Detection of point mutation in gene for LDL receptor

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Hyperlipidemia

• Detection of point mutation in gene for LDL
  receptor

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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

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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.
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• Levels of LDL-cholesterol in familial
  hypercholesterolemia
• Age LDL cholesterol
• gt 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
• gt 39 let 7,8 mmol/l and more
• physiological level of LDL-cholesterol
• lt 3,4 mmol/l

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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.
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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).
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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.
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SNP - single nuclotide polymorphism
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Human blood - DNA isolation leukocytes tromboc
ytes 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
-
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Genes

• -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.

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PCR
(Polymerase Chain Reaction)
The purpose of a PCR is to make a huge number of
copies of a specific DNA sequence.
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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 94C During the
denaturation, the double strand melts open to
single stranded DNA. Annealing at 50-65C The
primers are annealed. extension at 72C 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.
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PCR - reaction mixture
DNA
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Restriction analysis
RFLP refers to the variation among individuals in
the lengths of DNA fragments between normal and
mutant allels.
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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.
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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).
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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.
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After restriction analysis - fragments on gel
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Molecular diagnostics research and clinical
research applications
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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
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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
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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
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Minimal Residual Disease after Stem-Cell
Transplants Monitoring of follicular lymphoma by
PCR
Two methods - one detects an associated t(1418)
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
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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 (83158 bp) indicated, as
well as hGH (434 bp) as internal control. D2D10
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).