From Iusmgenetics

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Current revision as of 02:32, 14 December 2011

1 Disease Profiles
2 Inheritance Patterns
3 Molecular and Biochemical Genetics
4 Unusual Molecular Pathogenesis
5 Mapping of Mendelian Disorders
- 5.1 Mapping Definitions and Instruction
- 5.2 Applications of Mendelian Mapping
6 Genetic Mapping of Complex Disease
7 Gene Therapy
8 Cancer Genetics
9 Personalized Medicine
10 Stem Cells

[edit] Disease Profiles

Disease	Clinical Presentation	Mode of Inheritance	Relevant Gene / Defect	Pathogenesis	Treatment
Achondroplasia	Short limbs, frontal bossing, midface hypoplasia, normal intelligence, normal fertility, delayed motor dev, hydrocephalus	AD, Incomplete Dominance, advanced paternal age, 80% de novo	FGFR3	Constitutive activation decreases bone dev (stat / mapk, SOC)	Screen for hydroceph (shunt), spinal stenosis (fuse), kyphosis (fuse), bowed legs (osteotomy), ear infections (rx). Increase height by GH or sx.
Thanatophoric dysplasia	Perinatal death, severe bone deformation, excess skin folds. (Similar to homozygous achondroplasia.)	?	FGFR3	Excess production of FGFR3.	Did not address
Osteogenesis imperfecta	Types 1-4; Severity: 2 > 3 = 1 > 4; Type 2: perinatal death, dark sclerae; Type 1: brittle bones, tendency for fractures, fractures heal without deformity, deafness, blue sclerae; Type 4: tendency for fractures, normal sclerae.	Autosomal dominant	Pro-alpha1, Pro-alpha2	Normal collagen has 2 alpha1 and 1 alpha 2 chains and trimmed terminals. OI demonstrates dominant negative effect and haploinsufficiency when alpha1 or alpha2 make bad product or no product, respectively.	Did not address
Ehlers Danlos Syndrome	Hyper extensibility, increased skin fragility / thinness, joint laxity, fragility of major arteries, type 4: arteries and colon especially affected	AD, AR, XR; dominant negative	col5a (Type 1), col3a1 (Type 4), plod (Type 6), mnk (Type 9)	type 1 & 4: dominant negative to abnormal collagen (glycine mutations); type 6 & 9: decreased cross-linking (lysyl hydroxylase deficiency, copper binding / lysyl oxidase deficiency)	Did not address
Marfan Syndrome	Pleotropic (ocular, cardiovascular, skeletal): lens subluxation, myopia, detachment, catracts; mitral valve prolapse, aortic dilation; dolichostenomelia, pectus / spinal curvature deformations, narrow palate, joint laxity, arachnodactyly, Walker-Murdoch wrists, Steinberg thumbs	AD; dominant negative effect; 75/25% inherited / de novo	fbn1 (fibrillin-1); EGF-like molecule	Ca++ binding fails in EGF-like domain mutations; TGF-beta binding protein mutations fail to sequester TGF-beta; Up-regulation of TGF-beta causes malformed matrix.	Multidisciplinary management; Ocular: lens correction, screening, sc (cataracts, ectopic lens); CV: echochardiography to monitor valves / aorta, beta-blockers; Counseling: isometric exercise, impact sports, pregnancy.
Homocysteinuria	Pleiomorphic (skeletal, ocular, vascular; like Marfan): long / thing bones, lens dislocation (downward), thromboembolism	AR	Cystathionine beta synthase (CBS), with much locus heterogeneity	Homocysteine is the toxic substance that causes disease; homocysteine may impair disulfide bridges in FBN1 and thus cause Marfan like S&S.	B6 (pyridoxine) supplemenatation (a cofactor for CBS); low methionine diet (meth is the aa most often converted to homocysteine); betaine / folate / b12 supplements to augment the homocysteine -> methionine converstion (to reduce homocysteine levels)
Familial Hypercholesterolemia	Early-onset atherosclerosis, elevated serum cholesterol, elevated LDL, Xanthomas (tendons, skin, eyelids), childhood MIs in homozygotes	AD, AR; semi-dominant; locus heterogeneity	LDL receptor (binds APOB100 on LDL for metabolism), APOB100 (surrounds LDL, binds receptor), ARH adapter protein (binds LDL receptos with APOB100 / LDL into clathrin pits), PCSK9 protease (degrades LDL receptor); LDLR mutations are classified I-V from failure to synthesize to failure to remove from surface	Loss of function: LDL receptor, APOB100, ARH adapter protein; Gain of function: PCSK9 protease.	Deplete bile: bile acid binding resins allow bile (with cholesterol in it) to be passed; Inhibit HMG-CoA reductase: statins inhibit HMG-CoA reductase so it doesn't make cholesterol out of acetyl CoA (hepatocytes).
Cystic Fibrosis	Pleiomorphic: respiratory (cough, infection, bronchiectasis), pancreas (deficient enzyme secretion, fibrosis), endocrine (diabetes mellitus), GI (meconium ileum, failure to thrive, jaundice, cirrhosis, steatorrhea), reproductive (males lack vas deferens (congenital bilateral absence of vas deferens), females can be infertile, too), etc (clubbing, sweat chloride elevated)	AR	CFTR, deltaF508; also ORCC and ENaC; genotype strongly predicts pancreatic phenotype but poorly predicts pulmonary phenotype (and all other phenotypes); loss of exon 9 occurs when only 5 thiamines are found in intron 8; R117H is a mild form	Mutants fail to move chloride and thus to move water; mutants are classified 1-4 based on type of CFTR failure (synthesis, processing, regulation, function)	Aminoglycosides (inhibit p. aeruginosa, allow read through; gentamicin, ataluren, ptc124); antimicrobials; anti-inflammatory tx; mechanical clearing of airway; CFTR modulators: chaperones / correctors / PBA to increase CFTR fxn
Hemochromatosis	Vague vignette: lethargy, abd pain, hepatosplenomegaly, bronzing, diabetes, hypogonadism, loss of libido, amenorrhea, loss of body hair	Obfuscated: AR?, variable expressivity, incomplete penetrance; yet even homozygotes may have no phenotype	HFE, C282Y (especially common in caucasians, founder effect?); also TFR2, HAMP, and HJV	HFE mutants inhibit hepcidin which is supposed to inhibit Fe release from enterocytes; hence excessive absorption of Fe	Phlebotomy
Alpha-1-anti-trypsin Deficiency	ZZ: full disease state (early emphysema, pulmonary fibrosis, liver cirrhosis); MM, MS, and SS: no disease	AR	AAT; M: wild-type, S:50-60% activity, Z:10-15% activity; Null: 0% activity	Defective AAT (a serine protease inhibitor) doesn't inhibit elastase (from neutrophils); chronic destruction of ECM in lungs and liver	Enzyme inhibitor injections (like AAT); liver transplant (ZZ / nulls)
Phenylketonuria (PKU)	Pleomorphic: Neuro (dev delay, microcephaly, "severe" mental retardation, seizures, autistic-like behavior), Integumentary (pale skin, eczema, mousy odor (phenylacetate), maternity issues (mid-facial hypoplasia, growth deficiency, heart defects; affects the fetus regardless of its genotype)	AR, allelic heterogeneity (pts usually compound heterozygotes)	pah (phenylalanine hydroxylase, converts phenylalanine to tyrosine); dihydropteridine	Phe not converted to Tyr (<5% activity), toxicity of Phe; malignant PKU results from deficiency of biopterin recycling (mutant dihydropteridine)	Low Phe diet (250-500 mg); malignant PKU will still develop neuro issues (because biopterine / BH4 is cofactor for making dopamine / cats / serotonini); Sapropterine (kuvan) as BH4 supplementation
Tay-Sachs Disease	Ocular (cherry red macula), Neuro (hyperacusis, lose CNS function after 18 months, spaciticy, swallowing, seizures, hypotonia, demntia, paraylsis, vegetative and dead by age 5)	AR, allelic heterogeneity, homozygotes don't live to reproduction	hexa / hexb (hexB = Sandhoff disease), hexosaminidase A / B degrades GM2 sphingolipid in neurons	GM2 gangliosides (cell signaling glycosphingolipids) not broken down in lysosome, GM2 accumulation -> Neuron Cell body distension -> Neuronal cell death -> Eventual brain atrophy	Anti-seizure drugs, gene therapy?, stem cell transplant?, enzyme replacement therapy doesn't work b/c of BBB; test high school Ashkenazis for carrier status; screen in vitro embryos
Galactosemia	GALT deficiency (vomiting, f2t, liver disease--hepatomegally / jaundice, cataracts, edema, sepsis, encephalopathy, seizures, brain damage), Epimerase deficiency (similar to GALT deficiency), Galactokinase deficiency (cataracts, no liver / neuro involvement); IQ decreases with age; female gonadal dysfunction	AR, locus heterogeneity (GALT, GALK, GALE)	GALT (galactose 1-p uridyltransferase), galactokinase, epimerase; each required for galactose metabolism to glucose (UDP-glucose)	Accumulation of Gal-1-P and Galactitol and depletion of UDP lead to liver toxicity and cataracts, respectively.	Galactose-free diet (very challenging)
Maple Syrup Urine Disease	Classic (lethargy, weight loss, encephalopathy, acidosis, hyper-ammoniemia, sweet smelling urine; <2% BCKD activity), Intermediate (similar to classic; 2-30% BCKD activity), Intermittent (symptoms only upon comorbidity)	AR	BCKD (branched chain ketoacid dehydrogenase)	Branched chain amino acids and alpha keto acids accumulate leading to neurologic disorders and development disruption.	Low-leucine diet (isoleucine and valine, too; requires special medical food and measured amounts of natrual foods), liver transplant, thiamine (in E2 protein mutation pts)
Biotinidase Deficiency	Pleiomorphic: Derm (alopecia, perioral rash, conjuctivitis), Neuro (psychomotor retardation, ataxia, seizures, deafness, blindness), Metabolic (hypoglycemia, hyperammonemia, acidosis)	AR	Biotin (a cofactor, not a gene	Inability to release biotin for recycling	Biotin supplementation (cannot reverse hearing loss, visual abnormalities, dev delay)
Rett Syndrome	Progressive disease with 4 stages; disability in mobility, speech, repetitive movements, breathing difficulties, "severe" cognitive impairment; first signs (6-18 months): head growth deceleration, loss of words / hand skills	XD; sex dependent phenotype (affects mainly girls: some in utero lethality in males, fathers more likely to pass mutant); heterogenity and variable expressivity	MeCP2 (methyl CpG binding protein 2), binds / methylates dinucleotides, recruits factors, prevents HDAC, prevents transcription	MeCP2 fails to bind / inhibit gene expression, allows dedifferentiation, dysregulates brain dev	Symptomatic only; naticonvulsants, occupational therapy, hydrotherapy (scoliosis), IGF-1
Lesch-Nyhan Syndrome	Irritable, poor head support, impaired motor dev (spacticity, dystonia, hypotonia), uric acid overproduction (gout, gritty / orange urine, soft tissue sweeling in the olecranon bursa), self-mutilation	XR	HPRT (hypoxanthine-guanine phophoribosyltransferase) salvages purines (from hypoxanthine / guanine)	HPRT defect results in uric acid overproduction and in striatum deficiency (60-80% loss of dopamine)	Decrease uric acid (hydration; allopurinol inhibits xanthine oxidase from converting hypoxanthine and guanine to uric acid), motor impairment (assistive devices, benzodiazepines, baclofen), behavior modifying drugs
Duchenne Muscular Dystrophy	Gower sign, muscle wasting, hypertrophic gastrocs, walks on toes, inability to run, progressive muscle weakness, elevated serum creatine kinase (can ID female carriers), early death (14-20 yo) via respiratory failure / cardiomyopathy	XR	Dystrophin, binds actin to the sarcolemma membrane	Deletion (usually) of dystrophin means sarco-cytoskeleton is not connected to the membrane; contraction is futile	Did not address
Becker Muscular Dystrophy	Less severe than DMD, later onset (16yo); carriers may have skeletal / cardiac abnormalities	XR	Dystrophin, connects sarco-cytoskeleton to sarcolemma	Frameshifts or small deletions result in "reduced function"	Did not address
X-linked Dilated Cardiomyopathy	No dystrophin expression in cardiac muscle	XR	Dystrophin, ...	5' promotor mutations
Congenital Muscular Dystrophy	CNS involvement	AR	Merosin / Laminin, parts of the complex that connects the sarcolemma to ECM	Did not address	Did not address
Oculopharyngeal Muscular Dystrophy	Ptosis, Dysphagia, onset over 50	AD or AR	Polyadenylation binding protein 2	Did not address	Did not address
Myotonic Dystrophy	Progressive muscle weakness / wasting (begins in face, generalizes), myotonia (cannot relax after contraction; CIC-1 chloride channel defect), cataracts, cardiac conduction defects, insulin resistance, gonadal failure	AD; anticipation; excessive maternal expansion	dmpk (dystrophia myotonica protein kinase); znf9 (dm2; same mechanism as dmpk expansion)	CTG repeats accumulate in 3' UTR (protein sequence is normal), a disease of RNA accumulation (results in aberrant splicing of CIC-1, troponin, and insulin receptor: hyperexcitability of skeletal muscle, cardiac conduction defects, insulin resistance; trans-dominant effect)	Did not address
Fragile X syndrome	Mental retardation (males: moderate, females: mild), behavioral problems (hyperactivity, tantrums, autistic features), physical signs (large head, prominent ears / jaw / forehead, macroorchidism), premutation (ovarian failure, early menopause, FXTAX (fragile X associated tremor / ataxia syndrome), mild cognitive / behavioral deficits)	Unusual: some obligate carrier males have normal phenotype (normal transmitting males); usually of maternal source (fathers usually do not pass on large repeats; similar to myotonic dystrophy)	fmr1, a transporter of mRNA	CGG repeat in the 5' UTR of exon 1 of fmr1 cause decreased function and therefore decreased transport of mRNA; normal / premutation / full mutation: <50 / 50-200 / >200; promotor unmethylated / promotor unmethylated but protein unstable / methylated promotor, no transcription	Did not address
Huntington Disease	Motor (chorea converting to rigidity), Cognitive (all aspects, language later in disease), Behavioral (aggression, apathy, hypersexuality), Psychiatric (personality changes, affective psychosis, schizophrenia)	AD; 97% inherited; anticipation	Huntingtin, unknown function	Glutamine tract leads to nuclear / cytoplasmic huntingtin accumulation (a gain of negative function as with myotonic dystrophy); normal (10-26 repeats) / premutation (27-35) / variable penetrance (35-42+) / adult onset (40-55) / juvenile onset (60+);	Did not address
Polycystic Kidney Disease	Kidney cysts, hematuria, adenomas, hypertension, kidney stones, systemic signs (heart, liver, pancreas, cerebral vasculature)	AD;	pkd1, pkd2 (allelic heterogeneity); pkd1 (85% of cases) has high mutation rate; pkd2 is a Ca++ channel and pkd1 is part of the same Ca++ channel complex	Dysregulation of Ca++ transport via cilia-mediated activation leads to improper movement of Ca++ and H20, forming cysts.	Did not address
Neruofromatosis	Pfleiotropy:	AD, full penetrance, variable expressivity; 50% de novo, 80% paternally derived (but no paternal age variation)	NF1, a high-mutation rate tumor suppressor gene that regulates Ras; much allelic heterogeneity	Mutation inhibits NF1 as tumor suppressor	Did not address
Hemophilia A	External bleeding episodes: spontaneous bleeding, prolonged bleeding from normal procedures (circumcision, heel prick, bleeding); hemorrhage at joints, muscles, GI tract, brain;	XR; an example of unusual crossing over (40-50% of cases)	FVIII, makes clotting factor VIII	Inverted repeats allow for rearrangement (22->...->1->23->...->26), usually during male meiosis; functionality > 25%: norm, ~5%: disease, <1%: severe disease (spontaneous bleeding, joint bleeds)	Did not address
Charcot-Marie-Tooth Disease	Slowly progressinve distal wasting and weakness (legs, then arms; difficulting walking, loss of reflexes, loss of sensation)	AD, AR	pmp22, structural protein of PNS myelin	pmp22 duplication by flanking repeats; poorly formed pmp22 cannot be degraded, accumulation leads to cellular loss and PNS demyelination (altered nerve conduction velocities, onion bulb formation)	Did not address
HNPCC = Lynch Syndrome	Elevated risk of colorectal cancer (and other GI cancers), endometrial cancer, and ovarian cancer; usually proximal CRC (FAP is usually distal)	AD	hMLH 1/2/6, hPMS 1/2; DNA mismatch repair genes	Defective mismatch repair and microsatellite instability	Did not address

[edit] Inheritance Patterns

Auto-Dom	Auto-Rec	Other (Not X)
Oculopharyngeal Muscular Dystrophy	Oculopharyngeal Muscular Dystrophy
Ehlers Danlos	Ehlers Danlos
Familial Hypercholesterolemia	Familial Hypercholesterolemia
Charcot-Marie-Tooth	Charcot-Marie-Tooth
Acondroplasia	Homocysteinuria	Hemochromatosis
Osteogenesis Imperfecta	Cystic Fibrosis
Marfan Syndrome	A1 anti-trypsin Deficiency
Myotonic Dystrophy	Phenylketonuria
Huntington Disease	Tay-Sachs Disease
PCKD	Galactosemia
NF	Maple Syrup Urine Disease
HNPCC	Biotinidase Deficiency
	Congenital Muscular Dystrophy

Note that all the amino acid / metabolics are autosomal recessive.
The structure proteins are AD: OI, Marfan, Achondro, Myotonic Dystrophy (or X-Rec: DMD, BMD, X Dilated Cardiomyopathy).
- Ehler-Danlos is the odd one--it is all three: AD, AR, XR!

X-Dom	X-Rec
Rett Syndrome	Ehlers Danlos
Fragile X Syndrome (unusual)	Lesch-Nyhan Syndrome
	Hemophilia A
	Duchenne Muscular Dystrophy
	Becker Muscular Dystrophy
	X-linked Dilated Cardiomyopathy

[edit] Molecular and Biochemical Genetics

[edit] Objectives

Important terms:
- "Incomplete" dominance or "semi-dominant": homozygous individuals have a worse manifestation than heterozygous individuals (achondroplasia).
- "Distinct disorder": consistent clinical and radiological findings.

[edit] Dominant Diseases

Dominant disease are defined as those manifested when only one allele is mutated.
- Recall that some diseases can be both dominant and negative because of allelic heterogeneity and locus heterogeneity.

[edit] Recessive Diseases

Requires two mutant alleles to show the phenotype or disease state.

[edit] Newborn Screening

The following criteria for newborn screening assure that our screening has analytic validity, clinical validity, and clinical utility:
- The disorder must be well defined.
- The disorder must be fairly high in population frequency (to justify the cost of newborn screening by the cost saved in care for the true-positives).
- The disorder must be poorly clinically detected early in life (assymptomatic) (otherwise it is more cost effective to let physicians identify the disease at newborn checkups).
- The disorder must be significant in morbidity / mortality if left untreated (otherwise we might start treating things that have little consequence).
- The disorder must be treatable such that there is an improved condition (lest we start adding anxiety to insult).
- The test must be rapid, inexpensive, specific AND sensitive over an entire population.
- The test must be acceptable and cost-effective.
- The test must be appropriately administered.

In Indiana we screen for:
- Metabolic disorders:
- Endocrine disorders:
  - Hypothyroidism
  - Congenital Adrenal Hyperplasia
- Hemoglobinopathies:
  - Sickle Cell Disease
- Cystic Fibrosis
Using several methods: enzyme assays, radiommunoassays, electrophoresis, tandem mass spectrometry

[edit] General Molecular / Biochemical Pathogenesis Principles

Dominant diseases usually result from:
- Gain of abnormal function
- Haploinsufficiency
- Dominant-negative effects (think multi-subunit proteins)
Recall that dominant disease can be "incomplete" and thus have more severe phenotypes when presented as homozygous conditions.
Recessive diseases usually result from:
- Loss of normal function
Heterozygotes of recessive diseases (carriers) usually have enough wild-type gene product to function properly.

[edit] Unusual Molecular Pathogenesis

[edit] Objectives

Know the concepts:
- Two hit phenomenon
- Alterened gene structure / dose from unusual crossover
Know the standard profile of each disease

[edit] Unstable repeat expansion

Unstable repeat expansion refers to the phenomenon of nucleotide repeats accumulating in a certain region of the genome and causing disease.
- can be at any location in the genome: non-coding regions, regulatory regions, introns, or exons.
  - When repeats result in coding regions, they are called poly amino acid tracts.
  - It is estimated that 20% of human proteins contain a poly amino acid tract.
- usually triplets.
- repeats are added with each mitosis / meiosis.
- NB: instability can also result in decreases in repeats.
- even the germline cells have instability
  - leads to anticipation: earlier onset of disease with each generation.
The number of repeats usually correlates with severity of disease.
interrupted repeats are less likely to expand
- like in Fragile X syndrome, CGGs are interrupted by AGGs

Poly amino acid tracts tend to manifest as neurologic disorders.
Some poly amino acid tracts demonstrate anticipation.
Poly amino acids tracts include:
- Huntington disease
- Spinocerebellar ataxia
- Spinobulbar muscle atrophy
- Macado-Joseph disease
- Dentorubropallido dysplasia

[edit] Myotonic Dystrophy

[edit] Fragile-X Syndrome

[edit] Huntington Disease

[edit] Two Hit Phenomenon

The concept of the two hit phenomenon is the idea that if a pt inherits one mutant allele, it only takes one "hit" at that locus to cause dieases; whereas if a pt has not inherited a mutant allele, two hits are required at the locus (which is highly unlikely) to cause disease.
- The second hit can result from the mutation of the wild-type allele or the deletion of the wild-type allele.
Loss of heterozygosity is related to the two hit phenomenon; LOH is the case when a pt has the same alleles at both copies of a loci.
- LOH can result from uniparental disomy, abnormal crossover events, etc.

[edit] Polycystic Kidney Disease

[edit] Neurofibromatosis

[edit] Hemophilia A

[edit] Gene duplication

Gene duplication can lead to disease.
Gene duplication may be facilitated by homologous sequences and / or repeats flanking a loci causing misalignment during synthesis and an attempt at "repair" generating a new copy.

[edit] Charcot-Marie-Tooth Disease

[edit] Miscellaneous

Familial hypercholesterolemia is a product of LDL receptor issues.
Alpha globin deletions lead to alpha thalassemias.

[edit] Mapping of Mendelian Disorders

[edit] Mapping Definitions and Instruction

Recombination Fraction = theta = centimorgans; from 0-50
- 0 means the loci are so close together they are always inherited together.
- 50 means the loci are on different ch or are so far apart (on the same ch) that they segregate independently (no bias, no linkage).
The goals are to:
- Understand when (in a pedigree) recombination took place.
- Understand the proband's phase, not just genotype.
Knowing this
To follow disease alleles, we often use marker loci / alleles to understand inheritance.
- We choose marker loci that are close to the disease loci.
Phase: determining (among two loci and the four associated alleles) which two alleles (from two different loci) are found on each of the two chromosomes).
- Phase (along with recombination fraction, AKA linkage) helps us understand how inheritance is likely to occur as it tells us which alleles share the same chromosome.
- Phase diagrams are a drawing that describes the two chromosomes and the distribution of the four alleles.
- Phase diagrams do not distinguish which ch was maternally derived and which was paternally derived.
Once the phase has been determined for 3 generations, we can determine which offspring genotypes resulted from recombination.
When the phase of the first (of three) generations isn't known, deduce the possible phases and find out how many recombination events would have to occur to generate the (known) genotypes of the existing third generation.

[edit] Applications of Mendelian Mapping

Map disease genes to a particular chromosome.
Use these methods to then limit disease gene to a smaller chromosomal region.
Before the actual gene has been identified, linkage can be used in appropriate families to offer prenatal or presymptomatic testing.
For some disease with a wide variety of mutations, linkage analysis may still be necessary, even after the gene has been identified. In these cases, intragenic (within the gene) markers may be used in the linkage analysis (Duchenne muscular dystrophy)

[edit] Genetic Mapping of Complex Disease

[edit] Complex Disease

Rarely can we strongly predict manifestation in an individual

Examples of Complex Disease:
- Atherosclerosis
- Multiple Sclerosis
- Diabetes mellitus
- Alzheimer Disease
- Epilepsy
- Alcoholism
- Dementia
- Oral Clefts
- Schizophrenia
- Inflammatory Bowel Disease
- Neural Tube Defects
- Skeletal Disorders
- Hypertension
- Lung Cancer
- Obesity
- Parkinson Disease
- Breast Cancer
- Bipolar (manic depressive)
- Depression

[edit] Is it Genetic?

How do we quantify the genetic-ness of a disease?
- Twin Studies
- Familial Aggregation
- Mendelian Form of Disease
- Animal Models

[edit] Twin Studies

Compare concordance rates of MZ and DZ twins

[edit] Familial Aggregation

Familial aggregation is used when we can't get enough twin info.
Increased risk for disease among family members of an affected individual
Compare frequency of disease among first degree relatives of affected individuals with the frequency of the disease in the general population.
Bipolar, as an example, has a RR (relative risk) of 4 or 5.
- This is about as genetic as "complex disorders" get.
- By comparison, Huntington disease (a single-gene disorder) has a RR of 10,000.

[edit] Genetics of Complex Disease

Polygenic: determined by multiple genes
- No environmental factors known.
Multifactorial: determined by both genes and environmental factors
As an example, height is both polygenic and multifactorial:

[edit] Multifactorial Liability Threshold Model

Know that the point of this material is to be able to answer questions about an individuals increase or decrease in RR based on genetic and environmental information.

Multifactorial liability threshold model is a theoretical model designed to provide a means to explain gene interactions as well as possible environmental interaction with disease susceptibility

Genetic liability: an individual's inherent (genetic) risk for a disease.
- High genetic liability means you are (inhrently) closer to the threshold.
All multifactorial diseases we discus are binomial in nature: you either have it or you don't.
The threshold is the cutoff for the binomial state (having the disease or not).

How many genes must be involved for a disease to manifest as multifactorial (binomial with a bell curve)?
- Only two, really.
- Even with only two genes segregating independently, we see bell curve distributions in genotype.

Hypothesize that only those individuals who have inherited a sufficient number of susceptibility alleles at various genes will develop disease.
Postulate that milder forms of the disease might be due to smaller number of susceptibility alleles.

[edit] Risk Factor Concepts

There are four types of risk factors: sex, degree of relationship (to relation with disease state), severity of defect, number of individuals in pedigree.
These risks represent the type of information one would collect from patients to assess their risk for a given complex disease.

[edit] Risk factor 1: Sex differences

Some disorders have differing frequencies among the genders
- individuals of the more rarely affected sex would need more susceptibility alleles to manifest disease
- individuals of the more commonly affected sex would need fewer susceptibility alleles to manifest disease

For example:
- In a model, females are less often affected
- Consider relatives of an affected male, the liability is shifted to the right, therefore increased risk of both affected male and female relatives
  - The liability for the whole family is shifted to the right; they are no longer in the normal population (because of their affected member).
- Consider relatives of an affected female, the liability is shifted even further to the right, therefore even more increased risk of both affected males and female relatives
- More area (of the curve) above the threshold (which does not move) indicates more people of the shifted population will be affected (both male and female, though one gender may have a higher threshold and therefore a lower percentage of infected members).

[edit] Risk Factor 2: Degree of Relationship

First Degree: Share 1/2 of genes
- parents, children, siblings
- one line connects first degree relatives in a pedigree
Second Degree: Share 1/4 of genes
- uncles / aunts, nieces / nephews, g’parent, g’child, half-siblings
- two lines connect second degree relatives in a pedigree
Third Degree: Share 1/8 of genes
- 1st cousins, g-g’parent, g-g’child, half-uncle / aunts, half-nephew / nieces
- three lines connect third degree relatives in a pedigree
1/2^n

Risk drops for 2nd degree relatives
- It is expected that more closely related individuals would share more alleles in common than more distantly related relatives
- Individuals more closely related to an affected individual are at higher risk of also being affected
- The relative risk to a proband drops quickly as the affected relative moves from first degree to second, and on to third degree.

Recall that on disease distribution diagrams, the threshold does not change but the curve is shifted from the general population position to represent the proband's population.
When a first degree relative is affected, the proband's risk curve is shifted to the right.
- A second or third degree affected relative would shift the curve to the right, too, but less to the right.
Use this concept of shifting the curve to the right or left to consider risk in an individual.

[edit] Risk factor 3: severity of the defect

Recurrence risk increases if the trait is more severe
- More severe phenotype requires more susceptibility alleles
- Greater risk for any spectrum of the phenotype (mild to severe)
That is, we assume that if we observe a severe phenotype there is more susceptibility related genetic change underlying the phenotype.
- Therefore, a more severe phenotype means an increased risk (as compared to another, less severe phenotype).

Cleft Lip and Palate:
- 60-80% of affected are male
- Different causes:
  - isolated single gene (Mendelian inheritance)
  - part of a syndrome
  - teratogen
  - several genes/environment (Multifactorial)
After these factors are ruled out, clefts are a function of many factors, including race.

Different thresholds for different levels of disease severity
- Consider relatives of a less severely affected individual, the liability is shifted to the right, therefore increased risk of both males and female relatives to be affected with any form of disease (mild to severe).
- Consider relatives of a more severely affected individual, the liability is shifted even further to the right, therefore even more increased risk of both males and female relatives to be affected with any form of disease (mild to severe)
Shift the proband's population risk farther to the right with increased severity of defect in affected relative.

[edit] Risk Factor 4: Number of Affected Individuals

If there is more than 1 affected individual in the pedigree:
- recurrence risk increases
- suggests that the family might have even more susceptibility alleles segregating
Be sure to also consider whether the family might have a single gene disorder
- any other dysmorphism suggesting a syndrome?

[edit] Incidence vs. Prevalence

Incidence: Proportion of individuals who have a disorder at birth.
- That is: the proportion of the general population who get the disease.
Prevalence: Number of individuals with a disorder in a given population at a particular time.
- That is, the number of individuals of a particular population at a particular time.

It is important to know incidence and prevalence of a disease because our default risk in predicting recurrence risk in a pt with an affected relative is the square root of the population incidence.

[edit] Counseling in Multifactorial Traits

Recurrence risk for 1st degree relatives of an affected individual is square root of population incidence
Example: Ventricular septal defect:
- population incidence = 1/575
- square root of 575 = ~24
- recurrence risk for 1st degree relatives = 1/24
- convert percentages to decimals or fractions!
  - Don't take the square root of the percent! (one way to represent incidence)
  - If population incidence is 2%, convert it to 2/100 = 1/50 and then take the square root of 50 such that the default relative risk is 1/7.

[edit] New types of genetic data

Genome-wide SNP markers:
- ~1 million SNPs to tag all common variants in an individual’s genome (~400 USD)
Sequence data (whole-exome or -genome)
- Identify all 60,000-100,000 rare and common variants in an individual’s genome
- Rapidly becoming feasible (“$1,000 genome”)
- Sequencing error rates low with current technology, but large numbers of false variants remain in any one person’s sequence

[edit] (Genetically) Personalized Medicine

Challenge is interpretation
We expect to identify both:
- Rare variants that explain disease in a particular person / family
  - But it is hard to know which of these 90K are causing the disease.
- Variants that increase susceptibility to disease across an ethnic group or larger population
  - But it is hard to know which have real consequence in terms of treatment and correction.
How to translate this to prognosis, lifestyle modification recommendations, etc. in any one individual??

[edit] “Missing heritability”

GWAS studies typically detect common variants explaining <20% (combined) of total variation
- GWAS is capable of associating SNPs with variability but it doesn't always explain inheritiablity of the complex disease.
The rest of the difference (non SNP difference) is probably explained by:
- Gene-gene interactions (epistasis)
- Gene-environment interactions
Only a very small proportion of variation in trait or disease risk is explained by combined effects of common SNPs.

[edit] Gene Therapy

Integration is a rare event, perhaps 1 in 10K on the best cell line; so not very efficient.
How do we make this more efficient?

[edit] Hx of Gene Therapy

So we try to use viruses which are really good at integrating their DNA.
We remove the gag, pol, and env genes which make it infectious and put in our own genes of interest.
The trick now is to get our material into the virus:
- use packaging cell lines which are immortalized
- gag is expressed; capsid protein for virus
- pol is expressed; integrase gene
- put in the vector material (often rna for rna viruses)
So this cell is infected with a virus and is then a little factor that makes virus particles that are set up to infect our target cells.
They carry capsid for update, RT for converting rna to DNA and integrase for putting the DNA in the target cell.
These then infect the target cell.
Then when the target cell divides, all the daughter cell will have the delivered DNA, too.

So we tried this and even got it to trial.

[edit] New Viruses

But we wanted to try other viruses.
Retrovirus / Lentivirus:
- Murine retrovirus was the first tried.
  - Turns out murine virus can cause leukemia because of the enhancer region on the virus.
HIV is a lentivirus that doesn't have the enhancer region of murine retrovius so it will decrease the chance of leukemia.
- Used in bone marrow and T cells
- Used here because T cells and bone marrow have many, many offspring so the treatment must involve integration into the genome.

Adenovirus:
- Gives flu-like symptoms and that's it so you can give a high titer without much side effect.
- Can integrate without regard to the cell cycle of the target cell
- However, it doesn't integrate so it isn't so useful for tx with longevity
- Applications are cancer and immunization because they are short-term objectives that don't need integration.

Adeno-associated virus (AAV)
- Challenge is that it is a very small virus, so not much material fits in the virus
- While AAV doesn't integrate, you can get it into slow-dividing cells like muscle , liver, and brain
- We have many different serotypes that can be used to target specific tissues.

Herpes
- Huge virus so you can put tons of stuff in there
- Non integrating
- First application was for cancer pain syndrome: inject in skin, neruons will pick it up (because that's what herpes likes to infect) and then

express some anti-pain proteins

- Now being used for CNS tumor destruction.

The concept here is that there won't be one vector but many depending on the target cell and how long you want to express your material.

[edit] Gene Therapy and SCID

The first gene therapy that was approved was for the treatment of adenosine deaminase deficiency or bubble baby deficiency.
When T cells leave BM to visit thymus for education, there is rapid turnover of the T cells as they are educated to antigens.
Without ADA to breakdown the biproducts of cell apoptosis, the T cells die without making it out of the thymus.
Pts have very low T cells and without T cells you can't educate B cells so they have combined immune deficiency.
Usually die of infection in the first year.
Why was ADA picked for gene therapy?
- Can simply plug in the protein.
- Ex vivo can be done by extracting the pt's bone marrow, exposing to vector, put back in, hope that the protein is expressed.
- Selective advantage because T cells normally die in the thymus (especially in SCID pts) so there are not many cells to compete against.

Wide range of expression is important (in wildtype, non-disease state of target gene product) because it allows us to be generous in the expression we end up providing without causing a different

disease.

Selective advantage is important when you contrast with something like sickle cell diease:
- There must be a selective advantage for the transfected cells because we can only reasonable transfect a small proportion.
- In sickle cell, for example, the cells are only at a disadvantage once they get out into the peripheral blood--before that they are at the same

selectivity as transfected cells.

- Once in the peripheral blood, it's too late--that is the disease state.

This study shows
- 10 pts treated
- 9 pts have normal immune function
- T cell numbers are in the normal range
- Kids have normal immunization reaction
- kids went from being in bubble to going to school and stuff.
This project used lentivirus.

[edit] Gene Therapy Examples

This study:
- used AAV
- Leber's congenital amaurosis
- Causes blindness
- At birth, the children are pretty normal in terms of sight
- Without this gene, by 8 or 9 they are blind
- In this case, used AAV to express gene
- Injected in retina
- Showed improvement in sight
- Showed little disruption in the retina
- Legally blind to playing baseball.
- Putting in a good copy.

This study:
- Used lentivirus
- Treating adrenoleukodystrophy
- Major affect over time is deterioration of the brain
- Lack of enzyme that helps maintain myelin, thus bad demyelination
- Treated not the brain but treated the bone marrow because that's where glial cells come from
- Glial cells eventually ended up in the brain and helped.
- Much slower progression, perhaps arrested.
Lorenzo's oil was hogwash.

We can also think about this technology as drug delivery, too.

[edit] Hemophilia and Gene Therapy

Hemophilia:
- Very expensive to treat
- Disruptive to the pt's life to have to give themselves factor IX ir IV
Inject intravenously, let it get taken up by the liver, let it integrate, let the liver then make the clotting protein like it should.
When we did this, we saw that factor IX expression went up after being taken up by the liver
But then it went down after 4-6 weeks.
This happened at the same time that the liver function tests went high and back to normal.
So we wondered if this was an immune thing.
Perhaps the immune response is due to the transgene.
Two animal models are the evidence:
- One model: knockout mouse, treated with gene therapy, showed immune response on the transgenetic product
- Two model: non-fxnal factor IX mouse, treated with gene therapy, no immune response (because even though the factor wasn't functional it

was still present during immune cell training to present as an epitope and therefore generate central tolerance that leads to no immune reaction upon gene therapy correction)

So this is what we thought was going on.
Turns out it was that the vector (AAV) was leaving it's capsid protein around for weeks / months and upon detection the transfected cells were killed off one by one.
- Hence the reduction in factor IX expression
- Hence the hepatitis-like elevation of liver fxn tests.

Because of this reaction, they tried the eye because they knew it was an immune priviledged site.
The brain is protected, too, so they are trying to work on Parkinsons.
They are also thinking aobut tx profilactically with predisone or some immune supporessor.

[edit] Huntington and Gene Therapy

This work is on a mouse model of HD.
Here they put the mice on a rotorod and they look at how quickly the mice fall off.
- Faster fall off times in those with poorer neurological function.
Putting in the lentivirus causes delayed onset of the dieases.
They put in glial-cell derived neurotrophic factor
- A biologic factor that is endogenous
- This is truly drug therapy
- This is good because giving it systemically causes lots of bad systemic side effects.
So we aren't replacing HD gene but we delay the onset by making the microenvironment more suitable to handle the disease.

[edit] Gene Therapy as a mechanism of inhibition

We can inactive harmful genetic defects.
Triplex: we can try to make stable oligonucleotides that are not degraded that sit between the two strands of DNA at the site of the defunct gene such that when polymerase comes along it gets stuck and won't transcribe.
- Not to the clinic yet.
Antisense:
- Short, complimentary sequence to the defunction mRNA sequence
- This ends up making dsmRNA which is natveily rapidly degraded.
- In clinical trials.
Ribozymes
- short squences of RNA that you can make to compliment your target mRNA
- They pair and are actually enzymatic so they directly cut up the target mRNA.
- This is in clinical trials
RNAi
- can cause degredation at the RNA level or potentially even at the translational level
- In clinical trials

[edit] RNAi

Treating HIV
Usually BM stem cells are not infected with HIV.
So let's take out the bone marrow, get the stem cells, introduce a lentivirus with a RNA that will get turned into DNA that will get integrated, that will get expressed as RNA(i) which can interfere with the HIV's RNA products.
The lentivirus also has ribozymes in it to directly cut up the HIV RNA (before it has even integrated).

And we're thinking about using this on HepC, too.

[edit] miRNA

We realize that lots of gene regulation is via miRNAs.
- Probably lots of the "filler genome" is very important.
- Propably processed through RISC and DICER.
Basically, miRNA bind to RNA and cause cleavage or cause translation repression by preventing the ribosome from functioning.

[edit] Cell Engineering

How might you want to engineer a cell?
One idea is in chemotherapy.
- Chemo has lots of side effects, especially in BM.
*BM is usually the limiting factor as chemo will kill it to the pt of having bad infections and bleeding.
So, we'd like to make the bm resistant to BCNU therapy.
- BCNU is a methyl donor, it donates the methyl to guanine.
- After 18 hours, BCNU and guanine are irreversibly bound across cytosines.
- And that's how it kills tumor cells because upon division, DNA synthesis fails.
We know of a protein that can demethylate (MGMT); a methyl acceptor.
- It is a one-time use protein (and therefore not an enzyme).
- It is one of our endogenous DNA repair mechanisms.
So normally we give BCNU to the highest dose we can without losing the pt to bleeding or infection.
Now if we take out the BM cells and introduce the MGMT gene before we treat with BCNU, then the bone marrow cells will be more resilient to the chemotherapy and we will have better outcomes.

This can also be used as a selective advantage marker for other gt tx.
- For example, if we replace the HgA gene in thalassemia, we still need a way to give that 5% of BM stem cells a selective advantage.
- If we give them MGMT, also, then we can give chemo on top and select for the BM stem cells with both MGMT and the fixed HbA gene.

[edit] GT and suicide genes

This idea is the convers: suicide genes.
Rather than protecting the cell, we might want to kill the cells.
We can target bad cells and give them suicide genes.
One way is to use thymidine kinase
- Herpes virus has thymidine kinase.
- TK will help phosphorylate some compounds (like gancyclovir, etc.).
- These products only get phosed in infected cells because only infected cells have thymidine kinase.
  - It kill by integrating into DNA synthesis and screwing stuff up.
So we can put thymidine kinase in our vectors so that if the cells we've engineered start screwing up, we can kill them with ganciclovir, etc.

Trial here at IU:
- In pts with BM transplant we often give T cells from their donor to help with graft-verus-tumor.
- Problem is, at the doses at whcih one has to give T cells it can also cause GVHD which is often fatal.
So we put into the T cells TK we can turn them off if they start GVHD.
Used in the clinic.

[edit] GT, Engineering, and Melanoma

Melanoma
- There are many T cells in the tumor but they aren't doing anything.
- When he grew them and gave them back to the pt, they only sometimes did stuff.
- He saw that the T cells were clonal (so they all have the same receptor).
- He saw that the tumor has specific antigens.
- So he cloned the appropriate T cell receptor for the melanoma antigen and ptu it in the vectyor.
- Then he used retroviral to infect the T cells so they have the "right" receptor.
2 of 11 seem to have long-term affect.
Now we are trying with lots of different antigens like CEA and lung stuff.

This is similar to the melanoma slide.
This is against leukemia.
Reprogrammed T cells against a CD19 epitope.
BM decreased in B cell congestion.
Worked so well that there was tumor lysis syndrome.
3/3 had great response.
Pts will probably need Ig administration for life because they probably won't have B cells anymore.

[edit] Take away

Approved trials:
2/3 in cancer
but lots of other stuff, too

[edit] GT and Homologous Recombination

Homologous recombination
There are zinc finger nucleases that are used for DNA repair and sit down nicely on dNA.
Can be designed to sit down on a specific sequence.
Then it can remove a portion of DNA.
Then flood with an engineered template, it will get integrated.
Could be used for something like Sickle Cell where we know we only need to fix a single codon.
This is attractive because viral integration can't be targeted to a certain integration site.

[edit] GT and Germ Cell Lines

All things we have done are somatic cell repair.
- Must convince FDA when doing trials that there is little to no risk of altering germ line
- People just aren't really to alter the gene pool.

[edit] GT Adverse Events

JG had modest problems, had severe immune reaction and died.
FDA investigated
- Multiple protocol violations
- Press had a hay day
Trial approvals went way down
Congressed pushed for more regulation
Also, a volunteer study for an asthma drug resulted in several deaths.

[edit] GT Ethical Issues

Raised ethical issues for many types of treatment.
Most GT therapies are in phase 1 which is for toxicity
- We don't think this will help, but we need to find out if it will help.
- Increase dosage until you see harm.
Phase II:
- Safety is the number one criteria
Phase III:

So we said, OK, we'll only try these things on people who have really no hope of getting better.
- The problem is that ethically that doens't seem right; we should treat people who are likely to benefit.
- That's how JG got on the study.
But now we're much more cautious.
- Hard to balance the risk and benefit.

Therapeutic misconception
- Tell the pt over and over that it won't help them.
- Even still, 40% will believe that the study is helping them.
- Ethicists say this happens because of poor informed conscent.

[edit] GT pro / con

What are the pros and cons of GT?
Right to an open future:
- You shouldn't do gt in kids because they should be able to decide for themselves if they want changed.
- But parents will say that they are responsible for taking care of their children.

Sperm, egg, embryo manipulation

Unintended consequences
- Recall that SC has an advantage in some places
- What about other gene "defects"?
- Hypertension

Altered gene pool
- we aren't perfect at insertion and we might get it wrong

[edit] GT: the science fiction part

What about enhancement?
- Taller, smarter, etc.
- Splitting of populations; rich get richer, etc.

Resetting what is normal: small people of america say they aren't broken

Eugenics
- We used to sterilize people who we though weren't smart enough or strong enough or whatever enough to bear children
- If we weed them out we will become a better society, right?

There is a balance, though.
How do we define what is a disease and what is an enhancement
- Study on Rat impotence:
  - Used DNA to treat the impotent mice
  - Some said this was enhancement and not necessary
  - Some said that impotence is a real disease and it is right to treat it.

[edit] GT's future

Bone marrow transplants
- BM transplants took off after we knew more about the immune system
- Now an accepted therapy.
- Took 30-40 years.
We are 20 years into GT
Antibody therapy took is about 10 years ahead of GT.

[edit] Cancer Genetics

[edit] Objectives

Understand that cancer is a genetic disease and a multistep process.
Know the types of genes associated with cancer.
Know fundamentals about cancer pathogenesis, including the concepts of:
- oncogene action / activation;
- modes of disease pathogenesis associated with tumor suppressor genes (e.g., two hit);
- microsatellite instability
- potential role of epigenetics in cancer
Know features of the specific disorders discussed in class.

[edit] Cancer

Cancer is a heterogeneous disease that will claim more than 560,000 lives in our country this year.

Cancer INCIDENCE

Cancer	% of all cancers cases	Gender	Gender	% of all cancer cases	Cancer
Prostate	29%	Male	Female	26%	Breast
Lung / Bronchus	15%	Male	Female	15%	Lung / Bronchus
Colon / Rectum	10%	Male	Female	11%	Colon / Rectum
Bladder	7%	Male	Female	6%	Uterine

Cancer DEATHS

Cancer	% of all cancers cases	Male	Female	% of all cancer cases	Cancer
Lung / Bronchus	31%	Male	Female	26%	Lung / Bronchus
Prostate	9%	Male	Female	15%	Breast
Colon / Rectum	9%	Male	Female	10%	Colon / Rectum
Pancreas	6%	Male	Female	6%	Pancreas

More patients becoming (will be) aware of genetic considerations.
Take at least three generations of family history into account.

[edit] Features suggesting an inherited predisposition to cancer

Factors suggesting an inherited predisposition for cancer include (as in a single gene causing high susceptibility to cancer):
- Two or more close relatives affected.
- Early age of onset.
  - Or earlier onset in each generation
- Cancers of a specific type occurring together (e.g., breast and ovary).
- Multiple or bilateral cancers occurring in one person.
- Rare cancers

Factors suggesting familial clustering of cancer:
- A clear increase in cancer compared to expected, though no clear inheritance pattern
- Moderate age at diagnosis
- Absence of multiple primary cancers (as seen in hereditary predisposition)
- Absence of rare cancers

[edit] Cancer is a genetic disease

In addition to genes, there are other predisposing factors such as:
- Infection (virus)
- Radiation
- Carcinogens
- Immunological defects

There is increasing aneuploidy as the cell accumulates lesions.
Recall that lesions to DNA repair genes can accelerate the rate of lesion accumulation.

[edit] Cell Proliferation and Cell Death Genes

[edit] Oncogenes

To understand oncogenes, first understand that proto-oncogenes are normal genes that have something to do with the cell cycle or proliferation.
- Proto-oncogenes are found in the normal genome and are an important part of cellular function and organism development.
- Proto-oncogenes have many regulations upon them that cause them to function at the right time and place in the organism and in development.
- Examples of proto-oncogenes include:
  - Growth factors / receptors
  - Signal transduction molecules (nuclear proteins)
  - Transcriptional regulators (which affect the cell cycle)

An oncogene, then, is a proto-oncogene that is out of control--acting aberrantly in time or space.
Oncogenes are "dominantly acting" because only one copy of the gene need be turned on to induce it's pro-growth, pro-proliferation effect.

*Oncogenes are "dominant at the cellular level"

Oncogenes have been identified by their ability to convert non-neoplastic cells into neoplastic cells--that is they promote tumors / cancers.

Activation of a proto-oncogene (into an oncogene):
- Activation of a proto-oncogene generally requires a gain of function mutation.
- A gain of function occurs either through a change in protein structure or a change in expression.
- Proteins structure changes can occur in many ways, including point mutations and hybrid proteins:
  - Ras is commonly point mutated to gain function.
  - CML has the t9:22 to generate the function-gained bcr-abl gene.
- Protein expression changes can manifest as a change in expression level or a change in expression location (as in tissue):
  - Viral insertion can cause expression in a new location or a new level.
    - From gene therapy, think about the virus donating a novel promotor that upregulates downstream genes.
  - Gene amplification causes increased expression.
  - Translocation can cause increased or decreased expression depending on the promotor relavant to the translocation.

Common oncogenes include:
- ret, met, and ras: kinases and signaling genes
- fas: pro-apoptotic (in wildtype form)

[edit] Burkitt lymphoma

Burkitt lymphoma is the most common tumor in children of equatorial Africa (but is rare, elsewhere).
Burkitt lymphoma is a B-cell tumor of the jaw.
Burkitt lymphoma is characterized by translocations that convert proto-oncogenes to oncogenes.
myc conversion
- t(8;14) is the primary translocation associated with Burkitt lymphoma and the c-myc gene.
- Converts myc to an oncogene.
- t(8;22) and t(2;8) are also common translocations that convert myc to an oncogene.
Immunoglobulin genes
- Chromosomes 2, 15, and 22 carry genes required for immunoglobulin formation.
- Ch 2: kappa light chain
- Ch 14: heavy chain
- Ch 22: lambda light chain
c-myc is a proto-oncogene
- When translocation occurs, it brings c-myc from 8 to elements important for Ig expression and thus c-myc is upregulated in cells that function to make Ig (B cells).
- Elevated c-myc results in excessive proliferation and thus t(8;22) generates Burkitt lymphoma.

[edit] Inherited mutations in oncogenes

It is rare to inherit a mutation in an oncogene, but it does happen; MEN2 and HPRC are two examples.

MEN2 is associated with an inherited RET mutation
- MEN2 presents as an autosomal dominant cancer predilection syndrome.
  - This makes sense as we previously mentioned the dominant activity of oncogenes (only one has to be mutated to cause cancer).
- Thyroid carcinoma is usually the primary tumor culprit.
- RET is a tyrosine kinase receptor
- Gain-of-function mutations in RET (inherited, remember) lead to constitutive kinase activity.
- NB: loss-of-function mutations in RET lead to Hirschsprung disease

Hereditary papillary renal carcinoma (HPRC):
- HPRC, like MEN2, is inherited in an autosomal dominant fashion.
- HPRC is associated with gain-of-function mutations in MET.
- MET is a tyrosine kinase receptor.
- Gain-of-function mutation in MET lead to constitutive activation of the kinases to which MET is bound (even when the proper ligand isn't present).

[edit] Tumor suppressor genes

Tumor suppressor genes are those that normally serve to discourage growth and replication.
Tumor suppressor gene mutations act in a cellular recessive manner because though one copy of the gene may become defective, the other copy can usually maintain the proper cell cycle, and therefore cellular function is not usually affected by this first hit.
Because tumor suppressor gene mutations act in a recessive mannner, the two hit hypothesis suggests that at least two hits (genetic lesions) are required to "knock-out" the function of a tumor suppressor gene.
- The two hit hypothesis was developed by Knudson.
Loss-of-function lesions on tumor suppressor genes can act dominantly at the organismal level.
- Recall that something of a "dominant" nature always--or close to always--occurs; like brown eyes.
- Recall that a second "hit" that would cause cancer is unlikely.
- However, note that there are many, many cells in the body.
- Therefore, development of cancer always--or nearly always--occurs when all cells of the body start out with a single hit (and therefore, development of a tumor is considered "dominant" at the organism level).
  - This makes sense because even though the odds of a second hit are very low, there are trillions of cells that only need 1 more hit to develop cancer.
Mutation of only one copy of a tumor suppressor gene leads to loss of heterozygosity: the state of having two of the same allele for a gene (or having only one functional copy of the allele and therefore no hetergeneity in gene product).

Common tumor suppressor genes are:
- rb, p53: cell cycle regulators
- msh2, mlh1: DNA repair / inspection regulators
- bcl2, telomerase: anti-apoptotic

[edit] Cancer genes

Most mutations related to cancer occur in somatic cells that are not passed on to off-spring.
However, some mutations do occur in the germ line and can be passed to off-spring.
Inherited mutations can come from the egg, the sperm, or the zygote.
Inherited mutations are usually / often found in the genome of every cell of the body.

Alfred Knudson was the first to describe the phenotypic difference between somatic and inherited mutations in cancer.
Knudson observed that cases of retinoblastoma had distinct characteristics when there was a family history of retinoblastoma:
- tumors occurred earlier in life,
- tumors were more likely to be bilateral, and
- tumors were mutlifocal (have more than one site of origin).
So he reasoned that a family history suggests that the germline contains a "hit" or lesion against the rb gene and that offspring had earlier tumors that were more likely to be bilateral and multifocal because they had only to receive one more "hit" to develop retinoblastoma.
In comparison, with no family history, two independent "hits" had to occur to develop retinoblastoma and therefore the tumors occurred later in life and were less likely to be bilateral or multifocal.

[edit] Retinoblastoma

Rb is the most common eye tumor in early childhood.
Retinoblastoma is a tumor of the retina.
Retinoblastomas may actually begin forming in utero.
Average age of Rb onset is 18 months.
Treatment for retinoblastoma is to remove the entire orbit.

1/23k live births have retinoblastomas.
Retinoblastoma can be inherited as an autosomal dominant trait.
40% of retinoblastomas are inherited.
- Parent may be a carrier or there may have been a germline mutation.
- Bilateral state suggests inheritance.
- 15% of inherited retinoblastomas are unilateral.
60% of retinoblastomas are sporadic.

Retinoblastoma pts have elevated risk for other cancers, too.
- Including osteosarcoma

So, we say that rb is recessive at the cellular level because one loss makes no difference in cellular function but rb is dominant at the organismal level because it nearly always results in tumor development because of the large number of cells of the organims--all of which are at risk to develop cancer because they are starting with a single hit.

[edit] Loss of heterozygosity

Loss of heterozygosity: "loss of a normal allele from a region of one chromosome of a pair, allowing a defective allele on the homologous chromosome to be clinically manifest. A feature of many cases of retinoblastoma, breast cancer, and other tumors due to mutation in a tumor-suppressor gene."
That is, when a tumor suppressor gene is mutated, the organism is then relying on the second copy of the gene to maintain proper function; when a second loss occurs ("loss of heterozygosity") the deficiency is finally manifested since, now, neither copy works sufficiently well to maintain normal cellular function.
LOH occurs when the second, functional allele (that is allowing for proper function) is overwritten by the non-functional copy (perhaps because of complexity of DNA replication or because of repair at the allele that used the other allele as a template).
LOH can result from several mechanisms: somatic recombination, loss / duplication, chromosomal atresia.
- Note that a very small lesion directly at the site of the (second, still functional) allele will not change flanking / intragenic markers and is therefore not considered a loss of heterogeneity
- Linked markers are genetic markers that tend to be inherited with the locus of origin (that is, they are nearby the locus).

LOH (loss of heterozygosity) can also refer to a laboratory analysis technique that identifies the mechanism of the second hit.
LOH is a way to identify the existence of tumor suppressor genes.

[edit] Breast cancer

Breast cancer is common (#1 in incidence for women, #2 in deaths for women).
Lifetime risk of breast cancer is 1/8.
180k new cases each year.

Most breast cancer is sporadic.
5% of breast cancer is considered hereditary
15-20% of breast cancer is considered family-clustered
10% of ovarian cancer is considered hereditary

Susceptibility genes for breast cancer include brca1 and brca2.
Mutations in these genes account for 3-5% of all breast cancers
brca1 and brca2 were cloned in 1994 / 5.
brca1 and brca2 have autosomal dominant effects
brca1 and brca2 are on different chromosomes.

Inheriting a mutated brca1 gene allele increases one's lifetime risk of developing cancer:
- One primary breast cancer risk: 50-85% (compared to 12% population risk)
- Second primary breast cancer risk: 40-60% (compared to 2% population risk)
- Ovarian cancer risk: 15-45%
- Potentially elevated risk for other cancers like prostate and colon, too.

Inheriting a mutated brca2 gene allele increases one's lifetime risk of developing cancer:
- Breast cancer risk (female): 50-85% (same as brca1 mutation risk)
- Breast cancer risk (male): 6% (no increased risk with brca1 mutation)
- Ovarian cancer risk: 10-20% (compared to 15-45% with brca1 mutation)
- 'Increased risk of prostate, laryngeal, and pancreatic cancers, too.
  - Magnitude unknown.

45-60% of brca1 or brca2 mutant carriers develop breast cancer by the age of 70.

Ashkenazi Jews are more likely to be carriers of brca1 and brca2 mutant alleles than other populations: 1/40.

[edit] Colorectal Cancer (CRC)

150k new cases each year
57k deaths each year (#3 cancer killer in both men and women)
2-5% lifetime risk
The vast majority of colorectal cancer is sporadic in etiology.
About 25% of colorectal cancer is familial or HNPCC in etiology.
A small proportion of colorectal cancer is due to Familial adenomatous polyposis (FAP) and a subvariant called Gardner syndrome.

[edit] Familial adenomatous polyposis (FAP)

Familial adenomatous polyposis is also known as adenomatous polyposis coli.
- Hence the causative gene is called APC.
Inheritance of FAP is autosomal dominant.
Offspring with FAP are heterozygous at the APC locus for function.
Heterozygotes develop many polyps within the first two decades of life.
- These polyps are benign.
Because there are so many polyps (hundreds, thousands), there is always transformation to malignancy.
Treatment is by surgical removal of the colon.
Carriers and relatives of pts should be screened with regular colonoscopy.
70% of sporadic colon cancer cases have loss of APC at both alleles despite not having inherited a defunct copy as with FAP cases.

APC is a tumor suppressor gene.
Beta-catenin is a pro-proliferation transcription factor.
APC acts to inhibit cell proliferation by maintaining low beta-catenin levels.
- APC is a kinase that phosphorylates beta-catenin, thus targeting it for destruction.
When APC becomes non-functional, beta-catenin accumulates, migrates to the nucleus', and induces transcription of myc and other pro-proliferation genes.

As colon cancer develops, mutations accumulate in common sites:
- APC
- Mismatch repair genes
- RAS
- DCC (a tumor suppressor gene)
- SMAD4
- p53 (a tumor suppressor gene)
- TGF-beta receptor 2

[edit] HNPCC: Hereditary non-polyposis colon cancer (Lynch Syndrome)

HNPCC was first describe in 1913 by Alfred Warthin.
Warthin noticed that his seemstress's family had a string of stomach and endometrial cancers
Lynch (Henry) then characterized hereditary non-polyposis colorectal cancer
- HNPCC = Lynch Syndrome

HNPCC is autosomal dominant
HNPCC usually results in a proximal colon cancers.
HNPCC increases risk for endometrial cancer and ovarian cancer in women.
HNPCC increases urinary tract cancers (in the kidneys and ureter).
HNPCC also gives rise to cancers in non-colon areas of the GI tract: stomach, biliary tract, small intestine.
Finally, HNPCC is associated with increased brain cancer.

Average age of colorectal onset in HNPCC is 44 years old (as compared to the general population average age of 64).

[edit] Amsterdam criteria

The Amsterdam criteria identifies HNPCC families
- These are high risk individuals who are candidates for confirmatory molecular testing for APC mutations.
The criteria has a series of 3-2-1 requirements:
- 3 relatives with CRC (2 of them must be first degree relatives to the proband)
- 2 successive generations with CRC
- 1 case of CRC before 50 yo

[edit] MMR Defects in HNPCC and MSI

HNPCC is caused by a defect in mismatch repair.
There are 5 genes related to mismatch repair, each of which can cause HNPCC if mutated:
- hMLH 1/2/6
- hPMS 1/2
Mutations in these genes cause ineffective DNA repair and therefore microsatellite instability (MSI) and therefore HNPCC.

MSI (microsatellite instability) is the expansion or contraction of short, repeated segments of DNA secondary to MMR defects.
MSI is not normally found in properly functioning cells.
MSI is found in 90% of HNPCC tumors.
MSI is found in only 15% of sporadic colorectal cancers.

*What is the significance of MSI?  Diagnosis?

[edit] HNPCC Take-home Points

Mutations in MLH1 and MSH2 result in defective mismatch repair and thus cause HNPCC.
Mutations in MLH1 and MSH2 raise the risk of CRC (colorectal cancer) by the age of 70 to 70-85%.
- The general population risk is 2-5%.
Mutations in MLH1 and MSH2 raise the risk of endometrial cancer by the age of 70 to 42-60%.
- The general population is much lower.

[edit] Cancer cells and Epigenetics

Cancer cells demonstrate several epigenetic changes: hypo / hyper methylation.
Recall that methylation closes DNA off from expression.
Improper methylation (or improper epigenetics of any form, really) should be considered one way to implement a "hit".

Hypomethylation:
- Recall that hypomethylation will generally allow more expression of the DNA.
- Hypomethylation can be seen in both benign and malignant neoplasms.
- Hypomethylation usually occurs at repetitive sequences.
- Hypomethylation may have several effects:
  - genomic instability
  - activation of oncogenes
  - activation of retrotransposons
  - loss of imprinting
- For example, loss of imprinting on the IGF2 gene is seen in 40% of colorectal cancers.

Hypermethylation:
- Recall that hypermethylation will generally inhibit expression of the DNA.
- Hypermethylation is usually at CpG islands.
- Hypermethylation may contribute to cancer via promotor silencing of tumor suppressor genes.

[edit] Personalized Medicine

[edit] Lecture Objectives

Define/describe personalized medicine and how it could be and is used, including
- Direct to Consumer (DTC) testing
Describe factors contributing to drug responses
List examples of how genetic polymorphisms effect drug metabolism
Describe some of the genetic differences in the cytochrome P450 genes and the resulting effects on drug metabolism

[edit] “Personalized Medicine”

[edit] Assessing Risk

[edit] HER2 as a personalized medicine risk factor

HER2 is known to be expressed in excess in 20-30% of breast cancers (and also in many other cancers).
HER2 is know to be associated with more aggressive tumors.
- That is, HER2 status is personalized medicine.

Tx for HER2+ breast CA:
- Monoclonal Ab against HER2 (Trastuzumab = Herceptin)

HER2 overexpression is accomplished by gene amplification.
HER2 is karyotypically observed as:
- double minutes (small, accessory chromosomes)
- HSRs (homogenously staining regions)

Other examples of personalized medicine surrounding tumor-expression variation include EGFR and BCR-ABL.
EGFR variable expression in colorectal cancer:
- EGFR IHC test
- Erbitux
- c-kit expression in gastrointestinal stromal tumors
- c-kit IHC test
- Gleevec

[edit] Direct To Consumer (DTC) Testing

[edit] ACMG's Stance on DTC Testing

Suggestion of minimum requirements for DTC:
- A knowledgeable professional should be involved in the process of ordering and interpreting a genetic test
- The consumer should be fully informed regarding what the test can and cannot say about his or her health
- The scientific evidence on which a test is based should be clearly stated
- The clinical testing laboratory must be accredited by CLIA, the State and / or other applicable accrediting agencies
- Privacy concerns must be addressed

[edit] Some DTC Testing Questions

[edit] Pt example

[edit] Pharmacogenetics

Pharmacogenetics is the study of how genetics affects drug effects.
Pharmacogenomics: Application of genomic information or methods to pharmacogenetic problems
- Genomics looks at many genes at once, usually.

[edit] Drug Response

Response rate of most drugs is 25-75%.
Thus, use of drugs in “non-responders” increases occurrence of side effects and health care costs.
About 15% of prescribed drugs have severe adverse effects.

As specific examples (drug, disease, efficacious population):
ACE inhibitors, HTN, 10-30%.
Beta blockers, heart failure, 15-25%
Anti-depressants, depression, 20-50%
Statins, cholesterol, 30-70%
Beta-2-agonists, asthma drugs, 40-70%

[edit] Adverse Reactions

Over 2 million people are hospitalized each year for adverse reactions to prescription drugs, most of which are avoidable.
More than 100,000 people die from adverse reactions (~sixth leading cause of death).

[edit] Pharmacogenetic Questions

[edit] Drug Response Factors

[edit] Pharmacokinetics and Pharmacodynamics

Pharmacokinetics explores what the body does to the drug
Pharmacodynamics explores what a drug does to the body

These genetic changes that cause pharmacodynamic (target) and pharmacokinetic (metabolism) changes can change the drug responsiveness.

[edit] Drug Conversion

Drug conversion is the metabolic change of a prodrug to an active drug once inside the body.
- Synonyms for drug conversion include "transformation" and "biotransformation".
Complete biotransformation typically requires several different enzymes.

Upon deficiency, much of the prodrug is lost to excretion before it can be converted and act at the target site.

[edit] Drug Metabolism

The goal at the liver is usually to make them more water soluble for subsequent elimination through the kidneys.
In a similar manner, the liver can metabolize lipophilic drugs to an hydrophilic form so that they can be excreted.
Lipophilic drugs that reach the kidney are often reabsorbed (retained).

[edit] Drug target mutations (pharmacodynamics)

Recall that drug dynamics describe the drug's activity at the target site.

[edit] CYP2D6 and Drug Metabolism

Recall that drug metabolism is called pharmacokinetics.

CYP2D6 is predominantly expressed in the liver.
Metabolizes 20-25% of commonly prescribed drugs.
Major, mentionalable drugs include: codeine, oxycodone, amphetamines, lidocaine, haloperidol, risperidone, fluoxetine, and carvediolol.
Major classes include: beta blockers, anti-depressants, antipsychotics, anti-cancer agents, and opioids.
CYP2D6 catalyzes conversion of codeine to morphine.
- That means that 10% of the population (who have deficient CYP2D6 codeine->morphine conversion) have no pain relief from codeine.

[edit] CYP2D6 Variation

CYP2D6 is highly polymorphic: SNPs (of all sorts), copy number polymorphisms, etc.
We classify alleles as "functional", "dysfunctional", and "non-functional".

CYP2D6 can be divided into subpopulations (sub-allelic populations, sub-"functional/dysfunctional/non-functiona") of "metabolizers" based on the functional activity of the resulting protein: "poor", "extensive", "ultrarapid".
The normal CYP2D6 response is rapid conversion of codeine to morphine.

[edit] CYP2D6 Inadequate Expression Alleles

Alleles that lead to "inadequate expression" are poor metabolizers.
- Poor metabolizers, therefore, have poor pain relief from codeine.
5-10% of caucasians are homozygous for low / no-activity alleles.

[edit] CYP2D6 Overexpression Alleles

Overexpression of CYP2D6 leads to increased conversion of codeine to morphine--called ultrametabolizing.
Ultrametabolism results in excessive morphine at a given dose.
Alleles 1, 2, 4 and others have copy number polymorphisms that cause their overexpression.
- For example, variant *2 can have 1-13 copies.
- Recall that "normal" is to have two functional copies.
30% of East Africans have hyperactive CYP2D6.

[edit] Thiopurine Methyltransferase and Drug Metabolism

Thiopurine methytransferase is also called TPMT.
TPMT breaks down a class of chemotherapeutic compounds called thiopurines.
Thiopurines can reach toxic levels if the drug is eliminated too slowly.
Adjusting appropriately to the pt's TPMT variation has dramatically improved the survival rate of affected children.
The label for thiopurines even suggests “Recommendation to use pharmacogenetic testing to guide dosing”.
TPMT*3A is the primary allele responsible for the trimodal distribution of thipurine methyltransferase activity: normal, poor, and subpoor metabolizing activities.

[edit] TPMT: Normal metabolizers

[edit] TPMT: Poor metabolizers

Poor metabolizers
These pts metabolize thiopurines slowly.
Thiopurine toxicity is high in poor metabolizers.
Children are at risk of dying of the toxicicity of thiopurine.

[edit] TPMT: Supboor metabolizers

Subpoor metabolizers
These pts metabolize thiopurines via TPMT very slowly.
0.3% are homozygous for low activity variants
Mostly TPMT*3A allele homozygotes
These subpoor metabolizers (with TPMT*3A) are at high risk of myelosuppression.
These pts should be treated with 1/10 to 1/15 the standard dose.

[edit] Personalized Medicine Over Multiple Genes

[edit] Warfarin

One of the most widely prescribed oral anticoagulants.
Warfarin has a narrow therapeutic window (that is, the serum active metabolite level must fall within a tight window).
Warfarin has wide inter-individual variability.
These two factors make dosing warfarin very difficult, thus the many ER visits for adverse effects.

Warfarin stops the formation of active clotting factors by inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).
- VKORC1 reduces vitamin K epoxide so that it can be used as a cofactor in the activation of clotting factors 2, 7, 9, 10, and proteins C, S, and Z.
S-warfarin is the active form.
S-Warfarin is metabolized by CYP2C9.
- Recall that CYP2D6 is the enzyme that metabolizes codeine and about a ba-jillion other things.

[edit] CYP2C9 and Warfarin Metabolism (Pharmacokinetics)

CYP2C9 metabolizes warfarin.
- CYP2C9*1 is the wildtype allele with "normal" activity.
Variant alleles *2 and *3 have decreased activity than the wildtype allele.
Variation of CYP2C9 exists primarily in Caucasian patients.
- Variation is rare in African American and most Asian patients.
Recall that this is a metabolic issue and therefore is considered pharmacokinetics.

Lower activity means that warfarin is not metabolized as quickly for pts with the *2 and *3 variatns
- Therefore, *2 and *3 CYP2C9 variants require less warfarin to achieve the same anticoagulant effect.
- Therefore, *2 and *3 CYP2C9 variant pts have an increased risk for overdose and subsequent hemorrhage.

[edit] VKORC1 and Warfarin Target Activity (Pharmacodynamics)

VKORC1 (warfarin's target) also provides variability that affects dose response.
- This is a pharmacodynamic effect.

Recall that warfarin inhibits VKORC1 so that it cannot recycle (reduce) vitamin K for use as a cofactor in coagulation factor (2, 7, 9, 10, C, S, Z) activation.
VKORC1 has two main haplotypes: A and B.
Haplotype A has elevated sensitivity to warfarin; that is haplotype A binds warfarin very well.
- Therefore haplotype A requires a lower dose of warfarin to achieve anticoagulant effect.
- Asian Americans are often of haplotype A and therefore extra sensitive to warfarin.
Haplotype B has decreases sensitivity to warfarin; that is haplotype B binds warfarin poorly.
- Therefore haplotype B requires a higher dose of warfarin to achieve anticoagulant effect.
- African Americans are often haplotype B and therefore insensitive to warfarin.

[edit] Warfarin Dosing

Recall that CYP2C9 variation produces pharmacokinetic variation that affects warfarin dose response.
Recall that VKORC1 variation produces pharmacodynamic variation that affects warfarin dose response.
These two variation factors account for only 30-45% of the warfarin dose response differences.
Lower initial doses should be used for:
- CYP2C9 *2 and *3 pts
- VKORC1 haplotype A pts

[edit] Genetic Testing

Roche pharmaceuticals has been awarded the license from FDA to detect genetic variations in the activity of cytochrome P450 enzymes

CYP2D6 and CYP2C19.

- Recall that CYP2D6 is the enzyme that converts codeine to morphine.
About 25% of all drugs are substrates of either CYP2D6 and CYP2C19.

[edit] Ethical Issues

Not giving a nonresponder a certain drug which works in others, patient may feel discriminated against (direct to consumer

marketing)

Falsely accusing patient of being noncompliant or having drug-seeking behavior due to increased clearance.
Nonresponder status influencing risk calculation made by insurance companies (agreeing to cover expensive, toxic or ineffective drug)
“Do you want to be contacted if your genotype is found at a later time to be associated with a condition you weren’t originally tested for?”
1. Referrals
2. Privacy
3. Who contacts the patients?

[edit] Stem Cells

Stem cells have two features: self renewal and differentiation.

Early experiments in which we though HSCs could dediff into other tissue types were actually misinterpretations of HSC fusion with endogenous cells of other types.

One major limitation to using HSCs is the inability to expand the population ex-vivo.

The blastocyst is the center of ethical discussions on embryonic stem cell use; how much moral status does a blastocyst have?

SCNT: remove donor oocyte nucleus, place another nucleus in the oocyte, trick oocyte into feeling fertilized; develops into embryo.
Therapeutic cloning is SCNT with pt's own donated nucleus into an oocyte (unlikely to be from the donor).
Reproductive cloning is the same as therapeutic but you implant it in an organism.
- Not allowed in humans anywhere!
- Not allowed in any animals in some states.

Reasons reproductive cloning isn't allowed in humans:
- Most implants die soon after implantation.
- Many offspring have developmental abnormalities.
- Human cloning could then be performed simply for organ harvest.
Therapeutic and reproductive cloning are regulated only at the state level.
- Most states have no law at all.
- Indiana does not allow therapeutic cloning or reproductive cloning (in any animal).

Bush (2001) allowed use of 60 existing embryonic cell lines.
Obama (2009) allowed production of new embryonic cell lines:
- from well informed, freely consenting fertility-therapy donors
- with private funds

iPSCs can self renew and can differentiate.
iPSCs don't require a blastocyst stage and therefore avoid many ethical concerns.
One study has shown that Sickle Cell Disease could be fixed in a mouse by gene-correcting autologous iPSCs and reimplanting.

iPSCs are awesome because:
- immunologically match their pt
- can be repaired
- can be repeatedly differentiated into many different types of tissue
- avoid blastocyst stage and therefore ethical issues
iPSCs aren't perfect because:
- produced via C-myc which is an oncogene and therefore have a risk of oncogenesis
- produced via retroviral insertion of transcription factors and therefore have a risk of insertional mutagenesis
- still need lots of work to figure out how to purposefully differentiate them into specific tissue types

We are starting to figure out how to induce iPSCs without c-myc.
We are starting to figure out how to induce iPSCs only as pluripotent as needed:
- to get to the HSC stage, only, instead of going all the way "back" to the fully potent iPSC.
- means lower risk in teratomas in the pt.

@@ Line 23: / Line 23: @@
 |-
 !Osteogenesis imperfecta
-|Types 1-4; Severity: 2 > 3 = 1 < 4; Type 2: perinatal death, dark sclerae; Type 1: brittle bones, tendency for fractures, fractures heal without deformity, deafness, blue sclerae; Type 4: tendency for fractures, normal sclerae.
+|Types 1-4; Severity: 2 > 3 = 1 > 4; Type 2: perinatal death, dark sclerae; Type 1: brittle bones, tendency for fractures, fractures heal without deformity, deafness, blue sclerae; Type 4: tendency for fractures, normal sclerae.
 |Autosomal dominant
 |Pro-alpha1, Pro-alpha2
@@ Line 37: / Line 37: @@
 |-
 !Marfan Syndrome
-|AD; dominant negative effect; 75/25% inherited / ''de novo''
 |'''Pleotropic''' (ocular, cardiovascular, skeletal): lens subluxation, myopia, detachment, catracts; mitral valve prolapse, aortic dilation; dolichostenomelia, pectus / spinal curvature deformations, narrow palate, joint laxity, arachnodactyly, Walker-Murdoch wrists, Steinberg thumbs
+|AD; dominant negative effect; 75/25% inherited / ''de novo''
 |fbn1 (fibrillin-1); EGF-like molecule
 |Ca++ binding fails in EGF-like domain mutations; TGF-beta binding protein mutations fail to sequester TGF-beta; Up-regulation of TGF-beta causes malformed matrix.
 |''Multidisciplinary management''; Ocular: lens correction, screening, sc (cataracts, ectopic lens); CV: echochardiography to monitor valves / aorta, beta-blockers; Counseling: isometric exercise, impact sports, pregnancy.
-|-
-!Familial Hypercholesterolemia
-|Early-onset atherosclerosis, elevated serum cholesterol, elevated LDL, Xanthomas (tendons, skin, eyelids), '''childhood MIs in homozygotes'''
-|'''AD''', AR; '''semi-dominant'''
-|LDL receptor (binds APOB100 on LDL for metabolism), APOB100 (surrounds LDL, binds receptor), ARH adapter protein (binds LDL receptos with APOB100 / LDL into clathrin pits), PCSK9 protease (degrades LDL receptor); '''locus heterogeneity'''; LDLR mutations are classified I-V from failure to synthesize to failure to remove from surface
-|Loss of function: LDL receptor, APOB100, ARH adapter protein; Gain of function: PCSK9 protease.
-|'''Deplete bile''': bile acid binding resins allow bile (with cholesterol in it) to be passed; '''Inhibit HMG-CoA reductase''': '''statins''' inhibit HMG-CoA reductase so it doesn't make cholesterol out of acetyl CoA (hepatocytes).
 |-
 !Homocysteinuria
@@ Line 56: / Line 49: @@
 |Homocysteine is the toxic substance that causes disease; homocysteine may impair disulfide bridges in FBN1 and thus cause ''Marfan like S&S''.
 |B6 (pyridoxine) supplemenatation (a cofactor for CBS); '''low methionine diet''' (meth is the aa most often converted to homocysteine); '''betaine / folate / b12 supplements''' to augment the homocysteine -> methionine converstion (to reduce homocysteine levels)
+|-
+!Familial Hypercholesterolemia
+|Early-onset atherosclerosis, elevated serum cholesterol, elevated LDL, Xanthomas (tendons, skin, eyelids), '''childhood MIs in homozygotes'''
+|'''AD''', AR; '''semi-dominant'''; '''locus heterogeneity'''
+|LDL receptor (binds APOB100 on LDL for metabolism), APOB100 (surrounds LDL, binds receptor), ARH adapter protein (binds LDL receptos with APOB100 / LDL into clathrin pits), PCSK9 protease (degrades LDL receptor); LDLR mutations are classified I-V from failure to synthesize to failure to remove from surface
+|Loss of function: LDL receptor, APOB100, ARH adapter protein; Gain of function: PCSK9 protease.
+|'''Deplete bile''': bile acid binding resins allow bile (with cholesterol in it) to be passed; '''Inhibit HMG-CoA reductase''': '''statins''' inhibit HMG-CoA reductase so it doesn't make cholesterol out of acetyl CoA (hepatocytes).
 |-
 !Cystic Fibrosis
@@ Line 211: / Line 211: @@
 |Did not address
 |-
-!
+!HNPCC = Lynch Syndrome
+|Elevated risk of colorectal cancer (and other GI cancers), endometrial cancer, and ovarian cancer; usually proximal CRC (FAP is usually distal)
+|AD
+|hMLH 1/2/6, hPMS 1/2; DNA mismatch repair genes
+|Defective mismatch repair and '''microsatellite instability'''
+|Did not address
+|}
+==Inheritance Patterns==
+{|border=1
+!Auto-Dom
+!Auto-Rec
+!Other (Not X)
+|-
+|'''Oculopharyngeal Muscular Dystrophy'''
+|'''Oculopharyngeal Muscular Dystrophy'''
 |
+|-
+|'''Ehlers Danlos'''
+|'''Ehlers Danlos'''
 |
+|-
+|'''Familial Hypercholesterolemia'''
+|'''Familial Hypercholesterolemia'''
 |
+|-
+|'''Charcot-Marie-Tooth'''
+|'''Charcot-Marie-Tooth'''
 |
+|-
+|Acondroplasia
+|Homocysteinuria
+|Hemochromatosis
+|-
+|Osteogenesis Imperfecta
+|Cystic Fibrosis
 |
 |-
-!
+|Marfan Syndrome
+|A1 anti-trypsin Deficiency
 |
+|-
+|Myotonic Dystrophy
+|Phenylketonuria
 |
+|-
+|Huntington Disease
+|Tay-Sachs Disease
 |
+|-
+|PCKD
+|Galactosemia
 |
+|-
+|NF
+|Maple Syrup Urine Disease
 |
 |-
-!
+|HNPCC
-|
+|Biotinidase Deficiency
-|
-|
-|
 |
 |-
-!
-|
-|
-|
 |
+|Congenital Muscular Dystrophy
 |
+|}
+*Note that all the amino acid / metabolics are autosomal recessive.
+*The structure proteins are AD: OI, Marfan, Achondro, Myotonic Dystrophy (or X-Rec: DMD, BMD, X Dilated Cardiomyopathy).
+**Ehler-Danlos is the odd one--it is all three: AD, AR, XR!
+{|border=1
+!X-Dom
+!X-Rec
+|-
+|Rett Syndrome
+|Ehlers Danlos
+|-
+|Fragile X Syndrome ('''unusual''')
+|Lesch-Nyhan Syndrome
 |-
-!
-|
-|
-|
-|
 |
+|Hemophilia A
 |-
-!
-|
-|
-|
-|
 |
+|Duchenne Muscular Dystrophy
 |-
-!
-|
-|
-|
-|
 |
+|Becker Muscular Dystrophy
 |-
-!
-|
-|
-|
-|
 |
+|X-linked Dilated Cardiomyopathy
 |}
@@ Line 1,281: / Line 1,320: @@
 *Lynch (Henry) then characterized hereditary non-polyposis colorectal cancer
 **'''HNPCC = Lynch Syndrome'''
- HERE
@@ Line 1,325: / Line 1,362: @@
 ===Cancer cells and Epigenetics===
-*Cancer cells demonstrate several epigenetic changes: hypo / hyper methylation.
+*Cancer cells demonstrate several epigenetic changes: '''hypo / hyper methylation.'''
 *Recall that '''methylation closes DNA off from expression'''.
-*Improper methylation (or improper epigenetics of any form, really) should be considered one way to implment a "hit".
+*Improper methylation (or improper epigenetics of any form, really) should be considered one way to implement a "hit".
@@ Line 1,343: / Line 1,380: @@
 *Hypermethylation:
-**Recall that hypermethylation will generally inhibit expression of the DNA.
+**Recall that '''hypermethylation will generally inhibit expression of the DNA.'''
 **Hypermethylation is '''usually at CpG islands.'''
 **Hypermethylation may contribute to cancer via '''promotor silencing of tumor suppressor genes'''.
-==Indiana Familial Cancer Clinic==
-*Genetic Services for Familial Cancer
+=Personalized Medicine=
-*Referral
-*Intake Information
+==Lecture Objectives==
-*Family History Questionnaire
+*Define/describe personalized medicine and how it could be and is used, including
+**Direct to Consumer (DTC) testing
+*Describe factors contributing to drug responses
+*List examples of how genetic polymorphisms effect drug metabolism
+*Describe some of the genetic differences in the cytochrome P450 genes and the resulting effects on drug metabolism
+==“Personalized Medicine”==
+==Assessing Risk==
+===HER2 as a personalized medicine risk factor===
+*HER2 is known to be expressed in excess in 20-30% of breast cancers (and also in many other cancers).
+*HER2 is know to be associated with more aggressive tumors.
+**That is, HER2 status is personalized medicine.
+*Tx for HER2+ breast CA:
+**Monoclonal Ab against HER2 (Trastuzumab = Herceptin)
+*HER2 overexpression is accomplished by '''gene amplification'''.
+*HER2 is karyotypically observed as:
+**'''double minutes''' (small, accessory chromosomes)
+**'''HSRs''' (homogenously staining regions)
+*Other examples of personalized medicine surrounding tumor-expression variation include EGFR and BCR-ABL.
+*EGFR variable expression in colorectal cancer:
+**EGFR IHC test
+**'''Erbitux'''
+**c-kit expression in gastrointestinal stromal tumors
+**c-kit IHC test
+**Gleevec
+==Direct To Consumer (DTC) Testing==
+===ACMG's Stance on DTC Testing===
+*Suggestion of minimum requirements for DTC:
+**A knowledgeable '''professional should be involved in the process of ordering and interpreting''' a genetic test
+**The consumer should be fully informed regarding '''what the test can and cannot say''' about his or her health
+**The '''scientific evidence on which a test is based''' should be clearly stated
+**The clinical testing laboratory must be '''accredited by CLIA, the State and / or other''' applicable accrediting agencies
+**'''Privacy concerns''' must be addressed
+===Some DTC Testing Questions===
+===Pt example===
+==Pharmacogenetics==
+*Pharmacogenetics is the study of how genetics affects drug effects.
+*Pharmacogenomics: Application of genomic information or methods to pharmacogenetic problems
+**''Genomics'' looks at many genes at once, usually.
+===Drug Response===
+*Response rate of most drugs is 25-75%.
+*Thus, use of drugs in “non-responders” increases occurrence of side effects and health care costs.
+*About 15% of prescribed drugs have severe adverse effects.
+*As specific examples (drug, disease, efficacious population):
+*ACE inhibitors, HTN, 10-30%.
+*Beta blockers, heart failure, 15-25%
+*Anti-depressants, depression, 20-50%
+*Statins, cholesterol, 30-70%
+*Beta-2-agonists, asthma drugs, 40-70%
+===Adverse Reactions===
+*Over 2 million people are hospitalized each year for adverse reactions to prescription drugs, most of which are avoidable.
+*More than 100,000 people die from adverse reactions (~sixth leading cause of death).
+===Pharmacogenetic Questions===
+===Drug Response Factors===
+===Pharmacokinetics and Pharmacodynamics===
+*'''Pharmacokinetics explores what the body does to the drug'''
+*'''Pharmacodynamics explores what a drug does to the body'''
+*These genetic changes that cause pharmacodynamic (target) and pharmacokinetic (metabolism) changes can change the '''drug responsiveness'''.
+===Drug Conversion===
+*Drug conversion is the metabolic change of a prodrug to an active drug once inside the body.
+**Synonyms for drug conversion include "transformation" and "biotransformation".
+*Complete '''biotransformation typically requires several different enzymes.'''
+*Upon deficiency, much of the prodrug is lost to excretion before it can be converted and act at the target site.
+===Drug Metabolism===
+*The goal at the liver is usually to ''make them more water soluble for subsequent elimination through the kidneys''.
+*In a similar manner, the liver can metabolize lipophilic drugs to an hydrophilic form so that they can be excreted.
+*'''Lipophilic drugs that reach the kidney are often reabsorbed (retained).'''
+===Drug target mutations (pharmacodynamics)===
+*Recall that drug ''dynamics'' describe the drug's activity at the target site.
+===CYP2D6 and Drug Metabolism===
+*Recall that drug metabolism is called '''pharmacokinetics'''.
+*CYP2D6 is '''predominantly expressed in the liver'''.
+*Metabolizes 20-25% of commonly prescribed drugs.
+*Major, mentionalable drugs include: '''codeine''', oxycodone, amphetamines, lidocaine, haloperidol, risperidone, fluoxetine, and carvediolol.
+*Major classes include: beta blockers, anti-depressants, antipsychotics, anti-cancer agents, and '''opioids'''.
+*'''CYP2D6 catalyzes conversion of codeine to morphine.'''
+**That means that 10% of the population (who have deficient CYP2D6 codeine->morphine conversion) have no pain relief from codeine.
+====CYP2D6 Variation====
+*CYP2D6 is highly polymorphic: SNPs (of all sorts), copy number polymorphisms, etc.
+*We classify alleles as "functional", "dysfunctional", and "non-functional".
+*CYP2D6 can be divided into subpopulations (sub-allelic populations, sub-"functional/dysfunctional/non-functiona") of "metabolizers" based on the functional activity of the resulting protein: "poor", "extensive", "ultrarapid".
+*'''The normal CYP2D6 response is rapid conversion of codeine to morphine.'''
+====CYP2D6 Inadequate Expression Alleles====
+*Alleles that lead to "inadequate expression" are '''poor metabolizers'''.
+**Poor metabolizers, therefore, have poor pain relief from codeine.
+*'''5-10% of caucasians are homozygous for low / no-activity alleles.'''
+====CYP2D6 Overexpression Alleles====
+*'''Overexpression of CYP2D6 leads to increased conversion of codeine to morphine--called ''ultrametabolizing'''''.
+*Ultrametabolism results in excessive morphine at a given dose.
+*Alleles 1, 2, 4 and others have '''copy number polymorphisms that cause their overexpression'''.
+**For example, variant *2 can have 1-13 copies.
+**Recall that "normal" is to have ''two functional copies''.
+*'''30% of East Africans have hyperactive CYP2D6.'''
+===Thiopurine Methyltransferase and Drug Metabolism===
+*Thiopurine methytransferase is also called TPMT.
+*'''TPMT breaks down a class of chemotherapeutic compounds called thiopurines.'''
+*Thiopurines can reach toxic levels if the drug is eliminated too slowly.
+*Adjusting appropriately to the pt's TPMT variation '''has dramatically improved the survival rate of affected children.'''
+*The label for thiopurines even suggests “Recommendation to use pharmacogenetic testing to guide dosing”.
+*TPMT*3A is the primary allele responsible for the '''trimodal distribution''' of thipurine methyltransferase activity: normal, poor, and subpoor metabolizing activities.
+====TPMT: Normal metabolizers====
+====TPMT: Poor metabolizers====
+*Poor metabolizers
+*These pts metabolize thiopurines slowly.
+*Thiopurine toxicity is high in poor metabolizers.
+*Children are at risk of dying of the toxicicity of thiopurine.
+====TPMT: Supboor metabolizers====
+*Subpoor metabolizers
+*These pts metabolize thiopurines via TPMT very slowly.
+*0.3% are '''homozygous for low activity variants'''
+*Mostly '''TPMT*3A allele homozygotes'''
+*These subpoor metabolizers (with TPMT*3A) are at '''high risk of myelosuppression'''.
+*These pts should be '''treated with 1/10 to 1/15 the standard dose'''.
+===Personalized Medicine Over Multiple Genes===
+====Warfarin====
+*One of the most widely prescribed oral anticoagulants.
+*Warfarin has a '''narrow therapeutic window''' (that is, the serum active metabolite level must fall within a tight window).
+*Warfarin has '''wide inter-individual variability.'''
+*These two factors make dosing warfarin very difficult, thus the many ER visits for adverse effects.
+*Warfarin stops the formation of active clotting factors by '''inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).'''
+**VKORC1 reduces vitamin K epoxide so that it can be used as a cofactor in the '''activation of clotting factors 2, 7, 9, 10, and proteins C, S, and Z.'''
+*S-warfarin is the active form.
+*'''S-Warfarin is metabolized by CYP2C9'''.
+**Recall that CYP2D6 is the enzyme that metabolizes codeine and about a ba-jillion other things.
+====CYP2C9 and Warfarin Metabolism (Pharmacokinetics)====
+*CYP2C9 metabolizes warfarin.
+**CYP2C9*1 is the wildtype allele with "normal" activity.
+*Variant '''alleles *2 and *3 have decreased activity''' than the wildtype allele.
+*'''Variation of CYP2C9 exists primarily in Caucasian patients.'''
+**Variation is rare in African American and most Asian patients.
+*Recall that this is a metabolic issue and therefore is considered '''pharmacokinetics'''.
+*Lower activity means that '''warfarin is not metabolized as quickly for pts with the *2 and *3 variatns'''
+**Therefore, '''*2 and *3 CYP2C9 variants require less warfarin to achieve the same anticoagulant effect.'''
+**Therefore, *2 and *3 CYP2C9 variant pts have an ''increased risk for overdose and subsequent hemorrhage''.
+====VKORC1 and Warfarin Target Activity (Pharmacodynamics)====
+*VKORC1 (warfarin's target) also provides variability that affects dose response.
+**This is a '''pharmacodynamic effect'''.
+*Recall that '''warfarin inhibits VKORC1''' so that it cannot recycle (reduce) vitamin K for use as a cofactor in coagulation factor (2, 7, 9, 10, C, S, Z) activation.
+*VKORC1 has two main haplotypes: A and B.
+*Haplotype A has elevated sensitivity to warfarin; that is '''haplotype A binds warfarin very well'''.
+**Therefore ''haplotype A requires a lower dose of warfarin to achieve anticoagulant effect''.
+**'''Asian Americans''' are often of haplotype A and therefore extra sensitive to warfarin.
+*Haplotype B has decreases sensitivity to warfarin; that is '''haplotype B binds warfarin poorly'''.
+**Therefore ''haplotype B requires a higher dose of warfarin to achieve anticoagulant effect''.
+**'''African Americans''' are often haplotype B and therefore insensitive to warfarin.
+====Warfarin Dosing====
+*Recall that CYP2C9 variation produces pharmacokinetic variation that affects warfarin dose response.
+*Recall that VKORC1 variation produces pharmacodynamic variation that affects warfarin dose response.
+*'''These two variation factors account for only 30-45% of the warfarin dose response differences.'''
+*Lower initial doses should be used for:
+**CYP2C9 *2 and *3 pts
+**VKORC1 haplotype A pts
+==Genetic Testing==
+*Roche pharmaceuticals has been awarded the license from FDA to detect genetic variations in the activity of cytochrome P450 enzymes
+CYP2D6 and CYP2C19.
+**Recall that CYP2D6 is the enzyme that converts codeine to morphine.
+*About 25% of all drugs are substrates of either CYP2D6 and CYP2C19.
+==Ethical Issues==
+#Not giving a nonresponder a certain drug which works in others, patient may feel discriminated against (direct to consumer
+marketing)
+#Falsely accusing patient of being noncompliant or having drug-seeking behavior due to increased clearance.
+#Nonresponder status influencing risk calculation made by insurance companies (agreeing to cover expensive, toxic or ineffective drug)
+#“Do you want to be contacted if your genotype is found at a later time to be associated with a condition you weren’t originally tested for?”
+##Referrals
+##Privacy
+##Who contacts the patients?
+=Stem Cells=
+*Stem cells have two features: '''self renewal and differentiation.'''
+*Early experiments in which we though HSCs could dediff into other tissue types were actually misinterpretations of '''HSC fusion''' with endogenous cells of other types.
+*'''One major limitation to using HSCs is the inability to expand the population ex-vivo.'''
+*'''The blastocyst is the center of ethical discussions on embryonic stem cell use; how much moral status does a blastocyst have?'''
+*SCNT: remove donor oocyte nucleus, place another nucleus in the oocyte, trick oocyte into feeling fertilized; develops into embryo.
+*Therapeutic cloning is SCNT with pt's own donated nucleus into an oocyte (unlikely to be from the donor).
+*Reproductive cloning is the same as therapeutic but you implant it in an organism.
+**Not allowed in humans anywhere!
+**Not allowed in any animals in some states.
+*Reasons reproductive cloning isn't allowed in humans:
+**Most implants die soon after implantation.
+**Many offspring have developmental abnormalities.
+**Human cloning could then be performed simply for organ harvest.
+*'''Therapeutic and reproductive cloning are regulated only at the state level.'''
+**Most states have no law at all.
+**Indiana does not allow therapeutic cloning or reproductive cloning (in any animal).
+*Bush (2001) allowed use of 60 existing embryonic cell lines.
+*Obama (2009) allowed production of new embryonic cell lines:
+**from well informed, freely consenting fertility-therapy donors
+**with private funds
+*'''iPSCs can self renew and can differentiate.'''
+*iPSCs don't require a blastocyst stage and therefore avoid many ethical concerns.
+*One study has shown that Sickle Cell Disease could be fixed in a mouse by gene-correcting autologous iPSCs and reimplanting.
+*iPSCs are awesome because:
+**immunologically match their pt
+**can be repaired
+**can be repeatedly differentiated into many different types of tissue
+**avoid blastocyst stage and therefore ethical issues
+*iPSCs aren't perfect because:
+**produced via C-myc which is an oncogene and therefore have a risk of oncogenesis
+**produced via retroviral insertion of transcription factors and therefore have a risk of insertional mutagenesis
+**still need lots of work to figure out how to purposefully differentiate them into specific tissue types
+*We are starting to figure out how to induce iPSCs without c-myc.
+*We are starting to figure out how to induce iPSCs only as pluripotent as needed:
+**to get to the HSC stage, only, instead of going all the way "back" to the fully potent iPSC.
+**means lower risk in teratomas in the pt.

Final Review Sheet

From Iusmgenetics

Current revision as of 02:32, 14 December 2011

Contents

[edit] Disease Profiles

[edit] Inheritance Patterns

[edit] Molecular and Biochemical Genetics

[edit] Objectives

[edit] Dominant Diseases

[edit] Recessive Diseases

[edit] Newborn Screening

[edit] General Molecular / Biochemical Pathogenesis Principles

[edit] Unusual Molecular Pathogenesis

[edit] Objectives

[edit] Unstable repeat expansion

[edit] Myotonic Dystrophy

[edit] Fragile-X Syndrome

[edit] Huntington Disease

[edit] Two Hit Phenomenon

[edit] Polycystic Kidney Disease

[edit] Neurofibromatosis

[edit] Hemophilia A

[edit] Gene duplication

[edit] Charcot-Marie-Tooth Disease

[edit] Miscellaneous

[edit] Mapping of Mendelian Disorders

[edit] Mapping Definitions and Instruction

[edit] Applications of Mendelian Mapping

[edit] Genetic Mapping of Complex Disease

[edit] Complex Disease

[edit] Is it Genetic?

[edit] Twin Studies

[edit] Familial Aggregation

[edit] Genetics of Complex Disease

[edit] Multifactorial Liability Threshold Model

[edit] Risk Factor Concepts

[edit] Risk factor 1: Sex differences

[edit] Risk Factor 2: Degree of Relationship

[edit] Risk factor 3: severity of the defect

[edit] Risk Factor 4: Number of Affected Individuals

[edit] Incidence vs. Prevalence

[edit] Counseling in Multifactorial Traits

[edit] New types of genetic data

[edit] (Genetically) Personalized Medicine

[edit] “Missing heritability”

[edit] Gene Therapy

[edit] Hx of Gene Therapy

[edit] New Viruses

[edit] Gene Therapy and SCID

[edit] Gene Therapy Examples

[edit] Hemophilia and Gene Therapy

[edit] Huntington and Gene Therapy

[edit] Gene Therapy as a mechanism of inhibition

[edit] RNAi

[edit] miRNA

[edit] Cell Engineering

[edit] GT and suicide genes

[edit] GT, Engineering, and Melanoma

[edit] Take away

[edit] GT and Homologous Recombination

[edit] GT and Germ Cell Lines

[edit] GT Adverse Events

[edit] GT Ethical Issues

[edit] GT pro / con

[edit] GT: the science fiction part

[edit] GT's future

[edit] Cancer Genetics

[edit] Objectives

[edit] Cancer

[edit] Features suggesting an inherited predisposition to cancer

[edit] Cancer is a genetic disease

[edit] Cell Proliferation and Cell Death Genes

[edit] Oncogenes

[edit] Burkitt lymphoma

[edit] Inherited mutations in oncogenes

[edit] Tumor suppressor genes

[edit] Cancer genes

[edit] Retinoblastoma

[edit] Loss of heterozygosity

[edit] Breast cancer