Role of Interphase FISH Assay on Air-Dried Smears in Identifying Specific Structural Chromosomal Abnormalities among Pediatric Patients with Acute Leukemias.

Leukemia-associated structural chromosomal abnormalities (SCA) can be identified either by karyotyping or interphase-fluorescence in-situ hybridization (i-FISH) assays. Both karyotyping and i-FISH on mononuclear cell suspension are time, resource, and manpower-consuming assays. In this study, we have compared the results of specific leukemia-associated SCAs identified by i-FISH on air-dried bone marrow (BM)/peripheral blood (PB) smears and BM karyotyping. The study was conducted among pediatric patients (age ≤ 18 years) diagnosed with acute leukemias between January 2018 to December 2022. The results of i-FISH on air-dried BM/PB smears and BM-karyotyping for our SCA of interest (BCR::ABL1, ETV6::RUNX1, TCF3::PBX1, KMT2A rearrangement, RUNX1::RUNX1T1, CBFB::MYH11, and PML::RARA) were entered in a contingency table and the agreement of results was calculated. The strength of agreement was assessed by Cramer's V test. Among 270 patients, SCA of interest was identified among 26% and 17% of patients by i-FISH on air-dried smears and karyotyping, respectively. Excluding 53 patients with metaphase failure, the remaining 217 patients had 92% agreement (Cramer's V of 0.931 with p < 0.000) between the results for specific SCAs identified by both techniques. On excluding samples with cryptic cytogenetic aberrancies, there was 99% agreement (Cramer's V of 0.953 with p < 0.000) for gross SCA identified by both techniques. In addition, i-FISH on air-dried smears identified SCA in 30% of patients with metaphase failure. I-FISH on air-dried PB/BMA smears is a less-labor and  resource-consuming assay. It can be considered an efficient alternative to conventional karyotyping for  identifying specific SCA of interest in under-resourced laboratories. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01699-2.

Candidate Gene Expression in Regional Population and Its Relevance for Radiation Triage.

Quantification of gene expression signatures has been substantiated as a potential and rapid marker for radiation triage and biodosimetry during nuclear emergencies. Similar to the established biodosimetry assays, the gene expression assay has drawbacks such as being highly dynamic and transient, not specific to ionizing radiation, and also influenced by confounding factors such as gender, health status, lifestyle, and inflammation. In view of that, prior knowledge of baseline expression of certain candidate genes in a population could complement the discrimination of the unexposed from the exposed individuals without the need for individual pre-exposure controls. We intended to establish a baseline expression of reported radiation-responsive genes such as CDKN1A, DDB2, FDXR, and PCNA in the blood samples of healthy human participants and then compare it with diabetic/hypertension participants (as a chronic inflammatory condition) drawn from south Indian population. Further, we have examined the appropriateness of the assay for radiation triage-like situations; i.e., the expression profiles of those genes were examined in the participants who underwent X-ray-based medical imaging. Acute inflammation induced by lipopolysaccharide exposure in the blood significantly increased the fold expression of those genes (p < 0.0001) compared to the control. Whereas the basal expression level of those genes among the participants with the inflammatory condition is marginally higher than those observed in the healthy participants; despite the excess, the fold increase in those genes between the groups did not differ significantly. Consistent with the inflammatory participants, the basal expression level of those genes in the blood sample of participants who received X-radiation during neuro-interventional and computed tomography imaging is marginally higher than those observed in the pre-exposure of respective groups. Nevertheless, the fold increase in those genes did not differ significantly as the fold change fell within the two folds. Thus, overall results suggest that the utility of CDKN1A, DDB2, FDXR, and PCNA gene expression for radiation triage specific after very low-dose radiation exposure needs to be interpreted with caution for a much more reliable triage.

Mercaptopurine induced myelosuppression in a child with a NUDT15 rs116855232 homozygous variant.

INTRODUCTION: Mercaptopurine (6-MP) is the backbone of the consolidation and maintenance therapy for paediatric acute lymphoblastic leukaemia (ALL). Nevertheless, it can cause critical myelosuppression. Predicting adverse reactions to 6-MP often involves the investigation of pharmacogenetic variants; in particular thiopurine S-methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15). Lately, NUDT15 variants have been shown to play a significant pharmacogenetic role in predicting 6-MP intolerance in children of Asian descent. CASE REPORT: We present a six-year-old male child of Indian origin with persistent cytopenia after treatment. This prompted targeted sequencing of the genes TPMT and NUD15. The results revealed two copies of the variant of NUD15 rs116855232, that is, NUDT15*2 genotype. MANAGEMENT AND OUTCOME: Since the NUDT15*2 allele classified the patient as a poor metabolizer, he was restarted on a low dose of 6-MP, which he tolerated. DISCUSSION: Individuals with the NUDT15*2allele (*2/*2 genotype) are poor metabolizers of thiopurines which results in an adverse reaction to 6-MP. About 3.5% of Indians show variations in the TPMT gene as compared to 19.4% variations observed in NUDT15, which makes the latter a more reliable disease marker.

DHEA and polycystic ovarian syndrome: Meta-analysis of case-control studies.

BACKGROUND: Polycystic ovarian syndrome is a heterogenous endocrine disorder characterized by irregular menstrual cycles, hirsuitism and polycystic ovaries. It is further complicated by metabolic syndrome, infertility and psychological stress. Although the etiopathogenesis is unclear, many studies have pointed out the role of stress in this syndrome. DHEA, being a stress marker is being used by scientists to compare the stress levels between polycystic ovarian cases and healthy controls. However, the results obtained from previous studies are equivocal. OBJECTIVE: To perform meta-analysis and find the association between stress and the syndrome. DATA SOURCES: Relevant data till January 2021 were retrieved from PubMed, Scopus, Embase and Web of Science using MeSH terms. STUDY SELECTION: Case-control studies having PCOS subjects as cases and healthy women as controls were selected provided; their basal DHEA levels were mentioned in the published articles. DATA EXTRACTION: Two authors independently extracted the articles and qualified the final studies. DATA SYNTHESI: Pooled meta-analysis was done using random effect model and showed level of DHEA statistically significant in PCOS compared to healthy controls (SMD = 1.15, 95% CI = 0.59-1.71).Heterogeneity was statistically significant as well (I2 = 95%). CONCLUSION: Thismeta-analysis on DHEA and PCOS has helped in generating evidence regarding the involvement of stress in the pathogenesis of PCOS.

Cytogenetic and Fluorescence in situ Hybridization Profile of Pediatric Acute Lymphoblastic Leukemia in a University Hospital in South India.

OBJECTIVE: The purpose of this study was to evaluate the cytogenetic and fluorescent in situ hybridization (FISH) profile in children with acute lymphoblastic leukemia (ALL), referred to a university hospital in a 5-year 6-month period. SUBJECTS AND METHODS: Cytogenetic analysis of the bone marrow aspirate specimens of 91 patients was performed by standard Giemsa (G)-banding and interphase FISH (iFISH). RESULTS: The frequency of chromosomal abnormalities detected by G-banding was 29.5%, and the frequency of nonrandom abnormalities with independent prognostic significance identified by iFISH was 46.4%. The abnormality with the highest frequency was gain of RUNX1 (n = 18, 21.4%), followed by ETV6/RUNX1 fusion (n = 7, 8.3%), and gain of KMT2A (n = 6, 7.1%). Additionally, rarely reported gains of ETV6, PBX1, and ABL1 were observed at a frequency of 6% (n = 5), and the deletion of ETV6 and TCF3 was seen at a frequency of 3.6% (n = 3) and 2.3% (n = 2), respectively. A 10-year old with intrachromosomal amplification of chromosome 21 was also observed. CONCLUSIONS: This study strengthens and widens the current knowledge of the cytogenetic landscape of pediatric ALL.

Cortisol and polycystic ovarian syndrome - a systematic search and meta-analysis of case-control studies.

BACKGROUND: Polycystic ovarian syndrome (PCOS) has become an emerging disorder affecting women of reproductive age group. Its intricate presentation of signs and symptoms makes it a disease of interest to research. While there are varied hypotheses related to its cause and pathogenesis, role of stress in this disease is yet to be grounded. There is emerging body of evidence on cortisol and PCOS, although it is currently equivocal. METHODS: Medline, Embase, Pubmed, Science Direct, Google Scholar, and Scopus were searched from March 1985 to March 2020 using MeSH terms. After dual quality assessments and data abstraction, the final articles were included for meta-analysis. RESULTS: Forty-one studies qualified for the analysis. Pooled meta-analysis showed that the level of cortisol was significantly higher in PCOS when compared to healthy controls (standard mean difference [SMD] = 0.83, 95% confidence interval [CI] = 0.42-1.23) with highly significant heterogeneity (I2 = 94%). Subgroup analysis done based on type of sample stated high effect size for blood cortisol levels (SMD = 0.9, 95%CI = 0.32; 1.51) compared to overall effect. CONCLUSIONS: This systematic review and meta-analysis on cortisol and PCOS have helped in generating evidence regarding the role of cortisol in the pathogenesis of PCOS and the use of cortisol estimation as a potential stress marker in PCOS.

Blast size-specific flowcytometric ploidy assessment using FxCycleTM Violet dye and its correlation with conventional cytogenetic ploidy in pediatric precursor B-lineage acute lymphoblastic leukemia patients.

INTRODUCTION: Numerical chromosomal abnormalities (aneuploidies), present in approximately 30%-50% of pediatric precursor B-lineage acute lymphoblastic leukemia (B-ALL) patients, are commonly identified through a laborious conventional cytogenetic (CG) technique. Flow cytometry (FCM) can identify both physical and fluorescent properties of cells together, and by using fluorescent nucleic-acid-binding dyes, FCM can identify variations in total nucleic-acid content of cells. FxCycleTM Violet dye (FxCV) is a selective DNA-binding dye which permits simultaneous multiparametric immunophenotyping and cell-cycle/ploidy assessment in a single assay. To date, only two studies have demonstrated the feasibility of FxCV-aided FCM-ploidy analysis in B-ALL patients and only one of these studies have compared their results with CG-ploidy. METHODOLOGY: Blast size-specific FCM-ploidy was prospectively analyzed using FxCV-dye in 109 pediatric B-ALL patients, and the results were compared with concurrent CG-ploidy status. RESULTS: FCM-ploidy categorization was feasible in 98% of samples tested and the results were 82% concordant with CG-ploidy status. We observed significant correlation between DNA content and blast size (r = .823, P < .001) and could demonstrate size differences between diploid vs low-hyperdiploid (P = .025), diploid vs high-hyperdiploid (P < .001) and low- vs high-hyperdiploid blasts (P = .007). CONCLUSION: FCM-ploidy assessment using FxCV dye is a reliable assay and the results closely concur with CG-based ploidy stratification and risk assessment. Using blast size-assisted DNA content analysis, the results of FCM-ploidy analysis can be further fine-tuned.

Meta-analysis of association between il-6-174 g/c polymorphism and female infertility related disorders.

Female infertility continues to increase in prevalence annually and factors causing it need to be researched. As IL-6-174 G/C polymorphism is known to alter the plasma levels of IL-6, abnormal levels of IL-6 found in infertile females could be due to genetic reasons. With the understanding of the importance of IL-6 in reproductive physiology, several individual studies done so far to find the association of this polymorphism with female infertility related disorders were systematically combined for meta-analysis. Articles were searched using electronic data base sources and were included based on specific criteria. Finally, eight articles which includes polycystic ovarian syndrome (PCOS; n = 4), endometriosis (n = 3) and tubal damage (n = 1) were selected for the analysis. Results showed statistically significant heterogeneity across studies under the allele model (p < 0.0001, I2 = 78 %) and dominant model (p < 0.00001, I2 = 82%) but not under recessive model (p = 0.31, I2 = 16%). This difference could be possibly due to variation in ethnicity, lifestyle, age or BMI related factors. The pooled odds ratio under the three genetic models were 0.87(CI = 0.75-1.02), 0.77 (CI = 0.63-0.94) and 1.05 (CI = 0.76-1.46) respectively. Sub group analysis showed statistical significant (P < 0.01) for PCOS under allele and dominant model, but not for endometriosis and tubal damage. By this meta-analysis, we can say that IL-6-174 G/C polymorphism can be considered as a potential genetic marker for PCOS but not for endometriosis and tubal damage disorders. However, more studies with adequate sample sizes are required to be done in endometriosis, tubal disease and other female infertility disorders to arrive at a definite conclusion.

Evaluation of Cytogenetic Abnormalities in Patients with Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) accounts for 20% of all adult leukemias and is the most common leukemia during childhood (80%). We present data on cytogenetics of ALL from a tertiary centre in India correlating it with clinical factors. Karyotyping of bone marrow samples of 204 patients with newly diagnosed ALL was performed with standard G-banding technique. Clinical data of patients was obtained from case records. Survival was estimated using Kaplan-Meir curves and compared by the log-rank test. Univariate and multivariate analysis was done for survival with age, sex, immunophenotype, hyperleukocytosis, risk type, remission status and cytogenetics. The most common karyotypes observed were normal in 39.7% (N = 81), hyperdiploidy in 12.7% (N = 26), t(9;22) in 4.4% (N = 9), t(1;19) in 3.9% (N = 8). Adults with ALL had worse survival compared with pediatric patients (HR 3.62; 2.03-6.45 95% CI, p < 0.001). Patients not in morphologic remission after induction chemotherapy fared poorly (HR 4.86; 2.67-8.84 95% CI, p < 0.001). Patients with favourable cytogenetics had better overall survival (HR 0.36; 0.12-1.05 95% CI, p < 0.05). On multivariate analysis, achievement of morphologic remission emerged as single most significant predictor of survival (p < 0.001). MLL gene rearrangement and t(12;21) were seen less commonly as compared to Western data. However, incidence rates of various cytogenetic abnormalities were similar to that reported from other centres from India. Age, morphologic remission at end of induction chemotherapy and favourable cytogenetics correlated significantly with survival.

MicroRNA 146a Polymorphisms and Expression in Indian Children with Acute Lymphoblastic Leukemia.

BACKGROUND: MicroRNAs (miR) have been reported to be involved in hematopoiesis and in the pathogenesis of several hematological malignant neoplasms. Single-nucleotide polymorphisms (SNPs) in human miR genes may alter the expression of those genes and influence the predisposition to childhood leukemia. OBJECTIVE: To evaluate the association of rs2910164 G>C, rs57095329 A>G and the expression of miRNA-146a in ethnic South Asian children with acute lymphoblastic leukemia (ALL). METHOD: Genotyping and expression analysis using TaqMan Small RNA Assay was performed on 71 patients with pathologically confirmed ALL and 74 control individuals. RESULTS: No statistically significant association was found between the 2 SNPs, its expression levels, and ALL risk. CONCLUSION: Haplotype analysis indicated a combination of allele A of rs57095329 and allele G of rs2910164 could represent a risk haplotype and an allele combination of G of rs57095329 and G of rs2910164 could represent a protective haplotype for ALL.

Association of estrogen, progesterone and follicle stimulating hormone receptor polymorphisms with in vitro fertilization outcomes.

Despite the advances in in vitro fertilization (IVF), the implantation success rate for infertile women remains approximately only 15%. In this study, we sought to determine whether implantation failure after repeated IVF treatments is influenced by the presence of common variants in estrogen α, progesterone and follicle stimulating hormone receptor genes. The study population included three groups of women: group 1 were 50 women who had the transfer of ≥3 high-quality embryos during the IVF procedure without ever having had a clinical pregnancy; group 2 were 50 women who achieved a clinical pregnancy after ≤3 high-quality embryos transfers and group 3 were 50 control subjects who achieved a clinical pregnancy without any fertility therapy that resulted in a one live-born infant. Genotype analysis was performed using polymerase chain reaction and Sanger sequencing for rs6165, rs6166, rs2234693, rs9340799. While progesterone receptor single nucleotide polymorphism (SNP)  was genotyped based on the amplicon size, the repeats for the ESR1 TA-repeat polymorphism were calculated based on the fragment length. A higher frequency of the heterozygote AG genotype was observed in the infertile groups when compared to controls. Significantly, an allele combination of T of rs2234693, A of rs9340799; S of ESR1 (TA), A of rs6166, G of rs6165 and del of PROGINS had a higher frequency in women who had a successful IVF outcome compared to women who had an unsuccessful IVF outcome, indicating a possible protective combined genotype that could reduce a negative outcome during IVF. This study has demonstrated that combining several candidate genes is needed to assess which may play a role in fertility. ABBREVIATIONS: CI: confidence interval; COH: controlled ovarian hyperstimulation; DNA: deoxyribonucleic acid; ESR: estrogen receptors; FSH: follicle stimulating hormones; FSHR: FSH receptor; IVF: in vitro fertilization; PGR: progesterone receptors; SNP: single nucleotide polymorphism.

A retrospective exploratory study of fetal genetic invasive procedures at a University Hospital.

This is a retrospective analysis of the patient demographics and cytogenetic results of patients who underwent prenatal invasive testing for genetic analysis at the Foetal Medicine Division of the Department of Obstetrics and Gynecology, Sri Ramachandra Medical College and Research Institute. The main objective of this study was to characterise the changing trends in indications of pregnant women for foetal karyotyping in a 7-year period. A total of 257 procedures were performed in this period, and there was a significant change in the trend of indications for invasive prenatal diagnosis from an advanced maternal age in 2009 to a positive screen test by 2014. Chromosome abnormalities were observed in 9.8% of the cases, with trisomy 21 being the most frequent finding. The findings demonstrate the changing trends in screening and diagnostic testing in the tertiary care centre, with an acceptance of the first and second trimester maternal serum screening tests as a determinant for high-risk pregnancies. Impact statement What is already known on this subject? Despite the fact that India has one of the world's highest birth rates, there is still no public health care policy for the application of cytogenetic prenatal diagnosis. Nevertheless, we have been offering this test in our university teaching hospital since 2008, allowing us to characterise the changing trends in indications of pregnant women who sought invasive diagnostic procedures for foetal genetic studies. What do the results of this study add? The results of our study show that there were major changes in the common indications for prenatal diagnosis during the study period. In 2009, the main indication was an advanced maternal age, referred to in 31% of the cases, which declined steadily to 5% by 2014. In 2014, 51% of cases opted for a prenatal diagnosis because of a first trimester screen positive result, increasing from 12% in 2009. What are the implications of these findings for clinical practice and/or further research? This data is relevant as it would encourage other tertiary hospitals in developing countries like India to consider extending first trimester screening for all women, regardless of age and educate them on the options of prenatal genetic diagnosis for reassurance.

Differential expression of microRNAs let-7a, miR-125b, miR-100, and miR-21 and interaction with NF-kB pathway genes in periodontitis pathogenesis.

Periodontitis is a chronic inflammatory disease which is caused by destruction of the tissues that surrounds and supports the tooth. Deregulation of microRNAs has been reported to cause several inflammatory diseases such as autoimmune disease, chronic periodontitis, and cancer. In the present study, we have investigated the expression pattern of microRNAs let-7a, miR-125b, miR-100, miR-21, and RNA-binding protein LIN-28A among healthy individuals and chronic periodontitis patients. Total RNA was isolated from gingival tissue samples collected from 100 healthy individuals and 100 chronic periodontitis patients. The expression of microRNAs and LIN-28 was performed by qPCR. Target prediction for the microRNAs was done using miRWalk and miRTarbase online databases and the experimentally validated targets were analyzed for their molecular function, biological processes, and related pathways using gProfiler software. The expression analysis revealed that let-7a and miR-21 were upregulated, whereas, miR-100, miR-125b, and LIN-28 were down regulated. The age dependent expression analysis revealed that the expression levels of all the microRNAs and LIN-28 were found to increase with age (more than 50 years), thereby suggesting an increased risk to chronic periodontitis. Among the various targets predicted using miRWalk and miRTarbase databases, NFKB was found to be a common target among all the four microRNAs. gProfiler revealed several functions such as NF-ĸB signaling pathway, cytokine-cytokine receptor interaction, osteoclast differentiation, etc., all of which specific to inflammation and periodontitis.

Genetic variation in matrix metalloproteinase MMP2 and MMP9 as a risk factor for idiopathic recurrent spontaneous abortions in an Indian population.

PURPOSE: Matrix metalloproteinases, MMP2 and MMP9, are found to have an important role during ovulation and pregnancy because of their capacity to degrade components of the extracellular matrix (ECM) thereby facilitating cell migration and angiogenesis. In this respect, the aim of the present study was to evaluate the association of the promoter polymorphisms -1306 C > T and -1562 C/T in MMP2 and MMP9 respectively with couples diagnosed with idiopathic recurrent spontaneous abortions (IRSA). The expression levels of these two genes were also studied in fetal tissue. METHODS: In this case control study, a total of 35 couples with at least three consecutive IRSA and 35 fertile couples were included. Genotype analysis was performed using polymerase chain reaction and Sanger sequencing. RESULTS: No statistically significant differences were found in distribution of MMP2-1306C/T and MMP9-1562C/T genotypes in the three groups between the cases and controls. CONCLUSION: Further genetic association studies on a larger number of IRSA couples, as well as evaluation of more MMP polymorphisms and their expression profiling are needed to establish the potential role of MMP polymorphisms in IRSA.

A Study on the Role of Estrogen Receptor Gene Polymorphisms in Female Infertility.

BACKGROUND: Female infertility is often of unknown etiology and is a significant medical problem. It occurs when implantation does not occur; a fertilized embryo fails to survive after implantation; or when the egg cannot move from the ovary to the uterus. The aim of this study was to analyze the role of estrogen receptor 1 (ESR1) genotypes in female infertility. METHODS: Blood samples were collected from 114 women with infertility undergoing infertility treatment. Samples were also collected from 115 age-matched control women with at least one live child and with no history of infertility or abortions. Genomic DNA was isolated from the blood samples, and genotyping of the ESR1 gene was performed using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The study revealed the presence of two single nucleotide polymorphisms (SNPs) in the ESR1 gene, PvuII and XbaI. Individual analyses of these two polymorphisms showed that the XbaI heterozygote was significantly increased in controls compared to cases (odds ratio-0.39, confidence interval-0.21 to 0.74, p-0.005). The combined analysis of the PvuII and XbaI genotypes showed no significant difference between the case and control samples. CONCLUSION: Analysis of the Pvull and Xba1 polymorphisms of the ESR1 gene, demonstrated that the XbaI heterozygote was significantly increased in controls indicating a protective effect.

Isolation and evaluation of cytogenetic effect of Brahmi saponins on cultured human lymphocytes exposed in vitro.

Major saponins of Brahmi (Bacopa monniera, Fam: Scrophulariaceae) - bacosides A and B - were isolated from the total methanol extract and characterised based on melting point, TLC, IR, (1)H NMR and (13)C NMR. They were evaluated for their in vitro cytogenetic effects on human peripheral blood lymphocytes by chromosomal aberration (CA) assay and sister chromatid exchange (SCE) assay. The frequency of chromatid type aberrations and reciprocal interchanges between sister chromatids in the treated cells was scored in comparison to the untreated control. At 30 µg/mL dose, bacoside A showed a statistically significant increase in the frequency of both CA and SCE and bacoside B showed an increase only in SCE. Our report of the genotoxicity of the saponins is significant in view of the reports of anticancer activity of Brahmi extracts.