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.     

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.     

Zac1 (Lot1), a potential tumor suppressor gene, and the gene for epsilon-sarcoglycan are maternally imprinted genes: identification by a subtractive screen of novel uniparental fibroblast lines

Imprinted genes are expressed from one allele according to their parent of origin, and many are essential to mammalian embryogenesis. Here we show that the epsilon-sarcoglycan gene (Sgce) and Zac1 (Lot1) are both paternally expressed imprinted genes. They were identified in a subtractive screen for imprinted genes using a cDNA library made from novel parthenogenetic and wild-type fibroblast lines. Sgce is a component of the dystrophin-sarcoglycan complex, Zac1 is a nuclear protein inducing growth arrest and/or apoptosis, and Zac1 is a potential tumor suppressor gene. Sgce and Zac1 are expressed predominantly from their paternal alleles in all adult mouse tissues, except that Zac1 is biallelic in the liver and Sgce is weakly expressed from the maternal allele in the brain. Sgce and Zac1 are broadly expressed in embryos, with Zac1 being highly expressed in the liver primordium, the umbilical region, and the neural tube. Sgce, however, is strongly expressed in the allantoic region on day 9.5 but becomes more widely expressed throughout the embryo by day 11.5. Sgce is located at the proximal end of mouse chromosome 6 and is a candidate gene for embryonic lethality associated with uniparental maternal inheritance of this region. Zac1 maps to the proximal region of chromosome 10, identifying a new imprinted locus in the mouse, homologous with human chromosome 6q24-q25. In humans, unipaternal disomy for this region is associated with fetal growth retardation and transient neonatal diabetes mellitus. In addition, loss of expression of ZAC has been described for a number of breast and ovarian carcinomas, suggesting that ZAC is a potential tumor suppressor gene.     

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.     

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.     

Biparental expression of IGFBP1 and IGFBP3 renders their involvement in the etiology of Silver-Russell syndrome unlikely.

Maternal uniparental disomy (UPD) of chromosome 7 has recently been reported in about 10% of Silver-Russell (SRS) patients. It can therefore be concluded that at least one gene on chromosome 7 is imprinted and mutations in this gene/these genes might contribute to the phenotype of the disease. Two genes which are involved in growth and localised in 7p12-13 are the insulin-like growth factor binding proteins 1 and 3 (IGFBP1; IGFBP3). Comparison to the mouse genome shows that the syntenic region on mouse chromosome 11 is imprinted, UPD of this region leads to deviations in growth in mice. In the present study we investigated whether the genes for IGFBP1 and IGFBP3 might be involved in the etiology of SRS: after exclusion of SRS specific mutations we could demonstrate biparental expression of both genes in lymphocytes of an SRS patient without UPD7 as well as expression in a patient with maternal UPD7. Our results as well as those from other groups show biparental expression of IGFBP1 in fetal tissues and expression of IGFBP3 in nearly every tissue during puberty and adult life. Thus, no evidence is given for an involvement of the two genes in SRS.     

Developmental methylation program and concerted expression of Stx11 in mouse tissues

The human 6q24 region is involved in growth and development, transient neonatal diabetes (TND), cancer, and metabolic dysfunction. To further characterize this region, the developmental status of DNA methylation and expression of Zac1 and Stx11 genes located within the mouse 10A1 region ortholog of human 6q24 were determined. In mice, imprinted Zac1 and Stx11 were highly expressed at the end of fetal development but downregulated at 4 and 11 weeks in brain, pancreas, and heart. Postnatal Zac1 downregulation was independent from promoter methylation of the expressed allele, suggesting the mediation of age-dependent chromatin remodeling. Stx11 nonpromoter CpG island was methylated de novo from E18 to 1 year with tissue-specific kinetics. The high conservation in vertebrates of Stx11 CpG2 is suggestive of an important regulatory function in age-related regional epigenetic state and/or chromatin configuration. Stx11 alleles were unequally expressed in F1 mice tissues, reflecting the influence of cis-regulatory factors on its expression. These data suggest the presence of a methylation domain and a coordinated gene expression pattern in multiple tissues. Methylation variation and allelic regulation of expression may underlie genetic diversity and contribute to disease susceptibility at the 6q24 locus in humans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transienter neonataler Diabetes und Hypomethylierungssyndrome

Transient neonatal diabetes (TNDM) is manifested before the age of 6 weeks and typically resolves within 18 months. Main clinical features include intrauterine growth retardation, hyperglycemia and dehydration with absent ketoacidosis. Causes of TNDM are heterogeneous but 70% are due to a chromosomal aberration in the region 6q24 which contains the imprinted genes PLAGL1/ZAC and HYMAI. Paternal uniparental disomy 6 (upd(6)pat) or paternal duplications of the imprinted region as well as imprinting defects of the maternal allele all result in an overexpression of the paternally expressed gene PLAGL1. Imprinting defects in 6q24 can occur as isolated events or can affect more than one locus (hypomethylation syndrome). Hypomethylation at multiple loci has so far been observed in patients with TNDM, Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS).The risk of recurrence depends on the underlying cause of TNDM. Chromosomal aberrations in the parents affecting chromosome 6 increase the risk for UPD or duplication of the imprinted locus in 6q24. Nevertheless, UPD and duplication 6q24 are mostly de novo occurrences.     

Biallelic expression of<Emphasis Type="Italic"> HRAS</Emphasis> and<Emphasis Type="Italic"> MUCDHL</Emphasis> in human and mouse

At least eight genes clustered in 1 Mb of DNA on human chromosome (Chr) 11p15.5 are subject to parental imprinting, with monoallelic expression in one or more tissues. Orthologues of these genes show conserved linkage and imprinting on distal Chr 7 of mice. The extended imprinted region has a bipartite structure, with at least two differentially methylated DNA elements (DMRs) controlling the imprinting of two sub-domains. We previously described three biallelically expressed genes ( MRPL23, 2G7 and TNNT3) in 100 kb of DNA immediately downstream of the imprinted H19 gene, suggesting that H19 marks one border of the imprinted region. Here we extend this analysis to two additional downstream genes, HRAS and MUCDHL (mu-protocadherin). We find that these genes are biallelically expressed in multiple fetal and adult tissues, both in humans and in mice. The mouse orthologue of a third gene, DUSP8, located between H19 and MUCDHL, is also expressed biallelically. The DMR immediately upstream of H19 frequently shows a net gain of methylation in Wilms tumors, either via Chr 11p15.5 loss of heterozygosity (LOH) or loss of imprinting (LOI), but changes in methylation in CpG-rich sequences upstream and within the MUCDHL gene are rare in these tumors and do not correlate with LOH or LOI. These findings are further evidence for a border of the imprinted region immediately downstream of H19, and the data allow the construction of an imprinting map that includes more than 20 genes, distributed over 3 Mb of DNA on Chr 11p15.5.     

Linkage and sequence conservation of the X-linked genes DXS253E (P3) and DXS254E (GdX) in mouse and man

Lambda G28, a mouse genomic clone homologous to the human P3 gene and associated with a CpG island, also hybridizes to human probes for the neighboring GdX gene. The two genes, P3 and GdX (DXS253E and DXS254E), physically linked on the human X chromosome, lie within a similar physical distance on the mouse X chromosome. The CpG island corresponds to that at the 5' of the human GdX gene. The relative orientation of the two genes is the same. The DNA sequence in coding and noncoding regions is very conserved.     

Imprinted methylation profiles for proximal mouse Chromosomes 11 and 7 as revealed by methylation-sensitive representational difference analysis

Proximal mouse Chromosome (Chr) 11 shares regions of orthology with the candidate gene region for the imprinting growth disorder Silver-Russell syndrome (SRS) on human Chr 7p. It has previously been shown that mice with two maternal or two paternal copies (duplications, Dp) of proximal Chr 11 exhibit reciprocal growth phenotypes. Those with two paternal copies show fetal and placental overgrowth, while those with two maternal copies are growth retarded. The growth retardation observed in the latter is reminiscent of the intrauterine growth restriction (IUGR) observed in SRS patients with maternal uniparental disomy for Chr 7 (mUPD7). We have carried out a methylation-sensitive representational difference analysis (Me-RDA) screen to look for regions of differential methylation (DMRs) associated with imprinted genes. For these experiments, we have used mouse embryos with uniparental duplications of Chrs 11 and 7 proximal to the breakpoint of the reciprocal translocation T(7;11)40Ad. Two previously known imprinted loci associated with paternal allele hypomethylation were recovered on proximal mouse Chr 11, U2af1-rs1 and Meg1/Grb10. These two genes map 15 cM apart, so it seems likely that they are within separate imprinted domains that do not contain additional DMRs. The known imprinted gene Peg3, located on mouse proximal Chr 7, was also detected in our screen. The finding that Peg3 was differentially methylated in embryos with uniparental inheritance of proximal Chr 7 confirms that Peg3 is located proximal to the breakpoint of T40Ad in G-band 7A2. Because GRB10 has previously been reported to be a candidate gene for SRS, we analysed 22 patients for epimutations of the GRB10 differentially methylated region that could lead to the altered expression of this gene. No such mutations were found.     

Paradoxical methylation of the tyrosine hydroxylase gene in mouse preimplantation embryos

The mouse tyrosine hydroxylase (Th) gene is located in an evolutionarily conserved imprinted gene cluster on distal chromosome 7. It is associated with a CpG island that spans the promoter of the gene. Using a bisulfite sequencing method we show that the Th promoter is fully methylated in both male and female mouse germ cells and in human spermatozoa, suggesting that it belongs to the newly identified category of CpG islands, the similarly methylated regions (SMRs). Contrary to other tissue-specific gene sequences, the mouse Th promoter escapes the initial wave of genome demethylation during the first few cell cycles, but becomes demethylated between the morula and the blastocyst stages. This unusual methylation ontogeny may be a characteristic of the SMRs and/or related to the localization of the Th gene in an imprinted gene cluster.     

The neuronatin gene resides in a "micro-imprinted" domain on human chromosome 20q11.2.

A small fraction of the genome contains genes that are imprinted and thus expressed exclusively from one parental allele.We report here that the human neuronatin gene (NNAT) on chromosome 20q11.2 is imprinted and transcribed specifically from the paternal allele. The region containing NNAT has multiple CpG islands, and methylation analysis showed that a 1.8-kb CpG island in its promoter region exhibits differential methylation in all tissues examined. This finding is consistent with the island acting as a component of the NNAT imprint control domain. NNAT lies within the singular 8.5-kb intron of the gene encoding bladder cancer-associated protein (BLCAP), which, as we demonstrate, is not imprinted. This study provides the first example, to our knowledge, in humans of an imprinted gene contained within the genomic structure of a nonimprinted gene. Thus, NNAT is in an imprinted "microdomain," making this locus uniquely suited for the investigation of mechanisms of localized imprint regulation.