Short-term Lymphocyte Cultures Research Articles

Cytogenetic study of Meriz goat breeds in Iraqi Kurdistan region.

Goats are considered the leading farm animal that has a substantial role in the agricultural sector in the Kurdistan Region of Iraq. No cytological examination has been carried out on them. This experiment aims to identify the Karyotype of the local breeds of domestic goats. This experiment was conducted on the Karyotype and prepared the ideogram of Meriz goats. The determination of the relative length and centromeric index arm ratio of the chromosomes in the breed was achieved by the production of karyotypes. A total of (30)Meriz goats, consisting of (10) males and (20) females, were selected to collect blood samples for a short-term lymphocyte culture. The diploid chromosome count was observed to be (60), consisting of (29) pairs of acrocentric autosomes and one pair of allosomes, specifically the X and Y chromosomes. The acrocentric nature of the X-chromosome and the sub-metacentric nature of the Y-chromosome were identified through scientific investigation. The study observed a variation in the relative length of autosomal chromosomes in Meriz goats, with females ranging from 4.49% to 1.89% and males ranging from (4.53%) to (1.75%). The X-chromosome had a relative length of 3.96 in females, while the Y-chromosome displayed a relative length of (5.05). The findings of this karyological investigation suggest that the chromosomal composition seen in the Meriz goats under examination was within the expected range of normalcy. It is recommended that more cytogenetic analyses be conducted at the population level in order to identify individuals within the Meriz breed population who possesses numerical and/or structural chromosome abnormalities. This research is crucial for enhancing the efficiency of production and reproduction in this breed.

RABiT-III: an Automated Micronucleus Assay at a Non-Specialized Biodosimetry Facility.

Micronuclei, detected through the cytokinesis-block micronucleus assay, are valuable indicators of ionizing radiation exposure, especially in short-term lymphocyte cultures. The peripheral human blood lymphocyte assay is recognized as a prime candidate for automated biodosimetry. In a prior project at the Columbia University Center for Radiological Research, we automated this assay using the 96-well ANSI/SLAS microplate standard format and relied on established biotech robotic systems named Rapid Automated Biodosimetry Tool (RABiT). In this study, we present the application of a similar automated biotech setup at an external high-throughput facility (RABiT-III) to implement the same automated cytokinesis-block micronucleus assay. Specifically, we employed the Agilent BRAVO liquid-handling system and GE IN Cell Analyzer 6000 imaging system in conjunction with the PerkinElmer Columbus image data storage and analysis system. Notably, this analysis system features an embedded PhenoLOGIC machine learning module, simplifying the creation of cell classification algorithms for CBMN assay image analysis and enabling the generation of radiation dose-response curves. This investigation underscores the adaptability of the RABiT-II CBMN protocol to diverse RABiT-III biotech robotic platforms in non-specialized biodosimetry centers. Furthermore, it highlights the advantages of machine learning in rapidly developing algorithms crucial for the high-throughput automated analysis of RABiT-III images.

Penetrance of missense mutation of OR2AT4 and OR9G1/9 gene polymorphism increases risk factor in azoospermic patients

Objectives: In human, olfactory receptor (ORs) gene polymorphism regulate testicular development and physiological functions during spermatogenesis. However, the etiopathology of OR gene in male infertility is still restricted that required further studies. Basic mechanism involves the identification of peripheral signals based on specific chemoreceptors site that are expressed on the surface of mature spermatozoa inside the female reproductive tract. These receptors belong to G-protein couple receptors (GPCR) of superfamily and the roles of ORs gene linked to sperm chemotaxis through chemokinesis. In the present study, we have studied the role of Olfactory receptor family 2 subfamilies AT member 4 (OR2AT4) and Olfactory receptor family 9 subfamily G member 1/9 (OR9G1/9) genes polymorphism in clinically diagnosed azoospermic patients. Materials and Methods: Systematically, GTG banded karyotypes were developed using short term lymphocyte cultures, fluorescence in situ hybridization using SRY probe and PCR based microdeletion of Y-chromosome was characterized using STS marker (SY255). Whole exome sequencing (WES) based analysis was performed in azoospermic patients to identify the involvement of de-novo mutation in OR2AT4 and OR9G1/OR9G9 genes mutation followed by bioinformatics tools used for the analysis to identify changes in respective amino acids. Results: OR2AT4 showing missense mutation after substitution of nucleotide (G→A) followed by lack of change in amino acid (Gln→Gln), but in OR9G1/G9 gene showing changes of substitution of nucleotide thymidine into adenine (T→A), whereas valine is replaced by glutamine (Val→ Glu), similarly thymidine is substituted by cysteine (T→C), after decode phenylalanine is replaced by leucine (Phe→Leu). Further, 3D protein helical structure was developed with the help of bioinformatics on the basis of amino acids coded protein and compare with mutated amino acid or truncated protein residues with the help of iTASSER server. Conclusion: Present study demonstrate the role of OR2AT4 and OR9G1/9 gene polymorphism elucidates the functional role of protamine in ectopic receptors followed by increasing “risk” of developing male infertility that leads the clinical condition defined as non-obstructive azoospermia.

Valuating of sister chromatid exchange (SCE) in patients with type 2 diabetes mellitus

This study was established to test the cytogenetic parameter of sister chromatid exchange (SCE) in type 2 diabetic patients for its close relationship with mutations. In this study, cytogenetic analyses were done on peripheral blood lymphocytes obtained from 25 type 2 DM patients using short-term lymphocyte culture .Human lymphocytes have long been used to assess the gene damage occurring in vivo as well as in vitro. Results showed significant high frequencies of SCEs were scored in type 2 DM patients [2.25±0.08SCE/cell] compared with control [1.28±0.04SCE/cell] (P<0.01). We concluded that the DM patients have significantly more genetic damage (high SCE frequency), and there are no significant differences in diabetic patients in relation with demographic characteristics (age and gender).

Early Diagnosis of 60, XX/XY Chimerism in Cattle using RT-PCR

Background: Freemartinism is a special kind of cell chimerism occuring in female calf born co-twin with male calf. It is one of the most common forms of intersexuality found in cattle, arises as a consequence of vascular anastamosis between fetuses of different genders. Freemartinism causes alterations in the development of female reproductive organs and sterility. However, there were reports of fertile heterosexual twins also. Hence, the present study aimed at early diagnosis of 60, XX/XY chimerism in heterosexual pregnancies to prevent the unnecessary economic losses and preserve valuable genetic material. Methods: Chromosomal profile of female and male calves were analyzed through short term lymphocyte culture technique. The gene expression levels of ZFX and SRY genes were determined through SYBR green real-time PCR. Result: Both female and male co-twins were found to possess 60, XX/60, XY chimerism, yielding the PCR products of both the male (SRY) and female (ZFX) specific genes. A real-time PCR revealed higher levels of ZFX gene expression in female (81.66 per cent) and male (88.35 per cent) calves than SRY gene. Combination of karyotyping and real-time PCR gave a confirmative diagnosis of freemartinism, suggesting the early diagnosis soon after birth.

Cytogenetic study of type 2 diabetes mellitus

Type 2 of diabetic mellitus (DM) is characterized by chronic hyperglycemia with abnormalities of fat and protein metabolism resulting from defects in insulin secretion, insulin action or both. To understand cytogenetic changes associated with type 2 diabetic mellitus, cytogenetic analysis was done on peripheral blood lymphocytes collected from twenty five type 2 DM patients using short term lymphocyte culture, and 15 apparently healthy people with as control group. The present study includes evaluation the cell cycle changes by determining blast and mitotic indices (BI and MI), as well as study the cellular kinetics by scoring replicative index (RI). The present data revealed significant depressions in BI [41.69±2.28] in patient's group compare with healthy group [54.46±4.56] and in MI [0.52±0.04] compare with healthy group [1.2±0.11] (P<0.01)]. The rate of cell cycle progression was also a reduction in diabetic patients [1.44± 0.10] in comparison with control [1.88± 0.01](P<0.05). We conclude that cytogenetic indices (BI, MI, and RI) are sensitive tools to achieve the cytogenetic abnormalities in type-2 diabetic mellitus

Cytogenetic Characterisation of Nattukuttai - A Non-descript Cattle Population of Tamil Nadu

Background: Nattukuttai is a small-sized cattle population, native to north-eastern districts of Tamil Nadu. In light of the ongoing research on genetic characterisation of cattle genetic resources of India, the present study was undertaken with the objective of cytogenetic characterisation of Nattukuttai cattle, which is imperative for its conservation and genetic implications to breeding programs. Methods: Blood samples from ten Nattukuttai cattle (five males and five females) were utilized to study the chromosome profile through short-term lymphocyte culture method. Good metaphase spreads were selected for estimation of the relative length, arm ratio, centromeric index and morphological index. Result: The diploid number was 60. All the 29 pairs of autosomes and Y-chromosome were acrocentric while X-chromosome was sub-metacentric. The mean relative length of autosomes ranged from 5.24 ±0.08 to 1.90±0.06. X-chromosome was the largest in the karyotype (5.64±0.12), while the Y-chromosome was the smallest (1.85±0.03). The arm ratio, centromeric index and morphological index were 1.98±0.02, 0.33±0.03 and 4.06±0.4 respectively. The study revealed that the chromosome architecture of Nattukuttai cattle was similar to that of other breeds of Zebu cattle.

Profiling of Chromosomal Complement in Nandidurga Goats of Karnataka

Background: To characterize the chromosomal complement of Nandidurga goats. Methods: Blood samples for short term lymphocyte culture were collected from 5 bucks and 5 does from its breeding tract and then mitotic chromosomal spreads were accomplished. Result: The diploid chromosome number was found to be 60, consisting of 58 acrocentric autosomes and 2 sex chromosomes (X and Y). The X chromosome was found to be the longest acrocentric and Y chromosome was found to be sub metacentric. The mean mitotic drive was 64.5±2.01 and 63.15±1.30 percent in bucks and does, respectively. The mean relative length of autosomes varied from 2.08±0.24 to 4.81±0.12 in does and 2.09±0.09 to 4.70±0.11in bucks. The relative length of X chromosome in does was 5.14±0.17 and that in bucks was 4.92±0.21, whereas Y chromosome had a relative length of 1.99±0.20. The mean Arms Ratio, Centromeric Index and Morphological Index were 2.47, 31.62 and 396.14, respectively. This cytogenetic analysis indicates the normal chromosomal complement in the studied Nandidurga goats.

Improving Genetic Testing in Hereditary Cancer by RNA Analysis: Tools to Prioritize Splicing Studies and Challenges in Applying American College of Medical Genetics and Genomics Guidelines

RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants.

Identification and copy number profiling of sex chromosome specific gene fragments in crossbred pigs with numerically normal karyotype

We examined the copy number profile of sex chromosome specific genes in crossbred pigs with numerically normal karyotype. A total of 30 (15M + 15F) Landlly (Landrace crossbred) pigs from a research farm were cyto-screened by karyotyping to determine the number of chromosomes per diploid cell using short-term peripheral lymphocyte culture technique. All pigs had numerically normal karyotype with 38, XX (female) and 38, XY (male). Genomic DNA was extracted from cyto-screened pigs of both sexes. A set of sex chromosome (X and Y) specific single copy gene fragments along with an autosomal gene fragment were selected out of 12 pairs of primers on the basis of male specific PCR amplification, PCR and qPCR specificity. Quantitative real-time PCR was performed to study the relative copy number change of selected Phosphate repressible alkaline phosphatase X linked (PHOX) gene, using the Eukaryotic Translation Initiation Factor 1A Y-Linked (EIF1AY) gene as control and Breast Cancer Metastasis-Suppressor 1-Like (BRMS1 L- autosomal gene) as the reference gene. The relative copy number of the PHOX gene in females was found to be 1.873 times higher compared with the EIF1AY gene in males. The present study indicates that the real-time quantitative real-time PCR based copy number analysis can be helpful for the detection of sex chromosome ratio in pigs to aid as a preliminary screening for numerical sex chromosomal aneuploidies, adding to the rate of throughput of traditional cyto-screening.

EFFECTS OF FEMALE BLOOD AUTOSERUM ON ALLOGENIC INTERACTIONS IN SHORT-TERM LYMPHOCYTE CULTURES OF PARENTS HAVING CHILDREN WITH CONOTRUNCAL HEART MALFORMATIONS

Highlights The findings of this original study ensure the detection of violations in the humoral regulation of the maternal immune interactions with semiallogeneic fetus, considered as a risk factor for developing sporadic conotruncal heart malformations in the next generation.Aim To study the role of female autoserum blood in limiting allogeneic interactions in short-term lymphocyte cultures of parents having children with conotruncal heart malformations.Methods 21 married couples (the study group) with children suffering from conotrucnal heart malformations (Tetralogy of Fallot) without chromosomal diseases were examined. The control group consisted of 21 families with three or more healthy children. The immune response in a mixed lymphocyte culture of parents was assessed by the increase in HLA-DR expression in the mixed culture with respect to spontaneous lymphocyte cultures. Primary staining of female and male lymphocytes with monoclonal antibodies to CD45, conjugated with various fluorescent dyes (PC-5 and PC-7), allowed assessing the immune response of female lymphocytes to male and vice versa.Results The effects of female autoserum on the mixed lymphocyte culture of parents were assessed. The obtained results reported that the birth of children with conotruncal heart malformations is associated with the interfering effect of female autoserum on HLA-DR expression on subpopulations of female lymphocytes (CD3+, HLA-DR+) and the activating effect on subpopulations of female lymphocytes (CD3-, HLA-DR+). The observed role of female autoserum in the study group may be associated with the absence of HLA-DR-blocking autoantibodies and high synthesis of cytokines by T2 and T3 helper lymphocytes.Conclusion The effects of female autoserum on allogeneic lymphocyte interactions of parents may be observed in short-term mixed lymphocyte cultures. The evaluation of the activating and interfering effects ensures timely identification of any violations in the humoral regulation of the maternal immune interactions with the HLA semiallogenic fetus, considered as a risk factor for developing sporadic conotruncal heart malformations in the next generation.

PECULIARITIES OF ALLOGENIC INTERACTIONS IN THE SHORT-TERM CULTURE OF LYMPHOCYTES OF SPOUSES WHO HAVE CHILDREN WITH CONGENITAL HEART DISEASES OR EARLY REPRODUCTIVE LOSSES

We have studied HLA allogeneic interactions in short-term cultures of lymphocytes from the parents having children with congenital heart defects (CHD), or subject to early reproductive losses. Twentyone married couples (CHD as the main group) who had children with sporadic CHD (interventricular septal defect) without chromosomal diseases were observed. Fifty married couples (a comparison group) had two or more reproductive losses in early gestation (up to 9 weeks), denoted as PNPs (miscarriages, missed abortions, habitual miscarriages. Forty-one families with three or more healthy children represented a control group. Immune response in cell cultures was evaluated by increasing expression of HLA-DR in a mixed culture, as compared to spontaneous lymphocyte cultures. Initial labeling of female and male lymphocytes with monoclonal antibodies to CD45 conjugated to different fluorescent dyes (PC-5 and PC-7) allowed us to evaluate the immune response of female lymphocytes to males and vice versa. The suppressor effect of autologous female serum upon the mixed culture of the lymphocytes of the spouses was also evaluated. Results of the present study showed a difference in HLA allogeneic interactions in the short-term culture of lymphocytes registered for spouses with reproductive losses and children with congenital heart defects. Reproductive losses were associated with a low blocking effect of female auto-serum upon allogeneic HLA interactions in the short-term culture of the lymphocytes of the spouses. Congenital heart defects were associated with high activity of female B-lymphocytes (CD3-/HL-DR+) in short-term mixed culture of lymphocytes from the spouses.

Mitochondrial Morphological and Functional Reprogramming Following CD137 (4-1BB) Costimulation.

T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of T lymphocytes. Herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8+ T cells. Such mitochondrial changes increased T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in tumor-reactive CD8+ T cells from cancer-bearing mice were invigorated by agonist mAb to CD137, whereas mitochondrial baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD137 was critically dependent on OPA-1 expression in transferred CD8+ T cells. Moreover, stimulation of CD137 with CD137 mAb in short-term cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD137 costimulation of T cells. Cancer Immunol Res; 6(7); 798-811. ©2018 AACR.

In vitro study of human lymphocytes cytological and biochemical effects by zingiberene

In this study, the cytological and biochemical effects of zingiberene (ZBN) on human lymphocytes cultures were investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were used for viability and cytotoxic evaluations. Micronucleus (MN) and chromosomal aberration (CA) tests were used for genetic evaluations. Moreover, total antioxidant capacity (TAC) and total oxidative status (TOS) analyses were used for biochemical evaluations. Utilizing the MTT and LDH assays, the cytotoxicity of ZBN was determined on lymphocyte cultures and the short-term lymphocyte cultures were incubated with various doses of the ZBN; the results demonstrated that the growth of lymphocytes cells was inhibited in a dose-dependent manner. In addition, MNs and CAs in lymphocytes were not influenced by exposure to ZBN. Moreover, ZBN treatment caused increases in TAC levels in human lymphocytes without changing TOS levels. In conclusion, ZBN could be used as a suggested natural antioxidant for therapeutic, pharmaceutical and food applications.

Nuclear Anomalies, Chromosomal Aberrations and Proliferation Rates in Cultured Lymphocytes of Head and Neck Cancer Patients

Head and neck cancers (HNC) are extremely complex disease types and it is likely that chromosomal instability is involved in the genetic mechanisms of its genesis. However, there is little information regarding the background levels of chromosome instability in these patients. In this pilot study, we examined spontaneous chromosome instability in short-term lymphocyte cultures (72 hours) from 72 study subjects - 36 newly diagnosed HNC squamous cell carcinoma patients and 36 healthy ethnic controls. We estimated chromosome instability (CIN) using chromosomal aberration (CA) analysis and nuclear level anomalies using the Cytokinesis Block Micronucleus Cytome Assay (CBMN Cyt Assay). The proliferation rates in cultures of peripheral blood lymphocytes (PBL) were assessed by calculating the Cytokinesis Block Proliferation Index (CBPI). Our results showed a significantly higher mean level of spontaneous chromosome type aberrations (CSAs), chromatid type aberration (CTAs) dicentric chromosomes (DIC) and chromosome aneuploidy (CANEUP) in patients (CSAs, 0.0294±0.0038; CTAs, 0.0925±0.0060; DICs, 0.0213±0.0028; and CANEUPs, 0.0308±0.0035) compared to controls (CSAs, 0.0005±0.0003; CTAs, 0.0058±0.0015; DICs, 0.0005±0.0003; and CANEUPs, 0.0052±0.0013) where p<0.001. Similarly, spontaneous nuclear anomalies showed significantly higher mean level of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) among cases (MNi, 0.01867±0.00108; NPBs, 0.01561±0.00234; NBUDs, 0.00658±0.00068) compared with controls (MNi, 0.00027±0.00009; NPBs, 0.00002±0.00002; NBUDs, 0.00011±0.00007).The evaluation of CBPI supported genomic instability in the peripheral blood lymphocytes showing a significantly lower proliferation rate in HNC patients (1.525±0.005552) compared to healthy subjects (1.686±0.009520 ) (p<0.0001). In conclusion, our preliminary results showed that visible spontaneous genomic instability and low rate proliferation in the cultured peripheral lymphocytes of solid tumors could be biomarkers to predict malignancy in early stages.

Cytogenetic characterization of vizianagaram sheep

Cytogenetic studies were conducted on a total of 60 Vizianagaram sheep maintained at the Livestock Research Station, Garividi and sheep managed by farmers in and around Srikakulam District of Andhra Pradesh. Short term lymphocyte culture technique was adopted. The diploid chromosome number was found to be 54 (2n = 54, XY). The first three pairs of the autosomes were submetacentric and the remaining 23 pairs of autosomes were acrocentric. The X chromosome was the longest acrocentric, while the Y- chromosome was the smallest biarmed. The mean relative length for the autosomes ranged from 1.54 ± 0.04 to 9.59 ± 0.12 per cent, While the mean relative length of X and Y chromosomes were 5.06 ± 0.02 and 1.81 ± 0.17, percent respectively. The mean arm ratios, centromeric indices and morphological indices varied from 1.31 ± 0.07 to 1.24 ± 0.07, 0.545 ± 0.04 to 0.565 ± 0.07 and 7.59 ± 0.01 to 5.36 ± 0.01, respectively.

Dioxidine-Induced Changes in Genome-Wide DNA Methylation in a Culture of Peripheral Blood Lymphocytes

We studied the effect of dioxidine on genome-wide methylation in short-term cultures of peripheral blood lymphocytes derived from healthy donors. Methylation was evaluated in lymphocytes before culturing, after 25 h in culture, and 1 h after addition of dioxidine in two concentrations (0.1 and 0.01 mg/ml). The total time in culture was 25 h. The level of methylation was assessed using methyl-sensitive single-cell gel electrophoresis ("comet assay") with additional restriction with HpaII amd MspI. Significant individual differences were found in the levels of methylation in both native cells and in cells treated with dioxidine in both concentrations. Mean group indicators of methylation did not differ before culturing and after 25 h in culture (45.28 and 44.80%, respectively). The mean group rate of methylation increased to 46.14% (p<0.001) after dioxidine treatment in a concentration of 0.01 mg/ml. Dioxidine in 0.1 mg/ml reduced the level of methylation (mean group rate 42.31%; p<0.001).

Bleomycin-induced mutagen sensitivity, passive smoking, and risk of breast cancer in Chinese women: a case–control study

It is well recognized that genetic variation as well as environmental factors modulates breast cancer risk. Deficiencies in DNA repair capacity are thought to associate with breast cancer risk. The main aim of this study was to use the mutagen sensitivity assay as an indirect measure of DNA repair capacity to assess breast cancer risk and the relationship between passive smoking and breast cancer risk among women in China. We carried out a case-control study, involving 196 Chinese patients with breast cancer and 211 controls without the disease and with no history of cancer. We investigated the association between mutagen sensitivity and breast cancer risk using bleomycin as the mutagen. Mutagen sensitivity was measured by quantifying the chromatid breaks induced by mutagens in short-term cultures of peripheral blood lymphocytes. Nonparametric tests and the Fisher's exact test were used to determine the statistical significance of the crude case-control comparisons, followed by logistic regression to adjust for important covariates. The mean number of bleomycin-induced breaks per cell was 0.81 for cases compared with 0.73 for the controls (p = 0.016). A greater number of bleomycin-induced chromosomal breaks per cell was associated with an increased risk of breast cancer (adjusted odds ratio of 1.82, p trend <0.01). The association between bleomycin sensitivity and breast cancer risk was greater for women who were exposed to tobacco smoke (passive smokers). The combination of bleomycin sensitivity and exposure to tobacco smoke increased risk further; women passive smokers with high sensitivity to bleomycin had a 2.77-fold increased risk of breast cancer. Our data indicate that increased bleomycin-induced mutagen sensitivity is significantly associated with an increased risk of breast cancer among Chinese women. Exposure to passive smoke is also associated with increased breast cancer risk, and the correlation is even greater for women with both longer passive exposure to tobacco smoke and high sensitivity to bleomycin.

Cytogenetic study in natural hybrids of Callithrix (Callitrichidae: Primates) in the Atlantic forest of the state of Rio de Janeiro, Brazil

In the Atlantic forest of Rio de Janeiro, Callithrix aurita (É. Geoffroy in Humboldt, 1812) is a native species vulnerable to extinction and C. jacchus (Linnaeus, 1758) and C. penicillata (É. Geoffroy, 1812) are invasive species. The major threats to the native species are habitat degradation and hybridization, although there are currently no genetic data about natural hybrids available. Previous studies have revealed that species of the Callithrix genus are extremely homogeneous in their karyotypes with the exceptions of the morphology and size of the Y chromosome and its nucleolar organizer region (NOR) banding pattern. Three male marmosets captured in the wild in Guapimirim municipality, Rio de Janeiro, Brazil, considered as possible hybrids between C. aurita and C. jacchus or C. penicillata on the basis of pelage pattern, were cytogenetically studied. Metaphase chromosomes were obtained by using short-term lymphocyte cultures and Ag-NOR staining was performed. The hybrids karyotypes were 2n=46, 14 uni- and 30 bi-armed autosomes, a median size submetacentric X and NOR bearing autosomes, being compatible with that observed for the genus. In the three individuals studied, Y chromosomes were similar to those found for C. aurita, without NORs. The data obtained suggest the involvement of C. aurita in natural hybridization with one of the invasive species. We discuss the possible consequences of this hybridization.

A genetic variant near the PMAIP1/Noxa gene is associated with increased bleomycin sensitivity

Mutagen sensitivity, a measurement of chromatid breaks induced by various mutagens in short-term cultures of peripheral blood lymphocytes, is an established risk factor for a number of cancers and is highly heritable. The purpose of this study is to identify genetic predictors of mutagen sensitivity. Therefore, we conducted a multi-stage genome-wide association study. The primary scan analyzed 539,437 autosomal SNPs in 673 healthy individuals, followed by validations in two independent sets of 575 and 259 healthy individuals, respectively. One SNP, rs8093763, on chromosome 18q21 showed significant association with bleomycin (BLM) sensitivity (combined P = 2.64 × 10⁻⁸). We observed significantly lower BLM-induced chromotid breaks for genotypes containing wild-type allele compared with the homozygous variant genotype in the discovery set (0.71 versus 0.90, P= 3.77 × 10⁻⁵) and in replication phase 1 (0.61 versus 0.84, P= 7.00 × 10⁻⁵). The result of replication phase 2 was not statistically significant (0.65 versus 0.68, P= 0.44). This SNP is approximately 64 kb from PMAIP1/Noxa, which is a radiation-inducible gene and exhibits higher expression in BLM-sensitive lymphoblastoid cell lines than insensitive cell lines upon BLM treatment. In conclusion, we identified a biologically plausible genetic variant on 18q21 near the PMAIP1/Noxa gene that is associated with BLM sensitivity.