A novel imprinted gene, HYMAI, is located within an imprinted domain on human chromosome 6 containing ZAC

Transient neonatal diabetes mellitus (TNDM) is a rare disease characterized by intrauterine growth retardation, dehydration, and failure to thrive due to a lack of normal insulin secretion. This disease is associated with paternal uniparental disomy or paternal duplication of chromosome 6, suggesting that the causative gene(s) for TNDM is imprinted. Recently, Gardner et al. (1999, J. Med. Genet. 36: 192-196) proposed that a candidate gene for TNDM lies within chromosome 6q24.1-q24.3. To find human imprinted genes, we performed a database search for EST sequences that mapped to this region, followed by RT-PCR analysis using monochromosomal hybrid cells with a human chromosome 6 of defined parental origin. Here we report the identification of a novel imprinted gene, HYMAI. This gene exhibits differential DNA methylation between the two parental alleles at an adjacent CpG island and is expressed only from the paternal chromosome. A previously characterized imprinted gene, ZAC/LOT1, is located 70 kb downstream of HYMAI and is also expressed only from the paternal allele. In the pancreas, both genes are moderately expressed. HYMAI and ZAC/LOT1 are therefore candidate genes involved in TNDM. Furthermore, the human chromosome 6q24 region is syntenic to mouse chromosome 10 and represents a novel imprinted domain.     

Relaxation of imprinted expression of ZAC and HYMAI in a patient with transient neonatal diabetes mellitus

Transient neonatal diabetes mellitus (TNDM) is a rare disease believed to result from overexpression of a paternally expressed gene controlled by a differentially methylated CpG island on chromosome 6q24. Two genes partially overlap the island: the cell-cycle-control gene ZAC and the untranslated gene HYMAI, the function of which is currently unknown. Proof that either gene is involved in TNDM would require demonstration that imprinted expression is relaxed in TNDM patients; this has hitherto been lacking because of the rarity of the disease and the lack of imprinted expression in the lymphoblastoid cells that are generally the only resource available for study. Here, we show, for the first time, the aberrant expression of imprinted genes in a TNDM patient. In TNDM fibroblasts, the monoallelic expression of both ZAC and HYMAI is relaxed, providing strong supportive evidence that the presence of two unmethylated alleles of this locus is indeed associated with the inappropriate gene expression of neighbouring genes.     

A Novel Imprinted Gene, HYMAI, Is Located within an Imprinted Domain on Human Chromosome 6 Containing ZAC

Transient neonatal diabetes mellitus (TNDM) is a rare disease characterized by intrauterine growth retardation, dehydration, and failure to thrive due to a lack of normal insulin secretion. This disease is associated with paternal uniparental disomy or paternal duplication of chromosome 6, suggesting that the causative gene(s) for TNDM is imprinted. Recently, Gardner et al. (1999, J. Med. Genet. 36: 192–196) proposed that a candidate gene for TNDM lies within chromosome 6q24.1–q24.3. To find human imprinted genes, we performed a database search for EST sequences that mapped to this region, followed by RT-PCR analysis using monochromosomal hybrid cells with a human chromosome 6 of defined parental origin. Here we report the identification of a novel imprinted gene, HYMAI. This gene exhibits differential DNA methylation between the two parental alleles at an adjacent CpG island and is expressed only from the paternal chromosome. A previously characterized imprinted gene, ZAC/LOT1, is located 70 kb downstream of HYMAI and is also expressed only from the paternal allele. In the pancreas, both genes are moderately expressed. HYMAI and ZAC/LOT1 are therefore candidate genes involved in TNDM. Furthermore, the human chromosome 6q24 region is syntenic to mouse chromosome 10 and represents a novel imprinted domain.     

Transient neonatal diabetes mellitus gene Zac1 impairs insulin secretion in mice through Rasgrf1

The biallelic expression of the imprinted gene ZAC1/PLAGL1 underlies ≈ 60% of all cases of transient neonatal diabetes mellitus (TNDM) that present with low perinatal insulin secretion. Molecular targets of ZAC1 misexpression in pancreatic β cells are unknown. Here, we identified the guanine nucleotide exchange factor Rasgrf1 as a direct Zac1/Plagl1 target gene in murine β cells. Doubling Zac1 expression reduced Rasgrf1 expression, the stimulus-induced activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, and, ultimately, insulin secretion. Normalizing Rasgrf1 expression reversed this phenotype. Moreover, the transplantation of Zac1-overexpressing β cells failed to reinstate euglycemia in experimental diabetic mice. In contrast, Zac1 expression did not interfere with the signaling of the glucagon-like peptide 1 receptor (GLP-1R), and the GLP-1 analog liraglutide improved hyperglycemia in transplanted experimental diabetic mice. This study unravels a mechanism contributing to insufficient perinatal insulin secretion in TNDM and raises new prospects for therapy.     

Bisulphite sequencing of the transient neonatal diabetes mellitus DMR facilitates a novel diagnostic test but reveals no methylation anomalies in patients of unknown aetiology

Transient neonatal diabetes mellitus (TNDM) is associated with overexpression of an imprinted locus on chromosome 6q24; this locus contains a differentially methylated region (DMR) consisting of an imprinted CpG island that normally allows expression only from the paternal allele of genes under its control. Three types of abnormality involving 6q24 are known to cause TNDM: paternal uniparental disomy of chromosome 6 (pUPD6), an isolated methylation defect of the imprinted CpG island at chromosome 6q24 and a duplication of 6q24 of paternal origin. A fourth group of patients has no identifiable anomaly of 6q24. Bisulphite sequencing of the DMR has facilitated the development of a diagnostic test for TNDM based on ratiometric methylation-specific polymerase chain reaction. We have applied this method to 45 cases of TNDM, including 12 with pUPD6, 11 with an isolated methylation mutation at 6q24, 16 with a duplication of 6q24 and six of unknown aetiology, together with 29 normal controls. All were correctly assigned. The method is therefore capable of detecting all known genetic causes of TNDM at 6q24, although pUPD6 and methylation mutation cases are not distinguished from one another. In addition, we have carried out bisulphite sequencing of the DMR to compare its methylation status between six TNDM patients with a known methylation mutation, six patients with no identifiable 6q24 mutation and six normal controls. Whereas methylation mutation patients showed a near-total absence of DNA methylation at the TNDM locus, the patients with no identified molecular anomaly showed no marked methylation variation from controls.     

Epigenetic changes of CXCR4 and its ligand CXCL12 as prognostic factors for sporadic breast cancer

Chemokines and their receptors are involved in the development and cancer progression. The chemokine CXCL12 interacts with its receptor, CXCR4, to promote cellular adhesion, survival, proliferation and migration. The CXCR4 gene is upregulated in several types of cancers, including skin, lung, pancreas, brain and breast tumors. In pancreatic cancer and melanoma, CXCR4 expression is regulated by DNA methylation within its promoter region. In this study we examined the role of cytosine methylation in the regulation of CXCR4 expression in breast cancer cell lines and also correlated the methylation pattern with the clinicopathological aspects of sixty-nine primary breast tumors from a cohort of Brazilian women. RT-PCR showed that the PMC-42, MCF7 and MDA-MB-436 breast tumor cell lines expressed high levels of CXCR4. Conversely, the MDA-MB-435 cell line only expressed CXCR4 after treatment with 5-Aza-CdR, which suggests that CXCR4 expression is regulated by DNA methylation. To confirm this hypothesis, a 184 bp fragment of the CXCR4 gene promoter region was cloned after sodium bisulfite DNA treatment. Sequencing data showed that cell lines that expressed CXCR4 had only 15% of methylated CpG dinucleotides, while the cell line that not have CXCR4 expression, had a high density of methylation (91%). Loss of DNA methylation in the CXCR4 promoter was detected in 67% of the breast cancer analyzed. The absence of CXCR4 methylation was associated with the tumor stage, size, histological grade, lymph node status, ESR1 methylation and CXCL12 methylation, metastasis and patient death. Kaplan-Meier curves demonstrated that patients with an unmethylated CXCR4 promoter had a poorer overall survival and disease-free survival. Furthermore, patients with both CXCL12 methylation and unmethylated CXCR4 had a shorter overall survival and disease-free survival. These findings suggest that the DNA methylation status of both CXCR4 and CXCL12 genes could be used as a biomarker for prognosis in breast cancer.     

Epimutation of the TNDM locus and the Beckwith–Wiedemann syndrome centromeric locus in individuals with transient neonatal diabetes mellitus

Transient neonatal diabetes mellitus (TNDM) is characterised by intra-uterine growth retardation, while Beckwith–Wiedemann syndrome (BWS) is a clinically heterogeneous overgrowth syndrome. Both TNDM and BWS may be caused by aberrant loss of methylation (LOM) at imprinted loci on chromosomes 6q24 and 11p15.5 respectively. Here we describe two patients with a clinical diagnosis of TNDM caused by LOM at the maternally methylated imprinted domain on 6q24; in addition, these patients had LOM at the centromeric differentially methylated region of 11p15.5. This shows that imprinting anomalies can affect more than one imprinted locus and may alter the clinical presentation of imprinted disease.     

Monozygous triplets discordant for transient neonatal diabetes mellitus and for imprinting of the TNDM differentially methylated region

Transient neonatal diabetes mellitus (TNDM) is associated with paternal over-expression of an imprinted locus on chromosome 6q24, which contains one differentially methylated region (DMR); maternal demethylation at the DMR accounts for ~20% of cases. Here we report female monozygous triplets, two of whom have TNDM arising from loss of maternal methylation within the TNDM DMR.     

Degree of methylation of ZAC1 (PLAGL1) is associated with prenatal and post-natal growth in healthy infants of the EDEN mother child cohort

The ZAC1 gene, mapped to the 6q24 region, is part of a network of co-regulated imprinted genes involved in the control of embryonic growth. Loss of methylation at the ZAC1 differentially methylated region (DMR) is associated with transient neonatal diabetes mellitus, a developmental disorder involving growth retardation and diabetes in the first weeks of post-natal life. We assessed whether the degree of methylation of the ZAC1 DMR in leukocytes DNA extracted from cord blood is associated with fetal, birth and post-natal anthropometric measures or with C-peptide concentrations in cord serum. We also searched for an influence of dietary intake and maternal parameters on ZAC1 DMR methylation. We found positive correlations between the ZAC1 DMR methylation index (MI) and estimated fetal weight (EFW) at 32 weeks of gestation, weight at birth and weight at one year of age (respectively, r = 0.15, 0.09, 0.14; P values = 0.01, 0.15, 0.03). However, there were no significant correlations between the ZAC1 DMR MI and cord blood C-peptide levels. Maternal intakes of alcohol and of vitamins B2 were positively correlated with ZAC1 DMR methylation (respectively, r = 0.2 and 0.14; P = 0.004 and 0.04). The influence of ZAC1 seems to start in the second half of pregnancy and continue at least until the first year of life. The maternal environment also appears to contribute to the regulation of DNA methylation.     

Molecular Subtype-Specific Expression of MicroRNA-29c in Breast Cancer Is Associated with CpG Dinucleotide Methylation of the Promoter

Basal-like breast cancer is a molecularly distinct subtype of breast cancer that is highly aggressive and has a poor prognosis. MicroRNA-29c (miR-29c) has been shown to be significantly down-regulated in basal-like breast tumors and to be involved in cell invasion and sensitivity to chemotherapy. However, little is known about the genetic and regulatory factors contributing to the altered expression of miR-29c in basal-like breast cancer. We here report that epigenetic modifications at the miR-29c promoter, rather than copy number variation of the gene, may drive the lower expression of miR-29c in basal-like breast cancer. Bisulfite sequencing of CpG sites in the miR-29c promoter region showed higher methylation in basal-like breast cancer cell lines compared to luminal subtype cells with a significant inverse correlation between expression and methylation of miR-29c. Analysis of primary breast tumors using The Cancer Genome Atlas (TCGA) dataset confirmed significantly higher levels of methylation of the promoter in basal-like breast tumors compared to all other subtypes. Furthermore, inhibition of CpG methylation with 5-aza-CdR increases miR-29c expression in basal-like breast cancer cells. Flourescent In Situ Hybridization (FISH) revealed chromosomal abnormalities at miR-29c loci in breast cancer cell lines, but with no correlation between copy number variation and expression of miR-29c. Our data demonstrated that dysregulation of miR-29c in basal-like breast cancer cells may be in part driven by methylation at CpG sites. Epigenetic control of the miR-29c promoter by epigenetic modifiers may provide a potential therapeutic target to overcome the aggressive behavior of these cancers.     

Degree of methylation of ZAC1 (PLAGL1) is associated with prenatal and post-natal growth in healthy infants of the EDEN mother child cohort

The ZAC1 gene, mapped to the 6q24 region, is part of a network of co-regulated imprinted genes involved in the control of embryonic growth. Loss of methylation at the ZAC1 differentially methylated region (DMR) is associated with transient neonatal diabetes mellitus, a developmental disorder involving growth retardation and diabetes in the first weeks of post-natal life. We assessed whether the degree of methylation of the ZAC1 DMR in leukocytes DNA extracted from cord blood is associated with fetal, birth and post-natal anthropometric measures or with C-peptide concentrations in cord serum. We also searched for an influence of dietary intake and maternal parameters on ZAC1 DMR methylation. We found positive correlations between the ZAC1 DMR methylation index (MI) and estimated fetal weight (EFW) at 32 weeks of gestation, weight at birth and weight at one year of age (respectively, r = 0.15, 0.09, 0.14; P values = 0.01, 0.15, 0.03). However, there were no significant correlations between the ZAC1 DMR MI and cord blood C-peptide levels. Maternal intakes of alcohol and of vitamins B2 were positively correlated with ZAC1 DMR methylation (respectively, r = 0.2 and 0.14; P = 0.004 and 0.04). The influence of ZAC1 seems to start in the second half of pregnancy and continue at least until the first year of life. The maternal environment also appears to contribute to the regulation of DNA methylation.