Cryptic balanced chromosomal aberrations can be an underlying cause of infertility. In 2003 Cockwell and coworkers highlighted the relevance of euchromatic pericentric regions of acrocentric chromosomes that may be a yet ignored genomic region hosting cryptic rearrangements. Here we offer the first follow-up study to further explore this idea. Two specific molecular cytogenetic probe sets were established to elucidate such cryptic rearrangements together with chromosomal heteromorphisms of acrocentric centromeres. In 28 infertile couples and 20 controls, the rate of centromeric heteromorphisms was almost comparable in both groups and one heteromorphism was noted in ~30% of the cases, and two heteromorphisms in ~15% and three heteromorphisms 5%. However, none of the studied groups revealed any cryptic euchromatic pericentromeric abnormalities of the acrocentrics. Nonetheless, in parallel an infertile case with an inv(13)(p12q12.1?2) was uncovered, being not part of the systematically studied group of infertile. As unbalanced products of meiosis with such or similar karyotypes can potentially contribute to abortions, the existence of rare, cryptic pericentromeric euchromatic abnormalities in the acrocentrics thus needs to be still expected in banding cytogenetic diagnostics. Accordingly, this study reflected that suspicious acrocentric short arms in infertile need special attention and further characterization by fluorescence in situ hybridization.
The hepatic integration of human adipose tissue derived mesenchymal stem cells (hAT-MSCs) in vivo with or without prior differentiation to hepatocyte-like cells in vitro was investigated.Cells, isolated either from peritoneal or subcutaneous adipose tissue, expressed mesenchymal stem cell surface markers and featured multiple lineage differentiation. Under conditions favouring hepatocyte differentiation, hAT-MSCs gained hepatocytic functions in vitro including urea formation, glycogen synthesis, cytochrome P450 enzyme activity, and expression of hepatocyte-specific transcripts of carbamoylphosphate synthetase, albumin and cytochrome P450 type 3A4 (CYP3A4). Transgenic expression of green fluorescent protein emerged upon hepatocyte differentiation when driven by the hepatocyte-specific promoter of the cytosolic phosphoenolpyruvate carboxykinase gene but was constitutive from the ubiquitin gene promoter. Human AT-MSCs were transplanted into livers of immunodeficient Pfp/Rag2-/- mice with or without prior hepatocyte differentiation in vitro. Donor-derived human cells engrafted in the mouse host liver predominantly in the periportal region of the liver lobule. They expressed HepPar1 and albumin, typical features of differentiated human hepatocytes, in the otherwise negative mouse liver background. Engraftment was significantly more efficient using hAT-MSCs pre-differentiated to hepatocyte-like cells in vitro as compared with undifferentiated cells.Pre-differentiation of human MSCs from adipose tissue into hepatocyte-like cells in vitro facilitates long term functional hepatic integration in vivo.
Previous uranium mining in the "Wismut" region in Germany enhanced environmental distribution of heavy metals and radionuclides. Carryover effects may now lead to contamination of locally produced foods. Compounds of "Wismut" origin are probably genotoxic via their irradiating components (radon) or by interacting directly with cellular macromolecules. To assess possible hazards, we investigated the genotoxic effects of uranyl nitrilotriacetate (U-NTA) in human colon tumor cells (HT29 clone 19A), adenoma cells (LT97), and nontransformed primary colon cells. These are target cells of oral exposure to environmentally contaminated foods and represent different cellular stages during colorectal carcinogenesis. Colon cells were incubated with U-NTA. Cell survival, cytotoxicity, cellular glutathione (GSH) levels, genotoxicity, and DNA repair capacity (comet assay), as well as gene- and chromosome-specific damage combination of comet assay and fluorescence in situ hybridization [FISH], 24-color FISH) were determined. U-NTA inhibited growth of HT29 clone 19A cells (75–2000μM, 72 h) and increased GSH (125–2000μM, 24 h). U-NTA was genotoxic (1000μM, 30 min) but did not inhibit the repair of DNA damage caused by hydrogen peroxide (H2O2), 4-hydroxynonenal, and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]-pyridine. U-NTA was also genotoxic in LT97 cells and primary colon cells, where it additionally increased migration of TP53 into the comet tail. In LT97 cells, 0.5–2mM U-NTA increased chromosomal aberrations in chromosomes 5, 12, and 17, which harbor the tumor-related genes APC, KRAS, and TP53. It may be concluded that uranium compounds could increase alimentary genotoxic exposure in humans if they reach the food chain in sufficient amounts.
Molecular cytogenetic approaches applying smaller, locus-specific probes like cDNA, plasmids, cosmids, fosmids, P1 clones, bacterial artificial chromosomes (BACs), or yeast artificial chromosomes (YACs) sometimes may be hampered by inefficient hybridization. Also, especially in diagnostics, FISH results may be required within a few hours. Here a FISH protocol using microwave treatment is presented, leading to better hybridization efficiency in case of smaller probes and evaluable results within a few hours.
Xia-Gibbs syndrome (XGS) is a rare syndromic disorder characterized by developmental delay with intellectual disability, muscular hypotonia, brain anomalies, and nonspecific dysmorphic features. Different heterozygous variants in
The 4q deletion syndrome phenotype consists of growth failure and developmental delay, minor craniofacial dysmorphism, digital anomalies, and cardiac and skeletal defects. We have identified an inversion (inv(1)(q25.2q31.1)) and an interstitial deletion in a boy with developmental delay using array-comparative genomic hybridization. This de novo deletion is located at 4q31.21q31.22 (145,963,820- 147,044,764), its size is 0.9-1.1 Mb, and it contains 7 genes <i>(ABCE1, OTUD4, SMAD1, MMAA, C4orf51</i>, <i>ZNF827, </i>and<i> ANAPC10)</i> as well as 5 retrotransposon-derived pseudogenes. Bioinformatic analysis revealed that while small copy number variations seem to have no impact on the phenotype, larger deletions or duplications in the deleted region are associated with developmental delay. Additionally, we found a higher coverage in transposable element sequences in the 4q31.21q31.22 region compared to that of the expected repeat density when regarding any random genome region. Transposable elements might have contributed to the reshaping of the genome architecture and, most importantly, we identified 3 L1PA family members in the breakpoint regions, suggesting their possible contribution in the mechanism underlying the appearance of this deletion. In conclusion, this is one of the smallest deletions reported associated with developmental delay, and we discuss the possible role of genomic features having an impact on the phenotype.
Within cytogenetic preparations chromosomal breaks can be observed in patients suffering from Fanconi anemia (FA), a recessively inherited syndrome with an extremely elevated cancer risk, but also in healthy individuals as so-called fragile sites (FS). It is known that FS cytogenetically co-localize with tumor- and evolutionary-conserved chromosomal break-points. The also suggested co-localization of FS and FA associated break-points (FA-bp) was studied here for the first time systematically by molecular cytogenetics. Metaphase chromosomes were obtained from lymphocytes of two FA patients (FANC-A and FANC-C, respectively). Overall 50.58% of the investigated FA-bp correspond to cytogenetic regions with known FS. A detailed molecular cytogenetic study applying FS-spanning probes revealed that 24/29 (82.8%) of analyzed FS are in concordance with FA-bp. Notably, FA-bp show a distribution pattern deviating from that of Aphidicolin induced FS. FA-bp appear more frequently within GTG-light bands and additionally, a yet unreported correlation was observed between break rate and chromosomal banding level. In future, FA-bp might serve as model for the mapping and analysis of otherwise rarely observable FS.
