Neonato piccolo, ipotonico, con difficoltà di alimentazione: pensiamo anche alla sindrome di Temple

Temple syndrome is a rare imprinting disorder mainly due to maternal uniparental disomy of the chromosome 14. It represents the main differential diagnosis of Silver-Russell and Prader-Willi syndrome. This syndrome is characterized by growth retardation, hypotonia, difficult feeding, development delay and precocious puberty. The absence of congenital pathognomonic malformations and universally recognized screening methodologies make this pathology be underdiagnosed, so the analysis of 14q32 should be evaluated in all cases of intrauterine growth restriction, hypotonia and neonatal feeding difficulties. It should also be considered in cases of unexplained early puberty associated with poor stature growth. The paper presents the case of a girl with the final diagnosis of Temple syndrome, with an initial picture of intrauterine growth retardation, axial hypotonia and feeding difficulties. The initial diagnostic suspicion was a Silver-Russell syndrome.

ZNF445: a homozygous truncating variant in a patient with Temple syndrome and multilocus imprinting disturbance

Background ZNF445, as well as ZFP57, is involved in the postfertilization methylation maintenance of multiple imprinting-associated differentially methylated regions (iDMRs). Thus, ZNF445 pathogenic variants are predicted to cause multilocus imprinting disturbances (MLIDs), as do ZFP57 pathogenic variants. In particular, the MEG3/DLK1:IG-DMR would be affected, because the postzygotic methylation imprint of the MEG3/DLK1:IG-DMR is maintained primarily by ZNF445, whereas that of most iDMRs is preserved by both ZFP57 and ZNF445 or primarily by ZFP57. Results We searched for a ZNF445 variant(s) in six patients with various imprinting disorders (IDs) caused by epimutations and MLIDs revealed by pyrosequencing for nine iDMRs, without a selection for the original IDs. Re-analysis of the previously obtained whole exome sequencing data identified a homozygous ZNF445 variant (NM_181489.6:c.2803C>T:p.(Gln935*)) producing a truncated protein missing two of 14 zinc finger domains in a patient with Temple syndrome and MLID. In this patient, array-based genomewide methylation analysis revealed severe hypomethylation of most CpGs at the MEG3:TSS-DMR, moderate hypomethylation of roughly two-thirds of CpGs at the H19/IGF2:IG-DMR, and mild-to-moderate hypomethylation of a few CpGs at the DIRAS3:TSS-DMR, MEST:alt-TSS-DMR, IGF2:Ex9-DMR, IGF2:alt-TSS, and GNAS-AS1:TSS-DMR. Furthermore, bisulfite sequencing analysis for the MEG3/DLK1:IG-DMR delineated a markedly hypomethylated segment (CG-A). The heterozygous parents were clinically normal and had virtually no aberrant methylation pattern. Conclusions We identified a ZNF445 pathogenic variant for the first time. Since ZNF445 binds to the MEG3/DLK1:IG-DMR and other iDMRs affected in this patient, the development of Temple syndrome and MLID would primarily be explained by the ZNF445 variant. Furthermore, CG-A may be the target site for ZNF445 within the MEG3/DLK1:IG-DMR.

Preimplantation genetic testing for a chr14q32 microdeletion in a family with Kagami-Ogata syndrome and Temple syndrome

IntroductionKagami-Ogata syndrome (KOS14) and Temple syndrome (TS14) are two disorders associated with reciprocal alterations within the chr14q32 imprinted domain. Here, we present a work-up strategy for preimplantation genetic testing (PGT) to avoid the transmission of a causative micro-deletion.MethodsWe analysed DNA from the KOS14 index case and parents using methylation-sensitive ligation-mediated probe amplification and methylation pyrosequencing. The extent of the deletion was mapped using SNP arrays. PGT was performed in trophectoderm samples in order to identify unaffected embryos. Samples were amplified using multiple displacement amplification, followed by genome-wide SNP genotyping to determine the at-risk haplotype and next-generation sequencing to determine aneuploidies.ResultsA fully methylated pattern at the normally paternally methylated IG-DMR and MEG3 DMR in the KOS14 proband, accompanied by an unmethylated profile in the TS14 mother was consistent with maternal and paternal transmission of a deletion, respectively. Further analysis revealed a 108 kb deletion in both cases. The inheritance of the deletion on different parental alleles was consistent with the opposing phenotypes. In vitro fertilisation with intracytoplasmatic sperm injection and PGT were used to screen for deletion status and to transfer an unaffected embryo in this couple. A single euploid-unaffected embryo was identified resulting in a healthy baby born.DiscussionWe identify a microdeletion responsible for multigeneration KOS14 and TS14 within a single family where carriers have a 50% risk of transmitting the deletion to their offspring. We show that PGT can successfully be offered to couples with IDs caused by genetic anomalies.

Genome-wide methylation analysis in Silver–Russell syndrome, Temple syndrome, and Prader–Willi syndrome

Background Imprinting disorders (IDs) show overlapping phenotypes, particularly in Silver–Russell syndrome (SRS), Temple syndrome (TS14), and Prader–Willi syndrome (PWS). These three IDs include fetal and postnatal growth failure, feeding difficulty, and muscular hypotonia as major clinical features. However, the mechanism that causes overlapping phenotypes has not been clarified. To investigate the presence or absence of methylation signatures associated with overlapping phenotypes, we performed genome-wide methylation analysis (GWMA). Results GWMA was carried out on 36 patients with three IDs (SRS [n = 16], TS14 [n = 7], PWS [n = 13]) and 11 child controls using HumanMethylation450 BeadChip including 475,000 CpG sites across the human genome. To reveal an aberrantly methylated region shared by SRS, TS14, and PWS groups, we compared genome-wide methylation data of the three groups with those of control subjects. All the identified regions were known as SRS-, TS14-, and PWS-related imprinting-associated differentially methylated regions (iDMRs), and there was no hypermethylated or hypomethylated region shared by different ID groups. To examine the methylation pattern shared by SRS, TS14, and PWS groups, we performed clustering analysis based on GWMA data. The result focusing on 620 probes at the 62 known iDMRs (except for SRS-, TS14-, and PWS-related iDMRs) classified patients into two categories: (1) category A, grossly normal methylation patterns mainly consisting of SRS group patients; and (2) category B, broad and mild hypermethylation patterns mainly consisting of TS14 and PWS group patients. However, we found no obvious relationship between these methylation patterns and phenotypes of patients. Conclusions GWMA in three IDs found no methylation signatures shared by SRS, TS14, and PWS groups. Although clustering analysis showed similar mild hypermethylation patterns in TS14 and PWS groups, further study is needed to clarify the effect of methylation patterns on the overlapping phenotypes.

Temple syndrome and Kagami-Ogata syndrome: clinical presentations, genotypes, models and mechanisms

Temple syndrome (TS) and Kagami-Ogata syndrome (KOS) are imprinting disorders caused by absence or overexpression of genes within a single imprinted cluster on human chromosome 14q32. TS most frequently arises from maternal UPD14 or epimutations/deletions on the paternal chromosome, whereas KOS most frequently arises from paternal UPD14 or epimutations/deletions on the maternal chromosome. In this review, we describe the clinical symptoms and genetic/epigenetic features of this imprinted region. The locus encompasses paternally expressed protein-coding genes (DLK1, RTL1 and DIO3) and maternally expressed lncRNAs (MEG3/GTL2, RTL1as and MEG8), as well as numerous miRNAs and snoRNAs. Control of expression is complex, with three differentially methylated regions regulating germline, placental and tissue-specific transcription. The strong conserved synteny between mouse chromosome 12aF1 and human chromosome 14q32 has enabled the use of mouse models to elucidate imprinting mechanisms and decipher the contribution of genes to the symptoms of TS and KOS. In this review, we describe relevant mouse models and highlight their value to better inform treatment options for long-term management of TS and KOS patients.

SUN-090 Investigation of Imprinting Defects in MKRN3 and DLK1 in Children with Idiopathic Central Precocious Puberty Through Specific DNA Methylation Analysis

Background: Loss of imprinting has been implicated in the pathogenesis of several human diseases. Monogenic causes of central precocious puberty (CPP) were identified in families with loss-of-function mutations affecting mainly the coding region of two paternally expressed imprinted genes: Makorin ring finger 3 (MKRN3) and Delta-like 1 homolog (DLK1). The role of imprinting defects involving these two genes in CPP has not been described so far. Objective: To investigate the methylation status at primary differentially methylated regions (DMR) of MKRN3 and DLK1 in a cohort of children with idiopathic CPP. Patients and methods: One-hundred and twenty CPP patients (112 sporadic, 8 familial; 115 females, 5 males) were selected for analysis. Leukocyte DNA was obtained from all patients. MKRN3 and DLK1 pathogenic allelic variants were first excluded by DNA sequencing analysis. Bisulfite treatment followed by Allele-Specific Methylated Multiplex Real-Time Quantitative PCR was performed with leukocyte DNA, analyzing separately the methylation index (MI) of MKRN3:TSS-DMR and DLK1/MEG3:IG-DMR for each patient. The MI results were compared with controls with normal pubertal development. Results: Mean age at puberty onset was 5.8 ±1.9yr for girls and 7.2 ±2.6yr for boys. Hypomethylation at DLK1/MEG3:IG-DMR was identified in 3 patients (I, II and III) with sporadic CPP: MI 10%, 16% and 11%, respectively. Interestingly, cases II and III were both girls who had been firstly referred to pediatric endocrinology for presenting precocious menarche; while case I was a boy who had been referred for presenting mild growth retardation, and developed CPP during monitoring. In addition, during follow-up, other clinical findings were noticed: being born small for gestational age, prominent forehead, small hands/feet, overweight/obesity and early onset type 2 diabetes in case III. Additional genetic investigation included SNP array in cases I and II, identifying a maternal uniparental disomy at chromosome 14 (upd(14)mat). Meanwhile, case III had normal genomic microarray and microsatellites analysis, excluding copy number variants and upd(14)mat, and indicating a mechanism of epimutation at DLK1/MEG3:IG-DMR. Uniparental disomy and epimutation are molecular mechanisms associated with the imprinting disorder known as Temple syndrome. In the remaining cases, mean MI for DLK1/MEG3:IG-DMR was 49±2%. In all cases, mean MI for MKRN3:TSS-DMR was 49±6%. There were no significant correlations between age at puberty onset and MI for MKRN3 (p=0.69) and DLK1(p=0.45). Conclusion: There was no leukocyte DNA methylation defect at MKRN3 imprinting control region in the idiopathic CPP cohort. DLK1/MEG3:IG-DMR hypomethylation was identified in 3 patients with CPP and additional findings of Temple syndrome, indicating that loss of effective imprinting of DLK1 locus is a mechanism leading to CPP.