A novel pathogenic missense variant in CNNM4 underlying Jalili syndrome: Insights from molecular dynamics simulations

Abstract

Background: Jalili syndrome (JS) is a rare cone-rod dystrophy (CRD) associated with amelogenesis imperfecta (AI). The first clinical presentation of JS patients was published in 1988 by Jalili and Smith. Pathogenic mutations in the Cyclin and CBS Domain Divalent Metal Cation Transport Mediator 4 (CNNM4) magnesium transporter protein have been reported as the leading cause of this anomaly.

Methods: In the present study, a clinical and genetic investigation was performed in a consanguineous family of Pakistani origin, showing characteristic features of JS. Sanger sequencing was successfully used to identify the causative variant in CNNM4. Molecular dynamics (MD) simulations were performed to study the effect of amino acid change over CNNM4 protein.

Results: Sequence analysis of CNNM4 revealed a novel missense variant (c.1220G>T, p.Arg407Leu) in exon-1 encoding cystathionine-β-synthase (CBS) domain. To comprehend the mutational consequences in the structure, the mutant p.Arg407Leu was modeled together with a previously reported variant (c.1484C>T, p.Thr495Ile) in the same domain. Additionally, docking analysis deciphered the binding mode of the adenosine triphosphate (ATP) cofactor. Furthermore, 60ns MD simulations were carried out on wild type (p.Arg407/p.Thr495) and mutants (p.Arg407Leu/p.Thr495Ile) to understand the structural and energetic changes in protein structure and its dynamic behavior. An evident conformational shift of ATP in the binding site was observed in simulated mutants disrupting the native ATP-binding mode.

Conclusion: The novel identified variant in CNNM4 is the first report from the Pakistani population. Overall, the study is valuable and may give a novel insight into metal transport in visual function and biomineralization.

Keywords: CNNM4; CBS domain; Jalili Syndrome; MD Simulations; missense variant.

Figure 1

Sequence analysis of a novel variant (c.1220G>T, p.Arg407Leu) identified in Cyclin and CBS Domain Divalent Metal Cation Transport Mediator 4; (a) represents an affected individual (IV‐3) with the homozygous sequence variation (CGC‐Arg_CTC‐Leu) (b) the heterozygous carrier (III‐2) and (c) represents the wildtype nucleotide sequence in an unaffected individual (IV‐1). Panel (d) shows partial amino acid sequence comparison of human Cyclin and CBS Domain Divalent Metal Cation Transport Mediator 4 protein with other orthologs. A red box indicates arginine (R) 407 residue conserved across different species

Figure 2

. The RMSD trajectories of the Cyclin and CBS Domain Divalent Metal Cation Transport Mediator 4 backbone atoms (a) throughout 60ns for wt (black), Arg407Leu (turquoise), and Thr495Ile (red); and RMSF of backbone atoms relative to wt are displayed for Arg407Leu (b), and Thr495Ile (c) respectively. The comparison of conformational changes after every 20ns concerning the starting structure is interactively presented (in the ribbon) for wt (d), Arg407Leu (e) and Thr495Ile (f), complexed with ATP molecule (in the sticks), respectively

Figure 3

(a) The structural comparison of wt (in gray) and Arg407Leu (in blue) complexed with ATP molecule (with the same color code) after 60ns molecular dynamics simulation period. The formation of hydrogen bonds with distance in Angstrom (Å) between wt and Arg407Leu complexes are shown in close‐ups (b and c) while the movement of ATPtriphosphate is indicated with an arrow (in red)

Figure 4

The structural comparison of wt (in gray) and Thr495Ile (in blue) complexed with ATP molecule (with the same color code) after 60ns molecular dynamics simulation period. The formation of hydrogen bonds with distance in Angstrom (Å) between wt and Thr495Ile complexes is shown in the right close‐up