Schenkel et al. Clinical Epigenetics (2018) 10:21 https://doi.org/10.1186/s13148-018-0453-8 RESEARCH Open Access Peripheral blood epi-signature of Claes- Jensen syndrome enables sensitive and specific identification of patients and healthy carriers with pathogenic mutations in KDM5C Laila C. Schenkel1,2†, Erfan Aref-Eshghi1,2†, Cindy Skinner3, Peter Ainsworth1,2, Hanxin Lin1,2, Guillaume Paré4, David I. Rodenhiser5, Charles Schwartz3 and Bekim Sadikovic1,2,6* Abstract Background: Claes-Jensen syndrome is an X-linked inherited intellectual disability caused by mutations in the KDM5C gene. Kdm5c is a histone lysine demethylase involved in histone modifications and chromatin remodeling. Males with hemizygous mutations in KDM5C present with intellectual disability and facial dysmorphism, while most heterozygous female carriers are asymptomatic. We hypothesized that loss of Kdm5c function may influence other components of the epigenomic machinery including DNA methylation in affected patients. Results: Genome-wide DNA methylation analysis of 7 male patients affected with Claes-Jensen syndrome and 56 age- and sex-matched controls identified a specific DNA methylation defect (epi-signature) in the peripheral blood of these patients, including 1769 individual CpGs and 9 genomic regions. Six healthy female carriers showed less pronounced but distinctive changes in the same regions enabling their differentiation from both patients and controls. Highly specific computational model using the most significant methylation changes demonstrated 100% accuracy in differentiating patients, carriers, and controls in the training cohort, which was confirmed on a separate cohort of patients and carriers. The 100% specificity of this unique epi-signature was further confirmed on additional 500 unaffected controls and 600 patients with intellectual disability and developmental delay, including other patient cohorts with previously described epi-signatures. Conclusion: Peripheral blood epi-signature in Claes-Jensen syndrome can be used for molecular diagnosis and carrier identification and assist with interpretation of genetic variants of unknown clinical significance in the KDM5C gene. Keywords: KDM5C, DNA methylation, Variants of unknown significance, X-linked intellectual disability, Claes-Jensen * Correspondence: [email protected] †Equal contributors 1Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada 2Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, London, Ontario, Canada Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Schenkel et al. Clinical Epigenetics (2018) 10:21 Page 2 of 11 Background broad range of developmental delay and intellectual dis- DNA methylation and histone modifications are the ability disorders [14, 15]. most widely studied epigenetic mechanisms, involved in In this report, we used a high-resolution genome-wide the control of gene expression and maintenance of gen- methylation microarray analysis to describe a specific omic stability. Post-translational modifications of histone methylation profile in peripheral blood samples of a co- proteins are coordinated by a variety of enzymes that hort of affected males with loss-of-function KDM5C catalyze histone methylation, demethylation, acetylation, mutations and a similar but less pronounced methyla- deacetylation, phosphorylation, ubiquitination, SUMOy- tion change in healthy female carriers. We demonstrate lation, and ADP-ribosylation [1]. Mutations in the genes that the DNA methylation signature of Claes-Jensen syn- involved in the modification of histones cause a broad drome has the potential to be used as a diagnostic tool spectrum of Mendelian disorders [2]. Among these are for detection of both patients and healthy carriers with mutations in the X-linked gene KDM5C, which encodes KDM5C mutations. We further show that this epi- the histone H3 lysine 4 (H3K4) demethylase protein and signature is highly specific to KDM5C mutations, but causes Claes-Jensen syndrome [2]. not to the broad range of other Mendelian diseases The clinical manifestations in affected males carrying resulting from the disruption of genes in the epigenomic hemizygous KDM5C mutations include intellectual dis- machinery or other forms of developmental delay and ability, impairments in adaptive behavior, slowly progres- intellectual disabilities (DD/ID). sive spastic paraplegia, seizures, and facial dysmorphism [3, 4]. KDM5C is among the genes that escape X- Methods chromosome inactivation; therefore, female mutation Study cohort carriers usually remain unaffected but can demonstrate Peripheral blood samples from patients referred for gen- mild learning deficits [5]. etic testing at the Greenwood Genetic Center were col- Recent studies have suggested a bidirectional relation- lected for methylation study. All patients were screened ship between DNA methylation and histone modifica- for mutations in the KDM5C and variants assessed ac- tion which regulates locus-specific gene activity [6]. For cording to the American College of Medical Genetics instance, the DNA methyltransferase Dnmt3b1 select- Guidelines for interpretation of genomic sequence vari- ively binds to the bodies of transcribed genes, leading to ants [16]. The study included ten male patients affected their de novo methylation, and its recruitment requires with Claes-Jensen syndrome and eight healthy females co-transcriptional deposition of histone H3 trimethyla- carrying pathogenic mutations in KDM5C. All male sub- tion at lysine 36 [7]. These findings have encouraged jects were clinically confirmed to be affected by the Claes- global efforts to elucidate the specific underlying mo- Jensen syndrome. The age of the patients was recorded at lecular mechanisms that may be altered in epigenetic the time of blood draw. The mutation status and demo- syndromes and to identify epi-signatures that can be graphics of all of the patients are shown in Table 1. These used for diagnosis, particularly in those patients whose patients and carriers are members of three unrelated fam- clinical manifestations are associated with a phenotypic ilies for whom comprehensive clinical, molecular, and mu- spectrum shared across more than one syndrome, a situ- tation assessments have been previously reported by Abidi ation where a specific clinical diagnosis is difficult to et al. [17]. Controls were selected from our lab reference make. cohort of individuals with no known aberrant epigenomic Using a genome-wide DNA methylation analysis, we change, including examination for imprinting defects, and others have identified specific DNA methylation abnormal methylation pattern associated with other devel- epi-signatures in the peripheral blood of patients with opmental syndromes, and representation of outlier pro- a number of genetic diseases that result from the files in principle component analysis. This reference disruption of epigenomic machinery, including alpha- cohort includes individuals that were previously selected thalassemia/mental retardation X-linked (ATRX) syn- from a larger cohort of about 1000 individuals across the drome [8], Floating-Harbor syndrome [9], DNA methyl- broad range of age, sex, and ethnicity distribution. transferase 1 (DNMT1)-associated autosomal dominant cerebellar ataxia, deafness, and narcolepsy syndrome Methylation array and quality assessment [10], along with Kabuki [11, 12] and Sotos syndromes Genomic DNA was extracted from peripheral blood [13]. These epi-signatures from peripheral blood spec- using standard techniques. Following bisulfite conver- imens can be used to accurately identify patients with sion, DNA methylation analysis was performed using the these conditions, to assist with the interpretation of Illumina HumanMethylation450 bead chip (San Diego, genetic variants of unknown significance in related CA), according to the manufacturer’s protocol at the genes, and ultimately, to be used as part of the rou- Genetic and Molecular Epidemiology Laboratory at tine molecular screening protocols in patients with a McMaster University and the London Health Sciences Schenkel et al. Clinical Epigenetics (2018) 10:21 Page 3 of 11 Table 1 Clinical and molecular characteristics of male patients and female mutation carriers referred for methylation study Sample ID Sex Age (years) Disease status Mutation Cohort 3694a M 28 Patient c.4439_4440delAG; p.R1481GfsX9 Discovery/training 3695a M 26 Patient c.4439_4440delAG; p.R1481GfsX9 Discovery/training 3696a F 51 Carrier c.4439_4440delAG; p.R1481GfsX9 Training 12551D F 55 Carrier c.229G>A; p.A77T Training cms6013 M 37 Patient c.229G>A; p.A77T Testing cms13123A F 66 Carrier c.229G>A; p.A77T Testing cms13755 M 13 Patient c.229G>A; p.A77T Discovery/training cms13756 F 17 Carrier c.229G>A; p.A77T Testing cms13757 F 39 Carrier c.229G>A; p.A77T Training cms1179 F 54 Carrier c.1510G>A;