Megan Hitchins

Duplication of 7p11.2-p13, Including GRB10, in Silver-Russell Syndrome

Silver-Russell syndrome (SRS) is characterized by pre- and postnatal growth failure and other dysmorphic features. The syndrome is genetically heterogeneous, but maternal uniparental disomy of chromosome 7 has been demonstrated in approximately 7% of cases. This suggests that at least one gene on chromosome 7 is imprinted and involved in the pathogenesis of SRS. We have identified a de novo duplication of 7p11.2-p13 in a proband with features characteristic of SRS. FISH confirmed the presence of a tandem duplication encompassing the genes for growth factor receptor-binding protein 10 (GRB10) and insulin-like growth factor-binding proteins 1 and 3 (IGFBP1 and -3) but not that for epidermal growth factor-receptor (EGFR). Microsatellite markers showed that the duplication was of maternal origin. These findings provide the first evidence that SRS may result from overexpression of a maternally expressed imprinted gene, rather than from absent expression of a paternally expressed gene. GRB10 lies within the duplicated region and is a strong candidate, since it is a known growth suppressor. Furthermore, the mouse homologue (Grb10/Meg1) is reported to be maternally expressed and maps to the imprinted region of proximal mouse chromosome 11 that demonstrates prenatal growth failure when it is maternally disomic. We have demonstrated that the GRB10 genomic interval replicates asynchronously in human lymphocytes, suggestive of imprinting. An additional 36 SRS probands were investigated for duplication of GRB10, but none were found. However, it remains possible that GRB10 and/or other genes within 7p11.2-p13 are responsible for some cases of SRS.

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.

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

Abstract. 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.

Maternal repression of the human GRB10 gene in the developing central nervous system; evaluation of the role for GRB10 in Silver-Russell syndrome

The GRB10 gene encodes a growth suppressor and maps to human chromosome 7p11.2-p13. Maternal duplication (matdup) of this region has recently been associated with Silver-Russell syndrome (SRS), which is characterised by pre- and postnatal growth restriction, craniofacial dysmorphism and lateral asymmetry. Maternal uniparental disomy for chromosome 7 (mUPD7) occurs in approximately 7% of SRS patients. Exposure of a recessive allele due to isodisomy has been ruled out in five mUPD7 cases, suggesting genomic imprinting as the basis for disease. Assuming SRS patients with matdup of 7p11.2-p13 and mUPD7 share a common aetiology, this would implicate a maternally expressed gene from this interval, which is involved in growth inhibition. Murine Grb10 was identified as a maternally expressed gene by subtractive hybridisation using normal and androgenetic mouse embryos. Grb10 maps to the homologous region of proximal mouse chromosome 11, for which mUPD incurs reduced birthweight. A role for GRB10 in SRS was evaluated by determining its imprinting status in multiple human foetal tissues using expressed polymorphisms, and by screening the coding region for mutations in 18 classic non-mUPD7 SRS patients. Maternal repression of GRB10 was observed specifically in the developing central nervous system including brain and spinal cord, with biallelic expression in peripheral tissues. This is in contrast to mouse Grb10, and represents the first example of opposite imprinting in human and mouse homologues. While a role for GRB10 in mUPD7 SRS cases can not be ruled out on the basis of imprinting status, no mutations were identified in the patients screened.

Genomic imprinting in fetal growth and development

Each somatic cell of the human body contains 46 chromosomes consisting of two sets of 23; one inherited from each parent. These chromosomes can be categorised as 22 pairs of autosomes and two sex chromosomes; females are XX and males are XY. Similarly, at the molecular level, two copies of each autosomal gene exist; one copy derived from each parent. Until the mid-1980s, it was assumed that each copy of an autosome or gene was functionally equivalent, irrespective of which parent it was derived from. However, it is now clear from classical experiments in mice and from examples of human genetic disease that this is not the case. The functional activity of some genes or chromosomal regions is unequal, and dependent on whether they have been inherited maternally or paternally. This phenomenon is termed genomic imprinting and the activity or silence of an imprinted gene or chromosomal region is set during gametogenesis. Genomic imprinting involving the autosomes appears to be restricted to eutherian mammals, and has most likely evolved as a result of the conflicting concerns of the parental genomes in the growth and development of their offspring. When the normal pattern of imprinting is disrupted, the phenotypes observed in humans and mice are generally associated with abnormal fetal growth, development and behaviour, illustrating its importance for a normal intrauterine environment. The characteristics of imprinted genes, their regulation and the phenotypes associated with altered imprinting are discussed.

Characterisation of the growth regulating gene IMP3, a candidate for Silver-Russell syndrome

Silver-Russell syndrome (SRS) is characterised by pre- and postnatal growth restriction in association with other clinically recognised dysmorphic features such as triangular facies, asymmetry, and fifth finger clinodactyly.1–3 Since the major diagnostic features involve reduced growth, it is tempting to postulate that altered expression of a protein within a growth factor cascade may be causative.nnThere have been numerous documented defects of genes coding for proteins in the insulin-like growth factor (IGF) signalling pathways, whether the receptors, ligands, or signal modulators, which result in a SRS-like phenotype. Probands with ring chromosomes or deletions involving the 15q26-qter region present with growth failure and SRS-like features.4–11 It has been proposed that the phenotypes are the result of hemizygosity at the IGF1 receptor ( IGF1R ) gene. We have shown, however, that hemizygosity at this locus is not a common cause of SRS.9,11 In addition, it has been well documented that maternal uniparental disomy (UPD) of chromosome 7 is present in approximately 10% of SRS cases13–16 and no consistent regions of isodisomy have been shown for the full length of the chromosome.17 This suggests that there are imprinted genes on chromosome 7, which when disrupted are responsible for the phenotype. Recently two independently reported candidate gene regions on chromosome 7 containing imprinted genes defined by cytogenetic disruptions in SRS probands have been reported. Two unrelated probands with maternally transmitted duplications of 7p11.2-p13 define the first region.10,18 Recently, a number of other cytogenetic disruptions including balanced translocations and inversions within this region have been described in association with the SRS or SRS-like phenotype19 (Monk et al , manuscript submitted). This candidate gene region contains the growth related genes insulin-like growth factor binding proteins 1 and 3 ( IGFBP s) and growth factor binding protein 10 …

Evolutionary conservation, developmental expression, and genomic mapping of mammalian Twisted gastrulation

Abstract. The twisted gastrulation gene (tsg) encodes a secreted protein required for the correct specification of dorsal midline cell fate during gastrulation in Drosophila. We report that tsg homologs from human, mouse, zebrafish, and Xenopus share 72–98% identity at the amino acid level and retain all 24 cysteine residues from Drosophila. In contrast to Drosophila where tsg expression is limited to early embryos, expression is found throughout mouse and human development. In Drosophila, tsg acts in synergy with decapentaplegic (dpp), a member of the TGF-β family of secreted proteins. The vertebrate orthologs of dpp, BMP-2 and -4, are crucial for gastrulation and neural induction, and aberrant signaling by BMPs and other TGF-β family members results in developmental defects including holoprosencephaly (HPE). Interestingly, human TSG maps to the HPE4 locus on Chromosome 18p11.3, and our analysis places the gene within 5 Mbp of TG-interacting factor (TGIF).

Conflicting Reports of Imprinting Status of Human GRB10 in Developing Brain: How Reliable Are Somatic Cell Hybrids for Predicting Allelic Origin of Expression?

This study is supported by the START research program of the RWTH Aachen, and by the Wellcome Trust, the Dunhill Medical Trust, and Children Nationwide in London.

DDC and COBL, flanking the imprinted GRB10 gene on 7p12, are biallelically expressed

Maternal duplication of human 7p11.2-p13 has been associated with Silver-Russell syndrome (SRS) in two familial cases. GRB10 is the only imprinted gene identified within this region to date. GRB10 demonstrates an intricate tissue- and isoform-specific imprinting profile in humans, with paternal expression in fetal brain and maternal expression of one isoform in skeletal muscle. The mouse homolog is maternally transcribed. The GRB10 protein is a potent growth inhibitor and represents a candidate for SRS, which is characterized by pre- and postnatal growth retardation and a spectrum of additional dysmorphic features. Since imprinted genes tend to be grouped in clusters, we investigated the imprinting status of the dopa-decarboxylase gene (DDC) and the Cordon-bleu gene (COBL) which flank GRB10 within the 7p11.2-p13 SRS duplicated region. Although both genes were found to replicate asynchronously, suggestive of imprinting, SNP expression analyses showed that neither gene was imprinted in multiple human fetal tissues. The mouse homologues, Ddc and Cobl, which map to the homologous imprinted region on proximal Chr 11, were also biallelically expressed in mice with uniparental maternal or paternal inheritance of this region. With the intent of using mouse Grb10 as an imprinted control, biallelic expression was consistently observed in fetal, postnatal, and adult brain of these mice, in contrast to the maternal-specific transcription previously demonstrated in brain in inter-specific F1 progeny. This may be a further example of over-expression of maternally derived transcripts in inter-specific mouse crosses. GRB10 remains the only imprinted gene identified within 7p11.2-p13.n

Biallelic expression of IGFBP1 and IGFBP3, two candidate genes for the Silver-Russell syndrome.

Editor—Silver-Russell syndrome (SRS) is a condition characterised by intrauterine and postnatal growth retardation with relative sparing of cranial growth, triangular facies, fifth finger clinodactyly, and facial, limb, or truncal asymmetry.1 2 The molecular basis of SRS remains elusive and seems likely to be heterogeneous. However, maternal uniparental disomy of chromosome 7 (mUPD7) has been found in approximately 10% of SRS patients, suggesting that at least one gene on chromosome 7 is imprinted and involved in the pathogenesis of this condition.3 4 Interest has surrounded the human chromosomal region 7p12-13, which is homologous to mouse proximal chromosome 11, since mUPD for this region in mice leads to prenatal growth failure.5 Within this region lie the genes for insulin-like growth factor binding proteins 1 and 3 ( IGFBP1 and IGFBP3 ). Both are involved in regulation of fetal growth via the insulin-like growth factor axis.6 IGFBP1 and IGFBP3 are therefore obvious candidates for a role in the SRS phenotype associated with mUPD7.3 4 nnIn order to determine whether IGFBP1 and/or IGFBP3 are likely to be implicated in SRS, we have investigated their imprinting status in normal fetal tissues collected from termination of pregnancies. Samples were obtained from Queen Charlottes and Chelsea Hospital with the approval of the Research Ethics Committee of the Royal Postgraduate Medical School (96/4955) and from the MRC Tissue Bank at Hammersmith Hospital (L Wong). Genomic DNA and total RNA was extracted from these tissues and RT-PCR performed as previously described.7 The primers used in this study are listed in table 1. PCR was carried out for 35 cycles for all reactions. Products were sequenced on an ABI PRISM 377 automated DNA sequencer using the dRhodamine Ampli- Taq …