diagnostic strategy in genetically determined diseases ... karyotype analysis fish cgh mlpa qf-pcr...
TRANSCRIPT
Diagnostic strategy in geneticallydetermined diseasesdetermined diseases
Joanna Walczak-Sztulpa Ph.D.
Department of Medical GeneticsPoznan University of Medical Sciences
18.11.2014
Diagnostics
cytogenetic molecular
Human chromosomes DNA/RNA
Diagnostic strategy
Chromosomal abnormalities
Known gene(s) Unknown gene(s)abnormalities
Methods:
Karyotype analysis
FISH
CGH
MLPA
QF-PCR
Methods:
PCR
MLPA
Sequencing
PCR-RLFP
PCR-Multiplex
PCR-ASO
Methods:
Next generationsequencing (NGS)
Linkage analysis
Positional cloning
Functional cloning
Nucleic acid structure (1)
• The sugar:
– deoxyribose (DNA)
- ribose (RNA)
• Bases:
Purines (A and G) have two interlocked heterocycling rings
Pyrimidines (C and T or U in RNA) have one such ring
• Phosphate
Nucleic acid structure (2)
SUGAR+BASE = NUCLEOSIDE
NUCLEOSIDE + PHOSPHATE GROUP = NUCLEOTIDE
Nucleic acid structure (3)
• Genetic informationis defined by the order of the nucleotide bases: A, C, G and T
• The structure of DNA is a double-stranded, • The structure of DNA is a double-stranded, antiparalel helix
•The structure of RNA is single stranded
DNA/RNA extraction
Sources of DNA/RNA:
• Lymphocytes from the blood
• Fibroblasts
• AFC (amniotic fluid)
• CVS (chorionic villus biopsies)• CVS (chorionic villus biopsies)
• Fragments of the tissue
• Bone marrow
• Sperm cells
• Buccal mouthwash
DNA extraction
www.qiagen.com
Quality and quantity of DNA
1. Agarose gel electophoresis
2. NanoDrop mesaurments
RNA
RNA extraction
www.qiagen.com
Polimerase Chain Reaction - PCR
PCR permits selective amplification of specific target DNA sequence(s) within heterogeneous collection of DNA sequences, starting with quantities of 50ng or less of the initial target DNA
1. Template DNA
What do we need to amplify sequence by PCR method?
1. Template DNA
2. Synthetic oligonucleotide primers (primer forward, primer revers)
3. Heat stable DNA polymerase
4. dNTP’s - synthetic deoxynucleotides
5. Buffer
PCR method
Each cycle consists of three steps:
1. Heat denaturation
2. Primer anealing
3. Strand elongation
PCR primers are designed using Primer3
Primer design – Exon Primer
The positions of the exons within the genome assembly are provided by the UCSC genome browser
The user can define the maximum exon sizeThe user can define the maximum exon size
Exons larger than this size will be divided into several parts
Exons with small introns in-between are combined
All SNP positions in the human genome are masked with N's in order to avoid primers to be positioned across these sites
PCR in silico
cDNA synthesis and RT- PCR reaction
Electrophoresis
DNA fragments carry an electric charge, so small pieces of DNA can be separated by size if placed separated by size if placed in a gel and an electric current is applied across the gel
PCR reaction – PCR products
Marker (HyperLadder IV)
1. It is very rapid and easy to perform
Three major advantages of the PCR method
2. It is very sensitive – enabling amplification from minute amounts of target DNA – even DNA from a single cella single cell
3. It is very robust and it is often possible to amplify DNA from tissues or cells which are badly degraded, or embedded in some medium that makes it difficult to isolate DNA by standard methods (archaelogical/historical sites or formalin-fixed tissue samples)
Restriction Enzymes
• Restriction enzymes are DNA-cutting enzymes found in bacteria. Because they cut within the molecule, they are often called restriction endonucleasesrestriction endonucleases
• A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides
• The recognition site is commonly 4 or 6 bp in lengh
IFT122_ex1_seq (180 base pairs)
Restriction Fragment Length Polymorphism – RFLP
Results
Restriction Fragment Length Polymorphism – RFLP
PCR amplification of a portion of the ß-globin (HBB) gene and digestion with MstII in a sickle-cell disease
lane 1 – homozygotelane 1 – homozygote
lane 2 – heterozygote
lane 3 – normal homozygote
lane 4 – normal homozygote
lane 5 – control containing amplified but undigested DNA
Nucleic acid hybridization
Fundamental tool in molecular genetics which takes advantage of the ability of individual single-stranded nucleic acid molecule(s)to form double-stranded molecules
The fragment of interest is not amplified or purified in any way; instead it is specifically detected within a complex mixture of many different sequences
Hybridization techniques
Southern blot hybridization – the most popular method, DNA is digested with one or more restriction enzymes that cut DNA generating different fragments. Fragments are size-fractioned on agarose gel, transferred to a nylon membrane and hybridised with a specific probe
Northern blot hybridization – target nucleic acid Northern blot hybridization – target nucleic acid is undigested RNA - this method give the information about expression pattern of specific genes
FISH (fluorescence in situ hybridization) –prepared on microscopy slide
CGH (comparative genomic hybridization) - array-CGH
Southern blot technique (1)
Southern blot technique (2)
Northern blot technique
• Is used to evaluate the level of expression patterns of a gene
• Involves size-fractionation of samples of total RNA, transfer to a membrane and hybridization with a suitable labelled nucleic acid probe
The use of labelled cDNA probe from FMR1 (FraX syndrome) gene. Highest levels are detected in the brain and testis
Western blot technique
DNA polimorphisms
Polimorphism - the existance of two or more variants (allels, sequence variants) at the signifcant frequencies in the population
• Length polimorphism(Variable Number Tandem Repeats, VNTRs)
- Microsatellites, such as the frequently used (CA)n repeats, - Microsatellites, such as the frequently used (CA)n repeats, STR (Short Tandem Repeats)- Minisatellites (rarely used)
• Site polimorphismsDNA point variations or single nucleotide polimorphisms(SNPs) (by analysis of restriction fragment lenghpolimorphisms (RFLPs), by sequence-specific fluorescenceprobes or the use of allele-specific oligonucleotides (ASOs))
DNA fingerprint
Useful for identification of individuals by their respective/unique DNA profiles
- criminalistics/forensic - criminalistics/forensic investigation
- paternity testing
Quantitative Fluorescent PCR - QF-PCR
• In this technique, selected tetranucleotide repeats (STR)
are amlified by PCR using fluorescent primers and the
products analysed on an automated DNA sequencer
• Several markers are used for each chromosome
• Is rapid, sensitive, accurate and reliable diagnostic to • Is rapid, sensitive, accurate and reliable diagnostic to
detect aneuploidies for example in prenatal diagnostic
(DNA can be isolated from amniotic fluid) and spontaneous
miscarriages (DNA can be isolated from chorion)
• Allows the detection of trisomies 13, 18, 21 and the X, Y
chromosome abnormalities
Results
Detection of trisomy 18 using QF-PCR
Multiplex Ligation-Dependent Probe Amplification - MLPA
1. The MLPA analysis technique provides an additional method by which specific deletions or duplications can be identified
2. This technique permits, in a few hours, relative 2. This technique permits, in a few hours, relative quantification of more than 40 different nucleic acid sequences in a single reaction
3. A range of kits were designed to screen for subtelomeric microdeletions, for microdeletions affecting various regions with a single gene, such a dystrophin, or for interstitial deletions or duplications known to be responsible for a range of genetic syndromes
Multiplex Ligation-Dependent Probe Amplification - MLPA
Multiplex Ligation-Dependent Probe Amplification – MLPA
MLPA result for a patient affected by Williams syndrome
Homozygous and heterozygous deletion - ASPA gene
Detection of chromosome X copy number: 1, 2 or 3 copies per cell
Sequencing
1. Sanger sequencing
Is used for determining the order of the nucleotide bases — adenine, guanine, cytosine, and thymine —
in a molecule of DNA
1. Sanger sequencing
2. Next generation sequencing (NGS)/
Whole exome sequencing (WES)
Sanger sequencing
Sequencing results
Control DNA homozygote C/C
heterozygote C/T
homozygote T/T
Next generation sequencing - NGS
• Novel approch
• Very high throughput• Very high throughput
• Expensive (?)
• Not jet in routine use (?)
Next generation sequencing workflow
Karyotype analysis
Normal human male karyotype Normal human female karyotype
G-banding
Karyotype analysis
Triploidy detected at amiocentesis
Fluorescence in situ hybridization - FISH
Is widely used in the diagnosis of chromosome defects
Useful method of detecting microdeletions or microduplication of individual disease-associated chromosomal regions
Probes used in FISH
• Whole chromosome painting probes
• Alpha-satelite (centromeres, telomeres)
• Specific (unique) – hybridise to specific loci on chromosome
Multicolour FISH probes to determine chromosomecopy number in interphase nuclei
a - lymphocyte metaphase and interphase nuclei
• chromosome X
• chromosome Y
• chromosome 18
• chromosome 13
• chromosome 21
and interphase nuclei
b – uncultured amniotic fluid cell nucleus from a normal female fetus
c - uncultured amniotic fluid cell nucleus from a normal male fetus
d - uncultured amniotic fluid cell nucleus from a male fetus with trisomy 21 (Down syndrome)
Microdeletion detecting using specific FISH probes
Miller-Dieker syndrome
(probe 17p13.3)
Williams syndrome
(probe 7q11.2)
Microdeletion detecting using specific FISH probes
Prader Willi/Angelman syndrome
(probe 15q11-q13)
DiGeorge/velocardiofacial syndrome
(probe 22q11.2)
Multicolour FISH using a paint probe composed of a combination of all 24 chromosome-specific probes
Chromosome painting
• chromosome 1
• chromosome 2
• chromosome 6
is used to detect partial monosomies or trisomies, or chromosomal deletions or amplifications
Comparative Genomic Hybridization - CGH method
or chromosomal deletions or amplifications
Array - CGH
has the potential to allow genome-wide screens for microdeletions
and microduplications in patient screens for microdeletions
and microduplications in patient with congenital abnormalities
Array-CGH platform
Test DNAe.g. Cancer
Cot-1 DNA Reference DNA
Array-CGH
e.g. Cancer
Array-CGH
Sample Reference Sample Reference Sample Reference
Array-CGH
Array-CGH
Identifying human disease gene(s)
Next generation sequencing (NGS)/
Whole exome sequencing (WES)
Genetic linkage
Is the tendency of characters (phenotypes, marker alleles) to co-segregate (to be inherited together) in a pedigree
Genetic loci that are physically close to one another on the same chromosome tend to stay together during meiosis, and are thus genetically linked
Identifying human disease genes
genetically linked
Linkage analysis
Is a mathematical procedure that analyses meiotic recombination frequencies between pairs of genes to determine whether two loci are linked
Linkage analysis are familial studies
Once a candidate gene is confirmed, the next step is to understand its function – this can lead to insight related diseases and eventually to effective treatmentto effective treatment
Confirming a candidate gene
Mutation screening – the most popularmethod: rapid and generally applicable.Identyfying mutation in several unrelatedaffected individuals
Restoration of normal phenotype in vitro –if a mutant phenotype is demonstrable incells from patients, we can check whethercells from patients, we can check whethertransfection of a normal allele of thecandidate gene is able to „rescue” themutatant and restore the normal phenotype
Production of a mouse model of thedisease - loss of function phenotypes canbe modeled by knockouts made by genetargetig the mouse germline. For gain offunction phenotypes, the disease allelemust be introduced into the mouse germline
„Identification of causative gene defect(s) responsible for Sensenbrenner Syndrome
(Cranioectodermal dysplasia)”
An example………………
Deparment of Medical Genetics, Poznan University of Medical Sciences, Poland
Department of Human Molecular Genetics, Max Planck Institute for Molecular Genetics, Berlin, Germany
WHAT NEXT?
Functional genomics – global analysis of gene function on the biochemical level, cellular level and organism level
Proteomics – encompasses the analysis of protein expression, protein structure and protein interactions
Bioinformatics - concerns the development of new tools for the analysis of genomic and molecular biological data
Would you like to learn more???
General starting point for genetic data:http://www.ncbi.nlm.nih.gov
For genome data:www.ensembl.org; genome.cse.uscs.edu
For information on proteins:http://ca.expasy.orgFor information on proteins:http://ca.expasy.org
For information on any mendelian phenotype:http://www.ncbi.nlm.nih.gov/omim/
Access to biomedical literature:http://www.ncbi.nlm.nih.gov/entrez/
Thank you for your attention !!!