Multi-locus imprinting disturbance (MLID): interim joint statement for clinical and molecular diagnosis.

BACKGROUND: Imprinting disorders are rare diseases resulting from altered expression of imprinted genes, which exhibit parent-of-origin-specific expression patterns regulated through differential DNA methylation. A subgroup of patients with imprinting disorders have DNA methylation changes at multiple imprinted loci, a condition referred to as multi-locus imprinting disturbance (MLID). MLID is recognised in most but not all imprinting disorders and is also found in individuals with atypical clinical features; the presence of MLID often alters the management or prognosis of the affected person. Some cases of MLID are caused by trans-acting genetic variants, frequently not in the patients but their mothers, which have counselling implications. There is currently no consensus on the definition of MLID, clinical indications prompting testing, molecular procedures and methods for epigenetic and genetic diagnosis, recommendations for laboratory reporting, considerations for counselling, and implications for prognosis and management. The purpose of this study is thus to cover this unmet need. METHODS: A comprehensive literature search was conducted resulting in identification of more than 100 articles which formed the basis of discussions by two working groups focusing on clinical diagnosis (n = 12 members) and molecular testing (n = 19 members). Following eight months of preparations and regular online discussions, the experts from 11 countries compiled the preliminary documentation and determined the questions to be addressed during a face-to-face meeting which was held with the attendance of the experts together with four representatives of patient advocacy organisations. RESULTS: In light of available evidence and expert consensus, we formulated 16 propositions and 8 recommendations as interim guidance for the clinical and molecular diagnosis of MLID. CONCLUSIONS: MLID is a molecular designation, and for patients with MLID and atypical phenotypes, we propose the alternative term multi-locus imprinting syndrome. Due to the intrinsic variability of MLID, the guidelines underscore the importance of involving experts from various fields to ensure a confident approach to diagnosis, counselling, and care. The authors advocate for global, collaborative efforts in both basic and translational research to tackle numerous crucial questions that currently lack answers, and suggest reconvening within the next 3-5 years to evaluate the research advancements and update this guidance as needed.

The maternal protein NLRP5 stabilizes UHRF1 in the cytoplasm: implication for the pathogenesis of multilocus imprinting disturbance.

We have recently discovered that the so-called subcortical maternal complex (SCMC) proteins composing of cytoplasmic lattices are destabilized in Uhrf1 knockout murine fully grown oocytes (FGOs). Here we report that human UHRF1 interacts with human NLRP5 and OOEP, which are core components of the SCMC. Moreover, NLRP5 and OOEP interact with DPPA3, which is an essential factor for exporting UHRF1 from the nucleus to the cytoplasm in oocytes. We identify that NLRP5, not OOEP, stabilizes UHRF1 protein in the cytoplasm utilizing specifically engineered cell lines mimicking UHRF1 status in oocytes and preimplantation embryos. Further, UHRF1 is destabilized both in the cytoplasm and nucleus of Nlrp5 knockout murine FGOs. Since pathogenic variants of the SCMC components frequently cause multilocus imprinting disturbance and UHRF1 is essential for maintaining CpG methylation of imprinting control regions during preimplantation development, our results suggest possible pathogenesis behind the disease, which has been a long-standing mystery.

NLRP7 variants in spontaneous abortions with multilocus imprinting disturbances from women with recurrent pregnancy loss.

PURPOSE: Comparative analysis of multilocus imprinting disturbances (MLIDs) in miscarriages from women with sporadic (SPL) and recurrent pregnancy loss (RPL) and identification of variants in the imprinting control gene NLRP7 that may lead to MLIDs. METHODS: Chorionic cytotrophoblast and extraembryonic mesoderm samples from first-trimester miscarriages were evaluated in 120 women with RPL and 134 women with SPL; 100 induced abortions were analyzed as a control group. All miscarriages had a normal karyotype. Epimutations in 7 imprinted genes were detected using methyl-specific PCR and confirmed with DNA pyrosequencing. Sequencing of all 13 exons and adjusted intron regions of the NLRP7 gene was performed. RESULTS: Epimutations in imprinted genes were more frequently detected (p < 0.01) in the placental tissues of miscarriages from women with RPL (7.1%) than in those of women with SPL (2.7%). The predominant epimutation was postzygotic hypomethylation of maternal alleles of imprinted genes (RPL, 5.0%; SPL, 2.1%; p < 0.01). The frequency of MLID was higher among miscarriages from women with RPL than among miscarriages from women with SPL (1.7% and 0.4%, respectively, p < 0.01). Variants in NLRP7 were detected only in miscarriages from women with RPL. An analysis of the parental origin of NLRP7 variants revealed heterozygous carriers in families with RPL who exhibited spontaneous abortions with MLIDs and compound heterozygosity for NLRP7 variants. CONCLUSION: RPL is associated with NLRP7 variants that lead to germinal and postzygotic MLIDs that are incompatible with normal embryo development. TRIAL REGISTRATION: Not applicable.

Genome-wide methylation profiling of Beckwith-Wiedemann syndrome patients without molecular confirmation after routine diagnostics.

Beckwith-Wiedemann syndrome (BWS) is caused due to the disturbance of imprinted genes at chromosome 11p15. The molecular confirmation of this syndrome is possible in approximately 85% of the cases, whereas in the remaining 15% of the cases, the underlying defect remains unclear. The goal of our research was to identify new epigenetic loci related to BWS. We studied a group of 25 patients clinically diagnosed with BWS but without molecular conformation after DNA diagnostics and performed a whole genome methylation analysis using the HumanMethylation450 Array (Illumina).We found hypermethylation throughout the methylome in two BWS patients. The hypermethylated sites in these patients overlapped and included both non-imprinted and imprinted regions. This finding was not previously described in any BWS-diagnosed patient.Furthermore, one BWS patient exhibited aberrant methylation in four maternally methylated regions-IGF1R, NHP2L1, L3MBTL, and ZDBF2-that overlapped with the differentially methylated regions found in BWS patients with multi-locus imprinting disturbance (MLID). This finding suggests that the BWS phenotype can result from MLID without detectable methylation defects in the primarily disease-associated loci (11p15). Another patient manifested small but significant aberrant methylation in disease-associated loci at 11p near H19, possibly confirming the diagnosis in this patient.

Transient neonatal diabetes mellitus and hypomethylation at additional imprinted loci: novel ZFP57 mutation and review on the literature.

AIM: 6q24-related transient neonatal diabetes mellitus (6q24-TNDM) is a rare imprinting disorder characterized by uncontrolled hyperglycemia during the first 6 months of life. The molecular etiology of 6q24-TNDM is attributable to overexpression of the paternally inherited PLAGL1 and HYMAI genes located on the 6q24 locus. One of these major defects is maternal loss of methylation (LOM) at 6q24. In addition, approximately 50% of TNDM patients that present LOM at 6q24 can also display hypomethylation at additional imprinted loci (multilocus imprinting disturbances, MLID). Interestingly, the majority of these patients carry mutations in the ZFP57 gene, a transcription factor required for the adequate maintenance of methylation during early embryonic development. METHODS: Methylation analysis of 6q24 and additional imprinted loci was carried out by MS-MLPA in a Tunisian male patient with clinical diagnosis of TNMD. For the same patient, mutation analysis of the ZFP57 gene was conducted by direct Sanger sequencing. RESULTS: We report a novel nonsense mutation (c.373C > T; p.R125*; ENST00000376883.1) at the ZFP57 gene causing TNDM-MLID and describe detailed phenotype/epigenotype analysis of TNMD patients carrying ZFP57 mutations. CONCLUSION: We provide additional support to the role of ZFP57 as a genetic determinant cause of MLID in patients with TNMD.

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.