Paternally Inherited IGF2 Mutation and Growth Restriction.

In humans, mutations in IGF1 or IGF1R cause intrauterine and postnatal growth restriction; however, data on mutations in IGF2, encoding insulin-like growth factor (IGF) II, are lacking. We report an IGF2 variant (c.191C→A, p.Ser64Ter) with evidence of pathogenicity in a multigenerational family with four members who have growth restriction. The phenotype affects only family members who have inherited the variant through paternal transmission, a finding that is consistent with the maternal imprinting status of IGF2. The severe growth restriction in affected family members suggests that IGF-II affects postnatal growth in addition to prenatal growth. Furthermore, the dysmorphic features of affected family members are consistent with a role of deficient IGF-II levels in the cause of the Silver-Russell syndrome. (Funded by Bundesministerium für Bildung und Forschung and the European Union.).

NLRP genes and their role in preeclampsia and multi-locus imprinting disorders.

Preeclampsia (PE) affects 2-5% of all pregnancies. It is a multifactorial disease, but it has been estimated that 35% of the variance in liability of PE are attributable to maternal genetic effects and 20% to fetal genetic effects. PE has also been reported in women delivering children with Beckwith-Wiedemann syndrome (BWS, OMIM 130650), a disorder associated with aberrant methylation at genomically imprinted loci. Among others, members of the NLRP gene family are involved in the etiology of imprinting defects. Thus, a functional link between PE, NLRP gene mutations and aberrant imprinting can be assumed. Therefore we analyzed a cohort of 47 PE patients for NLRP gene mutations by next generation sequencing. In 25 fetuses where DNA was available we determined the methylation status at the imprinted locus. With the exception of one woman heterozygous for a missense variant in the NLRP7 gene (NM_001127255.1(NLRP7):c.542G>C) we could not identify further carriers, in the fetal DNA normal methylation patterns were observed. Thus, our negative screening results in a well-defined cohort indicate that NLRP mutations are not a relevant cause of PE, though strong evidence for a functional link between NLRP mutations, PE and aberrant methylation exist.

Targeted Next Generation Sequencing Approach in Patients Referred for Silver-Russell Syndrome Testing Increases the Mutation Detection Rate and Provides Decisive Information for Clinical Management.

OBJECTIVE: To investigate the contribution of differential diagnoses to the mutation spectrum of patients referred for Silver-Russell syndrome (SRS) testing. STUDY DESIGN: Forty-seven patients referred for molecular testing for SRS were examined after exclusion of one of the SRS-associated alterations. After clinical classification, a targeted next generation sequencing approach comprising 25 genes associated with other diagnoses or postulated as SRS candidate genes was performed. RESULTS: By applying the Netchine-Harbinson clinical scoring system, indication for molecular testing for SRS was confirmed in 15 out of 47 patients. In 4 out of these 15 patients, disease-causing variants were found in genes associated with other diagnoses. These patients carried mutations associated with Bloom syndrome, Mulibrey nanism, KBG syndrome, or IGF1R-associated short stature. We could not detect any pathogenic mutation in patients with a negative clinical score. CONCLUSIONS: Some of the differential diagnoses detected in the cohort presented here have a major impact on clinical management. Therefore, we emphasize that the molecular defects associated with these clinical pictures should be excluded before the clinical diagnosis "SRS" is made. Finally, we could show that a broad molecular approach including the differential diagnoses of SRS increases the detection rate.

Examinations of maternal uniparental disomy and epimutations for chromosomes 6, 14, 16 and 20 in Silver-Russell syndrome-like phenotypes.

BACKGROUND: Silver-Russell syndrome (SRS) is a growth retardation disorder with a very broad molecular and clinical spectrum. Whereas the association of SRS with imprinting disturbances of chromosomes 11p15.5 and 7 is generally accepted, there are controversial discussions on the involvement of other molecular changes. The recent reports on the occurrence of maternal uniparental disomies of chromosomes 6, 16 and 20 (upd(6, 16, 20)mat), as well as 14q32 imprint alterations in patients with SRS phenotypes raise the question on the involvement of these mutations in the etiology of SRS. METHODS: A cohort of 54 growth retarded patients with SRS features was screened for aberrant methylation patterns of chromsomes 6, 14, 16 and 20. RESULTS: One carrier of a 14q32 epimutation was identified whereas epimutations and maternal UPD for chromosomes 6, 16 and 20 were excluded. CONCLUSIONS: Our data and those from the literature confirm that 14q32 disturbances significantly contribute to the mutation spectrum in this cohort. Furthermore, maternal uniparental disomy of chromosomes 6, 16 and 20 can be observed, but are rare. In case they occur they can be regarded as causative for clinical features.

Microdeletions of the 7q32.2 imprinted region are associated with Silver-Russell syndrome features.

The association of maternal uniparental disomy of human chromosome 7 (upd(7) mat) and the growth retardation disorder Silver-Russell syndrome (SRS) is well established, but the causative gene or region is currently unknown. However, several observations indicate that molecular alterations of the genomically imprinted MEST region in 7q32.2 are associated with growth retardation and a phenotype reminiscent to SRS. We now report on a second patient with a similar phenotype and a de novo 7q32.2 microdeletion including MEST affecting the paternal allele. This confirms the central role of imprinted genes in 7q32.2 in the etiology of a growth retardation phenotype associated with SRS features.

A familial GLI2 deletion (2q14.2) not associated with the holoprosencephaly syndrome phenotype.

Molecular alterations of the GLI2 gene in 2q14.2 are associated with features from the holoprosencephaly spectrum. However, the phenotype is extremely variable, ranging from unaffected mutation heterozygotes to isolated or combined pituitary hormone deficiency, and to patients with a phenotype that overlaps with holoprosencephaly, including abnormal pituitary gland formation/function, craniofacial dysmorphisms, branchial arch anomalies, and polydactyly. Although many point mutations within the GLI2 gene have been identified, large (sub) microscopic deletions affecting 2q14.2 are rare. We report on a family with a 4.3 Mb deletion in 2q14 affecting GLI2 without any dysmorphologic features belonging to the holoprosencephaly spectrum. This family confirms the incomplete penetrance of genomic disturbances affecting the GLI2 gene. However, the family presented here is unique as none of the three identified individuals with a GLI2 deletion showed any typical signs of holoprosencephaly, whereas all patients reported so far were referred for genetic testing because at least one member exhibited holoprosencephaly and related features.

Congenital imprinting disorders: Application of multilocus and high throughput methods to decipher new pathomechanisms and improve their management.

Imprinting disorders (IDs) are a group of congenital diseases affecting growth, development and metabolism. They are caused by changes in the allele-specific regulation ("epigenetic mutation") or in the genomic sequence ("genetic mutation") of imprinted genes. Currently molecular tests in ID patients are generally restricted to single loci classically associated with the disease, but this approach limits diagnostic yield, because of the molecular and clinical heterogeneity between IDs. From the technical point of view, these limitations are aggravated by the lack of standardization in testing methodology, in the DNA sequences tested, and in clinical inclusion criteria prompting testing. However, an increasing number of new studies show that these problems can be addressed by the use of new tests targeting multiple loci and/or a total exome and genome analysis. The rapid development of efficient and high-throughput molecular techniques and their applications in research and diagnostics in the last decade have led to an impressive increase of knowledge on IDs and their basic pathomechanisms. In combination with the improvement of data recording and documentation, the diagnostic strategies are increasingly based on standardized protocols, and thereby provide the backbone for directed counseling, more personalized management, and new therapeutic approaches.

Search for cis-acting factors and maternal effect variants in Silver-Russell patients with ICR1 hypomethylation and their mothers.

Silver-Russell syndrome is an imprinting disorder characterized by severe intrauterine and postnatal growth retardation. The majority of patients show loss of methylation (LOM) of the H19/IGF2 IG-DMR (ICR1) in 11p15.5. In ~10% of these patients aberrant methylation of additional imprinted loci on other chromosomes than 11 can be observed (multilocus imprinting defect - MLID). Recently, genomic variations in the ICR1 have been associated with disturbed methylation of the ICR1. In addition, variants in factors contributing to the life cycle of imprinting are discussed to cause aberrant imprinting, including MLID. These variants can either be identified in the patients with imprinting disorders themselves or in their mothers. We performed comprehensive studies to elucidate the role of both cis-acting variants in 11p15.5 as well as of maternal effect variants in the etiology of ICR1 LOM. Whereas copy number analysis and next generation sequencing in the ICR1 did not provide any evidence for a variant, search for maternal effect variants in 21 mothers of patients with ICR1 LOM identified two carriers of NLRP5 variants. By considering our results as well as those from the literature, we conclude that the causes for epimutations are heterogeneous. MLID might be regarded as an own etiological subgroup, associated with maternal effect variants in NLRP and functionally related genes. In addition, these variants might also contribute to LOM of single imprinted loci. Furthermore, genomic variants in the patients themselves might result in aberrant methylation patterns and need further investigation.

De Novo Duplication of 11p15 Associated With Congenital Diaphragmatic Hernia.

Background: Congenital diaphragmatic hernia (CDH) is a rare defect of the diaphragm commonly associated with high morbidity and mortality due to lung hypoplasia and pulmonary hypertension. Although in 70% of patients the etiology of a CDH remains unknown, a multitude of causative chromosomal aberrations has been identified. Case presentation: We describe the first case of isolated 11p15 duplication with CDH. The 18.6 Mb large duplication affected 285 RefSeq genes and included the Beckwith-Wiedemann (BWS)-associated imprinting control region 2 (ICR2, KCNQ1OT1 TSS DMR), whereas the ICR1 (H19 TSS DMR) was not affected. We were able to demonstrate de novo occurrence of the duplication. The paternal origin of the chromosomal material was detected by methylation testing the ICR2. Corresponding to other patients with duplications of the paternal ICR2 copy, a BWS phenotype is not present. Conclusions: The patient presented here together with the review of four other cases from the literature indicate an association between duplications of the chromosomal region 11p15 and developmental defects of the diaphragm. Thus, we suggest duplications of 11p15 as a rare cause of CDH. This association may or may not appear in the context of BWS depending on the extent of the duplication and the imprinting status. Hence, a genetic workup should be performed in patients with CDH, particularly when other abnormalities are noted.

Maternal heterozygous NLRP7 variant results in recurrent reproductive failure and imprinting disturbances in the offspring.

It has been shown previously that homozygous and compound-heterozygous variants affecting protein function in the human NLRP genes impact reproduction and/or fetal imprinting patterns. These variants represent so-called 'maternal effect mutations', that is, although female variant carriers are healthy, they are at risk of reproductive failure, and their offspring may develop aberrant methylation and imprinting disorders. In contrast, the relevance to reproductive failure of maternal heterozygous NLRP7 variants remains unclear. The present report describes the identification of a heterozygous NLRP7 variant in a healthy 28-year-old woman with a history of recurrent reproductive failure, and the molecular findings in two of the deceased offspring. Next-generation sequencing (NGS) for NLRP variants was performed. In the tissues of two offspring (one fetus; one deceased premature neonate) methylation of imprinted loci was tested using methylation-specific assays. Both pregnancies had been characterized by the presence of elevated human chorionic gonadotropin (hCG) levels and ovarian cysts. In the mother, a heterozygous nonsense 2-bp deletion in exon 5 of the NLRP7 gene was identified (NM_001127255.1:c.2010_2011del, p.(Phe671Glnfs*18)). In the two investigated offspring, heterogeneous aberrant methylation patterns were detected at imprinted loci. The present data support the hypothesis that heterozygous NLRP7 variants contribute to reproductive wastage, and that these variants represent autosomal dominant maternal effect variants which lead to aberrant imprinting marks in the offspring. Specific screening and close prenatal monitoring of NLRP7 variant carriers is proposed. Egg donation might facilitate successful pregnancy in heterozygous NLRP7 variant carriers.

First report on concordant monozygotic twins with Silver-Russell syndrome and ICR1 hypomethylation.

Twin pairs with the imprinting disorder Silver-Russell syndrome (SRS) have rarely been reported. All six monozygotic (MZ) twin pairs described so far were clinically discordant. In two of the four SRS twin pairs with molecularly proven 11p15.5 epimutation, the healthy twin also showed the molecular alteration in blood cells, but not in the other tested tissues. The clinical discordance is a well-known but poorly understood observation because MZ twins derive from the same zygote. For the second 11p15.5-associated imprinting disorder, Beckwith-Wiedemann syndrome, a larger number of twins has been described, here the majority of pairs are MZ but clinically discordant as well. Interestingly, there is a considerable preponderance of females among the MZ twins with BWS, and a functional link between altered imprinting and X chromosome inactivation has been suggested. We now describe two further MZ SRS twins with H19/IGF2:IG-DMR hypomethylation, including the first clinically concordant pair. By summarizing the existing data, an excess of females in MZ twins with SRS is observed, thus confirming the hypothesis that X-chromosome inactivation might trigger the inaccurate methylation of imprinted loci at least in female twin conceptions. The occurrence of a MZ concordant SRS twin pair is exceptional. The detailed molecular characterization of both siblings of a twin pair enables a reliable diagnosis, furthermore it allows insights in the etiology of twinning in association with (aberrant) imprinting marking.

Phenotypic spectrum and extent of DNA methylation defects associated with multilocus imprinting disturbances.

AIM: To characterize the genotypic and phenotypic extent of multilocus imprinting disturbances (MLID). MATERIALS & METHODS: We analyzed 37 patients with imprinting disorders (explorative cohort) for DNA methylation changes using the Infinium HumanMethylation450 BeadChip. For validation, three independent cohorts with imprinting disorders or cardinal features thereof were analyzed (84 patients with imprinting disorders, 52 with growth disorder, 81 with developmental delay). RESULTS: In the explorative cohort 21 individuals showed array-based MLID with each one displaying an Angelman or Temple syndrome phenotype, respectively. Epimutations in ZDBF2 and FAM50B were associated with severe MLID regarding number of affected regions. By targeted analysis we identified methylation changes of ZDBF2 and FAM50B also in the three validation cohorts. CONCLUSION: We corroborate epimutations in ZDBF2 and FAM50B as frequent changes in MLID whereas these rarely occur in other patients with cardinal features of imprinting disorders. Moreover, we show cell lineage specific differences in the genomic extent of FAM50B epimutation.

EMQN best practice guidelines for the molecular genetic testing and reporting of chromosome 11p15 imprinting disorders: Silver-Russell and Beckwith-Wiedemann syndrome.

Molecular genetic testing for the 11p15-associated imprinting disorders Silver-Russell and Beckwith-Wiedemann syndrome (SRS, BWS) is challenging because of the molecular heterogeneity and complexity of the affected imprinted regions. With the growing knowledge on the molecular basis of these disorders and the demand for molecular testing, it turned out that there is an urgent need for a standardized molecular diagnostic testing and reporting strategy. Based on the results from the first external pilot quality assessment schemes organized by the European Molecular Quality Network (EMQN) in 2014 and in context with activities of the European Network of Imprinting Disorders (EUCID.net) towards a consensus in diagnostics and management of SRS and BWS, best practice guidelines have now been developed. Members of institutions working in the field of SRS and BWS diagnostics were invited to comment, and in the light of their feedback amendments were made. The final document was ratified in the course of an EMQN best practice guideline meeting and is in accordance with the general SRS and BWS consensus guidelines, which are in preparation. These guidelines are based on the knowledge acquired from peer-reviewed and published data, as well as observations of the authors in their practice. However, these guidelines can only provide a snapshot of current knowledge at the time of manuscript submission and readers are advised to keep up with the literature.

Prenatal molecular testing for Beckwith-Wiedemann and Silver-Russell syndromes: a challenge for molecular analysis and genetic counseling.

Beckwith-Wiedemann and Silver-Russell syndromes (BWS/SRS) are two imprinting disorders (IDs) associated with disturbances of the 11p15.5 chromosomal region. In BWS, epimutations and genomic alterations within 11p15.5 are observed in >70% of patients, whereas in SRS they are observed in about 60% of the cases. In addition, 10% of the SRS patients carry a maternal uniparental disomy of chromosome 7 11p15.5. There is an increasing demand for prenatal testing of these disorders owing to family history, indicative prenatal ultrasound findings or aberrations involving chromosomes 7 and 11. The complex molecular findings underlying these disorders are a challenge not only for laboratories offering these tests but also for geneticists counseling affected families. The scope of counseling must consider the range of detectable disturbances and their origin, the lack of precise quantitative knowledge concerning the inheritance and recurrence risks for the epigenetic abnormalities, which are hallmarks of these developmental disorders. In this paper, experts in the field of BWS and SRS, including members of the European network of congenital IDs (EUCID.net; www.imprinting-disorders.eu), put together their experience and work in the field of 11p15.5-associated IDs with a focus on prenatal testing. Altogether, prenatal tests of 160 fetuses (122 referred for BWS, 38 for SRS testing) from 5 centers were analyzed and reviewed. We summarize the current knowledge on BWS and SRS with respect to diagnostic testing, the consequences for prenatal genetic testing and counseling and our cumulative experience in dealing with these disorders.

Mosaicism and uniparental disomy in prenatal diagnosis.

Chromosomal mosaicism is the presence of numerous cell lines with different chromosomal complements in the same individual. Uniparental disomy (UPD) is the inheritance of two homologous chromosomes from the same parent. These genetic anomalies arise from errors in meiosis and/or mitosis and can occur independently or in combination. Due to the formation mechanisms of UPD, low-level or undetected mosaicisms are assumed for a significant number of UPD cases. The pre- and postnatal clinical consequences of mosaicism for chromosomal aberrations and/or UPD depend on the gene content of the involved chromosome. In prenatal evaluation of chromosomal mosaicism and UPD, genetic counseling should be offered before any laboratory testing.

The Frequency of Methylation Abnormalities Among Estonian Patients Selected by Clinical Diagnostic Scoring Systems for Silver-Russell Syndrome and Beckwith-Wiedemann Syndrome.

AIMS: To study the frequency of methylation abnormalities among Estonian patients selected according to published clinical diagnostic scoring systems for Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS). MATERIALS AND METHODS: Forty-eight patients with clinical suspicion of SRS (n = 20) or BWS (n = 28) were included in the study group, to whom methylation-specific multiplex ligation-dependant probe amplification analysis of 11p15 region was made. In addition, to patients with minimal diagnostic score for either SRS or BWS, multilocus methylation-specific single nucleotide primer extension assay was performed. RESULTS: Five (38%) SRS patients with positive clinical scoring had abnormal methylation pattern at chromosome 11p15, whereas in the BWS group, only one patient was diagnosed with imprinting control region 2 (ICR2) hypomethylation (8%). An unexpected hypomethylation of the PLAGL1 (6q24) and IGF2R (6q25) genes in the patient with the highest BWS scoring was found. CONCLUSIONS: Compared to BWS, diagnostic criteria used for selecting SRS patients gave us a similar detection rate of 11p15 imprinting disorders as seen in other studies. A more careful selection of patients with possible BWS should be considered to improve the detection of molecularly confirmed cases. Genome-wide multilocus methylation tests could be used in routine clinical practice as it increases the detection rates of imprinting disorders.

Mutations in NLRP5 are associated with reproductive wastage and multilocus imprinting disorders in humans.

Human-imprinting disorders are congenital disorders of growth, development and metabolism, associated with disturbance of parent of origin-specific DNA methylation at imprinted loci across the genome. Some imprinting disorders have higher than expected prevalence of monozygotic twinning, of assisted reproductive technology among parents, and of disturbance of multiple imprinted loci, for which few causative trans-acting mutations have been found. Here we report mutations in NLRP5 in five mothers of individuals affected by multilocus imprinting disturbance. Maternal-effect mutations of other human NLRP genes, NLRP7 and NLRP2, cause familial biparental hydatidiform mole and multilocus imprinting disturbance, respectively. Offspring of mothers with NLRP5 mutations have heterogenous clinical and epigenetic features, but cases include a discordant monozygotic twin pair, individuals with idiopathic developmental delay and autism, and families affected by infertility and reproductive wastage. NLRP5 mutations suggest connections between maternal reproductive fitness, early zygotic development and genomic imprinting.

Additional molecular findings in 11p15-associated imprinting disorders: an urgent need for multi-locus testing.

UNLABELLED: The chromosomal region 11p15 contains two imprinting control regions (ICRs) and is a key player in molecular processes regulated by genomic imprinting. Genomic as well as epigenetic changes affecting 11p15 are associated either with Silver-Russell syndrome (SRS) or Beckwith-Wiedemann syndrome (BWS). In the last years, a growing number of patients affected by imprinting disorders (IDs) have reported carrying the disease-specific 11p15 hypomethylation patterns as well as methylation changes at imprinted loci at other chromosomal sites (multi-locus methylation defects, MLMD). Furthermore, in several patients, molecular alterations (e.g., uniparental disomies, UPDs) additional to the primary epimutations have been reported. To determine the frequency and distribution of mutations and epimutations in patients referred as SRS or BWS for genetic testing, we retrospectively ascertained our routine patient cohort consisting of 711 patients (SRS, n = 571; BWS, n = 140). As this cohort represents the typical cohort in a routine diagnostic lab without clinical preselection, the detection rates were much lower than those reported from clinically characterized cohorts in the literature (SRS, 19.9%; BWS, 28.6%). Among the molecular subgroups known to be predisposed to MLMD, the frequencies corresponded to that in the literature (SRS, 7.1% in ICR1 hypomethylation carriers; BWS, 20.8% in ICR2 hypomethylation patients). In several patients, more than one epigenetic or genetic disturbance could be identified. Our study illustrates that the complex molecular alterations as well as the overlapping and sometimes unusual clinical findings in patients with imprinting disorders (IDs) often make the decision for a specific imprinting disorder test difficult. We therefore suggest to implement molecular assays in routine ID diagnostics which allow the detection of a broad range of (epi)mutation types (epimutations, UPDs, chromosomal imbalances) and cover the clinically most relevant known ID loci because of the following: (a) Multi-locus tests increase the detection rates as they cover numerous loci. (b) Patients with unexpected molecular alterations are detected. (c) The testing of rare imprinting disorders becomes more efficient and quality of molecular diagnosis increases. (d) The tests identify MLMDs. In the future, the detailed characterization of clinical and molecular findings in ID patients will help us to decipher the complex regulation of imprinting and thereby providing the basis for more directed genetic counseling and therapeutic managements in IDs. KEY MESSAGE: Molecular disturbances in patients with imprinting disorders are often not restricted to the disease-specific locus but also affect other chromosomal regions. These additional disturbances include methylation defects, uniparental disomies as well as chromosomal imbalances. The identification of these additional alterations is mandatory for a well-directed genetic counseling. Furthermore, these findings help to decipher the complex regulation of imprinting.