Structural Chromosomal Abnormalities Research Articles

Chromosome Aberration on Growth and Developmental Disorder

Growth and development in humans begin at conception, which shows progress and interdependence. Normal children show the characteristic of growth and development. In children with growth and development disorder, chromosomal abnormality may be found. This study aimed to determine chromosomal abnormality in patients with growth and development disorder. The samples were taken from karyotype results of patients in the Medical Genetics Laboratory, Faculty of Medicine, Universitas Wijaya Kusuma Surabaya, from 2010-2020. The inclusion criteria were infants to adolescents aged 24 years and unmarried who experienced impaired growth and development, including sexual development. Chromosomal abnormalities were obtained from cytogenetic analysis using the G-banding method. From 75 samples with growth and development disorders, there were abnormalities in the number of autosomal chromosomes and sex chromosomes found in patients with Down syndrome, Turner syndrome, Klinefelter syndrome, and Edwards syndrome. Chromosomal structural abnormalities found were deletion, translocation, inversion, duplication, marker chromosome, and heteromorphism. This study showed the importance of karyotyping in children and adolescents with growth and developmental disorders.

Whole-exome sequencing identified a homozygous novel RAG1 mutation in a child with omenn syndrome.

Omenn syndrome (OS) is a very rare type of severe combined immunodeficiencies manifested with erythroderma, eosinophilia, hepatosplenomegaly, lymph-adenopathy, and elevated level of serum IgE. OS is inherited with an autosomal recessive mode of inheritance. Germline mutations in the human RAG1 gene cause OS. In this study, we investigated a 2-month-old boy with cough, mild anaemia, pneumonia, immunodeficiency, repeated infection, feeding difficulties, hepatomegaly, growth retardation, and heart failure. Parents of the proband were phenotypically normal. Karyotype analysis and chromosomal microarray analysis found no chromosomal structural abnormalities (46, XY) and no pathogenic copy number variations (CNVs) in the proband. Whole-exome sequencing identified a novel homozygous single nucleotide deletion (c.2662delC) in exon 2 of the RAG1 gene in the proband. Sanger sequencing confirmed that both the proband parents were carrying this variant in a heterozygous state. This variant was not identified in two elder sisters and one elder brother of the proband and in the 100 ethnically matched normal healthy individuals. This novel homozygous deletion (c.2662delC) leads to the frameshift, which finally results in the formation of the truncated protein (p.Leu888Phefs*3) V(D)J recombination-activating protein 1 with 890 amino acids compared with the wildtype V(D)J recombination-activating protein 1 of 1043 amino acids. Hence, it is a loss-of-function variant. Our present study expands the mutational spectrum of the RAG1 gene associated with OS. We also strongly suggested the importance of whole-exome sequencing for the genetic screening of patients with OS.

Cytogenetic status of patients with congenital malformations or suspected chromosomal abnormalities in Turkey: a comprehensive cytogenetic survey of 11,420 patients.

Cytogenetic analysis is helpful in diagnostic workup of patients having prenatal or early postnatal medical problems and provides a basis for genetic counseling or deciding on clinical treatment options. Chromosomal abnormalities (CAs) constitute one of the most important category of genetic defects which have the potential to cause irreversible disorders. In this study, chromosome analysis results of 11,420 patients having congenital malformations or suspected of having chromosomal abnormalities, who were referred to Çukurova University Research and Training Hospital Cytogenetic Laboratory over a 16-year period, were investigated, retrospectively. Of all patients analyzed, CAs were found in 1768 cases, accounting for 15.5% of all cases. It was observed that 1175 (15.5%) of CAs were numerical (10.3%) and 593 (5.2%) were structural chromosome abnormalities. Among numerical CAs, Down syndrome (DS), Turner syndrome (TS) and Klinefelter syndrome (KS) constituted common categories which were observed in 7, 1.1 and 0.9% of all cases, respectively. Among the structural CAs, translocations, inversions, fragilities, deletions,, and others were the most common categories and constituted 2.2, 0.9, 0.9, 0.7, 0.3, and 0.3% of all cases, respectively. The sex ratio (male/female) of all cases was 1.01 and of DS cases was 1.6. Our results further confirmed that cytogenetic analysis is necessary in terms of making definite diagnosis of genetic disorders, providing proper genetic counseling and clinical treatment, assessing the recurrence risk, and preventing the hereditary genetic diseases and disorders. Besides, such studies will greatly assist in constituting national and international databases or records of genetic disorders.

The high toxicity of DSB-clusters modelling high-LET-DNA damage derives from inhibition of c-NHEJ and promotion of alt-EJ and SSA despite increases in HR.

Heavy-ion radiotherapy utilizing high linear energy transfer (high-LET) ionizing radiation (IR) is a promising cancer treatment modality owing to advantageous physical properties of energy deposition and associated toxicity over X-rays. Therapies utilizing high-LET radiation will benefit from a better understanding of the molecular mechanisms underpinning their increased biological efficacy. Towards this goal, we investigate here the biological consequences of well-defined clusters of DNA double-strand breaks (DSBs), a form of DNA damage, which on theoretical counts, has often been considered central to the enhanced toxicity of high-LET IR. We test clonal cell lines harboring in their genomes constructs with appropriately engineered I-SceI recognition sites that convert upon I-SceI expression to individual DSBs, or DSB-clusters comprising known numbers of DSBs with defined DNA-ends. We find that, similarly to high-LET IR, DSB-clusters of increasing complexity, i.e. increasing numbers of DSBs, with compatible or incompatible ends, compromise classical non-homologous end-joining, favor DNA end-resection and promote resection-dependent DSB-processing. Analysis of RAD51 foci shows increased engagement of error-free homologous recombination on DSB-clusters. Multicolor fluorescence in situ hybridization analysis shows that complex DSB-clusters markedly increase the incidence of structural chromosomal abnormalities (SCAs). Since RAD51-knockdown further increases SCAs-incidence, we conclude that homologous recombination suppresses SCAs-formation. Strikingly, CtIP-depletion inhibits SCAs-formation, suggesting that it relies on alternative end-joining or single-strand annealing. Indeed, ablation of RAD52 causes a marked reduction in SCAs, as does also inhibition of PARP1. We conclude that increased DSB-cluster formation that accompanies LET-increases, enhances IR-effectiveness by promoting DNA end-resection, which suppresses c-NHEJ and enhances utilization of alt-EJ or SSA. Although increased resection also favors HR, on balance, error-prone processing dominates, causing the generally observed increased toxicity of high-LET radiation. These findings offer new mechanistic insights into high-LET IR-toxicity and have translational potential in the clinical setting that may be harnessed by combining high-LET IR with inhibitors of PARP1 or RAD52.

Chromosomal abnormalities and clinical conditions associated with the male infertility among Emirati

Background: Male infertility is a global health issue that is poorly described in United Arab Emirates. Methods In this 10-year retrospective cross-sectional study, we retrieved data of 312 male patients attending Dubai Fertility Center in United Arab Emirates between January 2011 and January 2021. We identified the type and prevalence of chromosomal abnormalities and hormonal and semen abnormalities among Emirati infertile males as compared with regional and global populations. Results Total chromosomal abnormalities accounted for 13.9% and 8% among azoospermic Emiratis and total Emirati infertile males, respectively. Numerical chromosomal abnormalities causing male infertility were Klinefelter syndrome, 47,XXY (4.0%); Jacob syndrome, 47,XYY (0.8%); mosaic, 48,XXXY/47,XXY/46,XY (0.4%); and mosaic 47,XXY/46,XY (0.4%). Structural chromosomal abnormalities causing male infertility were Y chromosome microdeletion (1.2%), 46,XX/46,XY (0.4%), 46,XY,inv(5)(p15.1q11.2) (0.4%), and 45,XY,der(13;15)(q10;q10) (0.4%). About 59.0% of the Emirati cohort had azoospermia, whereas 28.46% were diagnosed with other conditions of spermatogenic failure as severe oligoasthenoteratozoospermia (7.63%), severe oligoasthenospermia (5.22%), severe oligozoospermia (4.41%), oligoasthenoteratozoospermia (3.6%), asthenozoospermia (2.4%), oligoasthenospermia (1.6%), oligozoospermia (2%), teratozoospermia (0.8%), asthenoteratozoospermia (0.4%), and aspermia (0.4%). As for male hormonal profile of the Emiratis, azoospermic males with chromosomal defects had higher testosterone abnormality (72.2% vs. 45.4%), interstitial-cell stimulating hormone abnormality (66.6% vs. 42.6%), follicle-stimulating hormone abnormality (72.2% vs. 41.5%), and inhibin B hormone abnormality (100% vs. 83.8%) as compared to azoospermic males without chromosomal abnormalities. Conclusion This is the first study to report conclusively the profiling of chromosomal abnormality among Emirati infertile males, which falls within the regional and global range, and to highlight the critical role of genetic testing and counseling for evaluating male infertility.

Evaluation of Genotoxicity of SU-Eohyeol Pharmacopuncture Using an In Vitro Chromosome Aberration Test in Chinese Hamster Lung Cell

ObjectivesThis study was conducted to evaluate the safety of SU-Eohyeol pharmacopuncture (SUEP) by assessing its potential to cause chromosomal abnormalities in Chinese hamster lung cells (CHL/IC).MethodsA dose-curve was conducted to determine the highest dose of SUEP. Doses of 10, 5, 2.5, 1.25, 0.625, and 0.313% were used, and no cytotoxicity or SUEP precipitation was observed. SUEP doses of 10, 5, and 2.5%, with positive and negative controls, were used in a chromosome aberration test.ResultsIn this study, the frequency of abnormal chromosomal cells in the SUEP group did not show a statistically significant difference from that of the negative control group in short-term treatments with and without metabolic activation and the continuous treatment without metabolic activation. Compared with the negative control group, the positive control group had a significantly higher frequency of cells with structural chromosomal abnormalities. This test’s results satisfied all conditions for determining the results.ConclusionSUEP did not induce chromosomal aberrations under the conditions of this study. Other toxicity evaluations, safety studies in humans, and various clinical trials are required to evaluate the safety and efficacy of SUEP.

Establishment and characteristics of GWH04, a new primary human glioblastoma cell line.

Glioblastoma multiforme (GBM) is a common and fatal disease of the central nervous system. GBM cell lines are fundamental tools used in GBM research. The establishment of novel continuous GBM cell lines with clear genetic backgrounds could facilitate the exploration of molecular mechanisms and the screening and evaluation of antitumor drugs in GBM studies. In the present study, a novel primary glioblastoma cell line was established, named GWH04, from a patient with GBM, and its STR genotype and various tumor parameters were examined. The STR information of GWH04 was identical to that of the original primary tumor tissue. Compared with existing cell lines, GWH04 had a similar in vitro proliferation rate as the U87 cell line, but a faster rate than the GL15 cell line, and substantial soft agar clone-formation capacity and subcutaneous and intracranial tumorigenic capacity. For drug sensitivity test, half maximal inhibitory concentration assays for multiple drugs were performed in these three cell lines, and GWH04 cells were found to be resistant to temozolomide. Aneuploid karyotype with a median of 84 chromosomes was possessed by GWH04, as well as chromosomal structural abnormalities, such as broken chromosomes, double centromere chromosomes, homogeneous staining regions, and double microbodies. Gene sequencing further revealed the mutational status of genes TP53, PTEN, PDGFRA, ERBB2, BRCA1, NF1, and MLH1 and the promoter region of telomerase reverse transcriptase (C228T) in this cell line. Altogether, these results indicated that GWH04 will be a useful tool for human GBM studies both in vitro and in vivo.

Recurring germline mosaicism in a family due to reversion of an inherited derivative chromosome 8 from an 8;21 translocation with interstitial telomeric sequences

BackgroundMosaicism for chromosomal structural abnormalities, other than marker or ring chromosomes, is rarely inherited.MethodsWe performed cytogenetics studies and breakpoint analyses on a family with transmission of mosaicism for a derivative...

Prenatal Diagnosis and Outcomes in Fetuses with Hemivertebra.

Background: There are few studies on the burden of chromosomal abnormalities and single gene disorders in fetal hemivertebra (HV). We aim to investigate the cytogenetic and monogenic risk and evaluate prenatal outcomes of fetal HV. Method: This study included fetuses diagnosed with HV divided into two groups: isolated HV and non-isolated HV. Data on other sonographic structural anomalies, chromosomal and sub-chromosomal abnormalities, monogenic variations detected by WES, and prenatal outcomes are recorded and reviewed. Results: Among 109 fetal HV cases, forty-seven (43.1%) non-isolated HV cases were associated with structural anomalies. Chromosomal test results were available in 58 cases, identifying six (10.3%) chromosomal aberrations involved in four isolated and two non-isolated HV. WES identified four (likely) pathogenic variants in three cases among 16 fetuses with HV, involving three novel variants, 1250G > T and c.1277G> inherited from parents, respectively, in DLL3 and c.7213C > A ** in the FLNB. The live birth rate (LB) was higher in the isolated fetal HV group than in the non-isolated group (67.7% (42/62) vs. 12.5% (12/47), p < 0.001). Conclusion: This study emphasizes the risk of cytogenetic abnormalities in isolated HV. WES yields a diagnostic rate of 18.3% in HV with normal CMA, probably aiding the prenatal counseling and management of fetal HV.

The effects of estrogen induction therapy on pubertal presentations in turner syndrome patients

ObjectiveIn this study, we investigated various pubertal presentations and progressions before and after estrogen induction therapy and the correlations with Turner syndrome karyotypes. Materials and methodsWe reviewed the medical records of patients with Turner syndrome diagnosed before the age of 18 years between 2000 and 2019. Sixty-six patients were enrolled and distributed into 45,X monosomy group, X chromosome structural abnormalities group and X mosaicism group. The pubertal presentations were classified into spontaneous puberty, arrested puberty and no spontaneous puberty. All patients’ karyotypes, pubertal progressions and laboratory data were collected and analyzed. ResultsThe karyotypes were highly correlated with pubertal presentations. No spontaneous puberty was noticed in 58.3% 45,X monosomy patients, 50% patients with X chromosome structural abnormalities had arrested puberty, whereas 70% patients with X mosaicism had spontaneous puberty. Estrogen induction therapy in patients with no spontaneous puberty could induce puberty and the tempo of puberty may approximate to the spontaneous puberty group (median, 2.3 vs. 2.2 years, P = 0.95). In both interventional groups, the FSH level was distinguishable before treatment (median, 65.1 vs. 100.4 mIU/mL, P = 0.02). After long term estrogen therapy, the FSH could be suppressed to similar level in both interventional groups (median, 37.5 vs 34.5 mIU/mL, P = 0.84). Neither LH nor E2 level provided valuable information before and after treatment. ConclusionThe karyotypes were highly correlated with pubertal presentations at Turner syndrome patients. The integrity of 2nd X chromosome plays an important role. Low dose estrogen could mimic the tempo of puberty even delay induction age at Taiwan. The FSH data could provide predictive information of pubertal induction for both interventional groups.

Chromosomal Anomalies in Bone Marrow Samples of Patients Diagnosed with Hematological Cancer: FISH Results of 109 Cases from one Center

Objective: Hematological cancers are a group of neoplasms that affect the bone marrow, blood and lymph nodes, usually associated with structural and numerical chromosomal abnormalities. Bone marrow examination in hematological cancers has an illuminating and guiding role in the diagnosis and prognosis of the disease. In our study, it was aimed to retrospectively evaluate the results obtained by using the Fluorescent In Situ Hybridization (FISH) method of certain genetic disorders in patients with a diagnosis of hematological cancer. Materials &amp;amp; Methods: In our study, between January 1, 2021 and November 30, 2021 The results obtained from bone marrow samples of 109 patients (KML n=14, AML n=40, KLL n=6, ALL n=27, M.Myelom n=22) with pre-diagnosis of hematological cancer, who were referred to Dicle University Faculty of Medicine, Department of Medical Biology and Genetics, Department of Hematology and other clinics, were analyzed retrospectively in terms of age, gender, and disease distribution. All of the patients included in the study group were evaluated in terms of chromosomal anomalies, numerical and structural changes, or presence of balanced translocation. Results: There were 47 female patients and 62 male patients, and the mean age was 48±21.6 years. As a result of the examination of the cases, known for AML; t(15;17), monosomy 7 and trisomy 8, t(9;22) for KML; del(13q14) and del(17p13) for KLL, t(11;14)(q13;q32) for M. Myeloma, (t[9;22]) (bcr-abl) and t(4;11) for ALL structural and numerical chromosome changes such as shown first. In addition, in the comparison of the groups, no significant difference was found between the materials participating in the study, male and female. Conclusion: In this study, which we conducted with our patients who were prediagnosed with hematological cancer with the FISH method, we evaluated our results regarding the prognostically important anomalies associated with the diseases. Rapid and accurate diagnosis of diseases is very important in terms of treatment planning and prognosis of the course of the disease. As a result, considering the difficulty of obtaining cells from bone marrow taken under limited and difficult conditions according to our study results, it was concluded that the FISH method is a powerful and effective technique used in cancer genetics, since it provides the opportunity to work even in interphase cells.

A case of complex balanced chromosomal translocations associated with adverse pregnancy outcomes

Complex chromosomal rearrangements (CCR) are rare chromosomal structural abnormalities. The chromosomal structural variants in CCR carriers are one of the factors contributing to a history of adverse pregnancy and childbirth. In this study, we report a patient with a history of adverse pregnancy and childbirth who exhibited complex balanced chromosomal translocations. The female patient was phenotypically and intellectually normal; in her first pregnancy, the embryo was damaged, and a histological examination of the chromosomes of the embryos revealed a deletion of approximately 4.66 Mb at 1p32.3p32.2, a duplication of approximately 1.02 Mb at 1p22.2p22.1, a duplication of approximately 1.46 Mb at 6q27 and a deletion of approximately 7.78 Mb at 9p24.3p24.1. Chromosomal examinations of the patient revealed the karyotype to be 46,XX,(1;9)(p32; p34). In the second pregnancy, the foetus was diagnosed prenatally with three or more positive ultrasound soft indicators. The patient's karyotype was re-examined and further confirmed by fluorescence in situ hybridisation as 46,XX,t(1;9;6)(p31;p22;q27), revealing this patient was a carrier of complex balanced chromosomal translocations. Carriers of CCR have a higher risk of spontaneous abortion, and genetic counselling clinicians should consider the karyotype analyses of such patients in clinical practice and recheck their chromosomes if necessary.

Pathophysiology of Nociception and Rare Genetic Disorders with Increased Pain Threshold or Pain Insensitivity.

Pain and nociception are different phenomena. Nociception is the result of complex activity in sensory pathways. On the other hand, pain is the effect of interactions between nociceptive processes, and cognition, emotions, as well as the social context of the individual. Alterations in the nociceptive route can have different genesis and affect the entire sensorial process. Genetic problems in nociception, clinically characterized by reduced or absent pain sensitivity, compose an important chapter within pain medicine. This chapter encompasses a wide range of very rare diseases. Several genes have been identified. These genes encode the Nav channels 1.7 and 1.9 (SCN9A, and SCN11A genes, respectively), NGFβ and its receptor tyrosine receptor kinase A, as well as the transcription factor PRDM12, and autophagy controllers (TECPR2). Monogenic disorders provoke hereditary sensory and autonomic neuropathies. Their clinical pictures are extremely variable, and a precise classification has yet to be established. Additionally, pain insensitivity is described in diverse numerical and structural chromosomal abnormalities, such as Angelman syndrome, Prader Willy syndrome, Chromosome 15q duplication syndrome, and Chromosome 4 interstitial deletion. Studying these conditions could be a practical strategy to better understand the mechanisms of nociception and investigate potential therapeutic targets against pain.

Analysis results of 169 cases of chorionic villus samples of missed abortion using high throughput sequencing.

This study aimed to evaluate the use of high-throughput sequencing (HTS) technology to detect chromosomes in chorionic villus samples of missed abortion embryos and investigate its utility in the genetic diagnosis of missed abortion. HTS was used to assess chorionic villus samples obtained from 169 patients with missed abortions from August 2020 to March 2022, at the Second Affiliated Hospital of Guangxi Medical University. The test results were statistically analyzed. To investigate the impact of advanced age on the incidence of chromosomal abnormalities, the patients were divided into two groups: elderly (≥35 years) and nonelderly pregnant women (<35 years). (1) Among the samples of 169 patients, 100 (59.17%) cases of chromosomal abnormalities were detected. Among these 100, 90 (90%) had chromosomal numerical abnormalities and 10 (10%) had chromosomal structural abnormalities. (2) Chromosomal numerical abnormality was abnormalities mainly included aneuploidy (92.22%, 83/90), with trisomy (62.22%, 56/90) and monosomy (22.22%, 20/90) accounting for the majority. The top three numerical abnormalities included 18 cases of Turner syndrome (monosomy X; 20%, 18/90), 10 cases of trisomy 16 (11.11%, 10/90), and 10 cases of trisomy 22 (11.11%, 10/90). (3) Villous chromosomal abnormalities were found in 48 (70.59%) elderly pregnant women, and 52 (51.48%) nonelderly pregnant women, with statistically significant differences (p < 0.05). (1) Chromosomal abnormality is an important cause of missed abortion, it majorly includes chromosomal numerical abnormality, of which most cases are of aneuploidy. (2) Advanced age may increase the risk of embryonic chromosomal abnormalities. (3) Villus chromosome detection using HTS has a positive value and can be used for analyzing and determining the causes of missed abortion.

Pregnancy Outcome and Postnatal Chromosome Analysis of the Cord Blood and Chorionic Villi in Two Cases after Intrauterine Transfer of Mosaic Aneuploid Blastocysts

The long-term prognosis and genetic mechanism of pregnancy after intrauterine mosaic aneuploid blastocyst transfer remain unknown. We report the case of two babies after the aforementioned procedure and chromosomal analysis of their cord blood and chorionic villi. Case Report 1. A 41-year-old primipara, with two previous spontaneous abortions, was pregnant after intrauterine transfer of a blastocyst carrying 40% mosaicism of long-arm monosomy of chromosome 5. The amniocentesis results were 46,XX. A cesarean section was performed at 39 weeks. The female infant was 3,315 g at birth. Case Report 2. A 44-year-old primipara, with two spontaneous abortions, was pregnant after intrauterine transfer of a blastocyst carrying 40% mosaicism of long-arm monosomy of chromosome 9 and monosomy of chromosome 14. After genetic counselling, she decided not to undergo amniocentesis. No abnormalities were found by ultrasound. A cesarean section was performed at 38 weeks. The male infant was 3,340 g at birth. Chromosome analyses of postnatal cord blood and chorionic villi were performed using SNP arrays. The cord blood and chorionic villi showed no chromosomal structural abnormalities or mosaicism. For both, no disorders were observed at 10 months of age. We experienced the birth of babies after intrauterine transfer of mosaic aneuploid blastocysts.

Labial Mucosa Stem Cells: Isolation, Characterization, and Their Potential for Corneal Epithelial Reconstruction.

PurposeThe purpose of this study was to characterize labial mucosa stem cells (LMSCs) and to investigate their potential for corneal epithelial reconstruction in a rabbit model of total limbal stem cell deficiency (LSCD).MethodsRabbit LMSCs (rLMSCs) and human (hLMSCs) LMSCs were derived from labial mucosa and characterized in terms of their proliferation activity by the evaluation of proliferation index (PI) and colony forming efficiency (CFE), cell senescence, and differentiation abilities. The expression of various limbus-specific, stem cell-specific, and epithelial markers was assessed via immunocytochemistry. Flow cytometry was used to evaluate mesenchymal and hematopoietic cell surface markers expression. Chromosomal stability of the derived cells was examined using the conventional GTG-banding technique. To assess the impact of LMSCs on corneal epithelial reconstruction, rLMSCs were seeded onto a decellularized human amniotic membrane (dHAM), thereafter their regeneration potential was examined in the rabbit model of total LSCD.ResultsBoth rLMSCs and hLMSCs showed high proliferation and differentiation abilities, entered senescence at later passages, and expressed different stem cell-specific (ABCB5, ALDH3A1, ABCG2, and p63α), mesenchymal (vimentin), and epithelial (CK3/12, CK15) markers. Cell surface antigen expression was similar to other described mesenchymal stem cells. No clonal structural chromosome abnormalities (CSCAs) and the low percentage of non-clonal structural chromosome abnormalities (NSCAs) were observed. Transplantation of rLMSCs promoted corneal epithelial reconstruction and enhanced corneal transparency.ConclusionsLMSCs have significant proliferation and differentiation abilities, display no detrimental chromosome aberrations, and demonstrate considerable potential for corneal repair.

Toxicological Properties of 7-Methylguanine, and Preliminary Data on its Anticancer Activity.

7-Methylguanine (7-MG) competitively inhibits the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) and RNA-modifying enzyme tRNA-guanine transglycosylase (TGT) and represents a potential anticancer drug candidate. Furthermore, as a natural compound, it could escape the serious side effects characteristic for approved synthetic PARP inhibitors. Here we present a comprehensive study of toxicological and carcinogenic properties of 7-MG. It was demonstrated that 7-MG does not induce mutations or structural chromosomal abnormalities, and has no blastomogenic activity. A treatment regimen with 7-MG has been established in mice (50 mg/kg per os, 3 times per week), exerting no adverse effects or changes in morphology. Preliminary data on the 7-MG anticancer activity obtained on transplantable tumor models support our conclusions that 7-MG can become a promising new component of chemotherapy.

Selection of a stable reference gene for relative copy number profiling of porcine chromosomal genes using SYBR green qPCR

Reference gene with stable copy number is essential for normalization in qPCR based copy number assay. Present study aims to identify a suitable reference gene in pigs for qPCR based relative copy number profiling of chromosomal genes. A total of 30 crossbred pigs of both sexes were cyto-screened and gDNA was extracted from the pigs having numerically normal karyotypes. The copy number stability was studied for 7 genes (FSHB, IL4, IGF1R, TCF24, BRMS1L, ARMC1 and SRSF4) selected on the basis of the chromosomal location, reports of single copy and lack of involvement in structural chromosomal abnormalities. The copy number was estimated from Ct values in 3 technical replicates using 6 animals from either sex for each gene. The stability was evaluated from the variations in Ct values using different (Delta Ct, geNorm, BestKeeper and normFinder) algorithms. While the moderate variation was observed among relative copy number stabilities among the genes, comprehensive ranking revealed the most stable gene for normalization (IGF1R > FSHB > TCF24 > IL4 > ARMC1> SRSF4 > BRMS1L) across the samples. The selected reference gene was validated using DNA of cyto-screened pigs to find out ratio of X and Y chromosome fragments using qPCR based copy number analysis.

P624: CHROMOTHRIPSIS IN PATIENTS WITH CLL AND COMPLEX KARYOTYPE: PATTERNS OF ABERRATIONS AND PROGNOSTIC VALUE

Background: Chromothripsis is a unique catastrophic event found in 1-5% of chronic lymphocytic leukemia (CLL) patients, associated with poor prognostic factors and short survival (Edelmann et al, 2012; Puente et al, 2015; Leeksma et al, 2021). It is characterized by the presence of multiple genomic rearrangements in one chromosome, resulting in oscillating switches between 2-3 copy number states by chromosomal microarrays (CMA) or next-generation sequencing (Stephens et al, 2011). Optical genome mapping (OGM) is a new method based on the imaging of long DNA molecules (>250kb) labelled at specific motifs, generating a unique pattern. This allows mapping of the genomic location of each molecule and detection of structural and numerical chromosomal abnormalities with high resolution and sensitivity. Aims: 1. To analyze the genome complexity associated with chromothripsis and the patterns of rearrangements detected by OGM; 2. To determine the prognostic impact of chromothripsis in a cohort of CLL patients enriched in complex karyotypes (CK). Methods: A total of 33 patients with chromothripsis detected by CMA were included: 11 with chromothripsis-like patterns (≥7 switches), 22 with classical chromothripsis (≥10 switches). Nine of the cases were studied with OGM (Bionano Genomics). Clinico-biological features and time to first treatment (TTFT) were analyzed. Results were compared with a cohort of 129 patients showing CK without chromothripsis (control group) (Ramos-Campoy et al, 2021). Results: All patients with chromothripsis showed a high genomic complexity (30/33 with CK by chromosome banding analysis and 3/33 classified as complex by CMA [range: 10-24 CNV, being at least three of them >5Mb (range: 3-6)]. Forty-six chromothriptic events were identified. These affected 1-4 chr/patient, but a single chromosome was involved in 25/33 patients (76%). Chromothripsis was distributed throughout all chromosomes, most frequently affecting 3 (n=5), 6 (n=5) and 13 (n=5). The events were mainly multiple losses (25/46) or alternated gains and losses (19/46). Size and coordinates of CNVs detected by OGM and CMA were highly concordant. OGM detected multiple rearrangements cryptic by CMA (median: 9/chromothriptic event). Two patterns of chromothripsis-related rearrangements were identified: one clustered in the chromothriptic region (3/9) and the other involving both chromothriptic and non-chromothriptic chromosomes (range: 1-6 chr) (6/9). One patient showed 36 rearrangements throughout the genome, suggesting the coexistence of chromothripsis and chromoplexy, characterized by the presence of multiple chained translocations (Figure). Patients with chromothripsis showed a higher frequency of TP53del/mut (70% vs 39%; p=0.001) and a shorter TTFT (15m vs 2m; p=0.013) than the control group. No differences were observed for IGHV and del(11q) status. When stratifying patients based on TP53 status, the presence of chromothripsis was not associated with shorter TTFT. In the multivariate analysis including TP53, genomic complexity by CMA and chromothripsis, only TP53 was statistically significant (HR=1.6; p=0.029). Image:Summary/Conclusion: 1. Chromothriptic events involve multiple loci in CLL patients; 2. OGM allows not only the detection of CNV but also the identification of two rearrangement patterns associated with chromothripsis (clustered or involving non-chromothriptic chromosomes) whose clinical impact should be further explored. 3. In CLL patients with CK, the adverse prognosis of chromothripsis is associated with a higher frequency of TP53del/mut.

Reliability of Cell-Free DNA and Targeted NGS in Predicting Chromosomal Abnormalities of Patients With Myeloid Neoplasms

IntroductionCytogenetic analysis is important for stratifying patients with various neoplasms. We explored the use of targeted next generation sequencing (NGS) in detecting chromosomal structural abnormalities or copy number variations (CNVs) in patients with myeloid neoplasms.MethodsPlasma cell-free DNA (cfDNA) from 2821 myeloid or lymphoid neoplasm patients were collected. cfDNA was sequenced using a 275 gene panel. CNVkit software was used for analyzing and visualizing CNVs. Cytogenetic data from corresponding bone marrow (BM) samples was available on 89 myeloid samples.ResultsOf the 2821 samples, 1539 (54.5%) showed evidence of mutations consistent with the presence of neoplastic clones in circulation. Of these 1539 samples, 906 (59%) showed abnormalities associated with myeloid neoplasms and 633 (41%) with lymphoid neoplasms. Chromosomal structural abnormalities in cfDNA were detected in 146 (16%) myeloid samples and 76 (12%) lymphoid samples. Upon comparison of the myeloid samples with 89 BM patients, NGS testing was able to reliably detect chromosomal gain or loss, except for fusion abnormalities. When cytogenetic abnormalities were classified according to prognostic classes, there was a complete (100%) concordance between cfDNA NGS data and cytogenetic data.ConclusionsThis data shows that liquid biopsy using targeted NGS is reliable in detecting chromosomal structural abnormalities in myeloid neoplasms. In specific circumstances, targeted NGS may be reliable and efficient to provide adequate information without the need for BM biopsy considering broad mutation profiling can be obtained through adequate sequencing within the same test. Overall, this study supports the use of liquid biopsy for early diagnosis and monitoring of patients with myeloid neoplasms.