Microdeletion syndromes disclose replication timing alterations of genes unrelated to the missing DNA

<p>Abstract</p> <p>Background</p> <p>The temporal order of allelic replication is interrelated to the epigenomic profile. A significant epigenetic marker is the asynchronous replication of monoallelically-expressed genes versus the synchronous replication of biallelically-expressed genes. The present study sought to determine whether a microdeletion in the genome affects epigenetic profiles of genes unrelated to the missing segment. In order to test this hypothesis, we checked the replication patterns of two genes – <it>SNRPN</it>, a normally monoallelically expressed gene (assigned to 15q11.13), and the <it>RB1</it>, an archetypic biallelically expressed gene (assigned to 13.q14) in the genomes of patients carrying the 22q11.2 deletion (DiGeorge/Velocardiofacial syndrome) and those carrying the 7q11.23 deletion (Williams syndrome).</p> <p>Results</p> <p>The allelic replication timing was determined by <it>fluorescence in situ hybridization </it>(FISH) technology performed on peripheral blood cells. As expected, in the cells of normal subjects the frequency of cells showing asynchronous replication for <it>SNRPN </it>was significantly (P < 10^-12) higher than the corresponding value for <it>RB1</it>. In contrast, cells of the deletion-carrying patients exhibited a reversal in this replication pattern: there was a significantly lower frequency of cells engaging in asynchronous replication for <it>SNRPN </it>than for <it>RB1 </it>(P < 10^-4and P < 10^-3for DiGeorge/Velocardiofacial and Williams syndromes, respectively). Accordingly, the significantly lower frequency of cells showing asynchronous replication for <it>SNRPN </it>than for <it>RB1 </it>is a new epigenetic marker distinguishing these deletion syndrome genotypes from normal ones.</p> <p>Conclusion</p> <p>In cell samples of each deletion-carrying individual, an aberrant, reversed pattern of replication is delineated, namely, where a monoallelic gene replicates more synchronously than a biallelic gene. This inverted pattern, which appears to be non-deletion-specific, clearly distinguishes cells of deletion-carriers from normal ones. As such, it offers a potential epigenetic marker for suspecting a hidden microdeletion that is too small to be detected by conventional karyotyping methods.</p

Epigenetic analyses in blood cells of men suspected of prostate cancer predict the outcome of biopsy better than serum PSA levels

Lymphocytes from the peripheral blood of patients with prostate cancer—the most frequent (noncutaneous) tumor in men—display epigenetic aberrations (altered modes of allelic replication) characteristic of the malignant phenotype. The present study aims to determine whether replication aberrations add certainty to the suspicion of prostate cancer provided by the prostate-specific antigen (PSA) blood test. The allelic replication mode (whether synchronous or asynchronous) was exemplified for RB1 and AML1. These two genes normally exhibit a synchronous mode of allelic replication. Fluorescence in situ hybridization (FISH) replication assay was used for replication analyses. The FISH assays were applied to PHA-stimulated lymphocytes, established from peripheral blood samples of 35 men referred to biopsy due to suspected prostate cancer. Following biopsy 13 out of these 35 men were found positive for prostate malignancy. The FISH assay—showing asynchronous or synchronous RB1 and AML1 replication—was able to predict, respectively, the results of all biopsy-positive men and in 18 out of the 22 biopsy-negative ones. These measurements, distinguishing biopsy-positive from biopsy-negative men, were highly significant (P < 10−8; 100% sensitivity and 81.8% specificity). Yet, distinguishing between the two groups of men based on the PSA measurements was nonsignificant (P > 0.70). The FISH replication assay applied to peripheral blood lymphocytes of 35 men referred for biopsy significantly predicted the outcome of the pathological examination, more precisely than the serum PSA test. As such, the epigenetic alteration offers a potential noninvasive blood marker, complementary to the PSA, for a preliminary prostate cancer diagnosis

The aberrant asynchronous replication — characterizing lymphocytes of cancer patients — is erased following stem cell transplantation

<p>Abstract</p> <p>Background</p> <p>Aberrations of allelic replication timing are epigenetic markers observed in peripheral blood cells of cancer patients. The aberrant markers are non-cancer-type-specific and are accompanied by increased levels of sporadic aneuploidy. The study aimed at following the epigenetic markers and aneuploidy levels in cells of patients with haematological malignancies from diagnosis to full remission, as achieved by allogeneic stem cell transplantation (alloSCT).</p> <p>Methods</p> <p><it>TP53 </it>(a tumor suppressor gene assigned to chromosome 17), <it>AML1 </it>(a gene assigned to chromosome 21 and involved in the leukaemia-abundant 8;21 translocation) and the pericentomeric satellite sequence of chromosome 17 (<it>CEN17</it>) were used for replication timing assessments. Aneuploidy was monitored by enumerating the copy numbers of chromosomes 17 and 21. Replication timing and aneuploidy were detected cytogenetically using fluorescence <it>in situ </it>hybridization (FISH) technology applied to phytohemagglutinin (PHA)-stimulated lymphocytes.</p> <p>Results</p> <p>We show that aberrant epigenetic markers are detected in patients with hematological malignancies from the time of diagnosis through to when they are scheduled to undergo alloSCT. These aberrations are unaffected by the clinical status of the disease and are displayed both during accelerated stages as well as in remission. Yet, these markers are eradicated completely following stem cell transplantation. In contrast, the increased levels of aneuploidy (irreversible genetic alterations) displayed in blood lymphocytes at various stages of disease are not eliminated following transplantation. However, they do not elevate and remain unchanged (stable state). A demethylating anti-cancer drug, 5-azacytidine, applied in vitro to lymphocytes of patients prior to transplantation mimics the effect of transplantation: the epigenetic aberrations disappear while aneuploidy stays unchanged.</p> <p>Conclusions</p> <p>The reversible nature of the replication aberrations may serve as potential epigenetic blood markers for evaluating the success of transplant or other treatments and for long-term follow up of the patients who have overcome a hematological malignancy.</p

Aberrant allele-specific replication, independent of parental origin, in blood cells of cancer patients

Abstract Background Allelic counterparts of biallelically expressed genes display an epigenetic symmetry normally manifested by synchronous replication, different from genes subjected to monoallelic expression, which normally are characterized by an asynchronous mode of replication (well exemplified by the SNRPN imprinted locus). Malignancy was documented to be associated with gross modifications in the inherent replication-timing coordination between allelic counterparts of imprinted genes as well as of biallelically expressed loci. The cancer-related allelic replication timing aberrations are non-disease specific and appear in peripheral blood cells of cancer patients, including those with solid tumors. As such they offer potential blood markers for non-invasive cancer test. The present study was aimed to gain some insight into the mechanism leading to the replication timing alterations of genes in blood lymphocytes of cancer patients. Methods Peripheral blood samples derived from patients with prostate cancer were chosen to represent the cancerous status, and samples taken from patients with no cancer but with benign prostate hyperplasia were used to portray the normal status. Fluorescence In Situ Hybridization (FISH) replication assay, applied to phytohemagglutinin (PHA)-stimulated blood lymphocytes, was used to evaluate the temporal order (either synchronous or asynchronous) of genes in the patients' cells. Results We demonstrated that: (i) the aberrant epigenetic profile, as delineated by the cancer status, is a reversible modification, evidenced by our ability to restore the normal patterns of replication in three unrelated loci (CEN15, SNRPN and RB1) by introducing an archetypical demethylating agent, 5-azacytidine; (ii) following the rehabilitating effect of demethylation, an imprinted gene (SNRPN) retains its original parental imprint; and (iii) the choice of an allele between early or late replication in the aberrant asynchronous replication, delineated by the cancer status, is not random but is independent of the parental origin. Conclusion The non-disease specific aberrant epigenetic profile displayed in peripheral blood cells of patients with a solid tumour (unlike genetic aberrations) can be reversed, by an epigenetic drug applied in vitro, to the normal. It appears that the cancerous status differentiates between two allelic counterparts in a non-random manner, but independent of the parental origin</p