Schenkel et al. Clin Epigenet (2021) 13:2 https://doi.org/10.1186/s13148-020-00990-7 RESEARCH Open Access DNA methylation epi-signature is associated with two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome L. C. Schenkel1,2, E. Aref‑Eshghi1, K. Rooney1, J. Kerkhof1, M. A. Levy1, H. McConkey1, R. C. Rogers4, K. Phelan5, S. M. Sarasua6, L. Jain3,6, R. Pauly3, L. Boccuto3,6, B. DuPont3, G. Cappuccio7,8, N. Brunetti‑Pierri7,8, C. E. Schwartz3* and B. Sadikovic1,2* Abstract Background: Phelan‑McDermid syndrome is characterized by a range of neurodevelopmental phenotypes with incomplete penetrance and variable expressivity. It is caused by a variable size and breakpoint microdeletions in the distal long arm of chromosome 22, referred to as 22q13.3 deletion syndrome, including the SHANK3 gene. Genetic defects in a growing number of neurodevelopmental genes have been shown to cause genome‑wide disruptions in epigenomic profles referred to as epi‑signatures in afected individuals. Results: In this study we assessed genome‑wide DNA methylation profles in a cohort of 22 individuals with Phelan‑ McDermid syndrome, including 11 individuals with large (2 to 5.8 Mb) 22q13.3 deletions, 10 with small deletions (< 1 Mb) or intragenic variants in SHANK3 and one mosaic case. We describe a novel genome‑wide DNA methylation epi‑signature in a subset of individuals with Phelan‑McDermid syndrome. Conclusion: We identifed the critical region including the BRD1 gene as responsible for the Phelan‑McDermid syndrome epi‑signature. Metabolomic profles of individuals with the DNA methylation epi‑signature showed sig‑ nifcantly diferent metabolomic profles indicating evidence of two molecularly and phenotypically distinct clinical subtypes of Phelan‑McDermid syndrome. Keywords: DNA methylation, Microdeletion, Epi‑signature, BRD1, Phelan‑McDermid syndrome Background as 22q13.3 deletion syndrome). Tis loss of genetic mate- Phelan-McDermid syndrome (PHMDS) is characterized rial can be caused by a terminal or interstitial deletion of by developmental delay, absent or impaired speech, neo- chromosome 22, an unbalanced translocation that can be natal hypotonia, autistic behaviors and mild dysmorphic inherited or de novo, or from other complex structural features [1, 2]. It is caused by a contiguous gene deletion rearrangements involving chromosome 22 [2]. Te size of the distal long arm of chromosome 22 (also referred to of the 22q13.3 deletion in PHMDS ranges from < 50 kb to > 9 Mb, including complete or partial deletion of the *Correspondence: [email protected]; [email protected] SHANK3 (SH3 and multiple ankyrin repeat domains 3) 1 Molecular Genetics Laboratory, Molecular Diagnostics Division, London gene in virtually all cases. Health Sciences Centre, London, ON N6A5W9, Canada A limited number of individuals with loss-of-func- 3 Greenwood Genetic Center, Greenwood, SC 29646, USA Full list of author information is available at the end of the article tion intragenic variants in the SHANK3 gene have been © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Schenkel et al. Clin Epigenet (2021) 13:2 Page 2 of 17 reported. Most individuals with SHANK3 intragenic vari- across diferent tissues. Genomic DNA methylation ants have been described as having autism spectrum dis- profles in peripheral blood, referred to as DNA meth- order (ASD) and/or intellectual disability [3–6]. A recent ylation epi-signatures, have been associated with many study had phenotypically characterized 17 individuals human traits, including age, sex and disease status with pathogenic variants in SHANK3, demonstrating that [18–21]. More recently, we and others have reported SHANK3 intragenic pathogenic variants are sufcient to evidence of highly sensitive and specifc peripheral cause a broad range of features associated with PHMDS blood epi-signatures associated with genetic mutations [7]. However, even though intellectual disability and ASD in a growing number of genes and genetic syndromes were present in the majority of individuals with SHANK3 associated with developmental delay/intellectual dis- mutations, speech impairment and motor defcits were abilities and multiple congenital anomalies [22–25]. less severe than in 22q13 deletions, and renal abnormali- In addition, microarray-based genomic DNA methyla- ties were absent [7]. Taken together, there is compelling tion assessment has enabled simultaneous detection of evidence, suggesting that SHANK3 haploinsufciency is imprinted disorders and Fragile X syndrome, which is responsible for the majority of neurological features, i.e., often within the spectrum of diferential diagnoses of intellectual disability and autism, but not the full spec- these neurodevelopmental syndromes [26, 27]. trum of phenotypes observed in PHMDS [8–10]. Tere In this study we assessed genome-wide DNA meth- are at least two cases reported with 22q13 interstitial ylation profles in a cohort of individuals with PHMDS, deletions sparing SHANK3 and phenotypes similar to including individuals with large (2 to 5.8 Mb) 22q13.3 those in PHMDS [11], suggesting an independent role for deletions, with small deletions (< 1 Mb) or with intra- genes in these genomic regions. Another candidate gene genic variants in SHANK3. Given the molecular and located near SHANK3, and deleted in most cases of the phenotypic complexity of this disease, we hypothesized syndrome, is the autism-linked gene IB2 (islet brain 2) a diferentiating DNA methylation epi-signature that that plays an important role in synaptic transmission and would enable assessment of variants of unknown clini- neuronal morphology [12]. cal signifcance, such as smaller deletions at 22q13 or Genotype–phenotype relationships in PHMDS have SHANK3 variants, as well as diagnosis of ambiguous been extensively studied [13–17]. For most pheno- clinical cases. We describe a highly sensitive and spe- types, the individuals with the smallest terminal dele- cifc DNA methylation signature associated with large tions (22q13.33) were less severely afected than those 22q13.3 deletions. Tis signature is not evident in indi- with the largest terminal deletions (22q13.2), except for viduals with small deletions or SHANK3 intragenic ASD which is commonly associated with smaller dele- variants, hence diferentiating these as separate molec- tions. Tese studies suggested that the efect of SHANK3, ular entities. Using these data, we build a classifca- a gene associated with ASD, may be attenuated as the tion algorithm for PHMDS and demonstrate its ability deletion size increases and additional genes are deleted. to specifcally identify individuals with PHMDS-large Tis may refect the difculty in diagnosing ASD in deletions diferentiating them from other neurodevel- individuals that are more severely afected. In addition, opmental conditions with overlapping phenotypes, as these studies mapped a large number of potential can- well as other genetic conditions with recognized DNA didate genes within 22q13 region, including genes asso- methylation epi-signature. Metabolic profling of cells ciated with dysmorphic features, speech and behavior. from individuals with large 22q13 deletions revealed a Taken together, these studies emphasize that PHMDS is functional impact of the disrupted DNA methylation a clinically heterogenous syndrome caused by haploinsuf- by showing, among other features, reduced capacity of fciency of multiple candidate genes. Te large range of cells to produce energy utilizing high-efciency aerobic deletion sizes observed resulting in a large range of phe- pathways, decreased ability to adjust to metabolic stress notypical features, together with the lack of knowledge of and abnormal response to hormones and cytokines all candidate genes within the deleted region, complicates regulating growth and infammation, as compared to the clinical and molecular diagnosis and/or prognosis for controls or cells with small 22q13 deletions or SHANK3 these individuals. variants. Te metabolic fndings provide in vitro vali- DNA methylation is one of the most widely studied dation of the hypothesis that abnormal genome-wide epigenetic mechanisms and is involved in many physi- DNA methylation and large 22q13 deletions in PHMDS ological and disease mechanisms through control of lead to a larger number of disrupted pathways and gene expression and genomic stability. Te establish- more severe clinical phenotypes than in cases with ment of the DNA methylation pattern occurs early dur-