Uncovering a Genetic Diagnosis in a Pediatric Patient by Whole Exome Sequencing: A Modeling Investigation in Wiedemann-Steiner Syndrome

Abstract

Background: Wiedemann-Steiner syndrome (WSS), a rare autosomal-dominant disorder caused by haploinsufficiency of the KMT2A gene product, is part of a group of disorders called chromatinopathies. Chromatinopathies are neurodevelopmental disorders caused by mutations affecting the proteins responsible for chromatin remodeling and transcriptional regulation. The resulting gene expression dysregulation mediates the onset of a series of clinical features such as developmental delay, intellectual disability, facial dysmorphism, and behavioral disorders. Aim of the Study: The aim of this study was to investigate a 10-year-old girl who presented with clinical features suggestive of WSS. Methods: Clinical and genetic investigations were performed. Whole exome sequencing (WES) was used for genetic testing, performed using Illumina technology. The bidirectional capillary Sanger resequencing technique was used in accordance with standard methodology to validate a mutation discovered by WES in all family members who were available. Utilizing computational protein modeling for structural and functional studies as well as in silico pathogenicity prediction models, the effect of the mutation was examined. Results: WES identified a de novo heterozygous missense variant in the KMT2A gene KMT2A(NM_001197104.2): c.3451C>G, p.(Arg1151Gly), absent in the gnomAD database. The variant was classified as Likely Pathogenetic (LP) according to the ACMG criteria and was predicted to affect the CXXC-type zinc finger domain functionality of the protein. Modeling of the resulting protein structure suggested that this variant changes the protein flexibility due to a variation in the Gibbs free energy and in the vibrational entropy energy difference between the wild-type and mutated domain, resulting in an alteration of the DNA binding affinity. Conclusions: A novel and de novo mutation discovered by the NGS approach, enhancing the mutation spectrum in the KMT2A gene, was characterized and associated with WSS. This novel KMT2A gene variant is suggested to modify the CXXC-type zinc finger domain functionality by affecting protein flexibility and DNA binding.

Keywords: KMT2A gene; Wiedemann–Steiner syndrome; modeling; whole exome sequencing.

Figure 1

Patient’s characteristics. Features include mild facial dysmorphisms (hypertelorism, epicanthus, wide nasal bridge, and low-set ears), stubby and puffy hands, and hypertrichosis.

Figure 2

(A) Electropherograms showing the c.3451C>G variant in the index case. The arrow shows the nucleotide substitution. (B) Schematic representation of the Histone lysine N-methyltransferase 2A (UniProt Q03164) showing the localization of the variant p.R1151G (highlighted with the arrow) found in the proband. The various domains are shown in sequential order from NH2 to COOH: CXXC-type zinc finger (CxxC), plant homeodomain (PHD), Bromodomain, extended plant homeodomain (ePHD), FY-rich N-terminal (FYRN), FY-rich C-terminal (FYRC), Su(Var)3-9 enhancer-of-zestetrithorax (SET) and post-SET. (C) Pedigree of proband with mutation in KMT2A and her family. Circles show females, squares show males. An arrowhead indicates the proband. (D) Alignment of the CXXC-type zinc finger domain (the amino acid location p.1151 is showed in red and indicated by a blue arrow) along different species boxed in light blue.

Figure 3

The figure displays the schematic conformation of the original (arginine, R, on the left) and the variant (glycine, G, on the right) amino acid. The backbone, which is the same for each amino acid, is colored in red. The side chain, unique for each amino acid, is colored in black.

Figure 4

Amino acids are color-coded based on the change in vibrational entropy after mutation, with blue indicating increased rigidity and red indicating enhanced flexibility of the structure.

Figure 5

Comparison of the predicted structures of both (left) wild-type (R1151) and (right) variant (G1151) proteins using the Swiss-Pdb Viewer (https://spdbv.unil.ch/ accessed on 15 May 2024). Hydrogen bonds are displayed in light blue dashed lines in the wild-type and mutant proteins. In the structure, the helices are highlighted in red and the strands in yellow. The R1151 (left panel) and G1151 (right panel) products are indicated as green sticks. White sticks indicate DNA.

Figure 6

Molecular characterization and 3D modeling of the KMT2A mutational profile. KMT2A is indicated by the white illustration, and R1151 or G1151 are depicted as white sticks. CpG DNA is indicated as orange and light pink lines. Zn ions are indicated by grey spheres. In the insert at the bottom of the figure, a slight perturbation of the local secondary structure of the CXXC domain is highlighted by comparing the variant (blue arrow) and wild-type (orange arrow) proteins.