The proband was a five-year-old female patient (deceased). The proband (II-1) had the clinical features such as chondrodysplasia, short stature, and global developmental delay (Figure 1A).
The midline structures, including the pituitary gland and suprasellar regions, were normal. The foramen magnum is unremarkable. There was no evidence of basilar invagination. The base of the skull angle measures 179 , suggestive of platybasia. The gray/white matter differentiation was preserved. There was delayed myelination for the patient's age. No definite cortical deformity was identified. Posterior fossa structures were normal with no evidence of Chiari 1 malformation. Prominent and extra-axial CSF spaces with prominent ventricular consistency were observed at infancy. There was no hydrocephalus, hemorrhage, or space-occupying lesion. Posterior fossa structures were normal with no Chiari I change. The visualized parts of the orbit, paranasal sinuses, and labyrinthine structures were normal. There was occurrence of platybasia at the skull base, and delayed myelination for the patient's age was observed (Figure 1B).
The right kidney measures 6.7 cm (previously 6.5 cm), and the left kidney measures 7 cm (previously 6 cm). Both kidneys demonstrated normal parenchymal echogenicity and good corticomedullary differentiation with multiple non-obstructed stones. Persistent bilateral non-obstructing renal stones were more on the right side. Grade 1 right hydronephrosis was observed, along with urinary bladder debris correlating with urinalysis laboratory values.
The enteric feeding tube was placed in the proper position within the stomach. A double stoma was observed in the left upper quadrant. The afferent and efferent loops were observed to be collapsed. The contrast was observed in the efferent loop opacifying the sigmoid colon. The significant proximal jejunum, duodenum, and gastric dilatation were related to high-grade mechanical bowel obstruction. The distal bowel loops were collapsed. The maximal diameter of the small bowel that courses behind the mesentery into the left abdomen was 3.1 cm. However, it was difficult to assess the proximal continuity of the bowel loop. This could be related to an internal hernia. The pelvis had minimal free fluid and several non-obstructive right renal stones. The largest stone recorded was of size .8 cm. The visualized chest parts showed bilateral subsegmental atelectasis/consolidation in the lower lobes. The liver, spleen, pancreas, and adrenals were unremarkable (Figures 1B-E).
The affected individual manifested widened irregular metaphysis with more destructive changes at the proximal femoral ends. In addition, there was a bilateral superolateral displacement of the proximal femur and stippled calcification along the knee joints. Levoscoliosis in the thoracolumbar spine and symmetrical limb shortening were observed. All these skeletal changes favor the chondrodysplasia punctata type of skeletal dysplasia (Figures 1B-E).
DNA of the affected member (II-1) was subjected to WES using standard methods. The variant filtration criteria were based on the gnomAD frequency of <=.0001, CADD-Phred score of >=15, exonic and splice site (+/- 12 bp) variants, and Kaviar allele count of <=10. A de novo nonsense variation in the TBX2 gene (c.529A>T; p.Lys177*; NM_005994.4) was detected which was validated by Sanger sequencing (Figures 1F, G). The variant had a CADD-Phred score of 39 and a GERP++ score of 4.81. Combined Annotation-Dependent Depletion (CADD) and other associated tools are widely used to measure the disease-causing nature of variants that can effectively prioritize causal variants in genetic analyses. These tools are integrative annotation built from several genomic features and can score human single-nucleotide variants and short insertion and deletions effectively. The Lys amino acid at position 177 was highly conserved across different species (Figures 1H, I). According to ACMG, the identified variant was classified as likely pathogenic (Class 2).
Structural analysis of TBX2WT and TBX2LYS177TER revealed several differences between the two proteins. Specifically, in TBX2LYS177TER, the alpha2 and alpha3 regions were fused together to form a single alpha2 region, while the alpha7 region was converted into a loop. Additionally, the loop between alpha7 and alpha8 was converted into a helix (designated alpha8 in TBX2LYS177TER), and the alpha11 region was extended from Lys166 to Ala175 (Figures 1J, K). These structural changes were unique to TBX2LYS177TER and were not present in TBX2WT.
The root mean square deviation (RMSD) is a measure of the stability of a protein structure over time. In this study, the observed RMSD values of 8-12 A for TBX2WT and 10-15 A for TBX2LYS177TER suggest that both systems displayed relatively stable behaviors during the simulation. However, the higher RMSD values for TBX2LYS177TER may indicate that this protein is less stable than TBX2WT. This difference in stability may be due to structural differences between the two proteins, such as the fusion of the alpha2 and alpha3 regions and the conversion of the alpha7 region into a loop in TBX2LYS177TER, which may introduce additional conformational flexibility into the protein. The analysis of fluctuating amino acids also indicates that TBX2LYS177TER is less stable than TBX2WT, as TBX2LYS177TER exhibits increased fluctuation in certain amino acids, including Pro21, Asp23, Ala63, Ala65, Lys112, Trp115, Phe118, Lys148, and Arg164. These structural changes, such as the extension of the alpha11 region and the formation of a new helical region (alpha8) in TBX2LYS177TER, may also contribute to the increased fluctuation in certain amino acids. Additionally, the radius of gyration, which is a measure of the compactness of a protein structure, indicates that TBX2WT has a more compact structure than TBX2LYS177TER. The observed radius of gyration values of 33-35 A for TBX2WT and 25-30 A for TBX2LYS177TER suggest that TBX2WT has a more tightly packed structure. This difference in the packing density may also contribute to the overall stability of the proteins, as a more compact structure may be less prone to conformational changes. In summary, the observed differences in RMSD values, fluctuation in amino acids, and radius of gyration values suggest that TBX2LYS177TER is less stable than TBX2WT. At the 0 ns time scale, both TBX2WT and its mutant form are in open conformations. However, upon reaching the 100 ns time scale, both proteins exhibit a shift toward close conformations. This change is likely due to the close contact between the N- and C-terminals over the course of the simulation. As a result of this interaction, TBX2WT assumes a circular shape, with the inner side forming a circular cavity (as depicted in Figures 2G, H). It is worth noting that the shift from open to close conformations may have different functional consequences for the wild-type and mutant forms of TBX2. Further investigation is necessary to fully understand the impact of these conformational changes on the function of these proteins.