Submicroscopic chromosomal imbalances in idiopathic Silver-Russell syndrome (SRS): the SRS phenotype overlaps with the 12q14 microdeletion syndrome.

Silver-Russell syndrome (SRS) is a heterogeneous disorder associated with intrauterine and postnatal growth restriction, body asymmetry, a relative macrocephaly, a characteristic triangular face and further dysmorphisms. In about 50% of patients, genetic/epigenetic alterations can be detected: >38% of patients show a hypomethylation of the IGF2/H19 imprinting region in 11p15, whereas the additional 10% carry a maternal uniparental disomy of chromosome 7. In single cases, cytogenetic aberrations can be detected. Nevertheless, there still remain 50% of SRS patients without known genetic/epigenetic alterations. To find out whether submicroscopic imbalances contribute to the aetiology of SRS, 20 idiopathic SRS patients were screened with the Affymetrix GeneChip Human Mapping 500 K array set. Apart from known apathogenic copy number variations, we identified one patient with a 12q14 microdeletion. The 12q14 microdeletion syndrome is characterised by dwarfism but it additionally includes mental retardation and osteopoikilosis. The deletion in our patient is smaller than those in the 12q14 microdeletion carriers but it also affects the LEMD3 and the HMGA2 genes. LEMD3 haploinsufficiency and point mutations have been previously associated with osteopoikilosis but radiographs of our patient at the age of 16 years did not reveal any hint for osteopoikilosis lesions. Haploinsufficiency of HMGA2 is probably responsible for aberrant growth in 12q14 microdeletion syndrome. However, in this study, a general role of HMGA2 mutations for SRS was excluded by sequencing of 20 idiopathic patients. In conclusion, our results exclude a common cryptic chromosomal imbalance in idiopathic SRS patients but show that chromosomal aberrations are relevant in this disease. Thus, molecular karyotyping is indicated in SRS and should be included in the diagnostic algorithm.

Use of multiplex ligation-dependent probe amplification increases the detection rate for 11p15 epigenetic alterations in Silver-Russell syndrome.

Silver-Russell syndrome (SRS) describes a malformation syndrome with severe intrauterine and postnatal growth retardation. Currently, two major (epi)mutations have been described: while approximately 10% of patients carry a maternal uniparental disomy of chromosome 7 (UPD7), 35-60% show a hypomethylation at the H19 differentially methylated regions (DMRs) in 11p15. Until recently, a Southern-blot based test was routinely used to identify epimutation carriers. Nevertheless, this test was time consuming and hampered by the huge amount of genomic DNA needed. With the methylation-specific multiplex ligation-dependent probe amplification assay (MLPA) for SRS, a PCR-based test is now available, allowing the analysis also of small amounts of DNA. Probes in this assay hybridize to the H19 DMRs but do not cover the genomic target of the Southern-blot probe. We now screened 72 patients with SRS by MLPA. Hypomethylation of the H19 DMRs was confirmed in all patients analyzed by Southern blot. In addition, we identified six individuals with hypomethylation of the H19 DMR who had previously normal blot results. This discrepancy can be explained by the observed generally lower degree of demethylation in this group, possibly not detectable by the less sensitive Southern-blot method but also with a varying degree of methylation at different DMRs in the same individual. Apart from hypomethylation in the H19 DMR, we observed a slight demethylation for one of the IGF2 probes. The total detection rate of 11p15 hypomethylation is now increased to >38%. Considering maternal UPD7 and chromosomal aberrations, (epi)genetic alterations now account for more than 50% of SRS patients. In summary, MLPA represents an easy, low cost and reliable system in the molecular diagnostics of SRS.

Epigenetic mutations in 11p15 in Silver-Russell syndrome are restricted to the telomeric imprinting domain.

INTRODUCTION: Silver-Russell syndrome (SRS; also know as Russell-Silver syndrome) is a heterogeneous syndrome which is characterised by severe intrauterine and postnatal growth retardation and typical dysmorphic features. Recently, the first SRS patients with (epi)genetic mutations in 11p15 affecting the telomeric imprinting domain have been identified. Interestingly, opposite mutations are associated with Beckwith-Wiedemann syndrome (BWS). However, the general significance of epigenetic mutations in 11p15 for the aetiology of SRS remained unclear. METHODS: We screened a cohort of 51 SRS patients for epimutations in ICR1 and KCNQ1OT1 by methylation sensitive Southern blot analyses. RESULTS: ICR1 demethylation could be observed in 16 of the 51 SRS patients, corresponding to a frequency of approximately 31%. Changes in methylation at the KCNQ1OT1 locus were not detected. DISCUSSION: Combining these data with those on maternal duplications in 11p15, nearly 35% of SRS cases are associated with detectable (epi)genetic disturbances in 11p15. We now have to also consider a general involvement of 11p15 alterations in growth retarded patients with only minor or without further dysmorphic features. SRS and BWS may now be regarded as two diseases caused by opposite (epi)genetic disturbances of the same chromosomal region displaying opposite clinical pictures.

IGF-II serum levels are normal in children with Silver-Russell syndrome who frequently carry epimutations at the IGF2 locus.

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 microg/liter (range, 238-875). This was significantly higher than in the SGA controls: 387 microg/liter (range, 265-596) (P < 0.03), but below the median value of the age-related reference, which was 532 microg/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.

(Epi)mutations in 11p15 significantly contribute to Silver-Russell syndrome: but are they generally involved in growth retardation?

(Epi)mutations affecting chromosome 11p15 are meanwhile well known to be associated with growth disturbances. The finding of 11p15 mutations in the overgrowth disease Beckwith-Wiedemann syndrome (BWS) led to the identification of imprinted growth-promoting genes which are expressed paternally and of imprinted growth-suppressing genes in the same region that are expressed maternally. Recently, the opposite (epi)mutations of the same region have been reported to result in growth retardation: maternal duplications of 11p15 as well as hypomethylation of the telomeric 11p15 imprinting domain (ICR1) could be identified in patients with Silver-Russell syndrome (SRS), a disease which is in particular characterised by intrauterine and postnatal growth retardation. To elucidate whether 11p15 mutations are generally involved in growth retardation we screened 125 growth retarded patients, among them 47 patients with SRS-like features and 20 with isolated growth retardation. Additional 58 patients were presented with clinical signs not consistent with SRS. We excluded 11p15 duplications in all 123 families by short tandem repeat typing. ICR1 hypomethylation was investigated by Southern-blot analyses and was therefore restricted to samples with a large amount of DNA. We identified ICR1 hypomethylation in 20% of the patients with SRS-like features (n=25). No further cases were detectable in the other two subgroups with isolated growth retardation (n=20) and with clinical signs not consistent with SRS (n=23), respectively. Our data show that 11p15 duplications are rare in growth retardation in general and that they seem to be restricted to patients with SRS features. Furthermore, testing for the ICR1 hypomethylation should also be focused on patients with SRS features. While the ICR1 epimutation is detectable with a significant frequency only in SRS patients, its role for isolated growth retardation remains to be elucidated.

Is maternal duplication of 11p15 associated with Silver-Russell syndrome?

BACKGROUND: Silver-Russell syndrome (SRS) is a heterogeneous malformation syndrome characterised by intrauterine and postnatal growth retardation (IUGR, PGR) and dysmorphisms. The basic causes are unknown, however in approximately 10% of patients a maternal uniparental disomy (UPD) of chromosome 7 or chromosomal aberrations can be detected. Four growth retarded children, two with SRS-like features, associated with maternal duplications of 11p15 have been described. Considering the involvement of this genomic region in Beckwith-Wiedemann overgrowth syndrome (BWS), we postulated that some cases of SRS--with an opposite phenotype to BWS--might also be caused by genomic disturbances in 11p15. METHODS: A total of 46 SRS patients were screened for genomic rearrangements in 11p15 by STR typing and FISH analysis. RESULTS: Two SRS patients with duplications of maternal 11p material in our study population (n = 46) were detected. In patient SR46, the duplicated region covered at least 9 Mb; FISH analysis revealed a translocation of 11p15 onto 10q. In patient SR90, additional 11p15 material (approximately 5 Mb) was translocated to the short arm of chromosome 15. CONCLUSIONS: We suggest that diagnostic testing for duplication in 11p15 should be offered to patients with severe IUGR and PGR with clinical signs reminiscent of SRS. SRS is a genetically heterogeneous condition and patients with a maternal duplication of 11p15.5 may form an important subgroup.

Identification of interstitial maternal uniparental disomy (UPD) (14) and complete maternal UPD(20) in a cohort of growth retarded patients.

The association of uniparental disomy (UPD) and short stature has been reported for different chromosomes and in several conditions. Therefore, we investigated a cohort of 21 patients referred because of intrauterine and postnatal growth retardation for UPD of chromosomes 2, 7, 9, 14, 16, and 20. Typing of short tandem repeats showed maternal UPD(14) and maternal UPD(20) in two cases. In the first case, an interstitial UPD(14) was detected and the growth retarded newborn showed some additional clinical signs in common with the putative "maternal UPD(14) syndrome". The maternal UPD(20) patient showed minor features. However, since it is only the second maternal UPD(20) case it is too early to delineate a specific syndrome and the role of this constitution in growth remains to be investigated. Our data suggest that searching for UPD in growth retarded patients is a helpful approach to getting more information on the role of UPD in growth retardation. Based on our results, general considerations and indications for UPD testing are discussed.

Growth hormone treatment of short children born small for gestational age: growth responses with continuous and discontinuous regimens over 6 years.

We report an epi-analysis of 6-yr growth responses obtained with GH treatment in short children born small for gestational age (SGA). Four randomized, multicenter studies explored the effects of continuous and discontinuous regimens of GH treatment in short, non-GH-deficient SGA children. A total of 49 untreated and 139 treated children were followed over 2 and 6 yr, respectively. At the start of the study, the age of these 188 children averaged 5.2 yr (range, 2-8 yr), height was -3.4 SD score, and height adjusted for parental height was -2.4 SD score. Onset of puberty was observed in 46% of the GH-treated cohort, on the average, at 10.7 yr in girls and 11.7 yr in boys. Two studies essentially investigated the effects of continuous GH treatment at a dose of 33 or 67 microg/kg, day, and two studies focused on the growth characteristics during an initial GH treatment for 2-3 yr (dose range, 33-100 microg/kg x day), followed by a withdrawal phase of 1-2 yr, and then by either no or 1 or more episodes of further GH treatment (33 or 67 microg/kg x day). Continuous GH treatment for 6 yr resulted in height increments of 2.0 +/- 0.2 SD (33 microg/kg x day; n = 35) and 2.7 +/- 0.2 SD (67 microg/kg x day; n = 27). Discontinuous GH treatment was given to 77 children, most of them experiencing only 1 (n = 47) or 2 (n = 26) treatment phases with an average duration of 2.0 yr. All these children received GH during the first 2 yr; the dose was only 32 microg/kg x day when averaged over 6 yr. Some individualization of treatment schedules was allowed, and the majority of investigators seemed to aim for a low normal height level, adjusted for parental height. After 2 yr, the mean adjusted height SD score had increased to -0.4 +/- 0.1 and stabilized thereafter. Bone maturation progressed similarly in all treatment subgroups, and after 6 yr of study, bone age remained slightly delayed compared to chronological age. Multivariate analysis identified the average GH dose over 6 yr, parental-adjusted height SD score, and age at start as prime predictors of the growth response. GH treatment was well tolerated. In conclusion, this epi-analysis of growth responses over 6 yr confirms the administration of GH as an effective approach to normalize the stature of short, non-GH-deficient SGA children, at least during childhood and early puberty. In addition, it is now increasingly apparent that a relatively broad spectrum of GH regimens is effective, and this experience should facilitate the design of more individualized treatment schedules in the future, in particular for young children.

Decreased expression of IGF-II and its binding protein, IGF-binding protein-2, in genital skin fibroblasts of patients with complete androgen insensitivity syndrome compared with normally virilized males.

The action of androgen by way of the AR is required for the development of male gonads and external genitalia. The interplay between androgens and the somatotropic axis, in particular the IGFs in sexual development, is currently under thorough investigation. The IGF system is thought to mediate the androgen action in androgen-responsive cells. To investigate the interaction of androgens with the IGF system, we compared the expression of IGFs and IGF-binding proteins in cultured genital skin fibroblasts from nine patients with the syndrome of complete androgen insensitivity with that in genital skin fibroblasts from 10 normally virilized males. Mutations in the AR gene and/or abnormalities of the AR protein in the immunoblot were detected in all complete androgen insensitivity genital skin fibroblast strains. They caused a complete failure of DHT binding. RIA and RT-PCR demonstrated that the genital skin fibroblast strains expressed IGF-II, IGF-binding protein-2, and IGF-binding protein-3, but no IGF-I. Most strikingly, complete androgen insensitivity genital skin fibroblast strains produced significantly lower IGF-II (P < 0.001; 42.2 +/- 9.7 vs. 106.9 +/- 11.8 ng/mg protein) and IGF-II mRNA (P < 0.01, by RT-PCR) than control genital skin fibroblast strains. The production of IGF-binding protein-2 was also decreased (P < 0.03) in complete androgen insensitivity genital skin fibroblasts, whereas that of IGF-binding protein-3 did not differ. Furthermore, high levels of IGF-binding protein-5 mRNA were detected in all genital skin fibroblast strains, whereby the 28-kDa band in the ligand blot, probably representing IGF-binding protein-5, was more abundant in complete androgen insensitivity genital skin fibroblasts. Exposure of the genital skin fibroblasts to T (5 x 10(-8) M) had only weak effects on the expression of IGFs and IGF-binding proteins. In conclusion, although the mechanism underlying these differences requires further study, it is conceivable that in addition to the endocrine actions of IGF-I, IGF-II and IGF-binding protein-2, as local growth factors, are involved in the mediation of androgen action and growth of genital tissues.

Gene dosage analysis in Silver-Russell syndrome: use of quantitative competitive PCR and dual-color FISH to estimate the frequency of duplications in 7p11.2-p13.

Silver-Russell syndrome (SRS) describes a heterogeneous malformation syndrome mainly characterized by intrauterine and postnatal growth retardation (IUGR/PNGR). Approximately 10% of SRS cases have been associated with maternal uniparental disomy (matUPD) 7. This suggests the involvement of at least one imprinted gene on chromosome 7 in the pathogenesis of SRS. Additionally, two familial and one single SRS patients have been published with an interstitial duplication in 7p11.2-p13, including the genes GRB10 and IGFBP1; IGFBP3 was investigated in only one case revealing duplication; conversely, double gene dosage of EGFR was excluded in all 3 patients. Two further cytogenetically abnormal cases, one with a paracentric inversion (7)(p14p12) and one with matUPD7/partial trisomy for 7p13-q11, confirmed that the proximal short arm of chromosome represents an interesting region possibly harboring (a) candidate gene(s) for SRS. Although previously published investigations on the genes GRB10, IGFBP1, IGFBP3, and EGFR report neither disease-relevant mutations nor abnormal imprinting patterns, the SRS cases with chromosomal duplications suggest that variation of gene copy number might be a further type of mutation. To obtain meaningful results on the frequency of duplications in proximal 7p, we screened 32 SRS patients using quantitative PCR assays for GRB10, IGFBP1, IGFBP3, and EGFR. The data were confirmed by dual-color fluorescence in situ hybridization (FISH) of spot check samples. Results obtained by both methods exclude duplications in all analyzed patients and indicate an overall percentage of duplication among SRS patients between 2.4% (GRB10) and 5% (IGFBP1). By testing and evaluating quantitative competitive PCR for various loci, we developed a practical approach for gene dosage analysis which can be easily established for routine purposes.

Short-term increments of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 predict the growth response to growth hormone (GH) therapy in GH-sensitive children.

The present study included a cohort of 42 children aged between 1.7 and 15.4 years, who presented with short stature and growth failure. Basal and generated serum levels of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3), measured in an IGF generation test following four or seven daily injections of growth hormone (GH), 0.1 IU/kg (0.033 mg/kg), were analysed in these patients. The growth response to 1 year of GH treatment, 0.6 IU/kg/week (0.2 mg/kg/week), was also investigated. Median height velocity of these patients increased from -1.6 SDS (range, -4.6 to -0.3 SDS) to 3.3 SDS (range, -0.2 to 7.1 SDS) after 1 year of GH treatment, and median height SDS increased by 0.7 SDS (range, 0.1 to 2.2 SDS). Strong correlations were observed between basal and generated IGF-I and IGFBP-3 levels. The increase in IGFBP-3 levels in response to GH in the generation test was a strong predictor of the growth response to GH therapy. All the patients in the present study could be differentiated from patients with GH insensitivity syndrome (GHIS) using the criteria of a diagnostic scoring system for GHIS. The most valuable parameters were the increases in IGF-I and IGFBP-3 levels in the generation test, which excluded 95.2% of the patients from a diagnosis of GHIS.

Short children with low birth weight born either small for gestational age or average for gestational age show similar growth response and changes in insulin-like growth factor-1 to growth hormone treatment during the first prepubertal year.

BACKGROUND/AIMS: Growth hormone (GH) is an accepted treatment for short children born small for gestational age (SGA). The aim of this analysis was to compare the growth response to GH in children with low birth weight born SGA or appropriate for gestational age (AGA). METHODS: This retrospective observational study is from one center. Of all the children with a birth weight <2,500 g treated, 50 were primarily diagnosed as having growth hormone deficiency ([A] SGA, n = 26; [B] AGA, n = 24) and 138 were originally diagnosed SGA or AGA (reclassified: [C] SGA, n = 102; [D] AGA, n = 36). RESULTS: [Median; A, B, C, D]: at an age of 4.9, 5.2, 5.8, 5.8 years, a height of -2.9, -2.4, -2.8, -2.9 SDS and a GH dose of 27, 28, 41, 39 µg/kg/day, the children grew 0.9, 0.9, 0.8, 0.9 SDS in height, respectively. Insulin-like growth factor-1 (IGF-1) at GH start was, respectively, -2.1, -2.2, -0.4, -0.9 SDS and rose to (delta IGF-1) 1.8, 2.0, 1.7, 1.5 SDS during the first year on GH. All differences were not significant. CONCLUSIONS: We show for the first time that short stature children with low birth weight born AGA experience the same increase in height and IGFs to GH treatment as those born SGA irrespective of actual GH secretory status.