Standard Karyotype Research Articles

Türkiye'ye endemik Squalius cappadocicus Özuluğ and Freyhof, 2011'un sitogenetik özellikleri

In this study, a detailed chromosome analysis of the endemic Cappadocian Chub, Squalius cappadocicus in Melendiz Stream (Aksaray) was performed. The standard Giemsa staining, C-banding (CBG and CB-DAPI), and Ag-NOR technique were applied. The diploid chromosome number was 50; its karyotype formula was 14M + 16Sm + 10St + 10A. Heteromorphic sex chromosomes weren’t detected in the karyotype of the studied specimens. The number of all chromosomal arms (NF) was 90. In the standard C-banded and CB-DAPI karyotype of the species, dark C-bands were observed in the centromeric region of some bi-armed and acrocentric chromosomes, while slightly centromeric or pericentromeric C-bands were detected in some chromosomes. Three different active Ag-NORs, which were hemizygous, were detected in all samples examined. Two of these active NORs were detected in the bi-armed and the other in the acrocentric chromosome short arm. The Ag-NOR number of this species was evaluated as a feature that distinguishes it from other Squalius species in Türkiye.

Comparative cytogenetics of Hemorrhois hippocrepis and Malpolon monspessulanus highlights divergent karyotypes in Colubridae and Psammophiidae (Squamata: Serpentes)

Despite the growing interest in the evolutionary cytogenetics of squamates, chromosomal data are lacking for most taxa. We performed a preliminary molecular taxonomic analysis and a comparative cytogenetic study on Hemorrhois hippocrepis and Malpolon monspessulanus. We used a combination of standard karyotyping, Chromomycin A3/Methyl green staining, C-banding, Ag-NOR staining and NOR-FISH to provide the first karyotype description of H. hippocrepis and a re-description of the karyotype of M. monspessulanus, including chromosome markers, heterochromatin patterns and sex chromosome systems. Our results show that H. hippocrepis has 2 n = 36 chromosomes, with 16 macro- and 20 microchromosomes and NORs on the 6th pair. The 4th pair represents homomorphic (metacentric) ZW sex chromosomes, but the W chromosome is completely heterochromatic. Malpolon monspessulanus has 2 n = 44 chromosomes, with 20 macro- and 24 microchromosomes, NORs on the 6th telocentric pair. The 4th pair represents the sex chromosomes (ZZ/ZW), with a W chromosome smaller than the Z and completely heterochromatic. Comparing our cytogenetic data to those available from the literature, we note the occurrence and distribution of primitive and derived chromosomal characteristics and discuss the chromosome diversification in two snake clades belonging to Colubridae and Psammophiidae, respectively. We highlight that these two families followed different chromosome diversification pathways, characterised by a highly conserved karyotype structure in Colubridae and a higher chromosome variability in Psammophiidae, mostly driven by a progressive reduction of the chromosome number by means of chromosome fusions. We also provide cytotaxonomic insights supporting the distinction between M. monspessulanus and M. insignitus.

Copy number variation analysis in 189 Romanian patients with global developmental delay/intellectual disability

BackgroundDevelopmental delay and intellectual disability represent a common pathology in general population, involving about 3% of the pediatric age population, the genetic etiology being often involved. The aim of this study was to determine the clinically relevant copy number variants in patients diagnosed with global developmental delay/intellectual disability in our population, using the chromosomal microarray analysis.MethodsWe analyzed 189 patients diagnosed with global developmental delay/intellectual disability, presented in Clinical Emergency Hospital for Children, Cluj-Napoca. The patients were completely clinically investigated, including dysmorphic and internal malformations evaluation, psychiatric, neuropsychological and metabolic evaluation, standard karyotyping. Genomic analysis was done using chromosomal microarray analysis.ResultsPathogenic findings (including uniparental disomy) and variants of unknown significance were detected in 53 of 189 patients (28.04%). Pathogenic copy number variants and uniparental disomy were observed in 35 of 189 patients (18.51%). Two patients presented uniparental disomy for chromosome 15, one with clinical phenotype of Prader-Willi syndrome and the other with clinical phenotype with Angelman syndrome. Within the category of pathogenic findings, the recurrent copy number variants were seen in 21 of 35 patients (60%).ConclusionsThe increased percentage of pathogenic structural variants observed in patients with global developmental delay/intellectual disability analyzed by chromosomal microarray technique supports its use in patients with a non-specific phenotype such as these neurodevelopmental disorders. The high percentage of recurrent pathogenic variants between these findings is a finding that support their initial evaluation when a genetic testing algorithm could be a useful option.

Relationship between methylation of promoters of apoptosis genes in blood lymphocytes with the frequency of chromosomal aberrations and the dose of radiation

BACKGROUND: Impaired apoptosis can have serious consequences: the accumulation of mutant cells, the development of teratogenic effects and malignant neoplasms. In this regard, the study of the mechanisms of changes in the activity of apoptosis due to methylation under the influence of long-term irradiation is urgent.
 AIM: The study of the degree of methylation of gene promoters involved in the induction of apoptosis in the personnel of the Siberian Chemical Plant, exposed to long-term technogenic irradiation of ionizing radiation in the course of their professional activities.
 MATERIALS AND METHODS: The study was performed on peripheral blood samples of employees of the Siberian Chemical Plant, with a total dose of external exposure from 100 to 300 mSv. Chromosomal aberrations were detected by standard karyotyping of cultured blood lymphocytes. The degree of gene promoters methylation was determined using MethylScreen technology.
 RESULTS: The degree of gene methylation BIRC2, CASP3, CASP9, CIDEB, CRADD, DAPK1, DFFA, FADD, GADD45A, LTBR, TNFRSF21, TNFRSF25 ranges from 0.31 to 41.75%. A strong negative correlation was found between the degree of methylation of GADD45A (r = 0.7364, р = 0.009) with an increased frequency of aberrant cells, moderate negative correlation GADD45A (r = 0.6347, р = 0.035) with an increased frequency of dicentric chromosomes, moderate negative correlation CASP9 (r = 0.6606, р = 0.026), and strong negative correlation CIDEB (r = 0.7982, р = 0.003) with an increased frequency of chromatid fragments. A moderate negative correlation of the methylation degree of CASP9 (r = 0.6636, р = 0.026), and CIDEB (r = 0.6636, р = 0.026) with the total dose of external exposure was shown.
 CONCLUSIONS: The decrease in the level of apoptosis at doses of 100300 mSv can be explained by the achievement of the demethylation threshold for the promoters of the proapoptotic genes GADD45A, CASP9, CIDEB. This once again testifies in favor of the threshold model of the dependence of the radiation effect on the radiation dose.

Genome-wide differential expression analysis of cell-free microRNAs in amniotic fluid of fetus with Down syndrome

BackgroundThe biological pathways related to the phenotypic development of trisomy 21 (Down syndrome, DS) have not been fully clarified. Cell-free microRNAs (miRNAs) in amniotic fluid are a valuable source of biological information for better understanding the pathogenesis of DS. MethodsIn the present study, small RNA sequencing was used to detect the genome-wide expression profile of cell-free miRNAs in amniotic fluid of standard DS versus normal karyotype (each n = 3). Unsupervised hierarchical clustering, differential expression analysis and KEGG pathway enrichment were performed using bioinformatics online tools. The differentially detected miRNAs were selectively validated by quantitative RT-PCR in another case-control cohort (each n = 6). ResultsA total of 75 miRNAs were detected in the amniotic fluid supernatants of the two study groups. Compared with normal karyotype, 33 miRNAs are significantly differentially expressed in DS, among which 9 are upregulated and 24 are downregulated. There are 329 coding genes predicted to be targeted by the differential miRNAs. The predicted target genes are mainly involved in cancer pathways, Cushing's syndrome, P13K-Akt, MAPK and AMPK signaling pathways, longevity regulating pathway and cellular senescence. Validation assay shows that miR-22-3p is significantly downregulated in the DS group compared to the normal group (Student's t-test, P < 0.05). ConclusionOur results provide new insights into the differential expression of cell-free miRNAs in amniotic fluid of fetus with Down syndrome.

Tracking Chromosomal Origins in the Northern Italy System of Metacentric Races of the House Mouse

The Western European house mouse is chromosomally diverse, with diploid karyotypes ranging from the standard 40 telocentric chromosomes down to 22 chromosomes. Karyotypes are modified through Robertsonian (Rb) fusion of 2 telocentrics into a single metacentric, occurring repeatedly with fixation, and whole-arm reciprocal translocations (WARTs) generating additional novel karyotypes. Over 100 metacentric populations (chromosomal races) have been identified, geographically clustered into “systems.” Chromosomal races within systems often hybridise, and new races may emerge through this hybridisation (“zonal raciation”). We wished to determine the degree to which chromosomal races in a system have evolved independently or share common ancestry. Recombination between chromosomes from hybridising chromosomal races can erase the signals associated with a particular metacentric of interest, making inferences challenging. However, reduced recombination near the centromeres of chromosomal race-specific metacentrics makes centromere-adjacent markers ideal for solving this problem. For the Northern Italy System (NIS), we used microsatellite markers near the centromere to test previous hypotheses about evolutionary relationships of 5 chromosomal races. We chose markers from chromosomes 1, 3, 4, and 6, all of which comprise one arm of a metacentric in at least 2 of these NIS metacentric populations. We used estimates of F_ST and R_ST, as well as principal components analyses and neighbour-joining phylogenetic analyses, to infer evolutionary relationships between these 5 chromosomal races and neighbouring mice with the standard karyotype. We showed that the metacentric populations form a single grouping distinct from the standard populations, consistent with their common origin and consistent with a parsimonious sequence of chromosomal rearrangements to explain the relationship of the chromosomal races. That origin and evolution of the chromosomal races in the system would have involved Rb fusions, explaining the occurrence of chromosomal races with diploid numbers as low as 22. However, WARTs and zonal raciation have also been inferred, and the rare occurrence of chromosome 1 in different metacentrics in closely related chromosomal races is almost certainly explained by a WART. Our results with centromeric microsatellites are consistent with the above scenarios, illustrating, once again, the value of markers in the centromeric region to test evolutionary hypotheses in house mouse chromosomal systems.

Comparative cytogenetics on Zamenis lineatus and Elaphe quatuorlineata (Serpentes: Colubridae)

Because of their peculiar genomic and chromosomal characteristics, reptiles are extraordinary model organisms to study karyotype and sex chromosome evolution, but despite the growing interest in their evolutionary cytogenetics, only a small fraction of species have a known karyotype. We performed a comparative cytogenetic analysis on Elaphe quatuorlineata and Zamenis lineatus, using classic and molecular techniques. We provide the karyotype of these two species and an assessment of their chromosomal features. Chromosome analysis was performed with standard karyotyping, C-banding, sequential C-banding + CMA3 + DAPI and Ag-NOR staining. On E. quatuorlineata, we also performed CMA3-methyl green staining and Fluorescence in situ Hybridization mapping NOR loci (NOR-FISH). Elaphe quatuorlineata and Z. lineatus show a very similar karyotype of 2n = 36, with 8 macro- and 10 microchromosome pairs, but differ in the morphology of the pair 8, which resulted submetacentric in the former and metacentric in the latter species. By comparing our data to those available from the literature on congeneric species, we analysed the occurrence of primitive and derivate chromosomal characters and provide cytotaxonomic insights, which further support the species status of Z. lineatus. In both species, the 4th pair was identified as the sex chromosome pair (ZZ/ZW) and NORs were localized on a microchromosome pair. We finally highlight in both genera Elaphe and Zamenis different stages of heterochromatinization of the W chromosome, in agreement with the progressive diversification model of sex chromosome as already shown in different reptile taxa.

110. Discovery and characterization of novel fusions in Pediatric B-Lymphoblastic Leukemia (B-ALL)

Newly discovered genetic drivers in B-lymphoblastic leukemia (B-ALL) frequently remain undetected by standard karyotype and FISH analyses. At our institution, comprehensive evaluation by karyotyping, FISH, chromosomal microarray and a custom sequencing panel (OncoKids®), identifies a primary driver in 85% of B-ALL cases. In this study we utilized a novel capture-based transcriptome sequencing (RNA-Seq) method and optical genome mapping (OGM) to detect driver fusions in the remaining 15% of B-ALL subtypes. OGM and RNASeq were performed on 19 and 53 leukemia cases, respectively, with undefined genetic drivers. Subtype-defining abnormalities were detected by RNAseq in 16/53 cases (30%). Twelve of the 16 demonstrated previously described genetic drivers, while 4 cases showed novel fusions at the time of testing. These included an RBM26::JAK2 fusion identified in a 1-year old with standard risk (SR) B-ALL, a STRBP::JAK2 fusion in a 23-year old with high-risk B-ALL; a PAX5::HIPK3 fusion in a 5-year old with SR B-ALL, and an ETV6::MFN2 fusion in a 2-year old with SR B-ALL. OGM demonstrated that the RBM26::JAK2, PAX5::HIPK3 and ETV6::MFN2 fusions originated from translocations, and the STRBP::JAK2 fusion from an inversion. Expression profiling showed a Philadelphia-like signature in the RBM26::JAK2 and STRBP::JAK2 cases, a PAX5Alt signature in the PAX5::HIPK3 case and an ETV6::RUNX1-like signature in the ETV6::MFN2 case. This study demonstrates clinical utility and discovery potential of RNAseq and OGM in B-ALL classification. Considering the genetic heterogeneity and high discovery rate of novel drivers, non-targeted genome and transcriptome interrogation may be superior to panel testing for clinical B-ALL evaluation.

Congenital hypopituitarism and multiple midline defects in a newborn with non-familial Cat Eye syndrome

BackgroundCat eye syndrome (CES) is a rare chromosomal disease, with estimated incidence of about 1 in 100,000 live newborns. The classic triad of iris coloboma, anorectal malformations, and auricular abnormalities is present in 40% of patients, and other congenital defects may also be observed. The typical associated cytogenetic anomaly relies on an extra chromosome, derived from an inverted duplication of short arm and proximal long arm of chromosome 22, resulting in partial trisomy or tetrasomy of such regions (inv dup 22pter-22q11.2).Case presentationWe report on a full-term newborn, referred to us soon after birth. Physical examination showed facial dysmorphisms, including hypertelorism, down slanted palpebral fissures, and dysplastic ears with tragus hypoplasia and pre-auricular pit. Ophthalmologic evaluation and heart ultrasound identified left chorioretinal and iris coloboma and ostium secundum type atrial septal defect, respectively. Based on the suspicion of cat eye syndrome, a standard karyotype analysis was performed, and detected an extra small marker chromosome confirming the CES diagnosis. The chromosomal abnormality was then defined by array comparative genome hybridization (a-CGH, performed also in the parents), which identified the size of the rearrangement (3 Mb), and its de novo occurrence. Postnatally, our newborn presented with persistent hypoglycemia and cholestatic jaundice. Endocrine tests revealed congenital hypothyroidism, cortisol and growth hormone (GH) deficiencies, which were treated with replacement therapies (levotiroxine and hydrocortisone). Brain magnetic resonance imaging, later performed, showed aplasia of the anterior pituitary gland, agenesis of the stalk and ectopic neurohypophysis, confirming the congenital hypopituitarism diagnosis. She was discharged at 2 months of age, and included in a multidisciplinary follow-up. She currently is 7 months old and shows a severe global growth failure, and developmental delay. She started GH replacement treatment, and continues oral hydrocortisone, along with ursodeoxycholic acid and levothyroxine, allowing an adequate control of glycemic and thyroid profiles as well as of cholestasis.ConclusionsCES phenotypic spectrum is wide and highly variable. Our report highlights how among the possible associated endocrine disorders, congenital hypopituitarism may occur, leading to persistent hypoglycemia and cholestasis. These patients should be promptly assessed for complete hormonal evaluations, in addition to major malformations and midline anomalies. Early recognition of such defects is necessary to decrease fatal events, as well as short and long-term related adverse outcomes.

First Insights on the Karyotype Diversification of the Endemic Malagasy Leaf-Toed Geckos (Squamata: Gekkonidae: Uroplatus).

Simple SummaryThe geckos of the genus Uroplatus include peculiar endemic species to Madagascar. Even though they have been the subject of several morphological and molecular studies, karyological analyses have been performed only on U. phantasticus, leaving the chromosomal diversity of the genus completely unexplored. In this study, we performed a preliminary molecular analysis and a comparative cytogenetic study providing the first karyotype description of eight species of Uroplatus and an assessment of their karyological variability. We found chromosome diversity in the species studied in terms of total chromosome number (2n = 34–38), localization of loci of Nucleolar Organizer Regions (NORs) (alternatively on the 2nd, 6th, 10th or 16th pair), heterochromatin composition and occurrence of heteromorphic sex chromosome pairs. Adding our newly generated data to those available from the literature, we show that in the genus Uroplatus, as well as in a larger group of phylogenetically related gecko genera, chromosome diversification mainly occurred toward a reduction in the chromosome number by means of chromosome fusions and translocation of NOR-bearing chromosomes. We also hypothesize that the diversification of sex chromosome systems occurred independently in different genera.We provide here the first karyotype description of eight Uroplatus species and a characterization of their chromosomal diversity. We performed a molecular taxonomic assessment of several Uroplatus samples using the mitochondrial 12S marker and a comparative cytogenetic analysis with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, +Chromomycin A3 (CMA3), +4′,6-diamidino-2-phenylindole (DAPI). We found chromosomal variability in terms of chromosome number (2n = 34–38), heterochromatin composition and number and localization of loci or Nucleolar Organizer Regions (NORs) (alternatively on the 2nd, 6th, 10th or 16th pair). Chromosome morphology is almost constant, with karyotypes composed of acrocentric chromosomes, gradually decreasing in length. C-banding evidenced a general low content of heterochromatin, mostly localized on pericentromeric and telomeric regions. Centromeric bands varied among the species studied, resulting in CMA3 positive and DAPI negative or positive to both fluorochromes. We also provide evidence of a first putative heteromorphic sex chromosome system in the genus. In fact, in U. alluaudi the 10th pair was highly heteromorphic, with a metacentric, largely heterochromatic W chromosome, which was much bigger than the Z. We propose an evolutionary scenario of chromosome reduction from 2n = 38 to 2n = 34, by means of translocations of microchromosomes on larger chromosomes (often involving the NOR-bearing microchromosomes). Adding our data to those available from the literature, we show that similar processes characterized the evolutionary radiation of a larger gecko clade. Finally, we hypothesize that sex chromosome diversification occurred independently in different genera.

PB1801: CHROMOSOMAL ABNORMALITIES DURING THE CLINICAL COURSE OF ADULT ACUTE MYELOID LEUKEMIA

Background: Using conventional cytogenetics, an abnormal karyotype can be detected in 60-70% of adult acute myeloid leukemia (AML) patients. Taking into consideration the identified cytogenetic abnormalities AML patients are currently classified into three risk groups: favorable, intermediate and adverse. These genetic abnormalities are the most important factors in determining response to chemotherapy as well as outcome in AML. Aims: Aim of this study was to detect the diagnostic and prognostic significance of chromosomal abnormalities in leukemic cells at different phases of AML. Methods: Cytogenetic investigations of bone marrow and/or peripheral blood cells from 140 adult patients were performed during clinical evolution of AML: in newly diagnosed patients, in remission and at relapse. The methods of conventional cytogenetics (GTG) and fluorescence in situ hybridization (FISH) were used. Cytogenetic methods were performed using standard techniques and karyotypes were described according to the International System for Human Cytogenetic Nomenclature. Results: Cytogenetic investigations of 140 adult AML patients were performed in newly diagnosed patients, in remission and at relapse. Structural (del(5q), del(7q), rearrangements of 3q, 12p, 17p and 11q23, t(8;21)(q22;q22), t(9;22)(q34;q11), t(15;17)(q22;q21), t(16;16)(p13;q22), inv(16)(p13q22), marker and ring chromosomes, acentric structures and others) and numerical (-5, -7, -Y, +8, monosomal karyotype and others) chromosomal abnormalities in leukemic cells were found. Some genetic abnormalities (BCR/ABL1, PML/RARa, AML1/ETO, CBFβ/MYH11 fusion genes) were detected by molecular genetic methods (FISH). Spectrum of cytogenetic abnormalities had an important diagnostic and prognostic significance. Diagnosis of AML was possible due to the presence of specific genetic markers that can confirm some types of AML (AML with t(8;21)(q22;q22), t(9;22)(q34;q11), t(15;17)(q22;q21), t(16;16)(p13;q22)/inv(16)(p13q22), 3q21q26 rearrangements, BCR/ABL1, PML/RARa, AML1/ETO, CBFβ/MYH11 fusion genes). Taking into consideration the identified cytogenetic abnormalities AML patients were classified by risk groups: the group of patients with adverse cytogenetic markers (3q21q26 rearrangements, -5, -7, t(9;22)(q34;q11), BCR/ABL1 fusion gene, multiple changes (≥3), monosomal karyotype), the intermediate risk group without significant prognostic markers and the group with favorable prognostic factors (t(8;21)(q22;q22), t(15;17)(q22;q21), t(16;16)(p13;q22)/inv(16)(p13q22), AML1/ETO, PML/RARa, CBFβ/MYH11 fusion genes). In remission cytogenetic investigations showed a normal karyotype without cytogenetically visible changes in all patients. Cytogenetic investigations at relapse were performed in 5 of 140 AML cases. Additional/secondary chromosomal aberrations in leukemic cells or new cells clones with multiple structural and numerical changes at relapse was not detected. Summary/Conclusion: Cytogenetic investigations had an important significance for diagnosis, prognosis and selection the optimal treatment strategy of AML. Besides the analysis of chromosome banding patterns it is necessary for patients with AML to apply molecular genetic studies, namely FISH and polymerase chain reaction (PCR). Taking into consideration the identified cytogenetic abnormalities AML patients were classified by risk groups. Presence of the additional/secondary chromosomal aberrations in leukemic cells or new cells clones with multiple structural and numerical changes at AML relapse is the one sign of clonal evolution and progression of disease.

P456: A LONG READ SEQUENCING AND CRISPR-CAS9 BASED APPROACH FOR RAPID COPY-NUMBER ALTERATION AND STRUCTURAL VARIATION DETECTION IN HEMATOLOGIC MALIGNANCIES

Background: Current standard karyotyping methods are labor-intensive and have severe limitations in terms of resolution and duration to results. For hematologic malignancies, but not restricted to, this represents a relevant obstacle in translating molecular findings into time critical treatment decisions. Aims: To develop a sequencing based approach for rapid and scalable cytogenetics that can be applied to a wide spectrum of hematologic malignancies to detect clinically relevant molecular markers within 48hrs Methods: For copy number alteration detection, whole genome sequencing was performed on a GridION sequencer (Oxford Nanopore Technologies, ONT, using the SQK-LSK109 kit) in a cohort of 53 hematologic neoplasms, including acute myeloid leukemia (AML, n=18), chronic lymphocytic leukemia (CLL, n=8), multiple myeloma (MM, n=17) and pediatric acute lymphocytic leukemia (ALL, n=10) samples. For the identification of balanced alterations, transcriptome sequencing data were generated using ONT (SQK-DCS109 kit), and we applied an in-house developed analysis pipeline based on minimap2 alignment followed by Blast with an ensuing filtering algorithm based on orientation, inter-read distance and length filtering. For targeted sequencing of structural aberrations, we developed a CRISPR-Cas9 based tiling approach using sgRNA pools (Integrated DNA Technologies, IDT). This approach allows to cover AML relevant targets such as t(8;21), the KMT2A (MLL) breakpoint area, and the FLT3-ITD region as well as lymphoma relevant regions such as the immunoglobulin heavy chain locus (library preparation was performed using SQK-CS9109 kit). Results: For copy number profiling a median whole-genome coverage of 2.6 was reached (range 0.27-7 fold). ONT sequencing and conventional karyotyping approaches showed a high concordance with Pearson correlation coefficients >0.95 for copy number alteration comparisons between conventional cytogenetics and ONT sequencing results for all investigated disease entities. Regarding the detection of structural aberrations transcriptome sequencing and fusion gene analysis using the above described analysis and filtering pipeline we could e.g. reliably detect the t(9;22) translocation from the K562 cell line and the t(8;21) translocation from Kasumi-1. Additionally, using this method we were able to correctly identify the primary AML sample harboring a t(8;21) translocation that was among the n=12 AML patient samples. In addition to an RNA-based work-flow, we established a CRISPR-Cas9 based DNA enrichment approach for the detection of recurrent structural aberrations from specified genomic DNA loci. The enrichment of genomic regions of interest using sgRNA libraries led e.g. to a median coverage of 44 reads (range 5-136-fold) of the t(8;21) region-of-interest in primary AML samples, which allow the detection of the exact coordinates of the breakpoints. Summary/Conclusion: Long read based sequencing based copy number alteration and structural variation detection based on combination of different long read sequencing approaches like low-coverage whole genome sequencing, transcriptome sequencing and CRISPR-Cas9 based sequencing of genomic loci of interest represents a highly promising tool for high resolution and high speed cytogenetics that has the potential to overcome many limitations of conventional cytogenetics.

Ring chromosome 9 and chromosome 9p deletion associated with neurological and malformative syndrome

Ring chromosome 9 is a rare chromosomal abnormality. The severity and nature of symptoms vary according to the structural abnormalities. Here, we report a case of a patient with neurological and malformative syndrome caused by a ring of chromosome 9 associated with partial 9p deletion. The 1-year-old child had a general hypotonia at birth regressed to peripheral hypotonia and normal axial tonus, psychomotor retardation, weight retardation, microcephaly and trigonocephaly, facial dysmorphia, flattened neck, abnormal nipple spread, dorsal kyphosis, white line hernia, ectopic testis and cardiac malformation cured at the age of 1 year, no other signs detectable. The standard constitutional karyotype revealed a ring of chromosome 9 with a deletion of the region pter-22p: 46,XY,r(9)(p22;q34). The chromosomal abnormality was homogeneous. The genetic counseling remains incomplete due to the absence of the parents’ karyotype. The cardinal traits reported in the ring chromosome 9 and chromosome 9p deletion syndrome were observed in our patient such as hypotonia, psychomotor retardation, microcephaly and trigonocephaly. Indeed, the deleted region contains numerous genes involved in the neurological manifestations and cranial dysmorphia including: CER1, FREM1, NFIB, GLDC, VLDLR, FOXD4, SMARCA2 and KANK1. Microarray analysis is important in order to detect the deleted segment and thereby the deleted genes.&#x0D; The ring chromosome 9 is characterized by a genotype heterogeneity and a phenotype variability that can be due to multiple factors: loss or gain of genetic material, mitotic disruption, ring chromosome instability. Genotype-phenotype correlation could provide reliable information for genetic diagnosis, genetic counseling and clinical management.&#x0D; &#x0D; Keywords: Chromosome 9, Ring, Deletion, Phenotype, Genotype.

Oligo-FISH Paints in Triticeae.

We developed seven oligonucleotide (oligo) pools based on single-copy sequences, targeting chromosomes 1 to 7 of barley (Hordeum vulgare L.) and wheat (Triticum aestivum) for chromosomal Oligo-FISH painting methods. The probes were applied to high-throughput karyotyping for the Triticeae tribe of over 350 species including 30 genera such as Triticum, Hordeum, Secale, Aegilops, Thinopyrum, and Dasypyrum, as well as several wheat alien-derived lines. In combination with other nondenaturing FISH (ND-FISH) procedures using tandem-repeat oligos, the newly developed Oligo-FISH painting technique provides an efficient tool for the identification of individual chromosomes with homologous linkage groups to establish standard karyotypes, particularly with any wild Triticeae species having nonsequenced genomes for chromosome evolutionary analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Oligo-pool probe development Basic Protocol 2: Nondenaturing FISH Basic Protocol 3: Oligo-FISH painting.

A review chromosome analysis comparison of swamp buffalo (Bubalus bubalis) with different phenotypic characters in East Java, Indonesia

Chromosomal data on swamp buffalo especially in East Java province, is still lacking. For breeders, chromosomal analysis needs to be done to detect the possibility of hereditary genetic defects. In Malang Area, farmers lack of care and breeding programs for swamp buffalo also caused a decline in the genetic quality and performance. The purpose of this study was to compare the results of the chromosome analysis of swamp buffalo in Malang and another area in East Java province, which were selected based on differences in phenotypic characters. Chromosome analysis is very important for breeders because the results of this study can be used as a consideration for strategies to improve the genetic quality of swamp buffalo. The method used is standard karyotyping using whole blood with G banding staining. Chromosomal preparation using Karyo MAX medium, Colcemic Solution, Giemsa Stain, and KCl solution. Cell culture was performed according to the standard karyotyping method in mammals. The minimum number of 5 spreading chromosomes is the best was chosen, microphotographed, and then chromosome analysis is performed using Cytovision Image Analysis software. The results of this study did not find swamp buffalo with an abnormal number of chromosomes in East Java Province, Indonesia.

Measurement of Chromosomal Arms and FISH Reveal Complex Genome Architecture and Standardized Karyotype of Model Fish, Genus Carassius.

The widely distributed ray-finned fish genus Carassius is very well known due to its unique biological characteristics such as polyploidy, clonality, and/or interspecies hybridization. These biological characteristics have enabled Carassius species to be successfully widespread over relatively short period of evolutionary time. Therefore, this fish model deserves to be the center of attention in the research field. Some studies have already described the Carassius karyotype, but results are inconsistent in the number of morphological categories for individual chromosomes. We investigated three focal species: Carassius auratus, C. carassius and C. gibelio with the aim to describe their standardized diploid karyotypes, and to study their evolutionary relationships using cytogenetic tools. We measured length ( ) of each chromosome and calculated centromeric index (i value). We found: (i) The relationship between and i value showed higher similarity of C. auratus and C. carassius. (ii) The variability of i value within each chromosome expressed by means of the first quartile () up to the third quartile () showed higher similarity of C. carassius and C. gibelio. (iii) The fluorescent in situ hybridization (FISH) analysis revealed higher similarity of C. auratus and C. gibelio. (iv) Standardized karyotype formula described using value () showed differentiation among all investigated species: C. auratus had 24 metacentric (m), 40 submetacentric (), 2 subtelocentric (), 2 acrocentric (a) and 32 telocentric (T) chromosomes (); C. carassius: ; and C. gibelio: . (v) We developed R scripts applicable for the description of standardized karyotype for any other species. The diverse results indicated unprecedented complex genomic and chromosomal architecture in the genus Carassius probably influenced by its unique biological characteristics which make the study of evolutionary relationships more difficult than it has been originally postulated.

Chromosome Diversity and Evolution in Helicoide a (Gastropoda: Stylommatophora): A Synthesis from Original and Literature Data.

Simple SummaryThe superfamily Helicoidea is a large and diverse group of Eupulmonata. The superfamily has been the subject of several molecular and phylogenetic studies which greatly improved our knowledge on the evolutionary relationships and historical biogeography of many families. In contrast, the available karyological information on Helicoidea still results in an obscure general picture, lacking a homogeneous methodological approach and a consistent taxonomic record. Nevertheless, the available karyological information highlights the occurrence of a significant chromosomal diversity in the superfamily in terms of chromosome number (varying from 2n = 40 to 2n = 62), chromosome morphology and the distribution of different karyological features among different taxonomic groups. Here we performed a molecular and a comparative cytogenetic analysis on of 15 Helicoidea species of three different families. Furthermore, to provide an updated assessment of the chromosomal diversity of the superfamily we reviewed all the available chromosome data. Finally, superimposing all the chromosome data gathered from different sources on the available phylogenetic relationships of the studied taxa, we discuss the overall observed chromosome diversity in Helicoidea and advance a hypothesis on its chromosomal evolution.We performed a molecular and a comparative cytogenetic analysis on different Helicoidea species and a review of all the available chromosome data on the superfamily to provide an updated assessment of its karyological diversity. Standard karyotyping, banding techniques, and Fluorescence in situ hybridization of Nucleolus Organizer Region loci (NOR-FISH) were performed on fifteen species of three families: two Geomitridae, four Hygromiidae and nine Helicidae. The karyotypes of the studied species varied from 2n = 44 to 2n = 60, highlighting a high karyological diversity. NORs were on a single chromosome pair in Cernuella virgata and on multiple pairs in four Helicidae, representing ancestral and derived conditions, respectively. Heterochromatic C-bands were found on pericentromeric regions of few chromosomes, being Q- and 4′,6-diamidino-2-phenylindole (DAPI) negative. NOR-associated heterochromatin was C-banding and chromomycin A3 (CMA3) positive. Considering the available karyological evidence on Helicoidea and superimposing the chromosome data gathered from different sources on available phylogenetic inferences, we describe a karyotype of 2n = 60 with all biarmed elements as the ancestral state in the superfamily. From this condition, an accumulation of chromosome translocations led to karyotypes with a lower chromosome number (2n = 50–44). This process occurred independently in different lineages, while an augment of the chromosome number was detectable in Polygyridae. Chromosome inversions were also relevant chromosome rearrangements in Helicoidea, leading to the formation of telocentric elements in karyotypes with a relatively low chromosome count.

High Mobility Group A1 Chromatin Remodeling Proteins: Potential Therapeutic Targets Involved in Polycythemia Vera Transformation to Acute Leukemia in Humans and JAK2 V617F Transgenic Mouse Models

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although the mechanisms driving disease progression remain elusive. Polycythemia vera (PV) is the most common clinical subtype caused by acquired mutations of JAK2. While PV generally presents as an indolent process, ~25% of patients will progress to MF or AML and JAK2 mutations alone do not account for MPN transformation.HMGA1 / 2 genes encode oncogenic chromatin binding proteins that are overexpressed in acute leukemia and drive clonal expansion in murine leukemia models. In preliminary studies, we found up-regulation in gene expression of both HMGA1/2 in HSC with disease progression in MPN. We therefore sought to test the hypothesis that HMGA proteins are critical drivers of transformation and rational therapeutic targets to treat or prevent PV progression.Methods: We examined clonal evolution in HSC from 49 JAK2V617F -positive PV patients using standard and SNP-array karyotyping and a targeted resequencing panel of 163 genes linked to myeloid cancers. HSC clonal burden was assessed via JAK2V617F genotype analysis on single cells. We also measured HMGA1 / 2 in JAK2V617F -positive human AML cell lines from MPN patients (PV-AML cells), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F / MPLSV ; Blood 2015;126:484) using real-time quantitative RT-PCR. To elucidate the role of HMGA1/2, we silenced HMGA1 or HMGA2 via short hairpin RNA and assessed proliferation in human PV-AML cells. Finally, to investigate Hmga1 function in vivo, we crossed mice deficient in Hmga1 onto murine models of PV and PV-AML. We also compared leukemic engraftment in JAK2V617F -positive PV-AML cells with or without HMGA1/2 silencing.Results: Both HMGA1/2 mRNA were up-regulated in all JAK2V617F -positive contexts. In primary human PV CD34+ cells, HMGA1 and HMGA2 were increased by 7- and 100-fold, respectively, compared to controls. There was a dramatic up-regulation in both HMGA1/2 in HSC of patients who transformed from PV to MF or AML compared to chronic phase PV when analyzed in cross-sectional samples from different patients or prospectively in selected patients. Overexpression of HMGA1 /2 also correlated with clonal dominance of JAK2V617F -homozygous stem cells, and additional mutations of epigenetic regulators, including EZH2 and SETBP1 . Similarly, when assessed in unfractionated bone marrow or tumor samples in transgenic mouse models for PV and PV-AML, Hmga1 / 2 were overexpressed compared to control littermates, with highest levels in the PV-AML mouse model. Silencing HMGA1 or HMGA2 in human PV-AML cells (DAMI, SET-2) dramatically halted proliferation. Moreover, leukemic engraftment was prevented in PV-AML cells after silencing HMGA1 or HMGA2. In preliminary studies with the transgenic PV-AML murine model, Hmga1 deficiency ameliorates the fulminant phenotype of aggressive leukemia and early mortality. PV-AML mice survived a median of 5 weeks (range 3-10; n=40) whereas PV-AML in Hmga1 deficient backgrounds survived a median of 8 weeks (range 7-16; n=7). The leukemic burden was decreased in mice with Hmga1 deficiency.Conclusions:HMGA1 and HMGA2 are overexpressed in MPN, and higher levels associate with disease progression to MF and AML, in both human PV and in transgenic murine models of PV. Strikingly, silencing HMGA1 or HMGA2 halted proliferation and prevented leukemic engraftment in vivo . Preliminary data in Hmga1 deficient models suggest that Hmga1 is required for PV progression to AML. These data highlight a key role for HMGA proteins as critical drivers of PV transformation and suggest that HMGA1 /2 overexpression is a consequence of aberrant JAK/STAT signaling and epigenetic dysregulation. Our findings indicate that HMGA1 /2 overexpression may function as a necessary molecular switch for PV leukemic transformation. Therefore, HMGA proteins and their transcriptional pathways offer novel therapeutic targets aimed at the prevention of PV progression to MF and AML. DisclosuresNo relevant conflicts of interest to declare.

Molecular Profiling in Extramedullary Acute Myeloid Leukemia Reveals Distinct Subgroups Defined By Mutations in KIT and RUNX1

Background: Between 2 to 23% of patients with acute myeloid leukemia manifest extramedullary involvement including myeloid sarcoma. While the genomic landscape of AML and the respective clinical and outcome correlations are well characterized, our understanding of the molecular underpinnings of extramedullary AML remains limited. Various cytogenetic abnormalities have been described in association with extramedullary AML, including core binding factor (CBF) translocations t(8;21) and inv(16). Recent studies have also applied targeted gene sequencing approaches (21 to 39 genes) in small patient cohorts (6-18) to generate limited mutational profiles of extramedullary AML [Li et al. Leukemia 2015; Pastoret et al. Leukemia & Lymphoma 2016; Kashofer et al. Leukemia & Lymphoma]. Here we utilize a comprehensive next generation sequencing (NGS) panel interrogating 585 genes implicated in hematological malignancies in 17 patients and provide the most comprehensive mutational profile of extramedullary AML to date.Methods: 17 pathologically confirmed cases of extramedullary AML were identified. Genomic DNA extraction was performed from formalin-fixed, paraffin-embedded (FFPE) tumor samples. Isolated DNA was sent for sequencing using the HemePACT panel, a custom hybridization capture-based NGS assay encompassing 585 genes related to hematologic malignancies. All HemePACT data were compared to an unmatched normal sample. Data were passed to standard pipelines used to detect candidate substitutions and indels (Caveman and pindel respectively). Candidate variants were manually reviewed to eliminate single nucleotide polymorphisms and artifacts and to find a subset of high-confidence putative oncogenic variants. Cytogenetic data was obtained from standard clinical karyotyping and fluorescence in-situ hybridization assays from bone marrow aspirates from patients with bone marrow AML involvement.Results: Demographic data for sequenced extramedullary AML patients is provided in Table 1. We identified a total of 80 mutations. The median number of mutations /sample is 4 (range:1-11). The most frequently mutated genes are KIT found in 7/17 patients (41%) and RUNX1 found in 6/17 (35%) patients. Intriguingly, KIT and RUNX1 mutations are mutually exclusive(p<0.05), and 13/17 (76.5%) sequenced extramedullary AML samples have mutations in either KIT or RUNX1 . (Figure 1) DNMT3A mutations are found in 3/17 (17%) patients. FLT3-tyrosine kinase domain (TKD) mutations are found in 4/17 (23%) of patients. DNMT3A and FLT3-TKD mutations co-segregate with RUNX1 (2/3 and 2/4 respectively) but not with KIT mutations. As only 2/17 patients underwent cytogenetic evaluation from extramedullary AML, mutational data from extramedullary AML was largely integrated with cytogenetic data from bone marrow aspirates. 7/17 (41.1%) of sequenced patients had CBF translocations (6/7 with inv(16)/t(16;16), and 1/7 with t(8;21) present in bone marrow cytogenetic evaluation by karyotype or FISH. Out of 7 patients with CBF translocations, 6/7 (85.7%) have KIT mutations present in extramedullary AML. 1 patient with inv(16) found by karyotyping from myeloid sarcoma did not have a KIT mutation in extramedullary AML. No patients with RUNX1 mutations in extramedullary AML (0/6) have CBF translocations. Kaplan-Meier analysis from this small cohort did not show a statistically significant difference in overall survival between patients with KIT and RUNX1 mutations (p=0.57).Overall, our data suggest the potential for distinct and specific mutational profiles associated with AML extramedullary involvement.Conclusions: Our study provides the most comprehensive mutational profiling of extramedullary AML to date. Combined molecular and cytogenetic profiling demonstrate that extramedullary AML is characterized by a distinct repertoire of acquired mutations enriched for mutations in KIT and RUNX1. Importantly our data identify two distinct and non-overlapping molecular subgroups. One subgroup is defined by CBF alterations and KIT mutations, while the second most prominent subtype is defined by mutations in RUNX1. Recognition of these subtypes and their co-mutations may form the basis for genotype-clinical correlations, as well as experimental models to explore the biology of extramedullary AML. Additional extramedullary AML patient samples are being sequenced and will be presented. [Display omitted] DisclosuresGoldberg:Genentech: Research Funding; Pfizer: Research Funding; ADC Therapeutics: Research Funding. Levine:Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.

Therapy‐Induced Senescence Is a Predictor of Treatment Outcome in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease, for which biologically grounded predictors of outcome remain an urgent clinical need. In addition to tumor cell death, antineoplastic drugs have been shown to mediate a long-lasting growth arrest of vital, metabolically active tumor cells termed therapy-induced senescence (TIS) in various cancer entities and model systems, but predictive investigations on TIS are lacking. We hypothesized here that an ex vivo assessment of TIS in blast samples from AML patients at diagnosis might serve as a biomarker of outcome.Methods: We established assays to quantitatively detect TIS in a primary culture setting of purified primary human AML specimens (n > 30) assessed through cytochemical and fluorescence-based analysis of senescence-associated β-galactosidase (SA-β-gal) activity and other senescence markers (such as p16INK4a, H3K9me3) in concert with downregulation of proliferation markers (Ki67, EdU). AML samples were subjected to standard karyotype and molecular genetics analyses, defining risk groups according to the European Leukemia Net (ELN) genetic risk stratification.Results: We found the individual fractions of senescent AML blasts in response to the standard anti-leukemic agents Daunorubicin and Cytarabine samples to be positively correlated with better disease-free (p= 0.042) and overall survival (p= 0.004) of the patients. Furthermore, a more favorable molecular risk group (p= 0.005), normal karyotype (p= 0.007), and NPM1 mutations (p= 0.018) as well as DNMT3A mutations (p < 0.001) were associated with higher TIS levels in our ex vivo senescence assay.Discussion: Our study is the first report providing direct evidence that cellular senescence induced in patient-derived AML blasts by chemotherapeutic drugs ex vivo serves as a predictor of patient long-term responsiveness to standard induction therapy. TIS might explain the underlying biology of current para-clinical risk indicators, and also seems to identify subgroups of superior outcome in pre-defined risk groups including patients with DNMT3A-mutant disease. Additional investigations exploring senescence in response to other AML-relevant agents and functional analyses expanding to patient-derived xenograft (PDX) AML models will be presented at the meeting. DisclosuresNo relevant conflicts of interest to declare.