Genomics: bridge from Lecture 8

1
Genomics bridge from Lecture 8 tools
2
Applications getting into the genomeapplying
recombinant DNA to genome

• Combining human genetics
• with genomics/molecular biology
• Populations, isolated especially
• deCODE Genomics
• Sequana Therapeutics
• Iceland-based
• 11 generations back to 17th century
• In general,
• Homogeneous population
• 146 families
• 5,136 markers
• 1,100 years geneology
• Ex., Asthma 100 patients
• Example of isolated populations
• Frequency of PKU
• 1 in 15,000
• 1 in 4,500 Ireland
• 1 in 100,000 Finland

• 2005

3
Human Genetics Human diseases genome-based
approaches Identifying and locating human
disease genes
4
Transmission patterns of three simple
disease-related genes

• Three patterns of transmission
• Auto dominant
• Auto recessive
• X-linked (dominance N/A)
• non gene-based, Epigenetic
• Determine mode of inheritance

5

X-linked genetic disease hemophilia

• Hemophilia, X-linked
• (1/10,000 males), across all populations
• (1/100,000,000 females)
• May skip for six generations
• Treat with factor VIII (hemA) or factor IX (hemB)
• Porcine factor VIII
• Recombinant human factor VII
• Prev, blood products-gt HepB, C and HIV (and ?)

• wikipedia

6

X-linked genetic disease hemophilia

• Genes, genomics and world history
• Hemophilia, 1/10,000 X-linked across all
  populations
• (1/100,000,000 females)
• Great-grandsons, King Alfonso and Gonzalo, Spain
• Grandson, Czar-to-be Alexis, Russia
• Rasputin, Bolshevik Revolution (October 17, 1917)
• Pax Tokugawa, Japan ca 1600-1850

• http//www.sciencecases.org/hemo/hemo.asp

7
Identification of disease locus

• Determine chromosomal position of disease allele
• Genetic linkage More markers, the finer the map
• Phenotypic markers allow identification and
  tracking of trait thru linkage, assortment
• Density of markers for high-resolution human
  genetic map is 1 per 5 cMorgan
• 1 cMorgan distance between two markers resulting
  in one recombination in 100 progeny

8
Restriction fragment length polymorphisms (RFLPs)

• RFLPs- first type of molecular markers in linkage
  studies
• RFLPs result from mutations in RE sites,
• Mutations at RE sites change pattern of RE digest
• Use as fingerprint
• Probe labeled complement, any size

9
Pedigree analysis of a trait

• RFLP analysis of region in Chrom5
• Taq1, Southern blot
• Three alleles 10, 7.7, 6.5 kb
• simple disease
• If dominant allele is 1, what phenotypes are
  expected?

10
Linkage disequilibrium

• Linkage analysis can localize a disease gene to
  within 1 cM
• In humans, 7.5 x105 nucleotides
• Linkage disequilibrium phenomenon may be used for
  higher resolution ( 0.1 cM)
• Haplotype set of polymorphisms
• Blue ancestral haplotype Pink (?) new
  haplotype
• After many generations, the disease allele is
  linked to different ancestral segments
• Position of disease allele is located by scanning
  for
• highly conserved polymorphisms of ancestral
  haplotype

11
Maps, relationship between genetic and physical
maps of a chromosome

• Linkage analysis can localize a disease gene to
  105 nucleotides
• 105 nucleotides could contain up to 10 different
  genes (or more)
• Other methods comparison of gene expression
  patterns in normal and afflicted

12
Genomics tools Chromosome walks

• Bottom up approach to physical map
• 50kb into cosmids, 250kb cloned into BAC or P1
  vector
• RE map, markers identification and probe
  hybridization analysis

13
Multigenic syndromes

• Monogenic traits vs multigenic
• Genetic heterogeneity, similar phenotypes caused
  by mutations in more than one gene
• ex., tuberous sclerosis (TSC) is a rare genetic
  disease
• causes benign tumors to grow in the brain
  and on other organs such as
• kidneys, heart, eyes, lungs, and skin it
  affects the central nervous system.
• Identical phenotype via mutation in TSC1, 9q34,
  or TSC2, 16p13
• Unrelated genes
• Presumably both genes act at different points in
  same biochemical or regulatory pathway
• one ligand, one receptor? TSC2- tuberin,
  suppresses tumor growth

• ex, retinitis pigmentosa
• Degeneration of retina leading to blindness
• Sixty different genes implicated

14
New areas in human genomics-based
diseases (better tools, better understanding. bet
ter diagnoses to better therapeutics)
15

Another perspective people and mutations

• Essay, NYTimes. Oct 9, 2007. BHLerner, MD
• Longevity reflects how the understanding of
  sickle cell disease has changed
• Initially misdiagnosed in early 1960s- found its
  way into the public consciousness.
• First complaints of joint aches- met with
  skepticism
• At 16, diagnosed with sickle cell anemia and
  ?-thalassemia
• Combined condition may be less severe than pure
  sickle cell disease, contribute to longevity ?
• Course of disease painful sickle cell crises,
  spleen removal, shoulder surgery, degeneration of
  hips
• Fear of promoting drug addiction under-prescribe
  pain meds, requiring blood tests first

16

Natural mutation HBB, beta hemoglobin gene

• Molecular, genetic and biochemical perspectives
• 1600 bp, three exons
• mRNA 626 bp 44 bp codes for AAc
• Normal rbc 120d sickle cell 10-20d
• Left hemoglobin, green and blue alpha chains
• Gold and aqua beta chains
• Gold spheres phosphates box Glu6
• Variant at 6 most common variant sickle cell
  HBS
• Right clumping of two hemoglobins with variant
  AAc
• Several 100 HBB variants
• Autosomal recessive

• http//www.ornl.gov/sci/techresources/Human_Genome
  /posters/chromosome/hbb.shtml

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Spontaneous chemical reactions

• Chemical basis of mutations
• Bases undergo random modifications
• If uncorrected, mutation is hardcopied
• SNP

18
Genomics Genetic vs epigenetic-unusual

• 10/28/06 Scott Adams. Rare example of recovery-
  largely but not totally
• Spasmodic Dysphonia, mysterious disease in which
  parts of the brain
• controlling speech shut down or go haywire.
• 30,000 Americans, typically in 40s and 50s
• Phenotype Typically unable to converse in
  normal voice, but under
• different circumstances, immediately after
  sneezing or laughing,
• can speak in exaggerated falsetto or baritone,
  or while reciting poetry.
• Off-label drug use Botox

19
Genomics Genetic vs epigenetic-unusual

• Diane Rehm, diagnosed in 98
• Cause unknown
• May run in families (inherited)
• Chrom 9 region/gene affecting spasms of the vocal
  cords.
• Also spasms in eyes, arms, legs and mouth.
• Also may have multiple dystonias, movement
  disorders
• Some cases, following upper respiratory
  infection, injury to larynx, overuse of voice, or
  stress

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Genomics Genetic vs epigeneticSporadic to
familial (heritable) mutation

• Dr. Ignazio Roiter is a country doctor.
• His wife, Elisabetta, is a descendant of a
  prominent Italian family with roots in Venice
  since the 1600s.
• Roiter's medical training hadn't prepared him for
  the sad and puzzling ailment that was suddenly
  overtaking
• Elizabetta's aunt, a woman in her 40s.

NYTimes 05/06/01. To sleep no more In 1791,
in a small town near Venice, a man named Giacomo
was born... one day in the fall of 1836, at the
age of 45, Giacomo fell mysteriously ill.. He
began to suffer from dementia. Eventually he was
confined to bed, lying awake in torment. Then
he died.

• Giacomo had three children who survived infancy.
• One had six, in turn.
• Over the next 150 yrs, his descendants
  flourished-
• becoming prominent doctors and businessmen,
• one owning 130 apts in Venice including a
  palazzo on the Grand Canal
• Running parallel with the familys affluence was
  a record of premature death and illnesses
• Parish books noted epilepsy and fever nervous
  gastric fever. Death certificates noted
  meningitis,
• Economos disease, presenile dementia,
  leukoencephalitis, alcoholic encephalopathy and
  ictus
• Identified as FFI in 1986.
• Six generations 288 relatives with 29 affected
• Vanishingly rare for a time this was the only
  case. Since then, 30 other families have been
  identified.

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Sporadic mutation Phenocopy/genocopy

• Fatal familial insomnia (FFI)
• Phenotype Loss of sleep (progressive),
  dysautonomia and motor signs
• Resources Case Studies in Virtual Genetics
  OMIM, Online Mendelian Inheritance in Man.
  JHU/NCBI
• VMcKusick 66, Mendelian Inheritance in Man
• Linked to mutation at codon 178 (C178) of prion
  protein gene (PRNP) Asp178Asn (D178N) AND
  homozygous Met 129
• Asn replaced by Asp subset of
  Creutzfeldt-Jakob disease
• Autosomal dominant localized to chr20
• Amyloid plaques in thalamus, center of
  communications from brain to body
• Prusiner Misfolded proteins can cause disease
• Expt Brain extrs of FFI and C-J inject into
  mice developed disease inject this into mice
  devd disease
• Note If Met129 to Val, it results in
  Creutzfeldt-Jacob disease (Mad Cow)

• JMastrianni May 99. NEJM Sporadic Fatal Insomnia
• non-inherited version occurs without the
  abnormal gene
• exact phenocopy not a genocopy of FFI
• Rapid progression of disease
• 44 yr old, recent sleeping problems pills
• 6 mos- walking difficulties
• 12 mos- wheelchair and memory probs, delucions
  and hallucinations
• 16 mos death

22
Inborn Errors of Metabolism (Human Genetics)

• Archibald Garrod, 1909.
• Inherited human diseases, as inborn errors of
  metabolism
• That is, rare genetic disorders involving
  metabolism
• Suggested alkaptonuria is an inherited condition
  caused by a deficiency
• in the enzyme that normally breaks down
  alkapton, resulting in dark
• red urine.
• Basis for one gene, one enzyme hypothesis.
• (ignored till rediscovered)
• 1940s Beadle and Tatum

23
Example alkaptonuria

• Autosomal recessive gene encoding enzyme in Tyr
  catabolic pathway
• Homogentisic acid oxidase acts upon homogentisic
  acid (alkapton)

• Alkapton harmful to bones and cartilage, usually
  excreted in urine
• Progressive arthritis, due to bones and cartilage
  degeneration

24
One pathway, multiple possible sites of mutations
25
Genome-based metabolic diseases

• Other examples,
• Defective Proteins and Disease
• Defects in Carbohydrate Metabolism
• Defects in Cholesterol and Lipoprotein Metabolism
• Mucopolysaccharide and Glycolipid Disorders
• Defects in Amino and Organic Acid Metabolism
• Porphyrias and Bilirubinemias
• Errors in Fatty Acid Metabolism
• Defects in Nucleotide Metabolism
• Disorders in Metal Metabolism and Transport
• Defects in Peroxisomes
• Diseases Associated with Defective DNA Repair

26
Gene expression, metabolic pathway regulation

• Biochemical pathways

27
Gene expression, metabolic pathway regulation

• Phenylketonuria or PKU
• IAFolling in 1934 observed hyperphenylalaninemia
  associated mental retardation
• Follings disease one of first to apply chemical
  analysis to disease- urine sampling
• Inherited error of metabolism
• Phenylalanine hydroxlase minus, so Phe
  accumulates
• Symptoms
• Mental retardation
• Organ damage
• Unusual posture
• Autosomal recessive, Chrom 12
• Population
• 1 in 15,000/ 1 in 4,500 Ireland/ 1 in 100,000
  Finland
• Mouse model

• Biochemical pathways

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Gene expression, metabolic pathway regulation

• Large neutral amino acid transporter
• Phe, Tyr, Trp compete for transport across
  blood-brain barrier
• Required for protein and neurotransmitter
  synthesis
• Reduction disrupts brain development and
  signaling
• Also for melanin production

• Biochemical pathways

29

• Cell cycle regulation
• Another example of gene expression function

• Cellular pathways
• also cell division

30
Developmental pathways

• Genetic pathways
• Most visual
• Most culturally impacted

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Lecture 9Genomics molecular cell biology/
recombinant DNA technology and.

• Molecular biology, recombinant DNA technology,
• human genetics
• Intro to genome-based diseases (hereditary)
• (Genomics/bioinformatics)
• basis for Genomics is genome data

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Genomics

• Origins biochemistry, enzymology, DNA
  replication (and repair)
• Discovery biology vs hypothesis driven
• Tools
• Technology
• Answers, more Questions
• Applications across other fields, eg human
  molecular biology and genetics

33

Human Genome Project (ongoing, still)

• 1977 DNA sequencing methodologies
• 1985 semi- automated DNA sequencing
  technology
• 1990 DOE and NIH present joint 5-year program to
  Congress 15-year project formally begins
• 1992 Low-resolution genetic linkage map
• 1995 First nonviral whole genome Hemophilus
  influenzae
• Smallest bacterium, Mycoplasma genitalium,
  minimum set of genes
• 1996 Methanococcus jannaschii, third branch of
  life
• Saccharomyces cerevisiae Human T-cell receptor
  region
• 1997 Escherichia coli bottleneckd Second
  Bermuda large-scale sequencing strategy meeting
• 1998 Caenorhabditis elegans
• 1999 First human chromosome (22) completely
  sequenced- 80k genes human genome
• 2000 Chromosome 21 (smallest and second
  completely sequenced) Draft 5, 16, 19
  Drosophila melanogaster
• 2001 Human chrom 20 finished (first 33.5Mb, 11
  gaps))
• Completion! Working drafts of human genome
  Sci (2/16/01) and Nat (2/15/01)
• 2002 Draft of mouse genome First draft of Fugu
  rubripes
• 2003 Chrom 14 (fourth chr completely sequenced),
  HGP done, Y, 7, 6
• 2004 Chrom 13, 19, 10, 9, 5
• Human gene count revised to 20k-25k (October)
• 2006 SGregory, et al. Nature 5/17. With
  publication of chromosome 1, HGP closes Final
  Chapter

34

Chromosome 22 completion

• Nature (Dec 2, 1999) Sanger Centre, Univ OK,
  Washington Univ and Keio Univ
• First human whole chromosome sequenced
• Longest, continuous stretch _at_23Mb
• 33.5 Mb total 11 gaps, mapped error rate
  150,000 bases
• because protein-coding genes do not seem to
  occur on short arm of 22
• 97 of long arm sequenced

35

Chromosome 22 completion

• Landscape
• 545 genes and 134 pseudogenes, possibly 200-300
  additional genes likely
• 247 hit as identical to previously identified
  human genes or proteins
• 150 additional genes with similarity to known
  genes
• 148 predicted genes homologous to known genetic
  markers (ESTs)
• Suggesting total number of genes 80,000
• Genes range in size from 1kb to 583kb, mean size
  190kb
• 39 of chromosome is copied into RNA as exons and
  introns
• 3 of chromosome encodes protein
• Several gene families (via gene duplication)
• Unexpected long-range complexity of repeat
  sequences near centromere
• chromosome rearrangement/reshuffles?
  (DiGeorge syndrome) Structure changes over time?
• Several regions of increased recombination
  several suppressed
• 160 human genes have counterparts in mouse
  genome, synteny mouse homologs in 8 regions

36

Chromosome 1 completion

• Largest and last published. SGregory, et al.
  Nature May06
• 60,000 pages or 223,569,564 bases (8 total)
• 99.9 accurate 1.3 Mb repetitive sequences
  gaps _at_99.4 complete
• 1000 new genes 3,141 genes 350 known diseases
• 4,500 SNPs

37
Semi-automated fluorescent DNA sequencing
Primer labelGenomics many sequences, genome
sequences

• Fred Sanger et. al., 1977.
• Maxam and Gilbert, 1977
• Leroy Hood et. al. 1986
• Applied Biosystems, Inc. 1987
• JM Prober et. al. _at_DuPont 2000
• H Swerdlow et. al. 1990, 1991
• BL Karger et. al. 1993

Note (Primer dye) 4 tubes-gt1 lane
38
Basecalling issues
39
ABI 377 data
40
Comparative genomics of loci and synteny
41
Applied Biosystems DNA analyzers(better
technology, better and more data -gtmore answers,
more questions)

• Capacity
• 1-cap 310
• 4-cap (3100), 3130
• 16-cap (3100), 3130xl
• 48-cap 3730
• 96-cap 3730xl

42
Improved methodology, technology -gt huge increase
in sequence data
43
Applied Genomics DNA sequencing and analysis
Ad4, Field Str 1-3, Ad7v, 4v, 6
QA, Assembly, re-Assembly, Analysis, Annotation,
Identification of probes (genome signatures)
Ad 4, 7, 7v, 3, 16, 21, Field Strains
1-3, Ad6, Ad4v
Ad16, Ad 6, Field Strain 5
Ad 7, 3, 21
44

• Bioinformatics
• BLAST (basic local alignment search tool)
• Stored sequences suggest functions of
• newly identified genes and proteins

45
BLAST analysis
46
BLAST analysis
47
BLAST analysis for the masses

• Limited to individuals, labs, companies
• Now, open access- eg, NIH BLAST, WU-BLAST

48
BLAST analysis of adenovirus hexon

• Individual proteins, genes, sequences

49
Alignment analysis of adenovirus hexon
50
The Elephant man

• Example of the power of BLAST analysis
• Neurofibromatosis
• Dominant mutation
• however, more likely
• Proteus Syndrome (?) (described 79)
• Partial gigantism, development of benign growths

51
inborn errors of metabolism Clinical to bench
genomics/bioinformatics

• Neurofibromatosis
• Dominant mutation

52
BLAST analysis to define function

• Neurofibromatosis
• Ira gp GTPase-accelerating protein -GAP-
• BLAST p value 10-28
• More examples of cross-species hits/relevance

several phenomena tying together
53
Genomics and bioinformatics -databases of
sequences Evolutionary relationships Paralogs
and orthologs

• Related sequences from different species give
• Clues to evolutionary relationships
• Comparison of proteins, genes, genomes
• Large-scale sequencing

54
Gene and protein relationships
55
Partial hits to sequences, protein domains

• Many times, protein shows no significant
  similarity to other proteins in db
• But, can identify short sequences that are
  functionally important
• Identify motifs, domains, repeats (strutural
  motifs)
• Take advantage of DNA/AAc sequences

56
Adenovirus- whole genome phylogeny

• Similar analysis, now, at whole genome level

57
Phylogenetic analysis of genes Zoonosis,
pathoepidemiology
National Geographic photo
58
Information paradox, ca 2003
59

• Examples of genes/proteins across species
• Importance of model organisms
• and others
• (you are not too different from the fly)

60
Genomics to Bioinformatics to Biochemistry to
Clinical

• Functional genomics
• Neurofibromatosis -gt line of NF1 mice
• UCLA study WLi, AJSilva, et al. Curr Biol Nov
  05
• Statins can reverse memory/learning deficit in
  NF1 mice
• Statins lower cholesterol by
• blocking effects of certain fats, by working
  on GAP
• Lovastatin, competitively inhibits HMG-CoA
  reductase
• Used commonly for treatment of hypercholesterolemi
  a
• AJSilva, RBourtchuladze, et al. Nat Gen 97
• Mouse model for the learning and memory
• deficits associated with NF1

several phenomena tying together
61
Developmental to Genomics to Biochemistry to
Clinical

• Another example SHH
• One of 3 proteins in mammalian hedgehog family
• Ligand for hh signaling pathway
• Vertebrate organogenesis (morphogen)
• Adult- controls cell division of adult stem cells
• -development of some cancers
• hh first identified from Dros screens
• EWieschaus and CNusslein-Volhard, 78 Nobel Prize
  95
• Controlled segmentation pattern

several phenomena tying together
62
Human mutation hedgehog, ex., single base change
to complex pathway changes

• F Umehara, et al. AmJHumGenet. Nov 00
• Desert hedgehog mutation, patient with 46,XY Yp
  to Xp
• Male phenotype, female karyotype
• Partial gonadal dysgenesis (PDG) with
  polyneuropathy
• CGD Swyers syndrome
• Sex reversal in XY female
• Premature female genitalia, blinded vagina and
  immature uterus plus
• Testis on one side and a streak gonad on other
• Homozygous missense ATG-gtACG at initiating Met of
  exon 1 DHH gene

several phenomena tying together
63
Human mutation hedgehog, ex., single base
phenotype

• Hedgehog gene
• pleiotrophic effects
• eg., digit formation, developmental pathways

several phenomena tying together
64
Model organisms and human disease

• Classes of human diseases
• Genes implicated
• Orthologous genes across species
• Genomics/ bioinformatics bridge disparate
  organisms

65
Model organisms value
66
Model organism, gt 100 yrs of Drosophila biology
67
Drosophila genomics

• ey, 47 alleles

• NCBI
• http//flybase.bio.indiana.edu

68
Homeostasis genes, development patterns

• single base change
• amino acid change
• protein change
• alters activity
• alters pathways
• organism-effect
• species-effect

69
art imitates

• The Fly
• 1958, 1986

70
Drosophila to Human

• PAX6, 11p13
• Encodes paired box gene 6
• Human homolog of D. melanogaster ey gp
• Conserved paired box domain and homeobox domain
• Both domains bind DNA, function as transcription
  regulators
• Expressed in developing nervous system and in
  developing eyes
• Causing Aniridia syndrome, as well as Peters
  anomaly (both ocular diseases)

• NCBI
• http//flybase.bio.indiana.edu

71
Functional genomics

• Databases
• organismbase
• Genetic stocks
• Mutations at each gene stocks

• NCBI
• http//flybase.bio.indiana.edu

72
Bioinformatics

• Complexity and simplicity things that work are
  re-used
• Its in the genome
• Model organisms
• Related and unrelated organisms
• Deep genomics
• Genome and environment
• Genome, environment and evolution

73
Alignment of maps different looks via tools

• Cytological, physical and genetic maps
• Distances between three are not uniform
• differences in recombination rates
• (reduced near telomere and centromere)

74
Comparative genomics Gene annotation of 50kb
75
Bioinformatics tools Orthologs

• NCI or ENSMBL browser
• Human IL13 (5)
• Ortholog Prediction mouse (11) and rat (10)
• 200kb view, above is 10kb resolution

76
Mouse-human synteny

• Conservation of gene order and DNA sequence
• Mouse 11
• Human 5q31
• 1-Mb with 23 putative genes, including four
  interleukins

77

• Applications of genomes and genome-based
  technology
• Better living through better science
• Environment
• Human condition
• Human genetic diseases
• Molecular diagnostics
• Molecular therapy
• Personalized medicine
• Evolution
• Deep evolution
• extensions of The Human Genome Program

78

Homo sapiens individual genome completion

• Vision personalized medicine, genome-based
• SLevy, et al. PLoS Biol (Sept 07). Diploid
  Genome Sequence of an Individual Human
• Higher quality sequence data vs Consortium data
  ca 2003
• 5 yrs, 10M to improve Celera draft genome
• Whole genome shotgun, modified Celera assembler
• 32M reads into 4,528 scaffolds, comprising 2,810
  Mb _at_7.5x coverage
• 4,500 gaps
• No technology for centromeric and telomeric
  sequences
• Individual variations greater than expected, 44
  genes differ maternal vs paternal
• Compared with Consortium genome, 4M variations
  incl 3M SNPs, 1M indels, 90 inversions
• Previously estimated two individuals similar at
  99.9 now, 99.5
• Adding phenotypic data- medical variants risk
  of disease

79

Pedigree confirmation with known history

• Present day medicine is based largely on patient
  and family history
• Three generation pedigree, documented cardiac
  arrest, hypertension, ovarian and skin cancer
• Genealogical record, 5 generations back to 1821
  (paternal) and 1700s (maternal) in England

• Genotyping and cluster analysis of 750 unique
  SNPs
• 99.5 similar to (N and W) European descent
• CEU -N and W European
• YRI- Yoruban, Ibadan, Nigerian
• JPT CHB- Japanese and Han Chinese

80

Individual gross chromosomal analysis

• old vs new technology
• G-banded karyotyping
• Spectral karyotypic chromosome imaging
• -gt no obvious chromosomal abnormalities

81

Individual ancestry

• Can reconstruct chromosome Y ethno-geneographic
  lineage (mitochondrial genome)
• Consistent with family history
• Haplogroup membership is R1b6- individuals from
  UK, Germany, Russia and US ???

82
Evolution of the Y chromosome

• X 165Mb Y 67Mb
• 1438 genes 45
• ca. 300Myr, SOX3 mutation to
• SRY Sex-determining Region of Y
• In human genetics, two haplogroups most commonly
  studied mtDNA and Y

83
Human Evolution and History embedded in the Y
chromosome

• First wave around Indian Ocean
• To Australia (150 mi open water)
• Second wave to Nursery of mankind

Mar06 Natl Geo
84
Human migration patterns embedded in the Y
chromosome

• Natl Geo Genographic Project
• gt2 waves of migration out of Africa

85
Use of genome data History embedded in the mt
chromosome

• Cann et al., Nature 87. RE mapping of 147
  individuals from five separate populations
• More data confirm

• Eve

86
One single Eve?

• Eve was not the first human
• Preceded by many mitochondrial Eves
• Member of an original human population comprising
• 2,000 interbreeding individuals

• http//home.nc.rr.com/ambiient/site/mtdna.htm

87

Homo sapiens re-sequencing and comparative
analysis

• Venter genome- Sanger method _at_800 nucs Watson
  genome- 454 Life Sci _at_200 nucs
• Compared with Consortium genome, 4M variations
  incl 3M SNPs, 1M indels, 90 inversions
• Previously est two individuals at 99.9 now,
  99.5
• Adding phenotypic data- medical variants risk
  of disease Both

88

Lessons from the genome evolution of behavior

• keys to the origins of human culture
• FOXP2 and language/speech
• KDMacDermot, SFisher, et al. 05. AJHG. 761074.
• 1996 TMonaco. Unique KE family, inherited in
  simple fashion with a defect in single gene
• Large London family, half of whose members had
  subtle defects in speech and understanding
• Three generations, problems controlling fine
  movements of lower half of face
• One of two copies inactivated by mutation chrom
  7 and localized with CS (unrelated)
• Echo-locating bats have the distinctive change in
  their FOXP2 as humans
• Non echo-locating do not (GLi and SZhang, PLoS
  One)

89

Lessons from the DNA FOXP2

• SNPs
• Only R328X and
• R553H found in
• affected members
• Expanded poly-Glu

90

Founder populations

• 1814 two British Navy vessels, Pitcairn Islands
• 10 women, 23 children and former able seaman John
  Adams
• Last surviving member of mutineers of the HMS
  Bounty, arrived 1790
• Founders 9 mutineers, 6 Tahitian men, 12 women
  and a baby
• May 1997 Tristan da Cunha
• 300 residents, volcanic island, S. Atlantic,
  descendants from Brit sailors 1817
• Half suffered from asthma Genes for asthma,
  wheeze 1 and 2
• Genetic data from worlds most remote outposts
  treasures of the lost world
• Amish, Eastern US- mental illness
• Pima Tibe, Arizona- diabetes
• Isolated Venezuela community- Huntingtons
  disease
• Sequana Therapeutics, et al. gene mining approach

91
Applications getting into the genome

• Populations
• deCODE Genomics
• Sequana Therapeutics
• Iceland-based
• 11 generations
• back to 17th century
• In general
• Homogeneous population
• 146 families
• 5,136 markers
• 1,100 years geneology
• Ex., Asthma 100 patients
• Population vs PKU
• 1 in 15,000
• 1 in 4,500 Ireland
• 1 in 100,000 Finland

2005
92
deCode Genomics

• Apr07 second major prostate cancer risk variant
  on 8q24. NatGen. US, Iceland, Spain,
  Netherlands
• Genome-wide association scan of 4500 Icelandic
  cases and controls, with Illumina Human Hap300
  Bead Chip typing, gt300,000 SNPs across genome
• Both variants acct for 11-13 of prostate cancer-
  Eur descent 30 of African-Am
• May06, first common genetic variant associated
  with increased prostate cancer in general pop on
  8q24
• Linkage analysis of men from gt300 families in
  Iceland Genotyping identifies region conferring
  60 increase in risk
• Twice as common among African-Americans than
  European ancestry
• Jun06 Phase 2 for DG041, for peripheral artery
  disease. Antagonist of EP3 receptor for PGE2.
  Phase 1- tolerated at dose levels. PAD is a
  vascular disorder affecting 10 of adults in
  industrialized world 1 in 5 people over 70.
  Narrow arteries due to atherosclerotic plaques in
  legs, reducing blood flow.
• Jun06 Population-based study. Impact of BARD1
  (BRCA1-associaed gene). Italy and Finland
  population /families with high incidence of
  breast cancer suggested BARD1 gene. This plus
  Icelandic founder risk mutation in BRCA2 gene
  increased risk. BARD1 is Cys557Ser mutation,
  Iceland. BRCA1 and 2 acct for 1-3 of all breast
  cancer cases 15-25 of familial component of
  breast cancer.
• May06 Phase 3 for DG031, for prevention of
  heart attacks. Inhibitor of 5-lipoxygenase
  activating protein (FLAP) linking FLAP and
  LTA4H, leukotriene A4 hydrolase, to risk of heart
  attack. Phase 2- reduced LTB4 in dose-dependent
  manner.
• CDKAL1 variant influences insulin response and
  risk of type 2 diabetes. Chr10 and CDKAL1.
  Significant risk factor 33 population carries
  one copy of at-risk variant at 45 increased risk
  of disease 7 carry two copies at 141 risk.
  Associated with reduced insulin secretion in
  homozygote. Iceland first, confirmed in Denmark
  and US (NatGen Feb 06 or Apr07?
  VSteinthorsdottirKStefansson). Confirmed two
  risk variants of CDKAL1 in European and (HK) Han
  Chinese ancestry. Population-based, genome-wide
  linkage scan in Iceland identified chr10, 228
  microsatellite markers in this region, 2000
  patients and controls. Diabetes- 200M worldwide,
  21M US. 7 population US- medical costs, ca.
  100B/year.

93

Gene mining Commercial aspect

• Sequana Therapeutics (Updated 7/11/2004)
• Genomics-based drug discovery company (gene
  mining).
• History
• Incorporated Feb 1997.
• Sequana Therapeutics was acquired by Arris
  Pharamceuticals, forming Axys Pharmaceuticals.
• Axys went on to form Axys Advanced Technologies,
  which got bought by
• ChemRx, becoming ChemRx Advanced Technologies.
  The remains of Axys were bought by
• Celera, operates as Celera South San Francisco
  and Rockville, merged
• Celera with Applied Biosystems becoming Applera
  Corp.
• Now Gene discovery phased out pharmaceutical
  phased out now, Molecular Diagnostics
• One scientific view wasting research
  resources
• Another non-sci view Biopiracy/Biocolonialism-
  stealing genome treasures
• loot the genes of Third World people to provide
  expensive cures for the privileged
• The Rural Advancement Foundation International
• Patenting a cell line from a member of Hagahai
  tribe Papua, New Guinea
• Human Genome Diversity Project (1991) collect
  biological samples, different populations
• LCavalli-Sforza
• Ownership- UCLA Hairy-cell leukemia. Late 70s
  (JM) splenectomy removing enlarged spleen
• Patent March 84, T-cell line derived from spleen

94

Lessons from Comparative Genomics of Homo sapiens
PHalpern, Whats Science Ever Done For Us

• What makes humans different from chimpanzees?
• TOOLS and data
• traditional comparisons, cited in textbooks-
  1.2 difference, based on single-base-pair
  changes
• Eichler, et al. Nat (Sept05), based on
  comparative genomics and variations in the whole
• genome-using draft genome- duplications (gt
  20kb), 2.7 difference
• Duplications of extensive segments of DNA occur
  during gametogenesis-
• Predisposition of certain chromosomal sites to
  undergo breakage and rearrangements
• Segmental duplications give rise to extra copies
  of genes- possible new traits
• Or to disease, two dozen genetic diseases
• Chimpanzee- ancestral reference 33.3 of
  duplications in humans, not in chimps
• Chimps- fewer sites of duplications
• Also, chimp-only duplications ex, human 4
  copies, chimp 400 copies
• Hyperexpansion, especially at ends of
  chromosomes.
• One massive duplication lies in a region of great
  apes that is broken into two chromosomes humans
  have a fused chromosome of the two
• different mechanisms for resolving chromosomal
  instability
• FOXP2 and speech/language

95

Lessons from Comparative Genomics of Homo sapiens
PHalpern, Whats Science Ever Done For Us

• What makes humans different from chimpanzees?

96

Lessons from the genome evolution of Homo
sapiens

• What makes humans different from chimpanzees?
  Amylase (?)
• GHPerry, et al. Nat Gen (Sept07)
• Genetic response (Adaptation) to Diet or Genome
  changes drives evolution, or
• What you eat makes you different from chimpanzees
• Old assumption is that meat eating, ca. 2.5M yrs
  ago, coincided with expansion of brain volume
• Brain and neural tissue requires large amounts of
  energy (3x chimp size)
• Populations high-starch diets have more copies
  of amylase gene than low-starch populations
• Amylase converts starch to simple sugars,
  beginning in the mouth
• If favored by Natural Selection, selective
  pressure could date to beginning of Neolithic Age
  when cereals were domesticated (10,000 yrs ago)
• Or date to split from other great apes, moving
  from primate diet of fruit and nuts
• Or alternative is that extra energy came from
  diet of tubers and plants that store starch
  underground, at 5M yrs ago human/chimp split
• Chimps- low starch-diet, have a single amylase
  gene
• Positive selection genomes of the Japanese
  (rice) versus the Yakut (reindeer) population
• Extra copies of genes is a widespread form of
  variation in the human genome
• Amount of amylase in saliva number of copies of
  amylase gene, ranging from 2 to 15
• Arranged as repeats of 120kb each on genome

97
Hominid evolution(paleoanthropology)

• 3.3 Myr old A. afarensis (3.8-3.0 Myr ago) 3 yr
  old female
• Selam, same species as Lucy, 150,000 yr apart
• One of most complete skeletons
• See transitional stage between ape and human, esp
  a child
• Legs like humans but shoulders like gorillas
• Teeth grew quickly like chimp
• Brains may have matured more slowly like humans
• Confirms human evolution was not straight line
  from
• ape to human different parts evolved at
  different times
• Expts of early evol had a combination of
  humanlike and apelike features
• Genomics for reading DNA history also
  biotechnology for reading old bones

98
Applications Deep history from genomes

• 1993 film, adapted from MCrichton
• Amusement park of cloned dinosaurs
• InGen extracts genetic material
• from dinosaur blood isolated from
• mosquitoes embedded in Dominican amber
• Splice with DNA from frogs to fill in gaps
• Generate all females to control population

99
Better Tools Paleogenomics

• T. rex related to birds
• First molecular proof of bird-dinosaur link
• Protein from 68 M yo thigh bone
• Jasara, Sci Apr07- Mass spec bone protein
  analysis, matching chicken, newt and frog
• MHSchweitzer reported soft tissue in a T. rex
  bone 05
• Reports Sci Apr07- extracts react with antibodies
  to chicken collagen
• Still looking for T. rex DNA
• Collaboration -better data with mastodon bones

100
Paleogenomics

• Poinar, HN, et al. Sci (Jan 06)
• Metagenomics to Paleogenomics Large-scale
  sequencing of Mammoth DNA
• Woolly mammoth, Mamuthus primigenius (Siberia),
  5-6MYr divergence from elephant
• PCR and pyrosequencing 28Mb 13Mb (45.4)
  identified as Mammoth DNA
• high percentage of endogenous DNA vs small
  diversity of environmental DNAs
• Comparative genomics 98.55 with African
  elephant (Loxodonta africana)
• Whole-genome approach from Ursus spelaeus
  (Pleistocene 40,000yr) 27kb of endogenous DNA
  (Noonan, JP. et al. Sci (July05))

101
Better Tools Paleogenomics

• July 97 first extr of Neanderthal DNA. Krings et
  al., Cell 97
• mtDNA upper arm, Feldhofer grotto/Neander Valley
• Second Neanderthal, 29k yo fossil baby, Mesmaisky
  cave in Russia
• Ovchinnikov et al., 00
• Third, Vindija cave in Croatia. Krings et al.,
  00
• Fourth plus four more in 02 and 04
• -no evidence gene transfer, separate
  branches

102

Lessons from the bones Neanderthals

• Oct 07. Sci. HRoempler, CLalueza-Fox,
  MHofreiter, et al.
• Neanderthal DNA. 400,000 years ago to 24,000 (?)
• Mutation in two individuals linked to skin and
  hair pigmentation
• 43,000 yo El Sidron, Spain 50,000 yo Monti
  Lessini, Italy
• Mutation in MC1R, melanocortin 1 receptor allele
• receptor/switch for red-yellow pheomelanin and
  black-brown eumelanin
• Recessive est 1/100 Neanderthals with two copies
  of this ginger gene
• In humans (3700), slightly different mutation
  yields red hair and fair skin
• SNP not seen in modern human in cell culture,
  impairs MC1R function
• 2006. HRoempler, et al, mutation in woolly
  mammoths- blond hair
• Similar to mouse work, 2006

• ScienceDaily

103

Lessons from the (deep) genome evolution of
behavior

• Curr Biology 2006. SPaabo, JKrause, et al.
• Ability to characterize specific gene
• Neanderthal, two individuals from El Sidron,
  northern Spain
• FOXP2, associated with language
• Human version differs from chimpanzee gene at two
  nucleotide,
• Suggesting these two result in human speech
• (many other genes are also involved in speech-gt
  data suggests but does not prove)
• SPaabo. Strain of mice with human FOXP2.
• Extra neuronal connections, and change in
  vocalization
• This contradicts earlier work, same lab,
• Humans have essentially same version of the gene
  (2002), and likely happened 200,000 yo
• Supports RKlein archaeological evidence that
  modern human underwent neurological
• Change around 50,000 yo enabling population to
  expand and emerge from Africa
• Neurological change could have been the
  perfection of modern language
• PLoS. JDWall and SKKim. These seqs differ from
  seq from JGCI (which is likely correct)

104
Applications of Genomics origins and spread of
HIV

• BKorber, et al. Africa as origin 1930s, 10 yrs
  to develop heterosexual transmission
• MWorobey, et al. PNAS Oct 07. Haiti 1966-gt US
  (?)
• US and most of world, subtype B developing
  countries non-B
• Cuts across politics and blame Data over
  speculation
• Disproves Patient Zero Hypothesis or single
  person vector

105
...of Sasquatch, Yeti, Nessie, chupacabra, ie,
cryptozoology
KENS-TV, San Antonio TX. July 31, 2007

• Blue-colored animal sucks blood, leaving flesh on
  bone
• Three dead found in southern Texas
• Myth origins in Mexico, Puerto Rico
• DNA matches coyote (Nov 07)
• (Still, carcasses of drained chickens)
• Coelacanth, megamouth shartk, Homo floresiensis
  (Hobbit Man)

106
Model organisms value (Human biology by proxy)
107

Felis catus genome

• Joins humans, chimps, dogs, cows, mice and rats
  _at_mammals
• DNA of Abyssinian cat, well-documented lineage
• Nov07 _at_2x coverage (light coverage) 20,285
  putative genes
• Development of the cat genome as
• Genome evolution
• Comparative genomics within family Felidae
• Animal model for human hereditary disease
• 250 naturally occurring feline hereditary
  diseases
• Cinnamons pedigree carries marker for retinitis
  pigmentosa humans 1 in 3,500 Americans
• Model for human infectious diseases, eg FIV HIV
• Forensics analysis using cat genetic markers
• First introduction of an animal DNA fingerprint
  into trial
• Development of simple tandem repeat (STR)
  forensic genotyping system

• http//home.ncifcrf.gov/ccr/lgd/comparative_genome
  /catgenome/index_n.asp

108
Whole genome analysis S. cerevisiae V

• 576,869 bp

109
Whole genome analysis S. cerevisiae
110
Whole genome analysis S. cerevisiae
111
Whole genome analysis S. cerevisiae
112
Whole genome analysis S. cerevisiaeGO

• Gene ontology
• Annotation

113
Genome sequence, now what?
Functional genomics Study gene function, by
gene inactivation in eukaryotes Each organism,
comprehensive database Genomic data, phenotypic
data Functional genomics, after the
sequencing Construction of mutant banks with
deletions across the genome, every
gene Bacterial genetics to functional
genomics Insertion/deletion/base mutation to
knock in/knock out
114
Inactivating the function of specific genes in
eukaryotes (deletion, knock-out)

• Yeast
• Homologous recombination inactivates gene with
  flanking complementary sequences
• kanMX as marker, resistance to G-418

115
S. cerevisiae sporulation for haploid cells

• One of two chromosomes has disruption (marker)
• Sporulation results in segregation
• Non-disrupted are not viable when challenged with
  G-418
• (However, if target gene is essential, disruption
  is lethal)

116
Disruptions in mouse cells

• Exogenous DNA in mouse embryonic stem cells
• Random insertion via nonhomologous recombination
  is more frequent event
• Use two markers neo and Herpes simplex virus
  thymidine kinase
• One targets homologous recombination
• neo gives G-418 resistance
• viral tk (unlike endogenous) kinases ganciclovir
  which is converted to PPP form
• inhibits DNA replication

117
Positive and negative selection of recombinant ES
cells
118
Production of gene-targeted knock-out mouse line

• ES cells heterozygous for knockout mutation in
  gene X
• Homozygous for a dominant allele of a marker
  (brown coat color here)
• Mice containing ES-derived cells are chimeric
• Backcross to homozygous black mouse
• Browns are homozygous or heterozygous X-
• Cross X heterozygotes to screen for homozygotes
  via tail tissue

119
loxP-Cre recombination system to knock out genes
in specific cell types

• Insert flanking loxP sites to essential exon 2
  of gene X
• loxP sites are introns, so do not disrupt
  function of gene X
• Cre mouse has cre from P1 and is heterozygous for
  gene x knockout
• cre protein mediates excision of lox element

120
Transgenic mice

• Injection of foreign gene into mouse gem line
• Random insertion into chromosome
• Nonhomologous recombination
• Does not disrupt endogenous genes

121
RNA interference (RNAi) Subtitle Can also do
knockouts with..

• C. elegans
• dsRNA blocks expression of corresponding
• mRNA, not other mRNAs
• disruption of gene
• dsRNA cleaved by Dicer into siRNA
• C. elegans mex3 RNA expression
• Left, expression scored by probe linked to enzyme
• that produces colored product
• Right, injected with ds mex3 mRNA

expressn of mex3 no expressn
122

Gene Therapy Super Mouse

• YXWong, REvans, et al. Oct 04 PLoSBiology.
  Marathon Mouse
• Can run twice as far endurance athlete
• Increase in slow-twitch muscle Resistant to
  weight gain
• PPAR-delta, master regulator gene-
  transformation in skeletal muscle fibers
• (peroxisome proliferator-activated receptor
  delta)
• Slow-twitch have larger numbers of mitochondria
  Energy from fat rather than glucose
• HansonRW, et al. Nov07 JBC. Super Mouse
• Engineered mouse with abilities of a super
  athlete- 10x more active
• Runs 6 km (3.7 mi) at 20 meters per minute for 5
  hrs without getting tired,, without stopping
• Equivalent of a human cycling up an Alpine
  mountain without a break
• Lives longer, increased sex drive, live longer,
  breeds into old age, eg 80 yo woman, eats more
  without getting fat
• Very aggressive and eats 2x (-60x?) more food
• Gene therapy for superior athletes?

123

One gene difference Mighty Mouse

• HansonRW, et al. Nov07 JBC. accident
  Engineered mouse with abilities of a super
  athlete- 10x more active
• Overexpression of PEPCK
• Runs 6 km (3.7 mi) at 20 meters per minute for 5
  hrs without getting tired, without stopping
• Equivalent of a human cycling up an Alpine
  mountain without a break
• Lives longer, increased sex drive, breeds into
  old age, eg 80yo woman
• Very aggressive and eats 60x more food (without
  gaining weight)
• Breeding colony of 500
• Same gene in human for more efficient fat
  metabolism, no lactic acid buildup
• Muscles with 10x number of mitochondria
• PEPCK-C, phosphoenolpyruvate carboxykinase
  glucose metabolism and stimulates fat for energy
• Glycolysis vs Krebs cycle Committed step in
  gluconeogenesis OAA to PEP when GTP is present

• http//www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/
  part2/gluconeo.htm

124
The Age of Enzymology
October 29, 2007 (Genome Technology Online) The
Father of Biotechnology Leaves Behind a
Legacy Biochemist and Noble laureate Arthur
Kornberg died on Friday he was 89. Kornberg won
the Nobel Prize in medicine for discovering DNA
polymerase, as well as was the first to
produce the active inner core of a virus in the
lab. His passion for enzymology and faith that
all metabolic reactions could be reproduced
in a test tube helped birth the field of genetic
engineering. His son, Roger, also won the Nobel
in 2006, making Kornberg one of only six
Nobel laureates whose sons also won Nobel Prizes.
125
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126
Better Tools Paleobiochemistry

• Nielsen-Marsh, CM, et al. PNAS (Mar 05)
• Osteocalcin protein sequences from two
  Neanderthal fossils
• MALDI-TOF/TOF sequenced
• gt27,000Yr, Shanidar Cave, Iraq
• Chimp, gorilla, man, orangutan ninth AAc is
  Pro, other mammals hydroxyPro
• May reflect vitC deficiency, as hydroxylation
  reqs vitC
• Suggest decline in vitC in the diet during
  omnivorous dietary adaptation

127
Better Tools Community genomics

• (2005-2006) Lindell, D et al., Nature Nov 05.
• Microbial community as a collection of
  genes,driving core metabolisms and
• in unique combinations in different microbes as
  prophage

• Prochlorococcus (Cyanobacteria), most abundant
  plankton species
• part of food chain
• oxygen
• climate regulation
• genome diversity key to population stability
  despite
• environmental variables, temperature, predators,
  light and nutrients
• include psbA, PSII core reaction center protein
  and hli, high-light-inducible genes

128
Better Tools Community genomics

• Genomic and bioinformatics approach Sequencing
  by JGI
• Allows niche exploitation

• Three Prochlorococcus phages
• Sequenced with no prior knowledge of genes
• P-SSP7, T7-like (45kb) infects high-light-adapted
  (HL) Prochlorococcus
• P-SSM2, P-SSM4, T4-like, (252kb, 178kb) infect
• three LL Prochlorococcus and two HL two LL
  Prochlorococcus, respectively
• PS-related embedded in viral genomes
• include psbA, PSII core reaction center protein
  and hli, high-light-inducible genes