The frequent and clinically benign anomalies of chromosomes 7 and 20 in Shwachman-diamond syndrome may be subject to further clonal variations

Background An isochromosome of the long arm of chromosome 7, i(7)(q10), and an interstitial deletion of the long arm of chromosome 20, del(20)(q), are the most frequent anomalies in the bone marrow of patients with Shwachman-Diamond syndrome, which is caused in most cases by mutations of the SBDS gene. These clonal changes imply milder haematological symptoms and lower risk of myelodysplastic syndromes and acute myeloid leukaemia, thanks to already postulated rescue mechanisms. Results Bone marrow from fourteen patients exhibiting either the i(7)(q10) or the del(20)(q) and coming from two large cohorts of patients, were subjected to chromosome analyses, Fluorescent In Situ Hybridization with informative probes and array-Comparative Genomic Hybridization. One patient with the i(7)(q10) showed a subsequent clonal rearrangement of the normal chromosome 7 across years. Four patients carrying the del(20)(q) evolved further different del(20)(q) independent clones, within a single bone marrow sample, or across sequential samples. One patient with the del(20)(q), developed a parallel different clone with a duplication of chromosome 3 long arm. Eight patients bore the del(20)(q) as the sole chromosomal abnormality. An overall overview of patients with the del(20)(q), also including cases already reported, confirmed that all the deletions were interstitial. The loss of material varied from 1.7 to 26.9 Mb and resulted in the loss of the EIF6 gene in all patients. Conclusions Although the i(7)(q) and the del(20)(q) clones are frequent and clinically benign in Shwachman Diamond-syndrome, in the present work we show that they may rearrange, may be lost and then reconstructed de novo, or may evolve with independent clones across years. These findings unravel a striking selective pressure exerted by SBDS deficiency driving to karyotype instability and to specific clonal abnormalities.

Genome and Karyotype Reorganization after Whole Genome Duplication in Free-Living Flatworms of the Genus Macrostomum

The genus Macrostomum represents a diverse group of rhabditophoran flatworms with >200 species occurring around the world. Earlier we uncovered karyotype instability linked to hidden polyploidy in both M. lignano (2n = 8) and its sibling species M. janickei (2n = 10), prompting interest in the karyotype organization of close relatives. In this study, we investigated chromosome organization in two recently described and closely related Macrostomum species, M. mirumnovem and M. cliftonensis, and explored karyotype instability in laboratory lines and cultures of M. lignano (DV1/10, 2n = 10) and M. janickei in more detail. We revealed that three of the four studied species are characterized by karyotype instability, while M. cliftonensis showed a stable 2n = 6 karyotype. Next, we performed comparative cytogenetics of these species using fluorescent in situ hybridization (FISH) with a set of DNA probes (including microdissected DNA probes generated from M. lignano chromosomes, rDNA, and telomeric DNA). To explore the chromosome organization of the unusual 2n = 9 karyotype discovered in M. mirumnovem, we then generated chromosome-specific DNA probes for all chromosomes of this species. Similar to M. lignano and M. janickei, our findings suggest that M. mirumnovem arose via whole genome duplication (WGD) followed by considerable chromosome reshuffling. We discuss possible evolutionary scenarios for the emergence and reorganization of the karyotypes of these Macrostomum species and consider their suitability as promising animal models for studying the mechanisms and regularities of karyotype and genome evolution after a recent WGD.

Cytogenetic and Transcriptomic Analysis of Human Endometrial MSC Retaining Proliferative Activity after Sublethal Heat Shock

Temperature is an important exogenous factor capable of leading to irreversible processes in the vital activity of cells. However, the long-term effects of heat shock (HS) on mesenchymal stromal cells (MSC) remain unstudied. We investigated the karyotype and DNA repair drivers and pathways in the human endometrium MSC (eMSC) survived progeny at passage 6 after sublethal heat stress (sublethal heat stress survived progeny (SHS-SP)). G-banding revealed an outbreak of random karyotype instability caused by chromosome breakages and aneuploidy. Molecular karyotyping confirmed the random nature of this instability. Transcriptome analysis found homologous recombination (HR) deficiency that most likely originated from the low thermostability of the AT-rich HR driving genes. SHS-SP protection from transformation is provided presumably by low oncogene expression maintained by tight co-regulation between thermosensitive HR drivers BRCA, ATM, ATR, and RAD51 (decreasing expression after SHS), and oncogenes mTOR, MDM2, KRAS, and EGFR. The cancer-related transcriptomic features previously identified in hTERT transformed MSC in culture were not found in SHS-SP, suggesting no traits of malignancy in them. The entrance of SHS-SP into replicative senescence after 25 passages confirms their mortality and absence of transformation features. Overall, our data indicate that SHS may trigger non-tumorigenic karyotypic instability due to HR deficiency and decrease of oncogene expression in progeny of SHS-survived MSC. These data can be helpful for the development of new therapeutic approaches in personalized medicine.

KARYOTYPЕ VARIABILITY OF THE COWS OF UKRAINIAN RED-AND-WHITE DAIRY CATTLE BREED WITH VARIED REPRODUCTIVE ABILITY

The results of the cytogenetic study of cows of Ukrainian red-bream dairy breeds with different reproductive ability are presented in the article. Breeding reproductive ability of cows is often and, as a rule, they include an increase in the duration of the service period, the occurrence of violations of embryonic development, stillbirth and miscarriages. Investigation of karyotype and thorough analysis of hereditary information of the herd population of herds will help to establish and eliminate the cause of reproductive failure of cows. The purpose of the study is to study the karyotype variability of cows with normal and violated reproductive qualities. The material for the research was the results of an individual assessment of the animals of the Ukrainian red-shingled breed of the DP DH "Khrystynivske" on the basis of zootechnical records and experimental cytogenetic data. To analyze the data of zootechnical accounting, the software package SUMS "Intesel Orsek" was used. Laboratory studies were conducted at the Laboratory of Biotechnology at the Institute of Animal Breeding and Genetics named after MV. Tooth The cultivation of lymphocytes, the preparation of cytogenetic drugs, the classification and recording of aberrations of chromosomes were carried out according to generally accepted techniques. The analysis of metaphase cells included cytogenetic parameters: the proportion of aneuploid and polyploid cells, the frequency of cells with structural aberrations of the chromosomes. For the cytogenetic study, based on the materials of the zootechnical account, three groups of cows have been formed, depending on their reproductive ability: I group, number 17, consists of animals with impaired reproductive ability. To II group included 33 cows, service period of which is not less than 150 days; Group ІІІ - 25 cows with a service period of 51-90 days. The study of blood lymphocytes of cows with different reproductive ability revealed a difference in the level of chromosomal instability. The results of the studies showed that in the karyotypes of animals with impaired reproductive ability, a significantly higher frequency of cells with aneuploid and polyploid chromosome sets, as well as cells with chromosomal aberrations was found to be significantly higher than that of cows with normal reproductive functions. It was established that a significantly larger proportion of cells with aneuploidy occurs in the karyotype of animals of group I, that is, in animals with a disturbed reproductive ability - 10.5 ± 2.38%. In cows of group ІІ, the frequency of cells with aneuploid number of chromosomes in blood cells decreased to 6.3 ± 1.45%. Less than twice the cows in this group and the frequency of cells with the polyploid chromosome set (from 1,0 ± 0,01 to 0,45 ± 0,16) and 15% with structural aberrations of the chromosomes (from 14,82 ± 2,87 up to 12.5 ± 2.87). In cows of group III (with normal reproductive ability), cells with aneuploid chromosomal set were found to be 2.3 times less than in cows with reproductive capacity (Group I) (P> 0.999). In the karyotypes of cows that were examined, no constitutional alterations of the chromosomes, including translocations of the Robertson type, were revealed. In animals of group I, the frequency of chromosomal aberrations was one third higher than that of animals of groups ІІ and ІІІ. The difference in frequencies between the higher and lower group values of this indicator was 4.15%. It has been established that in all groups of investigated animals there is a positive correlation between the service period and the main cytogenetic parameters. The highest positive correlation values (r = 0.70; r = 0.50; r = 0.44) are established between the duration of the service period and the frequency of structural aberrations, service period and polyploidy, service period and aneuploidy respectively in cows of the second group , In which the service period lasted 150 days or more. In cows of group I, the highest positive correlation (r = 0.48) is established between the duration of the service period and aneuploidy. For the third group, defined by us as a control, the positive relationship (r = 0.55) between the service period and aneuploidy is also established. Thus, the correlations established between the level of karyotype instability and one of the characteristics of reproductive ability (service period) indicate that the levels of karyotype instability in cows to a sufficient degree can characterize their reproductive qualities. The results of the studies show that the higher frequency of abnormal cells is found mainly in cows with broken reproductive functions. Consequently, we can conclude that for assessing the individual qualities of dairy cows, indicators of karyotype instability should be used as a criterion for evaluating the reproductive qualities of cows.

Centrosome Aberrations in Bone Marrow Cells From Patients with Myelodysplastic Syndromes Correlate with Chromosomal Instability

Abstract 3839 Introduction: Centrosomes play important roles in maintenance of genetic stability and centrosomal aberrations are a common hallmark of cancer. Deregulation of centriole duplication during the cell cycle leads to supernumerary centrosomes, sister chromatide missegregation and could result in chromosomal instability (CIN) and aneuploidy. CIN is a common feature in at least 50% of patients with myelodysplastic syndromes (MDS). Therefore, we sought to investigate the centrosomal status and its role for development of CIN in bone marrow (BM) cells of MDS patients. Furthermore, deregulation of the protease Separase is known as a driver of aneuploidy. It is considered as one of the master key players in centriole duplication and overexpression has been associated with the formation of supernumerary centrosomes in many cancers. Therefore, deregulated Separase could also serve as a marker for genetic instability and was investigated. Patients and methods: BM cells of 34 MDS patients were cytogenetically examined by G-banding technique. Furthermore, cells were immunostained with a centrosome-specific antibody to pericentrin followed by a Cy3-conjugated secondary antibody to analyze the centrosomal status. Umbilical cord blood specimens (CB; n=15) served as controls. In addition, Separase protein levels were analyzed in BM cells of four MDS patients and in CB cells of four healthy controls. Results: BM cells of all MDS patients displayed centrosome alterations as compared with corresponding controls. Centrosome abnormalities were detected in 10% (range, 4–17%) of analyzed cells of MDS patients but in only 2% (range, 0–4%) of cells of healthy donors (p≤0.0001). Normal karyotypes were found in all CB metaphases and in BM metaphases of 16/34 MDS patients. The incidence of centrosomal alterations was higher in bone marrow cells of patients with cytogenetic alterations (mean, 12%) compared to BM cells of patients without cytogenetic changes (mean, 7%). In BM cells of MDS patients Separase protein levels were lower (60% decrease) as compared to CB cells of the healthy control (p≤0.01). Conclusions: We could show that centrosome aberrations in BM cells of MDS patients occur before chromosomal changes are detectable. Therefore, centrosomal instability is an early step in MDS and may contribute to the acquisition of chromosomal alterations. Increase of aberrant centrosomes significantly correlates with karyotype instability and aneuploidy. It seems that centrosomal instability precedes karyotype instability via chromosomal missegregation and could contribute to the development of chromosomal changes and accelerate malignant transformation. Separase is one of the master key players in centriole duplication and chromatide segregation. Therefore, deregulated Separase could lead to genetic instability and malignant transformation. In future studies centrosomal alterations may serve as an additional prognostic biomarker for future diagnostics in MDS. Disclosures: No relevant conflicts of interest to declare.

Aneuploids in the shrub birch Betula humilis populations in Poland

Shrub birch (<em>Betula humilis </em>Schrk.) is endangered glacial relict growing in natural and drained fens and transitional mires. At present study we examined karyotypes of 103 individuals of <em>B. humilis</em>, collected in six populations from eastern and northeastern Poland. We found 60% of diploid individuals with 2n = 28. The rest of studied plants were aneuploids with 26, 27, 29, 30 and 31 chromosomes in their karyotypes. High frequencies of aneuploids in Polish populations of <em>B. humilis </em>could be a consequence of: (i) hybridization with congeneric species, (ii) stress resulting from range fragmentation, (iii) karyotype instability of individuals with 2n ≠ 28, or (iv) vegetative reproduction.