Increasing the Sensitivity of Single-Strand Conformation Polymorphism Analysis of the LDLR Gene Mutations in Brazilian Patients with Familial Hypercholesterolemia
-
Luis A. Salazar
Abstract
Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH), one of the most common single gene disorders. It is thought that FH affects approximately 1 of 500 individuals in most populations. Single-strand conformation polymorphism (SSCP) analysis is widely used to detect mutations in the LDLR gene. However, several factors such as temperature, pH, running time, gel composition and size of the DNA fragments can influence its sensitivity. We have optimized the electrophoretic conditions to screen mutations in the promoter region and exons 1–18 of the LDLR gene by varying temperature (5 °C, 8 °C, 12 °C and 15 °C), voltage (300 to 600 V), and running time (1 to 4 hours) in the semi-automated GenePhor™ system (Amersham Biosciences). The efficiency of the method was evaluated by using 30 positive controls (DNA samples with mutations and polymorphisms in the LDLR gene, previously characterized) and DNA samples from 90 Brazilian patients with FH. Our results show that the use of two temperatures (5 °C and 15 °C) in combination with other optimized conditions resulted in high mutation detection rate (97%), which was considered appropriate for routine screening. Therefore, this strategy could be useful for the diagnosis of genetic disorders, cancer, and for pharmacogenetic studies.
Copyright © 2002 by Walter de Gruyter GmbH & Co. KG
Articles in the same Issue
- XV Latin American Congress of Clinical Biochemistry, Florianópolis, Brazil, 1–5 July 2001
- A Method to Detect the G894T Polymorphism of the NOS3 Gene. Clinical Validation in Familial Hypercholesterolemia
- Increasing the Sensitivity of Single-Strand Conformation Polymorphism Analysis of the LDLR Gene Mutations in Brazilian Patients with Familial Hypercholesterolemia
- Hormone Replacement Therapy in Postmenopausal Women and Its Effects on Plasma Lipid Levels
- The Effect of N-Acetylcysteine Supplementation upon Viral Load, CD4, CD8, Total Lymphocyte Count and Hematocrit in Individuals Undergoing Antiretroviral Treatment
- An Evaluation of Antiretroviral Therapy Associated with α-Tocopherol Supplementation in HIV-infected Patients
- Effects of Re-education in Eating Habits and Physical Activity on the Lipid Profile of Obese Teenagers
- Myeloperoxidase-mediated Protein Oxidation: Its Possible Biological Functions
- Novel Haptoglobin Insertion/Deletion Polymorphism Is Associated with the Lipid Profile and C-Reactive Protein (CRP) Concentration
- Apolipoprotein E2/E2 Genotype in Combination with Mutations in the LDL Receptor Gene Causes Type III Hyperlipoproteinemia
- High-Sensitivity Human Thyroglobulin (hTG) Immunoradiometric Assay in the Follow-up of Patients with Differentiated Thyroid Cancer
- Tissue Transglutaminase-Serology Markers for Coeliac Disease
- Serum Adenosine Deaminase and Cytidine Deaminase Activities in Patients with Systemic Lupus Erythematosus
- Effects of Oral N-Acetylcysteine on Plasma Homocysteine and Whole Blood Glutathione Levels in Healthy, Non-pregnant Women
- Evaluating Sequential Values Using Time-adjusted Biological Variation
- High-dose Methylprednisolone Therapy in Multiple Sclerosis Increases Serum Uric Acid Levels
- Evaluation of Accuracy and Uncertainty of ELISA Assays for the Determination of Interleukin-4, Interleukin-5, Interferon-γ and Tumor Necrosis Factor-α
- Development of Immunoturbidimetric Assays for Fourteen Human Serum Proteins on the Hitachi 912™
- Multicenter Evaluation of a Fully Mechanized Soluble Transferrin Receptor Assay on the Hitachi and Cobas Integra Analyzers. The Determination of Reference Ranges
- IFCC News: May/June 2002
- Meetings and Awards
Articles in the same Issue
- XV Latin American Congress of Clinical Biochemistry, Florianópolis, Brazil, 1–5 July 2001
- A Method to Detect the G894T Polymorphism of the NOS3 Gene. Clinical Validation in Familial Hypercholesterolemia
- Increasing the Sensitivity of Single-Strand Conformation Polymorphism Analysis of the LDLR Gene Mutations in Brazilian Patients with Familial Hypercholesterolemia
- Hormone Replacement Therapy in Postmenopausal Women and Its Effects on Plasma Lipid Levels
- The Effect of N-Acetylcysteine Supplementation upon Viral Load, CD4, CD8, Total Lymphocyte Count and Hematocrit in Individuals Undergoing Antiretroviral Treatment
- An Evaluation of Antiretroviral Therapy Associated with α-Tocopherol Supplementation in HIV-infected Patients
- Effects of Re-education in Eating Habits and Physical Activity on the Lipid Profile of Obese Teenagers
- Myeloperoxidase-mediated Protein Oxidation: Its Possible Biological Functions
- Novel Haptoglobin Insertion/Deletion Polymorphism Is Associated with the Lipid Profile and C-Reactive Protein (CRP) Concentration
- Apolipoprotein E2/E2 Genotype in Combination with Mutations in the LDL Receptor Gene Causes Type III Hyperlipoproteinemia
- High-Sensitivity Human Thyroglobulin (hTG) Immunoradiometric Assay in the Follow-up of Patients with Differentiated Thyroid Cancer
- Tissue Transglutaminase-Serology Markers for Coeliac Disease
- Serum Adenosine Deaminase and Cytidine Deaminase Activities in Patients with Systemic Lupus Erythematosus
- Effects of Oral N-Acetylcysteine on Plasma Homocysteine and Whole Blood Glutathione Levels in Healthy, Non-pregnant Women
- Evaluating Sequential Values Using Time-adjusted Biological Variation
- High-dose Methylprednisolone Therapy in Multiple Sclerosis Increases Serum Uric Acid Levels
- Evaluation of Accuracy and Uncertainty of ELISA Assays for the Determination of Interleukin-4, Interleukin-5, Interferon-γ and Tumor Necrosis Factor-α
- Development of Immunoturbidimetric Assays for Fourteen Human Serum Proteins on the Hitachi 912™
- Multicenter Evaluation of a Fully Mechanized Soluble Transferrin Receptor Assay on the Hitachi and Cobas Integra Analyzers. The Determination of Reference Ranges
- IFCC News: May/June 2002
- Meetings and Awards
