Functional analysis of new variants at the Low Density Lipoprotein Receptor associated with familial hypercholesterolemia

Rodríguez-Jiménez, Carmen; Pernía, Olga; Mostaza, José María; Rodriguez-Antolín, Carlos; García-Díaz, Juan de Dios; Alonso-Cerezo, Concepción; García-Polo, Iluminada; Blanco, Agustín; Lahoz, Carlos; Arrieta, Francisco; Beltrán, Luis M.; Bustamante, Aránzazu Díaz de; Garzón-Lorenzo, Lucía; Álvarez-Sala, L.; Asenjo, A; Cáceres, Inmaculada Ibáñez de; Rodrı́guez-Nóvoa, Sonia

doi:10.1002/humu.23801

Cited by 9 publications

(7 citation statements)

References 35 publications

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“…Simultaneously expressing the variants and wild-type protein in cells to check the diversity of their function hinted at a pathogenesis role. Despite being widely used, several drawbacks exist, particularly the difficulty of normalizing the diversity of their function to their expression (24)(25)(26). CRISPR/Cas9 enables the editing of genomic DNA directly, excluding the impact of the aforementioned issues.…”

Section: Discussionmentioning

confidence: 99%

Integrative identification of the pathogenic role of a novel G6PD missense mutation c.697G>C

Zhang¹,

Peng²,

Shu³

et al. 2021

Ann Transl Med

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Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a hereditary disease caused by pathogenic mutations of G6PD. While most of the pathogenic variants of G6PD have been annotated, hemolysis of unknown etiology but analogous to that in G6PD deficiency persists, implying the existence of undocumented pathogenic variants. In our previous study, we reported four novel G6PD variants in China, for which the pathogenicity remains to be verified. Methods:The variants were verified by exogenous expression in HEK-293 cells, and their functions were predicted by PolyPhen-2 and SIFT. The CRISPR/Cas9 system was exploited to edit the G6PD c.697G>C variant in HEK-293 cells and K562 cells. The expression of G6PD was detected by quantitative PCR (qPCR) and western blotting. The cell growth capacity was detected by the CCK-8 assay and crystal violet staining. The G6PD enzyme activity was reflected by the G6P/6PG ratio test. The apoptosis of cells was detected by Annexin V-APC/7-AAD staining. The secondary and crystallographic structures were denoted according to the literature and PyMOL software. The G6PD protein was purified from lysis of transformed Escherichia coli (E. coli) cell with Ni-charged Resin Column. The enzymatic activity was detected at different temperatures.Results: The G6PD activity of exogenous G6PD c.697G>C in HEK-293 cells was significantly lower than that of wild type (WT) G6PD, a finding that was consistent with the observation in clinical samples. The functional predictions conducted by different algorithms indicated the damage role of the G6PD c.697G>C variant in its enzymatic activity. We recapitulated the G6PD c.697G>C variant both in HEK-293 cells and K562 cells by adapting the CRISPR/Cas9 strategy. Using distinct cell lines expressing the G6PD c.697G>C variant endogenously, we confirmed the deteriorative role of the G6PD c.697G>C variant in its enzymatic activity. Regarding the secondary and crystallographic structure, we found a mutated amino acid approaching the structural NADP + binding site. Finally, we demonstrated the c.697G>C variant compromised the thermal stability of G6PD protein.Conclusions: Our data delineated the pathogenic role of G6PD c.697G>C variant for G6PD deficiency,

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Section: Discussionmentioning

confidence: 99%

Integrative identification of the pathogenic role of a novel G6PD missense mutation c.697G>C

Zhang¹,

Peng²,

Shu³

et al. 2021

Ann Transl Med

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“…LDLR variants were classified as activity defective or activity negative ("null") according to variant type; nonsense, frameshift and copy number variants were classed as "null" and other variants classed as "defective" unless functional studies have demonstrated otherwise. 12,13 The association between GRS and CAD was investigated using logistic regression analyses that adjusted for the established predictors of increased CAD risk: age, sex, hypertension, type-2 diabetes, smoking status, Lp(a) levels and treatment-adjusted LDL-c. The association between GRS and coronary calcium score was investigated using linear regression adjusting for the same established predictors of CAD.…”

Section: Discussionmentioning

confidence: 99%

“…The association between GRS and clinical and anthropometric characteristics was assessed using linear and logistic regression as appropriate. LDLR variants were classified as activity defective or activity negative (“null”) according to variant type; nonsense, frameshift and copy number variants were classed as “null” and other variants classed as “defective” unless functional studies have demonstrated otherwise . The association between GRS and CAD was investigated using logistic regression analyses that adjusted for the established predictors of increased CAD risk: age, sex, hypertension, type‐2 diabetes, smoking status, Lp(a) levels and treatment‐adjusted LDL‐c.…”

Section: Methodsmentioning

confidence: 99%

A genetic risk score predicts coronary artery disease in familial hypercholesterolaemia: enhancing the precision of risk assessment

et al. 2019

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Familial hypercholesterolaemia (FH) is associated with increased risk of coronary artery disease (CAD); however, risk prediction and stratification remain a challenge.Genetic risk scores (GRS) may have utility in identifying FH patients at high CAD risk.The study included 811 patients attending the lipid disorders clinic at Royal Perth Hospital with mutation-positive (n = 251) and mutation-negative (n = 560) FH. Patients were genotyped for a GRS previously associated with CAD. Associations between the GRS, clinical characteristics, and CAD were assessed using regression analyses. The average age of patients was 49.6 years, and 44.1% were male. The GRS was associated with increased odds of a CAD event in mutation-positive [odds ratio (OR) = 3.3; 95% confidence interval (CI) = 1.3-8.2; P = .009] and mutationnegative FH patients (OR = 1.8; 95% CI = 1.0-3.3; P = .039) after adjusting for established predictors of CAD risk. The GRS was associated with greater subclinical atherosclerosis as assessed by coronary artery calcium score (P = .039). A high GRS was associated with CAD defined clinically and angiographically in FH patients. High GRS patients may benefit from more intensive management including lifestyle modification and aggressive lipid-lowering therapy. Further assessment of the utility of the GRS requires investigation in prospective cohorts, including its role in influencing the management of FH patients in the clinic.

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“…Traditionally, the uptake and degradation of 125 I‐labelled LDL in cultured skin fibroblasts was measured . A variety of methods is now used, including flow cytometry of transfected Chinese hamster ovary cells using fluorescent‐labelled LDL to determine receptor activity, with immunofluorescence detected by confocal laser scanning microscopy to assess LDL receptor expression and localisation within the cell; and mRNA studies …”

Section: Pathophysiology and Molecular Geneticsmentioning

confidence: 99%

“…12 A variety of methods is now used, including flow cytometry of transfected Chinese hamster ovary cells using fluorescent-labelled LDL to determine receptor activity, with immunofluorescence detected by confocal laser scanning microscopy to assess LDL receptor expression and localisation within the cell; and mRNA studies. [13][14][15][16] The reality of the contemporary investigation of FH has been an explosion in the detection of LDLR variants, many of which are labelled as causative, but functional assays of LDL receptor activity, representing the gold-standard confirmation of pathogenicity, are rarely performed. Generally, the pathogenicity of a new variant is assessed by a number of means, supported by guidelines from the American College of Medical Genetics and Genomics (ACMG).…”

Section: Variants In Ldlrmentioning

confidence: 99%

Widening the spectrum of genetic testing in familial hypercholesterolaemia: Will it translate into better patient and population outcomes?

2019

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Familial hypercholesterolaemia (FH) is caused by pathogenic variants in LDLR, APOB or PCSK9. Impaired low‐density lipoprotein (LDL) receptor function leads to decreased LDL catabolism and premature atherosclerotic cardiovascular disease (ASCVD). Thousands of LDLR variants are known, but assignation of pathogenicity requires accurate phenotyping, family studies and assessment of LDL receptor function. Precise, genetic diagnosis of FH using targeted next generation sequencing allows for optimal treatment, distinguishing FH from pathogenically distinct disorders requiring different treatment. Polygenic hypercholesterolaemia resulting from an accumulation of LDL cholesterol‐raising single nucleotide polymorphisms (SNPs) could also be suspected by this approach. Similarly, ASCVD risk could be estimated by broader sequencing of cholesterol and non‐cholesterol‐related genes. Both of these areas require further research. The clinical management of FH, focusing on the primary or secondary prevention of ASCVD, has been boosted by PCSK9 inhibitor therapy. The efficacy of PCSK9 inhibitors in homozygous FH may be partly predicted by the LDLR variants. While expanded genetic testing in FH is clinically useful in providing an accurate diagnosis and enabling cost‐effective testing of relatives, further research is needed to establish its value in improving clinical outcomes.

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Functional analysis of new variants at the Low Density Lipoprotein Receptor associated with familial hypercholesterolemia

Cited by 9 publications

References 35 publications

Integrative identification of the pathogenic role of a novel G6PD missense mutation c.697G>C

Integrative identification of the pathogenic role of a novel G6PD missense mutation c.697G>C

A genetic risk score predicts coronary artery disease in familial hypercholesterolaemia: enhancing the precision of risk assessment

Widening the spectrum of genetic testing in familial hypercholesterolaemia: Will it translate into better patient and population outcomes?

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