Panel A shows a heatmap uses a color gradient from blue to red to represent methylation levels, with blue indicating lower methylation and red indicating higher methylation. The samples are categorized into Patient, DEGCAGS, HVDAS T, and Control groups. Panel B presents a scatter plot with multidimensional scaling, displaying the DNA methylation profile of the case (red) compared to subjects with a confirmed DEGCAGS episignature (blue), HVDAS_T patients (purple), and controls (green). Panel C shows another scatter plot with multidimensional scaling, indicating the episignature clustering of parents (black for father and brown for mother) with heterozygous carriers. Panel D is a bar chart displaying the MVP score, a multi-class supervised classification system, with the elevated score for DEGCAGS indicating an episignature similar to the DEGCAGS syndrome reference. Panel E shows DNA electropherograms from Sanger sequencing, reporting the c.153C less than A mutation in the ZNF699 gene present in the case inherited from heterozygous parents. Panel F illustrates the domain architectures of the human ZNF699 protein, highlighting the Krppel-associated box (KRAB) domain and 16 zinc fingers (ZF). The compound heterozygous variants are highlighted in bold, with the variant described in this paper highlighted in bold red. Panel G visualizes the KRAB domain using the predicted structure of the mutated protein, modeled by AlphaFold2 and rendered in UCSF Chimera 1.18, showing the disruption of a hydrogen bond between the amino acid residues at positions 51 and 24 in the mutated protein compared to the wild-type protein.
Immunological evaluations at different time points and EpiSign (DNA methylation) analysis of peripheral blood from a case with a VUS missense ZNF699 variant, the causative gene for DEGCAGS syndrome. (A and B) (A) Hierarchical clustering showing the methylation levels (βvalues) of the most differentially methylated probes across the genome that best discriminate the disorder from all other reference conditions, and (B) multidimensional scaling plots indicate the case (red) has a DNA methylation profile similar to subjects with a confirmed DEGCAGS episignature (blue) and distinct from HVDAS_T patients (purple) and controls (green). (C) Parents’ (black, father, and brown, mother) episignature clusters with heterozygous carriers (gray). (D) MVP score, a multi-class supervised classification system capable of discerning between multiple episignatures by generating a probability score for each episignature. The elevated score for DEGCAGS shows an episignature similar to the DEGCAGS syndrome reference. (E) The DNA electropherogram from Sanger sequencing reports the c.153C>A mutation in the ZNF699 gene present in our case inherited from heterozygous parents. (F) KRAB domain and 16 ZFs. Domain architectures of human ZNF699 protein were given by UniProt Q32M78. The numbers showed up and down of the figure indicate which amino acid are defining the start and stop of the protein, the start and stop of the KRAB domain, and the start of the 16 ZF domains. The compound heterozygous variants are highlighted in bold. The variant described in this paper is highlighted in bold red. (G) Visualization of the KRAB domain using the predicted structure of the mutated protein, modeled by AlphaFold2 and rendered in UCSF Chimera 1.18. The mutation leads to the disruption of a hydrogen bond between the amino acid residues at positions 51 and 24, which is intact in the wild-type protein.