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IGF-II Serum Levels Are Normal in Children with Silver-Russell Syndrome Who Frequently Carry Epimutations at the IGF2 Locus

University Children’s Hospital, 72076 Tübingen, Germany; and Institute of Human Genetics, University Hospital, Rheinisch-Westfälische Technische Hochschule Aachen, 52074 Aachen, Germany

Address all correspondence and requests for reprints to: PD Dr. Gerhard Binder, Pediatric Endocrinology Section, University-Children’s Hospital, Hoppe-Seyler-Strasse1, 72076 Tübingen, Germany. E-mail: gerhard.binder{at}med.uni-tuebingen.de.

	Abstract

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Context: Epigenetic mutations of 11p15 encompassing IGF2 are present in short children with Silver-Russell syndrome (SRS) with high frequency (31–50%). It has been speculated that these mutations characterized by demethylation of ICR1 cause diminished IGF2 expression.

Objective: We aimed to determine the prevalence of pathologically low IGF-II serum levels in children with SRS.

Subjects: SRS was defined by birth weight or length below the 3rd percentile, lack of postnatal catch-up growth, and the presence of two of the following characteristics: typical face, relative macrocephaly, and skeletal asymmetry. Serum samples of 30 patients were available. Mean age was 5.4 ± 2.1 yr.

Methods: The serum levels of IGF-I, IGF-II, IGF binding protein (IGFBP)-2, and IGFBP-3 were measured by RIA and compared with age-related reference values and with serum concentrations measured in age- and gender-matched controls born small for gestational age (SGA), but lacking major dysmorphic features. Analysis of genomic DNA was possible in a subgroup of children with SRS: the methylation status of the ICR1 locus on 11p15 and the parental origin of chromosome 7 were analyzed in 9 and 23 children, respectively.

Results: Demethylation of ICR1 was found in 44% and uniparental disomy in 17% of the tested children with SRS. The median IGF-II serum level in SRS was 441 µg/liter (range, 238–875). This was significantly higher than in the SGA controls: 387 µg/liter (range, 265–596) (P < 0.03), but below the median value of the age-related reference, which was 532 µg/liter. The four children with SRS and ICR1 demethylation had high-normal and normal IGF-II serum levels that were higher than the levels of their SGA controls. IGF-I, IGFBP-2, and IGFBP-3 serum levels were not different between the SRS children and their SGA controls.

Conclusions: Our data render it unlikely that demethylation of ICR1 on 11p15 does cause diminished IGF-II serum levels in children with SRS. This observation does not exclude deficient IGF-II action before birth.

	Introduction

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APPROXIMATELY 75 GENES of the human genome are known to be imprinted by epigenetic mechanisms that allow expression from only one of the two parental alleles (1). This imprinting status can differ between species, but also between organs and developmental stages in the same subject (1). A major subset of imprinted genes is involved in fetal growth with opposing activities of their products: the paternally expressed ones promote, whereas the maternally expressed ones constrain fetal growth (2). Of these imprinted genes, the IGF2 gene is the only one encoding a hormone (2). IGF2 is located inside a complex cluster of at least eight imprinted genes on 11p15.5, which contains paternally expressed genes such as IGF2 and KCNQ1OT1 and maternally expressed genes such as H19 and CDKN1C (3). The mechanisms involved in paternal expression of IGF2 are complex and involve differential methylation of genomic loci that are located up- and downstream of IGF2 (3). The presence of different organ-specific IGF2 promotors and alternative splicing of primary IGF2 RNA add to the complexity of the picture envisioned to date (4, 5).

The effects of IGF-II on fetal growth are mediated through the IGF-I receptor and probably a second receptor not yet characterized (6). Mice knockout models indicate that loss of the paternal IGF2 causes intrauterine growth retardation with lack of 40% of the body weight in newborn mice, whereas experimental overexpression of the same allele results in hypertrophic mice (6). Growth of the mouse placenta and the differentiation of the labyrinthine trophoblasts were shown to be strongly promoted by IGF-II, for which placental expression is driven by a placenta-specific promotor (7). Overexpression of IGF2 in humans is one molecular basis of Beckwith-Wiedemann syndrome (BWS), which is characterized by neonatal hypertrophy, exomphalos, and macroglossia; some of the affected show duplication or uniparental disomy of the paternal 11p15 (3). Analysis of tissue samples from children affected with BWS revealed overexpression of IGF2 at the mRNA level (3, 8). In contrast, IGF-II serum levels were found to be normal in children with BWS (8).

Recently, the opposite of the mutations found in BWS, maternal duplications of 11p15 (9) as well as demethylation of the ICR1 locus on 11p15 (10, 11, 12), were described in children with Silver-Russell syndrome (SRS) with high frequency (31–50%). The SRS is defined by the presence of intrauterine growth retardation, lack of postnatal catch-up growth, a typical face, relative macrocephaly and skeletal asymmetry, a phenotype that is partially complementary to BWS (10). IGF2 mRNA in skin fibroblasts was found to be reduced in two SRS children with demethylation of the ICR1 locus (10). However, data on IGF-II serum levels in children with SRS are missing.

The aim of our study was to analyze whether epigenetic mutations of IGF2 are reflected by changes of the systemic level of the respective gene product in childhood. Therefore, IGF-II serum levels were measured in a well-characterized group of children with SRS and compared with reference data and with age- and gender-matched children who were born small for gestational age (SGA), but without major dysmorphic features.

	Subjects and Methods

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Subjects

The children studied are patients of the Pediatric Endocrinology Section Tübingen. We defined SRS by the presence of intrauterine growth retardation (birth weight or length below the 3rd percentile), lack of postnatal catch-up growth, and at least two of the following criteria: typical face, relative macrocephaly, and skeletal asymmetry (13). The majority were started on GH therapy. Height was measured using an electronic stadiometer and was expressed in height SD scores according to Prader et al. (14). Birth length and weight were expressed in SD scores according to Niklasson et al. (15). IGF-II serum levels before the start of GH therapy were available in 30 children with SRS. The clinical characteristics of these 30 children with SRS are shown in Table 1.

Hormone measurements

Blood was drawn in the morning. Fresh serum was assayed from 11 SRS children, and stored serum was assayed from 19 SRS children and from all 30 SGA controls. Serum levels of IGF-II were determined by RIA as previously described (16, 17). After extraction by acid-ethanol method, IGF-II levels were determined in the presence of an excess of IGF-I, which did not interfere with IGF-II measurements. Inter- and intraassay coefficients of variation were 12.2 and 3.6%, respectively. Serum levels of IGF-I and IGF binding protein (IGFBP)-3 were both measured by RIA as described by Blum et al. (18). The mean inter- and intraassay coefficients of the IGF-I assay were lower than 10%. For the IGFBP-3 assay, the intraassay coefficient of variation was 4.1%, and the interassay coefficient of variation was 9.7%. IGFBP-2 was measured by RIA as well; the interassay coefficient of variation was 8% (17)

Because IGF-II immunoreactivity may decrease with the time of storage, we compared the assay results obtained in the past with the results obtained after storage at –20 C for 1 to 4 yr in 14 samples. There was no indication of loss of immunoreactivity during this time span. The values measured after storage were in mean 10 ± 1.8% higher than those determined in fresh serum; this difference was within the interassay variation of 12.2%.

Molecular analysis

The methylation-sensitive Southern blot analysis was performed as described previously (10). Briefly, genomic DNA from peripheral lymphocytes was digested overnight with RsaI and HpaII for the locus ICR1 and with BamH1 and Not1 for KvDMR1 in KCNQ1OT1. The digested samples were electrophoresed on 1.2 and 0.7% agarose gels. The blots were hybridized with digoxigenin-labeled PCR products for ICR1 or KCNQ1OT1. Primer sequences for probe amplification were taken from previous publications (10, 11).

The methylation index was defined by the ratio of the amount of methylated genomic DNA to the amount of total genomic DNA and calculated using densitometry of autoradiographs in a GelDoc2000 system (Bio-Rad, München, Germany). The cutoff point indicating demethylation was defined by the normal mean index minus 2 SD. The number of control subjects was 40. The method needs genomic DNA at the microgram level; such an amount was available in nine SRS children studied (30%) who had been tested negative for uniparental disomy of chromosome 7.

For the detection of uniparental disomy of chromosome 7, DNA was analyzed by short tandem repeat typing of chromosome 7 in 23 SRS children as previously described (19). In seven cases, no genomic DNA was available.

Statistical analysis

Statistical analysis was performed using the two-tailed Student t test. P values < 0.05 were considered to indicate significance.

	Results

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The individual IGF-II serum levels of the children with SRS and their matched controls (SGA) as well as the 5th, 50th, and 95th percentiles of the reference are depicted in Fig. 1. In the SRS group, IGF-II serum levels showed a wider variation than in the SGA group and were significantly higher than in their matched controls (SGA group) (P < 0.03).

View larger version (22K): [in this window] [in a new window]   FIG. 1. The IGF-II serum levels of all tested individuals depicted in relation to age at serum sampling. Filled symbols indicate the measurements from SRS individuals, and open circles those from the SGA controls. SRS individuals carrying 11p15 epimutations are indicated by filled squares, and those with maternal uniparental disomy of chromosome 7 by filled diamonds. The given percentiles represent the normative data of the reference population.

Genotyping of chromosome 7 revealed maternal uniparental disomy in four of 23 children who were not tested further. Molecular analysis of the methylation status at the ICR1 and the KCNQ1OT1 locus was available for nine children with SRS who had been tested negative, and four of them were identified to carry an epimutation with diminished methylation of ICR1, whereas methylation at KCNQ1OT1 was found to be normal. The methylation indices of these four cases were 18, 21, 31, and 36%, respectively (normal controls, 52% ± 7%). Three of these four children had high-normal IGF-II levels as indicated in Fig. 1.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The high prevalence of methylation defects at the IGF2 locus in children with SRS and the finding of diminished IGF2 mRNA in fibroblasts of affected individuals were suggestive of a role for the hormone IGF-II in the pathogenesis of SRS (10, 11). Our study indicates that IGF-II serum levels are in fact not decreased in children with SRS, but higher than in age- and gender-matched children with a non-syndromic SGA condition where epigenetic mutations at the IGF2 locus were not detected (20).

Our study was retrospective, the number of children tested was relatively small, and to date only one third could be characterized at the IGF2 locus. In comparison to previous reports on the endocrine system in SRS, however, the group reported here is the largest one. Uniparental disomy of chromosome 7 was present in four SRS children who were not tested for epigenetic defects at the IGF2 locus. The prevalence of demethylations at this locus (4 of 9; 44%) is in agreement with recent reports (10, 11). Moreover, three of the four children with the epimutation at the IGF2 locus exhibited serum levels of IGF-II at the higher spectrum of the normal range. Therefore, our data renders it unlikely that serum levels of IGF-II in childhood are decreased in the presence of ICR1 demethylation at the IGF2 locus.

Our findings do not exclude a main functional role of IGF-II in SRS. There is good evidence that IGF-II is probably more important for fetal than for postnatal growth (6). One main source of IGF-II production during gestation is the placenta (21). This organ is also one of the main targets of IGF-II, being hypoplastic and incompletely differentiated in the absence of IGF-II in knockout mice (6, 7). Postnatally, this source of IGF-II is not available anymore. In addition, IGF-II is thought to act predominantly at the paracrine and autocrine levels, which may not necessarily be reflected by serum levels (6).

In conclusion, our data indicate that IGF-II serum levels during childhood are unlikely to reflect the methylation status at the IGF2 locus in SRS.

	Acknowledgments

 
We thank C. P. Schwarze for language editing.

	Footnotes

 
First Published Online August 29, 2006

Abbreviations: BWS, Beckwith-Wiedemann syndrome; IGFBP, IGF binding protein; SGA, small for gestational age; SRS, Silver-Russell syndrome.

Received May 24, 2006.

Accepted August 21, 2006.

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