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Clinically Distinct Epigenetic Subgroups in Silver-Russell Syndrome: The Degree of H19 Hypomethylation Associates with Phenotype Severity and Genital and Skeletal Anomalies

Department of Biosciences and Nutrition (S.B., J.K.), Karolinska Institutet, S-141 57 Huddinge, Sweden; Department of Medical Genetics (S.B., K.H.-J., J.K.), University of Helinski, 00014 Helinski, Finland; Folkhälsan Institute of Genetics (S.B., K.H.-J., J.K.), Biomedicum Helinski, 00014 Helinski, Finland; and Hospital for Children and Adolescents (J.P., M.L.-N.), University of Helinski, 00029 Helinski, Finland

Address all correspondence and requests for reprints to: Marita Lipsanen-Nyman, Hospital for Children and Adolescents, Pediatric Endocrinology PB 281, 00290 Helsinki, Finland. E-mail: Marita.Lipsanen{at}hus.fi.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: The H19 imprinting control region (ICR), located on chromosome 11p15.5, has been reported hypomethylated in 20–65% of Silver-Russell syndrome (SRS) patients.

Objective: We investigated the methylation status of 11p15.5 ICRs in SRS patients and children born small for gestational age (SGA) to clarify the relationship between phenotype and H19 methylation status.

Methods: We performed methylation screens of the H19 and KCNQ1OT1 ICRs in 42 SRS patients, including seven maternal uniparental disomy of chromosome 7 patients, and 90 SGA children without SRS. Clinical data were evaluated from patient records, and seven hypomethylated patients were clinically and radiologically reexamined.

Results: H19 ICR hypomethylation was found in 62% of SRS patients but in no SGA children. A clinical severity score demonstrated strong correlation between hypomethylation level and phenotype severity. Hypomethylation related to a more severe SRS phenotype, in which especially asymmetry and micrognathia were significantly more common. Extremely hypomethylated patients had abnormally high lumbar vertebrae, lumbar hypomobility, elbow subluxations, and distinct hand and foot anomalies. They also presented with congenital aplasia of the uterus and upper vagina, equivalent to the Mayer-Rokitansky-Küster-Hauser syndrome in females, and cryptorchidism and testicular agenesis in males.

Conclusions: We found a dose-response relationship between the degree of H19 hypomethylation and phenotype severity in SRS. We report for the first time the association of specific anomalies of the spine, elbows, hands and feet, and genital defects in SRS with severe H19 hypomethylation. Classical SRS features were found in H19 hypomethylation and milder symptoms in maternal uniparental disomy of chromosome 7, thus distinguishing two separate clinical and etiological subgroups.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Silver-Russell syndrome (SRS) [Online Mendelian Inheritance in Man (OMIM) no. 180860] features severe fetal onset growth restriction and variable dysmorphisms. Originally, the syndrome was independently described by Silver et al. (1) and Russell (2), but they had obviously described the same syndrome, each emphasizing different features. Classically, SRS includes low birth weight and length persisting throughout childhood, close to normal head circumference appearing as a large head, a triangular face, and skeletal asymmetry (3, 4). The typical SRS child is lean, with sparse sc fat and low muscle tone. Minor features include a bossed forehead, down-turned mouth corners, micrognathia, fifth finger clinodactyly, second to third toe syndactyly (3, 4), and many others (5, 6). Although SRS is well known, no universal diagnostic criteria are in use. The great variation of clinical characteristics makes the syndrome heterogeneous, and indeed, SRS might comprise different subgroups (4, 7).

Maternal uniparental disomy of chromosome 7 (matUPD7) is found in 5–10% of SRS patients, implying involvement of imprinted genes in SRS etiology (8). Imprinted genes are exclusively expressed from either the maternal or paternal chromosome, and are instrumental for growth and development (9, 10, 11). Recently, the H19 imprinting control region (ICR) on 11p15.5 was reported hypomethylated in 20–65% of SRS patients (12, 13, 14, 15, 16, 17, 18). One study suggested that H19 ICR hypomethylation co-occurred with isolated asymmetry and not necessarily SRS (14), whereas other studies did not confirm this (15, 16). The molecular findings of matUPD7 and H19 hypomethylation in subsets of SRS patients have revealed different (epi)genetic subgroups and opened the way to understanding the underlying pathogenesis. A corresponding clinical subgroup has been described for matUPD7, in which patients have a milder SRS phenotype, and lack some of the distinctive SRS features such as hemihypotrophy and down-turned mouth corners (4, 19, 20).

The 11p15.5 region harbors two well-studied imprinted gene clusters implicated in the overgrowth syndrome Beckwith-Wiedemann (OMIM no. 130650). The H19 ICR regulates IGF2 and H19, whereas the more centromeric KCNQ1OT1 ICR controls several imprinted genes, including KCNQ1, CDKN1C, and PHLDA2. Both regions are involved in Beckwith-Wiedemann syndrome pathogenesis (21), but methylation patterns appeared normal for KCNQ1OT1 ICR in SRS (12, 15, 16). However, 11p15.5 maternal duplications have been described in patients with SRS-like phenotypes, with one spanning only the KCNQ1OT1 ICR (22, 23, 24). The H19 ICR is paternally methylated, preventing H19 transcription, whereas IGF2 is expressed. Thus, hypomethylation will result in a maternal-only transcription profile with overexpression of H19 and down-regulation of IGF2. Serum IGF-II levels are normal, whereas the levels of IGF-I and IGF binding protein 3 are elevated in hypomethylated patients (16, 18, 25).

In this study we investigated the methylation status of the H19 and KCNQ1OT1 ICRs in 42 SRS patients, including seven matUPD7 patients, and 90 children born small for gestational age (SGA) without SRS. To understand if the molecular subgroups of SRS correspond to distinguishable clinical subgroups, a detailed (epi)genotype-phenotype analysis was performed with special attention on identifying associated skeletal and genital defects, dysmorphic features, and growth failure.

	Subjects and Methods

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Subjects

Patients were recruited from the endocrinological outpatient clinic at the Hospital for Children and Adolescents, Helsinki University, Finland, with SRS diagnoses spanning back to the 1970s. Six SRS patients were referred from other University and Central Hospitals in Finland (26). Twelve previously unreported patients (eight SRS, four SGA) have been included in this study. Detailed phenotypes of four matUPD7 patients were reported previously (19). All patients were evaluated by a pediatric endocrinologist. Moreover, all SRS patients visited a geneticist, and nearly all of them had at least one examination by an orthopedic surgeon. Patient data were gathered from medical records (K.H.-J. and M.L.-N.) and radiographs (J.P.). If a trait was neither mentioned in the patient records nor visible in photographs, it was assumed not present. Seven patients were reexamined clinically by an endocrinologist (M.L.-N.) and orthopedic surgeon (J.P.), and new radiographs were obtained. Patients and/or parents gave written consent for blood samples and DNA studies, and to publish photographs (when relevant). All patients have been screened for matUPD7 as previously described (26). The study was approved by the appropriate Ethical Review Boards at the University of Helsinki, Finland, and Karolinska Institutet, Sweden.

SRS was based on: SGA [birth length and/or weight 2.0 SD score (SDS) for gestational age]; postnatal growth retardation (height SDS below –2.5 at age 2 yr); relative macrocephaly (head circumference 1.5 SDS above length SDS); a typical SRS face with at least three characteristics, including triangular face, micrognathia (leading to down-slanting mouth corners and irregular teeth), frontal bossing, craniofacial disproportion, and at least one relative criteria, including asymmetry (limb length discrepancy and/or hemihypoplasia of skull, trunk, limbs), fifth finger clinodactyly and/or brachydactyly, low-set/dysmorphic ears, second to third toe syndactyly, cryptorchidism, feeding difficulties, speech delay/difficulties, and/or excessive sweating.

SGA patient inclusion criteria were SGA and postnatal short stature, as described previously, of unknown cause.

Molecular studies

Methylation measurements using real-time PCR The methodology has been described in detail (27). In brief, DNA was digested with the methylation-sensitive restriction enzyme HpaII or NotI (New England Biolabs, Ipswich, MA), and in parallel, DNA was incubated with restriction buffer only. An aliquot of -DNA was included as a control for digestion efficiency. DNA was digested overnight at 37 C. The digested DNA and corresponding undigested DNA were then assayed in parallel using SYBR green assays (Applied Biosystems, Foster City, CA) for HpaII site 25 in the H19 ICR, two HpaII sites, and the NotI site in the KCNQ1OT1 ICR and -DNA. The cycling conditions, primer sequences, mean methylation percentages, and SD values in a control population have been described (27). All PCR runs were inspected in the 7500 Fast System SDS software 1.3.1 (Applied Biosystems). Digestion of DNA and PCR measurements were repeated twice for the SRS patients with a very high correlation: R² = 0.97.

Microsatellite typing Four microsatellites (D11S2071, D11S1984, D11S922, and D11S4046) were used to screen for maternal duplications. Five nanograms of genomic DNA were amplified using a typical PCR program [95 C 15 min, 40x (95 C 30 sec, 51–58 C 15 sec, 72 C 30 sec), and extension 72 C for 7 min]. PCR products were ethanol precipitated, separated on MegaBACE 1000 instrument (Amersham Biosciences Inc., Piscataway, NJ/GE Healthcare Bio-Sciences Corp., Piscataway, NJ), and visualized using the Genetic Profiler version 2.0 software (Amersham Biosciences/GE Healthcare Bio-Sciences).

Sequencing reactions The H19 ICR (chr11:1972984-1985280, nonrepetitive portions) was sequenced on an ABI sequencer using the DYEnamic ET terminator Cycle Sequencing kit (GE Healthcare). Sequences were analyzed in the Pregap and Gap4 software (http://staden.sourceforge.net). There were 12 hypomethylated SRS patients sequenced. DNAs from 16 healthy Finnish blood donors were used as controls.

Statistical analyses

Methylation The relative methylation was calculated by subtracting the mean of the digested threshold cycle (Ct) values with the mean of the undigested DNA Ct values (controls for starting amount of DNA) and exponentiating the obtained difference with a base of two: P^M = 2^ [mean(Ct_digest) – mean(Ct_nondigest)]. When a nonmethylated restriction site was assayed, one minus the methylation percentage corresponded to the digestion efficiency. A cutoff for hypomethylation of less than –2 SD from the mean of a normal population was used, i.e. methylation less than 35% (27).

Clinical severity score A clinical severity score was created by assessing what percentage of the qualitative clinical characteristics described in Table 1 were considered positive in each patient, yielding a theoretical range between 0 and 100%, with 100% corresponding to the most severe phenotype.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
H19 and KCNQ1OT1 ICR methylation in SRS and SGA

We investigated the methylation status of the H19 and KCNQ1OT1 ICRs in 42 SRS and 90 SGA children. The mean and median digestion efficiencies were 96% ± 2 (SD) as measured by digestion of -DNA. Methylation levels of the H19 ICR were compared with reference values for individuals of normal stature (27). There were 26 SRS patients considered hypomethylated (methylation < 35%, < –2 SD), corresponding to a prevalence of 62%. The mean methylation percentage of H19 ± SD in the remaining SRS patients was 46 ± 5%, including seven matUPD7s and nine SRS patients. All KCNQ1OT1 methylation levels were within the normal range. The H19 ICR was sequenced in the 12 most hypomethylated patients, but no pathogenic mutations were found. Microsatellite analysis of SRS patients and parents revealed no 11p15 maternal duplications. Methylation profiles for all SGA children were within the normal range.

The degree of H19 methylation correlates with coexistence of SRS symptoms

To evaluate the clinical relevance of H19 hypomethylation, we focused on the most hypomethylated patients and, thus, divided the patients into four subgroups according to their epigenetic status: 1) extreme H19 hypomethylation ( –6 SD, <9%, n = 8); 2) moderate H19 hypomethylation (–2 SD, 9–35%, n = 18); 3) normal H19 methylation (> –2 SD, >35%, n = 9); and 4) matUPD7 (normal H19 methylation, n = 7). Table 1 summarizes the prevalence of clinical characteristics in these subgroups and SGA children. Several qualitative characteristics were present at a significantly higher proportion in the hypomethylated SRS patients compared with those with normal H19 methylation. We hypothesized that the degree of H19 hypomethylation might correlate with clinical severity. By creating a score from the qualitative clinical characteristics (Table 1), we found a strong correlation between clinical severity and H19 methylation, with the regression fit suggesting that 35% of the variation in clinical severity was explained by the degree of H19 hypomethylation (P value of the regression fit = 3.556e-05; Fig. 1).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Clinical severity correlates with the degree of H19 ICR methylation. A, Clinical severity score (see Subjects and Methods) plotted against H19 ICR methylation percent. The solid black line corresponds to the regression of the two variables in a linear model. B, Box plots of the clinical severity score for three groups of H19 methylation status: non-hypo, >35% (> –2 SD); hypo, 9–35% (–2 to –6 SD); and hypo, less than 9% (< –6 SD).

All extremely hypomethylated SRS patients (n = 8) had severe prenatal and postnatal growth retardation, relative macrocephaly, the typical triangular SRS face comprising of micrognathia and a bossed forehead, asymmetry, and fifth finger clinodactyly, and most of the confirmatory SRS features were observed more frequently (Fig. 2A and Table 1). They were born very small (average birth length and weight SDS for gestational age were –5.9 and –4.3, respectively), whereas the mean head circumference was close to Finnish standards at birth (–0.17 SDS). At 2 yr both the mean height SDS (–5.1) and the weight for height (–0.30%) were clearly below that of normally methylated SRS patients (Table 1).

View larger version (86K): [in this window] [in a new window]   FIG. 2. Severe/classical SRS phenotype in H19 hypomethylation is distinct from the milder phenotype in matUPD7. Typical overall appearance of SRS patient with severe H19 ICR hypomethylation (A) (<9%), at age 7 yr 9 months with asymmetry, macrocephaly, and the typical triangular face comprising of micrognathia and a bossed forehead, and extreme leanness, SRS patient with moderate H19 ICR hypomethylation (B), at age 8 yr 3 months, with similar findings but less striking features as with severe H19 ICR hypomethylation (9–35%), and SRS patient with matUPD7 (C), at age 3 yr 5 months with asymmetry, a round face, and a sturdy appearance.

Moderate H19 hypomethylation 9–35%

All SRS patients with hypomethylation between 9–35% (n = 18) presented with prenatal and postnatal growth retardation, relative macrocephaly, a triangular face, and micrognathia like the extremely hypomethylated group (Fig. 2B). Other typical SRS features, e.g. asymmetry (limb length discrepancy/hemihypoplasia) and fifth finger clinodactyly, were reported at slightly lower levels than the extremely hypomethylated group but at a higher frequency than the normally methylated SRS patients (Table 1). Moderate H19 hypomethylation patients were not significantly smaller at birth or 2 yr, although they tended to be thinner and shorter at 2 yr. They also had a larger head circumference at birth compared with the normally methylated SRS patients, similar to the extremely hypomethylated patients (Table 1). Of the six hypomethylated girls, five were postpubertal and had normal pubertal development with menarche at the age of 9.2–12.6 yr. The sixth girl was only 2.9 yr at evaluation. Cryptorchidism was present at a similar frequency as in the H19 normal methylation group.

Skeletal findings in H19 ICR hypomethylated (<35%) SRS patients

All SRS patients with H19 hypomethylation had a classical SRS phenotype of severe short stature, limb length discrepancy and asymmetry, and distinct skeletal malformations. Orthopedic reexamination revealed striking complications, specifically of the hands, elbows, spine, as well as limb length inequalities (Table 2). Radiohumeral joint incongruence and habitual or permanent luxation were evident in all four severely hypomethylated patients. Interestingly, upper-limb hypoplasia occurred on the same side as the elbow dysmorphology. None of the three moderately hypomethylated patients had radiologically visible abnormalities, but clinically prosupination restriction, upper-arm length discrepancy, and hypoplasia were noted in all. Clinodactyly of the fifth finger, ulnar ray hypoplasia, and ulna minus deformity (2–4 mm) were also common. Some patients had thumb hypoplasia and proximal syndactyly of the second through fifth digits. A mitt-like appearance of the hand (Fig. 3A and supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org) was found in five of eight of the severely hypomethylated and six of 18 of moderately hypomethylated patients, but not in any normally methylated SRS, matUPD7, or SGA patients. In addition, hypomethylated SRS patients had short and stubby toes, and syndactyly of the second to third toes (Fig. 3B). Scoliosis, lumbar hypolordosis with lumbar hypomobility, and abnormally high vertebrae (supplemental Fig. 1B) were found in all severely hypomethylated patients, and in most moderately hypomethylated patients. Clinical stiffness of the spine and difficulties in bending were typical for the hypomethylated patients. Klippel-Feil deformity, acetabular hypoplasia and hip subluxation, patellar luxations, and foot asymmetry were noted in single patients.

View larger version (66K): [in this window] [in a new window]   FIG. 3. Hand and foot abnormalities observed in H19 ICR hypomethylation. Mitt-like hand, with clinodactyly of the fifth fingers, syndactyly between second to fourth fingers and thenar hypoplasia (A) and typical short and stubby toes, and syndactyly of second to third toes (B) of a severely hypomethylated SRS patient.

The matUPD7 patients (n = 7) clearly differed from H19 hypomethylated SRS patients (Table 3). None of the matUPD7 patients showed a triangular face with micrognathia (Tables 1 and 3, and Fig. 2C). In addition, matUPD7 patients had a lower incidence of asymmetry, fifth finger clino- and brachydactyly, cryptorchidism, syndactyly of toes, muscle hypotonia, down-turned mouth corners, crowded teeth, and a high-arched palate (Table 3). On the other hand, matUPD7 patients presented more frequently with speech delay, feeding difficulties, a high-pitched voice, low-set/abnormal ears, and excessive sweating. Maternal and paternal ages at birth were significantly higher in matUPD7 patients. Advanced parental age increases the risk for nondisjunction events and the risk of uniparental disomy (28).

As seen in Table 1, many SRS features were also found in SGA patients, although less often. This emphasizes that these single features are not exclusive to SRS, but rather the accumulation of several characteristics suggests the diagnosis.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We confirmed that aberrant methylation of the H19 ICR is found in most SRS patients (62%), whereas it is absent in SGA children without SRS. We conclude that H19 methylation is clinically important as demonstrated by a strong correlation between the degree of hypomethylation and SRS phenotype severity. We focused especially on the extreme group of hypomethylated patients (arbitrary methylation cutoff <9%), and detected important genital and skeletal defects. Previous studies of H19 ICR hypomethylation in SRS have presented less-detailed clinical information. The lack of a clear phenotypical consensus for H19 hypomethylation is likely the result of small studies without systematic phenotyping. However, the studies seem to agree that hypomethylated children tend to be born smaller but with larger heads, and that asymmetry and clinodactyly V are typical (12, 16, 18).

All patients with H19 hypomethylation had a classical SRS phenotype, presenting with prenatal onset severe growth retardation, relative macrocephaly, lean appearance, a triangular face, asymmetry, and fifth finger clino-/brachydactyly. Their overall appearance was more severe than in SRS patients without hypomethylation and especially matUPD7 patients. Moreover, severe hypomethylation correlated with explicit orthopedic complications of the spine and extremities, which have not previously been specifically linked with SRS. Clinical and radiological reevaluation in seven hypomethylated patients revealed lumbar hypolordosis and hypomobility with pronounced stiffness in the spine and exceptionally high lumbar vertebrae, otherwise a rare abnormality. These distinct spine anomalies and the stiffness were not connected with limb asymmetry, and the etiology is unknown. A mitt-like hand was confined to hypomethylated patients, presenting as thumb and thenar hypoplasia, syndactyly of second to fifth digits, and ulnar ray hypoplasia. These patients had also short and stubby toes with syndactyly. Little attention has been drawn to the hands of SRS patients, apart from reports on fifth finger clino- and brachydactyly (29), and a range of sporadic hand anomalies, including syndactyly, camptodactyly, cleft hand, swan neck deformities, arthrogryposis, and a hypoplastic thumb (30). Price et al. (4) found that 10 of 50 SRS patients had camptodactyly of all fingers and arthrogryposis of the terminal interphalangeal joints, and one patient had bilateral dislocation of the radial heads. None of our SRS patients had camptodactyly or arthrogryposis. Overall, the extremely hypomethylated patients were from the severe end of the clinical spectrum, whereas the moderately hypomethylated patients presented with similar but milder deformities. Elbow deformities varied from permanent luxation and proximal radiohumeral joint incongruence in severe hypomethylation to a clinical rotation deficiency without radiological abnormalities in the moderately hypomethylated. In addition, the mitt-like hand and foot dysmorphology grew gradually milder as methylation level increased.

Asymmetry alone has been suggested as a good predictor of H19 hypomethylation, regardless of a SRS phenotype (14). In concordance with previous studies, we failed to identify H19 hypomethylation among 16 SGA patients with asymmetry and some slight dysmorphic features suggestive, but not diagnostic for SRS (15, 16).

We measured H19 ICR methylation on DNA extracted from blood. Importantly, the strong correlation between SRS severity and the degree of H19 methylation points to a clinical relevance of investigating methylation in blood. It is likely that the degree of hypomethylation in blood is a reflection of how early in development the imprinting error occurred, potentially contributing to varying degrees of mosaicism in different tissues. This could explain both the dose-response and the variable presentation of associated skeletal and genital anomalies observed. The mosaicism also raises the question whether normally methylated SRS patients have hypomethylation in other tissues and whether varying degrees of mosaicism in different tissues could in part explain why clinical severity and H19 methylation status were not perfectly correlated. However, other genes might also regulate SRS pathogenesis, resulting in its variable presentation.

The mechanism for how H19 hypomethylation causes growth retardation is not fully understood. Gicquel et al. (12) demonstrated down-regulation of IGF2 in hypomethylated patients, which together with the mouse knockout model of Igf2 suggests its important role (31). However, IGF2 is not imprinted in the adult liver, and normal IGF-II levels have been demonstrated in postnatally growth-restricted SRS patients (16, 18). These data suggest additional mechanisms such as para/autocrine effects of IGF-II, a role for H19, or trans-regulatory effects of the H19 ICR. Because no genetic explanation exists for the epigenetic defect at H19 ICR, we sequenced a region covering the H19 ICR but did not identify any causal mutations, in agreement with two other reports failing to identify mutations in the CTCF sites of the H19 ICR (14, 17).

Importantly, we found genital ambiguities in extremely hypomethylated SRS patients. The female genital defects were similar to Mayer-Rokitansky-Küster-Hauer (MRKH) (OMIM no. 277000) syndrome characterized by congenital aplasia of the uterus and upper part of the vagina. Bliek et al. (14) reported two girls with H19 hypomethylation and defects of the reproductive organs, such as neonatal vaginal prolapse, and one patient with ambiguous genitalia and a hypoplastic uterus. Price et al. (4) also reported one girl with a bicornuate uterus in a cohort of SRS and matUPD7 patients. We found cryptorchidism combined with primary testicular agenesis in four of five extremely hypomethylated males. The frequency of cryptorchidism has not been reported for H19 hypomethylated patients, but generally cryptorchidism is reported in 15–32% of SRS boys (4, 5, 6). Here, the incidence of cryptorchidism was somewhat higher in hypomethylated compared with normally methylated and matUPD7 patients (59 vs. 50%). It is unclear whether an equivalent MRKH syndrome might manifest in the male (32).

MRKH syndrome is frequently associated with skeletal, renal, and, to a lesser extent, auditory and cardiac defects (33). Skeletal anomalies of the hands and fingers (syndactyly), rib deformities, cleft palate, and cervical and thoracic vertebral abnormalities have been reported in MRKH syndrome (32, 33). Some skeletal anomalies observed in our severely hypomethylated patients have also been described in MRKH syndrome. MRKH syndrome has been reported in association with SRS (34), and also with skeletal dysplasias such as thrombocytopenia-absent radius syndrome (OMIM no. 274000), Klippel-Feil syndrome (OMIM no. 118100), and Holt-Oram syndrome (OMIM no. 142900) (35, 36, 37). Renal, auditory, and cardiac defects were only observed in single hypomethylated males. The spectrum of malformations encountered in both SRS and MRKH syndrome suggests a developmental field defect (32), involving organ systems closely related during embryogenesis.

Comparison of H19 hypomethylation and matUPD7 phenotypes showed that the classical SRS features in H19 hypomethylation are quite distinct from the milder phenotype in matUPD7 (19). These subgroups form distinct phenotypical and epigenetic entities among SRS patients, which should be recognized in future studies of the syndrome. Distinct major characteristics for the H19 hypomethylated-classical SRS subgroup include severe prenatal and postnatal growth retardation with an extremely lean appearance, macrocephaly, and a triangular face with a bossed forehead/macrocephaly and micrognathia, and at least two minor characteristics: asymmetry, fifth finger clino- and/or brachydactyly, low-set and abnormal ears, and genital abnormalities/cryptorchidism. Confirmatory characteristics include: syndactyly of the second-third toes, abnormal muscle tone, speech delay, feeding difficulties, and a high-pitched voice. Major characteristics of the milder matUPD7 subgroup include prenatal and postnatal growth retardation with relative macrocephaly, and at least three minor characteristics: asymmetry, fifth finger clino- and/or brachydactyly, low-set and abnormal ears, frontal bossing, feeding difficulties, and speech delay. Other confirmatory criteria include: cryptorchidism, excessive sweating, motor and neuropsychological delay, and a high-pitched voice.

In conclusion, we found a dose-response relationship between H19 hypomethylation and phenotype severity in SRS. Explicit spinal, elbow, hand, and foot anomalies were found in severely hypomethylated SRS patients, whereas seen milder in less hypomethylated patients. Reexamination of SRS patients revealed exceptionally high lumbar vertebrae, lumbar hypolordosis, and hypomobility, but also congenital aplasia of the uterus and the upper part of the vagina in females compatible with MRKH syndrome. Classical SRS features were found in H19 hypomethylation and a milder phenotype, with short stature and SRS-like features in matUPD7, distinguishing two separate clinical and etiological SRS subgroups.

	Acknowledgments

 
We thank all participating patients, families, and referring physicians, and express special gratitude to Professor Jaakko Perheentupa for informative Silver-Russell syndrome diagnoses. We thank Ms. Riita Lehtinen for excellent assistance.

	Footnotes

 
This work was supported by Magn Bergvalls stiftelse, the Swedish Research Council, Päivikki and Sakari Sohlberg Foundation, Sigrid Jusélius Foundation, Helsinki University Hospital research funds, Finnish Foundation for Pediatric Research, and the Academy of Finland.

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 18, 2008

¹ S.B. and K.H.-J. contributed equally to this work.

Abbreviations: Ct, Thershold cycle; ICR, imprinting control region; matUPD7, maternal uniparental disomy of chromosome 7; MRKH, Mayer-Rokitansky-Küster-Hauser; OMIM, Online Mendelian Inheritance in Man; SDS, SD score; SGA, small for gestational age; SRS, Silver-Russell syndrome.

Received August 15, 2008.

Accepted November 12, 2008.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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