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Imprinting of Human GRB10 and Its Mutations in Two Patients with Russell-Silver Syndrome

2000; Elsevier BV; Volume: 67; Issue: 2 Linguagem: Inglês

10.1086/302997

1537-6605

Hiroshi Yoshihashi, Katsuhiro Maeyama, Rika Kosaki, Tsutomu Ogata, Masato Tsukahara, Yu-ichi Goto, Jun-ichi Hata, Nobutake Matsuo, Robert J. Smith, Kenjiro Kosaki,

Epigenetics and DNA Methylation

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. 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. Russell-Silver syndrome (RSS [MIM 180860]) is characterized by prenatal and postnatal growth retardation accompanied by dysmorphic features, such as triangular facies and fifth-finger clinodactyly (Silver et al. Silver et al., 1953Silver H Kiyasu W George J Deamer W Syndrome of congenital hypertrophy, shortness of stature and elevated gonadotropins.Pediatrics. 1953; 12: 356-368Google Scholar; Russell Russell, 1954Russell A A syndrome of intrauterine dwarfism recognizable at birth with craniofacial dysostosis, disproportionate short arms and other anomalies.Proc R Soc Med. 1954; 47: 1040-1044PubMed Google Scholar; Price et al. 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Monk et al., 2000Monk D Wakeling EL Proud V Hitchins M Abu-Amero SN Stanier P Preece MA et al.Duplication of 7p11.2-p13, including GRB10, in Silver-Russell syndrome.Am J Hum Genet. 2000; 66: 36-46Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), a genetic etiology for RSS has been suggested. However, the gene responsible for RSS has not yet been identified. The recent discovery of maternal uniparental disomy of chromosome 7 [mUPD7] in 10% of patients with RSS has suggested the presence, on chromosome 7, of an imprinted gene whose mutation is responsible for the RSS phenotype (Kotzot et al. Kotzot et al., 1995Kotzot D Schmitt S Bernasconi F Robinson WP Lurie IW Ilyina H Mehes K et al.Uniparental disomy 7 in Silver-Russell syndrome and primordial growth retardation.Hum Mol Genet. 1995; 4: 583-587Crossref PubMed Scopus (247) Google Scholar; Eggermann et al. Eggermann et al., 1997Eggermann T Wollmann HA Kuner R Eggermann K Enders H Kaiser P Ranke MB Molecular studies in 37 Silver-Russell syndrome patients: frequency and etiology of uniparental disomy.Hum Genet. 1997; 100: 415-419Crossref PubMed Scopus (127) Google Scholar; Preece et al. Preece et al., 1997Preece MA Price SM Davies V Clough L Stanier P Trembath RC Moore GE Maternal uniparental disomy 7 in Silver-Russell syndrome.J Med Genet. 1997; 34: 6-9Crossref PubMed Scopus (118) Google Scholar; Bernard et al. Bernard et al., 1999Bernard LE Penaherrera MS Van Allen MI Wang MS Yong SL Gareis F Langlois S et al.Clinical and molecular findings in two patients with Russell-Silver syndrome and UPD7: comparison with non-UPD7 cases.Am J Med Genet. 1999; 87: 230-236Crossref PubMed Scopus (39) Google Scholar). The predicted function of this putative RSS gene is regulation of growth, since prenatal and postnatal growth retardation is the hallmark of RSS. Mutations in either a maternally repressed (i.e., paternally expressed) gene or a paternally repressed (i.e., maternally expressed) gene could account for the RSS phenotype. If we assume that the putative RSS gene is paternally expressed, the gene would not be expressed in mUPD7 cells. Therefore, the predicted function of the putative gene would be facilitation of growth. Loss-of-function mutations in the gene, when transmitted paternally, should lead to the RSS phenotype. The human PEG1 (paternally expressed gene-1) gene on 7q31 was considered to be an excellent candidate gene, because (1) human PEG1 and mouse Peg1 are imprinted and are expressed from the paternal allele (Kobayashi et al. Kobayashi et al., 1997Kobayashi S Kohda T Miyoshi N Kuroiwa Y Aisaka K Tsutsumi O Kaneko-Ishino T et al.Human PEG1/MEST, an imprinted gene on chromosome 7.Hum Mol Genet. 1997; 6: 781-786Crossref PubMed Scopus (134) Google Scholar; Riesewijk et al. Riesewijk et al., 1997Riesewijk AM Hu L Schulz U Tariverdian G Hoglund P Kere J Ropers HH et al.Monoallelic expression of human PEG1/MEST is paralleled by parent-specific methylation in fetuses.Genomics. 1997; 42: 236-244Crossref PubMed Scopus (76) Google Scholar) and (2) a loss-of-function mutation in mouse Peg1, when paternally transmitted, is associated with severe growth retardation (Kaneko-Ishino et al. Kaneko-Ishino et al., 1995Kaneko-Ishino T Kuroiwa Y Miyoshi N Kohda T Suzuki R Yokoyama M Viville S et al.Peg1/Mest imprinted gene on chromosome 6 identified by cDNA subtraction hybridization.Nat Genet. 1995; 11: 52-59Crossref PubMed Scopus (244) Google Scholar). However, analysis of PEG1 in 49 patients with RSS did not reveal any mutations (Riesewijk et al. Riesewijk et al., 1998Riesewijk AM Blagitko N Schinzel AA Hu L Schulz U Hamel BC Ropers HH et al.Evidence against a major role of PEG1/MEST in Silver-Russell syndrome.Eur J Hum Genet. 1998; 6: 114-120Crossref PubMed Scopus (47) Google Scholar). Alternatively, we could assume that the putative RSS gene is maternally expressed. In this scenario, the gene would be expressed in excess in mUPD7 cells. Hence, the predicted function of the putative RSS gene would be suppression rather than facilitation of growth. A gain-of-function mutation in such a gene, when transmitted maternally, would contribute to the RSS phenotype. The human growth factor receptor–bound protein 10/maternally expressed gene-1 (GRB10/MEG1) gene, on 7p11.2-p12 (Jerome et al. Jerome et al., 1997Jerome CA Scherer SW Tsui LC Gietz RD Triggs-Raine B Assignment of growth factor receptor-bound protein 10 (GRB10) to human chromosome 7p11.2-p12.Genomics. 1997; 40: 215-216Crossref PubMed Scopus (31) Google Scholar), is considered to be a prime candidate, because (1) mouse Grb10 is subject to imprinting and is maternally expressed (Miyoshi et al. Miyoshi et al., 1998Miyoshi N Kuroiwa Y Kohda T Shitara H Yonekawa H Kawabe T Hasegawa H et al.Identification of the Meg1/Grb10 imprinted gene on mouse proximal chromosome 11, a candidate for the Silver-Russell syndrome gene.Proc Natl Acad Sci USA. 1998; 95: 1102-1107Crossref PubMed Scopus (133) Google Scholar), (2) maternal uniparental disomy of the proximal region of mouse chromosome 11 encompassing Grb10 leads to prenatal growth retardation (Cattanach et al. Cattanach et al., 1998Cattanach BM Shibata H Hayashizaki Y Townsend KM Ball S Beechey CV Association of a redefined proximal mouse chromosome 11 imprinting region and U2afbp-rs/U2af1-rs1 expression.Cytogenet Cell Genet. 1998; 80: 41-47Crossref PubMed Google Scholar), and (3) in vitro studies indicate that, by interacting with either the insulin-like growth factor I (IGF-I) receptor (O'Neill et al. O'Neill et al., 1996O'Neill T Rose DW Pillay TS Hotta K Olefsky JM Gustafson TA Interaction of a GRB-IR splice variant (a human GRB10 homolog) with the insulin and insulin-like growth factor I receptors: evidence for a role in mitogenic signaling.J Biol Chem. 1996; 271: 22506-22513Crossref PubMed Scopus (128) Google Scholar; Morrione et al. Morrione et al., 1997Morrione A Valentinis B Resnicoff M Xu S Baserga R The role of mGrb10alpha in insulin-like growth factor I-mediated growth.J Biol Chem. 1997; 272: 26382-26387Crossref PubMed Scopus (83) Google Scholar) or the growth hormone receptor (Moutoussamy et al. Moutoussamy et al., 1998Moutoussamy S Renaudie F Lago F Kelly PA Finidori J Grb10 identified as a potential regulator of growth hormone (GH) signaling by cloning of GH receptor target proteins.J Biol Chem. 1998; 273: 15906-15912Crossref PubMed Scopus (60) Google Scholar), GRB10 has a suppressive effect on growth. Recent documentation of two patients with RSS who had a maternally derived interstitial duplication of 7p11-p13 encompassing GRB10 (Joyce et al. Joyce et al., 1999Joyce CA Sharp A Walker JM Bullman H Temple KI Duplication of 7p12.1-p13, including GRB10 and IGFBP1, in a mother and daughter with features of Silver-Russell syndrome.Hum Genet. 1999; 105: 273-280Crossref PubMed Scopus (93) Google Scholar; Monk et al. Monk et al., 2000Monk D Wakeling EL Proud V Hitchins M Abu-Amero SN Stanier P Preece MA et al.Duplication of 7p11.2-p13, including GRB10, in Silver-Russell syndrome.Am J Hum Genet. 2000; 66: 36-46Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) further strengthens arguments for GRB10 involvement in the pathogenesis of RSS. In the present study, we determined whether human GRB10 is imprinted, and we then screened 58 patients with RSS for GRB10 mutations. First, monoallelic expression of human GRB10 was investigated by reverse transcriptase (RT)–PCR analysis with a primer pair designed to amplify across a known G/A single-nucleotide polymorphism in exon 3 (Angrist et al. Angrist et al., 1998Angrist M Bolk S Bentley K Nallasamy S Halushka MK Chakravarti A Genomic structure of the gene for the SH2 and pleckstrin homology domain-containing protein GRB10 and evaluation of its role in Hirschsprung disease.Oncogene. 1998; 17: 3065-3070Crossref PubMed Scopus (19) Google Scholar). Human brain tissues for use in this analysis were obtained from the Research Resource Bank (Tokyo), with permission of the Ethics Committee at the National Center of Neurology and Psychiatry. Total RNA was extracted from brain by a commercial kit (QIAGEN) and was treated with RNase-free DNase. Then, cDNA was directly synthesized using the SuperScript II amplification kit (GIBCO/BRL) with an oligo-dT primer. RT-PCR analysis was done with nested primer pairs GRB76 (5′-GCAGAAGGAACCCATGGCTTTAG-3′) and GRB78 (5′-AGAGATGAGGTTCTAAACTGCTGGTC-3′) and nested primer pairs GRB83 (5′-CATCCGTACTACCAGGACAAGG-3′) and GRB44 (5′-TGACAGCGAGGATGTGCACAG-3′), which were designed on the basis of the published cDNA sequence of human GRB10 (GenBank accession number AF001534). Amplification was carried out in a 20-μl reaction volume containing 1 μl of reverse-transcription product, 1 × reaction buffer, 2.5 mM dNTP mix, 1.5 mM MgCl2, 10 nmol each primer, and 0.25 U of Taq Gold polymerase (PE Biosystems). Reaction conditions were denaturation at 95°C for 10 min; followed by 20 cycles at 95°C for 60 s, at 58°C for 45 s, and at 72°C for 90 s; and then a final extension step at 72°C for 10 min. A second round of PCR was performed under the same PCR conditions, with 1/20 of the first reaction product. The PCR products were directly sequenced by the dideoxy sequencing method (BigDye Dideoxy sequencing kit; PE Biosystems). Five fetal brain tissues (19–27 wk gestation), shown to be heterozygous for the G/A single-nucleotide polymorphisms within exon 3, were examined. Only one allele—g in two samples and a in three samples—was present in each of the five RT-PCR products (fig. 1A). Therefore, human GRB10 is monoallelically expressed in the fetal brain. The parental origin of the expressed allele of human GRB10 was determined by our taking advantage of the recent finding that imprinting of human genes is maintained in human-rodent somatic hybrid cells (Gabriel et al. Gabriel et al., 1998Gabriel JM Higgins MJ Gebuhr TC Shows TB Saitoh S Nicholls RD A model system to study genomic imprinting of human genes.Proc Natl Acad Sci USA. 1998; 95: 14857-14862Crossref PubMed Scopus (50) Google Scholar). Somatic hybrid cells produced by crossing CHO cells and human lymphocytes retaining a single human chromosome 7 (GM10791 and GM13302; Coriell Cell Repositories) were investigated by RT-PCR and methylation-specific PCR (Herman et al. Herman et al., 1996Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5072) Google Scholar; Kosaki et al. Kosaki et al., 1997Kosaki K McGinniss MJ Veraksa AN McGinnis WJ Jones KL Prader-Willi syndrome and Angelman syndromes: diagnosis with a bisulfite treated-methylation specific PCR method.Am J Med Genet. 1997; 73: 308-313Crossref PubMed Scopus (55) Google Scholar, Kosaki et al., in pressKosaki K, Kosaki R, Robinson W, Shaffer LS, Craigen WJ, Sato S, Matsuo N. Diagnosis of maternal UPD 7 by methylation specific PCR. J Med Genet (in press)Google Scholar; Kubota et al. Kubota et al., 1997Kubota T Das S Christian SL Baylin SB Herman JG Ledbetter DH Methylation-specific PCR simplifies imprinting analysis.Nat Genet. 1997; 16: 16-17Crossref PubMed Scopus (251) Google Scholar). GM10791 and GM13302 carry a single chromosome 7 and a derivative chromosome der(7)t(7;17)(q36;q22), respectively (fig. 1B). Standard tissue-culture techniques were used to propagate the hybrid cell lines according to the supplier's recommendations, and genomic DNA and cDNA were extracted from the cells by commercial kits (QIAGEN). Expression of PEG1, a human imprinted gene on chromosome 7, known to be transcribed from the paternal allele (Kobayashi et al. Kobayashi et al., 1997Kobayashi S Kohda T Miyoshi N Kuroiwa Y Aisaka K Tsutsumi O Kaneko-Ishino T et al.Human PEG1/MEST, an imprinted gene on chromosome 7.Hum Mol Genet. 1997; 6: 781-786Crossref PubMed Scopus (134) Google Scholar; Riesewijk et al. Riesewijk et al., 1997Riesewijk AM Hu L Schulz U Tariverdian G Hoglund P Kere J Ropers HH et al.Monoallelic expression of human PEG1/MEST is paralleled by parent-specific methylation in fetuses.Genomics. 1997; 42: 236-244Crossref PubMed Scopus (76) Google Scholar; Kosaki et al. Kosaki et al., 2000Kosaki K Kosaki R Craigen WJ Matsuo N Isoform-specific imprinting of the human PEG1/MEST gene.Am J Hum Genet. 2000; 66: 309-312Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), was evaluated by RT-PCR with the primer pair PEG33 (5′-ATGGGATAACGCGGCCATGGTG-3′) and PEG34 (5′-ATAGTGATGTGGTCTCGGTTTGTCACTG-3′) (Kosaki et al. Kosaki et al., 2000Kosaki K Kosaki R Craigen WJ Matsuo N Isoform-specific imprinting of the human PEG1/MEST gene.Am J Hum Genet. 2000; 66: 309-312Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The cycling conditions were denaturation at 95°C for 10 min; followed by 40 cycles at 95°C for 1 min, at 58°C for 1 min, and at 72°C for 2 min; and then a final extension step at 72°C for 10 min. PEG1 isoform 1 (Kosaki et al. Kosaki et al., 2000Kosaki K Kosaki R Craigen WJ Matsuo N Isoform-specific imprinting of the human PEG1/MEST gene.Am J Hum Genet. 2000; 66: 309-312Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), the imprinted isoform, was expressed in GM13302 but not in GM10791 (fig. 1C). Thus, the human chromosome 7 in the somatic hybrid cells was derived paternally in GM13302 and maternally in GM10791. To confirm this assignment of parental origin, the methylation status of the CpG island in the human PEG1 gene, which undergoes parent-of-origin–specific methylation (Kobayashi et al. Kobayashi et al., 1997Kobayashi S Kohda T Miyoshi N Kuroiwa Y Aisaka K Tsutsumi O Kaneko-Ishino T et al.Human PEG1/MEST, an imprinted gene on chromosome 7.Hum Mol Genet. 1997; 6: 781-786Crossref PubMed Scopus (134) Google Scholar; Riesewijk et al. Riesewijk et al., 1997Riesewijk AM Hu L Schulz U Tariverdian G Hoglund P Kere J Ropers HH et al.Monoallelic expression of human PEG1/MEST is paralleled by parent-specific methylation in fetuses.Genomics. 1997; 42: 236-244Crossref PubMed Scopus (76) Google Scholar), was evaluated by methylation-specific PCR, as described elsewhere (Kosaki et al., Kosaki et al., in pressKosaki K, Kosaki R, Robinson W, Shaffer LS, Craigen WJ, Sato S, Matsuo N. Diagnosis of maternal UPD 7 by methylation specific PCR. J Med Genet (in press)Google Scholar). PEGMF (5′-TAGTTGCGTTTCGTAAGCGTAGTGTC-3′) and PEGMR (5′-ACACAATCCTCCGCTCGCCTA-3′) were used to amplify the methylated allele, and PEG1UF (5′-GTGGTAGTTGTGTTTTGTAAGTGTAGTGTT-3′) and PEG1UR (5′-CACACAATCCTCCACTCACCTACA-3′) were used for the unmethylated allele. In GM13302 the promoter of PEG1 isoform 1 (Kosaki et al. Kosaki et al., 2000Kosaki K Kosaki R Craigen WJ Matsuo N Isoform-specific imprinting of the human PEG1/MEST gene.Am J Hum Genet. 2000; 66: 309-312Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), was exclusively unmethylated, whereas in GM10791 the promoter was exclusively methylated. Hence, expression of PEG1 and methylation of the promoter of PEG1 collectively indicated that the human chromosome 7 was derived paternally in GM13302 and maternally in GM10791. Last, expression of GRB10 was evaluated by RT-PCR, with the GRB83-GRB44 primers under PCR conditions the same as those used for amplification of PEG1. A GRB10 amplicon was present in GM10791, which contains a maternally derived chromosome 7, but not in GM13302, which contains a paternally derived chromosome 7. Hence, it can be concluded that human GRB10 is imprinted and expressed solely from the maternal allele. After demonstrating maternal allele–specific expression, we undertook a mutation analysis of GRB10 in two groups of patients with RSS, one from Japan and the other from Europe. The first group consisted of 30 unrelated Japanese patients with RSS who fulfilled at least three of the following diagnostic criteria (Price et al. Price et al., 1999Price SM Stanhope R Garrett C Preece MA Trembath RC The spectrum of Silver-Russell syndrome: a clinical and molecular genetic study and new diagnostic criteria.J Med Genet. 1999; 36: 837-842PubMed Google Scholar): low birth weight (<−2SD); short stature at the time of diagnosis (<−2SD); characteristic facial features; and facial, limb, or trunk asymmetry. Clinical information and material for analysis were provided by clinicians throughout Japan. Appropriate informed consent was obtained from all subjects. Karyotypes were normal, and mUPD7 was excluded by either genotyping analysis or methylation analysis of PEG1 (Riesewijk et al. Riesewijk et al., 1997Riesewijk AM Hu L Schulz U Tariverdian G Hoglund P Kere J Ropers HH et al.Monoallelic expression of human PEG1/MEST is paralleled by parent-specific methylation in fetuses.Genomics. 1997; 42: 236-244Crossref PubMed Scopus (76) Google Scholar; Kosaki et al., Kosaki et al., in pressKosaki K, Kosaki R, Robinson W, Shaffer LS, Craigen WJ, Sato S, Matsuo N. Diagnosis of maternal UPD 7 by methylation specific PCR. J Med Genet (in press)Google Scholar). Cell lines from the second group of patients with RSS were derived from the European Collection of Cell Cultures. A total of 28 RSS cell lines deposited by Richard Trembath, who defined the diagnostic criteria for RSS (Price et al. Price et al., 1999Price SM Stanhope R Garrett C Preece MA Trembath RC The spectrum of Silver-Russell syndrome: a clinical and molecular genetic study and new diagnostic criteria.J Med Genet. 1999; 36: 837-842PubMed Google Scholar), were analyzed. Using primers based on the reported genomic sequence of human GRB10 (GenBank accession numbers AF073363–AF073378; Angrist et al. Angrist et al., 1998Angrist M Bolk S Bentley K Nallasamy S Halushka MK Chakravarti A Genomic structure of the gene for the SH2 and pleckstrin homology domain-containing protein GRB10 and evaluation of its role in Hirschsprung disease.Oncogene. 1998; 17: 3065-3070Crossref PubMed Scopus (19) Google Scholar), we amplified each of the 16 exons of GRB10 from genomic DNA. The PCR conditions were denaturation at 95°C for 10 min; followed by 35 cycles at 95°C for 1 min, at 58°C for 1 min, and at 72°C for 1 min; and a final extension step at 72°C for 10 min. The primer sequences can be obtained from the corresponding author, on request. PCR products were subjected to denaturing high-performance liquid chromatography (WAVE; Transgenomic) (Liu et al. Liu et al., 1998Liu W Smith D Rechtzigel KJ Thibodeau SN James DC Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations.Nucleic Acids Res. 1998; 26: 1396-1400Crossref PubMed Scopus (227) Google Scholar; Wagner et al. Wagner et al., 1999Wagner TMU Hirtenlehner K Shen P Moeslinger R Muhr D Fleischmann E Concin H et al.Global sequence diversity of BRCA2: analysis of 71 breast cancer families and 95 control individuals of worldwide populations.Hum Mol Genet. 1999; 8: 413-423Crossref PubMed Google Scholar), and, when abnormal chromatographic patterns were detected, the PCR products were sequenced on an automated sequencer (ABI310; PE Biosystems). The mutation analysis revealed that two patients with RSS—RS1 and RS3—had a heterozygous C→T transition that was not present in 100 ethnically matched controls (200 chromosomes) (fig. 2). The base change was confirmed by EcoO109I digestion. This transition leads to a proline→serine substitution at codon 95 (P95S), which is located in the amino-terminal region of GRB10. Patient RS1, delivered at 39 wk gestation after an uneventful pregnancy, had a birth weight of 1.80 kg (−3.5 SD) and a length of 42.5 cm (−3.5 SD). At age 3.5 years, her weight was 10.5 kg and her height was 86.5 cm (both −3.5 SD). She had facial features that were characteristic of RSS, including triangular facies, downturned corners of the mouth, and fifth-finger clinodactyly. The P95S substitution was derived from the mother, who had normal height (163 cm) and no features of RSS. The unaffected mother received the mutant allele from her father (i.e., the maternal grandfather). RS3, who was unrelated to RS1 and was delivered at 39 wk gestation after an uneventful pregnancy, had a birth weight of 2.55 kg (−2SD) and a length of 45.5 cm (−2SD). He had the same characteristic physical features as patient RS1. The P95S substitution was derived from his mother, who had a height of 153 cm (−1SD). DNA samples from the maternal grandparents were not available. Both the identification of the P95S substitution in the proline-rich amino-terminal region of GRB10 in two of 58 patients with RSS and the absence of similar mutations in 100 normal individuals suggest that the amino acid substitution may result in functionally significant change in the critical RSS-related gene on chromosome 7. The documented paternal origin of the mutated P95S allele in the mother of patient RS1 is consistent with the normal phenotype observed in this individual, in spite of the inheritance of the mutant allele. Although she possesses an allele with the same mutation as her affected daughter, this paternally derived allele would not be expressed in the mother. Ultimately, direct assessment of the effects of the mutation on the functional properties of the GRB10 protein will be required in order to verify their causal role in RSS. Since GRB10 is a maternally expressed gene, it is expected to be overexpressed in patients with RSS who have maternal UPD7 or maternally derived duplication of 7p12 (Joyce et al. Joyce et al., 1999Joyce CA Sharp A Walker JM Bullman H Temple KI Duplication of 7p12.1-p13, including GRB10 and IGFBP1, in a mother and daughter with features of Silver-Russell syndrome.Hum Genet. 1999; 105: 273-280Crossref PubMed Scopus (93) Google Scholar; Monk et al. Monk et al., 2000Monk D Wakeling EL Proud V Hitchins M Abu-Amero SN Stanier P Preece MA et al.Duplication of 7p11.2-p13, including GRB10, in Silver-Russell syndrome.Am J Hum Genet. 2000; 66: 36-46Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). By analogy, the GRB10 mutations that we have identified are likely to result in a gain of function of the GRB10 protein. Functional studies are warranted to evaluate how the proline→serine substitution actually affects the activity of GRB10. Although several reports have provided evidence for inhibitory effects of GRB10 on insulin, IGF-I, and GH receptor signaling (O’Neill et al. O'Neill et al., 1996O'Neill T Rose DW Pillay TS Hotta K Olefsky JM Gustafson TA Interaction of a GRB-IR splice variant (a human GRB10 homolog) with the insulin and insulin-like growth factor I receptors: evidence for a role in mitogenic signaling.J Biol Chem. 1996; 271: 22506-22513Crossref PubMed Scopus (128) Google Scholar; Morrione et al. Morrione et al., 1997Morrione A Valentinis B Resnicoff M Xu S Baserga R The role of mGrb10alpha in insulin-like growth factor I-mediated growth.J Biol Chem. 1997; 272: 26382-26387Crossref PubMed Scopus (83) Google Scholar; Moutoussamy et al. Moutoussamy et al., 1998Moutoussamy S Renaudie F Lago F Kelly PA Finidori J Grb10 identified as a potential regulator of growth hormone (GH) signaling by cloning of GH receptor target proteins.J Biol Chem. 1998; 273: 15906-15912Crossref PubMed Scopus (60) Google Scholar), a recent study demonstrated positive growth-regulatory effects of GRB10 in cultured fibroblasts (Wang et al. Wang et al., 1999Wang J Dai H Yousaf N Moussaif M Deng Y Boufelliga A Swamy OR et al.Grb10, a positive, stimulatory signaling adapter in platelet-derived growth factor BB-, insulin-like growth factor I-, and insulin-mediated mitogenesis.Mol Cell Biol. 1999; 19: 6217-6228Crossref PubMed Scopus (100) Google Scholar). The investigation of both the functional significance of the GRB10 mutations that we have identified and their relevance to growth failure in patients with RSS should help to define the actions and physiological importance of GRB10. It is intriguing to note that the amino acid substitution is present in the proline-rich amino-terminal region of GRB10, which determines binding interactions with SH3-domain–containing proteins (Wojcik et al. Wojcik et al., 1999Wojcik J Girault JA Labesse G Chomilier J Mornon JP Callebaut I Sequence analysis identifies a ras-associating (RA)–like domain in the N-termini of band 4.1/JEF domains and in the Grb7/10/14 adapter family.Biochem Biophys Res Commun. 1999; 259: 113-120Crossref PubMed Scopus (45) Google Scholar). Although amino acids corresponding precisely to P95 are not evident in mouse Grb10 (Ooi et al. Ooi et al., 1995Ooi J Yajnik V Immanuel D Gordon M Moskow JJ Buchberg AM Margolis B The cloning of Grb10 reveals a new family of SH2 domain proteins.Oncogene. 1995; 10: 1621-1630PubMed Google Scholar; Laviola et al. Laviola et al., 1997Laviola L Giorgino F Chow JC Baquero JA Hansen H Ooi J Zhu J et al.The adapter protein Grb10 associates preferentially with the insulin receptor as compared with the IGF-I receptor in mouse fibroblasts.J Clin Invest. 1997; 99: 830-837Crossref PubMed Scopus (87) Google Scholar), the absence of sequence identity at this position does not rule out a functionally significant effect of the P95S mutation in humans. Indeed, multiple known disease-causing substitutions have been identified involving amino acid residues that are not identical in other homologous human and mouse genes. In the present study, we have demonstrated GRB10 coding-sequence mutations in only a small subset of patients with RSS. Such a low prevalence of GRB10 mutations is not unexpected, because the etiology of RSS is most likely genetically heterogeneous (Wakeling et al. Wakeling et al., 1998Wakeling EL Abu-Amero S Price SM Stanier P Trembath RC Moore GE Preece MA Genetics of Silver-Russell syndrome.Horm Res. 1998; 49: 32-36Crossref Scopus (22) Google Scholar). Studies have shown that the RSS phenotype is associated not only with chromosome 7 abnormalities but also with other chromosomal aberrations, including balanced translocation involving 17q (Midro et al. Midro et al., 1993Midro AT Debek K Sawicka A Marcinkiewicz D Rogowska M Second observation of Silver-Russell syndrome in a carrier of a reciprocal translocation with one breakpoint at site 17q25.Clin Genet. 1993; 44: 53-55Crossref PubMed Scopus (52) Google Scholar) and deletion of 15q26-qter (Wilson et al. Wilson et al., 1985Wilson GN Sauder SE Bush M Beitins IZ Phenotypic delineation of ring chromosome 15 and Russell-Silver syndromes.J Med Genet. 1985; 22: 233-236Crossref PubMed Scopus (38) Google Scholar; Rogan et al. Rogan et al., 1996Rogan PK Seip JR Driscoll DJ Papenhausen PR Johnson VP Raskin S Woodward AL et al.Distinct 15q genotypes in Russell-Silver and ring 15 syndromes.Am J Med Genet. 1996; 62: 10-15Crossref PubMed Scopus (40) Google Scholar). It has been proposed that the RSS phenotype in patients with 15q26-qter deletions results from hemizygosity for the IGF-I receptor (IGF1R) (Peoples et al. Peoples et al., 1995Peoples R Milatovich A Francke U Hemizygosity at the insulin-like growth factor I receptor (IGF1R) locus and growth failure in the ring chromosome 15 syndrome.Cytogenet Cell Genet. 1995; 70: 228-234Crossref PubMed Scopus (46) Google Scholar; Siebler et al. Siebler et al., 1995Siebler T Lopaczynski W Terry CL Casella SJ Munson P De Leon DD Phang L et al.Insulin-like growth factor I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15.J Clin Endocrinol Metab. 1995; 80: 3447-3457PubMed Google Scholar). Because GRB10 may negatively regulate IGF-I receptor–mediated growth and mitogenesis (O'Neill et al. O'Neill et al., 1996O'Neill T Rose DW Pillay TS Hotta K Olefsky JM Gustafson TA Interaction of a GRB-IR splice variant (a human GRB10 homolog) with the insulin and insulin-like growth factor I receptors: evidence for a role in mitogenic signaling.J Biol Chem. 1996; 271: 22506-22513Crossref PubMed Scopus (128) Google Scholar; Morrione et al. Morrione et al., 1997Morrione A Valentinis B Resnicoff M Xu S Baserga R The role of mGrb10alpha in insulin-like growth factor I-mediated growth.J Biol Chem. 1997; 272: 26382-26387Crossref PubMed Scopus (83) Google Scholar), it is conceivable that overexpression of GRB10 (e.g., mUPD7 and duplication of 7p11-13) and haploinsufficiency of the IGF-I receptor (e.g., 15q deletion) would confer a similar phenotype. Since hemizygosity for IGF-1 also has been associated with a phenotype similar to that of RSS (Woods et al. Woods et al., 1996Woods KA Camacho-Hubner C Savage MO Clark AJ Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene.N Engl J Med. 1996; 335: 1363-1367Crossref PubMed Scopus (908) Google Scholar), we suggest that loss-of-function mutations in IGF1 or its receptor and, most probably, gain-of-function mutations in GRB10, as a negative modulator of the IGF-I receptor, all lead to a common RSS phenotype. The documentation of GRB10 mutations in two patients with RSS in this study further supports the concept that defects in the insulin-like growth-factor axis all lead to the common RSS phenotype. In addition to its effects on the IGF-I receptor–signaling pathway, it recently has been shown that GRB10 can associate with the growth hormone (GH) receptor and inhibit transcription of a GH reporter gene (Moutoussamy et al. Moutoussamy et al., 1998Moutoussamy S Renaudie F Lago F Kelly PA Finidori J Grb10 identified as a potential regulator of growth hormone (GH) signaling by cloning of GH receptor target proteins.J Biol Chem. 1998; 273: 15906-15912Crossref PubMed Scopus (60) Google Scholar). This in vitro finding suggests that GRB10 mutations may affect GH as well as IGF-I signaling. Since some patients with RSS respond to GH therapy and others do not (Stanhope et al. Stanhope et al., 1998Stanhope R Albanese A Azcona C Growth hormone treatment of Russell-Silver syndrome.Horm Res. 1998; 49: 37-40Crossref Scopus (19) Google Scholar), it will be important ultimately to determine whether the presence of mutations in GRB10 correlates with the effectiveness of GH therapy. In summary, we have shown that the human GRB10 gene is subject to genomic imprinting. A mutation analysis of GRB10 in 58 unrelated patients with RSS identified two individuals with a P95S substitution in the amino-terminal region of the GRB10 protein. In both cases, the P95S 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 may contribute to the RSS phenotype. We thank Taichi Suzuki from Tokyo Technical College for excellent laboratory assistance. This work was supported in part by a grant from the Pharmacia-Upjohn Fund for Growth & Development Research and by the Foundation for Growth Science in Japan.