Detection of point mutation in gene for LDL receptor

1. Detection of point mutation in gene for LDL receptor Hyperlipidemia

2. Familial hypercholesterolemia • an inherited metabolic disorder • caused by a lack or malfunction of receptors for the low-density lipoproteins (LDL) that activate removal of cholesterol from the blood. • LDL receptors • on the cell membrane take cholesterol into the cell and break it down, • so that the HDL (high density lipoproteins) can carry • the cholesterol to the liver to be excreted from the body

3. People with FH have fewer receptors on their cell membranes Elevated cholesterol in their blood (because the cholesterol cannot get into the cell to be carried to the liver). Fewer receptors lead to elevated cholesterol which causes plaque formation and coronary artery disease = increased risk of early death secondary to heart disease.

4. Levels of LDL-cholesterol in familial hypercholesterolemia: • Age LDL cholesterol • > 20 years 6,2 mmol/l and more • 20-29 years 6,7 mmol/l and more • 30-39 years 7,2 mmol/l and more • > 39 let 7,8 mmol/l and more • physiological level of LDL-cholesterol • < 3,4 mmol/l

5. Gene for LDL receptor is lokated on chromosome 19, in position p3.1 -p3.3. 700 mutations were detected in gene for LDL receptor, allwith low frequence.

6. Detection of R395W mutation Mutation R395W, is one of the most frequent mutations in LDL receptor gene. Mutation is due to single nucleotide substitution of G to T, which leads to substitution of arginine (R) to thyrozine(W) in position 395 (GGG TGG).

7. SNP - single nuclotide polymorphism A key aspect of research in genetics is associating sequence variations with heritable phenotypes. The most common variations are single nucleotide polymorphisms (SNPs), which occur approximately once every 100 to 300 bases. Because SNPs are expected to facilitate large-scale association genetics studies, there has recently been great interest in SNP discovery and detection.

9. SNP - single nuclotide polymorphism

11. Human blood - DNA isolation leukocytes trombocytes erytrocytes Cells in ml 4-7 x 106 3- 4 x 108 5 x 109 DNA 30- 60 g/ml - - (6 pg/cell) RNA 1- 5 g/ml blood - - Hemoglobine - - 150 mg/ml Plasma proteins - 60- 80 mg/ml -

12. Genes • -carried on chromosomes • basic physical and functional units of heredity • specific sequences of bases that encode instructions on how • to make proteins – thegenetic information. • There are three types of genes : • 1) Protein-coding genes : these are transcribed into RNA and • then translated into proteins. • 2) RNA-specifying genes : these are only transcribed into RNA. • 3) Regulatory genes : these include only untranscribed sequences.

14. PCR (Polymerase Chain Reaction) The purpose of a PCR is to make a huge number of copies of a specific DNA sequence.

15. There are three major steps in a PCR, which are repeated for • 20 to 30 cycles on an automated cycler. • The tubes with the reaction mixture are heated and cooled • in a very short time. • Denaturation at 94°C : During the denaturation, the double strand melts open • to single stranded DNA. • Annealing at 50-65°C :The primers are annealed. • extension at 72°C :This is the ideal working temperature for the polymerase. • The polymerase adds dNTP's from 5' to 3', reading • the template from 3' to 5' side.

16. PCR - reaction mixture DNA

17. Restriction analysis RFLP refers to the variation among individuals in the lengths of DNA fragments between normal and mutant allels.

18. Restriction endonucleases are enzymes that cleave DNA molecules at specific nucleotide sequences depending on the particular enzyme used. Enzyme recognition sites are usually 4 to 6 base pairs in length.

19. ELFO Gel electrophoresis is a procedure for separating a mixture of molecules through a stationary material (gel) in an electrical field. DNA is negatively charged (the phosphates that form the sugar-phosphate backbone of a DNA molecule have a negative charge).

20. Samples containing DNA mixed with loading buffer are pipeted into the sample wells. DNA will migrate towards the anode. When adequate migration has occured, DNA fragments are visualized by staining with ethidium bromide. To visualize DNA, the gel is placed on a ultraviolet transilluminator.

21. After restriction analysis - fragments on gel

22. Molecular diagnostics research and clinical research applications

23. Bacteria Pathogenic enteric bacteriadetection in stool by multiplex PCR Chlamydia trachomatisin swabs and histological sections detected by PCR and sequencing Chlamydia pneumoniaeanalysis in monocytes by RT-PCR Bordetella pertussisdetection in nasopharyngeal swabs by PCR followed by immunoassay Legionella pneumophiladetection in bronchoalveolar lavage by PCR Periodontal bacteriaidentification of bacteria implicated in gingivitis by multiplex PCR Pneumocystis cariniigenotyping studies using samples isolated from bronchoalveolar specimens Various bacteriaidentification from 16S-rRNA gene fragments by RT-PCR and sequencing Mycobacteriadetection and identification in paraffin-embedded lung samples by PCR

24. Viruses Influenza A virusmolecular characterization of strains Human adenovirus subgeneradetection in clinical samples by multiplex PCR Enteroviruses and HSVdetection of viral nucleic acids in cerebrospinal fluid by multiplex PCR Viral genotypingmanual and automated isolation from plasma Viral load monitoringhighly sensitive quantification in peripheral blood mononuclear cells and biopsies

25. Fungi, parasites Candida and Aspergillus speciesisolation of DNA from cultures and blood Leishmania speciesdetection in tissue biopsies by PCR Trichomonas vaginalisdetection in cervical swabs and urine by PCR

26. Minimal Residual Disease after Stem-Cell Transplants Monitoring of follicular lymphoma by PCR Two methods - one detects an associated t(14:18) translocation, - the other a tumor- specific CDRIII sequence. Bone marrow and peripheral blood samples were collected from 15 patients with disseminated follicular lymphoma following autologous stem-cell transplantation

27. Blood-group incompatibilities between mother and fetus Detection in amniotic cell DNA by PCR Fetal DNA was isolated from two amniotic-fluid samples using the QIAamp Viral RNA Mini Kit. Individual PCRs contained primer sets specific for the RH sequences (83–158 bp) indicated, as well as hGH (434 bp) as internal control. D2–D10 refer to the specific exons targeted within the RHD gene. c(cyt48) refers to a sequence variant of the RHc allele. A RH genotype: CCD.ee; B RH genotype: ccddee. M: DNA molecular weight marker V (Boehringer Mannheim).