Genetic Spectrum of Familial Hypercholesterolaemia in the Malaysian Community: Identification of Pathogenic Gene Variants Using Targeted Next-Generation Sequencing

Razman, A.Z.; Chua, Yung-An; Kasim, Noor Alicezah Mohd; Al-Khateeb, Alyaa; Kadir, Siti Hamimah Sheikh Abdul; Jusoh, Siti Azma; Nawawi, Hapizah Md

doi:10.3390/ijms232314971

Cited by 6 publications

(8 citation statements)

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“…This variant is located at the second disulphide-rich repeat in the receptor protein binding domain and impacts the processing and intracellular transport of the generated protein ( 27 ). Moreover, some studies have suggested a damaging outcome of this variant ( 28 , 29 ).…”

Section: Discussionmentioning

confidence: 99%

Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia

Saidi,

Md Rani,

Sulaiman

et al. 2024

MJMS

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Background: The techniques for detecting single nucleotide polymorphisms (SNP) require lengthy and complex experimental procedures and expensive instruments that may only be available in some laboratories. Thus, a deoxyribonucleic acid (DNA)-based lateral flow assay (LFA) was developed as a point-of-care test (POCT) diagnostic tool for genotyping. In this study, single nucleotide variation (E101K) in the low-density lipoprotein receptor (LDLR) gene leading to familial hypercholesterolemia (FH) was chosen as a model. Methods: Hypercholesterolemic individuals (n = 103) were selected from the Malaysian Cohort project (UKM Medical Molecular Biology Institute) while the control samples were selected from the Biobank (UKM Medical Molecular Biology Institute). The DNA samples were isolated from whole blood. Polymerase chain reaction (PCR) amplification process was performed using bifunctional labelled primers specifically designed to correspond to the variant that differentiates wild-type and mutant DNA for visual detection on LFA. The variant was confirmed using Sanger sequencing, and the sensitivity and specificity of the LFA detection method were validated using the Agena MassARRAY® technique. Results: Out of 103 hypercholesterolemic individuals, 5 individuals (4.8%) tested positive for E101K, LDLR mutation and the rest, including healthy control individuals, tested negative. This result was concordant with Sanger sequencing and Agena MassARRAY®. These five individuals could be classified as Definite FH, as the DNA diagnosis was confirmed. The sensitivity and specificity of the variant detection by LFA is 100% compared to results using the genotyping method using Agena MassARRAY®. Conclusion: The developed LFA can potentially be used in the POC setting for detecting the E101K variant in the LDLR gene. This LFA can also be used to screen family members with E101K variant in the LDLR gene and is applicable for other SNP’s detection.

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Section: Discussionmentioning

confidence: 99%

Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia

Saidi,

Md Rani,

Sulaiman

et al. 2024

MJMS

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“…The PCSK9 variants, E498A and R499G were discovered by the study of Razman et al, (2022). Both novel variants were identified among 35 clinically diagnosed Malaysian FH patients and classified as likely pathogenic according to ACMG guidelines (Richards et al, 2015).…”

Section: Modeling Of the Pcsk9 Variantsmentioning

confidence: 99%

“…In this work, we employed molecular dynamics (MD) simulations to examine the dynamics of LDLR structure while interacting with PCSK9. Additionally, we investigated the impact of two novel PCSK9 variants, E498A and R499G, found in Malaysia FH patients (Razman et al, 2022) on the LDLR-PCSK9 complex and their interactions. The simulations captured the behavior and conformational changes of the LDLR structure while interacting with PCSK9.…”

Section: Introductionmentioning

confidence: 99%

Structural Dynamics of LDL Receptor Interactions with E498A and R499G Variants of PCSK9

Azhar,

Nawawi,

Chua

et al. 2024

Preprint

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The low-density lipoprotein receptor (LDLR) plays an integral role in cellular cholesterol uptake and lipid metabolism by primarily regulating hepatic clearance of plasma low-density lipoprotein cholesterol (LDL-C). Physiologically, proprotein convertase subtilisin/kexin type-9 (PCSK9) attenuates LDLR function by binding to the LDLR extracellular domain, leading to its lysosomal degradation and thereby preventing the total depletion of circulating LDL-C. However, pathogenic variants of PCSK9 are able to reduce the availability of LDLR, thus significantly increasing plasma LDL-C levels. Despite this understanding, the detailed molecular mechanism of LDLR-PCSK9 interaction remains elusive due to the lack of a full atomistic structure of LDLR. In this study, molecular dynamics (MD) simulations were employed to predict LDLR structural dynamics upon binding to PCSK9. Furthermore, two PCSK9 variants, E498A and R499G that were identified in clinically diagnosed Malaysian FH patients were investigated for their mutational effects. The simulations, spanning 500 ns, were conducted for three LDLR-PCSK9 complexes: LDLR-PCSK9 wild-type (WT), LDLR-PCSK9 (E498A), and LDLR-PCSK9 (R499G). Throughout the simulations, PCSK9 structure remained highly stable, in contrast to the LDLR structure that sampled large conformational space. The WT complex exhibited the least change, whereas the R499G complex displayed the most pronounced conformational rearrangement. During the simulations of WT and E498A complexes, the beta-propeller domain of LDLR formed interactions with the prodomain of PCSK9. Aligned with the observation, the MM/GBSA analysis revealed that the E498A complex exhibited the highest LDLR-PCSK9 binding affinity (-63.81 kcal/mol), followed by the WT complex (-33.07 kcal/mol), and the R499G complex (-24.21 kcal/mol). These findings provide novel insights into the dynamic interactions between LDLR and PCSK9, highlighting the importance of structural flexibility in mediating their functional relationship. Further studies with complete LDLR structures are required to fully elucidate the molecular mechanisms underlying LDLR-PCSK9-mediated cholesterol homeostasis.

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“…A recent systematic review and meta-analysis showed that the pooled prevalence of HeFH based on genetic testing or established clinical diagnostic criteria among the general adult population was 1 in 303 [ 14 ]. In Malaysia, the prevalence of clinically diagnosed HeFH was estimated to be 1 in 100 [ 15 ], while the prevalence of genetically confirmed HeFH was recently reported as 1 in 427 [ 16 ]. This is comparable to the prevalence in other countries [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Case Series of Genetically Confirmed Index Cases of Familial Hypercholesterolemia in Primary Care

et al. 2023

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Objective:Rare disease Background:In Malaysia, the prevalence of genetically confirmed heterozygous familial hypercholesterolemia (FH) was reported as 1 in 427. Despite this, FH remains largely underdiagnosed and undertreated in primary care. Case Reports:In this case series, we report 3 FH cases detected in primary care due to mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein-B (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. The mutations in case 1 (frameshift c.660del pathogenic variant in LDLR gene) and case 2 (missense c.10579C>T pathogenic variant in APOB gene) were confirmed as pathogenic, while the mutation in case 3 (missense c.277C>T mutation in PCSK9 gene) may have been benign. In case 1, the patient had the highest LDL-c level, 8.6 mmol/L, and prominent tendon xanthomas. In case 2, the patient had an LDL-c level of 5.7 mmol/L and premature corneal arcus. In case 3, the patient had an LDL-c level of 5.4 mmol/L but had neither of the classical physical findings. Genetic counseling and diagnosis were delivered by primary care physicians. These index cases were initially managed in primary care with statins and therapeutic lifestyle modifications. They were referred to the lipid specialists for up-titration of lipid lowering medications. First-degree relatives were identified and referred for cascade testing. Conclusions:This case series highlights different phenotypical expressions in patients with 3 different FH genetic mutations. Primary care physicians should play a pivotal role in the detection of FH index cases, genetic testing, management, and cascade screening of family members, in partnership with lipid specialists.

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Genetic Spectrum of Familial Hypercholesterolaemia in the Malaysian Community: Identification of Pathogenic Gene Variants Using Targeted Next-Generation Sequencing

Cited by 6 publications

References 104 publications

Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia

Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia

Structural Dynamics of LDL Receptor Interactions with E498A and R499G Variants of PCSK9

Case Series of Genetically Confirmed Index Cases of Familial Hypercholesterolemia in Primary Care

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