Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Genome-wide DNA methylation profiling and exome sequencing resolved a long-time misdiagnosed case


The search for aetiology of Mendelian disorders is traditionally based on the observation of clinical phenotypes and molecular screening of associated genes. However, a disease-specific diagnosis can be challenging. In this study we detail how the combinatorial genomic and epigenomic assessment allowed to find the underlying molecular event of a clinical case that remained misdiagnosed for years. The individual was referred as affected by an atypical form of Kabuki syndrome with a variant of uncertain significance in the KMT2D gene. However, significant inconsistencies with this diagnosis emerged upon familial segregation of the variant and after the clinical re-evaluation. Therefore, we applied an epigenomic strategy by studying the DNA methylation profile which resulted not consistent with the Kabuki syndrome episignature or with any other disorder-specific episignature described so far, providing strong evidence that the Kabuki syndrome diagnosis does not apply. This result led us to further investigate for epigenetic machinery diseases by using a multigene panel for chromatinopathies. Since this analysis yielded negative results, we applied a whole exome sequencing and identified a de novo pathogenic variant in the CTNNB1 gene associated to NEDSDV syndrome, a neurodevelopmental disorder characterized by intellectual disability and craniofacial anomalies. Based on molecular results and the updated clinical features, we confirmed the NEDSDV diagnosis. Our findings show that the combination of genomic and epigenomics strategies, along with a deeper analysis of clinical phenotype, may provide a significant improvement in the diagnostic protocols for rare genetic disorders and help resolve long-time misdiagnosed and unsolved case.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1


  1. Petersen BS, Fredrich B, Hoeppner MP, Ellinghaus D, Franke A. Opportunities and challenges of whole-genome and -exome sequencing. BMC Genet. 2017;18:14.

    Article  Google Scholar 

  2. Haghshenas S, Bhai P, Aref-Eshghi E, Sadikovic B. Diagnostic Utility of Genome-Wide DNA Methylation Analysis in Mendelian Neurodevelopmental Disorders. Int J Mol Sci. 2020;21:9303.

  3. Kerkhof J, Squeo GM, McConkey H, Levy MA, Piemontese MR, Castori M, et al. DNA methylation episignature testing improves molecular diagnosis of Mendelian chromatinopathies. Genet Med. 2022;24:51–60.

    Article  Google Scholar 

  4. Levy MA, McConkey H, Kerkhof J, Barat-Houari M, Bargiacchi S, Biamino E, et al. Novel diagnostic DNA methylation episignatures expand and refine the epigenetic landscapes of Mendelian disorders. HGG Adv. 2022;3:100075.

    PubMed  Google Scholar 

  5. Sadikovic B, Aref-Eshghi E, Levy MA, Rodenhiser D. DNA methylation signatures in mendelian developmental disorders as a diagnostic bridge between genotype and phenotype. Epigenomics 2019;11:563–75.

    CAS  Article  Google Scholar 

  6. Sadikovic B, Levy MA, Aref-Eshghi E. Functional annotation of genomic variation: DNA methylation episignatures in neurodevelopmental Mendelian disorders. Hum Mol Genet. 2020;29:R27–R32.

    CAS  Article  Google Scholar 

  7. Sadikovic B, Levy MA, Kerkhof J, Aref-Eshghi E, Schenkel L, Stuart A, et al. Correction: Clinical epigenomics: genome-wide DNA methylation analysis for the diagnosis of Mendelian disorders. Genet Med. 2021;23:2228.

    Article  Google Scholar 

  8. Priolo M, Micale L, Augello B, Fusco C, Zucchetti F, Prontera P, et al. Absence of deletion and duplication of MLL2 and KDM6A genes in a large cohort of patients with Kabuki syndrome. Mol Genet Metab. 2012;107:627–9.

    CAS  Article  Google Scholar 

  9. Squeo GM, Augello B, Massa V, Milani D, Colombo EA, Mazza T, et al. Customised next-generation sequencing multigene panel to screen a large cohort of individuals with chromatin-related disorder. J Med Genet. 2020;57:760–8.

    CAS  Article  Google Scholar 

  10. Alfaiz AA, Micale L, Mandriani B, Augello B, Pellico MT, Chrast J, et al. TBC1D7 mutations are associated with intellectual disability, macrocrania, patellar dislocation, and celiac disease. Hum Mutat. 2014;35:447–51.

    Article  Google Scholar 

  11. Lodder EM, De Nittis P, Koopman CD, Wiszniewski W, Moura de Souza CF, Lahrouchi N, et al. GNB5 Mutations Cause an Autosomal-Recessive Multisystem Syndrome with Sinus Bradycardia and Cognitive Disability. Am J Hum Genet. 2016;99:704–10.

    CAS  Article  Google Scholar 

  12. den Dunnen JT, Dalgleish R, Maglott DR, Hart RK, Greenblatt MS, McGowan-Jordan J, et al. HGVS Recommendations for the Description of Sequence Variants: 2016 Update. Hum Mutat. 2016;37:564–9.

    Article  Google Scholar 

  13. Biesecker LG, Harrison SM. The ACMG/AMP reputable source criteria for the interpretation of sequence variants. Genet Med. 2018;20:1687–8.

    Article  Google Scholar 

  14. Cocciadiferro D, Augello B, De Nittis P, Zhang J, Mandriani B, Malerba N, et al. Dissecting KMT2D missense mutations in Kabuki syndrome patients. Hum Mol Genet. 2018;27:3651–68.

    CAS  Article  Google Scholar 

  15. Micale L, Augello B, Fusco C, Selicorni A, Loviglio MN, Silengo MC, et al. Mutation spectrum of MLL2 in a cohort of Kabuki syndrome patients. Orphanet J Rare Dis. 2011;6:38.

    Article  Google Scholar 

  16. Sadikovic B, Levy MA, Kerkhof J, Aref-Eshghi E, Schenkel L, Stuart A, et al. Clinical epigenomics: genome-wide DNA methylation analysis for the diagnosis of Mendelian disorders. Genet Med. 2021;23:1065–74.

    CAS  Article  Google Scholar 

  17. Kopanos C, Tsiolkas V, Kouris A, Chapple CE, Albarca Aguilera M, Meyer R, et al. VarSome: the human genomic variant search engine. Bioinformatics 2019;35:1978–80.

    CAS  Article  Google Scholar 

  18. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–24.

    Article  Google Scholar 

  19. Aref-Eshghi E, Bourque DK, Kerkhof J, Carere DA, Ainsworth P, Sadikovic B, et al. Genome-wide DNA methylation and RNA analyses enable reclassification of two variants of uncertain significance in a patient with clinical Kabuki syndrome. Hum Mutat. 2019;40:1684–9.

    CAS  Article  Google Scholar 

  20. Xing Y, Takemaru K, Liu J, Berndt JD, Zheng JJ, Moon RT, et al. Crystal structure of a full-length beta-catenin. Structure 2008;16:478–87.

    CAS  Article  Google Scholar 

  21. MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell. 2009;17:9–26.

    CAS  Article  Google Scholar 

  22. Dong F, Jiang J, McSweeney C, Zou D, Liu L, Mao Y. Deletion of CTNNB1 in inhibitory circuitry contributes to autism-associated behavioral defects. Hum Mol Genet. 2016;25:2738–51.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Brault V, Moore R, Kutsch S, Ishibashi M, Rowitch DH, McMahon AP, et al. Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development 2001;128:1253–64.

    CAS  Article  Google Scholar 

  24. Huelsken J, Vogel R, Erdmann B, Cotsarelis G, Birchmeier W. beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 2001;105:533–45.

    CAS  Article  Google Scholar 

  25. Kuechler A, Willemsen MH, Albrecht B, Bacino CA, Bartholomew DW, van Bokhoven H, et al. De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause of intellectual disability: expanding the mutational and clinical spectrum. Hum Genet. 2015;134:97–109.

    CAS  Article  Google Scholar 

  26. Tucci V, Kleefstra T, Hardy A, Heise I, Maggi S, Willemsen MH, et al. Dominant beta-catenin mutations cause intellectual disability with recognizable syndromic features. J Clin Investig. 2014;124:1468–82.

    CAS  Article  Google Scholar 

  27. Kharbanda M, Pilz DT, Tomkins S, Chandler K, Saggar A, Fryer A, et al. Clinical features associated with CTNNB1 de novo loss of function mutations in ten individuals. Eur J Med Genet. 2017;60:130–5.

    Article  Google Scholar 

  28. Ke Z, Chen Y. Case Report: A de novo CTNNB1 Nonsense Mutation Associated With Neurodevelopmental Disorder, Retinal Detachment, Polydactyly. Front Pediatr. 2020;8:575673.

    Article  Google Scholar 

  29. Dubruc E, Putoux A, Labalme A, Rougeot C, Sanlaville D, Edery P. A new intellectual disability syndrome caused by CTNNB1 haploinsufficiency. Am J Med Genet A. 2014;164a:1571–5.

    Article  Google Scholar 

  30. Aref-Eshghi E, Bend EG, Colaiacovo S, Caudle M, Chakrabarti R, Napier M, et al. Diagnostic Utility of Genome-wide DNA Methylation Testing in Genetically Unsolved Individuals with Suspected Hereditary Conditions. Am J Hum Genet. 2019;104:685–700.

    CAS  Article  Google Scholar 

Download references


We are grateful to the patient and parents for their cooperation in this study and for giving consent to publish data and photographs. Funding for this study was provided by Telethon - Italy (Grant no. GGP13231), Italian Ministry of Health, Jerome Lejeune Foundation, Daunia Plast to GM; by the London Health Sciences Molecular Diagnostics Development Fund and Genome Canada Genomic Applications Partnership Program Grant (Beyond Genomics: Assessing the Improvement in Diagnosis of Rare Diseases using Clinical Epigenomics in Canada, EpiSign-CAN) awarded to BS. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

Author information

Authors and Affiliations



Conceptualization: AP, GM, GMS. Investigation and Methodology: AP, ED, EC, JK; Data curation and formal analysis: JK, HMC, BS, TM, SC; Patients and clinical follow-up: LS, MCD; Writing—original draft: AP, MCD, GM; Writing-review & editing: AP, MCD, BS, JK, GMS, HMC, TM, GM. Funding acquisition: GM, BS. All authors have read and agreed to the published version of the paper. The corresponding author has had full access to the data in the study and final responsibility for the decision to submit for publication.

Corresponding author

Correspondence to Giuseppe Merla.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval

Written informed consent was obtained from the mother of the child for the publication of any potentially identifiable images included in this paper.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Paparella, A., Squeo, G.M., Di Venere, E. et al. Genome-wide DNA methylation profiling and exome sequencing resolved a long-time misdiagnosed case. J Hum Genet 67, 547–551 (2022).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


Quick links