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

Abstract

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.

Figure 1

Imprinting of the human GRB10 gene. A, Monoallelic expression of human GRB10 in fetal brain. The genomic sequence of a normal fetal tissue heterozygous for a G/A polymorphism in exon 3 (top) and the corresponding cDNA sequence (bottom). Note that only the “A” allele is expressed in this sample. B, Determination of the parental origin of the human chromosome 7 in somatic cell hybrids GM10791 and GM13302, which carry a whole chromosome 7 and a derivative chromosome der(7)t(7;17)(q36;q22), respectively: (1) both hybrids harbor PEG1, as evidenced by positive amplification with primer pair 5′-caatagtccacttcttacc-3′ and 5′-ggccgagatcttttaatc-3′ (Riesewijk et al. 1998), corresponding to exon 6 of the human PEG1 genomic sequence; (2) expression of the imprinted PEG1 isoform 1 in GM10791, mUPD7 lymphoblastoid cell line GM11496, GM13302, and a paternal upd(7) (pUPD7) lymphoblastoid cell line (Pan et al. 1998) analyzed by RT-PCR with GRB83 and GRB44, where PEG1 is expressed in GM13302 and pUPD7 but not in GM10791 or mUPD7 and the housekeeping gene, HPRT, is used as a control; (3) methylation status of the promoter of human PEG1 in each cell line, where genomic DNA, treated with bisulfite, is amplified with PEGMF-PEGMR pair and PEG1UF-PEG1UR, specific for the methylated and the unmethylated allele, respectively, and the promoter is methylated in GM10791 and in mUPD7 but is unmethylated in GM13302 and pUPD7. Collectively, the human chromosome 7 is paternally derived in GM13302 and maternally derived in GM10791. C, Expression of GRB10 in GM10791 and GM13302, where (1) both hybrids harbor GRB10, as evidenced by positive amplification, with primer pair GRB7 and GRB8, and (2) GRB10 is expressed in GM10791, which carries a maternally derived chromosome 7, but not in GM13302, which carries a paternally derived chromosome 7.

Figure 2

Identification of the GRB10 mutation. Exon 3 of GRB10 was amplified from genomic DNA of patients affected with Russell-Silver syndrome. Two patients, RS1 and RS3, had a heterozygous missense mutation (cct→tct, Pro95Ser) that was maternally derived. Primers GRB7 and GRB8 were used. A, Automated sequencing of the PCR products. B, Restriction enzyme digest of the PCR products. The missense mutation ablates an Eco O-109 I site. The mutant allele is demonstrated as a band at 267 bp.