Genetic Differences Between Humans and Great Apes Ajit Varki Professor of Medicine and Cellular & Molecular Medicine Co-Director, Glycobiology Research

Genetic Differences

Between

Humans and Great Apes

Ajit Varki

Professor of Medicine and Cellular & Molecular Medicine Co-Director, Glycobiology Research and Training Center

University of California, San DiegoLa Jolla, CA, USA

Homo sapiens sapiens, a recent addition to life on earth

1011

1010

109

108

107

106

105

104

YEARS BEFORE PRESENT

Origin of Life

Cambrian Expansion

Origin of Primates

Ape/Human Commonancestor

ModernHumans

Origin of Universe

“Nothing in biology makes sense, except in the light of evolution”.

Theodosius Dobzhansky

TheProbableEvolution

of Life Forms on

Earth

Evolutionary Relationships of

Primates and Rodents

10

Mil

lio

ns

of

Yea

rs B

efo

re P

rese

nt *

Humans0

*Precise Timing

Uncertain

20

30

40

50

Great Apes

LesserApes

New WorldMonkeys Prosimians

Old WorldMonkeys

Rodents

“Great Apes”

Evolutionary Relationships

amongst Humans and the

Great Apes

10

5

Mill

ion

s o

f Y

ears

Bef

ore

Pre

sen

t*0

*Precise Timing Uncertain

Gorilla gorilla

GorillaPan paniscusBonobo

Pan troglodytes Chimpanzee

MEAN Amino AcidDifference

<1.0%

Homo sapiens

HumanPongo pygmaeus

Orangutan

Wright-stained Normal Human Blood Smear

Red Blood Cell

White Blood Cell

Platelet

White Blood Cell Blood Plasma

Protein

Two-Dimensional Separation of proteinsThe critical first step in Proteomic Analysis

Isoelectric focusing

Total Plasma Proteins

Proteomic Comparison of Human and Great Ape Blood Plasma Reveals Conserved Glycosylation and a Small Numbers of Differences Gagneux et al. American Journal of Physical Anthropology 115: 99-109, 2001

MW

PI

7

8

Orangutan

Chimpanzee Gorilla

BonoboHuman 2

2

1

Human 1

1

2

1 = Haptoglobin

2 = Transthyretin

Human-specific Blood Plasma Protein Differences from Great Apes

Gagneux et al. American Journal of Physical Anthropology 115: 99-109, 2001

Brain size relative to bodyRelative size of neocerebellum% of brain growth complete at birthAge of closure of major fontanelles/suturesSize of cerebral venous sinusesAge of final pelvic bone fusion Rotation of the neck of the astralagus boneBicondylar angle of the femur Presence of chinInner ear canal orientationForamen magnum relative to basal skull axisAdductive thumb Absence of Pharyngeal Air SacsDescended larynxAge of first rolling over Maximum protected life spanSkeletal muscle strengthBone cortex thicknessPenis size relative to body lengthPenile baculum(penis bone)

Earliest age of onset of menarcheMenstrual blood loss (amount per cycle)Concealed ovulationPeriod of sexual receptivity (% of total cycle)Average Breast tissue mass in virgin adult femaleIncrease in breast mass during lactationModal Chromosome NumberNeonatal Cephalhematoma/caput at birthMenopausePresence of Ear Lobes Presence of canine tooth diastemaThickness of tooth enamelAge of first molar eruptionPresence of retromolar gapFrequency of Third Molar ImpactionBody hair Eccrine/Apocrine sweat gland ratioDermal fat thickness (relative to total skin)Relative size of frontal air sinusesPostnatal % body water change

SOME FEATURES OF HUMANS THAT SEEM DIFFERENT FROM THE GREAT APES

Old WorldMonkeys

OrangutansGorillas

Chimpanzees

Humans

CommonAncestor

Are we really a “Third Chimpanzee?”

Analysis of 20 structural and functional features of hominoids

(C.Wills & A.Varki)presented IN

“Children of Prometheus”By Chris Wills

A-Z Examples of Human Uniqueness

AAbbreviatingBBag-makingCCalculusDDartsEEar-piercingFFace-liftingGGamblingHHacking(computers)IIllustratingJJet-skingKKarateLLacrosseMMachining

NNailing(wood) OOperating (Surgery)PPanning for goldQQuiltingRRacing (organized)SSacrificing (others)TTagging (systematic marking)UUmpiringVVacationingWWage-earningXXeroxingYYachtingZZeroing

Exercise: Take a dictionary and check entries under each letter from the top. Stop when you reach the first one you think is unique to humans

Examples of Human Uniqueness starting with the letter S

Exercise: Take a dictionary and scan all entries under the letter S. Record all the ones that you think are unique to humans.

SacrificingSack-makingSaddlingSailingSalt-makingSalutingSand-castle buildingSandwich-makingSawing (wood)Saxaphone playingSchedule makingSchoolingScienceScoring (points)Scuba-divingScullingSculptingSealing (wax)SellingSemen bankingSerum collectingSewingShampooingShaving

Ship-buildingShootingShoppingSignaturesSilver (trading etc.)Singing (e.g., opera)SkatingSketchingSkiingSkinningSkippingSky-divingSlang wordsSlaverySleddingSleighingSlimmingSmelteringSnowball fightingSnuff-takingSoapingSobbingSoccerSoliciting

Sowing (seeds)Soup makingSpayingSpear-throwingSpelunkingSpice collectionSpending _____________

_____________________

_____________________

_____________________

_____________________

Steel productionStitchingStory-tellingSun-tanningSurfing

DNA RNA PROTEIN CELL ORGANISM

DNA

ORGANISM

? ?

DNA

ORGANISM

PHYSICAL ENVIRONMENT

DNA

ORGANISM


BIOLOGICAL ENVIRONMENT

DNA

Microbes

DNA

ORGANISM


CULTURAL ENVIRONMENT


DNA

DNA

DNA

ORGANISM


CULTURAL ENVIRONMENT


DNA

HUMANS HUMANS

DNA DNA

LIPIDS

DNA

ORGANISM

RNA PROTEINS

MATRIXCELL

GLYCOPROTEINSPROTEOGLYCANSGLYCOLIPIDS

SIGNALLINGMOLECULES

TISSUES & ORGANS

TRANSCRIPTION FACTORS


MICROBESPARASITES

ENZYMES

DIET

DNA

SUGARS

Glycobiology: the study of the structure, biosynthesis and biology of saccharides (sugar chains or “glycans”) in nature.

DNA

Wright-stained Normal Human Blood Smear

Red Blood Cell

Scanning Electron Micrograph of Human Red Blood Cells

Red Blood CellMembrane

Sugar chains (Oligosaccharides)

(Glycans)

Sialic Acid

Neutral Sugars

Protein

Scale Model of 1/100,000 of Human Red Blood Cell Surface

Modified from Viitala & Järnefelt

Cell Membrane(Lipids)

Biological Roles of Sialic Acids

Structural/Physical Roles

ENDOGENOUS RECEPTOR

SELF

EndogenousRecognition

“Self”

SiglecsFactor HSelectins

Uterine AgglutininLaminins

=SIALIC ACID CARRYING SUGAR CHAIN

M

EXOGENOUS RECEPTOR

M = Micro-organism or Toxin

ExogenousRecognition“Non-self”

InfluenzaMalariaCholera

HelicobacterMycoplasma

RotavirusPolyoma virusCoronavirus

Pertussis Tetanus etc.

SELF

MolecularMimicry

E.ColiGonococcus

MeningococcusCampylobacterTrypanosomaStreptococcus

Etc.

HEMATOLOGY CONSULT

Case: A 22-year old woman with weakness and bleeding.

Diagnosis: Aplastic Anemia (bone marrow failure)

Treatment: Trial of Equine Anti-Thymocyte Globulin (Horse Serum)

Complication: “Serum Sickness Reaction” to Horse Serum

Immune Reaction partly against Sialic Acids in Horse Serum!

(1984) UCSD Medical Center

Sarah Anna Varki

Two major kinds of Sialic Acids in Mammals: Neu5Ac and Neu5Gc

Human

Neu5Ac

Neu5Gc

Great Ape orOther Mammal

Human Cells are Unusual:Excess of Neu5Ac and Loss of Neu5Gc

Neu5Ac Neu5GcHumans ++++ -Great Apes ++ ++

The Human Loss of Neu5Gc Expression

•HOW did it happen?

•WHY did it happen?

•WHEN did it happen?

•WHAT are the consequences for risk of infections in humans?

•WHAT are the consequences for human sialic acid receptors?

•HOW does Neu5Gc re-expression in tumors and fetuses occur?

•WHERE does the small amount of Neu5Gc in humans come from?

•WHAT are the consequences of human ingestion of Neu5Gc in food?

•WHAT are the consequences for biotechnology products?

•WHAT are the consequences for attempts at xenotransplantation?

•WHAT are the consequences for the human brain?

Sialic acids on Red Cells and Plasma Proteins from Humans and Great Apes Muchmore, E.A., Diaz, S. & Varki, A.: Amer.J.Physical Anthropology 107:187-198,1998

(Great Ape samples from Yerkes Primate Center, Atlanta, GA)

“Great Apes”

Evolutionary Relationships

amongst Humans and the

Great Apes

10

5

Mill

ion

s o

f Y

ears

Bef

ore

Pre

sen

t*0

*Precise Timing Uncertain

Gorilla gorilla

GorillaPan paniscusBonobo

Pan troglodytes Chimpanzee

MEAN Amino AcidDifference

~0.5%<1.0%

Homo sapiens

HumanPongo pygmaeus

Orangutan

Neu5Ac Neu5Gc

Genetic Mutation

Causing lossOf Neu5Gc














MANY BIOLOGISTS ASSUME THAT EVOLUTION USUALLY RESULTS IN OPTIMAL DESIGN

CREATIONISTS

EVOLUTIONISTS

“Intelligent Design”

“Optimal Design”Biologists

• Random mutation that drifted to fixation due to small effective population size?

• Selection due to Neu5Gc-recognizing pathogen(s)?

• Signature of other past “selective sweep” affecting hominid ancestors?

Regardless of the cause, what were the consequences for human evolution?

Human-Specific Loss of Neu5Gc(CMAH mutation)














CMAH Mutation found in Homozygous State in all Human Populations studied

4 million4000003000002000001000000

Bipedal gaitSimple stone tools

Home base(?) Control of Fire

(?) Organized huntingOrganized food gathering

Intermediate Stone tools(?) Burials

(?) ClothingConstructed dwellings

Preparation of pigmentsSettlements

Stone containersCrossing of Oceans

SculptureComplex stone tools

Bone toolsPersonal ornaments

DrawingLeather and Hide use

Complex projectile weaponsPottery

CeramicsMetal working

PaintingLamps

Drug usageReligious Symbolism

Animal domesticationAgriculture

WritingCompetitive sports

Musical instrumentsCities

PrintingComputers


3 million2 million1 million

Modern Humans - a very recent “success story”

4 million4000003000002000001000000

Bipedal gaitSimple stone tools

Home base(?) Control of Fire

(?) Organized huntingOrganized food gathering

Intermediate Stone tools(?) Burials

(?) ClothingConstructed dwellings

Preparation of pigmentsSettlements

Stone containersCrossing of Oceans

SculptureComplex stone tools

Bone toolsPersonal ornaments

DrawingLeather and Hide use

Complex projectile weaponsPottery

CeramicsMetal working

PaintingLamps

Drug usageReligious Symbolism

Animal domesticationAgriculture

WritingCompetitive sports

Musical instrumentsCities

PrintingComputers


3 million2 million1 million

Modern Humans - a very recent “success story”

Java Man

“Lucy”

Modified from: Bernard Wood, Nature, 418:133-5, 2002

Emergence of Moderns

Brain size

Brain sizeStone toolsHunting Meat Eating

BipedalPosture

Us Neandertals

Loss ofNeu5Gc

Collaboration with Meave Leakey, Svante Paabo, Yuki Takahata
















ENDOGENOUS RECEPTOR

SELF


“Self”



SIALYLATED OLIGOSACCHARIDE =

M

EXOGENOUS RECEPTOR







SELF

MolecularMimicry

E.ColiGonococcus


Etc.
















ENDOGENOUS RECEPTOR

SELF


“Self”



SIALYLATED OLIGOSACCHARIDE =

M

EXOGENOUS RECEPTOR







SELF

MolecularMimicry

E.ColiGonococcus


Etc.














Neu5Ac Neu5GcHumans ++++ traceGreat Apes ++ ++













Uptake and Excretion of Neu5Gc in Normal Humans

Detection by DMB-HPLC and Mass SpectrometryElaine Muchmore Sandra Diaz Pascal Gagneux

02040Time (hours)024681024Subject ASubject BSubject CIn

ten

sity

Tangvoranuntakul et al. Proc.Nat'l.Acad.Sci.U.S.A. 100:12045-12050, 2003

Distribution of Neu5Gc in Common Foods

• Fruits, vegetables and grains - none?

(no sialic acids)

• Chicken and Turkey - traces

• Fish - variable, but very low

• Bovine milk and milk products - low

• Lamb, Pork and Beef - high

Sandra Diaz Pascal Gagneux


Detection of Anti-Neu5Gc antibodies in Normal Human Sera

Frequency much higher than previously reportedLikely due to improved assay with lower background

and better negative controlsHigher values are in range for anti-alpha-Gal antibodies

Pam TangPascal Gagneux A

49

2















Neu5Ac Neu5GcHumans ++++ tracesGreat Apes ++ ++













Even Mammals With High Levels of Neu5Gc in Non-neural Tissues, it is Difficult to Detect Neu5Gc in the Brain

Neu5GC expression

No Neu5GC expression

CMAH gene expression is down-regulated in mammalian brains

PascalGagneux

•

•

•

•

•

• •

•

• •

•

• • •

•

• Random mutations that have drifted to fixation due to small effective population size? Unlikely, too many changes.

• Selection due to Neu5Gc-recognizing pathogen(s)?

• Signature of a past selective sweep affecting hominid ancestors? Likely - cause or consequence?

• What are consequences for humans today?

•Innate immune system?

•Susceptibility/resistance to pathogens?

•Brain development?

•Dietary ingestion of Neu5Gc?

•Biotechnology and Xenotransplantation?

Multiple Human-Specific Changes in Sialic Acid Biology -What do they mean?

Multiple Differences in Sialic Acid Biology Between Humans & Great ApesGenomic mutationmutation

in CMP-Neu5Ac Hydroxylase (CMAH)

Point mutation mutation Eliminating critical arginine residue in

Siglec-L1

Homozygosity of CMAH mutation Loss of

Neu5Gc Expression

Increased Expression of

Neu5Ac

Susceptibility to Neu5Ac-binding pathogens?

Increased Ligands for Siglec-1 (Sialoadhesin)

Resistance to Neu5Gc-requiring pathogens?

Dietary Neu5Gc as a foreign sugar

Traces of Neu5Gc in human tissues

Natural Anti- Neu5Gc Antibodies

Universal Expression of Siglec-1 in Tissue

Macrophages

Altered Distribution of

Siglec-1-positive Tissue

Macrophages

Altered regulation of ST6Gal-I gene expression (promoter mutationmutation?)

Sudden Unmasking of CD33-related

Siglecs

Multiple mutationsmutations in Sialic Acid-binding domains of CD33-

related Siglecs

CD33-related Siglec binding specificity relaxed

to include Neu5Ac

Loss of Sialic Acid Recognition by

Siglec-L1 (Neu5Gc preferred by chimp)

Human expression of 2-6-linked Sias on bronchial epithelium and certain other cell

types and loss of expression on goblet cells

Susceptibility to Human Influenza Strains

Human-specific Expression of Siglec-6

in Placenta(promoter mutation?mutation?)

Effects on timing of labor and birth?

DefiniteDifference: Probable Possible

No Neu5Gc in Brain ?

Epithelial excretion?

• McConkey E. H., and M. Goodman. 1997. A Human Genome Evolution Project is needed. Trends Genet. 13:350-351.

• Vigilant L., and S. Paabo. 1999. A Chimpanzee Millennium Biol Chem. 380:1353-1354.

• McConkey E. H., R. Fouts, M. Goodman, D. Nelson, D. Penny, M. Ruvolo, J. Sikela, C. B. Stewart, A. Varki, and S. Wise. 2000. Proposal for a Human Genome Evolution Project. Mol Phylogenet Evol. 15:1-4.

• McConkey E. H., and A. Varki. 2000. A Primate Genome Project deserves high priority. Science. 289:1295-1296. Co-signatories: John Allman, Kurt Benirschke, Terrence W. Deacon, Frans de Waal, Achilles Dugaiczyk, Pascal Gagneux, Morris Goodman, Lawrence I. Grossman, Deborah Gumucio, Thomas Insel, Kenneth K. Kidd, Mary-Claire King, Kenneth Krauter, Raju Kucherlapati, Arno G. Motulsky, David Nelson, Peter Oefner, George Palade, Maryellen Ruvolo, Oliver A. Ryder, James Sikela, Caro-Beth Stewart, Anne Stone , David Woodruff.

• Varki A. 2000. A chimpanzee genome project is a biomedical imperative. Genome Res. 10:1065-1070.

Apparent Differences between Humans and Great Apes in the Incidence or Severity of Medically Important Conditions

(Excluding those explained by obvious anatomical differences)

Olson, M. & Varki A. Nature Reviews Genetics., 4: 20-28, 2003 Varki, A. Genome Research 10:1065-1070, 2000.

MEDICAL CONDITION HUMANS GREAT APESHIV progression to AIDS Common Very rareP. falciparum Malaria Susceptible ResistantMenopause Universal RareSimian Foamy virus Infection Rare CommonAlzheimer’s Disease pathology Complete IncompleteInfluenza A symptomatology Moderate to Severe MildMyocardial Infarction (Heart Attack) Common UncommonHepatitis B/C late complications Moderate to Severe MildEpithelial Cancers Common Rare?E.coli K99 Gastroenteritis Resistant Sensitive?Menstrual Blood Loss Variable Lower amount?Early Fetal Wastage High Low?Bronchial Asthma Common Rare?Autoimmune Diseases Relatively Common Rare?Acne Vulgaris Common Rare?Major Psychoses Common Rare?

Reasons for Sequencing the Chimpanzee Genome

• Explaining Features of the Human Condition

• Explaining Biomedical Differences

between Humans and Chimpanzees

• Improving the Care & Conservation of Chimpanzees in Captivity

Need other Primate Genomes to fully interpret differences

Need attention to Ethical, Legal and Social Issues

Need to check “differences” in multiple individuals

• Primate Genomics Workshop, Seattle, WA, January, 2001 (Organizers William R. Morton, Michael G. Katze & Roger Bumgarner)

• GEMINI: Genes and Minds Initiative -- Workshop on Ape Genomics, March 14-15, 2001, Tokyo (organizers: Yoshiyuki Sakaki, Naruya Saitou, Nobuyuki Nukina)

• Olson, M. V., Eichler, E. E., Varki, A., Myers, R.M., Erwin, J. M., and McConkey, E. H. A White Paper Advocating Complete Sequencing of the Genome of the Common Chimpanzee, Pan Troglodytes (White paper submitted to NHGRI, February 2001).

• Reich, D.E., Lander, E. S., Waterston, R., Pääbo, S., Ruvolo, M., and Varki, A. Sequencing the Chimpanzee Genome (White paper submitted to NHGRI, February 2001).

• Olson, M. and Varki A. Sequencing the Chimpanzee Genome: Insights into Human Evolution and Disease Nature Reviews Genetics., 4: 20-28, 2003.

NHGRI News Release Dec. 10, 2003

Chimp Genome Assembled by Sequencing Centers

Draft Sequence Aligned With Human Genome

“The sequence of the chimpanzee, Pan troglodytes , was assembled by NHGRI-funded teams led by Eric Lander, Ph.D., at The Eli &

Edythe L. Broad Institute of the Massachusetts Institute of Technology and Harvard University,

Cambridge, Mass.; and Richard K. Wilson, Ph.D., at the Genome Sequencing Center, Washington

University School of Medicine, Saint Louis.” http://www.nhgri.nih.gov/11509418

http://www.nhgri.nih.gov/11509418

Great Ape Phenome Project? Science 282, 239-240 (1998)

Varki, A., Wills, C., Perlmutter, D., Woodruff, D., Gage, F., Moore, J., Semendeferi, K., Benirschke, K., Katzman, R., Doolittle, R. & Bullock, T.

• Much to be learned by comparing human genomic sequences with…..those of our closest evolutionary cousins, the great apes.

• Part of the value of the Human Genome Project lies in interpreting genomic data in the context of the large body of existing information about humans.

• Corresponding information about the great apes is limited. • Funding of a Great Ape Genome Project should be

complemented by a "Great Ape Phenome Project," …. comparative studies of humans and apes at all levels, from expression patterns of mRNA, to biochemistry and cell biology, all the way to neural systems and cognitive functions.

• Significance of most genomic sequence differences found between humans and apes will not be obvious unless such a detailed comparative phenotypic database is also available.

• Could be obtained without harm to primates, using ethical principles similar to those guiding human experimentation.

• Project would also heighten awareness of the urgent need to protect and conserve these endangered hominoids who are so closely related to us.

“Genome”A haploid set of chromosomes;

the sum-total of the genes in such a set(Oxford English Dictionary, 2003)

orthe complete genetic material of an

organism“Phenome”

(no entry in any Dictionary)

(but 27,200 entries now in Google)

Suggested Definition:

“Complete information about an organism’s

phenotype and the relevant environmental influences”

Chimp“Phenome”

Compare

Human“Phenome”

Interactions of Genotype and Phenotype

HumanGenome

(10X coverage)

ChimpGenome

(4X)

Genotype PhenotypeENVIRONMENT

Compare

Acknowledgments

• Members of the lab

• Collaborators

• UCSD

• NIH, Mathers Foundation

• Colleagues in:• Glycobiology Research and Training Center

• Project for Explaining the Origin of Humans

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Genetic Differences Between Humans and Great Apes Ajit Varki Professor of Medicine and Cellular & Molecular Medicine Co-Director, Glycobiology Research