Objective: Recently, DNA methylation episignature analysis has been introduced as a tool enabling re-classification of “variants of uncertain significance“ (VUS) in lysine-specific methyltransferase 2B (KMT2B), guiding molecular diagnosis [1,2]. We applied this new methylation biomarker to a two-generation KMT2B-VUS-carrying family in which affected individuals exhibited either dystonic or non-dystonic phenotypes.

Background: Variants of KMT2B are frequently detected in exome sequencing (ES) data of patients with early-onset dystonia. However, KMT2B variants inherited from non-dystonic parents and located outside established mutational hotspots are often classified as VUS. Reduced penetrance and variable expressivity have been described in association with KMT2B variants.

Method: ES followed by Sanger-based segregation testing was undertaken in a family with three affected and two unaffected members. A prioritized missense VUS in KMT2B was studied by episignature analysis. Blood DNA was analyzed with Illumina® MethylationEPIC BeadChip arrays, and an in-house-developed bioinformatics classifier was used to evaluate the resulting methylation profiles.

Results: The index patient presented with early-onset generalized dystonia, while the affected sibling and the affected father demonstrated features compatible with a mild non-motor developmental disorder. A novel KMT2B missense variant, located outside the previously reported mutation clusters, c.3229C>T, p.(Arg1077Trp), was found to segregate in affected family members. The variant was associated with an abnormal KMT2B episignature methylation pattern, clearly separating affected individuals from controls. Remarkably, the episignature pattern was also different from that of KMT2B variants readily classifiable as “pathogenic“ according to ACMG rules, showing an intermediate methylation level between typical loss-of-function variant cases and control subjects.

Conclusion: DNA episignature analysis in KMT2B-related disorders is sensitive and efficient, facilitating variant classification. Our observation of an intermediate abnormal methylation pattern raises the intriguing possibility that the Arg1077Trp variant may act as a hypomorphic allele, distinguishing it from classic pathogenic loss-of-function mutations. This might explain, at least to some degree, the highly variable clinical manifestation observed in this family.

References: 1) Ciolfi A, Foroutan A, Capuano A, Pedace L, Travaglini L, Pizzi S, et al. Childhood-onset dystonia-causing KMT2B variants result in a distinctive genomic hypermethylation profile. Clin Epigenetics. 2021 Aug 11;13(1):157.
2) Mirza-Schreiber N, Zech M, Wilson R, Brunet T, Wagner M, Jech R, et al. Blood DNA methylation provides an accurate biomarker of KMT2B-related dystonia and predicts onset. Brain. 2021 Sep 30:awab360. doi: 10.1093/brain/awab360.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/kmt2b-episignature-analysis-identifies-a-probably-hypomorphic-missense-variant-in-a-family-with-dystonic-and-non-dystonic-phenotypes/