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.

Selection of Hormonal Reference Values for Undescended Testicle Case in 8-Year-Old Boy with Abnormal Chromosome

Establishing the diagnosis of undescended testicles requires appropriate hormonal laboratory reference values basedon age and gender. An 8-year-old boy with an undescended testicle, mental retardation, and stunting had a blood test thatwas carried out at the Clinical Pathology Laboratory, dr. Cipto Mangunkusumo (RSCM) Hospital on February 6, 2020, withtestosterone levels of 0.69 nmol/L (N male: 4.94-32.01 nmol/L) indicating decreased testosterone levels. The patient wasconsulted from urological surgery to pediatric endocrinology to determine the presence or rudiment of the patient'stesticles. Using the reference range of testosterone values assists clinicians in determining the diagnosis, monitoringtherapy, and prognosis of a disease. There are some testosterone reference values, which are currently available, includingCanadian Laboratory Initiative on Pediatric Reference Intervals Database (CALIPER) and the Tanner stage reference value.Later is more applicable because it is based on chronological age and secondary sexual development in assessing pubertydevelopment. A case of an 8-year-old boy with a clinical diagnosis of an undescended testicle, the laboratory test resultsshowed normal-low testosterone levels using the CALIPER and Tanner stage ranges according to the patient's age. Noincrease of testosterone levels after the second HCG stimulation test might be due to differences in the HCG administrationprotocol; therefore, the diagnosis of anorchia had not been established, and chromosome abnormalities of 46 XY, +6 Mar,17 dmin on chromosome analysis suggested the suspected syndrome. These findings were consistent with the suspicion ofprimary hypogonadism in children with suspected syndrome caused by bilateral cryptorchidism with a suspectedseminiferous tubular defect.

An infant with 49XXXXY syndrome: a case report

Background 49XXXXY syndrome is the rarest X chromosome aneuploidy, with approximate incidence of 1:85,000–100,000 male births. Worldwide, around 100 cases have been reported. In this report, we describe one such case seen in Sri Lanka. Case presentation A 10-day-old Sri Lankan neonate born in a tertiary care center was referred to the pediatric endocrinology unit of Lady Ridgeway Hospital due to detection of ambiguous genitalia at birth. He was the first child born to nonconsanguineous healthy parents following an uncomplicated antenatal period. He was born at term via normal vaginal delivery, with a birth weight of 2.385 kg. The baby was active, and there was no documented hypoglycemia or alteration in basic biochemical investigations. On examination, the child had hypertelorism, upslanting palpebral fissures, flat occiput, and mild webbing of the neck. System examination was normal. Genitalia examination revealed bifid scrotum, perineal urethra, 2 cm phallus, and bilateral testis in situ. Hormonal analysis, including dehydroepiandrosterone sulfate, testosterone, and 17-OH progesterone levels, was normal except for an elevated level of follicle-stimulating hormone, indicating gonadal dysgenesis. Ultrasound of the abdomen detected testis located at bilateral inguinal canal, and no Müllerian structures were visible. Echocardiography showed a small patent foramen ovale with otherwise normal heart. Chromosome analysis revealed 49XXXXY syndrome. Conclusion 49XXXXY syndrome should be entertained as a rare possibility for ambiguous genitalia, and karyotyping is an essential investigation for evaluation of such patients.

Investigation of Mosaicism Detected in STR Typing

Short tandem repeats (STRs) are the most popular markers for human identification in forensics. These markers can be easily analyzed through a multiplex polymerase chain reaction and electrophoresis and provide high discrimination power. However, in STR analysis, several atypical phenomena can be observed such as allelic dropouts, drop-ins, or imbalance, which may be due to DNA polymerase slippage or DNA degradation effects. The observed atypical STR profiles can also provide information for mixed DNA samples or chromosomal abnormalities. In this study, we report a case of mosaicism detected in routine casework of paternity testing. Hair samples from a phenotypically normal male were tested, and the result presented a typical STR profile of a female for the amelogenin gene (XX). Through chromosome analysis using peripheral blood, it was found that 45,X/46,XY mosaicism resulted in the discrepancy between the genotype and the phenotype. In addition, the amount of Y chromosome detected was particularly low in hair compared to that in blood. This study shows that mosaicism can make interpretation difficult during STR analysis and suggests that sample types and repeated analysis should be considered even for routine STR testing.

Can Culture Media Impact Preimplantation Embryo Aneuploidy?

With continued improvements in blastocyst culture, cell sampling approaches, and genetic analysis platforms, the resulting improvements in embryo development and the resolution and accuracy of chromosome analysis have provided valuable insights into the preimplantation embryo. This includes the impact of in vitro culture conditions on chromosomal dynamics. Specifically, through analysis of embryo aneuploidy and mosaicism, a growing number of reports indicate that rates of chromosomal abnormalities can vary between IVF centers. Because differences in mosaicism reflect mitotic errors, this endpoint analysis suggests that IVF laboratory-controlled variables during embryo development may be influencing chromosome separation and segregation. A growing body of literature suggests that culture media may be one variable influencing preimplantation embryo aneuploidy and mosaicism. However, these data are far from definitive in demonstrating cause-and-effect. Whether reported differences may be due to media formulation, use of sequential media or single-step media, or uninterrupted culture approaches is unknown. Importantly, variables directly impacting media performance and embryo development, including pH, temperature, osmolality, and oxygen concentration, must also be considered and make it difficult to isolate the impact of culture media as the sole factor responsible. These IVF laboratory variables will be reviewed and literature suggesting a possible link to mitotic aneuploidy/mosaicism will be discussed.

Comparative Analysis of HSF Genes From Secale cereale and its Triticeae Relatives Reveal Ancient and Recent Gene Expansions

Plants have evolved sophisticated systems to cope with the environmental stresses, with the heat shock factor (HSF) family proteins composing an integral part of the transcriptional regulation system. Understanding the evolutionary history and functional diversity of HSFs will facilitate improving tolerance of crops to adverse environmental conditions. In this study, genome-wide analysis of Secale cereale identified 31 HSF genes. The total number of HSF genes in S. cereale is larger than that in barley and the three subgenomes of wheat, suggesting it is a valuable resource for mining functional HSFs. Chromosome analysis revealed an uneven distribution of HSF genes among the 7 S. cereale chromosomes, with no HSF gene was detected on chromosome 4. Further interspecies synteny analysis revealed that chromosome reorganization during species-speciation may lead to the escape of HSF genes from the S. cereale chromosome 4. Phylogenetic analysis revealed that S. cereale experienced more HSF gene duplications than barley and the three wheat subgenomes. Expression analysis demonstrated that S. cereale HSF genes showed diverse expression patterns across plant developmental stages and upon drought and freezing treatment, suggesting functional diversity of the gene family. Notably, we detected distinct expression patterns for a recently duplicated HSF gene pair, indicating functional divergence may have occurred between the two genes. The study presents the genome organization, evolutionary features and expression patterns of the S. cereale HSF genes. These results provide new insights into the evolution of HSF genes in Triticeae and may serve as a resource for Triticeae molecular breeding.

Establishment and Characterization of a Human Primary Myelofibrosis Cell Line

Aims: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by clonal proliferation of stem cells and myeloid cells. JAK2, CARL, and MPL mutations could be detected in most patients, but 15% of PMF patients could be absent of these three gene mutations. PMF has shorter median survival time and poorer prognosis when compared with closely related MPNs, polycythemia vera (PV) and essential thrombocythemia (ET). Allogenic hematopoietic stem cells (allo-HSCT) is the only curative therapy for PMF patients, however transplantation-related complications and deaths were observed in more than 50% patients. Hydroxyurea and Ruxolitinib are commonly used drugs to improve clinical symptoms of PMF patients, but lack of anti-tumor activities. These drugs could not reverse bone marrow fibrosis or induce cytogenetic remission. Basic research work of PMF is limited as to the lack of human PMF cell line. Here, we aimed to establish and characterize a patient-derived human PMF cell line, which would provide a useful tool for PMF research and screenings for novel drugs. Methods: The bone marrow cells were obtained from a 61-year-old male patient, who was diagnosed as PMF for 2 years. Mono-nuclear cells were isolated and cultured in a continuous culture system. The cells were characterized by different methods, including STR profiling, morphology observation with Giemsa staining, flow cytometry, chromosome analysis, Epstein-Barr virus (EBV) detection, mycoplasma detection, whole-genome-exon sequencing, and cell proliferation assay. Results: The cells have continued to grow in liquid culture for more than 50 passages using the same culture conditions. Doubling time was about 48-72 hours. The STR profiling identified that the cultured cells and the patient's bone marrow cells were from the same origin. The cells are free from EBV and mycoplasma. Giemsa-Wrights staining showed primitive erythrocytes (Figure A), which was consistent with CD71 positive detected by flow cytometry. Chromosome analysis revealed a hyperdiploid karyotype, which was 47, XY,+12[1]/48,idem,+9[9]. Whole-genome-exon sequencing identified mutations of ASXL1, TP53, IKZF1, IDH1, FLT3, and TET1 (Figure B). Now this cell line, designated ZYXY-M2, is collected by China center for type culture collection (CCTCC). Discussion: Established cell lines are invaluable tools in cell biology. Right now, HEL and SET-2 are two cell lines that are most widely used in MPN-related researches. HEL is an erythroleukemia cell line which was derived from a patient with leukemia transformation of Hodgkin's disease, and SET-2 is a megakaryoblastic cell line which was derived from a patient with leukemic transformation of ET. Here, we established a cell line, ZYXY-M2, derived from a PMF patient and it would prove to be a valuable model for the study of PMF. ZYXY-M2 could help to explore the pathogenesis and development of PMF, novel drug screening, and may also help to set up xenograft PMF mouse models. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Non-Age-Related Neoplastic Loss of Sex Chromosomecorrelated to Prolonged Survival in CBF-AML Patients in Real-World Clinical Setting

Introduction Since Core-binding factor acute myeloid leukemia (CBF-AML) is categorized as a favorable cytogenetic risk group, allogenic hematopoietic stem cell transplantation (HSCT) is generally not recommended during the first complete remission (CR1). However, approximately 30-50% of CBF-AML patients relapse, and about 50% of relapsed patients remain incurable. Therefore, in real-world clinical setting, the indication of HSCT even in CR1 is considered based on prognosis prediction and minimal residual disease (MRD) status. With respect to prognosis prediction by chromosomal abnormalities, the prognostic impact of loss of sex chromosome (LOS) in the patients with CBF-AML is still controversial. Previous studies suggest that the impact of LOS in predicting the prognosis of CBF-AML may depend on treatment strategies including HSCT. In addition, although CBF-AML develops LOS at a high rate of 20-40%, it is still unclear whether LOS is age-related or neoplastic. Methods Hokkaido Leukemia Net (HLN) is prospective cohort study collecting AML samples from hospitals of North Japan Hematology Study Group (NJHSG).Cases of CBF-AML that were registered in HLN from January 2010 to December 2019 were used for analysis in the present study. All the patients received either intensive or low-intensity chemotherapy. HSCT was performed at the discretion of the attending physicians even in CR1. Conventional cytogenetic analysis was performed at the time of diagnosis and after each treatment. FLT3-ITD and KIT exon17 mutations were analyzed using genome DNA template at the central laboratory. The results of genetic analysis were returned to physicians so that they could reflect the genetic result in their treatment choice. We evaluated the impacts of several factors including advanced age, poor response to chemotherapy, LOS, and KIT exon17 mutation on overall survival (OS) and relapse-free survival (RFS) in these patients. This study was conducted in compliance with ethical principles based on the Declaration of Helsinki and was approved by the institutional review board of Hokkaido University Hospital. Results A total 96 CBF-AML patients including 62 patients with RUNX1/RUNX1T1 and 34 patients with CBFβ/MYH11 were enrolled in the present study. Intensive induction chemotherapy using '7+3' cytarabine plus anthracycline and low-intensity induction chemotherapy were performed in 90 patients and 6 patients, respectively. In 90 patients who underwent intensive induction chemotherapy, the CR rate after one cycle of induction therapy was 73.3%. Total CR rate as the best response was 91.7% for all CBF-AML patients. HSCT in CR1 was performed in 15 patients (15.6%) at the discretion of the attending physician based on genetic alterations, poor response to chemotherapy, and sustained detection of MRD after chemotherapy. There was no significant difference in 5-year OS (RUNX1/RUNX1T1 64.9% vs CBFβ/MYH11 52.9%; P=0.546) or 5-year RFS (63.6% vs 53.3%; P=0.364) between the CBF subtypes. LOS was significantly more frequent in patients with RUNX1/RUNX1T1 than in patients with CBFβ/MYH11 (35.5% vs 5.9%; P=0.001). Multivariate analyses for 5-year OS in CBF-AML patients revealed that age of 50 years or older (HR: 3.46, 95% CI: 1.47-8.11, P=0.004) and receiving 2 or more induction cycles (HR: 3.55, 95% CI: 1.57-8.05, P=0.002) were independently associated with worse OS and that LOS was independently associated with better OS (HR: 0.09, 95% CI: 0.01-0.71, P=0.022). We next sought to clarify whether LOS was age-related or neoplastic based on the transition of chromosome results. All the 24 patients with LOS achieved CR, and chromosome analysis at CR showed a normal karyotype in all of the 21 patients for whom chromosome analysis was performed. In the remaining 3 patients, only FISH analysis for RUNX1/RUNX1T1 or CBFβ/MYH11 was performed. Furthermore, chromosome analysis showed mosaics of metaphase cells with and without t(8;21) in 9 patients at diagnosis. In all of those patients, metaphase cells without t(8;21) showed a normal karyotype. Conclusion In our real-world clinical setting in which '7+3' induction was performed and the indication of HSCT was determined by prognostic factors and MRD status, LOS correlated to prolonged survival in CBF-AML patients. Furthermore, the transition of chromosome results revealed that LOS was not age-related physiological loss but part of neoplastic chromosomal abnormalities. Figure 1 Figure 1. Disclosures Teshima: CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Janssen Pharmaceutical K.K.: Other; Novartis International AG: Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Gentium/Jazz Pharmaceuticals: Consultancy; Takeda Pharmaceutical Company: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co.,Ltd.: Honoraria, Research Funding; Astellas Pharma Inc.: Research Funding; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; TEIJIN PHARMA Limited: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Bristol Myers Squibb: Honoraria; Fuji pharma CO.,Ltd: Research Funding; Pfizer Inc.: Honoraria; Sanofi S.A.: Research Funding. Kondo: Astellas Pharma Inc.: Consultancy, Honoraria; Otsuka Pharmaceutical: Honoraria, Research Funding; Novartis Pharma KK: Honoraria; Bristol-Myers Squibb Company: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Sanwa Kagaku Kenkyusho CO.,LTD: Consultancy.