First molecular-cytogenetic characterization of Fanconi anemia fragile sites in primary lymphocytes of FA-D2 patients in different stages of the disease

Background: Fanconi anemia (FA) is a chromosomal instability syndrome characterized by increased frequency of chromosomal breakages, chromosomal radial figures and accelerated telomere shortening. In this work we performed detailed molecular-cytogenetic characterization of breakpoints in primary lymphocytes of FA-D2 patients in different stages of the disease using fluorescent in situ hybridization. Results: We found that chromosomal breakpoints co-localize on the molecular level with common fragile sites, whereas their distribution pattern depends on the severity of the disease. Telomere quantitative fluorescent in situ hybridization revealed that telomere fusions and radial figures, especially radials which involve telomere sequences are the consequence of critically shortened telomeres that increase with the disease progression and could be considered as a predictive parameter during the course of the disease. Sex chromosomes in FA cells are also involved in radial formation indicating that specific X chromosome regions share homology with autosomes and also could serve as repair templates in resolving DNA damage. Conclusions: FA-D2 chromosomal breakpoints co-localize with common fragile sites, but their distribution pattern depends on the disease stage. Telomere fusions and radials figures which involve telomere sequences are the consequence of shortened telomeres, increase with disease progression and could be of predictive value

Epispadias: recent techniques

Aim: This paper presents the latest surgical approaches for epispadias treatment in the pediatric population, as well as those for adolescent and adult populations after initial failed repair in childhood.Methods: The retrospective study was conducted between March 2005 and May 2020 and included 18 patients with the mean age of 21 months (range 11-48 months) (Group A), who underwent primary epispadias repair and 15 patients with the mean age of 18 years (range 13-29 years) (Group B), who underwent redo surgery after failed epispadias repair in childhood. In Group A, the surgery was performed as a one-stage procedure using complete penile disassembly technique, while, in Group B, the surgery was done as a two-stage procedure and included complete straightening and lengthening of the penis, followed by urethral reconstruction. Penile straightening and lengthening were achieved by tunica albuginea incision and grafting. In Group A, the urethral plate was mobilized, transposed ventrally, and tubularized and augmented with vascularized preputial skin flap where needed. In Group B, the urethra was reconstructed either using the buccal mucosa graft and genital skin flaps or with tubularization of genital skin flaps. Successful treatment was defined as a functional and esthetically acceptable penis without complications.Results: The mean follow-up was 88 months (range 15-197 months). Satisfactory results were achieved in 26/33 patients. Urethral fistula occurred in 4/18 patients from Group A and in 3/15 patients in Group B and was surgically repaired after four months. Skin dehiscence occurred in eight patients, five from Group A and three from Group B. Recurrent penile curvature was observed in 2/18 patients from Group A and required surgical correction and in 2/15 patients from Group B and was mild and did not need surgical repair. Eleven patients from Group B who filled out the International Index for Erectile Function reported satisfying erectile function, sexual desire, intercourse, and overall satisfaction.Conclusion: Primary or redo epispadias repair is challenging even for experienced reconstructive urologists. Only radical surgical approach can lead to complete correction of all deformities and provide successful outcome

Dysfunctional telomeres in primary cells from Fanconi anemia FANCD2 patients

Background: Fanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity. In this study we investigated telomeric abnormalities of non-immortalized primary cells (lymphocytes and fibroblasts) derived from FA patients of the FA-D2 complementation group, which provides a more accurate physiological assessment than is possible with transformed cells or animal models. Results: We analyzed telomere length, telomere dysfunction-induced foci (TIFs), sister chromatid exchanges (SCE), telomere sister chromatid exchanges (T-SCE), apoptosis and expression of shelterin components TRF1 and TRF2. FANCD2 lymphocytes exhibited multiple types of telomeric abnormalities, including premature telomere shortening, increase in telomeric recombination and aberrant telomeric structures ranging from fragile to long-string extended telomeres. The baseline incidence of SCE in FANCD2 lymphocytes was reduced when compared to control, but in response to diepoxybutane (DEB) the 2-fold higher rate of SCE was observed. In contrast, control lymphocytes showed decreased SCE incidence in response to DEB treatment. FANCD2 fibroblasts revealed a high percentage of TIFs, decreased expression of TRF1 and invariable expression of TRF2. The percentage of TIFs inversely correlated with telomere length, emphasizing that telomere shortening is the major reason for the loss of telomere capping function. Upon irradiation, a significant decrease of TIFs was observed at all recovery times. Surprisingly, a considerable percentage of TIF positive cells disappeared at the same time when incidence of γ-H2AX foci was maximal. Both FANCD2 leucocytes and fibroblasts appeared to die spontaneously at higher rate than control. This trend was more evident upon irradiation; the percentage of leucocytes underwent apoptosis was 2.59- fold higher than that in control, while fibroblasts exhibited a 2- h delay before entering apoptosis. Conclusion: The results of our study showed that primary cells originating from FA-D2 patients display shorten telomeres, elevated incidence of T-SCEs and high frequency of TIFs. Disappearance of TIFs in early response to irradiation represent distinctive feature of FANCD2 cells that should be examined further