Gene domain-specific DNA methylation episignatures highlight distinct molecular entities of ADNP syndrome

Abstract

Background: ADNP syndrome is a rare Mendelian disorder characterized by global developmental delay, intellectual disability, and autism. It is caused by truncating mutations in ADNP, which is involved in chromatin regulation. We hypothesized that the disruption of chromatin regulation might result in specific DNA methylation patterns that could be used in the molecular diagnosis of ADNP syndrome. Results: We identified two distinct and partially opposing genomic DNA methylation episignatures in the peripheral blood samples from 22 patients with ADNP syndrome. The "epi-ADNP-1" episignature included~6000 mostly hypomethylated CpGs, and the "epi-ADNP-2" episignature included~1000 predominantly hypermethylated CpGs. The two signatures correlated with the locations of the ADNP mutations. Epi-ADNP-1 mutations occupy the N-and C-terminus, and epi-ADNP-2 mutations are centered on the nuclear localization signal. The episignatures were enriched for genes involved in neuronal system development and function. A classifier trained on these profiles yielded full sensitivity and specificity in detecting patients with either of the two episignatures. Applying this model to seven patients with uncertain clinical diagnosis enabled reclassification of genetic variants of uncertain significance and assigned new diagnosis when the primary clinical suspicion was not correct. When applied to a large cohort of unresolved patients with developmental delay (N = 1150), the model predicted three additional previously undiagnosed patients to have ADNP syndrome. DNA sequencing of these subjects, wherever available, identified pathogenic mutations within the gene domains predicted by the model. Conclusions: We describe the first Mendelian condition with two distinct episignatures caused by mutations in a single gene. These highly sensitive and specific DNA methylation episignatures enable diagnosis, screening, and genetic variant classifications in ADNP syndrome.

Episignatures stratifying ADNP syndrome show modest correlation with phenotype

“…Differentially methylated genes in class I are enriched in neuronal and synaptic genes ("neuron cell-cell adhesion" and "receptor localization to synapse" as top Gene Ontology processes) and in risk genes for ASD 28 (26 genes, P<0.0001), DD/ID 32 (119 genes, P=4x10 -4 ), and congenital heart defects (CHD) (24 genes, P=0.005) (Supplemental Note). In line with earlier findings 16 , class II individuals show a much more modest episignature, with 2,582 differentially methylated autosomal CpGs (Figure 1C, Table S2), 1,007 of which overlap with the 1,374 previously described 16 (~73%). The 1,442 unique RefSeq genes with altered methylation in the promoter and/or gene body are enriched in DD/ID genes 32 (69 genes, P=5x10 -4 ), ASD 28 (11 genes, P=0.013) but not CHD (10 genes, P=0.13).…”

“…We identified 6,448 autosomal CpGs that were differentially methylated in class I individuals compared to controls (Table S2). Of these differentially methylated sites, 4,143 overlap with the 5,987 previously identified 16 (~69%). These sites map to the gene promoter (defined here as ± 2 kb from the transcriptional start sites) and/or gene body (transcription start to transcription end) of 2,802 autosomal Refseq genes, and most hypomethylated in ADNP cases (Figure 1C, Table S2).…”

“…Notably, this individual carries the most distal frameshift mutation (Table S1, Figure 1A). Similarly, Bend and colleagues found a carrier of a p.Tyr780* mutation having more modest changes compared to other class II mutations 16 . These independent observations suggest that more terminally truncated ADNP mutant proteins may retain partial function associated with an attenuated epigenetic signature.…”

“…These two groups were well separated from unaffected agematched or sibling controls. Specifically, class II mutations were located between c.2157 and c.2317 of NM_ 015339.4, within the interval defined by Bend and colleagues 16 . Eight class II participants carry the most recurrent mutation found in ADNP, p.Tyr719* (Figure 1A, Table S1, note that another six p.Tyr719* participants were included in the genotype-phenotype studies).…”

Genome‐wide DNA methylation and RNA analyses enable reclassification of two variants of uncertain significance in a patient with clinical Kabuki syndrome

“…The addition of epigenomic analysis allows for the detection of a genetic condition without any prior knowledge of sequence variants and will be particularly important when sequencing analysis results in a VUS or no candidate variant. We have successfully developed a similar technology for more than 20 constitutional disorders and cancer, routine application of which in the clinical setting will enable drastic improvements in variant interpretation and diagnosis (Aref‐Eshghi et al, 2017b; Aref‐Eshghi, Schenkel, et al, ; Aref‐Eshghi, Schenkel, Carere, Rodenhiser, & Sadikovic, ; Bend et al, ; Schenkel et al, ).…”