No evidence for isolated imprinting mutations in the PEG1/MEST locus in Silver-Russell patients

Imprinting defects have meanwhile been described in nearly all human imprinting disorders among them Silver-Russell syndrome (SRS). In this disorder, 11p15 epimutations and maternal Uniparental Disomy of chromosome 7 (UPD7) are detectable in approximately 50% of patients. To find out whether isolated imprinting defects on chromosome 7 play a role in the aetiology of SRS we screened a cohort of 54 SRS patients without 11p15 epimutations. Methylation-specific PCR was carried out for the PEG1/MEST locus in 7q31. This test detects all known segmental and complete UPD7 cases. The exclusion of isolated imprinting defects in our study population shows that this type of epimutation at the PEG1/MEST locus in 7q31 does not play a relevant role in SRS. However, the role of imprinting disturbances in other genes cannot be excluded.     

No evidence of PEG1/MEST gene mutations in Silver-Russell syndrome patients.

Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.     

Structural and sequence variants in patients with Silver‐Russell syndrome or similar features—Curation of a disease database

Silver‐Russell syndrome (SRS) is a clinically and molecularly heterogeneous disorder involving prenatal and postnatal growth retardation, and the term SRS‐like is broadly used to describe individuals with clinical features resembling SRS. The main molecular subgroups are loss of methylation of the distal imprinting control region (H19/IGF2:IG‐DMR) on 11p15.5 (50%) and maternal uniparental disomy of chromosome 7 (5%–10%). Through a comprehensive literature search, we identified 91 patients/families with various structural and small sequence variants, which were suggested as additional molecular defects leading to SRS/SRS‐like phenotypes. However, the molecular and phenotypic data of these patients were not standardized and therefore not comparable, rendering difficulties in phenotype–genotype comparisons. To overcome this challenge, we curated a disease database including (epi)genetic phenotypic data of these patients. The clinical features are scored according to the Netchine‐Harbison clinical scoring system (NH‐CSS), which has recently been accepted as standard by consensus. The structural and sequence variations are reviewed and where necessary redescribed according to recent recommendations. Our study provides a framework for both research and diagnostic purposes through facilitating a standardized comparison of (epi)genotypes with phenotypes of patients with structural/sequence variants.     

Increased incidence of aberrant DNA methylation within diverse imprinted gene loci outside of IGF2/H19 in Silver-Russell syndrome

Almost half of the patients with Silver-Russell syndrome (SRS) are affected by DNA hypomethylation of the Imprinting Center Region 1 (ICR1) at the IGF2/H19 locus on 11p15. We searched genome-wide for additional aberrant DNA methylation in such SRS patients that could account for the clinical variability of the disorder. For this purpose, 18 children with SRS (11 with ICR1 hypomethylation) and 9 children small for gestational age (SGA), serving as controls, were recruited. Genomic DNA from whole blood was subjected to microarray analysis with the HumanMethylation27 BeadChip. This array allows investigating 27,500 CpG sites mostly located in the promoter regions of 14,000 genes. Data were validated by the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) technique for the 11p15 region. SRS patients with ICR1 hypomethylation were significantly more frequently affected by DNA hypo- and hypermethylation of CpG sites from diverse imprinted loci than the SGA controls (p = 0.0048). There was no recurrent specific methylation defect outside of IGF2/H19. These findings suggest as causative in SRS a defective mechanism necessary for establishment or maintenance of imprinting marks, which affects imprinted loci in general with low specificity and the IGF2/H19 locus with high specificity, implying the existence of some structural peculiarities at the IGF2/H19 locus.     

Familial reciprocal translocation t(7;16) associated with maternal uniparental disomy 7 in a Silver‐Russell patient

We present the case of a maternal heterodisomy for chromosome 7 in the daughter of a t(7;16)(q21;q24) reciprocal translocation carrier. The proband was referred to the hospital for growth retardation and minor facial dysmorphism without mental retardation. A diagnosis of Silver‐Russell syndrome was suspected. Chromosomal analysis documented a 46,XX,t(7;16)(q21;q24)mat chromosome pattern. Microsatellite analysis showed a normal biparental inheritance of chromosome 16 but a maternal heterodisomy of chromosome 7. Occurrence of uniparental disomy (UPD) is a well‐recognized consequence of chromosomal abnormalities that increase the rate of meiotic nondisjunction, mainly Robertsonian translocations and supernumerary chromosomes. Although reciprocal translocations should, theoretically, be also at increased risk of UPD, only three cases have been reported so far. However, because the association between uniparental disomy and reciprocal translocation may exist with an underestimated frequency, prenatal diagnosis is recommended when clinically relevant chromosomes for UPD are involved. © 2002 Wiley‐Liss, Inc.     

A multiplex methylation PCR assay for identification of uniparental disomy of chromosome 7

Uniparental disomy of chromosome 7 (UPD7) is associated with abnormal phenotypic effects because of inappropriate expression of imprinted genes on chromosome 7. Based on the differential methylation of the promoter region of the imprinted PEG1/MEST locus at 7q32, we designed a multiplex methylation PCR (mPCR) assay to rapidly distinguish UPD7 from biparental inheritance of chromosome 7. Primers were designed to produce different sized PCR amplicons based on the parent of origin-specific methylation at this locus; electrophoresis of PCR amplicons showed a 189-bp product from the methylated maternal allele and a 109-bp product from the unmethylated paternal allele. This mPCR assay correctly predicted the chromosome 7 imprinting status in normal control and UPD7 samples. Previous assays for UPD7 required genotyping of the proband and parents, or separate maternal- and paternal-specific mPCR reactions. The advantage of this assay is that parental samples are not required and that amplification of both alleles in the same reaction is simpler and provides an internal control. This multiplex mPCR assay will be useful in screening for UPD7 in patients with Silver-Russell syndrome (SRS; also Russell-Sliver syndrome, RSS), primordial growth retardation, and in patients with supernumerary marker chromosomes or chromosome rearrangements of chromosome 7 origin.     

NSD1 duplication in Silver–Russell syndrome (SRS): molecular karyotyping in patients with SRS features

Silver–Russell syndrome (SRS) is a growth retardation syndrome characterized by intrauterine and postnatal growth retardation, relative macrocephaly and protruding forehead, body asymmetry and feeding difficulties. Nearly 50% of cases show a hypomethylation in 11p15.5, in 10% maternal uniparental disomy of chromosome 7 is present. A significant number of patients with SRS features also exhibit chromosomal aberrations. We analyzed 43 individuals referred for SRS genetic testing by molecular karyotyping. Pathogenic variants could be detected in five of them, including a NSD1 duplication in 5q35 and a 14q32 microdeletion. NSD1 deletions are detectable in overgrowth disorders (Sotos syndrome and Beckwith–Wiedemann syndrome), whereas NSD1 duplications are associated with growth retardation. The 14q32 deletion is typically associated with Temple syndrome (TS14), but the identification of a patient in our cohort reflects the clinical overlap between TS14 and SRS. As determination of molecular subtypes is the basis for a directed counseling and therapy, the identification of pathogenic variants in >10% of the total cohort of patients referred for SRS testing and in >16% of characteristic individuals with the characteristic SRS phenotype confirms the need to apply molecular karyotyping in this cohort.     

Silver–Russell syndrome due to paternal H19/IGF2 hypomethylation in a twin girl born after in vitro fertilization

Silver–Russell syndrome (SRS) is a clinically and genetically heterogeneous syndrome characterized by severe intrauterine and postnatal growth retardation, facial dysmorphism and body asymmetry. One of the main molecular mechanisms leading to the syndrome involves methylation abnormalities of chromosome 11p15. In the last decades, an increase of imprinting disorders have been reported in children born from assisted reproductive technology (ART); however there is currently little evidence linking SRS and ART. Only few infants with SRS born using ART, supported by molecular analysis, have been described. We report on a twin‐girl conceived using intracytoplasmic sperm injection (ICSI) diagnosed with SRS. Molecular studies revealed a hypomethylation of the paternal H19/IGF2 Imprinting Control Region. Her twin sister had a normal prenatal and postnatal growth and a normal methylation pattern of the chromosome 11p15. This is the second reported case of a twin infant with SRS conceived using ART with hypomethylation of H19/IGF2; it provides additional evidence of a possible relationship between ART procedures and methylation defects observed in SRS. Given the clinical heterogeneity of SRS, and the increased risk of multiple and preterm births in the ART‐conceived children, it is possible that a number of cases of SRS remains undiagnosed in this population. Future studies should investigate the possible link between ART and SRS, in order to better understand the causes of epimutations in ART pregnancies, and to help clinicians to adequately counsel parents who approach to ART and to assess the opportunity of a long‐term follow‐up of children conceived using ART. © 2013 Wiley Periodicals, Inc.     

11p15 ICR1 Partial Deletions Associated with IGF2/H19 DMR Hypomethylation and Silver–Russell Syndrome

The 11p15 region harbors the IGF2/H19 imprinted domain, implicated in fetal and postnatal growth. Silver–Russell syndrome (SRS) is characterized by fetal and postnatal growth failure, and is caused principally by hypomethylation of the 11p15 imprinting control region 1 (ICR1). However, the mechanisms leading to ICR1 hypomethylation remain unknown. Maternally inherited genetic defects affecting the ICR1 domain have been associated with ICR1 hypermethylation and Beckwith–Wiedemann syndrome (an overgrowth syndrome, the clinical and molecular mirror of SRS), and paternal deletions of IGF2 enhancers have been detected in four SRS patients. However, no paternal deletions of ICR1 have ever been associated with hypomethylation of the IGF2/H19 domain in SRS. We screened for new genetic defects within the ICR1 in a cohort of 234 SRS patients with hypomethylated IGF2/H19 domain. We report deletions close to the boundaries of ICR1 on the paternal allele in one familial and two sporadic cases of SRS with ICR1 hypomethylation. These deletions are associated with hypomethylation of the remaining CBS, and decreased IGF2 expression. These results suggest that these regions are most likely required to maintain methylation after fertilization. We estimate these anomalies to occur in about 1% of SRS cases with ICR1 hypomethylation.     

Myoclonus‐dystonia and Silver–Russell syndrome resulting from maternal uniparental disomy of chromosome 7

Myoclonus‐dystonia (M‐D) is a movement disorder that is often associated with mutations in epsilon‐sarcoglycan (SGCE), a maternally imprinted gene at 7q21.3. We report a 24‐year‐old male with short stature (<5th percentile) and a movement disorder clinically consistent with M‐D. Single nucleotide polymorphism (SNP) array did not identify significant copy number changes, but revealed three long continuous stretches of homozygosity on chromosome 7 suggestive of uniparental disomy. Parental SNP arrays confirmed that the proband had maternal uniparental disomy of chromosome 7 (mUPD7) with regions of heterodisomy and isodisomy. mUPD7 is the cause of approximately 5–10% of Silver–Russell syndrome (SRS), a disorder characterized by prenatal and postnatal growth retardation. Although SRS was not suspected in our patient, these findings explain his short stature. SGCE methylation testing showed loss of the unmethylated paternal allele. Our findings provide a unifying diagnosis for his short stature and M‐D and help to optimize his medication regimen. In conclusion, we show that M‐D is a clinical feature that may be associated with SRS due to mUPD7. Individuals with mUPD7 should be monitored for the development of movement disorders. Conversely, individuals with M‐D and short stature should be evaluated for SRS.     

De novo IGF2 mutation on the paternal allele in a patient with Silver–Russell syndrome and ectrodactyly

Although paternally expressed IGF2 is known to play a critical role in placental and body growth, only a single mutation has been found in IGF2. We identified, through whole‐exome sequencing, a de novo IGF2 indel mutation leading to frameshift (NM_000612.5:c.110_117delinsAGGTAA, p.(Leu37Glnfs*31)) in a patient with Silver–Russell syndrome, ectrodactyly, undermasculinized genitalia, developmental delay, and placental hypoplasia. Furthermore, we demonstrated that the mutation resided on the paternal allele by sequencing the long PCR product harboring the mutation‐ and methylation‐sensitive SmaI and SalI sites before and after SmaI/SalI digestion. The results, together with the previous findings in four cases from a single family with a paternally inherited IGF2 nonsense mutation and those in patients with variable H19 differentially methylated region epimutations leading to compromised IGF2 expression, suggest that the whole phenotype of this patient is explainable by the IGF2 mutation, and that phenotypic severity is primarily determined by the IGF2 expression level in target tissues.     

De novo mutations of the gene encoding the histone acetyltransferase KAT6B in two patients with Say‐Barber/Biesecker/Young‐Simpson syndrome

The Say‐Barber/Biesecker/Young‐Simpson (SBBYS) type of the blepharophimosis–mental retardation syndrome group (Ohdo‐like syndromes) is a multiple congenital malformation syndrome characterized by vertical narrowing and shortening of the palpebral fissures, ptosis, intellectual disability, hypothyroidism, hearing impairment, and dental anomalies. Mutations of the gene encoding the histone‐acetyltransferase KAT6B have been recently identified in individuals affected by SBBYS syndrome. SBBYS syndrome‐causing KAT6B mutations cluster in a ～1,700 basepair region in the 3′ part of the large exon 18, while mutations located in the 5′ region of the same exon have recently been identified to cause the genitopatellar syndrome (GPS), a clinically distinct although partially overlapping malformation‐intellectual disability syndrome. Here, we present two children with clinical features of SBBYS syndrome and de novo truncating KAT6B mutations, including a boy who was diagnosed at the age of 4 months. Our results confirm the implication of KAT6B mutations in typical SBBYS syndrome and emphasize the importance of genotype–phenotype correlations at the KAT6B locus where mutations truncating the KAT6B protein at the amino‐acid positions ～1,350–1,920 cause SBBYS syndrome. © 2013 Wiley Periodicals, Inc.     

Co‐existence of 9p deletion and Silver‐Russell syndromes in a patient with maternally inherited cryptic complex chromosome rearrangement involving chromosomes 4, 9, and 11

We report a patient with a maternally inherited unbalanced complex chromosomal rearrangement (CCR) involving chromosomes 4, 9, and 11 detected by microarray comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). This patient presents with clinical features of 9p deletion syndrome and Silver‐Russell syndrome (SRS). Chromosome analysis performed in 2000 showed what appeared to be a simple terminal deletion of chromosome 9p22.1. aCGH performed in 2010 revealed a 1.63 Mb duplication at 4q28.3, a 15.48 Mb deletion at 9p24.3p22.3, and a 1.95 Mb duplication at 11p15.5. FISH analysis revealed a derivative chromosome 9 resulting from an unbalanced translocation between chromosomes 9 and 11, a chromosome 4 fragment inserted near the breakpoint of the translocation. The 4q28.3 duplication does not contain any currently known genes. The 9p24.3p22.3 deletion region contains 36 OMIM genes including a 3.5 Mb critical region for the 9p‐phenotype. The 11p15.5 duplication contains 49 OMIM genes including H19 and IGF2. Maternal aCGH was normal. However, maternal chromosomal and FISH analyses revealed an apparently balanced CCR involving chromosomes 4, 9, and 11. To the best of our knowledge, this is the first report of a patient with maternally inherited trans‐duplication of the entire imprinting control region 1 (ICR1) among the 11p15.5 duplications reported in SRS patients. This report supports the hypothesis that the trans‐duplication of the maternal copy of ICR1 alone is sufficient for the clinical manifestation of SRS and demonstrates the usefulness of combining aCGH with karyotyping and FISH for detecting cryptic genomic imbalances. © 2012 Wiley Periodicals, Inc.