To our knowledge, this is the largest cohort to date to be used to characterize the genetic spectrum of the Vietnamese population, screening 110 FH index cases for LDLR, APOB, and PCSK9 mutations. Overall, we obtained an FH mutation detection rate of 43.6% and found four novel variants (14.3%), whereas 56.4% of the clinical FH index cases did not present any LDLR, APOB, or PCSK9 mutations. Notably, a recent study also reported that 52% of clinical FH index cases did not present an identifiable mutation in any of these genes 11, suggesting that some patients may have mutations in other genes and polygenic dyslipidemia. We discovered that most index cases were attributed to LDLR variants and rarely to PCSK9, similar to recent reports in Asian Indians 12. However, APOB mutations in the Vietnamese population appear to be common, accounting for 6/28 (21.4%) of mutations and 6/48 (12.5%) of index cases carrying mutations, whereas mutations in APOB accounted for 5% of cases in another study 13. Notably, the six index cases carrying APOB mutations in our study were identified using NGS. In recent reports, using NGS techniques, the index case rate carrying APOB mutations tended to be higher, for example, at 26.05% in FH Chinese populations 14. Furthermore, our findings confirmed the genotype–phenotype relationships, with a trend for more severe clinical symptoms in HoFH and cHeFH index cases, consistent with other studies 14,15.
Our study also observed relationships between the genotype and phenotype in FH patients. HoFH patients had a high prevalence of premature ASCVD and an average LDL-C level of 14.83 mmol/L, which are consistent with the typical clinical presentation of HoFH described in the 2023 updated clinical guidance of the European Atherosclerosis Society Consensus 16. The HoFH patients also exhibited higher levels of total cholesterol (TC) and LDL-C than did the HeFH patients, consistent with the previous data on the differences between HoFH and HeFH phenotypes 15. We observed LDL-C levels of 5–10 mmol/L in HeFH patients, consistent with recent global data on mean LDL-C levels in HeFH 17. These findings support the trend of using the lipid profile as an initial screening tool for FH. Furthermore, we noted slight differences in plasma LDL-C levels among patients with different mutation sites. Plasma LDL-C levels were higher in patients with LDLR-negative mutations than in patients with LDLR-defective mutations 15; however, comprehensive data comparing different mutant sites are currently limited and unclear. Notably, we identified that some HeFH patients have slightly increased LDL-C levels (< 5 mmol/L) despite testing positive for mutations, while some patients who tested negative for mutations had plasma LDL-C levels of > 10 mmol/L. This finding suggests that genetic testing should be still considered if FH is suspected to confirm the type of FH and to plan an appropriate management strategy.
Our study explored mutations in LDLR, APOB, and PCSK9 using homology modeling. We adopted a unique approach by collectively investigating all three proteins and examining mutations in different regions. In silico analysis allows for more efficient prediction of clinical severity compared with in vitro methods. Previous bioinformatics studies have also shed light on the association between the genotype and phenotype of altered residues 18–20. We conducted molecular dynamics simulations and observed the impact of several LDLR mutations on protein structure and function. For example, the Asp227Glu mutation leads to enhanced protein stability, as indicated by changes in the polar and apolar regions. This mutation also affected the surface and buried residues, potentially influencing protein interactions. These findings align with those of previous research highlighting the influence of surface properties on protein stability and interactions 21. Similarly, His583Tyr, Ser648Pro, Ala684Thr, and Pro476Arg mutations exhibited changes in polar and apolar regions, indicating potential impacts on protein stability and interactions 7,22–27. Mutations in non-conserved regions, such as Gln660Ter, Cys104Tyr, and Cys318Arg, also disrupted protein stability and interactions, which could have broader implications. Changes in hydrophobic and hydrophilic regions observed in mutations such as Ser130Ter and Arg257Trp may affect protein stability and folding 22,28. Furthermore, mutations such as Val797Met, Gly844Ser, Asp843Glufs*86, and Val806fs led to residue modifications, potentially influencing protein activity and stability [46–48]. Our study also highlighted the impact of these mutations on the interaction between mutant LDLR and APOB, which is crucial for lipoprotein transport and cholesterol metabolism 29–31.
In terms of the molecular dynamics of LDLR mutants, Asp227Glu showed structural differences from the wild-type, indicating its potential impact on protein function and stability. Similarly, His583Tyr and Ser648Pro exhibited structural dissimilarities and increased protein flexibility, potentially affecting domain structure and stability 7. Ala684Thr exhibited structural dissimilarity and increased flexibility with potential consequences for protein stability, interactions, and function, particularly in the context of its location within the EGF-like 3 domain 23. By contrast, Pro476Arg showed slight structural dissimilarity to the wild-type and increased flexibility. This mutation, located in the EGF spacer domain of LDLR, is conserved across species and may exert pathogenic effects upon binding to APOB. This study also highlighted the impact of mutations in non-conserved regions such as Gln660Ter, Cys104Tyr, and Cys318Arg on protein stability, flexibility, and potential disruptions in binding affinity with APOB 23. The Cys104Tyr mutation, located in the semi-conserved region of LDLR class A 2, may affect the binding between LDLR and APOB. By contrast, Ser130Ter and Arg257Trp mutations showed significant structural changes, with Arg257Trp being non-pathogenic owing to its location in a non-conserved region of the LDLR class A 6 domain. Val797Met and Gly844Ser mutations exhibited structural similarity, but increased protein flexibility, potentially influencing their interactions and stability. Meanwhile, Asp843Glufs*86 and Val806fs mutations, although causing structural changes, led to an increased stability and reduced flexibility 32. This comprehensive analysis of various LDLR mutations underscores the intricate relationships among genetic variation, protein structure, and function. These mutations can influence protein stability, interactions, and dynamics, ultimately affecting their role in lipid metabolism and associated diseases. FH mutations, especially novel mutations such as Pro476Arg, have been extensively studied and provide valuable insights into lipid metabolism and genetic causes of FH. These mutations serve as reference points for understanding FH. This study identified new mutations that shed light on the pathophysiology of FH and identified potential therapeutic targets. By examining the conserved, semi-conserved, and non-conserved regions, this study revealed how these mutations affect protein behavior and stability. The dynamic nature of genetic diversity in FH underscores the need for ongoing research to address the emerging clinical challenges.
When studying APOB mutations, several key observations were made. The Arg1386Trp mutation led to a decrease in buried residues, implying greater accessibility of the protein interior to solvent molecules. Accessibility influences protein stability and interactions. Additionally, there was an increase in polar, apolar, and surface residues, indicating enhanced interactions with the environment. An increase in hydrophobic (apolar) and surface residues suggests an increased stability 33. In the case of the Phe2469Cys mutation, no significant differences were observed between the buried and surface residues. However, a decrease in the apolar region indicates reduced stability, while an increase in the polar region indicates potential disruptions in interactions with water molecules and local microenvironment changes 34. Conversely, the Arg4297His mutation showed an increase in the apolar region and surface residues, suggesting an increased interaction with other molecules. Although an increase in hydrophobic residues indicated stability, the increased surface residues implied a greater likelihood of interaction with other proteins, possibly leading to protein aggregation. This aggregation can affect protein stability and cellular health 35. Structural dissimilarity was observed among the mutations, as indicated by the RMSD differences. Molecular dynamics simulations showed that the Arg1386Trp mutation increased stability and reduced flexibility. By contrast, the Phe2469Cys and Arg4297 mutations resulted in protein structural instability and increased flexibility. These mutations could affect the binding between APOB and LDLR, affecting the strength and specificity of their interactions, and potentially altering the stability and conformation of the complex.
Exon 1 of PCSK9, which codes the signal peptide domain, is polymorphic because it contains a repeat region, which may make the interpretation of variants in this region difficult. Differences in PCSK9 activity seem to depend on the number of Leucine residues in the signal peptide domain. For examples, the p.Leu23del mutation does not affect PCSK9 activity, but the p.Leu22_Leu23dup mutation increases intracellular degradation of LDLR 36. The Leu23_Gly24insLeu mutation, an in-frame insertion, was previously reported to be associated with low plasma LDL-C levels, but this has not yet been explained using molecular pathogenesis 37. However, our study observed plasma LDL-C levels of 8.21 mmol/L in a 9-year-old boy with the Leu23_Gly24insLeu mutation and did not identify other mutations to explain the hypercholesterolemia. In addition, for the Leu23_Gly24insLeu mutation in PCSK9 mutation, significant decreases in polar, apolar, surface, and buried residues were observed, indicating that these modifications led to major changes in the physicochemical characteristics of the protein, which may have an impact on its interactions, stability, and function. A slight increase in the superimposed RMSD was observed, indicating a slight structural dissimilarity between the wild-type and mutant models. The RMSD and RMSF values obtained from the molecular dynamics simulation analysis indicated that the protein became less stable and flexible than the wild-type after mutation. As this mutation lies in a non-conserved region it is possible that this mutation might affect the binding affinity, stability, and overall function of the PCSK9, APOB, and LDLR complex formed by these interactions. Moreover, this mutation could potentially disrupt normal protein–protein interactions and alter the structural integrity of the complex. This could, in turn, affect crucial biological processes, such as lipid metabolism and cholesterol regulation.
This study has some limitations. First, due to the absence of appropriate templates, the complete structures of LDLR, APOB, and PCSK9 were not fully modeled using homology techniques. Additionally, most studies rely on computational methods, such as simulations and homology modeling, which cannot accurately represent actual protein functions. Nonetheless, the findings of this study provide valuable insights into potential effects of various FH mutations, setting the stage for further exploration of the genetic and molecular factors contributing to this inherited condition.
In summary, our study confirmed the existence of FH mutations and genotype–phenotype relationships in Vietnamese populations. In this study, we investigated how these mutations affect protein stability, interactions, and dynamics, based on homology modeling and molecular dynamics simulations. These findings contribute to predicting disease severity and understanding the genotype–phenotype relationships. Novel mutations have expanded our knowledge on the genetic diversity of FH and its potential therapeutic targets. This study provides valuable insights into FH mutations and lays the foundation for the further understanding of molecular consequences and development of potential treatments.