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. 2019 Feb 20;5(2):eaau9425.
doi: 10.1126/sciadv.aau9425. eCollection 2019 Feb.

Transcriptional profiling at the DLK1/MEG3 domain explains clinical overlap between imprinting disorders

Walid Abi Habib  1   2 , Frédéric Brioude  1   2 , Salah Azzi  1   2 , Sylvie Rossignol  3 , Agnès Linglart  4   5 , Marie-Laure Sobrier  1 , Éloïse Giabicani  1   2 , Virginie Steunou  1 , Madeleine D Harbison  6 , Yves Le Bouc  1   2 , Irène Netchine  1   2
Affiliations

Transcriptional profiling at the DLK1/MEG3 domain explains clinical overlap between imprinting disorders

Walid Abi Habib et al. Sci Adv. .

Abstract

Imprinting disorders (IDs) often affect growth in humans, leading to diseases with overlapping features, regardless of the genomic region affected. IDs related to hypomethylation of the human 14q32.2 region and its DLK1/MEG3 domain are associated with Temple syndrome (TS14). TS14 is a rare type of growth retardation, the clinical signs of which overlap considerably with those of Silver-Russell syndrome (SRS), another ID related to IGF2 down-regulation at 11p15.5 region. We show that 14q32.2 hypomethylation affects expression, not only for genes at this locus but also for other imprinted genes, and especially lowers IGF2 levels at 11p15.5. Furthermore, expression of nonimprinted genes is also affected, some of which are also deregulated in SRS patients. These findings highlight the epigenetic regulation of gene expression at the DLK1/MEG3 domain. Expression profiling of TS14 and SRS patients highlights common signatures, which may account for the clinical overlap observed between TS14 and SRS.

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Figures

Fig. 1
Fig. 1. Overlap of clinical features between SRS, TS14, and PWS and DMRs with parent-specific gene expression from the DLK1/MEG3, IGF2/H19, and SNURF/IPW domains.
(A) Schematic representation of the overlapping clinical features in SRS, TS14, and PWS patients. Schematic diagram of the regions imprinted in humans (B) the IGF2/H19 domain on 11p15.5 and (C) the DLK1/MEG3 domain of the 14q32.2 region. The relative positions of hairpin-like [pre-microRNA (miRNA)] structures within the miR-379/miR-410 cluster are indicated in the enlargement in the inset and (D) the SNURF/IPW domain on 15q11-q13. PEGs are shown as blue rectangles, and MEGs are shown as pink rectangles. miRNAs and snoRNAs (small nucleolar RNAs) are depicted as stem loops and ovals, respectively. Arrows indicate the direction of transcription. The DMRs ICR1, IG-DMR, MEG3-DMR, and IC, which control monoallelic expression over the domains, are indicated by closed and open lollipops (methylated and unmethylated, respectively). SGA, small for gestational age.
Fig. 2
Fig. 2. Schematic presentation of the patients, biological materials, and strategies used in the study.
ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction; RNA-seq, RNA sequencing.
Fig. 3
Fig. 3. Expression profiling of 14q32.2 genes from the serum and fibroblasts of TS14 patients.
(A) DLK1 is absent from the serum of TS14 patients but present in that of age-matched controls. Boys and girls are indicated by open triangles and circles, respectively. TS14 patients are represented by black diamonds. (B to D) MEG3, MEG8, and miRNAs are up-regulated in TS14 patient fibroblasts. Relative levels of expression for MEG3, MEG8, and three miRNAs of the DLK1/MEG3 domain in skin-derived fibroblast cultures from TS14, SRS/TS14, and SRS patients, compared with control fibroblasts. (E and F) MEG3 and H19 are biallelically expressed upon the hypomethylation of 14q32.2 and 11p15.5, respectively. Electropherogram showing the informative SNPs rs11160608 and rs217727, within the MEG3 (E) and H19 (F) coding sequences, respectively, on genomic DNA (gDNA) and cDNA. The data shown are mean values ± SEM for five different passages for the SRS/TS14 patient skin-derived fibroblast cultures, four TS14 patients and five SRS patients, with comparison to five donors as a control. *P ≤ 0.05 and **P ≤ 0.01 versus controls, in Mann-Whitney tests.
Fig. 4
Fig. 4. 11p15.5 and 15q11-q13 PEGs are down-regulated upon 14q32.2 hypomethylation.
Relative levels of expression for IGF2, SNURF, and IPW in cultures of skin-derived fibroblasts from TS14, SRS/TS14, and SRS patients compared with control fibroblasts. The data shown are mean values ± SEM for five different passages for the skin-derived fibroblast cultures for the SRS/TS14 patient, four TS14 patients, and five SRS patients, with comparison to five different donors as a control. *P ≤ 0.05 and **P ≤ 0.01 versus controls, in Mann-Whitney tests.
Fig. 5
Fig. 5. 11p15.5 and 15q11-q13 PEGs are deregulated upon MEG3 and/or MEG8 overexpression or knockdown in experimental models.
(A) Relative levels of expression for IGF2, SNURF, and IPW in a control primary fibroblast line transfected with an empty, MEG3, and/or MEG8 expression vector. (B) Relative levels of expression for IGF2, SNURF, and IPW for a control primary fibroblast line transfected with a negative control, MEG3, and/or MEG8 small interfering RNA (siRNA). The data shown are the mean values ± SEM for four independent transfection assays (with at least four wells transfected per experiment). ***P ≤ 0.001 versus controls (transfected with an empty vector), in unpaired t tests.
Fig. 6
Fig. 6. PCA and DEGs from TS14, SRS, and control fibroblasts.
(A) Proportion of the variance accounted for by each principal component (PC). (B) Two-dimensional and (C) three-dimensional projection plots of the first three PCs: PC1, PC2, and PC3. (D and E) Volcano plots of the expression of all genes (with FPKM of ≥1) from TS14 and SRS, respectively, relative to controls. Green and red dots represent gene displaying statistically significant underexpression and overexpression, respectively [log2(±2) fold change, with a q value threshold of 0.05]. (F) The number of genes differentially regulated in both TS14 and SRS fibroblasts or in one of these types of fibroblasts only. (G) Schematic representation of gene ontology pathways from the GSEA displaying significant up- and down-regulation in patients relative to controls fibroblasts after correction for multiple testing (q < 0.05). GSEA reveals a dysregulated pathway signature common to TS14 and SRS.
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