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. 2025 Jan 9;6(1):100380.
doi: 10.1016/j.xhgg.2024.100380. Epub 2024 Nov 4.

CUL3-related neurodevelopmental disorder: Clinical phenotype of 20 new individuals and identification of a potential phenotype-associated episignature

Liselot van der Laan  1 , Ananília Silva  2 , Lotte Kleinendorst  3 , Kathleen Rooney  4 , Sadegheh Haghshenas  5 , Peter Lauffer  3 , Yasemin Alanay  6 , Pratibha Bhai  5 , Alfredo Brusco  7 , Sonja de Munnik  8 , Bert B A de Vries  8 , Angelica Delgado Vega  9 , Marc Engelen  10 , Johanna C Herkert  11 , Ron Hochstenbach  12 , Saskia Hopman  13 , Sarina G Kant  14 , Ryutaro Kira  15 , Mitsuhiro Kato  16 , Boris Keren  17 , Hester Y Kroes  13 , Michael A Levy  5 , Ngu Lock-Hock  18 , Saskia M Maas  3 , Grazia M S Mancini  14 , Carlo Marcelis  19 , Naomichi Matsumoto  20 , Takeshi Mizuguchi  20 , Alessandro Mussa  21 , Cyril Mignot  17 , Anu Närhi  22 , Ann Nordgren  23 , Rolph Pfundt  19 , Abeltje M Polstra  12 , Slavica Trajkova  24 , Yolande van Bever  14 , Marie José van den Boogaard  13 , Jasper J van der Smagt  13 , Tahsin Stefan Barakat  14 , Mariëlle Alders  3 , Marcel M A M Mannens  3 , Bekim Sadikovic  25 , Mieke M van Haelst  1 , Peter Henneman  26
Affiliations

CUL3-related neurodevelopmental disorder: Clinical phenotype of 20 new individuals and identification of a potential phenotype-associated episignature

Liselot van der Laan et al. HGG Adv. .

Abstract

Neurodevelopmental disorder with or without autism or seizures (NEDAUS) is a neurodevelopmental disorder characterized by global developmental delay, speech delay, seizures, autistic features, and/or behavior abnormalities. It is caused by CUL3 (Cullin-3 ubiquitin ligase) haploinsufficiency. We collected clinical and molecular data from 26 individuals carrying pathogenic variants and variants of uncertain significance (VUS) in the CUL3 gene, including 20 previously unreported cases. By comparing their DNA methylation (DNAm) classifiers with those of healthy controls and other neurodevelopmental disorders characterized by established episignatures, we aimed to create a diagnostic biomarker (episignature) and gain more knowledge of the molecular pathophysiology. We discovered a sensitive and specific DNAm episignature for patients with pathogenic variants in CUL3 and utilized it to reclassify patients carrying a VUS in the CUL3 gene. Comparative epigenomic analysis revealed similarities between NEDAUS and several other rare genetic neurodevelopmental disorders with previously identified episignatures, highlighting the broader implication of our findings. In addition, we performed genotype-phenotype correlation studies to explain the variety in clinical presentation between the cases. We discovered a highly accurate DNAm episignature serving as a robust diagnostic biomarker for NEDAUS. Furthermore, we broadened the phenotypic spectrum by identifying 20 new individuals and confirming five previously reported cases of NEDAUS.

Keywords: CUL3; DNA methylation; NEDAUS; episignature; genotype-phenotype correlation; intellectual disability.

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Conflict of interest statement

Declaration of interests B.S. is a shareholder in EpiSign Inc., a biotech firm involved in the commercial application of EpiSign technology.

Figures

Figure 1
Figure 1
Overview of our CUL3 cohort Cytogenetics band and known genes are represented in this figure using the UCSC genome browser 2009 (GRCh37/hg19) genome build. (A) Deletion of chromosome 2q36.1q36.2 in case 18 is represented by the horizontal red bar and the genes contained within the region listed below. (B) Overview of other CUL3 variants in this cohort. Image is edited from St. Jude Cloud protein paint image (https://pecan.stjude.cloud/proteinpaint).
Figure 2
Figure 2
Facial features of our NEDAUS cases (A and B) Participant 4. (C and D) Participant 7. (E and F) Participant 10. (G and H) Participant 12. (I and J) Participant 17.
Figure 3
Figure 3
Assessment of the NEDAUS episignature (A) The Euclidean hierarchical clustering heatmap shows each column as an NEDAUS discovery case (highlighted in red), along with negative samples (in purple), VUS (in orange), and a VUS+ sample (in yellow). Each row represents a specific probe chosen for this episignature. A clear distinction is evident between the cases (in red) and controls (in blue). (B) The multidimensional scaling (MDS) plot displays the separation between NEDAUS cases and controls, including the negative sample identified in (A). (C) In the SVM classifier model, the selected NEDAUS episignature probes were used for training. Seventy-five percent of controls and 75% of samples from other neurodevelopmental disorders (NDDs; shown in blue) were used for training, while the remaining 25% of controls and 25% of the other disorder samples (gray) were used for testing. The plot indicates that all NEDAUS samples have MVP scores near 1. Additionally, NEDAUS testing samples, including negatives and VUS, generally showed low MVP scores, except for a VUS sample that scored 0.16.
Figure 4
Figure 4
DMPs shared between the NEDAUS cohort and other EpiSign established episignatures (A) Methylation probe overlap, showing the percentage of DMPs shared between disorders on a color scale from white (0%) to red (100%). Each square in the graph indicates the percentage of common probes between two syndromes, with the percentage of DMPs from the syndrome on the bottom bar that also appears in the DMPs of the syndrome on the right-hand side bar. (B) A tree-and-leaf diagram represents each cohort as a node. Syndromes with more similar methylation levels are closer on the tree. Node size corresponds to the ratio of the number of DMPs to the total number of probes, while node color reflects the overall mean methylation difference in the corresponding cohort. (C) Comparison of global mean methylation differences between syndromes with known episignatures, highlighting the overall hypomethylation classifier of the NEDAUS episignature.
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