Genetic and Molecular Testing David Amor 2005
Genetic Tests •
Diagnostic testing to confirm presence of disease and/or for clinical prognosis • karyotyping for Down syndrome • DNA test for Duchenne muscular dystrophy • biochemical test for Tay Sachs disease
•
Carrier testing for heterozygous state for autosomal and X-linked recessive disorders • DNA test for common mutation (DF508) for cystic fibrosis in heterozygotes (unaffected)
•
Predictive testing for pre-symptomatic individuals at risk of developing genetic disorder • DNA test for mutation (triplet repeat expansion) in Huntington disease (autosomal dominant) • DNA test for BRCA1 and BRCA2 genes in autosomal dominant breast cancer
Cytogenetic Tests
Karyotype Patient cells (usually lymphocytes, amniocytes or chorionic villus cells) are cultured and held at metaphase. Chromosome spreads are prepared on slides, stained and photographed. Individual chromosomes are identified and arranged by size.
Possible questions: 1. sex chromosome aneuploidy. Eg. • Klinefelter synd. • Turner syndrome 2. Balanced translocations
Karyotype of a male with Klinefelter syndrome
FISH: Fluorescence In Situ Hybridisation A chromosome spread is prepared. DNA strands are denatured in the presence of a probe (fluorescently labelled complementary DNA sequence), reannealed and washed. Main role: To identify common chromosomal deletions that are below the resolution of standard cytogenetics. Prader-Willi syndrome due to deletion of 15q11.3.
Critical Region Probe Chromosome Reporter Probe
Note the absence of signal from the critical region probe on one chromosome 15
Sub-telomeres (TTAGGGG)n
Chromosome specific sequences
Subtelomere Telomere cap 50
4p, 5p, 9p, 16p, 17p
11–50
1p, 2q, 22q
2–10
1q, 2p, 3p, 4q, 5q, 6q, 7q, 8p, 9q, 10p, 10q, 11q, 12p, 13q, 14q, 18q, 20p
Single
3q, 6p, 7p, 11p, 16q, 17q
None
12q, 15q, 18p, 19p, 19q, 20q, 21q
Well defined sub-telomeric deletions • • •
• • • •
4p – Wolf Hirschhorn. 5p – Cri du chat. 1p36 – Severe MR, growth anomalies., large anteria fontanelle, prominent fore head, deep set eyes, self destructive behaviour. 2q37.3 - Albrights syndrome (osteodystrophy). 7q – Sonic hedgehog gene – holoprosencephaly. 9p – DMRT1 gene: male->female sex reversal with trigonocephaly. Xp - SHOX Short stature (8%)
Case • • • • • • • •
Presented at 14 months. Normal karyotype as neonate. Moderate MR. Polymicrogyria. Short stature. Palpable fissures. Dysmorphic toes and fingers. MLPA result indicated deletion 1p36 with duplication of 17pter (LIS1).
P070
del 1p(subtel), dup 17p(subtel) by MLPA
MEAN
P36 (All)
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4
22q
20q
18q
16q
14q
12q
10q
8q
6q
4q
2q
Xp
21p*
19p
17p
15p*
13p*
11p
9p
7p
5p
3p
0
1p
0.2
DNA tests
Testing for Faulty Genes Causing Disease • Direct tests – – – – –
for specific mutations gene must be cloned DNA sequence (at least part) must be known number of different techniques used many of these techniques use PCR to amplify DNA around the mutation site
• Indirect tests (linkage) – track genotype (DNA pattern) associated with presence of disease through family – usually gene, or specific mutation, not known
Direct Tests: DNA Sequencing Most techniques are based on DNA synthesis with base-specific chain terminators - called the enzymatic method. Older technique; gel separation using 4 separate reactions
Automated sequencing with fluorescent labels Homozygous sequence
. . . A G C G G C C
Read from bottom up (smallest fragment to largest)
Two peaks implies heterozygosity
*
PCR (Polymerase Chain Reaction) • Can rapidly amplify large amounts of target DNA in a specific region • Uses a thermostable DNA polymerase to synthesise new copies of specific DNA sequence that is flanked by two oligonucleotide primers (complementary to target sequence) • Can use minimal amount of target DNA as starting material, even a single cell
Common Mutation in Cystic Fibrosis Normal Gene
Cystic Fibrosis ∆F508 mutation
DNA
DNA
AT TA CG
507 Isoleucine
AT TA --
508 Phenylalanine
--TA
509 Glycine
GC GC TA
TA TA TA GC GC TA
507 Isoleucine
X 508 Glycine
Test for ∆F508 in Cystic Fibrosis Nucleotides 1653-1655 deleted in mutant allele
_ 300bp 200bp
Perform PCR and analyse products after electrophoresis
98bp 95bp
100bp
50bp M Wt N/N N/CF CF/CF markers
+
N = normal allele CF = allele with mutation
Genetic Testing:PCR Blot for Huntington disease The pedigree is drawn to correspond to the lanes.
PCR is used to amplify a DNA fragment containing the (CAG)n repeat in the Huntington gene. Bands are shown by a general DNA stain
86 55 -
Each individual is expect to have two bands, one for each Huntington gene. Affected individuals almost always have a normal sized allele as well as an abnormally large allele.
34 16 -
Line indicates the cut off between asymptomatic and possibly symptomatic (35-36 repeats).
Genetic testing: Blots All blots separate molecules by size (and charge) using electrophoresis – Southern Blot - separation of DNA fragments of different sizes – Northern Blot - separation of RNA fragments – Western Blot - separation of proteins
•
Most blots require a ‘probe’ to identify the specific molecule in question – Southern/northern blots - labelled complementary DNA sequence – Western blot - labelled antibodies – Blot to separate PCR fragments - DNA non-specifically labelled.
Approach • Assume the start of the gel is at the top • Larger molecules run more slowly. Therefore molecules closer to the top of the blot are larger in size • From first principles how many bands do you expect (less predictable with western blots for protein)?
Genetic Testing: Southern Blot A DNA sample is digested (chopped into fragments) by restriction endonuclease(s), run through an agarose gel, transferred to a membrane, probed with a labelled oligonucleotide complementary to the DNA sequence of interest. Can use 100 base pairs to 20 kb size fragments. Main role: Detecting large scale DNA changes such as large deletions, duplications, expansions or rearrangements.
Top
Markers
Larger DNA fragments
Smaller Control
1
2
3
4
Patient 2 has a smaller DNA fragment, suggesting there is a deletion in one allele.
Western Blot A protein sample is solubilised, run through polyacrylamide gel by electrophoresis (SDS-PAGE), transferred to a membrane, probed with an antibody.
Patient samples:
Control 1
2
3
4
Control
Patient 1 makes no dystrophin; Consistent with Duchenne muscular dystrophy
Normal dystrophin Patients 2 & 3 make a very small amount of dystrophin that may be smaller than normal. Suggestive of Becker muscular dystrophy
Dystrophin blot
Myosin staining shows roughly equivalent amounts of muscle protein were loaded in each lane
Patient 4’s dystrophin is significantly smaller in size compared with controls because it has run further in the gel. Consistent with Becker muscular dystrophy due to a large deletion.
Gels are now seldom used •
Sequencing – Used where need to detect non-recurrent mutations • e.g. FAP, HNPCC, BRCA, Rb
•
Fragment analysis – Used to size triplet repeat disorders • e.g. HD, Fragile X premutation, small myotonic dystophy alleles • Still need Southern Blot for large expansions
•
SNP analysis – Used for recurrent mutations • e.g. Cystic Fibrosis, Ashkenazi disorders
•
MLPA – Used to screen for large deletions • e.g. DMD, Rb, Cancer genes
Fragment analysis (HD)
SNP analysis (CF) A07 SNP_05_09_05_CF(12)Run01 2000 1000 0 0 100 B08 SNP_05_09_05_CF(12)Run01
200
300
400
500
600
Negative Control
700
800
900
2000
N1303K/N G>C
1000 0 0
100
200
300
400
500
600
700
800
900
B09 SNP_05_09_05_CF(12)Run01 2000
G542X/N C>A
1000 0 0 100 B10 SNP_05_09_05_CF(12)Run01
200
300
400
500
600
700
800
900
2000
621+1g>t/N
1000 0 0
100
200
300
400
500
600
700
800
900
B11 SNP_05_09_05_CF(12)Run01 3000 2000
∆F508/N C>T
1000 0 0
100
200
300
400
500
600
700
800
900
B12 SNP_05_09_05_CF(12)Run01 4000 3000 2000 1000 0 0 100 C07 SNP_05_09_05_CF(12)Run01
200
300
400
500
600
700
W1282X/N C>T 800
900
2000
3849+10kb/N G>A
1000 0 0
100
200
300
400
500
600
700
800
900
Indirect Tests (Linkage) • •
Principal is to use inherited DNA sequence variation (polymorphisms) to ‘track’ or follow a mutation within a family Two major types – Restriction fragment length polymorphisms (RFLPs) • now often referred to as Single Nucleotide Polymorphisms (SNPs)
– Variable tandem repeat DNA length polymorphisms (VNTRs) • minisatellites 9-25 nucleotide repeat units • microsatellites 1-6 nucleotide repeat units
•
Used in clinical testing if – Gene has been mapped but not cloned – Gene has been cloned but mutation mutation not detected (not if locus heterogeneity)
• •
Mutation tracking requires that the marker DNA used as a probe lies close to the faulty gene, ie linked If marker DNA is too far away from the faulty gene, then crossing over is more likely during meiosis, and so won’t track with the disease in the family
Crossing Over and Linkage A
a
A C
a c Loci close together
Loci far apart B
b
no cross over A
cross over a
A
no cross over A C
a possible gamete chromosomes
B
b
b
B
cross over a c
A C
a c
Example of Linkage Autosomal Dominant Pedigree
BB
Bb
BB
B - marker allele linked to normal gene b - marker allele linked to faulty gene, contains restriction enzyme site
Bb ?
Bb
BB
Bb
Bb
BB
4.5kb - allele B 3.0kb 1.5kb
allele b
Linkage Analysis in Gene Discovery Aims to determine the chromosomal position (locus) of a gene responsible for a Mendelian genetic trait or disorder. Most often the first step in identifying a causative gene. Principles 1. Cross-overs shuffle portions of chromosomes in a family. (On average there are 52 cross-overs per meiosis) 2. Naturally occurring variations (polymorphic sites) in DNA sequences are used to track chromosome regions through a family tree. Eg genotype 300 markers spaced over all chromosomes in 20 family members. 3. Which piece of chromosome tracks with the disease in a family? 4. A LOD score (Logarithm of the Odds) > 3 is deemed significant linkage. Lower values are suggestive.
For autosomal dominant disorders you need at least 10 informative family members (+ luck). Even in very large families linkage analysis often leaves sizeable candidate regions (eg often containing over 50 genes).
Linkage Disequilibrium The tendency for alleles of genes or genetic markers to be inherited together in a nonrandom fashion. Occurs when two genes or genetic markers are close in chromosomal position. Here particular patterns of markers are unlikely to be separated by the random assorting of chromosomes or by cross-overs at meiosis. The closer together two genes or genetic markers, the higher the linkage disequilibrium.
The investigation of suspected mitochondrial disease Clinical Investigations Blood: Lactate, glucose Urine: Amino and organic acids CSF: protein, lactate ECG, MRI Yes Specific point mutations syndrome? Eg MELAS, MERRF, LHON, NARP No Biopsies: Liver, Muscle, Skin Histology/EM
Molecular studies
Mutation analysis for known point mutations
Enzyme studies (Respiratory chain)