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 Subtelomere Telomere cap sequences <300kb 3-20kb • 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.

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 on derivative 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-telomeric deletions reported. • Majority are de novo although some have been inherited.

Frequency of specific submicroscopic sub-telomere deletions

>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). del 1p(subtel), dup 17p(subtel) by MLPA P070 MEAN P36 (All)

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1p 3p 5p 7p 9p 2q 4q 6q 8q Xp 11p 17p 19p 10q 12q 14q 16q 18q 20q 22q 13p* 15p* 21p* 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 Automated sequencing with fluorescent labels reactions Homozygous sequence Two peaks implies heterozygosity

. . . A G C G * G C C

Read from bottom up (smallest fragment to largest) 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

A T A T 507 T A 507 T A Isoleucine Isoleucine C G --

T A -- T A 508 -- T A Phenylalanine T A X

G C G C G C 509 G C 508 T A Glycine T A Glycine Test for ∆F508 in Cystic Fibrosis

Nucleotides 1653-1655 deleted in mutant allele

Perform PCR and _ analyse products after 300bp electrophoresis 200bp

100bp 98bp 95bp N = normal allele

50bp CF = allele with M Wt N/N N/CF CF/CF + mutation markers 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

Each individual is expect to have two bands, one for each 86 - Huntington gene. Affected individuals almost always have a 55 - normal sized allele as well as an abnormally large allele. 34 - Line indicates the cut off between 16 - asymptomatic and possibly symptomatic (35-36 repeats). Genetic testing: Blots All blots separate molecules by size (and charge) using electrophoresis – - separation of DNA fragments of different sizes – - separation of RNA fragments – - separation of

• Most blots require a ‘probe’ to identify the specific molecule in question – Southern/northern blots - labelled complementary DNA sequence – Western blot - labelled – 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 )? 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.

Markers Top 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 .

Patient Control 1 2 3 4 Control Patient 1 makes no dystrophin; samples: Consistent with Duchenne muscular dystrophy Normal dystrophin Patients 2 & 3 make a very small amount of dystrophin that may be smaller than normal. Dystrophin Suggestive of Becker muscular blot dystrophy 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 Myosin staining shows roughly large deletion. equivalent amounts of muscle protein were loaded in each lane 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 Negative

0 0 100 200 300 400 500 600 700Control800 900 B08 SNP_05_09_05_CF(12)Run01 2000 1000 N1303K/N G>C

0 0 100 200 300 400 500 600 700 800 900 B09 SNP_05_09_05_CF(12)Run01 2000

1000 G542X/N C>A

0 0 100 200 300 400 500 600 700 800 900 B10 SNP_05_09_05_CF(12)Run01 2000 1000 621+1g>t/N 0 0 100 200 300 400 500 600 700 800 900 B11 SNP_05_09_05_CF(12)Run01 3000

2000 1000 ∆F508/N C>T 0 0 100 200 300 400 500 600 700 800 900 B12 SNP_05_09_05_CF(12)Run01 4000 3000 2000 W1282X/N 1000 0 0 100 200 300 400 500 600 700 C>T 800 900 C07 SNP_05_09_05_CF(12)Run01 2000

1000 3849+10kb/N G>A

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 a C c Loci far Loci apart close together Bb no cross cross no cross cross over over over over

A a A a A a A a C c C c possible gamete chromosomes

B b bB Example of Linkage

Autosomal Dominant Pedigree B - marker allele linked to normal gene b - marker allele Bb BB linked to faulty gene, contains restriction site

BB Bb

?

BbBB Bb Bb BB

4.5kb - allele B

3.0kb allele b 1.5kb 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 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 Investigations Blood: Lactate, glucose Urine: Amino and organic acids CSF: protein, lactate ECG, MRI Yes Mutation Specific point mutations syndrome? analysis Eg MELAS, MERRF, LHON, NARP for known No point mutations Biopsies: Liver, Muscle, Skin

Histology/EM Enzyme studies Molecular studies (Respiratory chain)