Silver–Russell syndrome (Russell–Silver syn-drome; SRS; OMIM 180860) is mainly defined by severe intrauterine and postnatal growth restriction (<third percentile) associated with a variable spectrum of further features. Classical SRS includes a relative macrocephaly, a trian-gular-shaped face with a prominent forehead and a small chin, body and limb asymmetry, a fifth finger clinodactyly, feeding difficulties and hypoglycemia (for review see [1]). Some patients show mild motor and cognitive developmental delay, including learning difficulties and speech delay [2–7].
Genetic findings in SRSThe etiology of SRS is far from being understood. The contribution of genetic factors became evident with the reports on familial cases (for review see [8]), but a uniform pattern of molecular altera-tions has not been described. Three main types of genetic and epigenetic mutations have been reported: maternal uni parental disomy (UPD) of chromosome 7 (upd(7)mat); hypomethylation of the imprinting control region 1 (ICR1) in 11p15; and (sub)microscopic chromosomal aberrations (Figure 1, Table 1). As a result, the molecular diag-nostic work-up is complex.
Both upd(7)mat and hypomethylation of ICR1 lead to the definition of SRS as one of the congeni-tal imprinting disorders that also include inter alia
Prader–Willi syndrome, Angelman syndrome, Beckwith–Wiedemann syndrome (BWS) and transient neonatal diabetes mellitus (TNDM). The term genomic imprinting describes an epigen-etic marking of specific genes that allows expres-sion from only one of the two parental alleles (for review see [9]). So far, more than 60 human genes are considered to be imprinted by epigenetic mechanisms, but there are probably many more (for review see [10]). The imprinting marks are inherited from the parental gametes, and are then maintained in the somatic cells of an individual. Genes regulated by genomic imprinting mecha-nisms tend to cluster; therefore the imprinting control is often not restricted to a single gene at an imprinted locus but affects the expression of several factors. The regulation of imprinted gene expression is influenced by mechanisms on differ-ent molecular levels, and includes methyl ation of the DNA itself, changes in chromatin structure, post-translational histone modifications such as acetylation, ubiquitylation, phosphorylation and methylation, and noncoding RNAs (for review see [11,12]). In routine diagnosis, testing is focused on DNA methylation of differentially methylated regions, which represent sequences regulating the coordinated expression of clusters of imprinted genes. DNA methylation affects the 5 position of the cytosine pyrimidine ring, and typically occurs in a CpG dinucleotide context.
Thomas Eggermann*, Sabrina Spengler, Magdalena Gogiel, Matthias Begemann and Miriam ElbrachtInstitute of Human Genetics, University Hospital Aachen, Pauwelsstr. 30, D-52074 Aachen, Germany *Author for correspondence: Tel.: +49 241 8088008 Fax: +49 241 8082394 [email protected]
Silver–Russell syndrome (SRS) is a congenital imprinting disorder characterized by intrauterine and postnatal growth restriction and further characteristic features. SRS is genetically heterogenous: 7–10% of patients carry a maternal uniparental disomy of chromosome 7; >38% show a hypomethylation in imprinting control region 1 in 11p15; and a further class of mutations are copy number variations affecting different chromosomes, but mainly 11p15 and 7. The diagnostic work-up should thus aim to detect these three molecular subtypes. Numerous techniques are currently applied in genetic SRS testing, but none of them covers all known (epi)mutations, and they should therefore be used synergistically. However, future next-generation sequencing approaches will allow a comprehensive analysis of all types of alterations in SRS.
Upd(7)mat & SRSUPD is defined as the unusual inheritance of both homologs of a chromosomal pair from only one parent. Several modes of UPD formation have been suggested, the most prominent being
trisomic rescue (for review see [13,14]), where one of the super-numerary chromosomes in a trisomic zygote has been rescued. In one third of these rescues, the chromosome inherited from the parent not contributing the additional chromosome will be lost,
Table 1. Overview of techniques currently used in routine diagnosis of Silver–Russell syndrome and other imprinting disorders†.
Method Detection of UPD
CNVs Epimutation Number of loci in one assay
Advantage Disadvantage
Techniques focusing on genomic disturbances; aberrant methylation is not detectable
MSA Yes Single loci No Single Fast and easy; quantification may be possible
No differentation between UPD and CNVs; DNA sample of the patient and at least one parent required
Cytogenetics No >5 Mb No Whole genome
Genome-wide detection of large (balanced) rearrangements
Low resolution (>5 Mb); cell culture required
FISH No Single loci No Whole genome
Genome-wide detection of large (balanced) rearrangements
Microduplications are difficult to identify; information about the affected region is necessary; cell culture required
Molecular karyotyping (array CGH/SNP array)
(Isodisomy) Genome-wide
No Whole genome
High-resolution genome-wide detection of unbalanced rearrangements
No methylation; no balanced rearrangements
Methylation-specific tests
MS Southern blot
Yes Single loci Yes Single Quantitative Large amounts of DNA required; time-consuming; no differentation between different types of (epi)mutations
MS PCR Yes Single loci Yes Single Fast and easy No differentation between different types of (epi)mutations; only semiquantitative
MS pyrosequencing
Yes Single loci Yes Single Quantitative No differentation between different types of (epi)mutations
QAMA real-time PCR-based methylation assay
Yes Single loci Yes Single Quantitative No differentation between different types of (epi)mutations
Bisulfite sequencing
Yes Single loci Yes Single Quantitative Time-consuming, cloning necessary; no differentation between different types of (epi)mutations
MS MLPA Yes Several loci
Yes Up to 46 target sequences
Quantitative; differentiation between (epi)mutations; detection of MLMD
Sensitive for DNA quality
MS SNuPE Yes Several loci
Yes Multilocus Quantitative; detects MLMD
No differentation between different types of (epi)mutations
†Indeed, a huge number of techniques have been reported but we can present only those procedures widely applied by many laboratories.CGH: Comparative genome hybridization; CNV: Copy number variation; MLMD: Multilocus methylation defect; MS: Methylation-specific; MS MLPA: Methylation-specific multiplex ligation probe-dependent amplification; MS SNuPE: Methylation-specific single nucleotide primer extension; MSA: Microsatellite analysis; QAMA: Quantitative analysis of methylated alleles; SNP: Single nucleotide polymorphism; UPD: Uniparental disomy.
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Figure 2. Simplified overview of mutations and epimutations in Silver–Russell syndrome affecting the imprinting regions in 11p15. Only the major factors regulated by epigenetic mechanisms are shown. While CDKN1C, H19 and KCNQ1 are expressed from the maternal allele, IGF2 and the noncoding RNA KCNQ1OT1 are expressed from the paternal copy. The regulated expression is mediated by CpG methylation and chromatide organization (not shown). Adapted from [57].
Normal
Paternal
Maternal
Paternal
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ICR1 hypomethylation
upd(11p15)mat
11p15.5 duplication
H19 CDKN1C KCNQ1IGF2
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CDKN1C KCNQ1IGF2
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Telomeric CentromericICR1 ICR2
CDKN1C KCNQ1IGF2
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H19CH3
Telomeric CentromericICR1 ICR2
CDKN1C KCNQ1IGF2
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CH3 Methylation
Meso/endodermalenhancers
Regulation
Supposedregulation
Expressed
Silenced
Paternalgenes
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resulting in UPD. UPD mainly affects the whole chromosome, but several UPDs affecting only parts of a chromosome have been reported (‘segmental UPDs’). Depending on the mode of formation, two types of UPD can be distinguished, uniparental heterodisomy and uniparental isodisomy. Uniparental hetero-disomy is defined as the presence of the two different homolo-gous chromosomes from the same parent, whereas in uniparental isodisomy two copies of the identical chromosome are inherited.
With respect to the pathology in imprinting disorders, three mechanisms can be hypothesized:
• Common isodisomic segments in UPD carriers may indicate ‘simple’ recessive genes responsible for the phenotype. However, a common isodisomic segment in upd(7)mat patients/SRS has not been found [15];
• Chromosomal mosaicism originating from the postzygotic trisomic rescue and consisting of a trisomic and a UPD cell line, but in case of the established imprinting disorders, cases with chromosomal mosaicism have not yet been reported. However, as routine diagnostics are mainly restricted to peripheral lymphocytes, chromosomal mosaicism may escape detection;
• As the two aforementioned mechanisms have not yet been identified in patients with imprinting disorders, the most prob-able explanation for their phenotype is the disturbed expres-sion of imprinted genes on the affected chromosome (Figure 1). In case of a maternal UPD, it is conceivable that reduced expression of a paternally expressed gene or overexpression of a maternally expressed factor leads to the aberrant phenotype.
The concept of UPD was suggested for the first time by Engel in 1980 [16], and 8 years later the first case was described [17]. Indeed, this case was a upd(7)mat, and the patient was homozygous for a CFTR mutation due to isodisomy, but was also growth-retarded.
The significant contribution of upd(7)mat to the etiology of SRS was then confirmed by Kotzot et al., who reported on the association of upd(7)mat with the SRS phenotype [18]. A frequency of 7–10% among SRS patients is well established [1]. Upd(7)mat is generally associated with growth restriction and SRS-like features, whereas paternal UPD of chromosome 7 is not (for review see [19]).
In addition to upd(7)mat affecting the whole of chromosome 7, several patients have been reported carrying a UPD restricted to the long arm of chromosome 7 (upd(7q)mat; for review see [20]). These cases indicate that a SRS-relevant genomic region is located within this region. Indeed, several imprinted genes’ non-coding RNAs are located in 7q32. In particular, the MEST/PEG1 gene was regarded as a convincing candidate gene for SRS, since PEG1/MEST-knockout mice show pre- and post-natal growth failure when the mutant gene is transmitted from the father (for review see [21]). MEST/PEG1 has been discussed as a candidate gene for SRS, but screening studies for point mutations as well as methylation studies have not detected any pathogenic variants or aberrant methylation patterns so far (for review see [22]). However, the recent report on a growth-retarded patient with SRS features
and a 3.7 Mb deletion in 7q32 including the MEST/PEG1 gene provides further evidence for a central role of this region for the pathology of the disease [23].
The situation is complicated by several reports on SRS patients with duplications of 7p11.2p13 [24,25]. The region harbors at least one imprinted gene (GRB10) and several factors involved in human growth and development (IGFBP1, IGFBP3, PGK1, EGFR and GHRHR). The most prominent candidate in 7p is GRB10 encoding a cytoplasmic adaptor protein that interacts with tyrosine kinase receptors. GRB10 plays an essential role in growth and was therefore a good candidate for SRS, but the identification of patients with overgrowth or growth restriction carrying different genomic deletions/duplications does not pro-vide clear evidence for an involvement of GRB10 in the etiol-ogy of SRS [eggermann T, unpublished daTa]. It can be assumed that unknown factors relevant for SRS are also localized in the short arm of chromosome 7.
Chromosome 11p15.5 epimutations & rearrangementsThe first evidence for a contribution of 11p15 to the etiology of SRS was provided by the identification of maternal 11p15 duplica-tions in growth-restricted patients; some of them exhibited SRS features (for review see [22]). Likewise, duplication of paternal 11p15 as the opposite disturbance results in BWS, which is mainly characterized by overgrowth.
A cluster of imprinted genes crucial for the control of (fetal) growth is localized in 11p15. Two ICRs regulate the expression of these genes – the telomeric ICR1 and the centromeric ICR2 (Figure 2).
The reciprocal expression of H19 and IGF2 is regulated by the telomeric ICR1. The two genes are both expressed in endo-derm- and mesoderm-derived tissues during embryonic devel-opment and compete for the same enhancers in 11p15. IGF2 is expressed paternally and has a central role in fetal development and growth. By contrast, the function of H19 is unclear, although it was one of the first noncoding transcripts to be identified. The ICR1 consists of seven CCCTC-binding factor target sites in the H19 differentially methylated region and shows an allele-specific methylation. CCCTC-binding factor binds to the mater-nal unmethylated ICR1 copy and forms a chromatin boundary. Thereby the transcription of the maternal IGF2 allele is blocked and the transcription of the maternal H19 copy is promoted (for review see [1]).
The maternally methylated centromeric ICR2 regulates the (reciprocal) expression of CDKN1C and KCNQ1, as well as of other genes. Mutations in the maternal active allele of the CDKN1C gene account for up to 40% of familial and 5–10% of sporadic BWS patients (Table 1) [26]. The gene encodes a CDK inhibitor (p57KIP2) and is part of the p21CIP2 CDK inhibitor family. The gene of the noncoding RNA KCNQ1OT1 (LIT1) is localized in intron 9 of the KCNQ1 gene. KCNQ1OT1 is expressed from the paternal allele and represses the expression of the CDKN1C gene.
In SRS, the most frequent finding is hypomethylation at the ICR1 on 11p15, accounting for approximately 38% of patients
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(Figures 1 & 2) ([27]; for review see [22]) . This ICR1 hypomethylation should result in a suppressed expression of the human growth promotor IGF2. Indeed, in fibroblasts of SRS individuals, IGF2 mRNA is reduced [27] but, in serum, IGF2 levels in SRS patients with H19 hypomethylation are normal [3,28]. However, it must be considered that the liver, as the major organ of postnatal IGF2 secretion, expresses the factor from a nonimprinted promoter (for review see [28]). Owing to the negative findings in serum, it has been concluded that ICR1 hypomethylation does not directly influence IGF2 secretion in SRS children, but an altered IGF2 production probably leads to a diminished autoparacrine action in the fetus (for review see [28]).
Epigenetic and genetic alterations in 11p15 are also associated with the overgrowth disorder BWS, where they account for >70% of patients. However, in contrast to SRS, a preponderance of disturbances affect the centromeric ICR2 (for review see [26]).
In lymphocytes, the majority of SRS patients with 11p15 dis-turbances show a mosaic distribution of the epimutation [3–7] – that is, some of the cells in an individual show normal, and others aberrant, methylation patterns. Clinically, this mosaicism is reflected by hemihypoplasia/asymmetry, which is present in the majority of these patients [1]. The mosaic distribution makes the molecular diagnosis difficult and it requires sensitive quantitative assays. It is therefore not surprising that some cases of ICR1 hypo-methylation escape routine diagnostic detection [29,30], and the real frequency of ICR1 hypomethylation among SRS patients is therefore currently unclear. It has been assumed that epimutation mosaicism may show an unequal distribution in different tissues, resulting in nearly normal methylation patterns in lympho cytes and thereby giving false-negative results. However, early data from methylation-specific (MS) testing in tissues other than lympho-cytes do not indicate that there is a remarkable fraction of patients with undetected 11p15 epimutation [31].
In addition to ICR1 hypomethylation, genomic duplications within 11p15 contribute to the spectrum of molecular disturbances in SRS. Duplications of the maternal 11p15 copy including both ICRs result in a SRS phenotype, whereas duplications of the pater-nal copy are associated with BWS. Furthermore, the recent identi-fication of a SRS patient with a duplication restricted to the ICR2 suggests that both ICRs on 11p15 are involved in the etiology of the disease [32,33]. However, imbalances affecting only parts of one of the ICRs in 11p15.5 do not reflect this parent-of-origin effect in any case [34,35]; as a result, the interpretation of these unusual findings is difficult, but these cases allow interesting insights in the complex regulation mechanisms within the ICRs in 11p15.5.
Aberrant methylation at multiple imprinted loci in SRS (multilocus methylation defect)In the last 4 years, an increasing number of SRS patients have been reported with aberrant methylation at multiple imprinted loci (multilocus methylation defects; MLMD). In these patients, not only the disease-specific ICR1 hypomethylation was detected but also other imprinting domains showed an aberrant methyl ation. In 2005, MLMD was first reported in patients with TNDM, an imprinting disorder characterized by hypomethylation of the
maternal allele in 6q24 [36,37]. Two years later, Mackay et al. reported on seven TNDM/MLMD pedigrees with homo zygosity or compound heterozygosity for ZFP57 mutations as the cause of the first heritable global human imprinting disorder [38]. By contrast, ZFP57 mutations are probably not associated with aberrant imprinting in SRS or BWS [39,40]. In SRS and BWS, MLMD accounts for approximately 9% of ICR1 and 25% of ICR2 hypomethylation carriers, respectively; both paternally and maternally imprinted loci are affected [41–46].
Clinically, there is no difference between SRS or BWS patients carrying MLMD and patients with ‘isolated imprinting defects’ in ICR1 or ICR2 in 11p15, respectively. Another striking but unex-plained finding is that MLMD carriers with the same aberrant methylation patterns in lymphocytes may present with either BWS or SRS [41]. Azzi and colleagues suggested that the imprinted locus with the lowest level of methylation is dominant and determines the clinical outcome [41]. Furthermore, the studies published so far are based on lymphocyte DNA, thus a mosaic distribution of epimutations in other tissues influencing the phenotypic expression cannot be excluded.
Chromosomal aberrationsStructural chromosomal aberrations detected by conventional microscopic karyotyping have been reported in several patients with SRS features [1]: they affected numerous chromosomes, but only chromosomes 7, 11 and 15 were consistently involved in individuals fulfilling the diagnostic criteria of SRS.
With the application of high-resolution array technologies, a considerable increase in detection of pathogenic copy number variations (CNVs) in growth-retarded patients with SRS features could be observed [47,48]. Up to 19% of patients carry relevant pathogenic variations with a size ranging from 677 kb to 9.1 Mb [49]. However, similar to the observations from conventional cyto-genetic analyses, a heterogeneous pattern of variants is detectable. The broad clinical spectrum in the carriers of these submicro-scopic imbalances reflects this genetic heterogeneity: several of the SRS features are unspecific and overlap with different microdeletion syndromes [49]. As a result, molecular karyotyping generally not only enables us to identify genomic disturbances in growth-retarded patients with atypical SRS phenotypes or with only slight clinical features reminiscent of SRS, but also provides relevant insights into the molecular pathology of the disease as it has been shown for the 7q32 or the 11p15.5 loci.
Clinical findings & genotype–phenotype correlationOverall, SRS patients show a broad clinical spectrum with a less clear phenotype in adulthood. The clinical diagnosis is therefore not easy and requires careful anamnestic work, including pictures from early childhood. Therefore, several clinical scoring systems to assist the diagnosis have recently been suggested [3,5,6]. Indeed, these scores are helpful but just auxiliary, and do not replace the experience of the clinical investigator (Table 1). As a result, patients referred for molecular genetic testing show a broad clinical hetero-geneity, and the detection rate for the known molecular altera-tions in routine diagnostic cohorts is only approximately 19%,
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whereas they account for more than 50% in cohorts of patients with typical SRS features [7].
With the definition of molecular subgroups, a correlation between specific molecular alterations and the clinical outcome has been postulated [3,5,6,19,28]. With respect to the ICR1 hypomethylation, a correlation between the clinical severity and the level of epimutation is controversially discussed [2,5,6]. However, it becomes apparent that patients with 11p15 epimutation show the classical SRS phenotype, whereas upd(7)mat carriers often have a milder clinical expression. There is a considerable overlap between the two groups, thus it is hardly possible to clinically distinguish between them. Indeed, patients with ICR1 hypo methylation in 11p15 are generally shorter and leaner and body asymmetry is more frequent in comparison to patients with upd(7)mat or without molecular diagnosis [3,5,6]. Learning difficulties and the need for speech therapy are more likely in upd(7)mat [2–4]; the latter has been associated with the absence of FOXP2 expression. For the upd(7)mat subgroup, an increased probability for movement disorders can be assumed; it is conceivable that the imbalanced expression of the imprinted e-sarcoglycan gene (SGCE) in 7q21 is causative for these upd(7)mat-specific feature mutations, which are associated with myoclonus-dystonia.
Furthermore, an influence of the molecular disturbance on the response to growth hormone treatment has been suggested [28]: the first data indicate that upd(7)mat carriers show a trend toward more height gain than patients with 11p15 hypomethylation [28].
As already mentioned, patients carrying (sub)microscopic chromo somal aberrations show a broad range of clinical fea-tures, often overlapping with other microdeletions/duplication syndromes [49]. In a recent study, in patients referred to as SRS for routine diagnostic purposes, we detected that only half of the car-riers of chromosomal imbalances showed the typical phenotype, whereas the others revealed only slight clinical features. These findings reflect the nonspecificity of clinical features overlapping between SRS and the detected syndromes.
Genetic testing in SRSBased on the current knowledge on genomic and epigenetic muta-tions detectable in SRS, routine genetic testing strategies in SRS should focus on the detection of the following disturbances:
• Epimutations in 11p15
• Upd(7)mat
• (Submicroscopic) chromosomal disturbances
• MLMD
Nowadays, a broad panel of molecular tests for imprinting dis-orders is available, but none of them detects all known mutations and epimutations (Table 1, Figures 3–5). Generally, two groups of approaches can be differentiated: those focusing on DNA methyl-ation; and those aiming for the identification of genomic altera-tions (i.e., UPD and genomic imbalances [CNVs]). All currently used tests for routine diagnostics have their limitations. Therefore a step-wise or combined use of different tests is essential for a diagnostic work-up (Figure 5).
MS testsIn many of the currently applied MS approaches (e.g., MS PCR, MS pyrosequencing, MS single nucleotide primer extension, bisulfite sequencing and allele-specific methylated multiplex real-time quantitative PCR), bisulfite-treated DNA is used as a template: bisulfite converts cytosine residues in genomic DNA to uracil, but leaves 5-methylcytosine residues unaffected (for review see [50,51]). As a result, specific changes are introduced in the DNA that depend on the methylation status of the DNA sequence of interest. Today, numerous protocols and commercial kits for bisulfite conversion are available with no need for special equip-ment. Another possibility to analyze methylated CpG islands is the use of methylation-sensitive restriction enzymes like HpaII. These enzymes cut only unmethylated DNA sequences and leave the methylated alleles unaffected. The digested/undigested DNA sample then serves as a template for further MS tests like MS multiplex ligation probe-dependent amplification (MS-MLPA).
MS assays generally detect all currently known classes of epimutations and mutations resulting in an unusual methyl-ation pattern of the investigated locus, but with the exception of MS-MLPA they do not allow the differentiation between these three classes, therefore additional analyses are necessary (Figure 5).
Another problem with molecular SRS diagnostics is the putative mosaicism for ICR1 hypomethylation: the majority of patients reported so far are mosaics of the epimutation, therefore the appli-cation of quantitative MS tests is needed.
Furthermore, many of the MS tests applied in routine diag-nostics of SRS are techniques for analyzing single CpG islands or short sequences (Table 1). Considering the heterogeneity of the disease, which currently makes the analysis of at least three sepa-rated loci (7p13, 7q32 and 11p15.15) necessary, this limitation is indeed hampering. To circumvent this problem, and to detect MLMD as another challenge in SRS diagnostics, multilocus tests have been developed (e.g., MS-MLPA and MS single nucleotide primer extension) [30,52].
Figure 4. Genetic testing in Silver–Russell syndrome: chromosome 11p15 (see facing page). MS MLPA as well as MS SNuPE allow the identification of all three major mutation and epimutation types in one approach. (A) MS MLPA: an ICR1 hypomethylation in 11p15 corresponds to a reduced hybridization signal for the H19-specific methylation-sensitive MLPA probes. (B) MS SNuPE: the typical results of a duplication of paternal 11p15 material (a pattern consistent with a hypermethylation of the loci H19 and IGF2P0 and a hypomethylation of LIT1). However, this finding can not be differentiated from a upd(11p)pat, therefore STR typing is indicated (C). (D) MS SNuPE shows a disturbed methylation of imprinted loci on different chromosomes and thus allows the detection of aberrant methylation at multiple loci (MLMD). ICR1: Imprinting control region 1; MLMD: Multilocus methylation defect; MS MLPA: Methylation-specific multiplex ligation probe-dependent amplification; MS SNuPE: Methylation-specific single nucleotide primer extension; SPS: Single primer sequencing; STR: Short tandem repeat.
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Tests focused on genomic alterationsBased on its frequency of 7–10% in SRS, upd(7)mat testing is a key element of molecular diagnostics in patients with intrauterine and postnatal growth restriction and SRS features. The tradi-tional method to detect UPD is microsatellite typing using highly polymorphic short sequence repeat markers (short sequence repeat and short tandem repeat). Microsatellite analysis consists of a simple PCR amplification and subsequent sequencing electro-phoresis. By comparing the alleles of the patient and his parent, segregation analysis of the investigated locus is possible, and it is particularly useful to identify and confirm a UPD (Figures 3 & 4).
As mentioned earlier, a considerable number of patients pre-senting SRS features carry chromosomal imbalances; while large aberrations detectable by conventional cytogenetics are rare,
submicroscopic deletions or duplications significantly contribute to the etiology of this heterogeneous cohort of patients (Figure 1) [47–49]. Molecular karyotyping should therefore be included in the diagnostic work-up in patients with suspected SRS after exclu-sion of upd(7)mat and 11p15 epimutations. The application of the test may furthermore be indicated in patients with duplica-tions in 11p15, because it allows characterization of breakpoints, determination of the size of the affected region, and identification of pathogenic CNVs that would escape detection by single locus tests [23,34,53]. This characterization should be accomplished by conventional cytogenetics and/or FISH, which allow the genome-wide identification of imbalanced and balanced chromosomal rear-rangements as the basis of well-directed genetic counseling (see below). In addition to CNV detection, molecular karyotyping by
Confirmation by MSA Confirmation by MSA Normal result
Normal methylation in11p15
Normal karyotype
Confirmation by MSA
Testing for upd(7)mat
upd(7)mat(typical phenotype† 7–10%
SRS features‡ ∼2%)
Molecularkaryotyping
Confirmation by FISH,MLPA and so on
(Sub)microscopicimbalances
(typical phenotype† ∼22%,SRS features‡ ∼17%)
upd(11p15)mat(single cases)
11p15 duplications(1–2%)
Multilocus methylationdefects
(∼7–10%)
ObligateOptional
Figure 5. Diagnostic algorithm in Silver–Russell syndrome. Molecular karyotyping is indicated to exclude familial rearrangements.†Patients with classical SRS phenotype.‡Patients with features reminiscent of SRS.ICR: Imprinting control region; MLPA: Multiplex ligation probe-dependent amplification; MS: Methylation-specific; MSA: Microsatellite analysis; SRS: Silver–Russell syndrome. Adapted from [57].
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Key issues
• Silver–Russell syndrome (SRS) is associated with both epigenetic and genetic disturbances (i.e., on chromosomes 7 and 11p15).
• The genetic heterogeneity of SRS requires a complex molecular work-up.
• Numerous molecular techniques are applied in routine diagnostics of SRS, but none of the currently used tests covers all known mutations and epimutations.
• The identification of the molecular alterations in SRS contributes to an understanding of its pathophysiology.
• Next-generation sequencing approaches will allow a comprehensive analysis of both genomic and epigenetic disturbances in SRS, thereby making individualized patient management possible.
single nucleotide polymorphism typing allows the identification of UPD.
However, all of these ‘genomic’ tests do not allow the identi-fication of aberrant methylation, and some of them require the analysis of patients as well as of parental (DNA) samples. In case of microsatellites, deletions or duplications may be misinterpreted as uniparental isodisomy or as mosaic UPDs (Figures 3 & 4).
Genetic testing & genetic counselingIdentification of the molecular alterations in patients significantly helps to determine the recurrence probability for other children.
Isolated ICR1 hypomethylation mainly occurs sporadically, but single families with parent-to-child transition have been reported [6]. Upd(7)mat has also been regarded as a sporadic event, but recently a familial translocation 7;13 has been reported to predispose for this disturbance [54]. In case of MLMD, an increased recurrence probability cannot be excluded, as the identification of autosomal recessive ZFP57 mutations in patients with TNDM and MLMD show [38]. However, the majority of SRS cases occur sporadically, therefore a probability for a germline mutation and a recurrence risk of <1% can be delineated, even in the case of MLMD.
By contrast, the detection of a chromosomal aberration (e.g., 11p15 duplications) in a patient generally requires a chromosomal analysis in the family because one of the parents may carry a balanced trans-location. These families have a significantly increased probability for an offspring with a chromosomal imbalance; in these cases, both the affected chromosomal region and the parent inheriting the variant have to be considered (for example see [55]).
Expert commentary & five-year viewIn addition to their significance for routine diagnostics, the iden-tification and characterization of molecular disturbances in SRS provide us with the opportunity to identify the pathophysiological mechanisms in SRS and – in the future – to develop evidence-based diagnostic and individualized therapeutic measurements. Therefore it will be possible to significantly improve the clinical management of SRS patients who currently often remain without diagnosis and – as a result – are treated inappropriately or too late. Development
of novel diagnostic tests will result in a timely and satisfactory diagnosis and thereby allow an individualized treatment as early as possible. These improvements will not only enhance the quality of life of the patients and their families, but also avoid undirected, extensive therapies. In general, epigenetic testing will become as important a diagnostic tool as genetic testing is now, and will ensure the development of more cost-effective diagnostic tests, that may also be applicable to more common diseases.
This development will be strongly influenced by the newly estab-lished high-throughput deep sequencing techniques: the future next-generation sequencing (NGS)-based approaches for both genomic and epigenetic targets will result in a comprehensive characteriza-tion of both normal and pathogenic genomes and epigenomes. This knowledge will influence routine diagnostics in at least two ways:
• Targeted or whole epigenome array and/or NGS approaches will circumvent the current problem that only single and selected imprinted loci are analyzed, thus allowing the genome- and epigenome-wide detection of both qualitative and quantitative abnormalities;
• Deep NGS will not only result in the identification of mosaic cases, it will also include genome-wide analyses of point mutations caus-ing aberrant methylation and/or imprinting disorder phenotypes, as recently reported for single MLMD patients and families [38,56].
In the future, both genomic alterations – imbalanced as well as balanced chromosomal aberrations and point mutations – and aber-rant methylation will become detectable by application of one assay. The analysis, interpretation and translation to clinical application of these data require new strategies and represent a challenge for human geneticists.
Financial & competing interests disclosureThe authors are supported by the Bundesministerium für Bildung und Forschung (Network ‘Imprinting Diseases’, 01GM0884). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
ReferencesPapers of special note have been highlighted as:•ofinterest••ofconsiderableinterest
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