The effect of nsSNPs on protein functioning were analysed on 10
different prediction tools built on varying principals to obtain a
holistic evaluation. The combined effect of these predictions determined
pathogenicity of a nsSNP. It was declared as ‘Deleterious/Damaging’
‘(D)’ only if it was predicted so by > 90% of tools that
provided results as described below: -
Our analysis identified that CHD8 is an Intrinsically Disordered Protein
(IDP). For reliable identification, we set a probability/propensity
score cut-offs of ≈ ≥0.7 for tools MoRFCHiBi and IUPred2A, but selected
a probability score of ≈ ≥0.4 for tool MoRFPred relative to the first
two tools. Two distinct IDRs were detected at the N terminal (around aa
1-600) and C terminal regions (2500-2570 aa) of CHD8 separated by
exceptionally ordered, evolutionarily conserved domain region (Figure
4). Although IDRs predicted by different tools were in broad agreement,
we observed wide contradictions while detecting specific MoRF sites.
However, 9 high-confidence MoRF sites and 7 disordered binding sites
were predicted (with consensus across tools) within these two large
terminal IDRs (Table 4, Figure 4).
Compositional bias between disordered and ordered residues was analysed.
While no significant differences were observed among nonpolar residues,
polar aa Proline and Serine were the most common residues within
disordered regions. An overall significant depletion in aromatic and
positively charged aa and enrichment of polar uncharged aa were seen
within disordered regions (Supplementary Figure S1). Since PTMs and IDRs
commonly coincided, 36% residues within IDRs accumulated PTM sites;
with 50, 33, 4 and 3 Phosphorylation, Carboxylation, Ubiquitination,
Sulfation and Acetylation sites, respectively; but just two Methylation
sites that are known as prominent histone modifiers. The tool DISPHOS
detected 84 PTM residues within these terminal IDRs, only 34 PTM sites
contained nsSNPs and just one nsSNP (S1759G) was predicted to be
pathogenic SNP effect analysis. Additionally, these IDRs were found to
be prominent sites for DNA and protein binding (Figure 5A).
Mutation cluster analysis of the prioritised 42 severely pathogenic
nsSNPs identified two statistically significant clusters of aa
substitutions above the clustering MPQS threshold of ≥ 0.5
(Supplementary Table S11A and Figure S2). Tool Mutant3D auto-selected
PDB model 3mwy to evaluate the spatial arrangements of these variants.
The first significant cluster was identified within the Helicase
ATP-binding and SNF2_N domains involving residues 861, 920, 943 (Figure
5B, and Supplementary Figure S2- shown in yellow); whereas the second
cluster included residues 1051, 1264, 1325 and 1333- located around
SNF2_N and Helicase C-terminal domain (shown in red) indicating that
these three domains are central to the precise functioning of the
protein CHD8.
Additionally, we looked for statistically significant patterns of
association between the occurrence of deleterious and destabilising
variations, evolutionarily conserved and variable residues, as well as
PTM sites on residues located within or outside domains of the protein.
Our analysis revealed that there is a significant difference in the
occurrence of truncating SNPs between general and ASD population atP-value 0.0001 (Supplementary Table S11B); that domain residues
hosted severely deleterious aa substitutions than residues outside
(P-value 0.0001 ); however, nsSNPs localisation within domains was
moderately destabilising or stabilising (Supplementary Table S11C).
Evolutionarily conserved residues were prominently segregated within
signature regions (P-value 0.0001 ) and remarkably, PTMs were most
often located outside domains (P-value 0.0108 ).
Importantly, a detailed inspection of the 9 MoRFs identified that they
did not host any truncating SNPs, but contained 21 nsSNPs (2% of the
general population), which were not predicted to be deleterious, but
were destabilising in nature. ASD population did not contain any SNPs
within MoRF sites.
We thank all database and tools included in this study for making them
readily available research use. We acknowledge the support and thank
members of the Genetics and Genomics lab, Department of Studies in
Genetics and Genomics for their help and encouragement. We immensely
thank ICMR for senior-research fellowship provided to ASNM (Award: F.
No. 45/28/2018-HUM/BMS). This research did not receive any specific
research grant from funding agencies in the public, commercial, or
not-for-profit sectors.
The authors declare no competing financial interests.
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