Human Molecular Genetics Institute of Medical Genetics Yaoqin Gong 2003

Human Human Molecular Molecular GeneticsGenetics

Institute of Medical GeneticsYaoqin Gong

2003

Gene Mutation

DNA Polymorphism

Outline of this chapter

Gene MutationGene Mutation

Definition

Major Types

Mutation Detection

DefinitionDefinition A gene mutation is a change in the n

ucleotide sequence that composes a gene. This is a change or variation from the most common or wildtype sequence.

Somatic mutations

Germline mutations

Somatic mutationsSomatic mutations

are mutations that occur in cells of the body excluding the germline.

Affects subsequent somatic cell descendants

Limited to impact on the individual and not transmitted to offspring

Germline mutations

are mutations that occur in the germline cells

Possibility of transmission to offspring

Somatic vs. germline mutaSomatic vs. germline mutationstions

Mutations can occur in either somatic or germline tissue, but only germline mutations can be passed on to one’s offspring.

Causes of MutationCauses of Mutation

Spontaneous Due to naturally-occurring errors in DNA replication

Induced Due to exposures to radiation or chemical mutagens

Causes of MutationCauses of Mutation

Major TypesMajor Types Point mutations

silent mutation missense

mutation nonsense

mutation splicing

mutation

Rearrangements frameshift mutation codon deletion large deletion and

insertion deletions and

duplications trinucleotide

expansion

Point mutationPoint mutation substitution of one base with another

Point mutationPoint mutation Transition

Transversion

purine replaces purine

pyrimidine replaces pyrimidine

orA -> G or G -> A

C -> T or T -> C

purine replaces pyrimidine

pyrimidine replaces purine

orA or G -> T or C

T or C -> A or G

What are the What are the consequences?consequences?

Silent mutation

Missense mutation

Nonsense mutation

Splicing mutation

Silent MutationSilent Mutation changes one codon for an amino acid to an

other codon for that amino acid no change in amino acid

CGT - CGC

Missense mutationMissense mutation A point mutation that exchanges one codon for

another causing substitution of an amino acid

Missense mutations may affect protein function severely, mildly or not at all.

CGT AGT

HemoglobinHemoglobin Linus Pauling, 1949

Four globular proteins surrounding heme group with iron atom: two beta chains and two alpha chains

Function is to carry oxygen in red blood cells from lungs to body and carbon dioxide from cells to lungs

Single base change in Single base change in hemoglobin gene causes hemoglobin gene causes

sickle cell anemiasickle cell anemia

wildtypeallele

mutantallele

wildtypephenotype

mutantphenotype

Nonsense mutationNonsense mutation A point mutation changing a codon for an

amino acid into a stop codon (UAA, UAG or UGA).

5’ ATG GGA GCT CTA TTA ACC TAA 3’

met gly ala leu leu thr stop

5’ ATG GGA GCT CTA TGA ACC TAA 3’

met gly ala leu stop

Nonsense mutationNonsense mutation

Premature stop codons create truncated proteins.

Truncated proteins are often nonfunctional.

Some truncations have dominant effects due to interference with normal functions.

Splicing MutationsSplicing Mutations

“GT/AG rule” Disruption of existing splice sites

intron is not removed from mRNA Creation of novel splice sites in exons

HbE: missense mutation and splice error features of hemoglobinopathy and thalassemi

a

Hemoglobin E Hemoglobin E (Glu26Lys)(Glu26Lys)

GGT GGT GAG GCC bAGGT GGT AAG GC bE

Aberrant splicing

Splicing mutationSplicing mutation

6 7

Alu sequence

5

6 75 75

Normal transcript Transcript with Alu insertion: frameshift

Insertion or deletion Insertion or deletion mutationsmutations

The genetic code is read in triplet nucleotides during translation.

Addition or subtraction of nucleotides not in multiples of three lead to a change in the reading frame used for translation. Amino acids after that point are different, a phenomenon called a frameshift.

Addition or subtraction of nucleotides in multiples of three leads to addition or subtraction of entire amino acids but not a change in the reading frame.

Frameshift MutationFrameshift Mutation

5’ ATG GGA GCT CTA TTA ACC TAA 3’ met gly ala leu leu thr stop

5’ ATG GGG AGC TCT ATT AAC CTA A 3’

met gly ser ser ile asn leu ….

Insertion or deletion of codonsInsertion or deletion of codons

5’ ATG GGA GCT CTA TTA ACC TAA 3’ met gly ala leu leu thr stop

5’ ATG GGA TTA TTA GCT CTA TTA ACC TAA 3’ met gly leu leu ala leu leu thr

stop

Unequal Crossing Unequal Crossing OverOver

Unequal Crossing Unequal Crossing OverOver

Fusion gene

Triplet repeat expansion Triplet repeat expansion (Dynamic Mutation)(Dynamic Mutation)

Stretches of triplet repeats (i.e. CAGCAGCAG……) have variable lengths across individuals.

These lengths can vary to a small extent without consequence.

However, once a repeat length reaches the critical length, huge expansions can occur which will disrupt gene expression or function.

Triplet repeat diseases display “genetic anticipation” (the progressively earlier appearance and increased severity of a disease in successive generations) due to the continual expansion of the repeat as its passed from one generation to the next.

Trinucleotide Trinucleotide ExpansionExpansion

Fragile X Syndrome

CGGCGGCGGCGGCGGCGG Amplification

CpG island

FMR-1

(CGG)n

A Fragile X syndrome pedigree

The number below each individual indicates the number of CGG repeats in each copy of FMR1.

Myotonic dystrophy: a triplet repeMyotonic dystrophy: a triplet repeat diseaseat disease

5 -37 copies of CTG repeat normal phenotype 50-1000 repeats myotonic dystrophy Genes with 40+ copies are unstable and can gain

(or less commonly lose) repeat copies in successive generations.

Triplet repeat disordersTriplet repeat disorders

Disease Repeat

Normal

# of copies

Disease

# of copies

Fragile X syndrome CGG or CCG

6-50 200-2000

Freidreich ataxia GAA 6-29 200-900

Haw River syndrome CAG 7-25 49-75

Huntington disease CAG 10-34 40-121

Jacobsen syndrome CGG 11 100-1000

Myotonic dystrophy type 1 CTG 5-37 50-1000

Myotonic dystrophy type 2 CCTG < 10 > 100

Spinal and bulbar muscular atrophy CAG 14-32 40-55

Spinocerebellar ataxia CAG 4-44 40-130

Type I diseases(translated CAG repeat in ORF)

Huntington’s disease, Spinobulbar muscular atrophy, Spinocerebellar ataxia types 1, 2, 6 and 3 (MJD), Dentatopallidoluysian atrophy

All type I diseases are thought to be due to the presence of an expanded polyglutamine tract in the expressed proteins. This gain of function mutation leads to progressive neuronal dysfunction.

Type II diseases(triplet repeat outside of ORF)

Fragile X syndrome (CGG in 5’UTR hypermethylation tx)

Friedreich ataxia (GAA in intron triplex DNA structure tx)

Myotonic dystrophy (CTG in 3’UTR mechanism unknown)

Gene mutations Gene mutations (functional changes)(functional changes)

Loss-of-function (usually recessive)

Null – complete absence of a gene product or its function

Leaky – partial absence of a gene or its function

Gain-of-function (usually dominant)A mutation that confers a new function to the gene product

Misregulated (usually recessive)A mutations in a promoter, enhancer, or regulatory elements of the mRNA (untranslated regions or splice site junctions)

Wild Type Allele Null (loss-of-function) Allele

Leaky Allele Gain-of-function Allele

Gene mutations Gene mutations (functional changes)(functional changes)

Large

Small

- -

++Northern

Western

Mutation DetectionMutation Detection

Sequencing is “gold standard” Methods to detect known mutations Methods to detect unknown mutations

Detecting Known Detecting Known MutationsMutations

Insertion or deletion

large fragments – by Southern

small fragments – by PCR Point mutation

Restriction site altered by mutation RFLP or PCR/restriction enzyme digestion

No restriction site altered by mutation Allele specific oligonucleotide (ASO) probe

Southern Blot Application: Southern Blot Application: Fragile-X SyndromeFragile-X Syndrome

Fragile-X is most frequent genetic cause of MR after Down’s Syndrome

Unstable trinulceotide repeat in FRAXA Normal 6-54 repeats Premutation 55- ~200 repeats Affected > ~200 repeats

FRAXA

(CGG)n

EcoRI EcoRISacII

CpGIsland

ProbeTarget

5.2 kb

2.8 kb

Fragile-X DiagnosisFragile-X Diagnosis

Duchenne Muscular Duchenne Muscular DystrophyDystrophy

Xp21; gene is 2.3 Mb long; 79 exons 2/3 of cases due to deletions of some

exons Becker muscular dystrophy is allelic Diagnosis

Southern analysis PCR of exons

Duchenne Muscular Duchenne Muscular DystrophyDystrophy

17 18 19 44 45 46 47 48 49

1719444548

C P1 P2 P3 P4Patient1: exon 44 deleted

Patient2: exon 17deleted

Patient4: exon 19 deleted

Patient3: exon 45 deleted

DNA Diagnosis of HbDNA Diagnosis of HbS S

Hb bA

Hb bS

MstII MstII MstII

Glu

Val

… CCT GAG GAG … (Hb bA)… CCT GTG GAG … (Hb bS)

CCT NAG G (MstII)… CCT AAG GAG … (Hb C)

1.15 kb

1.35 kb

DNA Diagnosis of HbDNA Diagnosis of HbS S

By SouthernBy Southern

Genomic DNA

Digested DNA with Mst II

Seperated on agarose gel

denature

Transfer to membrance

Hybridize with the probe

Hb bA

Hb bS

MstII MstII MstII

Glu

Val

1.15 kb

1.35 kb

probe

Wild type: 1.15kb

Mutant: 1.35kb

HbHbSS Testing By Southern Testing By Southern AnalysisAnalysis

A/A A/S S/S

1.35 kb

1.15 kb

HbHbSS Testing By PCR-RFLP Testing By PCR-RFLP

Amplify region of -globin around codon 6

Cut with MstII Run products on gel Much faster than Southern analysis

Hb bA

Hb bS

MstII MstII MstII

Glu

Val

1.15 kb

1.35 kb

100

150

PCR product: 250bp

Sickle Cell DiseaseSickle Cell Disease

Sickle Cell Disease

HeterozygoteNormal

250

150100

Allele specific Allele specific oligonucleotide hybridization oligonucleotide hybridization

ASOASO Dot-blotting, apply and bind target DNA on

membrane (nylon, nitrocellulose) Denature and hybridize labeled probe, wash and

detect (radio-active or non radioactive techniques)

Can be used for the detection of point-mutations ASO-probe 15-20 nucleotides, difference central

in probe Also reverse dot-blot ASO, unlabeled probe is

bound to membrane, hybridize with labeled target

ASOASO

ASOASO

Detecting Unknown Detecting Unknown MutationsMutations

Mutation screening

- Chemical cleavage of mismatch

- SSCP

- Heteroduplex formation

- Southern analysis Mutation confirming

Generate specific Generate specific fragment by PCRfragment by PCR

Amplify exons from genomic sequences Amplify cDNA from mRNA


From genomic DNA

PCR amplification

PCR product analysis

Design PCR primers Extract genomic DNA

SIZE CHANGE?

SEQUENCE CHANGE?


From mRNA

RT-PCR

PCR product analysis

Design PCR primers Extract RNA

SIZE CHANGE?

SEQUENCE CHANGE?

In chemical mismatch cleavage, a test duplex is mixed with a wild type duplexand the mix is denatured and rehybridised.

If a mutation is present in the test duplex, amismatch occurs. Chemicals are used to break the DNA at the point of mismatch.

The sizes of the resulting fragments give anindication of the position of the mutationin the duplex (see next 3 slides).

CHEMICAL MISMATCH CLEAVAGE

*

*


Mutant Wild type

*

*

Heteroduplexeswith single nucleotide mismatches

*

*

*

Hydroxylamine Osmiumtetroxide

Piperidine

Chemical Mismatch Cleavage


Electrophorese

Denature

WT M


Hydroxylamine - mismatched ‘C’

Osmium tetroxide - mismatched ‘T’

The chemicals used cleave at specific nucleotides:

All possible mismatches can be detectedusing these two reagents.

Single strand conformation polymorphismanalysis (SSCP), takes advantage of the secondary structure (conformation) of single stranded DNA.A mutation in a DNA strand may changethe conformation of that strand. The mutantconformation may electrophorese differently to the wild type conformation (see next 2 slides).

SSCP

Denature

Renature rapidly

Wild type Mutant

SSCP

Electrophorese

ss DNA

ds DNAWT M

Wild type Mutant

SSCP

In classical heteroduplex analysis, test DNA is mixed with wild type DNA, the mix is denatured and allowed to rehybridise to give heteroduplexes. If a mutation is present, the heteroduplexes contain a mismatch which affects their electrophoretic mobility in certain gel systems (see next 2 slides).

Heteroduplex Analysis

*

*

Classical Heteroduplex Analysis

Mutant Wild type

*

*

Heteroduplexeswith single nucleotide mismatches

*

*

Classical Heteroduplex Analysis

WT MWT+M

heteroduplex

homoduplex

Large genetic changes

deletions

insertions

rearrangements

inversions

Southern Blot Analysis

Southern Blot Analysis

R R R RR

WT D1 D2 I

Hybridisationprobe

D1 = whole genedeletion

D2 = partial genedeletion

I = insertion

Hybridisation patternswhich differ to the wild type sequenceindicate a sequence

alteration.

Polymorphisms

Definition Major types Application

What makes us different from each other?

The answer is

POLYMORPHISMSPOLYMORPHISMS

Genetic polymorphism

The occurrence in a population of two or more genetically determined forms in such frequencies that the rarest of them could not be maintained by mutation alone

Definition

A polymorphic locus is one at which there are at least two alleles, each with a frequency greater than 1%. Alleles with frequencies less than 1% are referred to as mutants.

Mutations and Normal Mutations and Normal VariantsVariants

Gene A

Allele 1 (Normal) 99.5%

Allele 2 (Mutant) 0.5%

Gene B (Polymorphic)

Allele 1 (Nl Variant) 50%



Gene C (Polymorphic)Allele 1 (Nl Variant) 45.5%Allele 2 (Nl Variant) 30%Allele 3 (Nl Variant) 15%Allele 4 (Mutant) 0.2%Allele 5 (Mutant) 0.3%

PolymorphismPolymorphism Polymorphism presents at different level:

Immunoglobulins ABO blood groups Minor variants in chromosome structure DNA sequence polymorphism

Chromosomal Chromosomal PolymorphismPolymorphism

Human blood groupsHuman blood groups

It is variation of DNA sequence that is common in the general population (>1%)

Most are neutral, but some confer susceptibility or resistance to disease

It may be a single nucleotide change or variation in copy number of a repetitive sequence

DNA PolymorphismDNA Polymorphism

DNA PolymorphismDNA PolymorphismDifferent alleles of a polymorphic locus

can be tracked thru generations in a family

If the chromosomal location of a polymorphic locus is known, the locus can be used to track a chromosomal region thru the family. When used in this fashion, the polymorphic locus is called a polymorphic “marker” locus


(CA)7/ (CA)10 (CA)12/ (CA)14

(CA)7/ (CA)12 (CA)7/ (CA)14 (CA)10/ (CA)12 (CA)10/ (CA)14


(CA)7/ (CA)10 (CA)12/ (CA)14



Nomenclature for Human Nomenclature for Human Genetic MarkersGenetic Markers

D4S182

Segment 182 (numbered in order of discovery)Chromosome 4

DNA

*D4S182 may have multiple alleles defined by RFLP, SSR, etc.

Major types Major types Restriction fragment length polymorphism

(RFLP) Short tandem repeat polymorphism (STR)

Mini-satellite repeat polymorphismMicrosatellite repeat polymorphism

Single nucleotide polymorphism(SNP)

RFLPsRFLPs polyms that alter the length

of restriction fragments Result from

- changes (e.g. SNPs) that introduce or delete an restriction enzyme site

Two alleles Genotyping by Southern or PCR-

RFLP

Restriction Fragment Length Polymorphisms

Restriction Fragment Length Polymorphism (RFLP)

3.0kb 2.0kb

1

2

11 12 22

Genotyping-by Genotyping-by SouthernSouthern

Isolate DNA Digest DNA w/ restriction enzyme Size fractionate DNA Denature DNA Blot SS DNA to membrane

Genotyping-by Genotyping-by SouthernSouthern

Prepare a probe Label Denature

Hybridize probe with membrane Autoradiography

11 11 11 11 11 11 12 11 11 22 12 12 22 11 12 12

Genotyping-by PCR-Genotyping-by PCR-RFLPRFLP

Design primers PCR Digest PCR product with RE Gel electrophoresis

100 150

250

100 150

11 11 11 11 11 11 12 11 11 22 12 12 22 11 12 12

Short Tandem Repeats Short Tandem Repeats (STR)(STR)

Variable number of tandem repeats Multiple alleles Genotyping by Shouther or PCR-SSRP

Variable Number of Tandom Repeats (VNTR)

VNTRs - MinisatellitesVNTRs - Minisatellites

Short Tandem Repeats Short Tandem Repeats Polymorphism (STR)Polymorphism (STR)

....CACACACACACACA....

....CACACACACACACACACACA....

....CACACACACACACACACACACACA....

....CACACACACACACACACACACACACACACACA....

(CA)7

(CA)10

(CA)12

(CA)14

Microsatellite DNA PolymorphismPCR

Gel electrophoresis separates DNA by size

Single Nucleotide Single Nucleotide Polymorphism (SNP)Polymorphism (SNP)

Replacement at a single nucleotide in a genome Must occur in > 1% of the population Account for over 90% of genetic variation amon

g humans Occurrence frequency of once every ~1000 base pa

irs, in both in coding and noncoding regions Approximately 3-4 million SNPs in human genome About 2/3 of SNPs are C replaced by T

GATACAATGCATCATAGATGCAATGTATCATAGATGCAATGCATCATA

Single Nucleotide Polymorphism (SNP)

almost exclusively biallelic

SNP Genotyping vs Microsatellites

A microsatellite (tetranucleotide) with four alleles:

GATA GATA GATA GATA

GATA GATA GATA GATA GATA

GATA GATA GATA GATA GATA GATA

GATA GATA GATA GATA GATA GATA GATA

Allele 1

Allele 2

Allele 3

Allele 4

A single nucleotide polymorphism with two alleles (SNP):

ATG CAT CTG GCT GGA TCC TCG TAGA alleleA

ATG CAT CTG GCT GGG TCC TCG TAGG alleleG ***

Detection for SNPsMicroarray

One gene at a time before, now tens of thousands simultaneously

Gene expression Gene disease relation Gene-gene interaction Finding Co-Regulated Genes Understanding Gene Regulatory Networks Many, many more

Basic idea of MicroarrayBasic idea of Microarray Construction

Place array of probes on microchipProbe (for example) is oligonucleotide ~25

bases long that characterizes gene or genome

Chip is about 2cm by 2cm Application principle

Put (liquid) sample containing genes on microarray and allow probe and gene sequences to hybridize and wash away the rest

Analyze hybridization pattern

Application of DNAPApplication of DNAP

Linkage analysis Indirect gene diagnosis Paternity test Analyzing the origin of extra chromosome LOH

Mapping disease genes by LMapping disease genes by Linkage Analysisinkage Analysis

Disease Dd dd Dd dd dd Dd Dd Dd dd

Marker 1 12 34 13 23 24 14 13 14 23

Marker 2 12 34 13 14 23 24 13 14 23

Marker 1 : linked to disease locus

Marker 2 : unlinked to disease locus

Linkage AnalysisLinkage Analysis

D17S579 5/5 4/3 4/5 4/3 2/2 4/2 4/1 1/2

3 65421

1 2

II

III

D12S90 3/4 5/3 5/4 5/3 1/2 3/2 5/2 5/2

Disease resistant population Disease susceptible population

Genotype all individuals for thousands of SNPs

ATGATTATAG ATGTTTATAG

Resistant people all have an ‘A’ at position 4 in geneX, while susceptible people have a ‘T’

geneX

Indirect Gene DiagnosisIndirect Gene Diagnosis

Known disease genes

- large genes

- different disease-causing mutation Unknown disease genes

Indirect Gene DiagnosisIndirect Gene Diagnosis

M1 M2

M1 1 12 1 1 3 1 11 2

M2 2 13 3 3 2 3 23 1

XAY XAXa

XaY

XaY XAY XaY

? ?

XAXa

XAY

Paternity testing

ForensicsForensicsCrime sceneSample:Victim + perp

Victim AJ OJ DJ

Suspects

Down syndromeDown syndrome

karyotypekaryotype

Down’s Syndrome

Loss of Heterozygosity Loss of Heterozygosity (LOH)(LOH)

frequent somatic mutation associated with cancerfrequent somatic mutation associated with cancer

Normal allele Mutant allele

Chromosome loss

Deletion Unbalanced translocation

Loss and reduplication

Mitotic recombination

Point mutation

Loss of normal allele

LOH AnalysisLOH Analysis

Normal tissues

Tumor

M1

M2

M3

Documents

Human Molecular Genetics Institute of Medical Genetics Yaoqin Gong 2003