In Silico Analysis of Already Published DNAH6 Mutations: Correlating Structural, Mechanical, Microstructural, and Axonemal Assembly Impacts with Reproductive Health Phenotypes

Background/Objectives: Dynein axonemal heavy chain (DNAH) genes, including DNAH6, are implicated in male infertility, particularly multiple morphological abnormalities of the spermatozoa flagellum (MMAF). However, an underlying mechanism is unclear. Methods: This in silico study analyzed 19 previously reported DNAH6 mutations to elucidate their effects on the structural, mechanical, and microstructural aspects and axonemal assembly of flagellum and how these changes impact reproductive health, correlating with pathogenicity scores, ATP binding capacity, and protein interactions. Results: DNAH mutations were associated with CDGP (52.63%), male infertility (36.84%), and primary ovarian insufficiency (10.53%). MMAF-linked mutations exhibited higher SNAP2 scores (57.25 ± 5.68 vs. −32.58 ± 44.85, p = 0.002), reduced ATP binding affinity (−6.27 ± 4.20 vs. −8.92 ± 0.23 kcal/mol, p = 0.05), and smaller catalytic cavity size (17,646 ± 13,005 vs. 27190 ± 3485 Å3, p = 0.04). These mutations showed reduced DNAH6-CLIP4 binding affinity (−303.90 ± 5.23 vs. −313.60 ± 4.28 kcal/mol, p = 0.002). Literature-based semen analysis revealed correlations between Phred scores and absent flagella (r = 0.952, p = 0.012) and inverse correlations between ATP binding capacity and absent flagella (r = −0.902, p = 0.036) or irregular width (r = −0.949, p = 0.014). A mathematical model of ATP binding kinetics predicted reduced flagellar motility in MMAF mutants due to impaired dynein function. Ultrastructural analyses indicated that high pathogenicity scores and reduced ATP binding correlate with absent inner dynein arms and radial spokes, while impaired DNAH6-CLIP4 interactions disrupt axonemal assembly. Conclusions: In silico analyses, integrated with microstructural, axonemal, and mathematical modeling data, demonstrate that DNAH6 mutations cause MMAF by impairing ATP binding, protein interactions, and axonemal assembly, leading to severe flagellar dysfunction and thereby negatively affecting reproductive health.