Mutations are changes in DNA that may or may not affect phenotype. Some mutations affect a single gene, while others affect an entire chromosome. Mutations

A substitution (point) mutation substitutes one nucleotide for another. – a single point in the sequence is changed For instance a C is changed to an A. Compare this to reading a sentence: Cats eat big rat. --> point mutation --> Cats eat big hat – The sentence is still almost recognizable, the protein changed in this case might still function. Single Gene Mutations

Types and Effects of Substitution Mutations Missense mutation: causes a single amino acid change Silent mutation: causes no amino acid change Nonsense mutation: causes amino acid chain to be terminated early mutated base

A frameshift mutation is a point mutation that involves an addition or deletion –results in a shift in the reading frame –The reading frame is the way in which the codons are read – start with AUG and go from there Frameshift Mutations

–Cats eat big rat --> add an additional letter A, and the reading frame changes –Aca tse atb igr at - this protein does not resemble the original and will probably not be functional Frameshift Mutations

Mutations may or may not affect phenotype. Some gene mutations change phenotype. – A mutation may cause a premature stop codon. – A mutation may change protein shape or the active site. – A mutation may change gene regulation. blockage no blockage

Some gene mutations do not affect phenotype. –A mutation may be silent. –A mutation may occur in a noncoding region. –A mutation may not affect protein folding or the active site.

Chromosomal mutations affect many genes. Chromosomal mutations tend to have a bigger effect than single gene mutations. Chromosomal mutations may occur during crossing over Chromosomal Mutations

Gene duplication or deletion results from unequal crossing over between homologous chromosomes. Deletion – a segment of a chromosome has been deleted Duplication – a segment of a chromosome has been copied and added to the chromosome more than once

Inversion – a segment of a chromosome has been reversed

Translocation results from the exchange of DNA segments between nonhomologous chromosomes. Translocation– a segment of a chromosome has been moved to a different chromosome

Mutations in body cells do not affect offspring. Mutations in sex cells can be harmful or beneficial to offspring. Natural selection often removes mutant alleles from a population when they are less adaptive. Mutation Effects

Mutations can be caused by several factors. Replication errors can cause mutations. Unequal crossing over leads to chromosomal mutations. Mutagens, such as UV ray and chemicals, can cause mutations. Some cancer drugs use mutagenic properties to kill cancer cells.

Red blood cell phenotypes and blood flow in normal people and people with sickle cell anemia Sickle-shaped blood cells can occlude (block) capillaries. This leads to pain. RBC smear from normal person RBC smear from person with sickle cell anemia

Hemoglobin Beta (HBB) gene Gene Location: Human chromosome 11p15.5 *Chromosome 11 is an autosome Gene Structure: Three exons spanning 1,606 genomic base pairs Polypeptide Size: 147 amino acids

What is Hemoglobin? Hemoglobin is a protein found in red blood cells Hemoglobin is made up of 4 polypeptide subunits (2 alpha, 2 beta)- tetramer –HBB codes for β-globin (aka hemoglobin beta) –Hemoglobin carries oxygen which is needed in body tissues

Are all mutations harmful? Missense mutation: causes a single amino acid change Silent mutation: causes no amino acid change Nonsense mutation: causes amino acid chain to be terminated early

Hb A ALLELE (normal allele) Coding5’-…CAT CTG ACT CCT GAG GAG AAG TCT GCC GTT…-3’ Template3’-…GTA GAC TGA GGA CTC CTC TTC AGA CGG CAA…-5’ mRNA5’-…CAU CUG ACU CCU GAG GAG AAG UCU GCC GUU…-3’ Protein N-…His Leu Thr Pro Glu Glu Lys Ser Ala Val…-C Hb S ALLELE (mutant allele) Coding 5’-…CAT CTG ACT CCT GTG GAG AAG TCT GCC GTT…-3’ Template3’-…GTA GAC TGA GGA CAC CTC TTC AGA CGG CAA…-5’ mRNA5’-…CAU CUG ACU CCU GUG GAG AAG UCU GCC GUU…-3’ Protein N-…His Leu Thr Pro ? Glu Lys Ser Ala Val…-C TRANSCRIPTION TRANSLATION TRANSCRIPTION TRANSLATION Va l (Missense mutation)

Hb A ALLELE (normal allele) Coding 5’-…CAT CTG ACT CCT GAG GAG AAG TCT GCC GTT…-3’ Template3’-…GTA GAC TGA GGA CTC CTC TTC AGA CGG CAA…-5’ mRNA5’-…CAU CUG ACU CCU GAG GAG AAG UCU GCC GUU…-3’ Protein N-…His Leu Thr Pro Glu Glu Lys Ser Ala Val…-C New mutant allele… Coding 5’-…CAT CTG ACT CCT GAA GAG AAG TCT GCC GTT…-3’ Template3’-…GTA GAC TGA GGA CTT CTC TTC AGA CGG CAA…-5’ mRNA5’-…CAU CUG ACU CCU GAA GAG AAG UCU GCC GUU…-3’ Protein N-…His Leu Thr Pro ? Glu Lys Ser Ala Val…-C TRANSCRIPTION TRANSLATION TRANSCRIPTION TRANSLATION Glu(Silent mutation)

Hb A ALLELE (normal allele) Coding 5’-…CAT CTG ACT CCT GAG GAG AAG TCT GCC GTT…-3’ Template3’-…GTA GAC TGA GGA CTC CTC TTC AGA CGG CAA…-5’ mRNA5’-…CAU CUG ACU CCU GAG GAG AAG UCU GCC GUU…-3’ Protein N-…His Leu Thr Pro Glu Glu Lys Ser Ala Val…-C Another new mutant allele… Coding 5’-…CAT CTG ACT CCT TAG GAG AAG TCT GCC GTT…-3’ Template3’-…GTA GAC TGA GGA ATC CTC TTC AGA CGG CAA…-5’ mRNA5’-…CAU CUG ACU CCU TAG GAG AAG UCU GCC GUU…-3’ Protein N-…His Leu Thr Pro ? Glu Lys Ser Ala Val…-C TRANSCRIPTION TRANSLATION TRANSCRIPTION TRANSLATION -(Nonsense mutation)

What happens when the HBB gene is mutated? Most commonly, people with sickle cell anemia have two defective hemoglobin beta HbS alleles (instead of normal HbA alleles). –The HbS allele has a substitution of a single base pair –This mutation results in a glutamic acid to valine change in the protein –Valine is a hydrophobic amino acid Homozygous expression of the HbS allele results in hemoglobin tetramers with two mutant beta hemoglobin subunits. (view picture) The problem is that the hemoglobin tetramers made with HbS beta- globin are “sticky” and clump to form long fibers. These long fibers cause the red blood cell to have a sickle shape.

Is the structure of hemoglobin tetramers in normal people and people with sickle cell anemia different? Hb tetramer in normal people Hb tetramer in people with sickle cell anemia &page=0&pdbId=4HHB &page=0&pdbId=2HBS

Hemoglobin tetramers in people with the HbS/HbS genotype stick together!

How does the HbS/HbS genotype in people with sickle cell anemia cause sickle-shaped red blood cells? Hartwell et al Genetics: Genes to Genomes, McGraw-Hill

Genotype Phenotype Hemoglobin protein Red blood cellPerson

Genotype (HbA/HbA) Phenotype Hemoglobin protein Red blood cellPerson Normal tetramers Normal in shape (donut) and number Normal

Genotype (HbS/HbS) Phenotype Hemoglobin protein Red blood cell (RBC) Person Tetramers stick together and form long fibers Abnormal shape (sickle), fewer RBCs Sickle cell anemia

How is sickle cell anemia treated? Blood transfusions –Build-up of iron is a problem Hydroxyurea medication (causes switch to expression of fetal hemoglobin) –Can be toxic

Is sickle cell anemia found more often in certain ethnic groups? Ashley-Koch A, Yang Q, Olney RS. (2000) Sickle Hemoglobin (HbS) Allele and Sickle Cell Disease: A HuGE Review. Am J Epidemiol Vol. 151, No. 9.

Malaria Incidence, 2009 Cibulskis RE, Aregawi M, Williams R, Otten M, Dye C (2011) Worldwide Incidence of Malaria in 2009: Estimates, Time Trends, and a Critique of Methods. PLoS Med 8(12): e doi: /journal.pmed