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Biology 321 Start Reading Chapter 6 in text (both editions) Also look carefully again at: 10th edition pg 58-60 on polymorphism 9th edition 69-71 on polymorphism

NOTE: In this and subsequent lectures, we may or may not review and/or discuss every example or every page of lecture in class. NONETHELESS: You are responsible for all material in the posted lecture notes (unless otherwise informed)

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Can the rich phenotypic variation observed in the natural world be explained with Mendel’s basic principles? Yes, but we need to add various “complications” or layers of complexity to basic Mendelian principles

Discrete vs continuous trait – eye color and skin color in humans shows continuous variation as does fruit size in tomato culivars

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How to explain these crosses?

Plants: Truebreeding Dwarf Strain #1 X truebreeding Dwarf Strain #2 Tall

Cats: Truebreeding White x Black Orange stripped tabby

Parakeets: Truebreeding Blue X yellow Green

Human: Green eyes X Brown eyes Blue, Green & Brown

Can we apply the basic principles of Mendelian inheritance to traits that don’t seem to be easily explained by two discrete phenotypic alternatives specified by one gene with two alleles that exhibit a simple dominance relationship? Yes, but for each example, we need to tinker with one or more of these parameters

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Multiple allelism: the existence of three or more known alleles of a gene A striking example of multiple allelism of the white gene

• multiple alleles of the

Drosophila white gene • each of the different alleles

affects eye pigment deposition to a different degree resulting in different shades of red/orange/pink

• in this case a normal wild-type allele can be clearly designated and all other alleles can be considered mutant

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• In the case of multiple allelism in the white gene, one allele (for normal red color) can be designated wild-type and all of the rest as mutant alleles

• The multiple mutant alleles of the white gene were generated in the laboratory and would not be found in a wild population

• The examples on the next few pages show a different situation in which there is more than one normal phenotype commonly present in the wild-type population – that is, naturally occurring multiple allelic forms

•

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Multiple allelism involving 7 alleles of a gene controlling chevron pattern in clover

In this situation, one can designate the trait & gene as polymorphic and not use the term wild type allele

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In natural populations, a polymorphism is the coexistence of two or more common phenotypes of a character (definition from text)

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• In other cases, nearly the entire population is characterized

by one form of the gene or character, with rare exceptional individuals carrying an unusual variant.

• That extremely common form is called the wild type, in contrast with the rare mutation or mutant alleles.

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Polymorphism: a situation where more than one allele of a specific gene is common in a population: A gene or trait is polymorphic in a given population if: • more than one allele is found in the population • AND if the frequency of the rarer allele exceeds 1% (yes,

this is a somewhat arbitrary threshold) • A variant allele (representing less than 1% of the gene

copies in a population) is not considered a polymorphism

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Pharmacogenetics: Inherited differences in the metabolism of and response to drugs can have a great influence on how a person responds to a drug. • Most drug-metabolizing enzymes exhibit clinically relevant genetic polymorphisms • Inherited differences in drug metabolizing capacity are generally monogenic traits. • The graphs show the phenotypic effect of a common allele variation (m) on the

levels of a drug in the body. • Allele m+ specifies a highly active enzyme responsible for inactivating the drug.

Allele m results in a loss-of-function of the enzyme’s activity.

wt = m+ X axis = time after administration of the drug

Y axis = concentration of drug in body Science 286:487

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Science 308: 1858 June 24, 2005 Polymorphisms are often found in genes coding for enzymes that metabolize various types of drugs Drug-metabolizing enzymes, such as CYP2D6, CYP2C9 and CYP2C19* come in many versions: • People who are poor metabolizers break

down drugs slowly, increasing toxicity concerns.

• Ultrametabolizers break them down quickly, lowering the chance that the drug will work.

• Intermediate and extensive metabolizers fall in the middle.

Recommended doses of pharmaceuticals are based on what the poor metabolizers can tolerate! * Three different genes coding for three different enzymes CYP = Cytochrome P450 Gene Family

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Paraphrased from A Primer of Genome Science: See also last page of this lecture Confusion arises over the distinction between polymorphisms and mutations, largely due to the dual usage of the term mutation. All polymorphisms arise as mutations, in the sense that conversion of one allele into another (resulting, for example, from a conversion of one nucleotide into another) is a mutational event. But, by the time an allelic variation is observed in a population, the mutation event that created it is usually long past* so that the observed variation is no longer a mutation – but rather a rare allelic variant or a polymorphic allelic variant. However, mutation is also often used to describe an allele that deviates from the majority type, particularly where the aberrant allele affects the “normal” phenotype – that is, is associated with a disease. *exceptions will be addressed later

Genetical jargon demystified

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Major human polymorphic variant CYP2D6 alleles and their global distribution • CYP2D6 is of great importance for the metabolism of clinically used drugs • about 20–25% are metabolized by this enzyme

Important things to note about this data set: • A mutant allele that is rare (or absent) in one population may qualify as a

polymorphism in another population • Note polymorphic alleles below that are loss-of-function mutations

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See this article on Optional Reading Assignments: http://fire.biol.wwu.edu/trent/trent/2003pharmacogenetics.pdf In addition to detoxifying and eliminating drugs and metabolites, drug-metabolizing enzymes are often required for activation of “pro-drugs” Many opioid analgesics are activated by CYP2D6 Many people (see previous figure) are homozygous for non-functional mutant alleles of this gene These individuals are relatively resistant to opioid analgesic effects -- explaining the inter-individual variability in the adequacy of pain relief from a given dose of codeine (widely prescribed pain killer)

http://fire.biol.wwu.edu/trent/trent/2003pharmacogenetics.pdf

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New York Times 11/8/05

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Chevron patterns in clover

7 alleles and 22 phenotypes?

2 alleles & phenotypes

VhVh

VhVb

VbVb

Drug metabolism Genotypes

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Complete dominance: phenotype of heterozygote is identical to one of the homozygotes Incomplete dominance: phenotype of the heterozygote is intermediate between (or at least different from) the phenotypes of the two homozygotes Codominance: heterozygote exhibits the phentoype of both homozygotes

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Example of incomplete dominance where heterozygote is a quantitative intermediate between the two homozygotes

Bar mutation in flies: affects the number of facets in the eye: B+ = wt B= mutant B+ B+ wildtype 800 eye facets B+ B intermediate bar eye 250-500 facets B B severe bar eye ~60 facets

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Example of incomplete dominance where heterozygote is not a quantitative intermediate between the two homozygotes

Wildtype allele = m+ Manx mutation = m m+ m+ = cat has a tail m+ m = cat has no tail m m = inviable mm kittens usually die before birth (spontaneous abortions The Manx breed originated before the 1700s on the Isle of Man (hence the name), where they are common. They are called stubbin in the Manx language. Tail-less cats were common on the island as long as three hundred years ago. The tail-lessness arises from a genetic mutation that became common on the island (an example of the founder effect). Folk beliefs claim the Manx cats came from the Spanish Armada; a ship foundered on Spanish Rock on the coast of the Isle of Man. According to legend, the cats on the ship swam ashore and became an established breed. Legend has it that the cats originally went onboard the Spanish ship in the Far East.

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http://www.nytimes.com/2007/06/12/science/12dog.html

http://www.nytimes.com/2007/06/12/science/12dog.html

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Complete, incomplete and codominance can be somewhat relative terms depending on how the phenotype is being assessed The type of dominance observed for a particular pair of alleles often depends on the level of organization at which the phenotypic observations are being made: Are you looking at the net effect on the whole organism or at specific cells or more directly at the gene products themselves?

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e+ = wild-type allele codes for an enzyme e = loss-of-function mutant allele (no residual enzymatic activity seen in the mutant protein) Phenotype Phenotype Phenotype

Organismal level

Biochemical assay of enzyme activity

Molecular analysis of protein product (based on size and charge)

e+ e+ normal 100% wild-type only e+ e normal 50%* wild-type and

mutant protein** e e abnormal 0% mutant only complete incomplete codominance Most wild-type alleles are dominant to loss of function alleles because they are haplosufficient: 50% of wild-type activity is sufficient for a normal phenotype at the organismal level * assumes no compensatory up regulation of the wild-type allele ** assumes that mutant alleles produces a stable protein product

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Haploinsufficiency: If one wild-type copy of the gene is not sufficient for a normal phenotype, the gene is said to be haploinsufficient

Familial hypercholesterolemia in humans:

h+ = normal allele h = loss-of-function mutant allele FH gene codes for LDL receptor which is responsible for taking up cholesterol from the blood -- see next LDLR (LDL receptor) on the next page serum cholesterol average age of Genotype mg/deci-liter heart attack h+ h+ 150-250 >60 h+ h 250-450 30-40 h h > 500 very young h+ h = 1/500 individuals h h = 1/106 indivduals h allele frequency about 1/1000 or 0.1% So, by definition, a mutant allele but not a polymorphism

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NATURE CLINICAL PRACTICE CARDIOVASCULAR MEDICINE SOUTAR AND NAOUMOVA APRIL 2007 VOL 4 NO 4

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http://www.ncbi.nlm.nih.gov/books/NBK7574/

Which possibility or possibilities

1. represent(s) a recessive null allele? 2. represent(s) a dominant null allele? 3. is likely to represent a pleiotropic (see definition below) l-of-f mutation? 4. may represent a situation where different l-of-f alleles have variable

phenotypic effects

http://www.ncbi.nlm.nih.gov/books/NBK7574/

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5.

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Codominance typically involves a phenotype that is assayed at the molecular or biochemical level (although see exception on the next page): • assaying the presence of specific carbohydrates on a protein -- this is the ABO

blood group assay • in DNA-based tests, directly assaying the presence of specific DNA repeats or

DNA sequence differences (direct detection of genotype) • directly analyzing the protein products of different alleles using biochemical

methods -- separation of proteins based on size or charge or shape (See Figure 6.6 in text: electrophoresis of hemoglobin variants.)

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Dominance reflects the specific mechanisms by which alleles are expressed in the phenotype which depend on • the nature of the allelic variation • the specific role of the gene in the development or functioning of the

organism • the dosage requirements for the gene (which may vary from tissue to

tissue): haploinsufficiency is one explanation for dominance of a mutant allele over a wild-type allele

Whether an allele is dominant or recessive is unrelated to the abundance of the specific allele in the wild-type population Dominance is not an absolute truth: whether an allele is completely dominant, incompletely dominant or codominant may depend on how you are assessing the phenotype

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Recall our discussion of mutations in the connexin 26 gene: • homozygotes for a loss-of-function mutation in connexin26 are

profoundly deaf • there are no other phenotypic effects of this mutation • this mutation in not pleiotropic

Pleiotropy: the phenomenon in which a single gene locus affects two or more distinct phenotypic traits A pleiotropic allele: affects more than one property of an organism

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Frequency of trait: 1/40,000 autosomal dominant

Pleiotropic effects of Waardenburg’s syndrome hallmarks: • deafness (accounts for 5%

of congenital deafness) • widely spaced eyes • eye with mismatched colors • fused eyebrows • white forelock of hair How could multiple phenotypic effects from a single mutant allele be explained?

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A central question about pleiotropy is whether the pleiotropic effects of a gene are conferred • by multiple molecular functions of the gene

OR • by multiple consequences of a single molecular function

Pleiotropy is usually caused by a single molecular function (ie product of a gene) that is involved in multiple biological processes

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Neural crest cells • produce melanocytes (pigment in skin, eyes and hair) • cells make up part of the inner ear • other structures

• neural crest cells are found only in vertebrates

• during embryonic development these cells originate along the back mideline of the developing embryo near the neural tube

• during development these cells migrate extensively throughout the embryo

• generate a wide variety of differentiated cell types including melanocytes, sensory neurons, facial bone and cartilage

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Single gene product involved in more than one related or unrelated process (neural crest derivatives) Gene mutated in Waardenburg’s syndrome is called pax-3 (paired box 3) and is located on chromosome 2: http://ghr.nlm.nih.gov/gene=pax3 • Pax-3 is a transcription factor in the “paired box” family • Pax-3 acts at a critical stage of fetal development to control the

development of neural crest cells by controlling the activity of other genes that signal these cells to form specific types of cells or tissues

http://ghr.nlm.nih.gov/

http://ghr.nlm.nih.gov/gene=pax3http://ghr.nlm.nih.gov/

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Mechanisms to explain pleiotropy

Single primary defect in the HBB (beta hemoglobin gene) results in a cascade of physiological ramifications http://ghr.nlm.nih.gov/gene=hbb

http://ghr.nlm.nih.gov/gene=hbb

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Polymorphism and Mutation

The term polymorphism is used by geneticists to mean different things at different times. Needless to say, this can cause confusion.

• Molecular geneticists often describe a variant as a polymorphism if its frequency in the population is above some arbitrary value, often 0.01. Variants whose frequency is below the arbitrary threshold might be described as rare variants.

• Population geneticists may define a polymorphism as the stable coexistence in a population of more than one genotype at frequencies such that the rare type could not be maintained by recurrent mutation. With this definition, some pathogenic human mutations would count as polymorphisms. For example the commonest mutation that causes cystic fibrosis has a frequency of 0.01-02 in northern European populations.

• Clinical geneticists often use polymorphism to mean a non-pathogenic variant, regardless of its frequency in the population. Pathogenic variants, whether common or rare, would be described as mutations.

The word mutation can also be used in different senses, referring to either the process or the product

• An event that changes a DNA sequence: for example, UV radiation produced a mutation in the DNA • A DNA sequence change that may have happened along time ago: for example, she inherited a mutation

from her father

Paraphrased from: Human Molecular Genetics 4th edition by Strachan and Read