Whole genome sequencing analysis of small and large colony mutants from the mouse lymphoma assay.

The Thymidine kinase (Tk) gene forward mutation assay, known as the mouse lymphoma assay (MLA), has been widely used for evaluating the genotoxicity of chemical agents. A striking morphological feature of Tk mutant colonies is the bimodal distribution of their sizes, with cells from the large colonies growing at a normal rate and cells from the small colonies growing at a slower rate than normal. To understand the molecular distinction for the different growth rates, we performed whole genome sequencing (WGS) analysis of the large and small colony mutants generated from the MLA. Three large colony and three small colony mutants generated from cells treated with 4-nitroquinoline 1-oxide (4-NQO) or the vehicle control were selected for analysis. The WGS data were analyzed for loss of heterozygosity (LOH) and chromosome copy number along chromosome 11, where the Tk gene is located. Although there were LOH alterations in both large and small colony mutants, copy number changes near Tk locus were found only in small colony mutants produced by the vehicle control and 4-NQO treatments. The chromosome copy number in the regions near the Tk locus increased from two to three or four in the spontaneous small colony mutants and decreased from two to one in the 4-NQO-induced small colony mutants. These results suggest that chromosome damage was repaired differently in the large and small colony mutants, resulting in significant chromosome alterations in the small colony mutants, but not in the large colony mutants. Thus, chromosome alterations near the Tk locus may play a major role in the inhibition of cell growth in the Tk small colony mutants.

Histological and molecular glioblastoma, IDH-wildtype: a real-world landscape using the 2021 WHO classification of central nervous system tumors.

Introduction: Glioblastoma (GBM), the most lethal primary brain malignancy, is divided into histological (hist-GBM) and molecular (mol-GBM) subtypes according to the 2021 World Health Organization classification of central nervous system tumors. This study aimed to characterize the clinical, radiological, molecular, and survival features of GBM under the current classification scheme and explore survival determinants. Methods: We re-examined the genetic alterations of IDH-wildtype diffuse gliomas at our institute from 2011 to 2022, and enrolled GBMs for analysis after re-classification. Univariable and multivariable analyses were used to identify survival determinants. Results: Among 209 IDH-wildtype gliomas, 191 were GBMs, including 146 hist-GBMs (76%) and 45 mol-GBMs (24%). Patients with mol-GBMs were younger, less likely to develop preoperative motor dysfunction, and more likely to develop epilepsy than hist-GBMs. Mol-GBMs exhibited lower radiographic incidences of contrast enhancement and intratumoral necrosis. Common molecular features included copy-number changes in chromosomes 1, 7, 9, 10, and 19, as well as alterations in EGFR, TERT, CDKN2A/B, and PTEN, with distinct patterns observed between the two subtypes. The median overall survival (mOS) of GMB was 12.6 months. Mol-GBMs had a higher mOS than hist-GBMs, although not statistically significant (15.6 vs. 11.4 months, p=0.17). Older age, male sex, tumor involvement of deep brain structure or functional area, and genetic alterations in CDK4, CDK6, CIC, FGFR3, KMT5B, and MYB were predictors for a worse prognosis, while MGMT promoter methylation, maximal tumor resection, and treatment based on the Stupp protocol were predictive for better survival. Conclusion: The definition of GBM and its clinical, radiological, molecular, and prognostic characteristics have been altered under the current classification.

Comparative genomics of Leishmania isolates from Brazil confirms the presence of Leishmania major in the Americas.

Leishmania (Leishmania) major is an important agent of cutaneous leishmaniasis, having as a vector sandflies belonging to the genus Phlebotomus. Although this species has been described as restricted to the Old World, parasites similar to L. major have been isolated from South American patients who have never travelled abroad. These parasites were named "L. major-like", and several studies have been carried out to characterise them biochemically, molecularly, and biologically. However, the phylogenetic origin of these isolates is still unknown. In the present study we characterised three L. major-like isolates, named BH49, BH121 and BH129, using comparative genomics approaches. We evaluated the presence of gene and segmental duplications/deletions and the presence of aneuploidies that could explain the differences in infectivity observed in the BH49 and BH121 isolates. All isolates presented a pattern of mosaic aneuploidy and gene copy number variation, which are common in the genus Leishmania. Virulence factors such as phosphatases and peptidases were found to have increased gene copy numbers in the infective isolate, which could explain the difference in infectivity previously observed between BH121 and BH49. Phylogenetic analyses revealed that BH49, BH121 and BH129 L. major-like grouped with L. major isolates, and suggest they were imported from the Old World in at least two independent events. We suggest that new epidemiological inquiries should also evaluate L. major infections in South America, to assess the epidemiological importance of this species in the New World.

Case Report: Identification of a de novo Missense Mutation in the F8 Gene, p.(Phe690Leu)/c.2070C > A, Causing Hemophilia A: A Case Report.

Hemophilia A is an X-linked recessive bleeding disorder caused by various types of pathological defects in the factor VIII gene (F8/FVIII). Preimplantation genetic testing for monogenic disease (PGT-M) is a powerful tool to tackle the transmission of monogenic inherited disorders from generation to generation. In our case, a mutation in F8 had passed through female carriers in a hemophilia A family and resulted in two male patients with hemophilia A. To identify the etiological genetic variants of F8, next-generation sequencing (NGS) was used for chromosome copy number variation detection, Sanger sequencing to verify mutation sites, single nucleotide polymorphism (SNP) for site amplification, and sequencing to validate the genetic linkage. Finally, a novel missense mutation, p. (Phe690Leu)/c.2070C > A, occurring in exon 13 of F8, was screened out as a pathogenic mutation. Following this, an F8 normal euploid blastocyst was transferred. At the 18th week, the pregnant mother underwent amniocentesis, NGS, Sanger sequencing, and SNP typing that further confirmed that the fetus had a healthy genotype. After delivery, a neonatal blood sample was sent for FVIII concentration detection, and the result established that the FVIII protein was rescued to a nearly average level. We first identified a new type of pathogenic mutation in F8, which has not been previously reported, selected a genetically healthy progeny for an affected family, and provided valuable knowledge of the diagnosis and treatment of hemophilia A.

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma.

OBJECTIVE: This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. METHODS: Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). RESULTS: Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification (P=0.009) or deletion (P=0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly (P=1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. CONCLUSION: Chromosomal CNVs may contribute to their transcript expression in cervical cancer.

Parasexuality and mosaic aneuploidy in Leishmania: alternative genetics.

Reproduction as identical or similar organisms in most biological systems depends on the extreme accuracy of the mitotic (and meiotic) mechanisms involved in the transmission of the genetic material to the two daughter cells. Character recombination and genotype diversification are ensured by the alternation between haploidy and diploidy, which corresponds to the most predominant model in sexually reproducing organisms. In Leishmania, the unique association of high levels of automixis and of constitutive 'mosaic aneuploidy' unexpectedly does not lead to loss of heterozygosity but constitutes an alternative for genotype recombination, hence a source of adaptability.

Putative precursor cancer cells in human colorectal cancer tissue.

Multistage carcinogenesis is an important concept in cancer biology. Each new stage is triggered by the acquisition of an additional genetic aberration, leading to clonal expansion of the cancer cell. The resulting tumor mass consists of cancer cells with all genetic aberrations, but may include precursor cells at some point of carcinogenesis. We analyzed six colorectal cancer tissues with APC, K-ras, and p53 mutations. From each sample, 40-50 areas (100x100x40microm) consisting only of cancer cells were microdissected, and genomic DNA was purified. Ratios of mutated and normal alleles were quantitated by the SNaPshot assay, a primer extension assay. In five tumor tissues, we identified cancer cell subpopulations corresponding to putative precursors, i.e., cells with mutations in one or two of the three genes. All samples were likely to be of monoclonal origin, and temporal sequences of the mutations could be deduced from the mutation patterns of putative precursors. The orders of mutation events were variable. However, the two carcinoma tissues accompanying adenoma regions started with the APC mutation, not contradicting the previous studies. The analysis also revealed considerable heterogeneity in allele ratios of one or two of the chromosomes. The current findings are promising to uncover the process of carcinogenesis directly from the tumor tissue of the patient.