Human Genome Structure and Organization

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Human Genome Structure and Organization

• Bert Gold, Ph.D., F.A.C.M.G.

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Genetic Variation

• Phenotype
• Expression of the genotype (modified by the
  environment). The structural or functional nature
  of an individual. Includes
• appearance, physical features, organ structure
• biochemical, physiologic nature
• Genotype
• Genetic status, the alleles an individual carries.

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Learning Objectives

• Recap and Update Public and Private Human Genome
  Project Status
• Provide Reminders of Necessary Background for
  Genetic Disease Association and Linkage Studies

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Definitions

• Penetrance - The probability that an individual
  who is at-risk for the disorder (ie- carries
  the gene) develops (expresses) the condition.
  May be age dependent.
• Expression - The characteristics of a trait or
  disease that are outwardly expressed.
  Eg-myotonic dystrophy myotonia, cataracts,
  narcolepsy, frontal balding, infertility.
• Ascertainment The method used in gathering
  genetic data. Study conclusions differ depending
  on how affected individuals entered the study.
• Phenocopy Individuals whose phenotype, under
  the influence of non-genetic agents, has become
  like the one normally caused by a specific
  genotype in the absence of non-genetic agents.
• Pleiotropy - The quality of an allele to produce
  more than one effect ie- to manifest its
  expression in the structure and/or function of
  more than one organ system or tissue
• Recurrence Risk Likelihood that a relative of a
  proband for a rare disease will have the same
  disease.

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Penetrance and Expressivity

• Penetrance Proportion that expresses a trait
• Complete P1.0 or 100
• Incomplete (reduced) Plt1.0 or lt 100
• Expressivity Severity of the phenotype
• Expressivity may vary
• Between families (interfamilial) or
• Within families (intrafamilial)
• TRY NOT TO CONFUSE VARIABLE EXPRESSIVITY WITH
  INCOMPLETE PENETRANCE

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Chromosomes, Genes and Proteins

• Genes are on Chromosomes
• Genes may encode proteins or RNA

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Non-coding RNA genes

• tRNAs (497 were counted, 821 when count genes and
  pseudogenes)
• tRNAs found are consistent with Wobble
• Codon bias only roughly correlated with tRNA
  distribution
• rRNAs
• small nucleolar RNAs (snoRNAs)
• snRNAs (spliceosome constituents)
• 7SL RNA
• telomerase RNA
• Xist transcript
• Vault RNA

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tRNAs
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Some chromosomes are richer in genes than others
Number of Nucleotides in Exons
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HOXA, HOXB, HOXC and HOXD are in regions with a
particularly low density of repeats This is
believed to result from the presence of
Cis-acting elements in this vicinity.
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Proteins demonstrate patterns and similarity of
function
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Functionally and Structurally similar proteins
are organized into families
e.g.- E.C., SWISS-PROT, TrEMBL,
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In silico approaches to characterize genes
include

• PFAM, searchable via HMMER
• Other in silico collections include
• PRINTS
• PROSITE
• SMART
• BLOCKS
• Creation of an Integrated Protein Index (IPI)

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How many genes are there?

• Estimates from the Public Program
• RefSeq
• Exons
• Introns
• Average Sizes
• Coding Sequences (CDS)
• Alternative splice products (about 3)
• Creation of an Integrated Gene Index (IGI)
• Genscan to Ensembl to Pfam via GeneWise (31,778)
• Could be as low as 24,500 using overprediction
  corrections.

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Estimates from Celera25,086 in Assembly 3

• 25,086 in Assembly 3

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Pre-existing estimates

• W. Gilberts back of the envelope calculation
• Reassociation Kinetics
• Estimates from Double Twist using Promoter
  Inspector plus
• Unpublished estimates from Human Genome Sciences

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Size of Genes

• Largest Dystrophin 2.7 Mb
• Titin
• 80,780 bp coding
• 178 exons
• largest single exon 17,106

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GENE HOMOLOGS, ORTHOLOGS, PARALOGS

• Vaculolar sorting machinery in yeast
• ABC gene superfamily
• Ig gene superfamily
• FGF superfamily
• Intermediate filament superfamily
• PROTEIN FAMILY EXPANSION APPEARS TO BE A PRIMARY
  EVOUTIONARY MECHANISM

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The proteome

• Functional categories
• PRINTS
• Prosite
• Pfam
• Interpro (http//www.ebi.ac.uk/interpro/)

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GENE ONTOLOGY

• Standard Vocabulary
• Hierarchy of terms (Directed ACYCLIC Graph)
• Ashburner Nature Genetics 2525-29 (2000)
• Bushy model

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Horizontal Transfer controversy

• One of the major conclusions of the Public Genome
  effort, published in Feb. 15, 2001 Nature was
• Hundreds of human genes appear likely to have
  resulted from horizontal transfer from bacteria
  at some point in the vertebrate lineage. Dozens
  of genes appear to have been derived from
  transposable elements
• This has now been widely disputed and is believed
  to result from
• Microbial contaminants in the sequence.
• Bacterial gene integration into pre-vertebrates
• And
• The more probable explanation for the existence
  of genes shared by humans and prokaryotes, but
  missing in nonvertebrates, is a combination of
  evolutionary rate
• variation, the small sample of nonvertebrate
  genomes, and gene loss in the nonvertebrate
  lineages.
• -Salzberg et. al., Science

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Splice Pattern, 98 GT-AG
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Chromatin Structure

• Euchromatin
• Heterochromatin
• Nucleosomes

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Chromosome Facts

• Chromosomes replicate during S phase
• Chromosomes recombine during Pachytene
• Recombination is an obligate activity
• Sex chromosomes recombine with each other

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Cytogenetics is done by Karyotyping

• Chromosomes are chemically frozen in metaphase
• Must be carried out on dividing cells
• Microfilament inhibitors
• Microtubule inhibitors
• Membrane lysis
• Pronase, trypsin digest
• Giemsa stain
• G-bands correspond to regions of relatively low
  GC content
• http//genome.ucsc.edu/goldenPath/mapPlots/
• http//genome.ucsc.edu/goldenPath/hgTracks.html

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Cell Division Meiosis

• Segregation
• Defined Alleles are paired gametes receive one
  of each.
• Exceptions trisomy and uniparental disomy
• Independent Assortment
• Gene Pairs segregate independently
• Exception linkage

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Meiosis Creates Gametes

• And provides a basis for genetic recombination!

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Genetic Recombination

• Crossing Over
• Resolution
• Recombinant Chromosomes
• OBLIGATE ACTIVITY
• FEMALE RECOMB. RATES HIGHER THAN MALE
• INCREASED RATES AT TELOMERES
• PARADOX SHORT ARMS SHOW MORE THAN LONG ARMS
• 1cM is 1 Mb on long arms, but short arms are 2 cM
  per Mb and the Yp-Xp pseudoautosomal region is 20
  cM per Mb.

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INCREASED RATES AT TELOMERES
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PARADOX SHORT ARMS SHOW MORE THAN LONG ARMS
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Genes

• Units of heredity
• Encode proteins (and some RNAs)
• Human genetics is the study of gene variation in
  humans
• Gene as a term is used ambiguously to refer
  both to the locus and the allele ie- There is
  only one locus but two alleles in a given
  individual.
• Sequencing in both genome projects took place
  upon multiple alleles this has led to some
  assembly confusions.
• Ultimately want a haploid genome map.

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The Human Genome Project

• International public effort commencing in 1990 to
  sequence the entire human genome by 2005.
• STS approach chosen in 1991
• Private effort launched in 1996 by Celera using
  Shotgun cloning

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BAC clones, sequenced into BAC end reads, and
assembled into contigs
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Markerless contigs in the Celera assembly are
called Scaffolds
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Markers are BAC ends in the shotgun
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Mate pair reads provided the core of Celera
sequence
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Draft human genome sequences complete by February
2001.

• Published simultaneously in Feb. 2001
• Public Sequence in NATURE (409 745-964)
• Celera Sequence in SCIENCE (291 1145-1434)

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Greater than 50 of sequence is repetitive
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45 of the human genome is derived from
transposable elements

• Long Interspersed Elements LINEs (21 of genome)
• LINE1 Some Still Active, Autonomous, consist of
  two ORFs (one is a pol).
• LINE2
• LINE3
• Short Interspersed Elements SINEs (13 of
  genome)
• ALU Some still active, use L1 enzymes to
  replicate
• MIR
• Ther2/MIR3
• LTR Retroposons
• Consist of gag and pol
• Protease, rt, RNAseH, integrase all encoded
• Reverse transcription occurs cytoplasmically,
  using a tRNA to prime replication
• DNA Transposons

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98.5 of sequence is non-coding.

• Approximately 1/3 of the human genome is
  transcribed (public guess).

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Allelism

• Alternate forms of a gene
• e.g.- Sickle Cell, CFTR
• Recessive disease
• e.g. Achondroplasia, Tuberous Sclerosis
• Dominant Disease

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Heterozygote or Homozygote

• 1,2 or 1,1
• homogeneity of alleles at a locus

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Genetic Markers

• RFLPs
• VNTRs (STRs)
• Microsatellites
• STSs
• SNPs
• Tools used to find disease genes
• Flags with locations throughout the genome

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Polymorphism Information Content versus
Heterozygosity (PIC vs. het)

• Determining heterozygosity from SNP rare allele
  frequency
• Information Content in SNPs versus STRs

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Typology of SNPs

• Type I- Coding, non-synonymous, non-conservative
• Type II- Coding, non-synonymous, conservative
• Type III- Coding, synonymous
• Type IV- Non-coding, 5-UTR
• Type V- Non-coding, 3UTR
• Type VI- Other non-coding
• Type I and Type II SNPs have lower heterozygosity
  than other SNPs, presumably as a result of
  selective pressure.
• About 25 of type I and type II SNPs have minor
  allele frequencies gt 15
• About 60 have minor allele frequencies lt 5

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Mutation

• Occurs more often during male meiosis
• Occurs more often in long genes
• More easily detected in Dominant Diseases
• Achondroplasia
• Duchenne Muscular Dystrophy
• May often involve CpG mutating to TpG

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Autosomal Recessive Inheritance

• Two copies of a gene required to be affected
• Carriers have one copy of the mutation and are
  unaffected
• 25 of offspring of two carriers will be affected
• Males and females affected in equal number
• Eg. Sickle Cell, beta-thal., CF

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X Linked Recessive (Sex Linked)

• Females rarely affected
• No male to male transmission
• Affected males transmit gene to all daughters
• Eg- Duchenne Muscular Dystrophy, Hemophilia A

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Autosomal Dominant Inheritance

• Each child at 50 risk
• Does not skip generations
• Often, lethal in double dose
• Large genetic load

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X-linked Dominant Pedigree

• Example is Hypophosphatemic, Vitamin D Resistant
  Rickets
• Distinguished from Autosomal Dominant by
• No male-to-male transmission
• All daughters of affected fathers are affected

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IMPORTANT NOTE

• Dominant and Recessive refer to the phenotypic
  expression of alleles, NOT to intrinsic
  characteristics of gene loci.

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Inheritance Pattern Complexities

• Pseudodominant Transmission of a Recessive
• Pseudorecessive Transmission of a Dominant
• Misassigned paternity, causal heterogeneity,
  incomplete penetrance, germline mosaicisim
• Mosaicism
• Mitochondrial Inheritance
• Penetrance and Expressivity
• Semi-dominant, gender- influenced, age-related,
  transmission-related, imprinting
• Uniparental Disomy (UPD)
• Environmental effects, phenocopies

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Preview of linkage analysis

• Characterizing Human Genetics
• Long generation time
• Inability to control matings
• Inability to control study population
• Inability to control exposures to environmental
  conditions
• It is possible to define phenotypes well!
• Can study genetic structures through family
  history
• Link phenotypes and genetic structures through
  statistical methods