BackgroundCell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases and acts upon the cells. Response to cfDNA depends on concentrations and levels of the damage within cfDNA. Oxidized extracellular DNA acts as a stress signal and elicits an adaptive response.Principal FindingsHere we show that oxidized extracellular DNA stimulates the survival of MCF-7 tumor cells. Importantly, in cells exposed to oxidized DNA, the suppression of cell death is accompanied by an increase in the markers of genome instability. Short-term exposure to oxidized DNA results in both single- and double strand DNA breaks. Longer treatments evoke a compensatory response that leads to a decrease in the levels of chromatin fragmentations across cell populations. Exposure to oxidized DNA leads to a decrease in the activity of NRF2 and an increase in the activity of NF-kB and STAT3. A model that describes the role of oxidized DNA released from apoptotic cells in tumor biology is proposed.Conclusions/SignificanceSurvival of cells with an unstable genome may substantially augment progression of malignancy. Further studies of the effects of extracellular DNA on malignant and normal cells are warranted.

Background The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in nonuniform genomic coverage. Using sequence-agnostic restriction enzymes, such as DNAse I, could help to overcome this limitation. Results In this study, we compare different DNAse Hi-C protocols and identify the critical steps that significantly affect the efficiency of the protocol. In particular, we show that the SDS quenching strategy strongly affects subsequent chromatin digestion. The presence of biotinylated oligonucleotide adapters may lead to ligase reaction by-products, which can be avoided by rational design of the adapter sequences. Moreover, the use of nucleotide-exchange enzymes for biotin fill-in enables simultaneous labelling and repair of DNA ends, similar to the conventional Hi-C protocol. These improvements simplify the protocol, making it less expensive and time-consuming. Conclusions We propose a new robust protocol for the preparation of DNAse Hi-C libraries from cultured human cells and blood samples supplemented with experimental controls and computational tools for the evaluation of library quality.

Sixty blood samples from pregnant women during gestational weeks 9-28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.

We report on a 37-year-old XX male with complex hidden X chromosomal mosaicism. The patient had fully mature male genitalia with hypoplastic testes descended in the scrotum and no sign of undervirilization. Hormonal examination demonstrated hypergonadotropic hypogonadism, semen analysis showed severe oligoasthenoteratozoospermia. In situ hybridization revealed the presence of 3 SRY-positive cell lines bearing 1, 2 or 3 X chromosomes. Skewed inactivation of the paternal SRY-bearing X chromosome was detected by molecular analysis of the androgen receptor gene.

We report on a 45,X male with hydrocephaly, lobar holoprosencephaly and ichthyosis. In situ hybridization and molecular analysis have demonstrated the presence of a mosaic SRY-bearing derivative X chromosome that included Yp and heterochromatic Yq fragments.

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Objective:We report a male patient with ovotesticular disorder of sex development (OTDSD), resulting from structurally abnormal Y chromosome. Case report: A 3-year-old boy was admitted to the Surgical Pediatric Department for masculinizing reconstruction. He had a clitorophallus, bifi d scrotum, perineal hypospadias and bilateral impalpable gonads. Pelvic ultrasound and laparoscopy showed a uterus and two gonads with primary ovarian follicles. Chromosome analysis detected a mos 47,XX,mar/46,XX karyotype. Complex genetic evaluation revealed that the marker was Yp isochromosome. Surgical care included a feminizing genitoplasty and separation of the gonads with total excision of testicular tissue. Conclusions: The presented case emphasizes the importance of a systematic approach to the investigation and management of the patients with ovotesticular DSD. It also raises the important issue about gender reassignment in intersex individuals in mid-childhood.

Background Because of the significant occurrence of “WAGR-region” deletions among de novo mutations detected in congenital aniridia, DNA diagnosis is critical for all sporadic cases of aniridia due to its help in making an early diagnosis of WAGR syndrome. Standard cytogenetic karyotype study is a necessary step of molecular diagnostics in patients with deletions and in the patients’ parents as it reveals complex chromosomal rearrangements and the risk of having another affected child, as well as to provide prenatal and/or preimplantation diagnostics. Case presentation DNA samples were obtained from the proband (a 2-year-old boy) and his two healthy parents. Molecular analysis revealed a 977.065 kb deletion that removed loci of the ELP4, PAX6, and RCN1 genes but did not affect the coding sequence of the WT1 gene. The deletion occurred de novo on the paternal allele. The patient had normal karyotype 46,XY and a de novo pericentric inversion of chromosome 11, inv(11)(p13q14). Conclusions We confirmed the diagnosis of congenital aniridia at the molecular level. For the patient, the risk of developing Wilms’ tumor is similar to that in the general population. The recurrence risk for sibs in the family is low, but considering the possibility of gonadal mosaicism, it is higher than in the general population.