Detection of point mutation in gene for LDL receptor

-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

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.

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

Gene for LDL receptor is lokated on chromosome 19, in position p3.1 -p3.3.

700 mutations were detected in gene for LDL receptor, all with low frequence.

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).

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.

SNP - single nuclotide polymorphism

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 -

-carried on chromosomes-basic physical and functional units of heredity

-specific sequences of bases that encode instructions on how

to make proteins – the genetic 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.

translation="MDTKHFLPLDFSTQVNSSLTSPTGRGSMAAPSLHPSLGPGIGSP GQLHSPISTLSSPINGMGPPFSVISSPMGPHSMSVPTTPTLGFSTGSPQLSSPMNPVS SSEDIKPPLGLNGVLKVPAHPSGNMASFTKHICAICGDRSSGKHYGVYSCEGCKGFFK RTVRKDLTYTCRDNKDCLIDKRQRNRCQYCRYQKCLAMGMKREAVQEERQRGKDRNEN EVESTSSANEDMPVERILEAELAVEPKTETYVEANMGLNPSSPNDPVTNICQAADKQL FTLVEWAKRIPHFSELPLDDQVILLRAGWNELLIASFSHRSIAVKDGILLATGLHVHR NSAHSAGVGAIFDRVLTELVSKMRDMQMDKTELGCLRAIVLFNPDSKGLSNPAEVEAL REKVYASLEAYCKHKYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTF LMEMLEAPHQMT"

BASE COUNT 1010 a 1643 c 1645 g 1151 tORIGIN 1 gcgccggggg ccgccgcgcc cgccgcccgc tgcctgcgcc gccggccggg catgagttag 61 tcgcagacat ggacaccaaa catttcctgc cgctcgattt ctccacccag gtgaactcct 121 ccctcacctc cccgacgggg cgaggctcca tggctgcccc ctcgctgcac ccgtccctgg 181 ggcctggcat cggctccccg ggacagctgc attctcccat cagcaccctg agctccccca 241 tcaacggcat gggcccgcct ttctcggtca tcagctcccc catgggcccc cactccatgt 301 cggtgcccac cacacccacc ctgggcttca gcactggcag cccccagctc agctcaccta 361 tgaaccccgt cagcagcagc gaggacatca agccccccct gggcctcaat ggcgtcctca 421 aggtccccgc ccacccctca ggaaacatgg cttccttcac caagcacatc tgcgccatct 481 gcggggaccg ctcctcaggc aagcactatg gagtgtacag ctgcgagggg tgcaagggct 541 tcttcaagcg gacggtgcgc aaggacctga cctacacctg ccgcgacaac aaggactgcc 601 tgattgacaa gcggcagcgg aaccggtgcc agtactgccg ctaccagaag tgcctggcca 661 tgggcatgaa gcgggaagcc gtgcaggagg agcggcagcg tggcaaggac cggaacgaga 721 atgaggtgga gtcgaccagc agcgccaacg aggacatgcc ggtggagagg atcctggagg 781 ctgagctggc cgtggagccc aagaccgaga cctacgtgga ggcaaacatg gggctgaacc 841 ccagctcgcc gaacgaccct gtcaccaaca tttgccaagc agccgacaaa cagcttttca 901 ccctggtgga gtgggccaag cggatcccac acttctcaga gctgcccctg gacgaccagg 961 tcatcctgct gcgggcaggc tggaatgagc tgctcatcgc ctccttctcc caccgctcca 1021 tcgccgtgaa ggacgggatc ctcctggcca ccgggctgca cgtccaccgg aacagcgccc 1081 acagcgcagg ggtgggcgcc atctttgaca gggtgctgac ggagcttgtg tccaagatgc

(Polymerase Chain Reaction)

The purpose of a PCR is to make a huge number of copies of a specific DNA sequence.

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.

DNA

PCR - reaction mixture

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

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.

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).

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.

After restriction analysis - fragments on gel

Molecular diagnostics researchand clinical research

applications

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

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

Fungi, parasites

Candida and Aspergillus speciesisolation of DNA from cultures and blood

Leishmania speciesdetection in tissue biopsies by PCR

Trichomonas vaginalis

detection in cervical swabs and urine 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

Minimal Residual Disease after Stem-Cell Transplants

Monitoring of follicular lymphoma 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).

Blood-group incompatibilities between mother and fetus

Detection in amniotic cell DNA by PCR