FGFR3 gene mutation plus GRB10 gene duplication in a patient with achondroplasia plus growth delay with prenatal onset
- Haiming Yuan†1, 2,
- Linhuan Huang†3,
- Xizi Hu4,
- Qian Li5,
- Xiaofang Sun6,
- Yingjun Xie6Email authorView ORCID ID profile,
- Shu Kong6 and
- Xiaoman Wang6
© The Author(s). 2016
Received: 29 March 2016
Accepted: 9 June 2016
Published: 2 July 2016
Achondroplasia is a well-defined and common bone dysplasia. Genotype- and phenotype-level correlations have been found between the clinical symptoms of achondroplasia and achondroplasia-specific FGFR3 mutations.
A 2-year-old boy with clinical features consistent with achondroplasia and Silver-Russell syndrome-like symptoms was found to carry a mutation in the fibroblast growth factor receptor-3 (FGFR3) gene at c.1138G > A (p.Gly380Arg) and a de novo 574 kb duplication at chromosome 7p12.1 that involved the entire growth-factor receptor bound protein 10 (GRB10) gene. Using quantitative real-time PCR analysis, GRB10 was over-expressed, and, using enzyme-linked immunosorbent assays for IGF1 and IGF-binding protein-3 (IGFBP3), we found that IGF1 and IGFBP3 were low-expressed in this patient.
We demonstrate that a combination of uncommon, rare and exceptional molecular defects related to the molecular bases of particular birth defects can be analyzed and diagnosed to potentially explain the observed variability in the combination of molecular defects.
Achondroplasia (ACH, OMIM #100800) is the most common non-lethal skeletal dysplasia. It is an autosomal-dominant disorder with an incidence of approximately 5–15 per 100,000 live births. Mutations in FGFR3 at c.1138 G > A (p.Gly380Arg) are known to cause ACH, with 97 % of cases involving this mutation [8, 22]. Gain-of-function mutations in FGFR3 have been shown to cause both chondrodysplasias and craniosynostoses and to result in impaired endochondral ossification .
While patients with maternal uniparental disomy of chromosome 7 (mUPD7) that involves GRB10 are consistently diagnosed with Silver–Russell syndrome (SRS, OMIM #180860) [4, 7]. SRS is a rare congenital developmental disorder that is characterized by severe intrauterine and postnatal growth restriction, a triangular-shaped face, and congenital malformations that include relative macrocephaly, hemihypotrophy, and fifth-finger clinodactyly . However, there is no conclusive evidence regarding the pathogenic mechanism by which GRB10 (i.e., imprinting vs. dosage) contributes to the etiology of SRS.
Review genomic and clinical information on patients with duplication involving GRB10
Patients TB/LB from Joyce et al. 
Patient DP from Monk et al. 
Patients HC/AC from Monk et al. 
Patient from Eggermann et al.  (decipher 285981)
Genomic location (hg19)
overgrowth, DD, microcephaly, seizure
FTT, GDD, large eyes, microcephaly, short stature, triangular face
Genes involved (Refseq)
GRB10 and part of COBL
Part of GRB10
Here, we report a patient who displayed prenatal onset growth delay and ACH and carried a small de novo duplication at 7p12.1 that involved the GRB10 gene in addition to a heterozygous point mutation at FGFR3. We detected the smallest known duplication in a patient who displayed one of the typical clinical features of SRS. This duplication was restricted to the GRB10 gene at 7p12.1. This case provides direct evidence showing that a GRB10 duplication is sufficient to cause the prenatal onset growth delay phenotype in SRS and the combination of these two molecular defects may explain the so-called variable expressivity dominant traits.
The patient was the fourth child of healthy unrelated parents with a negative family history. His siblings were all healthy. Fetal short limbs were noticed on the ultrasound examination at 8 months of pregnancy, indicating intrauterine growth restriction (date not provided). He was born by vaginal delivery at 38 weeks of gestation after an uneventful pregnancy. His birth weight was 1900 g (<1st percentile), his length was 43 cm (<1st percentile) and his head circumference was 34 cm. His Apgar scores were all 9 s. Feeding difficulty was noted during the first two months after birth.
Karyotyping and chromosomal microarray analysis
A standard chromosome analysis at 550-band resolution was performed using cultured peripheral blood from the patient according to standard procedures.
A chromosomal microarray analysis (CMA) of the patient and his parents was performed using an Affymetrix Cytoscan HD Array (Affymetrix, Santa Clara, CA, USA). Genomic DNA was extracted from peripheral blood using a commercial kit (Qiagen). The labeling and hybridization procedures were performed according to the manufacturer’s instructions. The raw CMA data were analyzed using Affymetrix Chromosome Analysis Suite software. The microarray data for the family trio were then processed using the standard protocols provided by the manufacturer to perform a basic analysis using Chromosome Analysis Suite software (NetAffx 33.1, Affymetrix, USA).
Confirmation of GRB10 duplication
The GRB10 copy number was further confirmed using quantitative real-time PCR analysis. The primer sequences that were used and their descriptions are included in Additional file 1: Table S1.
GRB10 expression level
GRB10 expression levels were detected in blood samples using quantitative real-time PCR analysis. Total RNA was extracted from whole blood, and reverse transcription was performed using a commercial kit (Promega, USA). A blood sample from the proband’s mother was used as the control. The primer sequences that were used and their descriptions are included in Additional file 1: Table S2.
Measurement of IGF1 and IGFBP3
We measured the levels of IGF1 and IGF binding protein-3 (IGFBP3) in the patient’s serum using enzyme-linked immunosorbent assay (ELISA) kits (Diagnostic Systems Laboratories, Webster, TX, USA) according to the manufacturer’s instructions.
Sequencing of the fibroblast growth factor receptor-3 gene (FGFR3)
DNA samples were tested for common disease-associated mutations in FGFR3 (achondroplasia and hypochondroplasia: p.Gly380Arg and p.Asn540Lys, respectively; thanatophoric dysplasia type I: p.Arg248Cys, p.Tyr373Cys, p.Ser249Cys, and p.X807; and thanatophoric dysplasia type II: p.Lys650Glu) using Sanger sequencing. The primer sequences are shown in Additional file 1: Table S3 (reference sequence for the FGFR3 gene: NM_000142.4). Sanger sequencing was performed according to the manufacturer’s instructions using a Big Dye Terminator Cycle Sequencing Kit (version 3.1) and analyzed using an ABI Prism 3130xl Genetic DNA analyzer.
Comparison of clinical abnormalities with Achondroplasia (ACH) and Silver-Russell syndrome (SRS)
Silver-Russell syndrome (SRS)
nose bridge collapse
a trident configuration of the hands
fifth finger clinodactyly
asymmetry of limbs
Scoliosis and thoracic kyphosis
Feeding difficulties in early childhood
Snoring (coarse breath sounds)
In our patient, we detected a three-fold increase in GRB10 expression. GRB10 is an imprinted gene on 7p12.1. Because it is a key genetic component of developmental programming, recent studies have also suggested that placental GRB10 plays a role in regulating fetoplacental growth and that it is thereby implicated in the pathophysiology of fetal growth restriction in the context of fetal gender [2, 26]. Studies have consistently supported the notion that increases in gene dosage are the main contributors to the over-production of the GRB10 gene product. The GRB10 gene encodes an adaptor protein that interacts with IGF1 and insulin receptors to negatively regulate the IGF1 growth cascade [5, 18]. A previous study reported that endogenous GRB10 expression was increased in the hippocampus of rats with diabetic encephalopathy and that this increase might result in damaged nerve functions and cognitive impairments. In addition, the insulin/IGF1 signaling pathways have been implicated in dysregulated synaptic maturation and may play key roles in brain aging and dementia as well as learning and cognitive functions in rodent models [14, 19]. In the present study, the IGF1 and IGFBP3 levels in the patient were far below normal levels, consistent with a model in which the duplication of GRB10 directly inhibits the phosphorylation of the IGF1R substrate and reduces the level of Grb10 expression to below normal limits, which may result in nervous system impairments such as growth delays and intellectual disabilities (ID) . These findings suggest that the duplication of GRB10 contributes to the prenatal onset growth delay phenotype that was observed in our patient and may also explain the ID features in SRS.
Previous studies of mice with uniparental disomy of Grb10 demonstrated that Grb10 is a typical imprinted gene and that it affects growth mainly via an imprinting mechanism [1, 20, 21, 25]. However, the imprinting behavior of human GRB10, which is located on chromosome 7, has been shown to be different from that of its mouse homolog . Human GRB10 is biallelically expressed in most tissues, with the exception of the placental villus trophoblast and fetal brain tissues [10, 15]. Because of the known complexity of the 7p12.1 imprinting regions and their interactions, interpretations of copy number variations in this region are complicated. The clinical outcomes in cases of micro-duplications are influenced by the size, breakpoint positions and parental inheritance of the imbalance and by the imprinting status of the affected genes.
Though, the duplication that was identified in our propositus could potentially have disrupted the genomic structure of the cordon-bleu WH2 repeat protein (COBL) gene, which is specifically expressed in the node and its derivatives until organogenesis, we could not exclude the possibility that a disruption in COBL might have been involved in the patient’s phenotype. In addition, there are no known correlations between mutations in FGFR3 and the duplication of GRB10.
The data related to the patient described in the present study at least suggest that mutations in FGFR3 cause ACH but do not influence the effects of the duplication of GRB10 on prenatal onset growth delay in SRS. The results of our study also suggest that phenotypes are rarely “simple” or directly related to specific gene defects and that combinations of uncommon, rare and exceptional molecular defects, which can be explored and used in diagnoses, may explain the so-called variability observed in the expression of dominant traits.
CMA, chromosomal microarray analysis; COBL, cordon-bleu WH2 repeat protein; ELISA, enzyme-linked immunosorbent assay; FGFR3, fibroblast growth factor receptor-3; FGFR3, fibroblast growth factor receptor-3; GRB10, growth-factor receptor-bound protein 10; H19DMR, H19 promoter; HMGA2, high-mobility AT-hook 2; ICR1, Imprinted Center Region 1; ID, intellectual disability; IGFBP3, IGF binding protein-3; mUPD7, maternal uniparental disomy of chromosome 7; SRS, Silver–Russell syndrome
We would like to thank the family of the proband for their cooperation during this study.
This study was supported by the National Natural Science Foundation Committee of China (NSFC-81500974 to Xie Yingjun).
Availability of data and materials
Affymetrix cytoscan HD (http://media.affymetrix.com/support/technical/datasheets/cytoscan_hd_datasheet.pdf).
Chromosome Analysis Suite Software, Version 3.1 (http://www.affymetrix.com/support/technical/software_downloads.affx).
YH and HL contributed equally to the manuscript. XY was responsible for the design of the project, data analysis, and manuscript write-up. XY also facilitated the panel with assistance from YH and HL. YH and XY drafted the first versions of the manuscript. SX, KS and WX were the neurologists on the team, and they reviewed the relevant abstracts and articles and contributed to the final critical review of the write-up. HL, HX and LQ assisted in the literature review, performed the experiments, data entry, and data analysis phases of the project. All authors read and approved the final manuscript.
The authors declare that there are no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Consent for publication
Consent to publish has been obtained from the guardians of all involved children.
Ethics approval and consent to participate
Ethical approval was obtained for this study from the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University (IRB 201301).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Charalambous M, Smith FM, Bennett WR, Crew TE, Mackenzie F, Ward A. Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism. Proc Natl Acad Sci U S A. 2003;100:8292–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Cowley M, Garfield AS, Madon-Simon M, Charalambous M, Clarkson RW, Smalley MJ, Kendrick H, Isles AR, Parry AJ, Carney S, Oakey RJ, Heisler LK, Moorwood K, Wolf JB, Ward A. Developmental programming mediated by complementary roles of imprinted Grb10 in mother and pup. PLoS Biol. 2014;12:e1001799.View ArticlePubMedPubMed CentralGoogle Scholar
- Di Rocco F, Biosse Duplan M, Heuze Y, Kaci N, Komla-Ebri D, Munnich A, Mugniery E, Benoist-Lasselin C, Legeai-Mallet L. FGFR3 mutation causes abnormal membranous ossification in achondroplasia. Hum Mol Genet. 2014;23:2914–25.View ArticlePubMedGoogle Scholar
- Dias RP, Bogdarina I, Cazier JB, Buchanan C, Donaldson MC, Johnston LB, Hokken-Koelega AC, Clark AJ. Multiple segmental uniparental disomy associated with abnormal DNA methylation of imprinted Loci in silver-russell syndrome. J Clin Endocrinol Metab. 2012;97:E2188–93.View ArticlePubMedGoogle Scholar
- Dufresne AM, Smith RJ. The adapter protein GRB10 is an endogenous negative regulator of insulin-like growth factor signaling. Endocrinology. 2005;146:4399–409.View ArticlePubMedGoogle Scholar
- Eggermann T, Begemann M, Gogiel M, Palomares M, Vallespin E, Fernandez L, Cazorla R, Spengler S, Garcia-Minaur S. Heterogeneous growth patterns in carriers of chromosome 7p12.2 imbalances affecting GRB10. Am J Med Genet A. 2012;158A:2815–9.View ArticlePubMedGoogle Scholar
- Hannula-Jouppi K, Muurinen M, Lipsanen-Nyman M, Reinius LE, Ezer S, Greco D, Kere J. Differentially methylated regions in maternal and paternal uniparental disomy for chromosome 7. Epigenetics. 2014;9:351–65.View ArticlePubMedGoogle Scholar
- He X, Xie F, Ren ZR. Rapid detection of G1138A and G1138C mutations of the FGFR3 gene in patients with achondroplasia using high-resolution melting analysis. Genet Test Mol Biomarkers. 2012;16:297–301.View ArticlePubMedGoogle Scholar
- Hecht JT, Bodensteiner JB, Butler IJ. Neurologic manifestations of achondroplasia. Handb Clin Neurol. 2014;119:551–63.View ArticlePubMedGoogle Scholar
- Hikichi T, Kohda T, Kaneko-Ishino T, Ishino F. Imprinting regulation of the murine Meg1/Grb10 and human GRB10 genes; roles of brain-specific promoters and mouse-specific CTCF-binding sites. Nucleic Acids Res. 2003;31:1398–406.View ArticlePubMedPubMed CentralGoogle Scholar
- Hitchins MP, Monk D, Bell GM, Ali Z, Preece MA, Stanier P, Moore GE. Maternal repression of the human GRB10 gene in the developing central nervous system; evaluation of the role for GRB10 in Silver-Russell syndrome. Eur J Hum Genet. 2001;9:82–90.View ArticlePubMedGoogle Scholar
- Ireland PJ, Donaghey S, McGill J, Zankl A, Ware RS, Pacey V, Ault J, Savarirayan R, Sillence D, Thompson E, Townshend S, Johnston LM. Development in children with achondroplasia: a prospective clinical cohort study. Dev Med Child Neurol. 2012;54:532–7.View ArticlePubMedGoogle Scholar
- Joyce CA, Sharp A, Walker JM, Bullman H, Temple IK. Duplication of 7p12.1-p13, including GRB10 and IGFBP1, in a mother and daughter with features of Silver-Russell syndrome. Hum Genet. 1999;105:273–80.View ArticlePubMedGoogle Scholar
- Ma L, Wei Q, Deng H, Zhang Q, Li G, Tang N, Xie J, Chen Y. Growth factor receptor-bound protein 10-mediated negative regulation of the insulin-like growth factor-1 receptor-activated signalling pathway results in cognitive disorder in diabetic rats. J Neuroendocrinol. 2013;25:626–34.View ArticlePubMedGoogle Scholar
- Monk D, Arnaud P, Frost J, Hills FA, Stanier P, Feil R, Moore GE. Reciprocal imprinting of human GRB10 in placental trophoblast and brain: evolutionary conservation of reversed allelic expression. Hum Mol Genet. 2009;18:3066–74.View ArticlePubMedGoogle Scholar
- Monk D, Bentley L, Hitchins M, Myler RA, Clayton-Smith J, Ismail S, Price SM, Preece MA, Stanier P, Moore GE. Chromosome 7p disruptions in Silver Russell syndrome: delineating an imprinted candidate gene region. Hum Genet. 2002;111:376–87.View ArticlePubMedGoogle Scholar
- Monk D, Wakeling EL, Proud V, Hitchins M, Abu-Amero SN, Stanier P, Preece MA, Moore GE. Duplication of 7p11.2-p13, including GRB10, in Silver-Russell syndrome. Am J Hum Genet. 2000;66:36–46.View ArticlePubMedGoogle Scholar
- Morrione A. Grb10 adapter protein as regulator of insulin-like growth factor receptor signaling. J Cell Physiol. 2003;197:307–11.View ArticlePubMedGoogle Scholar
- Saenger S, Goeldner C, Frey JR, Ozmen L, Ostrowitzki S, Spooren W, Ballard TM, Prinssen E, Borroni E, Metzger F. PEGylation enhances the therapeutic potential for insulin-like growth factor I in central nervous system disorders. Growth Horm IGF Res. 2011;21:292–303.View ArticlePubMedGoogle Scholar
- Shiura H, Miyoshi N, Konishi A, Wakisaka-Saito N, Suzuki R, Muguruma K, Kohda T, Wakana S, Yokoyama M, Ishino F, Kaneko-Ishino T. Meg1/Grb10 overexpression causes postnatal growth retardation and insulin resistance via negative modulation of the IGF1R and IR cascades. Biochem Biophys Res Commun. 2005;329:909–16.View ArticlePubMedGoogle Scholar
- Shiura H, Nakamura K, Hikichi T, Hino T, Oda K, Suzuki-Migishima R, Kohda T, Kaneko-ishino T, Ishino F. Paternal deletion of Meg1/Grb10 DMR causes maternalization of the Meg1/Grb10 cluster in mouse proximal Chromosome 11 leading to severe pre- and postnatal growth retardation. Hum Mol Genet. 2009;18:1424–38.View ArticlePubMedGoogle Scholar
- Sobetzko D, Braga S, Rudeberg A, Superti-Furga A. Achondroplasia with the FGFR3 1138 g-- > a (G380R) mutation in two sibs sharing a 4p haplotype derived from their unaffected father. J Med Genet. 2000;37:958–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Stein EG, Gustafson TA, Hubbard SR. The BPS domain of Grb10 inhibits the catalytic activity of the insulin and IGF1 receptors. FEBS Lett. 2001;493:106–11.View ArticlePubMedGoogle Scholar
- Varma SN, Varma BR. Clinical spectrum of Silver - Russell syndrome. Contemp Clin Dent. 2013;4:363–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang L, Balas B, Christ-Roberts CY, Kim RY, Ramos FJ, Kikani CK, Li C, Deng C, Reyna S, Musi N, Dong LQ, DeFronzo RA, Liu F. Peripheral disruption of the Grb10 gene enhances insulin signaling and sensitivity in vivo. Mol Cell Biol. 2007;27:6497–505.View ArticlePubMedPubMed CentralGoogle Scholar
- Xue Y, Shankar S, Cornell K, Dai Z, Wang CE, Rudd MK, Coffee B. Paternal duplication of the 11p15 centromeric imprinting control region is associated with increased expression of CDKN1C in a child with Russell-Silver syndrome. Am J Med Genet A. 2015;167A(12):3229–33.View ArticlePubMedGoogle Scholar