Conserved methylation imprints in the human and mouse GRB10 genes with divergent allelic expression suggests differential reading of the same mark

Grb10/GRB10 encodes a cytoplasmic adapter protein which modulates coupling of a number of cell surface receptor tyrosine kinases with specific signalling pathways. Mouse Grb10 is an imprinted gene with maternal-specific expression. In contrast, human GRB10 is expressed biallelically in most tissues, except for maternal-specific expression of one isoform in muscle and paternal expression in fetal brain. Owing to its location in 7p11.2-p12, GRB10 has been considered a candidate gene for the imprinted growth disorder, the Silver-Russell syndrome (SRS), but its predominantly biallelic expression argues against involvement in the syndrome. To investigate the discrepant imprinting between mouse and human, we compared the sequence organization of their upstream regions, and examined their allelic methylation patterns and the splice variant organization of the mouse locus. Contrary to expectation, we detected both maternal and paternal expression of mouse Grb10. Expression of the paternal allele arises from a different promoter region than the maternal and, as in human, is restricted to the brain. The upstream regions are well conserved, especially the presence of two CpG islands. Surprisingly, both genes have a similar imprinted methylation pattern, the second CpG island is a differentially methylated region (DMR) with maternal methylation in both species. Analysis of 24 SRS patients did not reveal methylation anomalies in the DMR. In the mouse this DMR is a gametic methylation mark. Our results suggest that the difference in imprinted expression in mouse and human is not due to acquisition of an imprint mark but in differences in the reading of this mark.     

Silver-Russell syndrome: a dissection of the genetic aetiology and candidate chromosomal regions

The main features of Silver-Russell syndrome (SRS) are pre- and postnatal growth restriction and a characteristic small, triangular face. SRS is also accompanied by other dysmorphic features including fifth finger clinodactyly and skeletal asymmetry. The disorder is clinically and genetically heterogeneous, and various modes of inheritance and abnormalities involving chromosomes 7, 8, 15, 17, and 18 have been associated with SRS and SRS-like cases. However, only chromosomes 7 and 17 have been consistently implicated in patients with a strict clinical diagnosis of SRS. Two cases of balanced translocations with breakpoints in 17q23.3-q25 and two cases with a hemizygous deletion of the chorionic somatomammatropin gene (CSH1) on 17q24.1 have been associated with SRS, strongly implicating this region. Maternal uniparental disomy for chromosome 7 (mUPD(7)) occurs in up to 10% of SRS patients, with disruption of genomic imprinting underlying the disease status in these cases. Recently, two SRS patients with a maternal duplication of 7p11.2-p13, and a single proband with segmental mUPD for the region 7q31-qter, were described. These key patients define two separate candidate regions for SRS on both the p and q arms of chromosome 7. Both the 7p11.2-p13 and 7q31-qter regions are subject to genomic imprinting and the homologous regions in the mouse are associated with imprinted growth phenotypes. This review provides an overview of the genetics of SRS, and focuses on the newly defined candidate regions on chromosome 7. The analyses of imprinted candidate genes within 7p11.2-p13 and 7q31-qter, and gene candidates on distal 17q, are discussed.


Keywords: Silver-Russell syndrome; imprinting; mUPD(7); candidates     

Maternally inherited duplication of chromosome 7, dup(7)(p11.2p12), associated with mild cognitive deficit without features of Silver-Russell syndrome

We report on a familial duplication in the short arm of chromosome 7, dup(7)(p11.2p12), present in three generations. The duplication was identified by GTG-banding and fluorescence in situ hybridization (FISH) with a whole chromosome 7 DNA painting probe that verified that the duplicated material originated from chromosome 7. The multicolor banding (mBAND) was used to refine the breakpoint assignment. The duplication identified in the proband was also present in her son and mother. All three carriers have mild cognitive deficiencies. Interstitial duplications of the short arm of chromosome 7, although relatively uncommon, have been described in association with a variety of clinical features, including mental retardation of varying severity. Duplication of the p11.2p13 region on chromosome 7 was reported in association with Silver-Russell syndrome (SRS), and an overlapping dup(7)(p11.2p14.1)dn was described in an individual with autistic disorder. Furthermore, a potentially overlapping maternally transmitted inverted duplication, dup(7)(p13p12.2), was reported in patients with cognitive delay. These observations and the phenotype of our duplication carriers suggest that partial trisomy of the proximal 7p region causes cognitive deficiency. The maternal origin of the duplication is of special interest in light of genomic imprinting and implication of the 7p11-p13 region in the SRS etiology. Locus-specific FISH targeting a growth factor receptor binding protein 10 (GRB10), the strong candidate for SRS residing at 7p12.2, showed that it is not duplicated in our patients. Our study helps refine the SRS critical region on 7p and extends our understanding of the clinical manifestations associated with 7p duplications.     

Contribution of GRB10 to the prenatal phenotype in Silver-Russell syndrome? Lessons from 7p12 copy number variations

The growth factor binding protein 10 (GRB10) has been suggested as a candidate gene for Silver-Russell syndrome because of its localization in 7p12, its imprinting status, data from mice models and its putative role in growth. Based on a new patient with normal growth carrying a GRB10 deletion affecting the paternal allele and data from the literature, we conclude that the heterogeneous clinical findings in patients with copy number variations (CNVs) of GRB10 gene depend on the size and the gene content of the CNV. However, evidence from mouse and human cases indicate a growth suppressing role of GRB10 in prenatal development. As a result, an increase of active maternal GRB10 copies, e.g. by maternal uniparental disomy of chromosome 7 or duplications of the region results in intrauterine growth retardation. In contrast, a defective GRB10 copy might result in prenatal overgrowth, whereas the paternal GRB10 allele is not required for proper prenatal growth.     

Trisomy 7 mosaicism, maternal uniparental heterodisomy 7 and Hirschsprung's disease in a child with Silver-Russell syndrome

Prenatal trisomy 7 is usually a cell culture artifact in amniocytes with normal diploid karyotype at birth and normal fetal outcome. In the same way, true prenatal trisomy 7 mosaicism usually results in a normal child except when trisomic cells persist after birth or when trisomy rescue leads to maternal uniparental disomy, which is responsible for 5.5-7% of patients with Silver-Russell syndrome (SRS). We report here on the unusual association of SRS and Hirschsprung's disease (HSCR) in a patient with maternal uniparental heterodisomy 7 and trisomy 7 mosaicism in intestine and skin fibroblasts. HSCR may be fortuitous given its frequency, multifactorial inheritance and genetic heterogeneity. However, the presence of the trisomy 7 mosaicism in intestine as well as in skin fibroblasts suggests that SRS and HSCR might possibly be related. Such an association might result from either an increased dosage of a nonimprinted gene due to trisomy 7 mosaicism in skin fibroblasts (leading to SRS) and in intestine (leading to HSCR), or from an overexpression, through genomic imprinting, of maternally expressed imprinted allele(s) in skin fibroblasts and intestine or from a combination of trisomy 7 mosaicism and genomic imprinting. This report suggests that the SRS phenotype observed in maternal uniparental disomy 7 (mUPD(7)) patients might also result from an undetected low level of trisomy 7 mosaicism. In order to validate this hypothesis, we propose to perform a conventional and molecular cytogenetic analysis in different tissues every time mUPD7 is displayed.     

Mosaic maternal uniparental disomy of chromosome 11 in a patient with Silver-Russell syndrome

Silver-Russell syndrome (SRS) is a clinically heterogeneous disorder characterised mainly by intrauterine and postnatal growth retardation. While maternal uniparental disomy of chromosome 7 is found in 5-10% of SRS patients, recently genetic and epigenetic mutations affecting the imprinting centres on chromosome 11p15 have been reported in up to 64% of patients. Chromosome 11p15 abnormalities reported in SRS include methylation defects in the imprinting centre 1 (ICR1) and maternally inherited duplications involving all or part of the imprinted region of 11p15. Here we report the first published case of SRS with mosaic maternal uniparental disomy of chromosome 11.     

No evidence of PEG1/MEST gene mutations in Silver-Russell syndrome patients.

Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.     

47,XX,UPD(7)mat,+r(7)pat/46,XX,UPD(7)mat mosaicism in a girl with Silver-Russell syndrome (SRS): possible exclusion of the putative SRS gene from a 7p13-q11 region

Maternal uniparental disomy for chromosome 7 (UPD7) may present with a characteristic phenotype reminiscent of Silver-Russell syndrome (SRS). Previous studies have suggested that approximately 10% of SRS patients have maternal UPD7. We describe a girl with a mos47,XX,+mar/46,XX karyotype associated with the features of SRS. Chromosome painting using a chromosome 7 specific probe pool showed that the small marker was a ring chromosome 7 (r(7)). PCR based microsatellite marker analysis of the patient detected only one maternal allele at each of 16 telomeric loci examined on chromosome 7, but showed both paternal and maternal alleles at four centromeric loci. Considering her mosaic karyotype composed ofdiploid cells and cells with partial trisomy for 7p13-q11, the allele types obtained at the telomeric loci may reflect the transmission of one maternal allele in duplicate, that is, maternal UPD7 (complete isodisomy or homodisomy 7), whereas those at the centromeric loci were consistent with biparental contribution to the trisomic region. It is most likely that the patient originated in a 46,XX,r(7) zygote, followed by duplication of the maternally derived whole chromosome 7 in an early mitosis, and subsequent loss of the paternally derived ring chromosome 7 in a subset of somatic cells. The cell with 46,XX,r(7) did not survive thereafter because of the monosomy for most of chromosome 7. If the putative SRS gene is imprinted, it can be ruled out from the 7p11-q11 region, because biparental alleles contribute to the region in our patient.     

Myoclonus‐dystonia and Silver–Russell syndrome resulting from maternal uniparental disomy of chromosome 7

Myoclonus‐dystonia (M‐D) is a movement disorder that is often associated with mutations in epsilon‐sarcoglycan (SGCE), a maternally imprinted gene at 7q21.3. We report a 24‐year‐old male with short stature (<5th percentile) and a movement disorder clinically consistent with M‐D. Single nucleotide polymorphism (SNP) array did not identify significant copy number changes, but revealed three long continuous stretches of homozygosity on chromosome 7 suggestive of uniparental disomy. Parental SNP arrays confirmed that the proband had maternal uniparental disomy of chromosome 7 (mUPD7) with regions of heterodisomy and isodisomy. mUPD7 is the cause of approximately 5–10% of Silver–Russell syndrome (SRS), a disorder characterized by prenatal and postnatal growth retardation. Although SRS was not suspected in our patient, these findings explain his short stature. SGCE methylation testing showed loss of the unmethylated paternal allele. Our findings provide a unifying diagnosis for his short stature and M‐D and help to optimize his medication regimen. In conclusion, we show that M‐D is a clinical feature that may be associated with SRS due to mUPD7. Individuals with mUPD7 should be monitored for the development of movement disorders. Conversely, individuals with M‐D and short stature should be evaluated for SRS.     

An analysis of the distribution of hetero- and isodisomic regions of chromosome 7 in five mUPD7 Silver-Russell syndrome probands

Silver-Russell syndrome (SRS) shares common features of intrauterine growth retardation (IUGR) and a number of dysmorphic features including lateral asymmetry in about 50% of subjects. Its genetic aetiology is complex and most probably heterogeneous. Approximately 7% of patients with SRS have been found to have maternal uniparental disomy of chromosome 7 (mUPD7). Genomic DNA samples from five SRS patients with mUPD7 have been analysed for common regions of isodisomy using 40 polymorphic markers distributed along the length of chromosome 7. No regions of common isodisomy were found among the five patients. It is most likely that imprinted gene(s) rather than recessive mutations cause the common phenotype. Heterodisomy of markers around the centromere indicated that the underlying cause of the mUPD7 is a maternal meiosis I non-disjunction error in these five subjects.     

Dlx5, the mouse homologue of the human-imprinted DLX5 gene,is biallelically expressed in the mouse brain

The mouse Dlx5 gene encodes a distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of mouse embryo and is located on chromosome 6, which is the syntenic region to the human chromosome 7q21–q31 imprinting cluster. Recently, its human homologue, DLX5, was identified to be imprinted and maternally expressed, at least in normal human lymphoblasts and in brain tissues. In our study, we analyzed the imprinting status of mouse Dlx5 by RT-PCR, first in the F1 of a reciprocal cross between two different mouse strains, and second in heterozygous Dlx5 mutant mice. Both approaches revealed that mouse Dlx5 followed a biallelic pattern of expression in brain tissue and in testis. Our findings suggest that the Dlx5 gene escapes genomic imprinting, at least in mice of certain genetic backgrounds.     

No evidence of PEG1/MEST gene mutations in Silver‐Russell syndrome patients

Silver‐Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (α and β) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST α coding region, and there were no significant mutations in the 5′‐flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST α were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS. © 2001 Wiley‐Liss, Inc.     

Two sisters with Silver-Russell phenotype

Silver-Russell syndrome (SRS) is a well recognizable syndrome, but the etiology of SRS seems to be heterogeneous. SRS is listed in Mendelian Inheritance in Man as an autosomal dominant disorder because most described cases have been of sporadic occurrence, and most likely were caused by de novo autosomal dominant mutation, and because families with apparent dominant transmission of a SRS phenotype have been described. Still, in a few families, autosomal recessive inheritance has been suggested. We describe two sisters who meet the criteria for SRS proposed by Price et al. [1999]. The parents had normal facial features, normal height, and normal post-natal growth. This is the second well-documented case of familial recurrence of SRS that resembles an autosomal recessive inheritance pattern. Since sib recurrence is so rare in SRS, other modes of inheritance should be considered. The finding of maternal uniparental disomy 7 (mUPD7) in 10% of SRS cases suggests that lack of paternally expressed imprinted gene(s) or overexpression of maternal imprinted gene(s) on chromosome 7 cause SRS. The recurrence in sibs could be caused by a mutation in the imprinted gene or imprinting center carried by one parent. Alternatively, recurrence in sibs could represent germ line mosaicism for a dominant mutation in one of the parents.     

The genetic aetiology of Silver-Russell syndrome

Silver-Russell syndrome (SRS MIM180860) is a disorder characterised by intrauterine and/or postnatal growth restriction and typical facies. However, the clinical picture is extremely diverse due to numerous diagnostic features reflecting a heterogeneous genetic disorder. The mode of inheritance is variable with sporadic cases also being described. Maternal uniparental disomy (mUPD) of chromosome 7 accounts for 10% of SRS cases and many candidate imprinted genes on 7 have been investigated. Chromosome 11 has moved to the forefront as the key chromosome in the aetiology, with reports of methylation defects in the H19 imprinted domain associated with the phenotype in 35-65% of SRS patients. Methylation aberrations have been described in a number of other imprinted growth related disorders such as Beckwith-Wiedmann syndrome. This review discusses these recent developments as well as the previous work on chromosome 7. Other candidate genes/chromosomal regions previously investigated are tabled.     

The imprinted region on human chromosome 7q32 extends to the carboxypeptidase A gene cluster: an imprinted candidate for Silver-Russell syndrome

Imprinted gene(s) on human chromosome 7q32-qter have been postulated to be involved in intrauterine growth restriction associated with Silver-Russell syndrome (SRS) as 7–10% of patients have mUPD(7). Three imprinted genes, MEST, MESTIT1, and COPG2IT1 on chromosome 7q32, are unlikely to cause SRS since epigenetic and sequence mutation analyses have not shown any changes. One hundred kilobases proximal to MEST lies a group of four carboxypeptidase A (CPA) genes. Since most imprinted genes are found in clusters, this study focuses on analysing these CPAs for imprinting effects based on their proximity to an established imprinted domain. Firstly, a replication timing study across 7q32 showed that an extensive genomic region including the CPAs, MEST, MESTIT1, and COPG2IT1 replicates asynchronously. Subsequently, SNP analysis by sequencing RT-PCR products of CPA1, CPA2, CPA4, and CPA5 indicated preferential expression of CPA4. Pyrosequencing was used as a quantitative approach, which confirmed predominantly preferential expression of the maternal allele and biallelic expression in brain. CPA5 expression levels were too low to allow reliable evaluation of allelic expression, while CPA1 and CPA2 both showed biallelic expression. CPA4 was the only gene from this family in which an imprinting effect was shown despite the location of this family of genes next to an imprinted cluster. As CPA4 has a potential role in cell proliferation and differentiation, two preferentially expressed copies in mUPD patients with SRS syndrome would result in excess expression and could alter the growth profiles of these subjects and give rise to intrauterine growth restriction.     

Identification of an imprinted gene, Meg3/Gtl2 and its human homologue MEG3, first mapped on mouse distal chromosome 12 and human chromosome 14q

BACKGROUND: The paternal duplication of mouse distal chromosome 12 leads to late embryonal/neonatal lethality and growth promotion, whereas maternal duplication leads to late embryonal lethality and growth retardation. Human paternal or maternal uniparental disomies of chromosome 14q that are syntenic to mouse distal chromosome 12 have also been reported to show some imprinting effects on growth, mental activity and musculoskeletal morphology. For the isolation of imprinted genes in this region, a systematic screen of maternally expressed genes (Megs) was carried out by our subtraction-hybridization method using androgenetic and normally fertilized embryos. RESULTS: We have isolated seven candidate clones of the mouse Meg gene. Among them, we identified a novel maternally expressed imprinted gene, Meg3, on mouse distal chromosome 12 and showed that it was identical to the Gtl2 gene. We also found that the human homologue MEG3 on chromosome 14q was also monoallelically expressed. CONCLUSIONS: This is the first identification of the imprinting gene, both on mouse distal chromosome 12 and on human chromosome 14q, respectively. Because there are no obvious open reading frames in either the mouse Meg3/Gtl2 or human MEG3, the function of these genes remains unclear. However, this result will provide a good basis for the further investigation of several important imprinted genes in this chromosomal region.     

Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5

In human and mouse, most imprinted genes are arranged in chromosomal clusters. Their linked organization suggests co-ordinated mechanisms controlling imprinting and gene expression. The identification of local and regional elements responsible for the epigenetic control of imprinted gene expression will be important in understanding the molecular basis of diseases associated with imprinting such as Beckwith- Wiedemann syndrome. We have established a complete contig of clones along the murine imprinting cluster on distal chromosome 7 syntenic with the human imprinting region at 11p15.5 associated with Beckwith- Wiedemann syndrome. The cluster comprises approximately 1 Mb of DNA, contains at least eight imprinted genes and is demarcated by the two maternally expressed genes Tssc3 (Ipl) and H19 which are directly flanked by the non-imprinted genes Nap1l4 (Nap2) and Rpl23l (L23mrp), respectively. We also localized Kcnq1 (Kvlqt1) and Cd81 (Tapa-1) between Cdkn1c (p57(Kip2)) and Mash2. The mouse Kcnq1 gene is maternally expressed in most fetal but biallelically transcribed in most neonatal tissues, suggesting relaxation of imprinting during development. Our findings indicate conserved control mechanisms between mouse and human, but also reveal some structural and functional differences. Our study opens the way for a systematic analysis of the cluster by genetic manipulation in the mouse which will lead to animal models of Beckwith-Wiedemann syndrome and childhood tumours.