Editing Final Review Sheet

==Disease Profiles==
{|border=1
!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
|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
|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
|'''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)
|-
!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'''
|Hunting''tin'', 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
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==Molecular and Biochemical Genetics==

===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.

===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.

===Recessive Diseases===
*Requires two mutant alleles to show the phenotype or disease state.

===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:
***[[Phenylketonuria]]
***[[Galactosemia]]
***[[Maple Syrup Urine Disease]]
***[[Homocysteinuria]]
***[[Biotinidase Deficiency]]
**Endocrine disorders:
***Hypothyroidism
***Congenital Adrenal Hyperplasia
**Hemoglobinopathies:
***Sickle Cell Disease
**[[Cystic Fibrosis]]
*Using several methods: enzyme assays, radiommunoassays, electrophoresis, tandem mass spectrometry

===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.


==Unusual Molecular Pathogenesis==

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

===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

====[[Myotonic Dystrophy]]====

====[[Fragile-X Syndrome]]====

====[[Huntington Disease]]====

===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.'''

====[[Polycystic Kidney Disease]]====

====[[Neurofibromatosis]]====

====[[Hemophilia A]]====

===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.'''

====[[Charcot-Marie-Tooth Disease]]====

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


==Mapping of Mendelian Disorders==

===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.

===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)



=Genetic Mapping of Complex Disease=

==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

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

===Twin Studies===
*Compare concordance rates of MZ and DZ twins

===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.

==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:

===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.

==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.

===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).

===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.

===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.


===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?


==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'''''.

==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.

==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

===(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??

===“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.



=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? 

==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. 

==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. 

==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. 

==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. 

===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. 

===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. 


==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 

===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. 

===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. 

==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. '''

===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. 


==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. 


===Take away===
*Approved trials: 
*2/3 in cancer 
*but lots of other stuff, too 

==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. '''

==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. 

==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.

==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. 

===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 


===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.

==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.



=Cancer Genetics=

==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.

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

*Cancer '''INCIDENCE'''
{|border=1
!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'''
{|border=1
!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.

==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

==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.

==Cell Proliferation and Cell Death Genes==

===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)

====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.

===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).

===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'''

===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.

====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.

====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 <u>will not change flanking / intragenic markers</u> and is <u>therefore not considered a loss of heterogeneity</u>'''
**'''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.

===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 <u>brca1</u> 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 <u>brca2</u> 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.

===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'''.

====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

====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'''

 HERE 


*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).

=====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

=====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?

====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''.

===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 implment 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'''.


==Indiana Familial Cancer Clinic==
*Genetic Services for Familial Cancer
*Referral
*Intake Information
*Family History Questionnaire