Dysregulation of Gene ExpressionIn

Disease States

or

How alterations in gene expression affect the function of our organs and organ systems

December 7, 2009Dr. Coffman

Q: What all is covered by the term “gene expression”?

A: The entire process from transcribing the DNA sequence to the final, modified, biologically active end product

Components of Gene Expression

• Transcription: synthesis of pre-mRNA, transcription factors, epigenetics

• Post-transcriptional modifications: splicing, capping, tailing, transcript stability, transport

• Translation: protein synthesis, protein folding and stability

• Post-translational modifications: covalent additions (phosphorylation, glycoslyation, methylation, etc.), proteolytic cleavage

• Protein secretion and localization

Common dysregulations of gene expression in human diseases

• Overexpression of RNA or protein

• Underexpression of RNA or protein

• Expression of mutant RNA or protein

• Altered processing of unmutated RNA or protein

• Incorrect localization or trafficking

Complications - no gene acts alone

• Signaling pathways are interrelated• Transcription and translation of individual

genes linked to the transcription and translation of other genes

• A change in the expression or activity of one gene results in changes in the expression and activities of many other genes

Causes of dysregulated gene expression

• Mutations - can be drastic changes that completely change gene activity, or subtle polymorphisms that slightly alter responses

• Disease states - caused by mutations, infectious organisms, toxins

• Responses to disease states - at times the response is as dangerous as the disease (e.g. septic shock)

Causes of mutations

• Inheritance

• UV and other high energy radiation

• Chemical carcinogens

• Defects in DNA repair

• Defects in DNA replication and mitosis

• Defects in gene expression processes

Dysregulation of Transcription

• Transcription of mutant genes

• Abnormal transcription of normal genes– Mutations in promoter regions– Molecular misreading of normal gene

sequence

Mutant Receptors

• Normally, receptors are inactive until bound by a ligand (external signal)

• Binding by ligand causes change(s) in the receptor, often conformational changes that allow association with other molecules or that uncovers an active site, that lead to signals being sent inside the cell

• Transient signaling: bound receptors internalized and degraded or inactivated

• Normal situation: no ligand = no signal• Mutant receptors bypass the requirement for ligand

and can send signals inside the cell constitutively

Her-2 (wt)

Mutation: Val-Gln(point mutation in transmembrane helix)

Result:Neu oncoprotein

EGF receptor (wt)

Mutation: Deletionof ligand-bindingdomain

Result:ErbB oncoprotein

Result: Ligand-independent dimerization & signaling

Cystic Fibrosis chloride channel

The CF chloride channelin mucosal epithelial allowschloride ions to leave thecell and build up in the mucus,creating an ion gradient thatpulls water from the cellsand keeps the mucus moist.Defects in the CF channelallow the mucus to becomedried out (very viscous). Inthe airways, this makesbreathing difficult and createsan environment that promotesbacterial growth.

There are over 800 different mutations in the CF channelthat can cause cystic fibrosis.These mutations totally orpartially inactivate the CFchloride channel.

Chromosomal Translocations

• Common in cancer cells (genetic instability)• Part of one chromosome exchanges with part

of another• Result in gene inactivations, gene

overexpressions, and the creation of chimeric genes and gene products

• One potential cause: errors in repair of DNA double strand breaks

Slide 8.31

In CML, a 9:22 translocation creates the PhiladelphiaChromosome (produces a chimeric protein kinase)

(abl tyrosine kinaseis constitutively active)

Chromosometranslocations are common inhuman promyelocyticleukemias. Markersfor each chromosomehave been linked todifferent colored dyemolecules - look forchromosomes with>1 color.

Cancer-related mutations in p53

Most cancer-related mutations are in DNA-binding region

Sickle Cell Anemia

Single amino acid substitution: Val for Glu at beta 6Results in increased aggregation of deoxyhemoglobinRBCs irreversibly deform (sickle) when [glutathione] is low and disulfide bond forms in spectrinLeads to hemolytic anemia and occlusion of capillariesPrevalent in African populations (malaria resistance)

Normal hemoglobin Hemoglobin S

Normal erythrocyte Deoxygenated HbSaggregates into rods

Sickle cell erythrocytes

When sickle cell rbc glutathionelevels are low, disulfide bondscan form in spectrin filamentsand “freeze” the cell in thecharacteristic sickle shape

Duchenne’s Muscular Dystrophy

Due to mutation(s) in dystrophin genePart of T tubule network involved in calcium release

Progressive and fatal muscle degeneration

Scoliosis in Duchenne’s Muscular Dystrophy

Severe scoliosis in DMD patient

Postoperative (same patient)

Weakening muscles allowsabnormal spinal curvature

Marfan Syndrome

• Connective tissue disorder• Caused by mutation or deletion in fibrillin 1

gene (over 300 known mutations)• Causes decreased strength in elastic fibers• Multiple symptoms in bone and connective

tissues• Weakness in aorta can lead to sudden

rupture in aortic arch

Fibrillin (mutated in Marfan syndrome)

Marfan Syndrome

Werner Syndrome

• Characterized by premature aging (cells from Werner’s patients have a shorter lifespan in culture than normal cells)

• Autosomal recessive disease• Caused by mutation in RecQ family

helicase protein (WRN)• How a mutation in this protein causes

premature aging is currently unknown

Werner Syndrome - helicase mutation

Slide 8.30

Reciprocal translocation

Normal c-myc gene istightly regulatedAfter translocation,myc gene now underthe control of the B cellimmunoglobulin promoterwhich is extremely active

Dysregulated expression due to promoter mutation

Slide 8.32

NeuroblastomaAmplification of N-myc

Dysregulation due to gene amplification

Allelic exclusion(two alleles in the cell, but only one of them is expressed)

Diseases related to epigenetics

Prader-Willi Syndrome

• Complex genetic disorder; symptoms include short stature, mental retardation or learning disabilities, incomplete sexual development, behavioral problems, low muscle tone (floppy body), involuntary urge to eat constantly

• Caused by absence of segment 11-13 on long arm of paternal chromosome 15

• Genes on this segment of maternal chromosome 15 are normally suppressed

• One gene in this region is small ribonucleoprotein N (SNRPN), which is involved in mRNA processing

Fragile X syndrome(an epigenetic disease)

FMR1 gene normally contains6-50 CGG repeats. During replication, DNA polymerasecan “slip” and add more CGGrepeats. When the number ofrepeats exceeds 200, the genebecomes methylated and isloses transcriptional activity.

Fragile X

CGG triplet repeats

Fragile X disease status determined by the number of repeats within the gene locus

Fragile X traits

• Most common inherited disease that causes mental retardation

• Wide range of symptoms from mild behavioral syndromes to severe retardation

• Facial dysmorphology - long face, prominent forehead, large jaw, large ears

• Those with between 50-200 repeats are carriers

Molecular misreading

• Inaccurate conversion of gene sequence to pre-mRNA

• Usually result in small deletions• Dinucleotide deletions result in +1 proteins

(frameshifted from that point on)• Dinucleotide deletions in beta amyloid protein

(beta-APP) and ubiquitin B (UBB) have been found to accumulate in Alzheimer’s Disease plaques

Loss of dinucleotide GAin mRNA that codes forbeta amyloid precursorprotein (APP) leads toa “+1 mRNA” that isframeshifted from the point of the deletion, resulting in a +1 proteinthat has an altered Cterminus, doesn’t turnover properly, and leadsto protein aggregates

Molecular misreading in Alzheimers Disease

Between transcription and translation

• Pre-mRNA splicing - removal of selected sequences from the primary RNA transcript to generate a mature mRNA

• Message stability - processes and sequence elements that affect RNA half life within the nucleus

Alternative Splicing

• Human genome contains ~25,000 genes• ESTs suggest 100,000-150,000 genes• Reason: over 70% of human genes can

undergo alternative splicing and produce more than one gene product

• Alternative splicing occurs in all eukaryotes examined and is not directly correlated to the complexity of the organism

Drosophila Dscam - #1 in Alternative Splicing

• Homolog of human Down Syndrome Cell Adhesion Molecule

• Has 4 exon clusters, each with multiple splicing sites (12, 48, 33, 2 respectively)

• Can make over 38,000 different gene products using various combinations of these sites

Specific DNA sequences are required for pre-mRNA splicing

Pre-mRNA Splicing Sites and Protein Complexes

4 classes of pre-mRNAsplicing defects thatcause disease

Mutation in constitutive splice site

Mutation in alternative splice site

Mutation in basal splicing machinery

Mutation in splicing regulation

Mutations that disrupt alternative splice sites cause diseases

A. GH deficiency type II Mutations in GH gene

B. Frasier syndrome Mutations in Wilms Tumor suppressor gene

C. Dementia/ParkinsonismMutations in microtubule-associated tau protein (MAPT)

A. Atypical Cystic Fibrosis

Relationships between alternatively spliced mRNAs and diseases

Splicing mutations associated with human diseases thatpresent with variable phenotypes

Myotonic Dystrophy (DM)

• Directly linked to dysregulation of alternative splicing

• Most common form of the disease (Type I) caused by CTG expansion in 3’ UTR of the DM protein kinase (DMPK)

• Normal individuals have ~40 CTG repeats; affected individuals have from 80 to several thousand repeats (disease severity correlates with repeat length)

• DM Type II caused by CCTG expansion in intron 1 of ZNF9 gene

Myotonic Dystrophy

Expanded CUGrepeats; oftencalled “toxicRNA”

Large CUG repeats arebound by a dsRNA-binding protein calledMuscleblind, keepingit from binding to othersites and leading toincreased expressionof CUG binding protein(CUG-BP). ElevatedCUG-BP alters thesplicing of manypre-mRNA species and contributes to thepathology of myotonicdystrophy

mRNA DecayPathways

DeadenylationDependent (deadenylation= removal ofPoly-A tail)

DeadenylationIndependent

mRNA decay

• AU motifs in 3’ untranslated region• Bound by specific proteins (AUF1,

ELAV protein, TTP)• Common in cytokine genes to insure

transient response• JNK response element in 5’ UTR of at

least one cytokine (IL-2) also regulates decay

Deregulated mRNA stability

• Carcinomas - inducible COX2 gene contains multiple copies of AUUUA in 3’ UTR (stabilized mRNA)

• Alzheimer’s Disease - alterations in 3’ UTR regions of transferrin and other proteins involved in iron metabolism may be responsible for aberrant iron accumulation in brains of AD patients

Binding to mRNA cap structure

• mRNA cap binding protein eIF4E

• Binding of cap structure by eIF4E is a rate-limiting step in protein synthesis

• eIF4E synthesis is upregulated by c-myc

• C-myc commonly upregulated in human cancers

Translation and beyond

• Protein translation

• Protein folding and misfolding

• Post-translational covalent modifications

• Protein transport and localization

Protein synthesis

• Translation error rate approximately 1 in 5000 (for a 33,000 MW protein of 300 aa’s, one error per 16.7 proteins synthesized)

• Misincorporation leads to misfolded protein• More efficient to allow completion of protein

synthesis and then degrade mutant protein than to halt protein synthesis immediately after the misincorporation of an amino acid

Chaperone-assisted protein folding

Chaperones protect against toxic protein aggregates(neurodegenerative diseases)

Diseases of protein misfolding

Chaperone correction of or protection from mutated proteins

Chaperones protects against huntingtin fibril formation

Post-translational modifications

• Normal patterns of phosphorylation often disrupted in disease states

• Absence of glycosylation or fatty acid addition reduces or eliminates activities

• Disease states often produce signature patterns of changes in post-translational modifications

Protein localization and disease

• Proteins mutant in nuclear localization signal remain in the cytoplasm

• Overexpressed proteins often appear in abnormal places (in cells and organisms)

• Transformed cells express proteins that are inappropriate for the tissue or the developmental stage (many cancers express fetal antigens)

Changes in nuclear structure andnuclear compartmentalization

affect gene expression andare linked with disease states

Mutations in the human lamin A gene are associated withthree inherited diseases caused by defects in tissue-specific gene expression; evidence that nuclear structurecan regulate the transcription of specific genes

Extra copies of normal chromosomals

• Trisomy 21 (Down Syndrome)

• Trisomy 18 (Edward’s Syndrome)

• Trisomy 13 (Patau Syndrome)

• Klinefelter’s Syndrome (XXY)

Trisomy 21 (Down Syndrome)

Down Syndrome critical region on chromosome 21

Studies on individuals with partial trisomy 21 have limited theregion of chromosome 21 necessary for the full expression ofDown Syndrome to a relatively small region, termed 21q22. Among the genes located in this region that may be involvedin the development of Down Syndrome are: superoxide dismutasecollagen type VI,ETS2 transcription factorchromatin assembly factor 1 (CAF1),cystathione beta synthasedual-specificity tyrosine phosphorylated kinase 1 (DYRK1)alpha crystallinglycinamide phosphoribosyltransferase (GART)alpha/beta interferon receptor

Consequences of having an extra copy of chromosome 21

Mental retardation (IQ 40-45)Cranial and facial abnormalitiesEpicanthic foldsShort fingersAbsence of whorl on ball of footCardiac murmurMitral valve prolapseSeizure disorderConduct disorders (notably self-talk)Prevalence of Alzheimer’s diseaseIncreased risk of leukemia

Trisomy 18 (Edward’s Syndrome)

• Extra copy of chromosome 18

• Occurs once per 3,000 births

• 90% die within first year

• Numerous organ defects (heart, lungs, diaphragm, kidneys, ureter

• Microcephaly

Trisomy 13 (Patau Syndrome)

• Extra copy of chromosome 13

• 50% die within first month

• Severe mental retardation

• Microcephaly

• Seizures

Klinefelter’s Syndrome

• Usually XXY sex chromosome content (20% have higher grade aneuploidies, such as XXXY, XXYY etc.)

• Most common genetic cause of male infertility• Most common human chromosomal

aneuploidy• Symptoms: gynecomastia, hypogonadism,

elevated FSH

Genesis of Klinefelter’s Syndrome (XXY)

Abnormal hormonelevels in Klinefelter’sSyndrome patients

Septic Shockabnormal gene expression in response to infection

Normal inflammationLocal TNF induces local IL-1 and IL-6

Histamine, seratonin, prostaglandins and leukotrienes are released, result in local vasodilation

Chemokines recruit inflammatory and repair cells

SepsisMacrophages release large amounts of TNF

(LPS causes 3-5x increase in TNF mRNA and 100,000x increase in released TNF protein)Proinflammatory mediators released systemically

Increased vascular permeability, coagulation cascade activated, complement cascade activated

Low blood pressure, DIC, ARDS, multiple organ failure

Septic shock

is a condition

caused by the

over-reaction

of a defense

response

Summary

• Transcription of mutant genes• Abnormal transcription of normal genes• Abnormal mRNA splicing• Changes in mRNA stability• Abnormal protein folding• Extra copies of normal chromosomes• Sepsis: over-response to bacterial products