Mutations II

T/F: Most human diseases are polygenic

True

• Influenced by several genes

• mutations in different genes can result in disease spectrum

• disease state sometimes requires mutations in more than one gene, especially cancer

Polygenic diseases

• influenced primarily by a single gene 

• few examples (Huntington’s disease, sickle cell anemia) 

• usually obeys Mendelian inheritance (reliably passed to offspring) 

Monogenic diseases 

• result from extra or missing chromosomes

• few examples (down syndrome) 

Chromosomal diseases

• inherited autosomal recessive blood disorder

• most common single gene disease (HBB)

• results from reduction or absence of hemoglobin

• disease phenotype can arise from many different mutations

B thalassemia

Online Mendelian Inheritance in Man (OMIM)

comprehensive database of human genes, mutations, and disorders

What makes up 30% of mutations causing human disease?

Single base pair changes that cause nonsense mutations

Where are common disease associated mutations?

Located In:

• gene promoters

• mRNA splicing signals

• noncoding sequences

What kind of mutations often result in abnormal mRNA splicing?

Point mutations

What test is used to assess the mutagenicity of compounds? 

Ames test 

Ames test

• uses his- strains of salmonella typhimurium

• mutants are unable to synthesize histidine for growth

• liver extract growth media stimulates cell cytoplasm (promoting mutagenicity)

Assay measures frequency of mutations that revert his- to his+

Trinucleotide repeat sequences

• found throughout genome, clusters found in or near important genes

• coding or non coding

• unaffected individuals have a low number of repeats

• affected individuals have many repeats

Fragile X syndrome

230+ CGG repeats in the 5’ UTR of FMR1 gene 

• silencing methylation of FMR1 

• females display 50% penetrance 

Huntington disease

30+ CAG repeats within the HTT gene

• dominant negative; resulting gene product has a toxic gain of function effect

Myotonic dystrophy

50+ CTG repeats in the 3’ UTR of DMPK gene

• repeats interfere with mRNA splicing

What do all cells use to counteract mutations? 

DNA repair systems 

DNA repair systems used for replication errors?

Proofreading and Mismatch repair (MMR)

Repair system for post replication DNA lesions? (3)

1. photoreactivation repair: thymine dimers

2. base excision repair (BER): mismatch bases left after replication

3. Nucleotide excision repair (NER): bulky lesions (dimers, adducts)

Repair system for double strand breaks? 

• Homologous recombination dependent repair (HDR) 

• Nonhomologous end joining (NHEJ)

• Alternative nonhomologous end joining (Alt NHEJ) 

DNA damage types: On a scale of least to most severe

1. Replication Errors

2. Post Replication DNA Lesions

3. Double Strand Breaks

3 basic mechanisms of DNA repair

1. Direct repair

2. Excision repair

3. Double strand break repair

Direct repair

• does not require strand cleavage

• proofreading

• photoreactivation repair: thymine dimers

Types of Excision repair

• Mismatch repair (MMR)

• Base excision repair (BER): mismatch bases

• Nucleotide excision repair (NER): bulky lesions (dimers, adducts)

Double strand break repair 

• Homology directed repair (HDR)

• Nonhomologous end joining (NHEJ)

• Alternative nonhomologous end joining (Alt NHEJ)

Proofreading

• DNA polymerase detects, removes, and replaces incorrect nucleotide

• Polymerase must have 3’to 5’ exo activity; Pol III (bacteria), Pol δ (eukaryotes)

Mismatch repair (MMR)

• mismatch repair activated if proofreading fails

• incorrect nucleotide is replaced after the replication fork has passed

• correct nucleotide made by DNA polymerase, ends ligated

Strand discrimination - how the repair proteins know which base is wrong

• Adenines at GATC sequences are eventually methylated by DNA adenine methylase

• newly synthesized strands are unmethylated for several minutes

• in the event of a mismatch, MMR replaces nucleotide on the unmethylated strand

Photoreactivation repair

• direct repair of thymine dimers

• cleaves bonds between thymine dimers, reversing the damage

Base Excision Repair 

• repairs a single damaged DNA base 

• mismatched bases, basses modified by deamination/depurination 

• AP endonuclease nicks AP site

Nucleotide Excision Repair

• repairs bulky lesions that alter/distort double helix

• nuclease excises the lesion and ligase seals the nick

Xeroderma pigmentosum (XP)

• Cannot repair thymine dimers

• caused by genetic defects in NER pathway

Direct (primary) DSB

caused by ionizing radiation and chemical mutagens that break both strands

Indirect (secondary) DSB

base modifications create nicks and are converted to DSBs by replication 

Transposable elements 

jumping genes, can move throughout the genome