Genetic and Molecular Testing

David Amor2005

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

KaryotypePatient 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.

Critical Region ProbeChromosome Reporter Probe

Prader-Willi syndrome due to deletion of 15q11.3.

Note the absence of signal from the critical region probe on one chromosome 15

Sub-telomeres

• Region that lies approx 20-300kb below the telomere.• Often gene rich.• Difficult to detect abnormality microscopically due to g-

banding variation.• Deletions and rearrangements often sub-microscopic.

(TTAGGGG)n

Telomere cap 3-20kb

Subtelomere<300kb

Chromosome specificsequences

Uses for MLPA

• Qualitative and quantitative detection of dosage differences at any loci.

• Detection of microdeletions (eg. VCF & other microdeletion syndromes or known mutation within exons of genes eg BRACA or CF mutations).

• Identification of unknown genetic material onderivative chromosomes

• Detection of submicroscopic changes in telomeres (chromosome ends) which make up 6-11% of MR population.

Variable phenotype

• Phenotypes can range from mild to severe mental retardation to multiple congenital malformations, dysmorphic features and organ defects.

• Highest percentage of sub-telomeric deletions occur in the moderate-severe MR group.

• Almost all chromosomes have had sub-telomericdeletions reported.

• Majority are de novo although some have been inherited.

Frequency of specific submicroscopic sub-telomere

deletions

12q, 15q, 18p, 19p, 19q, 20q, 21qNone

3q, 6p, 7p, 11p, 16q, 17qSingle

1q, 2p, 3p, 4q, 5q, 6q, 7q, 8p, 9q, 10p, 10q, 11q, 12p, 13q, 14q, 18q, 20p

2–10

1p, 2q, 22q11–50

4p, 5p, 9p, 16p, 17p>50

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 destructivebehaviour.

• 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).

del 1p(subtel), dup 17p(subtel) by MLPA

00.20.40.60.8

11.21.41.61.8

21

p

3p

5p

7p

9p

11

p

13

p*

15

p*

17

p

19

p

21

p*

Xp

2q

4q

6q

8q

10

q

12

q

14

q

16

q

18

q

20

q

22

q

P070 MEAN P36 (All)

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 diseasethrough family

– usually gene, or specific mutation, not known

Direct Tests: DNA SequencingMost techniques are based on DNA synthesis with base-specific chain

terminators - called the enzymatic method.

.

.

.AGCGGCC

Older technique; gel separation using 4 separate reactions

Read from bottom up (smallest fragment to largest)

Two peaks implies heterozygosity

Automated sequencing with fluorescent labels

Homozygous sequence

*

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

A TT AC G

T AT AT A

G CG CT A

A TT A- -

- -- -T A

G CG CT A

Normal Gene Cystic Fibrosis∆F508 mutation

DNA DNA

507Isoleucine

508Phenylalanine

509Glycine

507Isoleucine

508Glycine

X

Common Mutation in Cystic Fibrosis

Test for ∆F508 in Cystic Fibrosis

Nucleotides 1653-1655 deleted in mutant allele

300bp200bp

100bp

50bpM Wt N/N N/CF CF/CF markers

_

+

Perform PCR and analyse products after electrophoresis

N = normal allele

CF = allele with mutation

98bp95bp

Genetic Testing:PCR Blot for Huntington disease

The pedigree is drawn to correspond to the lanes.

16 -

86 -

55 -

34 -Line indicates the cut off between asymptomatic and possibly symptomatic (35-36 repeats).

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.

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

Genetic testing: BlotsAll 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)?

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.

Genetic Testing: Southern Blot

Control 1 2 3 4

TopLarger DNA fragments

Smaller

Markers

Patient 2 has a smaller DNA fragment, suggesting there is a deletion in one allele.

Western BlotA protein sample is solubilised, run through polyacrylamide gel by electrophoresis

(SDS-PAGE), transferred to a membrane, probed with an antibody.

Patient samples:

Dystrophin blot

Control 1 2 3 4 Control

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.

Patient 1 makes no dystrophin; Consistent with Duchenne muscular dystrophy

Patients 2 & 3 make a very small amount of dystrophin that may be smaller than normal. Suggestive of Becker muscular dystrophy

Normal dystrophin

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)

A07 SNP_05_09_05_CF(12)Run01

0 100 200 300 400 500 600 700 800 9000

1000

2000

B08 SNP_05_09_05_CF(12)Run01

0 100 200 300 400 500 600 700 800 9000

1000

2000

B09 SNP_05_09_05_CF(12)Run01

0 100 200 300 400 500 600 700 800 9000

1000

2000

B10 SNP_05_09_05_CF(12)Run01

0 100 200 300 400 500 600 700 800 9000

1000

2000

B11 SNP_05_09_05_CF(12)Run01

0 100 200 300 400 500 600 700 800 9000

1000

2000

3000

B12 SNP_05_09_05_CF(12)Run01

0 100 200 300 400 500 600 700 800 9000

1000200030004000

C07 SNP_05_09_05_CF(12)Run01

0 100 200 300 400 500 600 700 800 9000

1000

2000

Negative Control

N1303K/N G>C

G542X/N C>A

621+1g>t/N

∆F508/N C>T

W1282X/N C>T

3849+10kb/N G>A

SNP analysis (CF)

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

Lociclosetogether

A a

B b

Loci farapart

no crossover

crossover

A

B

a

b

A

b B

a

AC

ac

no crossover

crossover

AC

ac

AC

ac

possiblegamete

chromosomes

4.5kb - allele B

3.0kb

1.5kballele b

Autosomal Dominant PedigreeB - marker allele

linked to normal geneb - marker allele

linked to faulty gene, contains restriction enzyme site

Bb

Bb

Bb Bb Bb

BB

BB

BB BB

?

Example of Linkage

Linkage Analysis in Gene DiscoveryAims 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.

Principles1. 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 non-random 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 InvestigationsBlood: Lactate, glucose

Urine: Amino and organic acidsCSF: protein, lactate

ECG, MRI

Specific point mutations syndrome?Eg MELAS, MERRF, LHON, NARP

Biopsies:Liver, Muscle, Skin

Histology/EM Enzyme studies(Respiratory chain)Molecular studies

Mutationanalysis

for knownpoint

mutations

Yes

No