Epigenetic modulation of the IGF2/H19 imprinted domain in human embryonic and extra-embryonic compartments and its possible role in fetal growth restriction.

Genomic imprinting, resulting in parent-of-origin-dependent gene expression, is mainly achieved by DNA methylation. IGF2 and H19, belonging to the same cluster of imprinted genes and regulated by ICR1, DMR2 and H19 promoter elements, play a major role in fetal/placental growth. Using quantitative approaches, we explored the epigenetic modulation of IGF2/H19 during human development in 60 normal and 66 idiopathic IUGR (Intrauterine Growth Restriction) pregnancies, studying embryonic (cord blood) and extraembryonic (placenta and umbilical cord) tissues. We found ICR1 normal methylation levels ( approximately 50%) and H19 promoter/DMR2 hypomethylation in extra-embryonic tissues. In contrast, in embryonic samples the three loci displayed normal methylation values comparable to those in postnatal blood. This feature is stably maintained throughout gestation and does not vary in IUGR cases. We reported asymmetric allelic expression of H19 and IGF2 as a common feature in pre- and post-natal tissues, independent of H19 promoter and DMR2 methylation levels. In addition, we excluded in IUGR post-transcriptional IGF2 interference possibly related to miRNA 483-3p (IGF2, intron 2) expression defects. Through LINE1 methylation analysis, we observed a methylation gradient with increasing methylation from pre- to post-natal life. The involvement of UPD (Uniparental Disomy) in IUGR aetiology was excluded. Our data indicate that: (1) ICR1 methylation status is a necessary and sufficient condition to drive the imprinting of IGF2 and H19 present in embryonic as well as in extra-embryonic tissues; (2) hypomethylation of H19 promoter and DMR2 does not influence the expression pattern of IGF2 and H19; (3) there is a gradient of global methylation, increasing from extra-embryonic to embryonic and adult tissues. Finally, because of placental hypomethylation, cautions should be exercised in diagnosis of imprinting diseases using chorionic villi.

Differential cytogenomics and miRNA signature of the Acute Myeloid Leukaemia Kasumi-1 cell line CD34+38- compartment.

The t(8;21) Acute Myeloid Leukaemia (AML) Kasumi-1 cell line with N822K KIT mutation, is a model system for leukemogenesis. As AML initiating cells reside in the CD34(+)CD38(-) fraction, we addressed the refined cytogenomic characterization and miRNA expression of Kasumi-1 cell line and its FACS-sorted subpopulations focussing on this compartment. By conventional cytogenetics, Spectral-Karyotyping and array-CGH the cytogenomic profile of Kasumi-1 cells evidenced only subtle regions differentially represented in CD34(+)CD38(-) cells. Expression profiling by a miRNA platform showed a set of miRNA differentially expressed in paired subpopulations and the signature of miR-584 and miR-182 upregulation in the CD34(+)CD38(-) fraction.

Three cases with de novo 6q imbalance and variable prenatal phenotype.

We describe two families in which three fetuses had a de novo 6q imbalance and abnormal phenotypes. We determined the boundaries and the parental origin of the chromosomal alterations by segregation analysis using a panel of short tandem repeats (STRs) located on 6q. Cases 1 and 2 (family A) were two sibs with 6q imbalance involving different regions. Case 1 was a female fetus with arthrogryposis, who had a complex rearrangement resulting in two deleted regions (6q22 and 6q25.1-q25.2) and a duplication of 6q23-q25.1. This latter imbalance was reported previously and is associated with joint contractures and short neck, also present in this fetus. The sib (case 2) had intrauterine growth restriction (IUGR) and agenesis of the ductus venosus. This male died shortly after birth; postnatal karyotype and molecular investigations showed a 6q21 de novo deletion. Case 3 (family B) had a prenatally detected deletion of 6q14-q16. Autopsy of the fetus documented minor facial anomalies and contractures of the limbs. All rearrangements were de novo and of paternal origin. Our data and the consistent number of cases of de novo 6q alterations previously reported suggest that chromosome arm 6q could be prone to rearrangements resulting in heterogeneous phenotypes. In family A, chromosome 6q imbalances involving different chromosomal regions were present in two consecutive pregnancies. In such cases counseling should suggest the impossibility of excluding recurrence of a chromosomal imbalance, and should discuss the option of early prenatal diagnosis in subsequent pregnancies.

Loss of the inactive X chromosome and replication of the active X in BRCA1-defective and wild-type breast cancer cells.

In females, X chromosome inactivation (XCI) begins with the expression of the XIST gene from the X chromosome destined to be inactivated (Xi) and the coating of XIST RNA in cis. It has recently been reported that this process is supported by the product of the BRCA1 tumor suppressor gene and that BRCA1-/- cancers show Xi chromatin structure defects, thus suggesting a role of XCI perturbation in BRCA1-mediated tumorigenesis. Using a combined genetic and epigenetic approach, we verified the occurrence of XCI in BRCA1-/- and BRCA1wt breast cancer cell lines. It was ascertained that the Xi was lost in all cancer cell lines, irrespective of the BRCA1 status and that more than one active X (Xa) was present. In addition, no epigenetic silencing of genes normally subjected to XCI was observed. We also evaluated XIST expression and found that XIST may be occasionally transcribed also from Xa. Moreover, in one of the BRCA1wt cell line the restoring of XIST expression using a histone deacetylase inhibitor, did not lead to XCI. To verify these findings in primary tumors, chromosome X behavior was investigated in a few BRCA1-associated and BRCA1-not associated primary noncultured breast carcinomas and the results mirrored those obtained in cancer cell lines. Our findings indicate that the lack of XCI may be a frequent phenomenon in breast tumorigenesis, which occurs independently of BRCA1 status and XIST expression and is due to the loss of Xi and replication of Xa and not to the reactivation of the native Xi.

Loss of heterozygosity on chromosome 4q32-35 in sporadic basal cell carcinomas: evidence for the involvement of p33ING2/ING1L and SAP30 genes.

BACKGROUND: Studies on basal cell carcinoma (BCC) have demonstrated that patched gene and p53 gene located at 9q22.3 and 17p13 are the main genes responsible for the onset of this tumor. In order to identify a possible involvement of other tumor suppressor genes, we screened 19 cases of BCCs for loss of heterozygosity (LOH). METHODS: The analysis was performed on tumoral tissue and on corresponding normal tissue by using a panel of 37 polymorphic markers spanning 26 chromosomal regions. RESULTS: We observed that only four chromosomal regions, 4q32 (30.00%), 4q35 (27.27%), 9q21-22 (28.57%), and 9q22-qter (35.71%), demonstrated a significative LOH (>20%), even if others show a borderline percentage (15-20%) of imbalance (1q32, 3p24, 10p22.1, and 17q21.3). CONCLUSIONS: Our findings suggest that a new chromosomal region mapping in the long arm of chromosome 4 could be involved in sporadic BCC carcinogenesis. Further analyses indicate that the minimal deleted region in 4q32-35 includes p33ING2/ING1L and SAP30, whose deletion could impair the G1-phase checkpoint control and favor gene silencing, respectively.

Biparental expression of ESX1L gene in placentas from normal and intrauterine growth-restricted pregnancies.

Equivalent levels of X-linked gene products between males and females are reached by means of X chromosome inactivation (XCI). In the human and murine embryonic tissues, both the paternally and maternally derived X chromosomes (X(P) and X(M)) may be inactivated. In murine extra-embryonic tissues, X(P) is imprinted and always silenced; humans, unlike mice, can inactivate the X(M) in extra-embryonic lineages without an adverse outcome. This difference is probably due to the presence of imprinted placental genes on the murine X chromosome, but not on the human homologue, essential for placental development and function. An example is the paternally imprinted Esx1 gene; mice with a null maternally derived Esx1 allele show intrauterine growth restriction (IUGR) because of placental insufficiency. We investigated the imprinting status of the human orthologous Esx1 gene (ESX1L) in placental samples of four normal full-term and 13 IUGR female fetuses, in which we determined the XCI pattern. Our findings demonstrated that IUGR as well as normal placentas display XCI heterogeneity, thus indicating that the IUGR phenotype is not correlated with a preferential pattern of XCI in placentas. Moreover, ESX1L is equally expressed in IUGR and normal placentas, and shows the same methylation pattern in the presence of both random and skewed XCI. These findings provide evidence that ESX1L is not imprinted in human third-trimester placentas and there is no parent-of-origin effect of chromosome X associated with placental insufficiency.