08.13.08: dna sequence variation
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DNA Sequence Variation
David Ginsburg, MD August 13, 2008
M1 Patients and Populations
Fall 2008
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms: – RFLP, VNTR, STR, SNP, CNV
• Know the different classes of DNA mutation: – Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements • Understand how to distinguish a disease-causing
mutation from a neutral DNA sequence variation
Learning Objectives
Chromosomes, DNA, and Genes Proteins
\ Jfdwolff (wikimedia)
Wikimedia Commons
TRANSCRIPTION
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.1
1944 DNA is the
genetic material
1949 Abnl Hemoglobin
in sickle cell anemia
1953 Double helix
1956 Glu 6 Val in sickle hemoglobin
1966 Completion of the
genetic code
1970 First restriction
enzyme
1972 Recombinant
plasmids
1975 Southern blotting
1981 Transgenic mice
1983 Huntington
Disease gene
mapped
1985 PCR
1986 Positional
cloning (CGD, muscular
dystrophy, retinoblastoma
1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000
1987 Knockout
mice
1989 Positional cloning without deletion (CF)
1990 First NIH-approved gene therapy experiment
1996 Complete yeast genome sequence
1995 1st complete bacterial genome sequence
2001 Draft human
genome sequence
Since 2001: Complete human genome complete; human hapmap; Other genomes: mouse, rat, dog, chimp, chicken, zebrafish…18 vertebrates, 8 invertebrates, >500 microbial genomes.
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.4
DNA Samples and PCR
Blood sample
DNA for analysis cheek swab urine sample Forensic specimens
DNA Repeat cycles
Amplified product
Target sequence
primers
Source: Dennis Myts (wikimedia)
D. Ginsburg
SINGLE-STRANDEDDNA PROBESFOR GENE A
MIXTURE OF SINGLE-STRANDEDDNA MOLECULES
+ B
B B
A
A
C
CC
D
D
D
E
EE
F
F F
ONLY A FORMS A STABLEDOUBLE-STRANDED COMPLEXES
A, C, E ALL FORMSTABLE COMPLEXES
STRINGENT HYBRIDIZATION REDUCED-STRINGENCY HYBRIDIZATION
A
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.8
Hybridization array: gene chip
DNA chip v6.0: ~1 million SNPs RNA expression: All ~ 20,000 genes CGH: Survey whole genome
for large deletions, insertions, rearrangements
Ricardipus (wikipedia)
Image of DNA sequencing
process removed
DNA Sequencing
Source Undetermined
New Sequencing Technologies
Source Undetermined
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms: – RFLP, VNTR, STR, SNP, CNV
• Know the different classes of DNA mutation: – Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements • Understand how to distinguish a disease-causing
mutation from a neutral DNA sequence variation
Learning Objectives
DNA Sequence Variation
• DNA Sequence Variation: – Human to human: ~0.1% (1:1000 bp)
• Human genome = 3X109 bp X 0.1% =~3X106 DNA common variants
– Human to chimp: ~1-2% – More common in “junk” DNA: introns, intergenic regions
• poly·mor·phism Pronunciation: "päl-i-'mor-"fiz-&m Function: noun : the quality or state of existing in or assuming different forms: as a (1) : existence of a species in several forms independent of the variations of sex (2) : existence of a gene in several allelic forms (3) : existence of a molecule (as an enzyme) in several forms in a single species
Mutations A mutation is a change in the “normal” base pair sequence
• Can be: – a single base pair substitution – a deletion or insertions of 1 or more base pairs (indel) – a larger deletion/insertion or rearrangement
Dennis Myts (wikimedia)
Polymorphisms and Mutations
• Genetic polymorphism: – Common variation in the population:
• Phenotype (eye color, height, etc) • genotype (DNA sequence polymorphism)
– Minor allele frequency > 1%
• DNA (and amino acid) sequence variation: – Most common allele < 0.99 = polymorphism
(minor allele > 1%) – Variant alleles < 0.01 = rare variant
• Mutation-- any change in DNA sequence – Silent vs. amino acid substitution vs. other – neutral vs. disease-causing
• Common but incorrect usage: – “mutation vs. polymorphism”
• balanced polymorphism= disease + polymorphism
Common but incorrect usage: “a disease-causing mutation” OR “a polymorphism”
All DNA sequence variation arises via mutation of an ancestral sequence
“Normal”
“Disease”
< 1% > 1%
Rare variant or “private”
polymorphism polymorphism
Disease mutation Example: Factor V Leiden
(thrombosis) 5% allele frequency
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms: – RFLP, VNTR, STR, SNP, CNV
• Know the different classes of DNA mutation: – Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements • Understand how to distinguish a disease-causing
mutation from a neutral DNA sequence variation
Learning Objectives
Types of DNA Sequence Variation • RFLP: Restriction Fragment Length Polymorphism • VNTR: Variable Number of Tandem Repeats
– or minisatellite – ~10-100 bp core unit
• SSR : Simple Sequence Repeat – or STR (simple tandem repeat) – or microsatellite – ~1-5 bp core unit
• SNP: Single Nucleotide Polymorphism – Commonly used to also include rare variants
• Insertions or deletions – INDEL – small (few nucleotides) insertion or deletion
• Rearrangement (inversion, duplication, complex rearrangement)
• CNV: Copy Number Variation
VNTR
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.19
STR
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.22
SNP
• Most are “silent” • Intragenic • Promoters and other regulatory sequences • Introns • Exons
– 5’ and 3’ untranslated regions – Coding sequence (~1-2% of genome)
Allele 1 A U G A A G U U U G G C G C A U U G A A C
Allele 2 A U G A A G U U U G G U G C A U U G A A C
Adapted from ASCO teaching slides
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms: – RFLP, VNTR, STR, SNP, CNV
• Know the different classes of DNA mutation: – Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements • Understand how to distinguish a disease-causing
mutation from a neutral DNA sequence variation
Learning Objectives
Silent Sequence Variants (Synonymous SNP)
Normal mRNA Protein
A U G
Met
A A G
Lys
U U U
Phe
G G C
Gly
G C A
Ala
U U G
Leu
A A
Gln
C
Sequence variant
mRNA Protein
A U G
Met
A A G
Lys
U U U
Phe
G G U
Gly
G C A
Ala
U U G
Leu
A A
Gln
C
G
Changes that do not alter the encoded amino acid
Adapted from ASCO teaching slides
Missense Mutations
Missense
Missense: changes to a codon for another amino acid (can be harmful mutation or neutral variant)
(nonsynonymous SNP)
mRNA Protein
Normal mRNA Protein
A U G
Met
A A G
Lys
U U U
Phe
G G C
Gly
G C A
Ala
U U G
Leu
A U G
Met
A A G
Lys
U U U
Phe
A G C
Ser
G C A
Ala
U U G
Leu
A A
Gln
C
A A
Gln
C
Adapted from ASCO teaching slides
Nonsense: change from an amino acid codon to a stop codon, producing a shortened protein
(nonsynonymous SNP)
Nonsense mRNA Protein
Normal mRNA Protein
A U G
Met
A A G
Lys
U U U
Phe
G G C
Gly
G C A
Ala
U U G
Leu
A U G
Met
U A G U U U G G C G C A U U G
A A
Gln
C
A A C
Nonsense Mutations
STOP Adapted from ASCO teaching slides
Frameshift U G C A A A U G
Met
A A G
Lys
G C G
Ala
C A U U U G
Leu
Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and (usually) shortened protein
mRNA Protein
Normal mRNA Protein
A U G
Met
A A G
Lys
U U U
Phe
G G C
Gly
G C A
Ala
U U G
Leu
A A
Gln
C
Frameshift Mutations
STOP Adapted from ASCO teaching slides
Exon 1 Intron Exon 2 Intron Exon 3
Exon 1 Exon 3 Altered mRNA
Splice-site mutation: a change that results in altered RNA sequence
Exon 2
Splice-site Mutations
Adapted from ASCO teaching slides
Other Types of Mutations
• Mutations in regulatory regions of the gene • Large deletions or insertions • Chromosomal translocations or inversions
Copy Number Variation (CNV) • Kb to Mb in size (average ~250 Kb) • >>2000 known, affect ~12% of human genome • ? ~100 / person • ? Role in human disease/normal traits
Nature 444:444, Nov 2006.
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms: – RFLP, VNTR, STR, SNP, CNV
• Know the different classes of DNA mutation: – Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements • Understand how to distinguish a disease-causing
mutation from a neutral DNA sequence variation
Learning Objectives
Tests to Detect Mutations
• Many methods/technologies • Rapidly changing
• DNA sequencing – Most direct and informative – The gold standard – Targeted region (known mutation) – “Whole” gene (unknown mutation) – … Whole genome
How do we distinguish a disease causing mutation from a silent sequence variation?
• Obvious disruption of gene – large deletion or rearrangement – frameshift – nonsense mutation
• Functional analysis of gene product – expression of recombinant protein – transgenic mice
• New mutation by phenotype and genotype
• Computer predictions • Disease-specific mutation databases
– Same/similar mutation in other patients, not in controls
• Rare disease-causing mutation vs. private “polymorphism” (rare variant)
X
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms: – RFLP, VNTR, STR, SNP, CNV
• Know the different classes of DNA mutation: – Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements • Understand how to distinguish a disease-causing
mutation from a neutral DNA sequence variation
Learning Objectives
Slide 5: Jfdwolff (wikimedia) http://creativecommons.org/licenses/by-sa/3.0/; http://commons.wikimedia.org/wiki/File:Chromosome_zh-tw.svg
Slide 6: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.1 Slide 7: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.4 Slide 8: D. Ginsburg Slide 9: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.8 Slide 10: CC: BY-SA: Ricardipus (wikipedia) http://creativecommons.org/licenses/by-sa/2.0/deed.en Slide 11: Image of DNA sequencing process removed Slide 12: Undetermined Slide 13: Undertirmined Slide 16: Dennis Myts (wikimedia) Slide 21: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.19 Slide 22: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.22 Slide 23: Adapted from ASCO teaching slides Slide 25: Adapted from ASCO teaching slides Slide 26: Adapted from ASCO teaching slides Slide 27: Adapted from ASCO teaching slides Slide 28: Adapted from ASCO teaching slides Slide 29: Adapted from ASCO teaching slides Slide 31: Nature 444:444, Nov 2006.
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