Imprinting of human GRB10 and its mutations in two patients with Russell-Silver syndrome

Documentation of maternal uniparental disomy of chromosome 7 in 10% of patients with Russell-Silver syndrome (RSS), characterized by prenatal and postnatal growth retardation and dysmorphic features, has suggested the presence of an imprinted gene on chromosome 7 whose mutation is responsible for the RSS phenotype. Human GRB10 on chromosome 7, a homologue of the mouse imprinted gene Grb10, is a candidate, because GRB10 has a suppressive effect on growth, through its interaction with either the IGF-I receptor or the GH receptor, and two patients with RSS were shown to have a maternally derived duplication of 7p11-p13, encompassing GRB10. In the present study, we first demonstrated that the GRB10 gene is also monoallelically expressed in human fetal brain tissues and is transcribed from the maternally derived allele in somatic-cell hybrids. Hence, human GRB10 is imprinted. A mutation analysis of GRB10 in 58 unrelated patients with RSS identified, within the N-terminal domain of the protein, a P95S substitution in two patients with RSS. In these two cases, the mutant allele was inherited from the mother. The fact that monoallelic GRB10 expression was observed from the maternal allele in this study suggests but does not prove that these maternally transmitted mutant alleles contribute to the RSS phenotype.     

Duplication of 7p12.1-p13, including GRB10 and IGFBP1, in a mother and daughter with features of Silver-Russell syndrome

Silver-Russell syndrome (SRS) has been associated with maternal uniparental disomy (UPD) of chromosome 7 in approximately 10% of cases, suggesting that at least one imprinted gene on chromosome 7 is involved in the pathogenesis of the disease. We report a proximal 7p interstitial inverted duplication in a mother and daughter both of whom have features of SRS, including marked short stature, low birth weight, facial asymmetry and 5th finger clinodactyly. Fluorescence in situ hybridisation (FISH) with YAC probes enabled delineation of the duplicated region to 7p12.1-p13. This region of proximal chromosome 7 is known to be homologous to an imprinted region in the mouse chromosome 11 and contains the growth-related genes GRB10 (growth factor receptor-bound protein 10), EGFR (epidermal growth factor receptor) and IGFBP1 (insulin-like growth factor binding protein 1), all of which have been suggested as candidate genes for SRS. Molecular analysis showed that the duplication in both mother and daughter spanned a distance of approximately 10 cM and included GRB10 and IGFBP1 but not EGFR. The de novo duplication in the proband's mother was shown to be of paternal origin. In order to test the hypothesis that sub-microscopic duplications of 7p, whether maternal or paternal in origin, are responsible for at least some cases of SRS, we screened a further eight patients referred to our laboratory for SRS. None were found to have duplications of either GRB10 or IGFBP1. The hypothesis that sub-microscopic duplications including GRB10 and IGFBP1 is a cause of SRS remains a possibility and warrants further investigation. Importantly, in contrast to current thinking, our results suggest that imprinted genes may not underlie the SRS phenotype, and we propose an alternative hypothesis to explain the occurrence of maternal UPD 7 seen in some cases of SRS.     

Chromosome 7p disruptions in Silver Russell syndrome: delineating an imprinted candidate gene region

Silver-Russell syndrome (SRS) is characterised by pre- and postnatal growth restriction (PNGR) and additional dysmorphic features including body asymmetry and fifth finger clinodactyly. The syndrome is genetically heterogeneous, with a number of chromosomes implicated. However, maternal uniparental disomy for chromosome 7 has been demonstrated in up to 10% of all cases. Three SRS probands have previously been described with a maternally inherited duplication of 7p11.2-p13, defining this as a candidate region. Over-expression of a maternally transcribed, imprinted gene with growth-suppressing activity located within the duplicated region, or breakpoint disruption of genes or regulatory sequences, may account for the phenotype in these cases. Here we describe two additional SRS patients and four probands with PNGR with a range of cytogenetic disruptions of 7p, including duplications, pericentric inversions and a translocation. An incomplete contig consisting of 80 PACs and BACs from the centromere to 7p14 was constructed. Individual clones from this contig were used as FISH probes to map the breakpoints in the six new cases and the three duplication probands previously described. Our data provide further evidence for a candidate SRS region at 7p11.1-p14. A common breakpoint region was identified within 7p11.2 in all nine cases, pinpointing this specific interval. The imprinting status of genes within the 7p11.1-p14 region flanked by the most extreme breakpoints have been analysed using both somatic cell hybrids containing a single full-length maternally or paternally derived chromosome 7 and expressed single nucleotide polymorphisms in paired fetal and maternal samples.     

A narrow segment of maternal uniparental disomy of chromosome 7q31-qter in Silver-Russell syndrome delimits a candidate gene region

Maternal uniparental disomy of chromosome 7 (matUPD7), the inheritance of both chromosomes from only the mother, is observed in approximately 10% of patients with Silver-Russell syndrome (SRS). It has been suggested that at least one imprinted gene that regulates growth and development resides on human chromosome 7. To date, three imprinted genes-PEG1/MEST, gamma2-COP, and GRB10-have been identified on chromosome 7, but their role in the etiology of SRS remains uncertain. In a systematic screening with microsatellite markers, for matUPD7 cases among patients with SRS, we identified a patient who had a small segment of matUPD7 and biparental inheritance of the remainder of chromosome 7. Such a pattern may be explained by somatic recombination in the zygote. The matUPD7 segment at 7q31-qter extends for 35 Mb and includes the imprinted gene cluster of PEG1/MEST and gamma2-COP at 7q32. GRB10 at 7p11.2-p12 is located within a region of biparental inheritance. Although partial UPD has previously been reported for chromosomes 6, 11, 14, and 15, this is the first report of a patient with SRS who has segmental matUPD7. Our findings delimit a candidate imprinted region sufficient to cause SRS.     

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.     

Imprinting of a small nucleolar RNA gene on mouse chromosome 12

We have identified a novel, maternally expressed imprinted gene encoding a C/D-box small nucleolar RNA (snoRNA) called MBII-343, which may regulate RNA editing or alternative splicing of an as yet unknown target gene. This gene is closely linked to an imprinted gene, Meg3, on mouse distal chromosome 12, which is syntenic to human chromosome 14. The paternal duplication of mouse distal chromosome 12 leads to late embryonal/neonatal lethality, growth promotion, and cardiomyopathy, whereas maternal duplication leads to late embryonal lethality and growth retardation. Human paternal uniparental disomy for chromosome 14 leads to musculoskeletal problems and mental retardation, whereas maternal uniparental disomy leads to intrauterine growth retardation, motor developmental delay, premature puberty, hypotonia, joint laxity, macrocephaly, short statue, neonatal poor sucking, skill with jigsaw puzzles, skin picking, obesity, and maturity onset diabetes of the young.     

An unexpected function of the Prader-Willi syndrome imprinting center in maternal imprinting in mice

Genomic imprinting is a phenomenon that some genes are expressed differentially according to the parent of origin. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders caused by deficiency of imprinted gene expression from paternal and maternal chromosome 15q11-q13, respectively. Imprinted genes at the PWS/AS domain are regulated through a bipartite imprinting center, the PWS-IC and AS-IC. The PWS-IC activates paternal-specific gene expression and is responsible for the paternal imprint, whereas the AS-IC functions in the maternal imprint by allele-specific repression of the PWS-IC to prevent the paternal imprinting program. Although mouse chromosome 7C has a conserved PWS/AS imprinted domain, the mouse equivalent of the human AS-IC element has not yet been identified. Here, we suggest another dimension that the PWS-IC also functions in maternal imprinting by negatively regulating the paternally expressed imprinted genes in mice, in contrast to its known function as a positive regulator for paternal-specific gene expression. Using a mouse model carrying a 4.8-kb deletion at the PWS-IC, we demonstrated that maternal transmission of the PWS-IC deletion resulted in a maternal imprinting defect with activation of the paternally expressed imprinted genes and decreased expression of the maternally expressed imprinted gene on the maternal chromosome, accompanied by alteration of the maternal epigenotype toward a paternal state spread over the PWS/AS domain. The functional significance of this acquired paternal pattern of gene expression was demonstrated by the ability to complement PWS phenotypes by maternal inheritance of the PWS-IC deletion, which is in stark contrast to paternal inheritance of the PWS-IC deletion that resulted in the PWS phenotypes. Importantly, low levels of expression of the paternally expressed imprinted genes are sufficient to rescue postnatal lethality and growth retardation in two PWS mouse models. These findings open the opportunity for a novel approach to the treatment of PWS.     

Differentially methylated regions in maternal and paternal uniparental disomy for chromosome 7

DNA methylation is a hallmark of genomic imprinting and differentially methylated regions (DMRs) are found near and in imprinted genes. Imprinted genes are expressed only from the maternal or paternal allele and their normal balance can be disrupted by uniparental disomy (UPD), the inheritance of both chromosomes of a chromosome pair exclusively from only either the mother or the father. Maternal UPD for chromosome 7 (matUPD7) results in Silver-Russell syndrome (SRS) with typical features and growth retardation, but no gene has been conclusively implicated in SRS. In order to identify novel DMRs and putative imprinted genes on chromosome 7, we analyzed eight matUPD7 patients, a segmental matUPD7q31-qter, a rare patUPD7 case and ten controls on the Infinium HumanMethylation450K BeadChip with 30?017 CpG methylation probes for chromosome 7. Genome-scale analysis showed highly significant clustering of DMRs only on chromosome 7, including the known imprinted loci GRB10, SGCE/PEG10, and PEG/MEST. We found ten novel DMRs on chromosome 7, two DMRs for the predicted imprinted genes HOXA4 and GLI3 and one for the disputed imprinted gene PON1. Quantitative RT-PCR on blood RNA samples comparing matUPD7, patUPD7, and controls showed differential expression for three genes with novel DMRs, HOXA4, GLI3, and SVOPL. Allele specific expression analysis confirmed maternal only expression of SVOPL and imprinting of HOXA4 was supported by monoallelic expression. These results present the first comprehensive map of parent-of-origin specific DMRs on human chromosome 7, suggesting many new imprinted sites.     

Differentially methylated regions in maternal and paternal uniparental disomy for chromosome 7

DNA methylation is a hallmark of genomic imprinting and differentially methylated regions (DMRs) are found near and in imprinted genes. Imprinted genes are expressed only from the maternal or paternal allele and their normal balance can be disrupted by uniparental disomy (UPD), the inheritance of both chromosomes of a chromosome pair exclusively from only either the mother or the father. Maternal UPD for chromosome 7 (matUPD7) results in Silver-Russell syndrome (SRS) with typical features and growth retardation, but no gene has been conclusively implicated in SRS. In order to identify novel DMRs and putative imprinted genes on chromosome 7, we analyzed eight matUPD7 patients, a segmental matUPD7q31-qter, a rare patUPD7 case and ten controls on the Infinium HumanMethylation450K BeadChip with 30 017 CpG methylation probes for chromosome 7. Genome-scale analysis showed highly significant clustering of DMRs only on chromosome 7, including the known imprinted loci GRB10, SGCE/PEG10, and PEG/MEST. We found ten novel DMRs on chromosome 7, two DMRs for the predicted imprinted genes HOXA4 and GLI3 and one for the disputed imprinted gene PON1. Quantitative RT-PCR on blood RNA samples comparing matUPD7, patUPD7, and controls showed differential expression for three genes with novel DMRs, HOXA4, GLI3, and SVOPL. Allele specific expression analysis confirmed maternal only expression of SVOPL and imprinting of HOXA4 was supported by monoallelic expression. These results present the first comprehensive map of parent-of-origin specific DMRs on human chromosome 7, suggesting many new imprinted sites.     

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.     

Mouse imprinting defect mutations that model Angelman syndrome

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders resulting from deficiency of imprinted gene expression from paternal or maternal chromosome 15q11-15q13, respectively. In humans, expression of the imprinted genes is under control of a bipartite cis-acting imprinting center (IC). Families with deletions causing PWS imprinting defects localize the PWS-IC to 4.3 kb overlapping with SNRPN exon 1. Families with deletions causing AS imprinting defects localize the AS-IC to 880 bp 35 kb upstream of the PWS-IC. We report two mouse mutations resulting in defects similar to that seen in AS patients with deletion of the AS-IC. An insertion/duplication mutation 13 kb upstream of Snrpn exon 1 resulted in lack of methylation at the maternal Snrpn promoter, activation of maternally repressed genes, and decreased expression of paternally repressed genes. The acquisition of a paternal epigenotype on the maternal chromosome in the mutant mice was demonstrated by the ability to rescue the lethality and growth retardation in a mouse model of a PWS imprinting defect. A second mutation, an 80-kb deletion extending upstream of the first mutation, caused a similar imprinting defect with variable penetrance. These results suggest that there is a mouse functional equivalent to the human AS-IC.     

The human aminophospholipid-transporting ATPase gene ATP10C maps adjacent to UBE3A and exhibits similar imprinted expression

Maternal duplications of the imprinted 15q11-13 domain result in an estimated 1%-2% of autism-spectrum disorders, and linkage to autism has been identified within 15q12-13. UBE3A, the Angelman syndrome gene, has, to date, been the only maternally expressed, imprinted gene identified within this region, but mutations have not been found in autistic patients. Here we describe the characterization of ATP10C, a new human imprinted gene, which encodes a putative protein homologous to the mouse aminophospholipid-transporting ATPase Atp10c. ATP10C maps within 200 kb distal to UBE3A and, like UBE3A, also demonstrates imprinted, preferential maternal expression in human brain. The location and imprinted expression of ATP10C thus make it a candidate for chromosome 15-associated autism and suggest that it may contribute to the Angelman syndrome phenotype.     

Imprinting analysis of the mouse chromosome 7C region in DNMT1-null embryos

The mouse chromosome 7C, orthologous to the human 15q11–q13 has an imprinted domain, where most of the genes are expressed only from the paternal allele. The imprinted domain contains paternally expressed genes, Snurf/Snrpn, Ndn, Magel2, Mkrn3, and Frat3, C/D-box small nucleolar RNAs (snoRNAs), and the maternally expressed gene, Ube3a. Imprinted expression in this large (approximately 3–4 Mb) domain is coordinated by a bipartite cis-acting imprinting center (IC), located upstream of the Snurf/Snrpn gene. The molecular mechanism how IC regulates gene expression of the whole domain remains partially understood. Here we analyzed the relationship between imprinted gene expression and DNA methylation in the mouse chromosome 7C using DNA methyltransferase 1 (DNMT1)-null mutant embryos carrying Dnmt1^ps alleles, which show global loss of DNA methylation and embryonic lethality. In the DNMT1-null embryos at embryonic day 9.5, the paternally expressed genes were biallelically expressed. Bisulfite DNA methylation analysis revealed loss of methylation on the maternal allele in the promoter regions of the genes. These results demonstrate that DNMT1 is necessary for monoallelic expression of the imprinted genes in the chromosome 7C domain, suggesting that DNA methylation in the secondary differentially methylated regions (DMRs), which are acquired during development serves primarily to control the imprinted expression from the maternal allele in the mouse chromosome 7C.     

The novel imprinted carboxypeptidase A4 gene ( CPA4) in the 7q32 imprinting domain

By a search for novel human imprinted genes in the vicinity of the imprinted gene MEST, at chromosome 7q32, we identified the carboxypeptidase A4 gene ( CPA4) in a gene cluster of the carboxypeptidase family, 200 kb centromeric to MEST. Because CPA4 was originally identified as a protein induced in a prostate cancer cell line (PC-3) by histone deacetylase inhibitors, and was located at the putative prostate cancer-aggressiveness locus at 7q32, we investigated its imprinting status in fetal tissues and in adult benign hypertrophic prostate (BPH). RT-PCR using four intragenic polymorphisms as markers showed that CPA4 was expressed preferentially from the maternal allele in the fetal heart, lung, liver, intestine, kidney, adrenal gland, and spleen, but not in the fetal brain. It was also preferentially expressed in the BPH. These findings support that CPA4 is imprinted and may become a strong candidate gene for prostate cancer-aggressiveness. As a Silver-Russell syndrome (SRS) locus has been proposed to be located to a region near MEST and to be involved in imprinting, CPA4 would have been a candidate gene for SRS. However, analysis of ten SRS patients revealed no mutations in CPA4.