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 !!!