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.


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


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






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






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


STR


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






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


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


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


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.






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






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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08.13.08: DNA Sequence Variation