University of Michigan



GENETICS OF CANCER

Carcinomas: cancer of epithelial cells

*environmental agents have greater causative effect

*e.g. lung, stomach, skin, colon cancer

Sarcomas: cancer of supportive cells (e.g. bone, blood vessels, muscle, fibrous tissue)

*e.g. osteosarcomas, fibrosarcomas

Leukemias: cancer of blood cells or blood progenitor cells

*e.g. lymphomas (T or B cells), megakaryoblastic leukemia

NORMAL & TRANSFORMED CELL LINES

Generation of normal and transformed cell lines

1. generation of primary cell strain

-mincing tissue (or tumor) to generate single cell suspension

-placed in dish w/ nutrients where they will normally adhere and grow

2. normal cell strain generated by maintaining primary cell strain (non tumor) for generations

-normal cells have finite lifetime—reach senescence and die

-with continued culturing (~40 generations), normal cell line will become established

-transformed (tumor) cells do not reach senesence

3. established lines are immortal and can be normal (non-tumor) or transformed (tumor)

Differences between normal and transformed cell lines

Normal Transformed

-cause tumors in nude mice no yes

-growth in soft agar (adhesion no yes

to substrate not required)

-% serum required for growth 10% 1%

-expression of LETS (fibronectin— high low (in 10% tumors studied)

cell surface protein)

-agglutinated by lectins no yes (at least 6 forms preferentially

(bind specific surface sugars— bind transformed cells)

e.g. concanavalin A binds mannose

and wheat germ agglutinin binds

NAc-Glucosamine)

-CHOs on cell surface normal fetal forms (e.g. less sialic acid)

-enzyme expression adult fetal isozymes (more active)

-hexokinase

(w/ low glucokinase levels)

-pyruvate kinase

-PFK-1

*glucose transport increased

*dedifferentiated features

-globoside AB binding normal increased (CHO determinant likely

a fetal AG)

Differences between normal and transformed cell lines (cont.)

-major difference is loss of growth regulation in transformed cells

*adherent cells grow exponentially, doubling in ~20h, until confluent monolayer reached and

growth stops (density dependent inhibition or contact inhibition of cell growth)—cells

in G0 (quiescence, a part of G1 w/ no DNA synth., reduced rate of protein synth., reduced

rate of amino acid & glucose transport, increased expression of growth factor receptors on

cell surface)

*transformed cells do not stop growing when confluent monolayer reached

-G0 ignored

-transit time in G1 about 2-3h shorter than normal cells

-cells form multilayers (until nutrient deprivation)

-reduced serum requirement

-cells appear more rounded

MODELS OF GROWTH CONTROL

1. Media depletion

Against:

-cells grown on coverslips: one w/ high density, other low density

-place both slips into same dish (same nutrients available to both)

-high density ( no growth; low density ( growth

2. Diffusion boundary layer: medium directly above cells contains diffusion boundary layer through which

all nutrients have to pass to reach cells; once conc. of nutrients in general media falls below critical level,

rate of diffusion of nutrients becomes rate limiting for growth

For:

-if medium pumped at slow rate, cells will grow directly below flow path of medium (same results w/ gentle shaking)

Against:

-if statement is true, growth should be dependent on viscosity of medium—it is not

Reconciliation:

-pump or agitation leads to alterations in cell-cell contact, which may be important in growth control

3. Cell shape: “height” of cell on culture dish will predict its growth rate; higher cells (not strongly attached

to substratum) grow more slowly and flatter cells grow more rapidly

For:

-tissue cultures covered w/ varying thicknesses of plastic adhesive (decreases cells ability to adhere to plate) ( direct correlation b/w height of cell and doubling time

-thus, when cells are in exponential growth in a sparse culture, they are relatively flat and grow at a

good pace; when density increases, cell height increases and proliferation slows

Against:

1) above experiments were done w/ cells which will not grow unless adhering to substratum;

therefore, if they are manipulated to not adhere well to culture dish then they will not grow well,

and it may have nothing to do w/ cell height

2) 3T3 (mouse fibroblast cell line) mutant (AD-6) is deficient in synthesizing NAc-Glc; glycoproteins

in cell membrane are thus under glycosylated and cell grows as a sphere w/ minimal adhesion to

plate; however, it grows at same rate as wild type and reaches same saturation density; cells also do not cause tumors in nude mice

4. Cell-cell contact: cells signal each other to stop growing once they are touching each other

For:

1) in confluent monolayer, cells in “wounded” area began growing upon addition of serum, while cells in remaining areas did not

2) isolated plasma membranes added to cells in exponential growth ( growth inhibited; experiment also performed on transformed cells w/ following results:

a) transformed cells carry inhibitory activity on their membranes but do not respond to factor

b) normal cells carry inhibitory factor and respond to it

c) transformed cells may not have receptor for inhibitory factor, or signal that putative receptor sends is not sufficient to overcome growth signal in transformed cells

5. Cultured cells produce and secrete a growth inhibitory molecule which arrests cells in G0

For:

-isolation and characterization of such substances

GROWTH FACTORS

-induce cells to leave G0 and enter cell cycle

-specificity of growth factor action resides in cell stimulated

*specificity for response lies w/in receptor, and receptor can couple binding event to other events

related to cell proliferation, even if cell does not normally express that receptor

1. Epidermal growth factor (EGF): 6045 D 2-5 ng/ml

-stimulates fibroblast, hepatocyte and glial cell proliferation

-highest conc. in male mouse submaxillary glands

-human version is urogastrone, which inhibits gastric acid secretion

2. Platelet-derived growth factor (PDGF): 32,000 D 2-5 ng/ml

-stimulates fibroblast, glial cell and smooth m. cell proliferation

-composed of 2 non-identical subunits linked by disulfide bonds (AA, AB or BB dimers)

-highest conc. in α-granules of platelets

-2 PDGF receptors: α and β

3. Fibroblast growth factor (FGF): 15,000 D 10-15 pg/ml

-stimulates fibroblast and endothelial cell proliferation

-angiogenic (stimulates new blood vessel formation)

-at least 6 forms of FGF: acidic FGF (aFGF) and basic FGF (bFGF) most common

-highest conc. in pituitary and brain (although every tissue contains some form of FGF)

-may play important role in embryonic growth and differentiation

-at least 3 different receptors isolated and cloned

4. Nerve growth factor (NGF): 26,000 D 5-10 ng/ml

-stimulates neuronal proliferation

-highest conc. in submaxillary glands

-essential for neuronal development and well being

5. Transforming growth factors α and β (TGF-α, TGF-β): TGFα 6-10,000 D 2-5 ng/ml

-in combination, will allow normal cell to exhibit a transformed cell phenotype

-TGF-α is very similar to EGF and will stimulate a cell by binding to EGF receptor

*in absence of TGF-β, TGF-α will act as EGF

*EGF + TGF-β will act as well as 2 TGFs in stimulating cell growth in soft agar

-TGF-β not well understood

*will do different things depending on which growth factors are present when TGF-β interacts

w/ cell

*will stimulate or inhibit cellular proliferation and terminal cell differentiation

-both believed to have roles in developmental process

-found in almost every tissue examined, transformed and untransformed

-highest conc. of TGF-α in α-granule; TGF-β in platelet

-at least 6 different forms of TGF-β and 3 different receptors

GROWTH FACTORS (cont.)

-growth factors act in endocrine, paracrine and autocrine manners

Development of atherosclerotic plaques

1) endothelial cell lining damaged and smooth m. layer exposed; circulating monocytes bind to and infiltrate denuded area, synthesizing and releasing a growth factor similar to PDGF

2) growth factor stimulates smooth m. cells to proliferate (paracrine action)

3) monocytes accumulate lipid and will eventually turn into foam cells which are found in atherosclerotic plaque

4) endothelial cells stimulated to produce 2 types of growth factors

a) one is similar to PDGF and will enhance smooth m. cell proliferation

b) other enhances endothelial cell proliferation (autocrine)

5) by this time, platelets have adhered to site of injury, and have released their granule contents, which include PDGF and TGF-β

6) PDGF and TGF-β continue to enhance smooth m. proliferation until endothelial cell layer regenerated, w/ partial occlusion of artery

Regulation of smooth m. proliferation in developing rat aorta

-smooth m. cells from newborn rat aortas secrete PDGF which stimulates smooth m. proliferation

(autocrine)

-rats > 3 mo of age do not secrete PDGF

*”switch” generated somewhere other than smooth m. cells themselves, as cells derived

from newborn rats cultured for 6 mo in laboratory will still secrete PDGF

GROWTH FACTOR FUNCTION

1. Growth factor (GF) binds to a specific receptor on cell surface

2. Receptor’s internal tyrosine kinase domain (cytoplasmic) is activated, leading to autophosphorylation and phosphorylation of some cytoplasmic components; receptor complex is formed, which contains some of substrates of kinase, which catalyze important secondary effects of growth factor binding

-EGF receptor has 3 important domains: extracellular hormone binding domain,

transmembrane domain, and intracellular tyrosine kinase domain

-phosphorylation of receptor is required for cell proliferation to be observed

-overall percentage of phosphotyrosine amongst all phosphorylated amino acids is very

small, however, lack of kinase activity blocks cellular proliferation

-GF merely “tweaks” receptor to initiate proliferation; ability to stimulate

proliferation lies w/in receptor alone (ABs can be made to “tweak” receptors)

-activated GF receptor forms complex w/ a number of its substrates, all of which are

phosphorylated on tyrosine residue; e.g.

*phospholipase C-γ

*phosphatidyl inositol 3’ kinase (distinct from normal PI kinase)

*raf, a serine/threonine kinase

*GAP

-thus, activation of GF receptor leads to activation of a number of intracellular enzymes, all

of which appear to be involved in intracellular response to GF

3. Receptor is internalized into cell, leading to down-regulation of receptor on cell surface

-adsorptive endocytosis via clathrin coated pits

-internal vesicles form a CURL (compartment of uncoupling of ligand and receptor), which is

acidified prior to fusion w/ lysosome, dissociating ligand and receptor

-EGF receptor is degraded; transferrin receptor is recycled

-possible that a degraded product of receptor or ligand may play a role in signaling

proliferation

-internalization is dependent on functional kinase activity

GROWTH FACTOR FUNCTION (cont.)

4. Na+-H+ antiporter is activated, leading to Na+ entering cell, H+ leaving cell and a cytoplasmic alkalinization (pH increased by 0.2)

5. Increased intracellular [Na+] activates Na+-K+ ATPase, leading to influx of K+

-K+ required for a number of cell processes, including protein synthesis, and thus prepares

cell to begin DNA synthesis

6. PI turnover enhanced, producing IP3 and DAG; occurs by action of phospholipase C by tyrosine phosphorylation

7. IP3 produced leads to release of intracellular Ca++ from internal stores, leading to elevation of free Ca++ inside cell

-PDGF and EGF will stimulate increase in free Ca++, while FGF does not, indicating at least

2 distinct pathways exist for growth stimulation, w/ an intersection later down pathway

8. Increased Ca++ and DAG both activate protein kinase C (PKC), which phosphorylates a number of target proteins, including EGF receptor, which acts as a brake on EGF stimulated pathway; protein kinase C was also show to be a receptor for tumor promoters as well

-increased Ca++ levels required for activation of PKC

-DAG directly binds to cytoplasmic, inactive PKC, which then activated, is found

bound to membrane

-PKC phosphorylates many substrates, primarily on serine residues

-PKC phosphorylates EGF receptor after EGF binds to receptor, leading to receptor having a

reduced affinity for EGF and bringing about dissociation of EGF from receptor (acting as

brake on EFG pathway)

*suggests only a transient signal required to initiate proliferation, and that constant

stimulation of receptor w/ GF may be detrimental

-tumor promoters are a class of compounds which can bind to and activate PKC (requiring

prior initiator exposure to produce tumor); PKC then remains active for extended period of

time

9. Certain genes are expressed in temporal fashion, and are transiently expressed as well; expression of some of these genes is required for cell cycle progression; these genes appear to be required even for GFs which do not stimulate PI turnover or exhibit increased levels of intracellular Ca++

-following addition of GFs to cells, certain genes are transcribed in temporal pattern

-mRNA of such genes is classified according to temporal expression as early, intermediate

or late

MITOGEN ACTIVATED PROTEIN (MAP) KINASES

-kinases activated by mitogens (GFs); in absence of mitogenic signal, kinases are inactive

-MAP kinase cascade

1. GF binds receptor, receptor activates (mediated by G-protein receptors or tyrosine kinase receptors) ras (GTP binding protein), causing it to bind GTP

2. Ras-GTP binds to raf (serine/threonine kinase); ras binding, coupled w/ raf’s phosphorylation directly by GF receptor, activates raf

3. Raf (MAP kinase kinase kinase—MAPKKK) phosphorylates and activates MAPKK (serine/threonine/tyrosine kinase)

4. MAPKK phosphorylates and activates MAP kinase (serine/threonine kinase)

-MAP kinase must be phosphorylated on both a threonine and a tyrosine residue to be active

5. MAP kinase phosphorylates transcription factors (e.g. jun) which then alter gene regulation; MAP kinase also phosphorylates phospholipase A2, activation of which leads to arachidonic acid release, for second messenger production

-MAP kinase might have feedback inhibition on MAPKK and MAPKKK

-this pathway provides a direct link b/w GF binding to cell surface and alteration of gene transcription, which is required for cells to proliferate

INSULIN SIGNALING & STIMULATION OF ISPK BY MAP KINASE

1. Insulin binds receptor, activating receptor’s tyrosine phosphorylase activity

2. Tyrosine phosphorylase phosphorylates and activates IRS-1

3. IRS-1 binds to GRB-2 (growth factor receptor binding protein)

4. IRS-1:GRB-2 complex interacts w/ SOS which functions as a guanine nucleotide exchange factor

5. SOS accelerates activation of ras, through exchange of bound GDP on ras for GTP

6. Ras-GTP in conjugation w/ bound IRS-1 activate raf (MAPKKK)

7. Active raf activates MAP kinase (via MAPKK)

8. Active MAP kinase leads to eventual activation of ISPK (insulin-stimulated protein kinase) which can lead to alterations in glycogen metabolism

-GRB-2 and SOS appear to be used by most tyrosine kinase receptors, except for receptors other than

insulin receptor, GRB-2 binds directly to activated receptor, w/o need for intermediary protein such as IRS-1

-only insulin signaling requires IRS-1 for cascade activation

CYCLINS

-proteins whose synthesis is regulated throughout cell cycle and whose synthesis peaks at certain times

during cycle

-predominantly identified for G2/M transition (though many also function in G1)

-appear to work by interacting w/ protein kinases and phosphatases whose activity appear to be required for

cell cycle progression

-best characterized is p34cdc2 (ser/thr protein kinase) which is inactive until associated w/ cyclin B

*required primarily for G2/M transition

*kinase subunit will associate w/ cyclin B (which peaks at G2/M boundary) to form functional kinase

*also regulated by phosphorylation on 2 residues: thr14 and tyr15

-phosphorylation suppresses kinase activity

-wee1 is kinase which phosphorylates p34cdc2; wee1 cannot phosphorylate p34cdc2 unless

p34cdc2 is associated w/ cyclin B

-kinase activity of p34cdc2/cyclin B activated by p80cdc25, a phosphatase which removes

both phosphates, activating the p34 kinase and allows cell to enter M phase

*active kinase phosphorylates a wide variety of nuclear proteins (e.g. lamins, vimentin) which may

play a role in allowing nuclear division to occur

RADICAL NITRIC OXIDE (NO)

-second messenger

- Arginine + O2 + NADPH ( N-ω-hydroxyarginine + NADP+ ( NO + Citrulline

*both reactions catalyzed by nitric oxide synthase (NOS)

*actual e- donor appears to be a bound FADH2 or FMNH2 which is then reduced by NADPH

-NO binds to a specific guanyl cyclase (enzyme which produces cGMP) which contains a heme molecule

and will activate guanyl cyclase; leads to increase in cGMP levels, which relaxes heart m. through

alterations in Ca-pump activity

*nitroglycerin, given to reduce angina, slowly decomposes to NO

*cGMP also activates a cGMP-dependent protein kinase (protein kinase G) which in other tissues

has other regulatory effects

ROLE OF VIRUSES IN CELL GROWTH & TRANSFORMATION: ONCOGENES

-a number of viruses can bring about cell transformation when introduced to normal cells

*DNA viruses

-transform cells by integrating viral DNA into host chromosome leading to transformation

-e.g. polyoma virus (T antigen), SV40 (large T antigen), adenovirus (E1A factor)

*RNA viruses (retroviruses)

-RNA in these viruses typically only codes for 3 proteins:

*env: envelope glycoprotein found in membrane

*gag: protein important in forming structural core of virus

*pol: reverse transcriptase

-transformation event due to addition of 1 gene to viral RNA, sometimes at expense of

one or more viral genes; if this one gene (oncogene) is removed or altered, virus will be no

longer able to transform cell

-retroviral oncogenes

*a fair number of retroviral transforming proteins code for protein tyrosine kinases

*for every retroviral oncogene, there is a normal cellular homolog, a proto-oncogene

-level of proto-oncogene expression is much lower than that of viral oncogene in transformed

cells

*origin of oncogene-containing retroviruses

1. viral DNA at some point integrated into chromosome adjacent to proto-oncogene

2. if rare event were to occur, such as deletion in viral DNA, or in DNA b/w viral DNA and proto-oncogene, adjacent proto-oncogene may then be transcribed in tandem w/ viral DNA

3. proto-oncogene then under control of viral promoters

4. RNA produced from fusion event then introduced into new viral particles as virus replicates; when new virus infects cell, viral DNA incorporated into host chromosome and all genes under control of viral promoters will be expressed, including proto-oncogene; since viral promoters are always active, proto-oncogene will be continually expressed

5. if protein product is important in regulation of cell growth, its inappropriate expression could lead to uncontrolled cellular proliferation

-b/c event which created oncogene was due to deletion, virus particle cannot normally

replicate itself; in order to replicate, a helper virus is required which will produce necessary

proteins to package transforming viruses genetic material

-retroviral oncogenes (cont.)

*examples

-v-erbB: homologous to EGF receptor but lacks EGF binding site; tyrosine kinase domain

locked in “on” position, leading to cellular transformation

-v-fms: similar to CSF-1 receptor but its kinase activity is always on

*many other oncogenes appear to code for GF receptors

-neu and ros: ligand unidentified

-bek and fig: code for FGF receptors

-v-sis: codes for B chain of PDGF; will react w/ anti-PDGF ABs and will bind PDGF receptor

*cells transformed by Simian sarcoma virus will alwys make PDGF-like molecules,

which can interact w/ internal PDGF receptors and stimulate uncontrolled growth

(autocrine)

*cell must produce own GF to be transformed (unaffected by addition of extrinsic

PDGF); may be due to interaction w/ receptor internally that leads to cell

transformation

-addition of external neutralizing ABs to sis transformed cells does not

reverse transformation event

*hst and int-2 also code for GFs, except both of these oncogenes encode FGF-like

factors

-v-raf: oncogene of c-raf (ser/thr kinase associated w/ activated GF receptors)

*if either c-raf or c-ras become mutated to constitutively active states, this would

lead to constant activation of MAP kinases which would always prime cell for growth

-v-myc: homolog of cellular protein c-myc, which is found in nucleus and is produced w/in

1 hr of adding a growth factor to a quiescent cell; it is transiently expressed and decreases

in intensity beginning at 6 hr after stimulation of cells

*in v-myc transformed cells, myc levels are high throughout cell cycle, meaning that

normal regulatory events due to c-myc are not restricted to G1 phase of cell cycle

and uncontrolled expression of v-myc leads to uncontrolled cell growth

-v-fos: similar in function to c-myc, except that c-fos is expressed w/in 10 min of adding

serum to quiescent cultures and a rapid decrease in c-fos mRNA is observed w/in 30 min

after adding serum

*in v-fos transformed cells, v-fos levels are always high which leads to uncontrolled

cellular proliferation

*c-fos protein interacts w/ DNA in a complex w/ c-jun proto-oncogene to regulate

gene expression, w/ complex acting as large transcription factor

-v-ras: v-ras gene products bind GTP and are similar to G proteins which regulate adenylate

cyclase activity; ras proteins, however, do not display GTPase activity as do G proteins;

manner in which ras alterations transform cells is unknown

*c-ras is a membrane bound protein w/ slow GTPase activity

*when ras interacts w/ activated GAP (GTPase activating protein), ras’ GTPase

activity is stimulated

-GAP is activated by phosphorylation on tyrosine residues, and upon

activation is found associated w/ GF receptors which contain tyrosine

kinase activity

-GAP cannot stimulate v-ras GTPase activity

*over-expression of either c-ras or v-ras results in transformation, however c-ras

transformation can b overcome w/ GAP

*SOS accelerates GDP:GTP exchange for ras protein

*cancer causing retroviruses have yet to be found in humans (w/ possible exception of HTLV-1), but

humans do possess proto-ongogenes related to retroviral oncogenes

TUMOR SUPPRESSORS

-oncogenes discussed previously were all “dominant” oncogenes—presence of mutated or altered form of

proto-oncogene, even in presence of normal gene, resulted in transformation

-“recessive” oncogenes require that both copies of gene be altered for transformation to occur; aka tumor

suppressors, b/c in their active state they suppress formation of tumor, and when function is lost, a tumor

develops (absence of function, not altered function, leads to tumor—negatively acting factors)

*Retinoblastoma (Rb) gene product

-regulates activity of E2F (transcription factors) by binding to E2F and preventing it from

activating transcription genes required for cell to leave G1 and enter S phase

-Rb activity regulated by phosphorylation: when phosphorylated, it can no longer bind E2F

*cyclin-cyclin dependent kinase complexes regulate phosphorylation of Rb such that

it is only phosphorylated at one particular point in cell cycle

-if Rb is absent, E2F is not controlled and cell experiences uncontrolled growth

*p53

-recognizes damaged DNA and halts cell cycle progression until damage is repaired

-acts like transcription factor, allowing cyclin-cyclin dependent kinase inhibitors to be

produced, which block G1 cyclins from working

-in absence of p53, damaged DNA is replicated, which increases frequency of mutation rate

and DNA rearrangements, which contribute to development of metastatic cells

-over half of human tumors have mutated version of p53

HUMAN GENETICS

Factors complicating inheritance patterns

1. new mutation

2. phenocopy (environmentally induced mimic of a genetic disorder)

3. sporadic disease not inherited

4. germline mosaicism

5. delayed age of onset

6. reduced penetrance

7. variable expressivity (variation in phenotype produced by penetrant gene)

8. non-paternity

AUTOSOMAL DOMINANT INHERITANCE

-paternal age effect: increased number of affected offspring w/ autosomal dominant traits in children of

older men

-diseases

Achondroplasia

Huntington disease (homozygotes have ~same degree severity as heterozygotes)

Hypercholesterolemia type II (homozygotes much more seriously affected than heterozygotes)

Marfan syndrome

Neurofibromatosis

AUTOSOMAL RECESSIVE INHERITANCE

-affected person may possess 2 different mutant alleles at locus involved

-diseases

Albinism

Cystic fibrosis (1/2000, most frequent genetic disease in Caucasians)

Phenylketonuria (PKU)

Sickle cell disease (1/400, most frequent genetic disease in Blacks)

X-LINKED INHERITANCE

X-linked recessive

-affected females are 45,X daughters of carrier mothers or 46,XX daughters of carrier mother and

carrier father

-diseases

Fabry disease

Hemophilia A & B

Hunter syndrome

Duchenne muscular dystrophy

X-linked dominant

-affected males transmit mutant gene to all daughters and no sons

-frequency in females ~twice males

-diseases

Hypophosphatemic rickets

Ornithine transcarbamoylase deficiency

CHROMOSOMES

-short arm = p long arm = q

-metacentric (centromere in middle), submetacentric (centromere b/w middle and end), acrocentric

(centromere at end w/ stalk and satellite)

ABNORMALITIES OF CHROMOSOME NUMBER

-aneuploid conditions consist primarily of monosomies and trisomies and are usually caused by

nondisjunction

-major chr aneuploidy syndromes compatible w/ live birth

Patau’s syndrome trisomy 13

Edward’s syndrome trisomy 18

Down syndrome trisomy 21

Turner syndrome monosomy X

Klinefelter’s syndrome XXY

Triple-X XXX

XYY

-frequency w/ which maternal meiotic non-disjunction occurs increases w/ age

*abnormalities in chr number can be caused by non-disjunction events in either meiosis or mitosis

*mitotic non-disjunction can lead to mosaicism in somatic tissue

ABNORMALITIES OF CHROMOSOME STRUCTURE

-types

pericentric inversion

paracentric inversion

duplication

interstitial deletion

terminal deletion

isochromosome

ring

robertsonian translocation

reciprocal translocation

-alternate segregation: alternate chromosomes segregate to same pole, daughter cells get

either normal chromosomes or balanced translocation

-adjacent I segregation: homologous centromeres separate, gametes w/ unbalanced

chromosomes result

-adjacent II segregation: non-homologous centromeres separate, gametes w/ unbalanced

chromosomes result

-chromosome microdeletion syndromes (w/ site of deletion)

Langer-Giedion syndrome 8q24.1 α-Thalassemia 16p13.3

WAGR 11p13 Miller-Dieker syndrome 17p13.3

Retinoblastoma 13q14.1 Smith-Magenis syndrome 17p11.2

Prader-Willi syndrome 15q11 (paternal) Alagille’s syndrome 20p11

Angelman’s syndrome 15q11 (maternal) DiGeorge’s syndrome 22q11

Velocardiofacial syndrome 22q11

MITOSIS & MEIOSIS

-2 major contributions to reassortment of genetic material that occurs during meiosis

*independent assortment of maternal and paternal homologues during meiosis I

*crossing-over that occurs during meiotic prophase I where segments of homologous

chromosomes are exchanged

POSITIONAL CLONING OF CYSTIC FIBROSIS GENE

Cystic fibrosis (CF): autosomal recessive; 1/2500 in N. Europeans; carrier freq. of 1/25; founder effect

Localization of CF gene to chr 7

-first found w/ anonymous DNA marker linked to gene for paraoxonase (other markers then found)

-closest marker found to recombine w/ CF less than 1% of time (markers < 1 cM from CF gene)

PHYSICAL MAPPING & CLONING CF GENE

-crucial step in positional cloning involves finding polymorphic markers that strongly segregate w/

and bracket disease gene to “candidate” region (typically, minimum bracketed region is 1 Mb)

-at this point, very few meiotic recombination events are seen b/w flanking markers so further genetic

mapping is not possible

-at this stage, physical techniques of mapping are used

1. Isolation of DNA from candidate region

-genomic libraries: made using YACs (yeast artificial chr) and plasmids

-chromosome walking: isolation of overlapping DNA clones, each containing 50-1000

kb of DNA, that collectively span region where disease-causing gene is known to occur

2. Identification of expressed sequences

-zoo blot: used in identification of protein-coding sequences; if probe contains coding

DNA, it will usually react w/ several of non-human DNAs on zoo blot; if it does not

contain any coding sequences, it will usually hybridize only to human DNA

3. Identification of mRNAs transcribed from each candidate gene

-demonstrate that candidate gene is expressed in normal persons in those tissues that

are primarily affected in patients w/ disease

-detection of mRNAs done w/ Northern blots

4. Correlate abnormal mRNAs w/ presence of disease

-absence of mRNA or presence of abnormally sized mRNA in some patients is evidence

for gene being responsible for disease (not necessary that all affected persons have

abnormal mRNA; many mutations that lead to non-functional proteins do not affect

synthesis of mRNA)

5. Correlate abnormal DNA segments in affected individuals

-correlate occurrence of submicroscopic DNA deletions w/ presence of disease

-deletions too small to be detected cytologically are detected w/ Southern blots of DNA

from affected and normal persons

-deletion indicated by change in size of 1+ restriction fragments in DNA from affected person

-most patients will not have deletions (only around 5-10% do) in gene responsible for

disease; in some cases (e.g. DMD) 50% of patients have deletions, in others (e.g. CF)

deletions large enough to be detected on Southern blots are virtually nonexistent

-demonstration that candidate gene contains deletions in at least some patients, but not in

normal controls, is an important aspect of positional analysis investigation

6. Identification of mutations in candidate gene from affected individuals

-obtain sequence of normal mRNA by cloning cDNA from normal donors and sequencing

it, and compare w/ cDNA from affected persons

-if disease causing mutations abolish synthesis of detectable mRNA, it may be necessary to

clone and sequence entire gene from genomic DNA and from normal and affected persons

to reveal any differences

CF MUTATIONS

-4 classes of mutation w/ differing effects on CF transmembrane conductance regulator (CFTR)

1. Failure of production of CFTR—generally due to chain termination or frame shift mutations

2. Disrupt processing of CFTR—defective folding of protein, interfering w/ normal processing in Golgi and ultimate failure to appear on cell surface

3. Disrupt regulatory domains in CFTR—regulated by binding of ATP and by phosphorylation w/ cAMP-dependent protein kinase

4. Point mutations—disrupt pore of chloride channel (cannot conduct chlorine)

-ΔF508 mutation accounts for 70% of CF mutations in N. European population

*hundreds of diverse mutations account for additional 30% (no single one being more than a few %)

*assay for ΔF508 exists (test for 30+ mutations for $200—uses cheek epithelial cells and PCR)

-mutations differ in different populations

-mutations widely distributed in gene and represent various types of mutations: deletion or insertion of 1 or 2

bases, or single base substitutions are the rule

-various mutations are not equivalent in their effects on function of CFTR (e.g. ΔF508 homozygotes

generally suffer pancreatic insufficiency w/ consequent malabsorption; R117H/ΔF508 compound

heterozygotes usually have normal pancreatic function)

DIAGNOSIS OF CF BY GENETIC LINKAGE

-definitive testing requires that gene for disorder be cloned

-linkage analysis can permit prenatal diagnosis and presymptomatic diagnosis

-pitfalls of linkage-based diagnoses include genetic recombination, genetic heterogeneity and it requires

DNA from many members of family

TREATMENT OF CF

-life expectancy has improved in CF patients due to antibiotics and pancreatic enzyme replacement therapy

-gene therapy: adenoviruses w/ CFTR cDNA have produced CFTR production in CF knockout mice

-liposomes have been used to transfect respiratory epithelial cells of transgenic mice homozygous for CFTR

mutation resulting in restored levels of chloride channel activity

GAUCHER DISEASE

LYSOSOMAL STORAGE DISEASES (LSDs)

-result of a genetic alteration of a specific lysosomal hydrolases producing defective intralysosomal digestion

and consequent accumulation of substrate in lysosomes of certain cells and tissues

-hydrolases normally encoded…

1. at a single locus as single polypeptide chains and are monomeric or homomeric as mature proteins

2. at 2 loci as 2 different polypeptide chains and form heteromeric complexes

3. as either 1 or 2 above and w/ participation of an additional locus for an activator or cofactor locus

-phenotype for LSD can be genetically heterogeneous at allelic or locus level

-deficiency of hydrolytic activity leads to progressive engorgement of lysosomes and leads to hypertrophy

and hyperplasia of lysosomal apparatus; eventually cellular functions become impaired; when functions of

many cells in a particular tissue are impaired, both structural and functional abnormalities result and

phenotype is manifested

*e.g. Gaucher disease: progressive glucosyl ceramide accumulation in MΦs, due to deficiency of

acid β-glucosidase, leads to eventual loss of liver, spleen and bone marrow function

-differences in severity or age of onset result from variation in amount of hydrolytic activity, i.e. there is a

threshold level of flux through the pathway to establish disease severity

-disease phenotype (i.e. severity) is established by effect of mutation; other factors (allelic polymorphisms,

modifier genes) produce variation in phenotype of patients w/ same genotype at disease locus; thus,

primary locus sets stage for disease (establishes susceptibility) but does not necessarily “cause” disease

phenotype

-LSDs classified by chemical nature of accumulated substrate

1) sphingolipidoses

2) mucopolysaccharidoses

3) glycoproteinoses

GAUCHER DISEASE

-acid β-glucosidase is a homodimer that requires as activator protein, saposin C, for catalytic function

acid β-glucosidase

glucosyl ceramide glucose + ceramide

saposin C

-allelic and locus heterogeneity for each of clinical variants

*defective glucosylceramide hydrolysis—major biochemical abnormality

*deficient acid β-glucosidase (glucosylceramidase, glucocerebrosidase) activity—major locus

involved

*diminished saposin C activity

*3 clinical variants

-type 1: 62% w/ 439/704 N370S mutant allele

-type 2: 67% w/ 12/18 mutant allele

-type 3: 69% w/ 55/80 L444P mutant allele

-numerous molecular defects have been defined and all types of mutations (missense, nonsense,

rearrangements and deletions) have been found in acid β-glucosidase gene

*relationship b/w mutation and type or variant of Gaucher disease; permits prediction of variant

type based on genotype (i.e. a susceptibility gene)

-Ashkenazi Jewish population has high freq. of type 1 variant (68-75% w/ 1226 N370S

mutation)

ENZYME THERAPY IN GAUCHER DISEASE

-IV administration of α-mannosyl terminated glucocerebrosidase (acid β-glucosidase)

-based on pathophysiology of major visceral manifestations resulting primarily from accumulation of

glucosylceramide in monocyte/MΦ lineage cells; since MΦs have α-mannose recognition receptors on

their surfaces, modification of oligosaccharides, attached to acid β-glucosidase to expose α-mannosyl

moieties will facilitate targeting to sites of pathology

RETINOBLASTOMA:

Positional cloning of a cancer causing gene

GENETICS OF RB

-hereditray Rb: autosomal dominant; bilateral involvenment; high incidence of other tumors (e.g.

osteosarcomas); some patients have abnormal karyotype

*one Rb allele already mutated; over time, other allele mutates in one of millions of cells in retina;

“good copy” lost ( Rb

-non-hereditary (sporadic) Rb: unilateral; no multiple independent tumors

*both Rb alleles normal; single somatic cell inactivates one of its Rb copies and mistake propogated

in that cell’s lineage; another somatic cell mutation in a cell derived from that lineage occurs ( Rb

(Knudson “two hit” hypothesis)

POSITIONAL CLONING OF RB GENE

-linkage analysis of markers in band q14 of chr 13 showed linkage in families w/ hereditary Rb

-positional cloning used to identify gene routinely mutated in tumors

MOLECULAR GENETICS OF RB: LOSS OF HETEROZYGOSITY (LOH) ANALYSIS

-DNA derived from Rb tumor shows homozygous deletions; this reduction of homozygosity is common in

tumor suppressor oncogenes (sometimes referred to as loss of heterozygosity or hemizygosity); this may

occur in various ways: (+ = normal allele, Rb = dysfunctionally mutated allele)

*nondisjuction (Rb/+ ( Rb/__ )

*nondisjunction reduplication (Rb/+ ( Rb/Rb)

*mitotic recombination (Rb/+ ( Rb/Rb)

*gene conversion (Rb/+ ( Rb/Rb)

*deletion (Rb/+ ( Rb/__ )

*point mutation (Rb/+ ( Rb/Rb)

RB GENE PRODUCT

-p105 (or RB), a 105,000 D protein

-protein is phosphorylated (many oncogenes are phosphorylated and/or can phosphorylate other cellular

proteins and affect cell cycle)

-over 85% of p105 found in nuclear fraction (a nuclear protein)

NEW AVENUES IN CANCER RESEARCH OPENED UP BY CLONING RB GENE

-using Rb protein, AB can be raised; opens up possibility of simple diagnostic procedures using Western

blot technique similar to that used for dystrophin in DMD

-w/ cDNA clones, site-directed mutagenesis has become increasingly important in determining importance

of regions of gene (e.g. in lysine 131 important? Knock it out and see)

MOST FREQUENTLY MUTATED TUMOR SUPPRESSOR GENE YET: p53

-p53, when in binds DNA, can activate transcription; tumor-derived, mutated p53 cannot

-if wild-type and mutant p53 proteins mixed, mix is unable to activate transcription, indicating that binding of

p53 protein in mediated through formation of hetero-oligomers: p53 probably binds to DNA as a tetramer;

p53 thought to be a dominant negative oncogene

-when DNA id damaged, p53 accumulates in cytoplasm, is transported to nucleus where it mediates cell

cycle arrest (at G1), presumably by interfering w/ synthesis of particular proteins; alternatively, it may bind

those proteins in cytoplasm and modulate their activity

*replication is switched off, giving cell time to repair DNA

*if repair can’t be effected, protein may trigger cell to destroy itself

-tumor cells which carry a mutated (inactive) p53, or bind up all of available p53 via overproduction of protein

w/ which it interacts, are not arrested; cells become genetically unstable, and when mutation or chr

rearrangement does occur, it will not be selected against—cells will accumulate ( tumor

INHERITED SUSCEPTIBILITY TO CANCER:

THE GENETICS OF COLON CANCER

BIOLOGY & HISTOLOGY OF COLON CANCER

-colon carcinoma develops through benign intermediate tumor known as adenoma or adenomatous polyp

-tumors of colon, both adenomas and carcinomas, are common in general population, are acessible at a

variety of stages and can be found at an increased incidence in some inherited syndromes

-any portion of colon or rectum can form adenomas and can be affected by carcinoma; in general, more

carcinomas occur in distal colon and rectum

-adenomas result from an abnormality of cell proliferation and a failure of cellular maturation; they are

benign and non-invasive; defining characteristic is presence of dysplastic glands

-adenomas of 2 broad types: sessile (close to wall of colon and more flat) and pedunculated (having stalk)

-adenomas are thought to be precursors of most colonic adenocarcinomas; adenomas that are large,

severely dysplastic or villous are more likely to give rise to an invasive adenocarcinoma

DISEASES THAT PREDISPOSE TO COLON CANCER

INHERITED DISEASES—all autosomal dominant

A. Hereditary polyposis colorectal cancer syndromes (1% of colon cancers)

1. Adenomatous syndromes

a. Adenomatous polyposis coli (APC)

*autosomal dominant; complete penetrance

*presence in early adulthood of 100s-1000s of adenomatous polyps

*adenocarcinoma inevitable outcome

*incidence of tumors outside colon and rectum is elevated

*2 patterns: carpeting and discrete

*heterogeneity in polyp number has been observed in some kindreds

showing an attenuated form of APC, known as AAPC (characterized

by finding of < 100 polyps)

b. Gardner syndrome

*APC w/ extracolonic lesions (osteomas, dental abnormalities, benign

soft tumors and cysts)

c. Turcot syndrome

*brain tumors and polyposis occurring together

-APC, Gardner syndrome and AAPC are each associated w/ disruption of APC

gene, which is at 5q21 (mutations clustered at 5’ end in AAPC)

*mutations include: frameshifts by small insertions and deletions, point

mutations that introduce premature stops, and mutations that affect splice

junctions

*most APC families carry unique mutations; only 2 mutations are known to

have occurred in more than a few kindreds (each is a 5 bp deletion in open

reading frame; together they account for < 10% APC mutations)

-6% of Ashkenazi Jewish population has polymorphism mutational hotspot in APC (series of As) which mutates more frequently

(frameshift); acounts for 28% of colon cancers in this population

2. Hamartomatous syndromes: multiple hamartomatous polyps in GI tract; affected

Individuals carry significant risk of colonic and other types of cancer

a. Cowden syndrome

b. Familial juvenile polyposis

c. Peutz-Jaegher syndrome

B. Hereditary non-polyposis colorectal cancer syndromes (HNPCC): 5-6% of colon cancers

1. Lynch syndrome I: site specific (usually right-sided)

2. Lynch syndrome II (cancer family syndrome): also characterized by endometrial,

ovarian, gastric and breast cancers

-germline mutation on any one allele of these 4 genes can lead to HNPCC:

*hMSH2 2p22-p21 & hMLH1 3p21.3: homologues of 2 bacterial DNA mismatch

repair genes, mutS and mutL

*hPMS1 2q & hPMS2 7p: involved in DNA mismatch repair

NON-INHERITED DISEASES

A. Ulcerative colitis (inflammatory bowel disease)

B. Cronkite-Canada syndrome: rare

MOLECULAR ANALYSIS OF COLON CANCERS

-Gain-of-function mutations

*Ki-ras mutations: in 50% colorectal tumors (both adenomas and carcinomas); amino acid changes

at codons 12 or 13; confer a gain-of-function in cell culture assays that measure tumor-forming

ability

-Loss-of-function mutations

*losses of genetic information by allelic deletion in tumors can mark physical location of specific

genes that normally function in suppression of tumorigenicity (tumor suppressor genes)

*colonic tumors frequently show LOH affecting chr arms 5q, 8p, 17p and 18q

*LOH at increasing number of chr sites in a tumor correlates w/ poor clinical prognosis

-Other molecular alterations

*nonspecific lessening of DNA methylation: generally associated w/ increase in gene expression,

and a higher than normal expression of c-myc oncogene

*amplification of cyclin genes

*microscopic changes in chromosomes of colorectal tumors

GENES INVOLVED IN FORMATION OF COLON CANCERS

-APC gene (5q21)—tumor suppressor

*at least one copy mutated in most carcinomas and adenomas

*freq. of mutation does not increase w/ tumor progression

*most mutations are chain terminating

*other tumor types showing LOH at 5q: small-cell lung cancer, gastric and esophogeal cancers, foci

of dysplastic (cancerous ulcerative colitis) (APC mutations found directly in gastric carcinomas)

*truncated proteins can be produced from some mutant APC alleles in tumors, acting in dominant

negative fashion on normal APC proteins in APC and AAPC patients

-AAPC characterized by APC mutations affecting exons 3 and 4, earlier in coding seq. than

any known mutations in APC (producing shorter proteins)

-p53 gene (17p53)—tumor suppressor (17p13—site of Li-Fraumeni mutation)

*(see pg 15 for function)

*targets of p53 include genes regulating genomic stability, response of cell to DNA damage, cell

cycle progression, and induction of cell death by radiation or chemotherapeutic DNA-damaging

drugs

*inactivation of p53 by…

-mutation of p53

-binding of p53 to other oncogenic proteins: MDM2 (mdm2 often amplified in human sarcomas), E1B gene product of adenovirus, E6 gene product of HPV, large T AG of SV40

*cancers involving p53 mutations found in: brain, breast, colon, esophagus, liver, lung, leukemias,

and lymphomas (spectrum of mutations differs slightly among these tumors)

*some mutant forms of p53 also acquire a gain-of-function and appear to stimulate cell growth (act

in dominant negative fashion)

*germline mutations have been described in kindreds w/ Li-Fraumeni cancer syndrome, in patients

multiple sarcomas or multiple primary tumors and in some patients w/ strong family history of cancer

(colon cancers not commonly found in Li-Fraumeni families, again suggesting that p53 mutation is

not an early event in colon-tumor formation)

-DCC & DPC4 genes (18q)—not well understood

-Ki-ras gene

*~50% of large adenomas and colon carcinomas carry mutations in Ki-ras (only 10% of small

adenomas)

*dominant gain-of-function mutations; majority occur in codon 12 or 13

*oncogene

-NF1 gene

*gene product is neurofibromin, protein containing GTPase-activating domain (GRD) involved in

catalysis of ras-GTP to ras-GDP

*disruption results in deregulation of cell growth

GENOMIC INSTABILITY IN COLON CANCER

-genomic instability syndromes (chr breakage syndromes) predispose affected individuals to malignancies

*Bloom syndrome, Fanconi anemia, Werner syndrome, ataxia telangiectasia

*these autosomal recessive disorders have an elevated mutation rate at specific loci

*inherited susceptibility to genomic instability could be involved in tumor initiation or promotion is

supported by high cancer incidence in all these syndromes (especially Bloom syndrome)

DIAGNOSIS OF COLON CANCER

-blood in stool and colonoscopy

-mutations in Ki-ras can be detected in stool of patients w/ curable colorectal tumors

INHERITED SUSCEPTIBILITY TO CANCER:

THE GENETICS OF BREAST CANCER

BRCA1 GENE

-located at 17q12-21

-mutations: 11 bp deletion, 1 bp insertion, stop codon, missense substitution, inferred regulatory mutation

-BRCA1 product

*expressed in numerous tissues, including breast and ovary

*encodes predicted protein of 1863 amino acids, containing zinc-finger domain in N-terminus

*encoded 220 kD nuclear phosphoprotein in normal cells w/ some similarities to transcription

factors

-sporadic tumor formation

*mutations in BRCA1 not found in high percentage of sporadic breast cancers

-BRCA1 mRNA levels markedly decreased during transition from carcinoma in situ to

invasive cancer in sporadic breast cancer

*in sporadic ovarian cancers, many tumor DNAs carried BRCA1 mutations

-tumor suppressor: somatic mutation in one chr and LOH on other may result in

inactivation of BRCA1 in some sporadic ovarian cancer

*BRCA1 may normally serve as negative regulator of mammary epithelial cell growth and that this

function is compromised in breast cancer either by direct mutation or by alterations in gene

expression

-women carrying BRCA1 mutation have 80% risk of breast cancer and 40% risk of ovarian cancer by age 70

*modifier of this risk is HRAS1 proto-oncogene (or closely linked gene); individuals w/ rare variants

of this polymorphism have increased risk for certain cancers, including breast cancer

*Ashkenazi women have increased freq. of 185delAG (frameshift mutation) of BRCA1

-men w/ BRCA1 mutations

*contribution of germline BRCA1 mutations to overall incidence of prostate cancer appears to be

small, at most, and may be limited to specific subgroups of patients

BRCA2 GENE

-located at 13q12-13

-shown to include 6 different germline mutations in breast cancer families; each mutation causes serious

disruption of open reading frame

-BRCA2 product

*present in normal breast epithelia, placenta and thymus, w/ slightly lower levels in lung, ovary,

testes and spleen

*encodes protein of 3418 amino acids w/ no similarity to known proteins

*like BRCA1 protein, BRCA2 protein is highly charged, w/ ~1/4 residues acidic or basic

-sporadic tumor formation

*mutations in BRCA2 appeared to be infrequent in all cancers including breast carcinoma; however,

homozygous mutation in a pancreatic tumor suggested a role for BRCA2 in that tumor type

*mutation confers a high rate of breast cancer but does not confer a substantially elevated risk for

ovarian cancer

*LOH on 13q in sporadic breast and other cancers suggests BRCA2 is tumor suppressor

*in Icelandic population, 999del5 mutation has been found in individuals w/ different tumor types

including cancer of prostate, pancreas, ovary, colon, stomach, thyroid, cervix and endometrium

-men w/ BRCA2 mutations

*participates in male breast cancer

MYOTONIC DYSTROPHY (DM):

GENOMIC EXPANSION OF TRIPLET NUCLEOTIDES

GENETICS OF DM

-autosomal dominant disorder w/ variable expressivity but high penetrance; affects 1/20,000

-tendency to become more severe from generation to generation (anticipation)

POSITIONAL CLONING OF DM GENE

-near 3’ end of gene, in a region transcribed but not translated, is a stretch of triplet bases CTG which is

repeated 5-27 times

-individuals w/ DM have 50+ copies of CTG repeat representing a mutation that upsets function of gene

*expansions also render region unstable and prone to further expansion

*e.g. parent w/ 60 repeats may transmit gene to child w/ 80 repeats

*accounts for genetic anticipation

*contractions of CTG region have also been noted on rare occasions (indicates genetic linkage

analysis can give erroneous diagnostic results even in absence of genetic recombination)

DM PROTEIN

-DM gene codes for DM kinase (phosphorylates on ser and thr residues)

-expressed in brain, muscle, heart and testes

-CTG repeat occurs near 3’ end (transcribed but not translated)

-expanded region can be detected by protocols involving Southern hybridization or by PCR

GENOMIC IMPRINTING:

CONTROL OF GENE EXPRESSION BY OTHER MEANS

IMPRINTING

-implies modification in expression of a gene or allele

-maternal imprinting: no phenotypic expression of mutant allele when transmitted from the mother

(presumably b/c it’s switched off by methylation)

-paternal imprinting: no phenotypic expression when mutant allele is transmitted from father

-why imprinting? dosage compensation and gene control

CLUES TO IMPRINTING

-hydratidiform mole: 2 complete sets of paternal chromosomes; no embryonic tissue; androgenetic

-teratoma: 2 complete sets of maternal chromosomes; no placental tissue; gynogenetic

-uniparental disomies: chromosome or chromosomes derived from one parent

*isodisomy: exactly same chr inherited

*heterodisomy: different copies of same chr inherited

-pathogenesis

*disease arises from deficiency (absence of at least 1 chr from each parent)

-methylation may play a role in imprinting by indicating and identifying imprinted genes

HUMAN DISEASES

-Prader Willi syndrome: deletion of paternal 15q11-13; those w/o deletions result from maternal chr

15 disomies, isomies and heterodisomies

-Angelman syndrome: deletion of maternal 15q11-13; paternal hemizygosity

-Down syndrome & cystic fibrosis: may be influenced by parentage of chromosomes and genes

-Cancer: in many cases familial tumors are dominantly inherited and associated w/ loss of wild type

allele during tumorigenesis

-Wilms tumor: chr 11 deletions; almost always of maternal origin

-Retinoblastoma: LOH producing Rb may be paternally derived

-Myotonic dystrophy: 10-20% of affected families where gene is maternally derived have very severe

lethal congenital form of disease

-Huntington chorea: 5-10% of paternal derived genes lead to severe, rigid juvenile form; never been

observed for maternal derived genes

CHANGES WITH GENERATION OF PASSAGE—REQUIREMENTS FOR IMPRINTING

-erase previous imprinting

-new modifications in germ cells of each sex

-new imprinting of genes from maternal or paternal lineage

-differential tissue specific expression of imprinted genes

MULTIFACTORAL DISEASE, POPULATION GENETICS & RELATIVE RISK

MULTIFACTORIAL TRAITS

-most common etiology for common diseases is “polygenic” or “multifactorial”

-“threshold” model predicts that cumulative risk for phenotype is additive and that either predisposition

to disease or phenotype becomes manifest at a given threshold

*continuous traits, e.g. hypertension (propensity to disease manifestation increases as BP

increases)

*quantum traits, e.g. cleft palate (risk of having offspring w/ phenotype is cumulative)

GENERAL PRINCIPLES OF MULTIFACTORIAL INHERITANCE

1. threshold model: multifactorial disorders caused by a number of minor genes and 1+ major genes; these genes produce small phenotypic effects which are usually additive; environmental factors are important to expression which results from interaction of genetic and environmental effects

2. sharp decrease in freq. of trait from monozygotic twins to 1st degree relatives and from 1st degree relatives to 2nd degree relatives

3. more freq. a trait in general population, smaller is risk to MZ twins and 1st and 2nd degree relatives

4. concordance in MZ twins is markedly greater than in nongenetic traits, but less than in traits w/ dominant or recessive transmission

5. where sex ratio of affected individuals is altered (one sex more frequently affected) risk for children of proband to be affected is higher if proband is of more rarely affected sex

6. if proband is of more rarely affected sex, risk to be affected is greater for his/her children of opposite sex; if proband is of more frequently affected sex, risk is greater for his/her children of same sex

7. more severly proband affected, higher risk for children to be affected

8. greater number of individuals in a family who are affected, higher risk component becomes (i.e. concentration of genetic risk factors)

MULTIFACTORIAL INHERITED DISORDERS PRESENT AT BIRTH

-Neural tube defects (spina bifida w/ meningomyelocele, spina bifida occulta, anencephaly, cranium

bifidum, midline encephalocele)

-Cleft lip w/ or w/o cleft palate, cleft palate

-Congenital heart defects

-Club foot, congenitally dislocated hips, pyloric stenosis, upper urinary tract malformations,

hypospadias

-Hirschsprung’s disease

-Scoliosis and multiple hemivertebrae

-Craniosynostosis (premature sagital fusion)

-Microtia and cryptorchidism

HARDY-WEINBURG EQUILIBRIUM

- p2 + 2pq + q2 = 1 p + q = 1

p2 = freq. of AA 2pq = freq. of Aa q2 = freq. of aa

-assumptions

*random mating

*large population (several hundred)

*negligible mutation b/w A and a

*negligible migration

*little selection and all genotypes viable and fertile

NON-MENDELIAN FORMS OF INHERITANCE: MITOCHONDRIAL INHERITANCE

CHARACTERISTICS OF MITOCHONDRIA AND THEIR INHERITANCE PATTERN

-entire mitochondrial genome ~16,569 bp and circular

*encodes 22 tRNAs and 13 other open reading frames (ORFs)

-genetic code different than for nuclear genes (e.g. UGA for trp not stop)

-almost all mitochondria inherited from mother since sperm mitochondria lost during fertilization

-mitochondrial inheritance is thus maternal (mother to all offspring)

-mitochondrial genome is highly polymorphic

-mitochondrial DNA mutations show threshold effect:: b/c there are hundreds of mito. per cell and only

some will have particular mutation, number of mutant mito. per cell would be critical in determining

phenotypic manifestation at a cell and organism level

-homoplasmy: indicates presence in all cellular mito. of a mutation or mutant mitochondrial gene seq.;

if these are deleterious mutations, it must be mild since a sever mutation would be lethal

-heteroplasmy: indicates presence of 2 different classes of mito.—usually normal and mutant; mutation

in this class could be maintained only below a certain threshold since larger percentages of mutant mito.

could be lethal to organism or to particular cells in organism

DISEASES CUASED BY MUTATION OF MITOCHONDRIAL GENES

-Lebner’s hereditary optic neuropathy

-infantile bilateral strial necrosis (BSN)

-myoclonic epilepsy w/ ragged red fibers (MERRF)

-Kearns-Sayre

-MELAS

-Leigh’s syndrome

-familial diabetes mellitus

-infantile encephalomyopathies

HUMAN GENOME PROJECT:

LINKAGE, GENE MAPPING & POSITIONAL CLONING

STRUCTURE OF CHROMOSOMES

-chr contain 2 main components in appx. equal mass: proteins and DNA

- ~108 bp DNA per chr; ~200 bp per nucleosome

- ~3 billion bp in genome

-classes of DNA sequences according to freq.

*unique class: found only once in entire genome

*middle repetitive class: repeated 100-1000 times per genome

*highly repetitive class: repeated 10,000-300,000 times per genome

-2 common repetitive elements in human genome are ALU and LINE sequences

STRUCTURE OF GENES

-gene: region of DNA that directs synthesis of gene product, either RNA itself, or mRNA then protein

*such a region includes signals for gene expression that may be more extensive than regions

that actually code for gene product

- ~104-105 bp DNA per gene; ~106 genes in genome

LINKAGE ANALYSIS

-basis of linkage analysis is homologous crossing over during meiosis since all chromosomes recombine

w/ a certain freq.; closer 2 loci are to each other, less frequently they will recombine

*loci can be DNA segments or DNA protein or protein marker and a disease phenotype

*when recombination is < 50%, loci are said to be linked

-distances measured in Morgans or centi-Morgans (cM)

*1 cM = 1% recombination freq. = 106 bp

-LOD (log of odds of linkage)

• Y = likelihood ratio (θ) = likelihood of linkage/likelihood no linkage

• log10Y = LOD score (Z)

• if LOD = 3, then 1000:1 that linkage did not occur by chance

• if LOD = -2, then 100:1 odds against linkage

SOMATIC CELL HYBRIDIZATION & HUMAN GENOME PROJECT

**see XXXI 5-10

GENE THERAPY

DELIVERY SYSTEM FOR GENES

-retroviruses

*can transfer genetic material into dividing cells w/ stable integration, although random, into genome

*viral genome contains 5 major elements: long terminal repeats (LTRs, promoters), ψ sequence

(signals packaging events), GAG (gene for encapsidation proteins), POL (gene for reverse

transcriptase) and ENV (gene for envelope proteins)

*retrovirus that contains LTRs, ψ seq. and endogenous promoter in cis w/ gene of interest could be

transfected into packaging cell line

-transfection: single integration event w/o production of additional viral particles

*IV administered retroviruses are rapidly inactivated presumably by C’ and are rendered useless in

a few circulation times; consequently, constructed retroviruses must be delivered in high titer to

appropriate target cells either by direct local application via physical injection or by ex vivo

manipulation of stem cells and transduction w/ retrovirus

*retroviral genome can only incorporate genes to about 4 kb

-adenoviruses

*stable, broad infectivity of cells, ability to express genes transiently in localized areas of body (e.g.

lungs)

*major limitation is inability of exogenous DNA to be integrated at any significant level into human

genome

*large capacity for exogenous DNA and accommodates transcripts up to ~7.5-10 kb

-adeno-associated viruses (AAV)

*ability to site-specifically integrate into genome and potentially integrate into non-dividing cells

*strictly non-pathogenic in humans

*potential for site-specific integration on chr 19

*small capacity for exogenous DNA, < 4 kb

*potentially restricted cell host range

ALTERNATIVE STRATEGIES USING GENE THERAPY

-organoids: cells genetically altered to produce high levels of secretable gene product are expanded ex

vivo; these cells then transplanted back into recipient so that gene product is secreted and delivered locally

in very high concentrations or systemically to a variety of cell types

*factor VIII for hemophilia

*β-glucuronidase for mucopolysaccharidosis (MPS) type VII

*cytokines for cancer

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