromosomes of the four species of Polygonatum Miller, 1754, used in traditional Chinese medicine, P.cyrtonema Hua, 1892, P.kingianum Collett et Hemsley, 1890, P.odoratum (Miller, 1768) Druce, 1906, and P.sibiricum Redouté, 1811, and have an insight into the karyotype variation of the genus Polygonatum, fluorescence in situ hybridization (FISH) with 5S and 45S rDNA oligonucleotide probes was applied to analyze the karyotypes of 9 populations of the four species. Detailed molecular cytogenetic karyotypes of the 9 populations were established for the first time using the dataset of chromosome measurements and FISH signals of 5S and 45S rDNA. Four karyotype asymmetry indices, CVCI, CVCL, MCA and Stebbins' category, were measured to elucidate the asymmetry of the karyotypes and karyological relationships among species. Comparison of their karyotypes revealed distinct variations in the karyotypic parameters and rDNA patterns among and within species. The basic chromosome numbers detected were x = 9, 11 and 13 for P.cyrtonema, x = 15 for P.kingianum, x = 10 and 11 for P.odoratum, and x = 12 for P.sibiricum. The original basic chromosome numbers of the four species were inferred on the basis of the data of this study and previous reports. All the 9 karyotypes were of moderate asymmetry and composed of metacentric, submetacentric and subtelocentric chromosomes or consisted of two of these types of chromosomes. Seven populations have one locus of 5S rDNA and two loci of 45S rDNA, and two populations added one 5S or 45S locus. The karyological relationships among the four species revealed by comparison of rDNA patterns and PCoA based on x, 2n, TCL, CVCI, MCA and CVCL were basically accordant with the phylogenetic relationships revealed by molecular phylogenetic studies. The mechanisms of both intra- and inter-specific dysploidy in Polygonatum were discussed based on the data of this study and literature.

The value of chromosomal microarray analysis and fluorescence in situ hybridization for the prenatal diagnosis of chromosomal mosaicisms

To assess the value of chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) for the prenatal diagnosis of chromosomal mosaicisms. A total of 775 pregnant women who had visited the Prenatal Diagnosis Center of Yancheng Maternal and Child Health Care Hospital from January 2018 to December 2020 were selected as study subjects. Chromosome karyotyping analysis and CMA were carried out for all women, and FISH was used to validate the suspected mosaicism cases. Among the 775 amniotic fluid samples, karyotyping has identified 13 mosaicism cases, which yielded a detection rate of 1.55%. Respectively, there were 4, 3, 4 and 2 cases for sex chromosome number mosaicisms, abnormal sex chromosome structure mosaicisms, abnormal autosomal number mosaicisms and abnormal autosomal structure mosaicisms. CMA has only detected only 6 of the 13 cases. Among 3 cases verified by FISH, 2 cases were consistent with the karyotyping and CMA results, and clearly showed low proportion mosaicism, and 1 case was consistent with the result of karyotyping but with a normal result by CMA. Eight pregnant women had chosen to terminate the pregnancy (5 with sex chromosome mosaicisms and 3 with autosomal mosaicisms). For fetuses suspected for chromosomal mosaicisms, CMA, FISH and G-banding karyotyping should be combined to determine the type and proportion of mosaicisms more precisely in order to provide more information for genetic counseling.

Contribution of amniocentesis in fetuses small for gestational age without other sonographic signs.

Our study evaluated the contribution of amniocentesis in the diagnostic approach of small-for-gestational-age fetuses (SGA) without morphological abnormality identified on ultrasound by studying FISH (fluorescence in situ hybridization) for chromosomes 13, 18 and 21, CMV PCR (cytomegalovirus polymerase chain reaction), karyotype and CGH (genomic hybridization array) METHODS: Our single-center retrospective cohort study included pregnant women referred for prenatal diagnosis for a SGA fetus in whom amniocentesis was performed between 2016 and 2019. A SGA fetus was defined as a fetus with an estimated fetal weight (EFW) below the 10th percentile according to referral growth curves in use. We evaluated the number of amniocenteses with an abnormal result and identified factors that may be associated with this outcome. Among the 79 amniocenteses performed, there were 5 (6.3%) abnormalities: karyotype (1.3%) and CGH (5.1%). No complications were described. We did not find any statistically significant factors associated with abnormal amniocentesis findings even if some elements seemed reassuring such as late discovery (p=0.31), moderate SGA (p=0.18), normal head, abdomen and femur measurements (p=0.57), but without statistically significant difference. Our study found 6.3% pathological analysis of amniocenteses, of which several would have been missed by conventional karyotyping. Patients must be informed about the risk of detecting abnormalities of low severity, with low penetrance or with unknown fetal consequences that could be source of anxiety.

Comparison of detection rates of chromosome G-banding karyotype analysis and fluorescence in situ hybridization among children with sex chromosome mosaicisms

To explore the coincidence rate of G-banding karyotype analysis and fluorescence in situ hybridization (FISH) for the diagnosis of children with sex chromosome mosaicisms. A retrospective analysis was carried out for 157 children with suspected sex chromosome abnormalities who had presented at Shenzhen Children's Hospital from April 2021 to May 2022. Interphase sex chromosome FISH and G-banding karyotyping results were collected. The coincidence rate of the two methods in children with sex chromosome mosaicisms was compared. The detection rates of G-banding karyotype analysis and FISH were 26.1% (41/157) and 22.9% (36/157) , respectively (P > 0.05). The results of G-banding karyotype analysis showed that 141 cases (89.8%) were in the sex chromosome homogeneity group, of which only 5 cases (3.5%) were inconsistent with the results of FISH. There were 16 cases (10.2%) in the sex chromosome mosaicism group, of which 11 cases (68.8%) were inconsistent with the results of FISH. There was a statistical difference between the two groups in the coincidence rate of the results of the two methods (P < 0.05). No significant difference was found between G-banding karyotype analysis and FISH in the detection rate of chromosome abnormalities. The coincidence rate in the mosaicism group was lower than that in the homogeneity group, and the difference was statistically significant. The two methods should be combined for clinical diagnosis.

Assessment of Combined Karyotype Analysis and Chromosome Microarray Analysis in Prenatal Diagnosis: A Cohort Study of 3710 Pregnancies.

The current study aimed to compare the characteristics of chromosome abnormalities detected by conventional G-banding karyotyping, chromosome microarray analysis (CMA), or fluorescence in situ hybridization (FISH)/CNVplex analysis and further explore the application value of combined karyotype analysis and CMA in prenatal diagnosis with a larger sample size. From March 2019 to March 2021, 3710 amniocentesis samples were retrospectively collected from women who accepted prenatal diagnosis at 16 to 22 + 6 weeks of pregnancy. The pregnant women underwent karyotype analysis and CMA. In the case of fetal chromosomal mosaicism, FISH or CNVplex analysis was utilized for validation. In total, 3710 G-banding karyotype results and CMA results from invasive prenatal diagnosis were collected. Of these, 201 (5.41%) fetuses with an abnormal karyotype were observed. The CMA analysis showed that the abnormality rate was 9.14% (340/3710). The detection rate of CMA combined with karyotype analysis was 0.35% higher than that of CMA alone and 4.08% higher than that of karyotyping alone. Additionally, 12 cases had abnormal karyotype analysis, despite normal CMA results. To further detect the chromosome mosaicism, we used FISH analysis to correct the karyotype results of case 1. Correspondingly, a total of 157 cases showed abnormal CMA results but normal karyotype analysis. We also found chromosomal mosaicism in 4 cases using CMA. Moreover, CNVplex and CMA demonstrated that representative case 15 was mosaicism for trisomy 2. Conventional G-banding karyotyping and CMA have their own advantages and limitations. A combination of karyotype analysis and CMA can increase the detection rate of chromosome abnormalities and make up for the limitation of signal detection.

Evaluation Criteria for Chromosome Instability Detection by FISH to Predict Malignant Progression in Premalignant Glottic Laryngeal Lesions.

Simple SummaryHead and neck cancer develops from premalignant lesions. The key issue is to recognize potentially harmful precursor lesions. Fluorescence in Situ Hybridization (FISH)-based detection of copy number variations (CNV) of chromosomes 1 and 7 can help to differentiate between low-risk and high-risk lesions. Dual-target FISH for chromosomes 1 and 7 in 87 glottic, laryngeal premalignancies was performed. Lesions were evaluated by (1) establishing the chromosome 7/1 ratio (CR-FISH), based on the absolute number of signals of both chromosomes in 100 nuclei, and (2) by the assessment of the percentage of aberrant nuclei, counted in a total of 100 (PAN-FISH). The latter approach combined with histopathological assessment was the best predictive model for progression. The defined evaluation criteria for FISH diagnostics and the proposed prognostic model may help in clinical decision making on treatment strategies in patients with laryngeal precursor lesions.Background: The definition of objective, clinically applicable evaluation criteria for FISH 1c/7c in laryngeal precursor lesions for the detection of chromosome instability (CI). Copy Number Variations (CNV) for chromosomes 1 and 7 reflect the general ploidy status of premalignant head and neck lesions and can therefore be used as a marker for CI. Methods: We performed dual-target FISH for chromosomes 1 and 7 centromeres on 4 µm formalin-fixed, paraffin-embedded tissue sections of 87 laryngeal premalignancies to detect CNVs. Thirty-five normal head and neck squamous cell samples were used as a control. First, the chromosome 7:1 ratio (CR) was evaluated per lesion. The normal range of CRs (≥0.84 ≤ 1.16) was based on the mean CR +/− 3 x SD found in the normal population. Second, the percentage of aberrant nuclei, harboring > 2 chromosomes of chromosome 1 and/or 7 (PAN), was established (cut-off value for abnormal PAN ≥ 10%). Results: PAN showed a stronger correlation with malignant progression than CR (resp. OR 5.6, p = 0.001 and OR 3.8, p = 0.009). PAN combined with histopathology resulted in a prognostic model with an area under the ROC curve (AUC) of 0.75 (s.e. 0.061, sensitivity 71%, specificity 70%). Conclusions: evaluation criteria for FISH 1c/7c based on PAN ≥ 10% provide the best prognostic information on the risk of malignant progression of premalignant laryngeal lesions as compared with criteria based on the CR. FISH 1c/7c detection can be applied in combination with histopathological assessment.

Analysis of complex chromosomal rearrangements using a combination of current molecular cytogenetic techniques

BackgroundUsing combined fluorescence in situ hybridization (FISH) and high-throughput whole-genome sequencing (WGS) molecular cytogenetic technology, we aim to analyze the junction breakpoints of complex chromosome rearrangements (CCR) that were difficult to identify by conventional karyotyping analysis and further characterize the genetic causes of recurrent spontaneous abortion.ResultsBy leveraging a combination of current molecular techniques, including chromosome karyotype analysis, FISH, and WGS, we comprehensively characterized the extremely complex chromosomal abnormalities in this patient with recurrent spontaneous abortions. Here, we demonstrated that combining these current established molecular techniques is an effective and efficient workflow to identify the structural abnormalities of complex chromosomes and locate the rearrangement of DNA fragments.ConclusionsIn conclusion, leveraging results from multiple molecular and cytogenetic techniques can provide the most comprehensive genetic analysis for genetic etiology research, diagnosis, and genetic counseling for patients with recurrent spontaneous abortion and embryonic abortion.

Karyotypic analysis and isolation of four DNA markers of the scleractinian coral Favitespentagona (Esper, 1795) (Scleractinia, Anthozoa, Cnidaria).

We performed conventional and molecular cytogenetic studies on the Favitespentagona Esper, 1795, a scleractinian coral mostly found along the west coast of Japan. Karyotype analysis of F.pentagona by G-banding revealed a karyogram containing a homogenously staining region (HSR) on chromosome 10 in more than 50% of the examined metaphase spreads. This HSR consisted of sequences from 18S ribosomal RNA (rRNA) genes, as demonstrated by fluorescence in situ hybridization (FISH) and DNA sequencing. We highlighted the development of four chromosomal FISH markers from repetitive genes such as U2 small nuclear RNA linked to 5S rRNA sequence (U2 snRNA-5S), 18S rRNA, histone H3, and uncharacterized gene FP-9X. The chromosomal locations of the U2 snRNA-5S and 18S RNA were on the terminal end of long arm of chromosomes 2 and 10, respectively, while the histone H3 and the uncharacterized gene were located near the centromeres of chromosomes 1 and 9, respectively. These FISH markers will improve the karyotyping of F.pentagona from mitotic preparations which helps in widening our understanding of coral genetic structure and chromosome organization. In addition, these improvements in karyotyping will provide the basis in constructing of chromosome-level genome assembly for F.pentagona.

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.

Characterization of a rare mosaic unbalanced translocation of t(3;12) in a patient with neurodevelopmental disorders

BackgroundUnbalanced translocations may be de novo or inherited from one parent carrying the balanced form and are usually present in all cells. Mosaic unbalanced translocations are extremely rare with a highly variable phenotype depending on the tissue distribution and level of mosaicism. Mosaicism for structural chromosomal abnormalities is clinically challenging for diagnosis and counseling due to the limitation of technical platforms and complex mechanisms, respectively. Here we report a case with a tremendously rare maternally-derived mosaic unbalanced translocation of t(3;12), and we illustrate the unreported complicated mechanism using single nucleotide polymorphism (SNP) array, fluorescence in situ hybridization (FISH), and chromosome analyses.Case presentationAn 18-year-old female with a history of microcephaly, pervasive developmental disorder, intellectual disability, sensory integration disorder, gastroparesis, and hypotonia presented to our genetics clinic. She had negative karyotype by parental report but no other genetic testing performed previously. SNP microarray analysis revealed a complex genotype including 8.4 Mb terminal mosaic duplication on chromosome 3 (3p26.3->3p26.1) with the distal 5.7 Mb involving two parental haplotypes and the proximal 2.7 Mb involving three parental haplotypes, and a 6.1 Mb terminal mosaic deletion on chromosome 12 (12p13.33->12p13.31) with no evidence for a second haplotype. Adjacent to the mosaic deletion is an interstitial mosaic copy-neutral region of homozygosity (1.9 Mb, 12p13.31). The mother of this individual was confirmed by chromosome analysis and FISH that she carries a balanced translocation, t(3;12)(p26.1;p13.31).ConclusionTaken together, the proband, when at the stage of a zygote, likely carried the derivative chromosome 12 from this translocation, and a postzygotic mitotic recombination event occurred between the normal paternal chromosome 12 and maternal derivative chromosome 12 to “correct” the partial 3p trisomy and partial deletion of 12p. To the best of our knowledge, it is the first time to report the mechanism utilizing a combined cytogenetic and cytogenomic approach, and we believe it expands our knowledge of mosaic structural chromosomal disorders and provides new insight into clinical management and genetic counseling.

High Grade B Cell Lymphoma with MYC and BCL2 and/or BCL6 Rearrangements Depict a High Complexity on the Cytogenetic, but Not on the Molecular Genetic Level and Show MYC Mutations As Prognostic Marker

Background: In the revised WHO classification of 2016, cases of large B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements were categorized as “HGBL (high grade B cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements)”, except for cases that fulfill the criteria for a follicular or lymphoblastic lymphoma. A detailed molecular analysis of these cases and comparison of accompanying aberrations has not been available to date.Aims: Evaluation of associated genetic alterations and prognosis in HGBL cases with rearrangement of (i) MYC + BCL2 , (ii) MYC + BCL6 or (iii) MYC + BCL2+BCL6.Patient cohorts and methods: The total cohort comprised 70 cases with MYC and BCL2 and/or BCL6 rearrangements (39 male, 31 female). Cases were selected by detection of the rearrangements by FISH (fluorescence in situ hybridization), cases diagnosed as CLL, follicular or lymphoblastic lymphoma were excluded. Median age was 67 years (range: 44-87 years). All patients were investigated using chromosome banding analysis (CBA) and FISH using probes for MYC , IGH-BCL2 and BCL6 . Additionally, mutation analyses of ATM , BCL2, BIRC3, BRAF, BTK, CBL, CSF3R, CSNK1A1, CXCR4, EGR2, ETNK1, FBXW7, FLT3, ID3, IDH1, IDH2, KIT, KLF2, KRAS, MAP2K1, MYC, MYD88, NFKBIE, NOTCH2, PHF6, PLCG2, POT1, PTPN11, SAMHD1, SETBP1, SF3B1, SRSF2, STAT3, STAT5B, TET2, TP53, U2AF1, UBR5, WT1, XPO1 and ZRSR2 was performed. Variants of unknown significance were excluded from statistical analysis.Results: We identified 33/70 cases with rearrangement of MYC + BCL2 , 10/70 cases with MYC + BCL6 and 27/70 cases with MYC + BCL2 + BCL6 (47%, 14% and 39%, respectively). In the total cohort, CBA revealed a complex karyotype (>3 abnormalities) in 59/70 (84%) of patients, 10/70 (14%) harbored an aberrant but non-complex karyotype, whereas in 2/70 (3%) of cases, CBA analyses failed due to insufficient cell proliferation. Regarding the subgroups, a complex karyotype was observed in 82%, 80% and 89% of cases, respectively (for MYC + BCL2 , MYC + BCL6 and MYC + BCL2 + BCL6 rearrangement). If only aberrations additional to MYC / BCL2 / BCL6 rearrangements were calculated, >3 aberrations were detected in 64% of cases (67%, 70% and 60%, respectively). The most frequent additional aberrations included 13q deletions, +12, gain of 1q and 6q deletions. Median number of additional cytogenetic aberrations was n=8 (range: 0-36). Despite the high cytogenetic complexity, the frequency of TP53 deletions was relatively low (6% in the total cohort), three of them harboured a concomitant TP53 mutation in the other allele. Mutation analyses of 40 genes revealed a median number of n=1 mutations in the total cohort with no differences between the three subgroups. The range of the number of molecular mutations was 0-3 (0 mutations: 31%; 1 mutation: 44%; 2 mutations: 20%; 3 mutations: 5%). Interestingly, out of the 40 genes analysed, mutations were almost exclusively detected in BCL2 (40%), MYC (21%) and TP53 (21%) in the total cohort. Other recurrent mutations were observed for ID3 and PHF6 only (3%, respectively). Cases with MYC+BCL6 rearrangement showed a higher - although not statistically significant - frequency of MYC mutations compared to the other cases (40% vs. 18%), while BCL2 mutations were rarely detected in this subgroup (10% vs. 45%, p=0.033). Of note, mutations in TP53 were found to show a trend towards a lower frequency in cases with a “triple hit” (MYC+BCL2+BCL6 ) in comparison to the “double hit” cases (MYC+BCL2 and MYC+BCL6 ) (11% vs. 28%).Median overall survival (OS) in the total cohort was 61 months. Cases with MYC + BCL2 showed a shorter OS compared to cases with MYC+BCL2+BCL6 rearrangements, although this was not statistically significant (37 vs.61 months). Unfortunately, OS could not be determined for cases with MYC+BCL6 due to the low number of cases. Mutations in MYC, but not in BCL2 or TP53 , showed a negative impact on OS in the total cohort (16 vs. 61 months, p=0.046).Conclusion: 1) Cases with rearrangements in MYC , BCL2 and/or BCL6 depict a high complexity on the cytogenetic, but not on the molecular genetic level. 2) Molecular mutations are almost exclusively detected in BCL2 , MYC and TP53. 3) Cases with MYC+BCL6 rearrangements differ from the other subgroups by showing a higher frequency of MYC mutations and a lower frequency of mutations in BCL2 . 4) Mutations in MYC negatively impact on OS. 5) Thus, our data support the current WHO classification and complement it on a molecular level. DisclosuresStengel:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Study of pregnant women with high risk of fetus abnormalities by routine cytogenetics method (karyotyping) and molecular method (FISH) by using X and Y probs and comparing the advantages and disadvantages of these methods in the northwest of Iran's patients

Background: The health of the fetus during the 9 months of pregnancy is very important for every pregnant couple. Identifying carriers of the genetic diseases and their diagnosis before birth, controls the disease's prevalence and does not impose huge costs on the patient's family and community. This study aimed to evaluate the rapid prenatal diagnosis importance in the chromosomal abnormalities identification. Methods: 50 amniotic fluid samples were studied by karyotyping and fluorescence in situ hybridization (FISH). Karyotyping was performed on me taphase chromosomes to identify all the chromosomal abnormalities and FISH detected chromosomal abnormalities by using X and Y probs, as the rapid method. Results: We identified one cases of Down syndrome (2%), three cases of extension in the polymorphism region of P arms of chromosome 15(15p+) (6%), one cases of extension in the polymorphism region of chromosome 9 (9 qh+) (2%), one case of peristaltic inversion in chromosome Y (2%), one case of XYY mosaic embryo, 46, XY /47, XYY variant (2%) and one case with the extra unknown segment on P arms of chromosome 15 (2%). Conclusion: FISH is a useful method with high sensitivity to provide rapid results for couples who don't have enough time to end their pregnancy legally. In cases of X-linked diseases, it is a reliable method to learn the sex of the fetus. FISH is not able to detect structural anomalies, therefore karyotyping is required for absolute right outcomes of chromosome abnormalities.

Molecular Cytogenetic and Agronomic Characterization of the Similarities and Differences Between Wheat-Leymus mollis Trin. and Wheat-Psathyrostachys huashanica Keng 3Ns (3D) Substitution Lines.

Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) and Leymus mollis Trin. (2n = 4x = 28, NsNsXmXm) are valuable resources for wheat breeding improvement as they share the Ns genome, which contains diverse resistance genes. To explore the behaviors and traits of Ns chromosomes from the two species in wheat background, a series of wheat–P. huashanica and wheat–L. mollis substitution lines were developed. In the present study, line DH109 (F7 progeny of wheat–P. huashanica heptaploid line H8911 × durum wheat Trs-372) and line DM131 (F8 progeny of wheat–L. mollis octoploid line M842 × durum wheat Trs-372) were selected. Cytological observation combined with genomic in situ hybridization experiments showed that DH109 and DM131 each had 20 pairs of wheat chromosomes plus a pair of alien chromosomes (Ns chromosome), and the pair of alien chromosomes showed stable inheritance. Multiple molecular markers and wheat 55K SNP array demonstrated that a pair of wheat 3D chromosome in DH109 and in DM131 was substituted by a pair of P. huashanica 3Ns chromosome and a pair of L. mollis 3Ns chromosome, respectively. Fluorescence in situ hybridization (FISH) analysis confirmed that wheat 3D chromosomes were absent from DH109 and DM131, and chromosomal FISH karyotypes of wheat 3D, P. huashanica 3Ns, and L. mollis 3Ns were different. Moreover, the two lines had many differences in agronomic traits. Comparing with their wheat parents, DH109 expressed superior resistance to powdery mildew and fusarium head blight, whereas DM131 had powdery mildew resistance, longer spike, and more tiller number. Therefore, Ns genome from P. huashanica and L. mollis might have some different effects. The two novel wheat–alien substitution lines provide new ideas and resources for disease resistance and high-yield breeding on further utilization of 3Ns chromosomes of P. huashanica or L. mollis.

Clinical Cytogenetics of the Dog: A Review.

Simple SummaryThe cytogenetic analysis of dogs is mainly focused on the diagnosis of disorders of sex development (DSD) and cancers. Unfortunately, the study of canine chromosomes is a challenging task due a high chromosome number (2n = 78) and the one-arm morphology of all autosomes. For years, the application of conventional cytogenetic techniques, Giemsa staining and G and DAPI (4′,6-diamidino-2-phenylindole) bandings, allowed the identification of sex chromosome aneuploidies and centric fusions. An advanced clinical cytogenetic diagnosis is also needed due to the fact that the dog is a valuable animal model in biomedical research. The application of hybridization methods, such as fluorescence in situ hybridization (FISH) and array comparative genome hybridization (aCGH), facilitated the detection of other chromosomal rearrangements. It can be foreseen that a wide use of modern molecular techniques (e.g., SNP microarray and next generation sequencing) will substantially extend the knowledge on canine chromosome mutations.The dog is an important companion animal and has been recognized as a model in biomedical research. Its karyotype is characterized by a high chromosome number (2n = 78) and by the presence of one-arm autosomes, which are mostly small in size. This makes the dog a difficult subject for cytogenetic studies. However, there are some chromosome abnormalities that can be easily identified, such as sex chromosome aneuploidies, XX/XY leukocyte chimerism, and centric fusions (Robertsonian translocations). Fluorescence in situ hybridization (FISH) with the use of whole-chromosome painting or locus-specific probes has improved our ability to identify and characterize chromosomal abnormalities, including reciprocal translocations. The evaluation of sex chromosome complement is an important diagnostic step in dogs with disorders of sex development (DSD). In such cases, FISH can detect the copy number variants (CNVs) associated with the DSD phenotype. Since cancers are frequently diagnosed in dogs, cytogenetic evaluation of tumors has also been undertaken and specific chromosome mutations for some cancers have been reported. However, the study of meiotic, gamete, and embryo chromosomes is not very advanced. Knowledge of canine genome organization and new molecular tools, such as aCGH (array comparative genome hybridization), SNP (single nucleotide polymorphism) microarray, and ddPCR (droplet digital PCR) allow the identification of chromosomal rearrangements. It is anticipated that the comprehensive use of chromosome banding, FISH, and molecular techniques will substantially improve the diagnosis of chromosome abnormalities in dogs.

Clinical Utility of Targeted Next-Generation Sequencing Assay to Detect Copy Number Variants Associated with Myelodysplastic Syndrome in Myeloid Malignancies

Copy number variants (CNVs) and gene mutations are important for diagnosis and treatment of myeloid malignancies. In a routine clinical setting, somatic gene mutations are detected by targeted next-generation sequencing (NGS) assay, but CNVs are commonly detected by conventional chromosome analysis and fluorescence in situ hybridization (FISH). The aim of this proof-of-principle study was to investigate the feasibility of using targeted NGS to simultaneously detect both somatic mutations and CNVs. Herein, we sequenced 406 consecutive patients with myeloid malignancies by targeted NGS and performed a head-to-head comparison with the results from a myelodysplastic syndrome (MDS) FISH and conventional chromosome analysis to detect CNVs. Among 91 patients with abnormal MDS FISH results, the targeted NGS revealed all 120 CNVs detected by MDS FISH (including -5/5q-, -7/7q-, +8, and 20q-) and 193 extra CNVs detected by conventional chromosome analysis. The targeted NGS achieved 100% concordance with the MDS FISH. The lower limit of detection of MDS CNVs by the targeted NGS was generally 5% variant allele fraction for DNA, based on the lowest percentages of abnormal cells detected by MDS FISH in this study. This proof-of-principle study demonstrated that the targeted NGS assay can simultaneously detect both MDS CNVs and somatic mutations, which can provide a more comprehensive genetic profiling for patients with myeloid malignancies using a single assay in a clinical setting.

Cytogenetic characterization of periodical cicadas (Hemiptera: Cicadidae: Magicicada)

The periodical cicadas of the genus Magicicada Davis, 1925 inhabiting eastern United States are characterized by a long juvenile period of 17 or 13 years and periodical mass emergence of adults. In this genus, only seven species are distinguished and are distributed into three species groups, Decim, Cassini and Decula. We performed comparative cytogenetic study of all seven Magicicada species, and 6 of them were explored for the first time. The techniques used included standard chromosome staining, differential chromosome staining techniques (C-banding, AgNOR-banding and fluorochrome DAPI-staining) and FISH (fluorescence in situ hybridization) with 18S rDNA and (TTAGG)n-telomeric probes. All these species have holokinetic chromosomes, like other Hemiptera. Karyotypes of all species appeared to be remarkably conserved with 2n = 19/20 (18 + X0/XX) (male/female); one very large pair of autosomes and all other chromosomes constituting a decreasing size series; X chromosomes were indistinguishable, at least, at mitotic metaphases. The nucleolus organizer regions (NORs) revealed on one of the medium-sized chromosome pairs in all species and were shown to coincide with the rDNA clusters revealed by FISH. In all species, very small blocks of C-heterochromatin were variously located on chromosomes clearly using DAPI but less distinctly by C-banding. The chromosomes of all the species studied were confirmed to contain the "insect type" motif of telomeres (TTAGG)n.

13q deletion syndrome resulting from balanced chromosomal rearrangement in father: the significance of parental karyotyping

BackgroundRetinoblastoma is a malignancy of the eye in children characterized by biallelic inactivation of the retinoblastoma 1 gene (RB1), located at chromosome 13q14.2. Children with interstitial chromosome 13q deletions that include the RB1 gene show a predisposition to develop retinoblastoma and variable other features. Large 13q deletions with severe clinical phenotype are nearly always the result of a de novo mutation, i.e. the pathogenic alteration is not detected in parents. This results in a low risk for siblings to develop 13q deletion syndrome.ResultHere, we describe a patient with profound muscle hypotonia, severe developmental delay and bilateral retinoblastoma carrying a large deletion in 13q13.3q14 with the size of 16 Mb, involving the RB1 gene. Neither parent showed retinoblastoma, muscle hypotonia or developmental delay. Chromosome analysis and Fluorescence in situ hybridization (FISH) showed a balanced complex chromosomal rearrangement (CCR) between chromosome 12 and 13 [ins(12;13)(q21.2;q12.3q14.3)] and an additional balanced translocation of chromosome 7 and 15 [t(7;15)(q31.2;q25.3)] in the healthy father. Malsegregation of the paternal insertional translocation involving chromosome 12 and 13 resulted in a 13q deletion syndrome of the child [46,XY,ins(12;13)(q21.2;q12.3q14.3)].ConclusionBalanced translocations in parents are a rare cause of de novo RB1 deletions in offspring. This case report emphasizes the need for parental chromosomal analysis and FISH in parents of children diagnosed with 13q deletion syndrome or large RB1 gene deletions to precisely determine the recurrence risk in siblings. Guidelines for genetic testing should be revised accordingly.

Prenatal genetic analysis and differential pregnancy outcomes of two de novo cases showing mosaic isodicentric Y chromosome

BackgroundFetal cells collected from the amniotic fluid of two pregnant women indicated sex chromosome abnormalities. Therefore, we performed G-banded chromosome karyotype analysis, single nucleotide polymorphism array (SNP array), fluorescence in situ hybridization (FISH), and sequence-tagged sites (STS) analysis of the Y chromosome to determine the rare molecular genetics of the two fetuses.Case presentationThe karyotypes of the fetuses from patients 1 and 2 were mos 45,X[92]/46,X,+idic(Y)(q11.21)[8] and mos 45,X[20]/46,X,+idic(Y)(q11.223)[80], respectively. Fetus 1 had a 7.76 Mb deletion in Yq11.222q11.23 and a 15.68 Mb duplication in Yp11.2q11.21. Fetus 2 had 21 Mb of repetitive segments in Yp11.3q11.223. Azoospermia factor (AZF) detection by STS analysis revealed a missing AZFb+c region in fetus 1 and three functional AZF regions in fetus 2. The isodicentric Y chromosome (idic (Y)) in both fetuses arose de novo. The pregnancy of patient 1 was terminated, whereas the fetus of patient 2 was delivered and is now 10 months old with normal appearance and growth.ConclusionA combination of technologies such as chromosome karyotyping, FISH, SNP arrays, and STS analysis of the Y chromosome is important in prenatal diagnosis to reduce birth defect rates and improve the health of the Chinese population.

Rare Double Aneuploidy in Down Syndrome (Down-Klinefelter Syndrome)

Background: The chromosomal aneuploidy described as Cytogenetic condition characterized by abnormality in numbers of the chromosome. Aneuploid patient either trisomy or monosomy, can occur in both sex chromosomes as well as autosome chromosomes. However, double aneuploidies involving both sex and autosome chromosomes relatively a rare phenomenon. In present study, we reported a double aneuploidy (Down-Klinefelter syndrome) in infant from Saudi Arabia. Materials and Methods: In the present investigation, chromosomal analysis (standard chromosomal karyotyping) and fluorescence in situ hybridization (FISH) were performed according to the standard protocols. Results: Here, we report a single affected individual (boy) having Saudi origin, suffering from double chromosomal aneuploidy. The main presenting complaint is the obvious dysmorphic features suggesting Down syndrome. Chromosomal analysis and FISH revealed 48,XXY,+21, show the presence of three copies of chromosome 21, two copies of X chromosome and one copy of Y chromosome chromosomes. Conclusion: Patients with Down syndrome must be tested for other associated sex chromosome aneuploidies. Hence, proper diagnosis is needed for proper management and the cytogenetic tests should be performed as the first diagnostic approach.

Genetic analysis of a child with mental retardation and hypospadia

To carry out genetic testing for a boy presenting with mental retardation and hypoplasia. Conventional karyotyping, fluorescence in situ hybridization (FISH) and single nucleotide polymorphism based array (SNP-array) were used to analyze the boy and his parents. SNP-array has detected a 25.7 Mb microduplication at 2q33.3q36.3 in the boy. Chromosomal karyotyping and FISH analysis indicated that his mother had a karyotype of 46,XX,ish ins(11;2) (p15;q33q36), and that the boy has carried an abnormal chromosome 11 derived from the maternal translocation. The karyotype of the boy was ascertained as 46,XY,ish der(11)ins(11;2) (p15;q33q36)mat. SNP-array combined with G-banding and FISH can delineate the cryptic translocation and is valuable for the assessment of recurrence risk for subsequent pregnancies.