Quantitative DNA Methylation Analysis and Epigenotype-Phenotype Correlations in Taiwanese Patients with Silver-Russell Syndrome.

Background: Silver-Russell syndrome (SRS; OMIM #180860) is a clinically and genetically heterogeneous imprinting disorder characterized by prenatal and postnatal growth failure. The aim of this study was to identify the epigenotype-phenotype correlations in these patients using quantitative DNA methylation analysis. Methods: One hundred and eighty-three subjects clinically suspected of having SRS were referred for diagnostic testing by the methylation profiling of H19-associated imprinting center (IC) 1 and imprinted PEG1/MEST regions using methylation-specific high-resolution melting analysis and methylation quantification with the MassARRAY assay. Correlations between quantitative DNA methylation status and clinical manifestations of the subjects according to the Netchine-Harbison (N-H) clinical scoring system for SRS were analyzed. Results: Among the 183 subjects, 90 had a clinical diagnosis of SRS [N-H score ≥ 4 (maximum = 6)] and 93 had an SRS score < 4. Molecular lesions were detected in 41% (37/90) of the subjects with a clinical diagnosis of SRS, compared with 3% (3/93) of those with an N-H score < 4. The IC1 methylation level was negatively correlated with the N-H score. The molecular diagnosis rate was positively correlated with the N-H score. Thirty-one subjects had IC1 hypomethylation (IC1 methylation level <35% by the MassARRAY assay), seven had maternal uniparental disomy 7, and two had pathogenic copy number variants. Among the 90 subjects with an N-H score ≥ 4, the IC1 methylation level was significantly different between those with or without some clinical SRS features, including birth length ≤ 10th centile, relative macrocephaly at birth, normal cognitive development, body asymmetry, clinodactyly of the fifth finger, and genital abnormalities. Conclusions: This study confirmed the suitability of the N-H clinical scoring system as clinical diagnostic criteria for SRS. Quantitative DNA methylation analysis using the MassARRAY assay can improve the detection of epigenotype-phenotype correlations, further promoting better genetic counseling and multidisciplinary management for these patients.

Further heterogeneity in Silver-Russell syndrome: PLAG1 deletion in association with a complex chromosomal rearrangement.

Silver-Russell syndrome (SRS) is a rare genetic condition primarily characterized by growth restriction and facial dysmorphisms. While hypomethylation of H19/IGF2:IG-DMR (imprinting control region 1 [IC1]) located at 11p15.5 and maternal uniparental disomy of chromosome 7 (upd[7]mat) are the most common genetic mechanisms responsible for SRS, the expanding body of literature describing alternative causative variants suggests SRS is a highly heterogeneous condition, also involving variation in the HMGA2-PLAG1-IGF2 pathway. We report a familial PLAG1 deletion in association with a complex chromosomal rearrangement. We describe two siblings with differing unbalanced chromosomal rearrangements inherited from a mother with a 5-breakpoint balanced complex rearrangement involving chromosomes 2, 8, and 21. The overlapping but diverse phenotypes in the siblings were characterized by shared SRS-like features, underlined by a PLAG1 whole gene deletion. Genetic analysis and interpretation was further complicated by a meiotic recombination event occurring in one of the siblings. This family adds to the limited literature available on PLAG1-related SRS. We have reviewed all currently known cases aiming to define the associated phenotype and guide future genetic testing strategies. The heterogeneity of SRS is further expanded by the involvement of complex cytogenomic abnormalities, imposing requirements for a comprehensive approach to testing and genetic counseling.

New euchromatic variant dup(11)(p15.3p15.1) transmitted through two generations defined by low coverage whole genome sequencing.

We report on a 14-year old boy, his father, and his paternal uncle, all three carriers of a duplication of chromosomal region 11p15.3-p15.1. The aberration was transmitted by the grandmother, who is carrier of a balanced insertion 46,XX,ins(14;11)(q32.1;p15.3p15.1). In order to determine the precise molecular basis of this structural variant, we performed low-coverage whole genome sequencing on the boy's father. This approach allowed precise determination of the genomic breakpoints and revealed a duplication of 6.9 Mb, centromeric to the Beckwith-Wiedemann/Silver-Russell syndrome critical region in 11p15.5, that inserted in inverse orientation into 14q32.12 (according to HGVS nomenclature: NC_000014.8:g.92871000_92871001ins[NC_000011.9:g.12250642_19165928inv;T]). To our knowledge, this is the first report of a duplication of 11p15.3-p15.1 involving more than 40 genes and transmitted through two generations without apparent clinical effects.

Paternal 132 bp deletion affecting KCNQ1OT1 in 11p15.5 is associated with growth retardation but does not affect imprinting.

BACKGROUND: The chromosomal region 11p15.5 harbours two imprinting centres (H19/IGF2:IG-DMR/IC1, KCNQ1OT1:TSS-DMR/IC2). Molecular alterations of the IC2 are associated with Beckwith-Wiedemann syndrome (BWS), whereas only single patients with growth retardation and Silver-Russell syndrome (SRS) features have been reported. CNVs in 11p15.5 account for less than 1% of patients with BWS and SRS, and they mainly consist of duplications of both ICs either affecting the maternal (SRS) or the paternal (BWS) allele. However, this correlation does not apply to smaller CNVs, which are associated with diverse clinical outcomes. METHODS AND RESULTS: We identified a family with a 132 bp deletion within the KCNQ1OT1 gene, associated with growth retardation in case of paternal transmission but a normal phenotype when maternally inherited. Comparison of molecular and clinical data with cases from the literature helped to delineate its functional relevance. CONCLUSION: Microdeletions within the paternal IC2 affecting the KCNQ1OT1 gene have been described in only five families, and they all include the differentially methylated region KCNQ1OT1:TSS-DMR/IC2 and parts of the KCNQ1 gene. However, these deletions have different impacts on the expression of both genes and the cell-cycle inhibitor CDKN1C. They thereby cause different phenotypes. The 132 bp deletion is the smallest deletion in the IC2 reported so far. It does not affect the IC2 methylation in general and the coding sequence of the KCNQ1 gene. Thus, the deletion is only associated with a growth retardation phenotype when paternally transmitted but not with other clinical features in case of maternal inheritance as observed for larger deletions.

A boy with Silver-Russell syndrome and Sotos syndrome.

Silver-Russell syndrome (SRS) is characterized by pre- and postnatal growth deficiency. It is most often caused by hypomethylation of the paternal imprinting center 1 of chromosome 11p15.5. In contrast, Sotos syndrome is an overgrowth syndrome that results either from pathogenic NSD1 gene variants or copy number variations affecting the NSD1 gene. Here, we report on a 6 month-old boy with severe short stature, relative macrocephaly, severe feeding difficulties with underweight, muscular hypotonia, motor delay, medullary nephrocalcinosis, bilateral sensorineural hearing impairment and facial dysmorphisms. SNP array revealed a 2.1 Mb de novo interstitial deletion of 5q35.2q35.3 encompassing the NSD1 gene. As Sotos syndrome could not satisfactorily explain his symptoms, diagnostic testing for SRS was initiated. It demonstrated hypomethylation of the imprinting center 1 of chromosome 11p15.5 confirming the clinically suspected SRS. We compared the symptoms of our patient with the typical clinical features of individuals with SRS and Sotos syndrome, respectively. To our knowledge, this is the first study reporting the very unusual coincidence of both Sotos syndrome and SRS in the same patient.

A paternally inherited 1.4 kb deletion of the 11p15.5 imprinting center 2 is associated with a mild familial Silver-Russell syndrome phenotype.

The Silver-Russell syndrome (SRS) is a rare disorder characterized by heterogeneous clinical features, including growth retardation, typical facial dysmorphisms, and body asymmetry. Genetic alterations causative of SRS mostly affect imprinted genes located on chromosomes 7 or 11. Hypomethylation of the Imprinting Center 1 (IC1) of the chromosome 11p15.5 is the most common cause of SRS, while the Imprinting Center 2 (IC2) has been more rarely involved. Specifically, maternally inherited 11p15.5 deletions including the IC2 have been associated with the Beckwith-Wiedemann Syndrome (BWS), while paternal deletions with a variable spectrum of phenotypes. Here, we describe the case of a girl with a mild SRS phenotype associated with a paternally inherited 1.4 kb deletion of IC2. The father of the proband inherited the deletion from his mother and showed normal growth, while the paternal grandmother had the deletion on her paternal chromosome and exhibited short stature. Together with previous findings obtained in mouse and humans, our data support the notion that deletion of the paternal copy of IC2 can cause SRS.

Heterogeneous phenotypes in families with duplications of the paternal allele within the imprinting center 1 (H19/IGF2:TSS-DMR) in 11p15.5.

The clinical impact of duplications affecting the 11p15.5 region is difficult to predict, and depends on the parent-of-origin of the affected allele as well as on the type (deletion, duplication), the extent and genomic content of the variant. Three unrelated families with inheritance of duplications affecting the IC1 region in 11p15.5 through two generations but different phenotypes (Beckwith-Wiedemann and Silver-Russell syndromes, normal phenotype) are reported. The inconsistent phenotypic patterns of carriers of the same variant strongly indicate the impact of cis- and/or trans-acting modifiers on the clinical outcome of IC1 duplication carriers.

Patient with an autosomal-recessive MBTPS1-linked phenotype and clinical features of Silver-Russell syndrome.

Pathogenic variants in the MBTPS1 gene encoding the Site 1 protease have been described so far only in one growth retarded patients with skeletal deformities, large ears, a triangular face reminiscent to Silver-Russell syndrome (SRS), and elevated blood lysosomal enzymes. We now report on the identification of a second adult patient homozygous for one of the two published pathogenic MBTPS1 variants (p.Asp365Gly) by Whole Exome Sequencing (WES), and a comparable phenotype. With this case, the association of pathogenic variants in MBTPS1 with a recognizable disorder could be confirmed, and the autosomal recessive inheritance is further established. As the variant was identified after a long diagnostic odyssey of the family, this example illustrates the need to apply WES in the diagnostic workup in case of growth retardation as early as possible. By compiling the clinical data of this new patient with those of the already reported patient, a better prognosis for future patients with MBTPS1 variants can be issued, and clinical management can be adjusted.

Maternal UPD of chromosome 7 in a patient with Silver-Russell syndrome and Pendred syndrome.

BACKGROUND: Silver-Russell syndrome (SRS) is a heterogeneous imprinting disorder featuring severe intrauterine and postnatal growth retardation and dysmorphic features. Pendred syndrome (PDS) is an autosomal recessive disorder caused by mutations in the SLC26A4 gene characterized by sensorineural hearing loss. METHODS: Karyotyping analysis was performed to investigate any chromosomal abnormalities. Whole-genome copy number variation and loss of heterozygosity were analyzed using an Affymetrix CytoScan 750 K Microarray. Variant screening was performed by targeted next-generation sequencing on all known deafness-causing genes. RESULTS: The proband was a patient with SRS caused by maternal uniparental disomy 7. The PDS of the proband was caused by homozygous variant c.919-2A > G of SLC26A4; both mutated alleles were inherited from his mother. CONCLUSION: This is the first report of uniparental disomy 7 leading to SRS and Pendred syndrome. Patients with intrauterine growth retardation or those born small for gestational age and exhibiting postnatal growth failure should undergo molecular testing to reach a clinical diagnosis.

Silver-Russell syndrome. Clinical and etiopathological aspects of a model genomic imprinting entity. / Síndrome de Silver-Russell. Aspectos clínicos y etiopatológicos de una entidad ejemplo de impronta genómica.

Silver-Russell syndrome is characterized by asymmetrical intrauterine growth retardation, with normal head circumference and small, pointed chin, which results in a triangular face. It can also include body asymmetry, among other characteristics. Its global incidence is estimated at 1 in 30 000-100 000 births, even though this figure may be underestimated. In approximately 60 % of cases, a molecular cause can be identified, and the main one is hypomethylation of the paternal allele at the imprinting control region 1 located at 11p15.5-p15.4. It is necessary to make the diagnosis of this entity, exclude differential diagnoses, and know (epi)genotype-phenotype correlations in order to ensure an adequate follow-up, provide available therapeutic options, and offer a timely family genetic counseling. The objective of this article is to describe the current status of the Silver-Russell syndrome, a model of genomic imprinting disorder.

El síndrome de Silver-Russell se caracteriza por retraso del crecimiento intrauterino asimétrico, con circunferencia craneal normal, barbilla pequeña y puntiaguda, que proporciona un aspecto de rostro triangular. Puede, además, presentar asimetría corporal, entre otros. Tiene una incidencia mundial estimada de 1 en 30 000- 100 000 nacimientos, aunque este número es, probablemente, subestimado. En alrededor del 60 % de los casos, se puede identificar una causa molecular y la principal es la hipometilación del alelo paterno en la región de control de impresión 1 localizado en 11p15.5-p15.4. Realizar el diagnóstico de esta entidad, excluir los diagnósticos diferenciales y conocer las correlaciones (epi)genotipo-fenotipo son necesarios para realizar el adecuado seguimiento, brindar las opciones terapéuticas disponibles y el oportuno asesoramiento genético familiar. El objetivo del presente artículo es mostrar el estado actual del síndrome de Silver-Russell, un ejemplo de trastorno de impronta genómica.

Phenotype of genetically confirmed Silver-Russell syndrome beyond childhood.

BACKGROUND: Silver-Russell syndrome is an imprinting disorder that restricts growth, resulting in short adult stature that may be ameliorated by treatment. Approximately 50% of patients have loss of methylation of the imprinting control region (H19/IGF2:IG-DMR) on 11p15.5 and 5%-10% have maternal uniparental disomy of chromosome 7. Most published research focuses on the childhood phenotype. Our aim was to describe the phenotypic characteristics of older patients with SRS. METHODS: A retrospective cohort of 33 individuals with a confirmed molecular diagnosis of SRS aged 13 years or above were carefully phenotyped. RESULTS: The median age of the cohort was 29.6 years; 60.6% had a height SD score (SDS) ≤-2 SDS despite 70% having received growth hormone treatment. Relative macrocephaly, feeding difficulties and a facial appearance typical of children with SRS were no longer discriminatory diagnostic features. In those aged ≥18 years, impaired glucose tolerance in 25%, hypertension in 33% and hypercholesterolaemia in 52% were noted. While 9/33 accessed special education support, university degrees were completed in 40.0% (>21 years). There was no significant correlation between quality of life and height SDS. 9/25 were parents and none of the 17 offsprings had SRS. CONCLUSION: Historical treatment regimens for SRS were not sufficient for normal adult growth and further research to optimise treatment is justified. Clinical childhood diagnostic scoring systems are not applicable to patients presenting in adulthood and SRS diagnosis requires molecular confirmation. Metabolic ill-health warrants further investigation but SRS is compatible with a normal quality of life including normal fertility in many cases.

upd(20)mat is a rare cause of the Silver-Russell-syndrome-like phenotype: Two unrelated cases and screening of large cohorts.

Silver-Russell syndrome (SRS) is an imprinting disorder characterized by prenatal and postnatal growth retardation, relative macrocephaly, feeding difficulties and body asymmetry. Recently, upd(20)mat has been identified in few patients with SRS-like features, suggestive of a new imprinting disorder characterized by prenatal and postnatal growth failure. Here, we describe two male patients with upd(20) and feeding difficulties, prenatal and postnatal growth retardation and normal cognitive development. During pregnancy, confined placental mosaicism for trisomy 20 was detected in one of the patients but was not investigated further until identification of upd(20)mat in the neonatal period. To evaluate whether upd(20)mat should be part of the first trier genetic diagnostic in patients with growth retardation, we screened a large cohort of patients (n = 673) referred to our laboratories for SRS-testing without detecting any upd(20). Our results, along with the existing evidence, indicate that upd(20)mat is a very rare cause of growth retardation, but should be followed up when confined placental mosaicism for trisomy 20 mosaicism is observed during pregnancy.

IGF2 Mutations.

OBJECTIVE: IGF2 is a paternally expressed growth-promoting gene. Here, we report five cases with IGF2 mutations and review IGF2 mutation-positive patients described in the literature. We also compare clinical features between patients with IGF2 mutations and those with H19/IGF2:IG-DMR epimutations. RESULTS: We recruited five cases with IGF2 mutations: case 1 with a splice site mutation (c.-6-1G>C) leading to skipping of exon 2 and cases 2-5 with different missense mutations (p.(Cys70Tyr), p.(Cys71Arg), p.(Cys33Ser), and p.(Cys45Ser)) affecting cysteine residues involved in the S-S bindings. All the mutations resided on the paternally inherited allele, and the mutation of case 5 was present in a mosaic condition. Clinical assessment revealed Silver-Russell syndrome (SRS) phenotype with Netchine-Harbison scores of ≥5/6 in all the apparently nonmosaic 14 patients with IGF2 mutations (cases 1-4 described in this study and 10 patients reported in the literature). Furthermore, compared with H19/IGF2:IG-DMR epimutations, IGF2 mutations were associated with low frequency of hemihypoplasia, high frequency of feeding difficulty and/or reduced body mass index, and mild degree of relative macrocephaly, together with occasional development of severe limb malformations, high frequency of cardiovascular anomalies and developmental delay, and low serum IGF-II values. CONCLUSIONS: This study indicates that IGF2 mutations constitute a rare but important cause of SRS. Furthermore, while both IGF2 mutations and H19/IGF2:IG-DMR epimutations lead to SRS, a certain degree of phenotypic difference is observed between the two groups, probably due to the different IGF2 expression pattern in target tissues.

Contribution of GRB10 to the prenatal phenotype in Silver-Russell syndrome? Lessons from 7p12 copy number variations.

The growth factor binding protein 10 (GRB10) has been suggested as a candidate gene for Silver-Russell syndrome because of its localization in 7p12, its imprinting status, data from mice models and its putative role in growth. Based on a new patient with normal growth carrying a GRB10 deletion affecting the paternal allele and data from the literature, we conclude that the heterogeneous clinical findings in patients with copy number variations (CNVs) of GRB10 gene depend on the size and the gene content of the CNV. However, evidence from mouse and human cases indicate a growth suppressing role of GRB10 in prenatal development. As a result, an increase of active maternal GRB10 copies, e.g. by maternal uniparental disomy of chromosome 7 or duplications of the region results in intrauterine growth retardation. In contrast, a defective GRB10 copy might result in prenatal overgrowth, whereas the paternal GRB10 allele is not required for proper prenatal growth.

The role of long non-coding RNAs in the pathogenesis of hereditary diseases.

BACKGROUND: Thousands of long non-coding RNA (lncRNA) genes are annotated in the human genome. Recent studies showed the key role of lncRNAs in a variety of fundamental cellular processes. Dysregulation of lncRNAs can drive tumorigenesis and they are now considered to be a promising therapeutic target in cancer. However, how lncRNAs contribute to the development of hereditary diseases in human is still mostly unknown. RESULTS: This review is focused on hereditary diseases in the pathogenesis of which long non-coding RNAs play an important role. CONCLUSIONS: Fundamental research in the field of molecular genetics of lncRNA is necessary for a more complete understanding of their significance. Future research will help translate this knowledge into clinical practice which will not only lead to an increase in the diagnostic rate but also in the future can help with the development of etiotropic treatments for hereditary diseases.

Could a chimeric condition be responsible for unexpected genetic syndromes? The role of the single nucleotide polymorphism-array analysis.

In this paper, is reported the identification of two chimeric patients, a rare finding if sexual abnormalities are absent. However, their chimeric condition is responsible at least for the Silver-Russell phenotype observed in one of the two patients. By single nucleotide polymorphism-array analyses, it was possible to clearly define the mechanism responsible for this unusual finding, underlining the importance of this technique in bringing out the perhaps submerged world of chimeras.

Severe in utero under-virilization in a 46,XY patient with Silver-Russell syndrome with 11p15 loss of methylation.

Background Silver-Russell syndrome (SRS) is characterized by growth retardation and variable features including macrocephaly, body asymmetry, and genital manifestations such as cryptorchidism in 46,XY patients. Case presentation The patient was born at 39 weeks with a birth weight of 1344 g. Subtle clitoromegaly warranted a thorough evaluation, which disclosed 46,XY karyotype, bilateral undescended testes, and a rudimentary uterus. Because of severe under-virilization, the patient was assigned as female. Failure to thrive, macrocephaly, and body asymmetry led to the diagnosis of SRS, confirmed by marked hypomethylation of H19/IGF2 intergenic differentially methylated region (IG-DMR). From age 9 years, progressive virilization occurred, which necessitated luteinizing hormone-releasing hormone analog (LHRHa) treatment. Gonadal resection at 15 years revealed immature testes with mostly Sertoli-cell-only tubules. Panel analysis for 46,XY-differences of sex development (DSD) failed to detect any pathogenic variants. Conclusions This is the second reported case of molecularly proven 46,XY SRS accompanied by severe under-virilization. SRS should be included in the differential diagnosis of 46,XY-DSD.

Search for altered imprinting marks in Mayer-Rokitansky-Küster-Hauser patients.

BACKGROUND: Mayer-Rokitansky-Küster-Hauser syndrome (MRKH) is the second most common cause of primary amenorrhea and characterized by absence of the uterus and the upper part of the vagina. The etiology of MRKH is mainly unknown but a contribution of genomic alterations is probable. A molecular disturbance so far neglected in MRKH research is aberrant methylation at imprinted loci. In fact, MRKH has been reported in patients with the imprinting disorder Silver-Russell syndrome. METHODS: We report on a rare patient with MRKH and SRS due to an ICR1 hypomethylation in 11p15.5. On the basis of this observation we screened a large cohort of MRKH patients (n > 100) for aberrant methylation at nine imprinted loci. RESULTS: We failed to detect any epimutation, thus we conclude that imprinting defects at least at the currently known disease-relevant imprinted loci do not contribute to the isolated MRKH phenotype. However, it cannot be excluded that altered methylation marks at other loci are involved in the etiology of MRKH. CONCLUSION: The molecular basis for MRKH remains unclear in the majority of patients, but future studies on the association between MRKH and ICR1 hypomethylation/SRS will to enlighten the role of epigenetics in the etiology of MRKH.

12q14 microdeletion syndrome: A family with short stature and Silver-Russell syndrome (SRS)-like phenotype and review of the literature.

We report here on the first family with short stature and Silver-Russell-like phenotype due to a microdeletion in 12q14.3. The Netchine-Harbison clinical scoring system was used for the clinical diagnosis of Silver-Russell syndrome (SRS). The three affected first-degree relatives (index patient, mother and brother) presented with prenatal and postnatal growth retardation, feeding difficulties, a prominent forehead and a failure to thrive, but did not show relative macrocephaly. In addition, our index patient showed dysmorphic facial features, periodically increased sweating, and scoliosis. Learning problems and cardiac arrhythmia presented as additional features of her brother. Using high-resolution array-CGH, heterozygosity for a 1.67 Mb deletion in 12q14.3 was detected in the index patient. The heterozygous loss was confirmed by MLPA in the index patient and the other two affected family members. The deletion includes the genes HMGA2, LLPH, TMBIM4, IRAK3, HELB, GRIP1, and the pseudogene RPSAP52. We conclude from these results and from the data of other patients reported in the literature that haploinsufficiency of HMGA2 leads to the short stature in this family.

Chromosomal rearrangements in the 11p15 imprinted region: 17 new 11p15.5 duplications with associated phenotypes and putative functional consequences.

BACKGROUND: The 11p15 region contains two clusters of imprinted genes. Opposite genetic and epigenetic anomalies of this region result in two distinct growth disturbance syndromes: Beckwith-Wiedemann (BWS) and Silver-Russell syndromes (SRS). Cytogenetic rearrangements within this region represent less than 3% of SRS and BWS cases. Among these, 11p15 duplications were infrequently reported and interpretation of their pathogenic effects is complex. OBJECTIVES: To report cytogenetic and methylation analyses in a cohort of patients with SRS/BWS carrying 11p15 duplications and establish genotype/phenotype correlations. METHODS: From a cohort of patients with SRS/BWS with an abnormal methylation profile (using ASMM-RTQ-PCR), we used SNP-arrays to identify and map the 11p15 duplications. We report 19 new patients with SRS (n=9) and BWS (n=10) carrying de novo or familial 11p15 duplications, which completely or partially span either both telomeric and centromeric domains or only one domain. RESULTS: Large duplications involving one complete domain or both domains are associated with either SRS or BWS, depending on the parental origin of the duplication. Genotype-phenotype correlation studies of partial duplications within the telomeric domain demonstrate the prominent role of IGF2, rather than H19, in the control of growth. Furthermore, it highlights the role of CDKN1C within the centromeric domain and suggests that the expected overexpression of KCNQ1OT1 from the paternal allele (in partial paternal duplications, excluding CDKN1C) does not affect the expression of CDKN1C. CONCLUSIONS: The phenotype associated with 11p15 duplications depends on the size, genetic content, parental inheritance and imprinting status. Identification of these rare duplications is crucial for genetic counselling.