Transcript LDL receptors

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
-
Plasma proteins
-
-
-
-
60- 80 mg/ml
150 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
ORIGIN
1 gcgccggggg ccgccgcgcc
61 tcgcagacat ggacaccaaa
121 ccctcacctc cccgacgggg
181 ggcctggcat cggctccccg
241 tcaacggcat gggcccgcct
301 cggtgcccac cacacccacc
361 tgaaccccgt cagcagcagc
421 aggtccccgc ccacccctca
481 gcggggaccg ctcctcaggc
541 tcttcaagcg gacggtgcgc
601 tgattgacaa gcggcagcgg
661 tgggcatgaa gcgggaagcc
721 atgaggtgga gtcgaccagc
781 ctgagctggc cgtggagccc
841 ccagctcgcc gaacgaccct
901 ccctggtgga gtgggccaag
961 tcatcctgct gcgggcaggc
1021 tcgccgtgaa ggacgggatc
1081 acagcgcagg ggtgggcgcc
1645 g
1151 t
cgccgcccgc
catttcctgc
cgaggctcca
ggacagctgc
ttctcggtca
ctgggcttca
gaggacatca
ggaaacatgg
aagcactatg
aaggacctga
aaccggtgcc
gtgcaggagg
agcgccaacg
aagaccgaga
gtcaccaaca
cggatcccac
tggaatgagc
ctcctggcca
atctttgaca
tgcctgcgcc
cgctcgattt
tggctgcccc
attctcccat
tcagctcccc
gcactggcag
agccccccct
cttccttcac
gagtgtacag
cctacacctg
agtactgccg
agcggcagcg
aggacatgcc
cctacgtgga
tttgccaagc
acttctcaga
tgctcatcgc
ccgggctgca
gggtgctgac
gccggccggg
ctccacccag
ctcgctgcac
cagcaccctg
catgggcccc
cccccagctc
gggcctcaat
caagcacatc
ctgcgagggg
ccgcgacaac
ctaccagaag
tggcaaggac
ggtggagagg
ggcaaacatg
agccgacaaa
gctgcccctg
ctccttctcc
cgtccaccgg
ggagcttgtg
catgagttag
gtgaactcct
ccgtccctgg
agctccccca
cactccatgt
agctcaccta
ggcgtcctca
tgcgccatct
tgcaagggct
aaggactgcc
tgcctggcca
cggaacgaga
atcctggagg
gggctgaacc
cagcttttca
gacgaccagg
caccgctcca
aacagcgccc
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.
PCR - reaction mixture
DNA
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 research
and clinical research
applications
Bacteria
Pathogenic enteric bacteria
detection in stool by multiplex PCR
Chlamydia trachomatis
in swabs and histological sections detected by PCR and sequencing
Chlamydia pneumoniae
analysis in monocytes by RT-PCR
Bordetella pertussis
detection in nasopharyngeal swabs by PCR followed by immunoassay
Legionella pneumophila
detection in bronchoalveolar lavage by PCR
Periodontal bacteria
identification of bacteria implicated in gingivitis by multiplex PCR
Pneumocystis carinii
genotyping studies using samples isolated from bronchoalveolar specimens
Various bacteria
identification from 16S-rRNA gene fragments by RT-PCR and sequencing
Mycobacteria
detection and identification in paraffin-embedded lung samples by PCR
Viruses
Influenza A virus
molecular characterization of strains
Human adenovirus subgenera
detection in clinical samples by multiplex PCR
Enteroviruses and HSV
detection of viral nucleic acids in cerebrospinal fluid by multiplex PCR
Viral genotyping
manual and automated isolation from plasma
Viral load monitoring
highly sensitive quantification in peripheral blood mononuclear cells and biopsies
Fungi, parasites
Candida and Aspergillus species
isolation of DNA from cultures and blood
Leishmania species
detection in tissue biopsies by PCR
Trichomonas vaginalis
detection in cervical swabs and urine by PCR
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
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).