Genetics F2016 RKM Term2 SGU

miRNA

"Natural" RNA interference - inhibits translation of mRNA - short RNA (~22 nucleotides) in eukaryotes - Binds to complementary sequences on target mRNA => translational repression & gene silencing. • Dysregulation has been implicated in numerous disease states Important: specificity is not complete and one miRNA can silence many different mRNA species

Common Mutation that Causes DMD

(all cause FRAMESHIFT) 1. Large deletions (in 2/3 of cases) 2. Duplication 2. Single base pair mutation 3. Small insertion or deletion (which lead to frame shifts) 4. Mutation of small number of base pairs

imatinib mesylate

(formerly known as STI571) - Treatment in chronic myeloid leukemia = powerful tyrosine kinase inhibitor specific for a few TKs including ABL -very effective against the BCR/ABL fusion protein -ABL is stuck in its activated form - imatinib binds to the active site of the fusion protein bcr-abl (tyrosine kinase) and prevents its activity

p53 mutation

**Occurs in >50% of all cancers** Loss of function => uncontrolled cell growth + increased rate of mutation

siRNA

- "Forced" RNA interference - Binds to complementary sequences on target mRNA => targets gene for degradation & destroy gene function **compare to miRNA which only causes inhibition of mRNA translation

BRCA2

- "single gene" mutation associated with Familial BC and ovarian cancer 1 in 740 - % of hereditary BC: 32% (~1% of all) - BC risk by age 70: 30-90% - Changes in sporadic BC: Mutations and loss of expression are rare

BRCA1

- "single gene" mutation associated with Familial BC and ovarian cancer 1 in 860 - % of hereditary BC: 52% (~2% of all) - BC risk by age 70: 40-90% - Changes in sporadic BC: Mutations rare, inactivated in 50% of some sub-types

COLA1 Gene

- 18 kilobases - 52 Exons - 1464-amino acid pro α 1 chain that will be used to construct type I collagen

Gendicine

- 1st marketed and licensed Gene Therapy for head and neck squamous cell carcinoma patients - uses an adenovirus as a vector for p53 VECTOR DELIVERY METHOD: --> Virus is injected directly into the tumor, reinstating endogenous production of p53 from cells that lost it during cancer progression - and therefore tumor suppression. - The viral vector is an engineered replication- incompetent human adenovirus • Gendicine will not replicate in the infected cells and is incapable of multi-cycle infection and of spreading to the neighboring cells.

Rate of new mutation for DMD

- 33% of cases results from new mutation - The rate of new mutation is high

Disruption of APC Pathway in FAP

- 88% of colon cancer patients have mutations in APC; - 7% in β-catenin; none have mutations in both BOTH mutations NEVER selected for concurrently Mutation => transcription and growth in absence of WNT signal

Testis Determining Factor (TDF)

- A transcription factor - DNA-bindingproteins - Contains a zinc finger domain - protein responsible for the initiation of male gonadal differentiation - induces the medulla of the embryonic gonads to develop into testes => produce testosterone (=> male sexual characteristics)

b-thalassemia

- AR - decreased or absent β-globin production leads to decreased or absent HbA levels. - Excess α-globin accumulates and precipitates in erythroid cells and causes damage due to the action of ROS and apoptosis of the damaged cells. Severe anemia results - Point mutations in the b-globin gene or loss of regulation of b-globin through the corfu deletion removes cis regulatory sequences are mechanisms responsible for the disease

Ryanodine Receptor Mutation

- AUTOSOMAL DOMINANT!!! -defect in this channel => malignant hyperthermia Mutation leads to excessive release of calcium - large scale membrane depolarization - Sustained muscle contraction and generation of heat -> inc. respiration, inc. CO2 acidosis, inc. lactic acidosis - depletion of energy stores -> death of muscle fibers -> rhabdomyolysis => hyperkalemia and myoglobinemia - Main cause of death by anesthetics

Y Linked Inheritance

- Affected males only - Male to Male (Son) transmission ONLY male CANNOT transmit to daughters - HINT: If a disease allele is on the Y, it has to have come from the father, as the mother is XX.

Roche P450 CYP Chip

- An Array that can be used to determine genotypes for ales' of selected CYP genes. - Main family members of cytochrome P45-, CYP2C8, 2C9, 2C18 and 2C19 are collectively responsible for Phase I metabolism of 20% f prescribed drugs

Genome-wide association studies (GWAS)

- Association studies do not concern familial inheritance patterns at all. - They are instead case-control studies. - To test the co-occurrence of a specific allele at a marker locus and a trait in a population by comparing the frequency of an allele in patients and controls - Usually the SNPs associated with the disease, share the same haplotype as the disease LIMITATIONS: Genetic Studies dont get the full picture because diseases are found in Low frequency varients which are hard to capture.

Marfan Syndrome

- Autosomal Dominant Displays Pleiotropy: - Mutation in the fibrillin gene - Skeletal abnormalities (long limbs, pectus excavatum) - Hypermobile joints - Ocular abnormalities (myopia, lens dislocation) - Cardiovascular disease (Mitral valve prolapse, Aortic aneurysm) Clinical Symptoms - Pectus Excavatum from dilated aorta - Scoliosis - Arachnodactyly (long, thin fingers/toes) - Subluxation of lens

Hemochromatosis

- Autosomal Recessive - HFE Gene Mutation => Most Common: C282Y - substitution of tyrosine for cysteine at amino acid position 282 - Delayed Age of Onset - Allelic Heterogeneity => e.g H63D-aspartic acid is substituted for histidine in position 63; and S65C

Reasons for Regulation of the Operon

- Bacteria have to be efficient: only produce the proteins they need. - The metabolic load of producing unneeded proteins will slow the bacteria's rate of cell division (b-galactosidase can make up a sizeable fraction of the cellular protein~20%) - Even a small reduction in division rate will result in the bacteria being out completed by quicker growing bacteria - Competitive environment with other bacteria, need to be good at growing quickly and monopolising resources

Fragile X syndrome

- CGG repeat is present at 5' end of the FMR1 gene - resulting in increased methylation of this region and silencing of the FMR1 gene - Fragile X Mental Retardation Protein (FMRP) is involved in RISC complex which process some miRNAs. - Mis- processing due to the low levels of FMRP found in fragile X mutations may be responsible for mental retardation. Normal Repeat#: 10-50 Pathogenic Repeat#: 200-2000 - anticipation in successive generations

FISH

- Can detect deletions at about the ~100,000 bp level - Can detect whole genome level rearrangements if whole chromosomes are painted (SKY FISH)

Array CGH

- Can detect very small deletions and insertions (CNV) - Cannot detect balanced rearrangements (discussed again during cytogenetics) - Resolution limit is determined by the construction of the microarray

Disadvantages of PCR

- Carriers in deletion mutations look identical to Normal Controls since bands are not quantitative

Effectors

- Caspase components to the Apoptotic Pathway - caspases are the proteases that serve to execute apoptosis by targeting various cellular components and enzymes such as nuclear lamins (nuclear envelope), inhibitor of caspase-activated Dnase (causing DNA fragmentation), DNA repair enzymes, cytoskeleton components, other procaspases etc etc.

Cri du chat syndrome

- Chromosome loses some of its genetic material - 46, XX, del(5p) or 46, XY, del(5p) - Diagnosis: FISH - Features: • Characteristic cry of affected infants, which is similar to that of a meowing kitten, due to problems with the larynx and nervous system • Mental retardation, Speech problems • Microcephaly

Common Clinical Features of Cystic fibrosis.

- Chronic bacterial infection of obstructed airways and sinuses including chronic obstructive lung disease and later by bronchiectasis - Fat malabsorbtion due to pancreatic exocrine insufficiency - Infertility in males due to obstructive azoospermia (CAVD) - Postnatal lower intestinal tract obstruction (meconium ileus) - Elevated sweat chloride levels - Abnormally thick sticky mucus by - several types of epithelial cell Carrier Frequency: 1 in 20 caucasians Incidence: 1 in 2,500 (most common fatal inherited disease)

OI type I

- Classic OI - predisposition to fractures, non-deforming, & with blue sclera - Typically, these are a result of loss of expression (haploinsufficiency) from one allele of COL1A1. -50% reduction of normal collagen NOTE: the collagen that actually gets produced is indeed perfectly normal.

Effect of inversions during crossover in meiosis

- Crossover during meiosis results in duplication or deletion of segments of the chromosome (unbalanced genetic material). - The gametes with acentric and dicentric chromosomes are not viable. - Carriers of inversions may be asymptomatic, but they have a high risk of spontaneous abortions.

histone deacetylase (HDAC)

- Deacetylates histones - Deactylated histones are positively charges and tightly associated with DNA => transcriptionally inactive DNA - Methylation player

X-linked SCID

- Defect in the gamma chain of the receptor for several different interleukins (IL2RG) - If T-cells lack this receptor they cannot mature => results in a deficiency of normal B-cell function

Advantages if Mutation-Based Genetic Testing

- Definitive diagnosis => mitigate parental anxiety - Early diagnosis -Diagnosis of less severe disorder (e.g. Legius syndrome diagnosis due to SPRED1 mutation, indicating that the child is at low risk of development of NF-1 tumors) - Can use any tissue source - doesn't require affected tissue

Type of Mutation of BMD

- Deletions or duplications in Dystrophin gene which often result in in-frame mutations i.e. maintenance of the codon reading frame

Interphase FISH

- Diagnosis tool for Trisomy 21 - can be done in interphase =>allows quick, high throughput, computerized screening of patients samples without the need for culture of samples

Disadvantages of Linkage-based Genetic Testing

- Diagnostic Error from Genetic Recombination - Requires study of entire family's genome >2 generations MUST be available - Fairly high incidence of discovering misattributed parentage - Not informative for all families - Does not account for genetic heterogeneity

Allelic heterogeneity

- Different mutant alleles at a single locus. i.e. different mutations in the same gene (most often presenting with same phenotypic manifestation) e.g. in CF

Retinitis Pigmentosa

- Digenic Disorders - a disease of progressive visual impairment - a result of a mutation in two independent genetic loci (ROM1 and peripherin)

replacement therapy in Hemophilia A

- Direct injection of factor VIII - Cryoprecipitate/factor VIII

Questions to consider in determining Clinical Utility

- Does the mutation diagnose disease, or indicate risk that an individual will someday develop disease? -Does it facilitate family planning or prenatal diagnosis? - Is there a medical intervention available that will modify the course of disease or prevent the onset of symptoms that can be implemented based on the results of testing?

cAMP

- E. Coli Hunger Signal - This molecule is the hunger signal that allows the expression of genes that break down other sugars including lactose - binds Catabolite gene Activator Protein (CAP) and activates it.

Hexoaminidase A

- Enzyme associated with Tay-Sachs Disease

Disadvantages if Mutation-Based Genetic Testing

- Expensive (>10,00USD) - Genetic Test Does not Predict Severity or Specific Complications - Mutations that reside deep within introns and copy number changes such as deletions, would be missed by mRNA-derived cDNA sequencing - No simple functional assay that can be performed on a clinical basis to validate new missense mutations.

Familial Adenomatous Polyposis (FAP)

- Familial Colorectal Cancer -**multiple (>100) adenomatous polyps develop throughout distal colon** - Many polyps, but progress slowly - Inherited as autosomal dominant (but loss-of-function) - Mutations in the APC (Adenomatous Polyposis Coli) gene on chromosome 5 (5q21) - Many different mutations of the APC gene are reported (Allelic heterogeneity) =Tumor suppressor gene (affects β- catenin involved in growth control pathway) ~1% of colorectal cancers -very high penetrance

Hereditary Non-Polyposis Colon Cancer

- Familial Colorectal Cancers - defects in mismatch DNA repair - Few polyps, but progress rapidly • Mutation of DNA mismatch repair (MMR) genes. • Inherited as autosomal dominant (but loss-of-function) • Five genes - MSH2 chromosome 2 ~60% of known mutations - MLH1 chromosome 3 ~30-35% of known mutations - Other MMR genes combined account for 5-8% • Genes/proteins not directly involved in control of cell division. = mutator genes • Cells accumulate mutations at 1000 times higher than normal • Tumors also exhibit MICROSATELLITE INSTABILITY.

SERPINA1

- Gene associated with A1AT Deficiency

FGFR3

- Gene associated with Achondroplasia

COL1A1

- Gene associated with Osteogenesis Imperfecta I

SCIDX1

- Gene deficienct in X-linked SCID

Eukaryote

- Has a nucleus and DNA is arranged in linear chromosomes. - Most cells also contain membrane-bound organelles such as mitochondria, chloroplasts and Golgi bodies.

Diseases with variable expression

- Hemochromatosis (autosomal recessive disorder) - more severe in males (females menstruate) - Xeroderma pigmentosum (autosomal recessive) - more severe in individuals exposed more frequently to environmental UV radiation - Osteogenesis imperfecta (blue sclera only vs deaf )

The Glucocorticoid Receptor

- In inactive state, the protein is located in the cytoplasm bound to multi-protein complex - In its active state it dissociates from regulatory complex, dimerizes and translocates to the nucleus where it is a zinc-finger transcription factor that binds to the the DNA sequence: 5' AGAACAnnnTGTTCT 3' 3' TCTTGTnnnACAAGA 5'

New Mutation Pedigree

- In pedigree The mutation is transmitted from an unaffected parent to an affected offspring - Typically the risk of recurrence for the couple is low • In some cases, increased age of the father is observed • There is NO family history of the disease • Most frequent examples due to hot spot for mutation: 1. Neurofibromatosis (NF1) 2. Achondroplasia (FGFR3) 3. Duchenne muscular dystrophy (Dystrophin gene) - About 33% (1/3) of patients have a new mutation • Other examples: osteogenesis imperfecta, Marfan syndrome, and some X-linked disorders (such as Duchenne muscular dystrophy) • Typically the risk of recurrence for the couple is low

compound heterozygote

- Inheritance of both the paternal and maternal mutation (different due to allelic heterogeneity)

Li-Fraumeni

- Inherited mutation in p53 - ** >50% all families with syndrome have inherited mutations in the TP53 gene Rare disorder that greatly increases the risk of cancer at a young age -can result in several kinds of cancer including breast, bone, brain, adrenocortical tumors and soft-tissue carcinomas) -1st hit is inherited from mum -2nd hit is somatic (responsible for LOH) - % of hereditary BC: 3% (<1% of all) - BC risk by age 70: >90% - Changes in sporadic BC: Mutations in 20%, LOH in 30-42%

Process of Genetic Counseling

- Interpretation of family and medical histories to assess the chance of disease occurrence or recurrence (in offspring) - Education about inheritance, testing, management, prevention, resources and research. - Counseling to promote informed choices and adaptations to the risk or condition.

Glucose 6-Phosphaste Dehydrogenase (G6PD)

- Key Enzyme of the oxidative site of the PPP - Key component of the Oxidative Stress Protection in RBCs: 1. G6PD required to make NADPH 2. NADPH required to maintain reduced glutathione pool 3. Glutathione required for detoxification of free radicals and peroxides

Prokaryote

- Lack a nucleus - Lack specialized membrane-bound organelles

Northern Blot

- Like southern blot but using mRNA

Clinical Manifestations of DMD

- Marked and progressive weakness of upper and lower limb muscles - Early onset: -->wheelchair-bound by 12yrs --> CARDIOMYOPATHY by 14-18yrs - Starts with Large muscles of the pelvic girdle => GOWER SIGN - Calf muscle hypertrophy (Pseudohypertrophy) caused by muscles growing to try to support weight but most is fatty tissue. - Genetic Fitness (Lethal <30yrs) --> common cause of death = respiratory problems & cardiomyopathy

Molecular Basis for the effect of smoking on A1AT Deficiency

- Methionine 358 forms part of the site where A1AT contact protease. - Methionine 358 oxidation reduces the affinity of A1AT for elastase by about 2000 fold. - The active site of A1AT at methionine 358 is oxidized by cigarette smoke and inflammatory cells thus decreasing its affinity for elastase by approx. 2000 folds.

Trans regulatory elements

- Modify the expression of genes distant from the gene that was originally transcribed to create them - Typically proteins like transcription factors / enhancer binding proteins

Role of Loss of Function in A1AT Lung Pathology

- Mutation in the SERPINA1 gene =>shortage (deficiency) of A1AT OR => abnormal form of the protein that can not control neutrophil elastase - Without enough functional A1AT, neutrophil elastase destroys alveoli and causes lung disease.

Germaine (Gonadal) Mosaicism

- Mutation is present in a proportion of the gremlin cells - Presence of two affected children is in favor this kind of phenomenon (compare to new mutation, where there is a single child with a disorder and no family history of the disorder)

Triplet repeat expansion Disorders that display Anticipation

- Myotonic dystrophy (autosomal dominant disorder)- CTG repeat expansion takes place in the mother - Fragile X syndrome (X linked disorder)- CGG repeat expansion in the mother - Huntington disease (autosomal dominant)- CAG repeat expansion transmitted in father

red/green color blindness

- Non-Lethal Sex-linked Diseases - Males are Hemizygous - 8% of the male population - Homozygous females (1 in 150) exist but are much rarer than males

Idiosyncratic Adverse Drug Effects

- Not associated with drug metabolism or drug targets - arise from interaction of drug with a unique aspect of patients physiology e.g. G6PD Deficiency

General categories of Small-Scale Mutations

- Nucleotide-pair substitutions - One or more nucleotide-pair insertions or deletions

Daughters of a man affected with an X-linked Disorder

- Obligate carriers in X-linked recessive disorders (heterozygous carriers of the mutation)

Rate of new mutation for BMD

- Only about 10% of cases represent new mutation.

Bacterial Operon

- Operon = polycistronic - Gene organisation in bacteria in which several proteins are coded by one mRNA - Allows all proteins to be controlled together

N-acetyltransferase 2 Deficiency

- Phase II drug metabolizing enzyme - metabolizes anti-mycobacterial - isoniazid (tuberculosis therapy) and other drugs via acetylation **Acetylation results in INACTIVATION of drug!** Patients classified as: 1. Slow acetylators - high blood con, slow metabolism => toxic buildup of drug 2. Fast Acetylators - low blood conc, fast metabolism toxic levels of isoniazid results in: --> liver damage and -->neuropathy. Other drugs affected: 1. hydralazine (antihypertensive) and 2. procainamide (antiarrhythmic) - systemic lupus erythematosus. -Test Acetylator Status using Sulfimidime -Modify isoniazid dose once determined

Dysplastic Nevus Syndrome

- Precursor to malignant melanoma - Inherited mutation in p16 (CDKN2A) CDK inhibitor Loss of p16/ mutant p16, allows active ClnD/CDK4, which phosphorylates and inactivates Rb → Increased transcription of S phase genes (Unregulated cell division) -90% penetrance by the age of 70yrs (90% likelihood of 2nd hit)

Dystrophin

- Protein associated with DMD and BMD

Azoospermia Factor

- Region on Y gene that may be deleted in an infertile man - Has active X- chromosome homologues (a, b, or, c - AZF)

Proof-reading

- Replication is very accurate but errors occur - DNA polymerase error rate =1/100,000 bases - Actual error rate = 1 in 10,000,000 - Because enzymes can detect and repair errors - Because enzyme has 5'-3' polymerase activity AND 3' to 5' exonuclease activity

Extrinsic Death Pathway

- Responsible for elimination of unwanted cells during development. - This apoptotic pathway is also important for termination of immune responses and for immunosurveillance. - Initiated by activation of members of the Tumor Necrosis Factor (TNF) receptor family of "cell death receptors", such as the Fas receptor. -Uses CASPASE 8 and 10 for initiation of apoptosis

Befits of using mRNA-derived cDNA Genetic Testing

- Reveals exon skips or insertions of intron sequence into the mRNA indicative of splicing mutations. - These may be due to mutations within introns that would be difficult to detect with genomic DNA sequencing unless the entire genomic sequence, including introns, were studied.

Disorders that demonstrate locus heterogeneity

- Sensorineural hearing impairment - Retinitis pigmentosa - Charcot Marie Tooth disease (AD, AR, or X-linked) - SCID (AR and X-linked) - Osteogenesis Imperfecta (COLA1/2) - Breast Cancer Susceptibility Genes (BRCA-1/2)

Adoption studies

- Separation of identical twins = same genes but different environment. Can yield good data, but logistically very difficult.

Dysplasias

- Single Abnormality Classification of Developmental Defects that are abnormalities in tissue organization, e.g. thanatophoric dysplasia (FGF3R mutation) affects almost all the skeleton; most are monogenic.

Deformations

- Single Abnormality Classification of Developmental Defects that are mechanical distortions, e.g.clubfoot that is caused by lack of amniotic fluid or intra-uterine crowding. They frequently resolve completely soon after birth.

Disruptions

- Single Abnormality Classification of Developmental Defects that is due to disturbances after an organ has been formed; abnormal structure of an organ or tissue as a result of external forces • Typically not genetics. E.g. Phocomelia (shortened arms or legs) resulting from a vascular problem.

Malformations

- Single Abnormality Classification of Developmental Defects that is due to errors occuring in the initial formation of structures, a primary structural defect of an organ or part of an organ • e.g. Congenital heart abnormalities, cleft lip or cleft palate, polydactyly • most are multifactorial

Role of Gain of Function in A1AT Pathology

- Some patients homozygous for the Z allele also have liver involvement. - Abnormal A1AT can accumulate intracellularly in the RER of liver hepatocytes. => damages hepatocytes => cirrhosis. NOTE: Disorder caused by attainment of novel property of protein

Survival Rates for A1AT

- Survival rate for Z/Z smokers is the worst - Survival rate for Z/Z non-smokers is better since they don't smoke - For M/M individuals - survival is better

MAP Kinase Pathway

- The Major Cell Proliferation Pathway -initiated by growth factors interacting with receptor -triggers cascade of activation of kinases = phosphorylation of serine/threonine residues -activating genes involved in driving cell division Most of the components of this signalling pathway have been implicated in various cancers

Negative regulation of Lac Operon

- The Normal Situation i.e. glucose present - Lac Operon System Turned OFF Mechanism: 1. Lac I gene expression => Lac I Repressor protein 2. Lac I Repressor protein binds LacO DNA sequence 3. Binding physically blocks RNA polymerase initiating transcription => RNA pol cannot move along the DNA and transcribe the structural proteins (LacZ LacY LacA)

Reason why Bacteria mainly use transcriptional control

- The amount of mRNA dictates protein synthesis and thus amount of protein, controlling its activity. - In bacteria the mRNA has a short half (minutes) therefore the amount of transcription taking place dictates how much protein is produced. - Transcription and Translation occur simultaneously

miRNA in cancer

- The genes that encode these miRNA are amplified (DNA sequence duplicated) in tumors. - Cancerous cells then lose cell growth regulation and the cancer gets larger.

Quantitative Trait

- The number of contributing (dominant) alleles determines phenotype, not the specific combination (e.g. AaBb and AAbb have the same phenotype; mixed dominance)

Globin Loci

- There are multiple of these genes found on chromosomes 11 and 16. - They must have arisen due to gene duplication in the evolutionary past. In early fetal life, the α- and γ-protein chains combine to form HbF (α2γ2), In infant/adults this switches to HbA (α2β2)

mass spectrometer

- Tool of the proteomics lab - measures the mass-to-charge ratio of charged particles. - Knowledge of mass and charge allows the elemental composition of a sample or molecule to be determined and thus the chemical structures of molecules, such as peptides

2D Gel electrophoresis

- Tool of the proteomics lab • This was one of the first technologies utilized for proteomics research. • Relies on separating proteins by charge and then (First dimension, 1D) and then by size (second dimension 2D) • The spots can be extracted and then indentified with MALDI-TOF

Skewed X-inactivation (Asymmetric X-inactivation)

- Unequal inactivation of X-chromosomes, this is seen in the Manifesting heterozygote (carriers display symptoms of recessive disease)

G6PD Deficiency

- X-linked Disease - 140mutations found, mostly SINGLE base SUBSTITUTIONS Leads to: 1. maintenance of sulfhydral groups in reduced state - heinz bodies 2. oxidation of membrane proteins => Rigid membranes ==> HEMOLYTIC ANEMIA!!!! Most Common Mutation: cSNP, A- 90-95% loss of activity => asymptomatic until precipitating factors i.e. oxidants (antimalarial drugs), lava beans, etc

Gene Therapy

- a means of providing a gene function to a patient suffering from a genetic disorder in order to counteract the effects of a non- functional one i.e. to provide a "cure" for genetic diseases -it means taking a "good" gene (one that is not mutated) that has been cloned and getting it into the cells of an individual with a defective gene => replacing the missing function -it is not actually replacing the bad gene (although that is possible to do) but the good gene is inserted somewhere else in the genome, or just introduced into the cell

Nonsense Mutation

- a point mutation that results in the formation of a stop codon in the mRNA -mRNA is usually processed normally (there are some exceptions) and the mRNA may be of normal length, however TRUNCATED PROTEIN produced is degraded Can be due to: => Frame Shift Mutation (from insertion or deletion of nucleotides) => splice site mutation (leading to ex

histone acetyl transferase (HAT)

- acetylates histones - Actylated histones neutrally changed and are not tightly attracted to DNA=> transcriptionally active DNA

Deamination

- also very common, ~500/cell/day - loss of amine group from base, particularly cytosine - cytosine deaminates to form uracil (pairs with A) - easy to fix => Base Excision Repair - However, 5-methyl cytosine deaminates to thymine = T-G pair both normal bases, could be repaired to TA or CG = mutational hotspot -spontaneous endogenous lesion

Warfarin

- anticoagulant (anti-thrombotic agent) - variation in response due to pharmacokinetic and pharmacodynamic variation - NARROW THERAPEUTIC WINDOW!!!! - Racemic mixture od R and S isoforms - S isoform is 3-5X more potent than the R.

Proto-oncogenes

- are genes that produce proteins which promote cell growth or prevent apoptosis - mutation and/or over- or mis-expression causes cell growth - Usually a gain of function mutation results in cancer e.g. BCL2 - Functions like a 'gas pedal/ accelerator' of a carProto-oncogenes - only requires one bad allele

Human Epigenome Project

- attempting to map all of the cytidines that are methylated in the human genome and where aberrant methylation may be pathogenic

Mechanism of Spinocerebellar Ataxia

- autosomal dominant, autosomal recessive, OR X-linked - variable age of onset (repeat size dependent) and life span - involves trinucleotide repeat expansion (not sex biased) - currently 29 different genes have been identified (LOCUS HETEROGENEITY) - CAG trinucleotide repeat expansion that occurs in the exons of at least 29 different genes (polyglutamine disorder due to polyglutamine tracts) - large polyglutamine tracts can affect protein function, alter protein interaction, causes protein aggregation, and make cytotoxic proteins

Phenylketonuria (PKU)

- autosomal recessive - usually engage in assortative mating

Sequences

- cascades of effects, e.g. Potter sequence (atypical physical appearance of a fetus due to Oligohydramnios sufficient to cause disruptions in morphogenesis of the fetus.) - type of Multiple Abnormality Classification of Developmental Defects

Sickle Cell Crisis

- caused by anaemia, haemolysis and vaso- occulsive ischaemia around the abdomen and long bones. NOTE: Splenic infarction can increase bacterial infections such as Streptococcus pneumonia and Haemophilus influence - Low O2 tension (<85%) => crystalizes AND forms FIBER

Recombination frequency

- chances of recombination - LINKED LOCI = low

Haplotype

- combination of alleles (DNA sequences) at different places (loci) on the chromosome that are TRANSMITTED TOGETHER!. - may be one locus, several loci, or an entire chromosome depending on the number of recombination events that have occurred between a given set of loci.

Heterochromatin

- condensed and transcriptionally inactive chromatin

Euchromatin

- decondensed and transcriptionally active chromatin

Ataxia telangiectasia

- defect is in ATM - AR = a serine/threonine kinase with a number of functions including: -detecting DNA damage (i.e. sensor) and activating cell cycle arrest and DNA repair proteins (e.g. p53) -rare disease -affects cerebellum (= ataxia) and immune system -increased incidence of cancer -ocular telangiectasia common

Hemophilia A

- deficiency of clotting factor VIII --> increased tendency to bleed - Gene map locus: Xq28 - meaning X chromosome long arm. - Most severe mutations involve inversions of an intron sequence However, OTHER types of mutations of Factor VIII gene result in the disease = Allelic Heterogeneity Clinical Features: subcutaneous Hematoma -develop a variable phenotype of haemorrhage into joints and muscles, easy bruising, and prolonged bleeding from wounds. -rare - Incidence: 1 in every 10,000 births (or 1 in 5,000 male births)

PMP22

- flanked by two Low copy repeats that can lead to unequal crossover repeats. - Duplication of Gene = CMT1A - Deletion of Gene = Hereditary Neuropathy with Liability to Pressure Palsies (HNPP)

UBE3A

- gene always and only expressed by maternal chromosome 15 - when maternal gene absent causes Angelman syndrome

SNRPN

- gene always and only expressed by paternal chromosome 15 - when paternal gene absent causes Prader-Willi Syndrome

Hox genes

- genes contain the homeodomain - humans have about 40 (39) located on four chromosomes as clusters in linear arrangement - code for transcription factors with a Helix-turn-helix DNA binding domain that allow multiple genes to be regulated together • Embryonic Stage specific expression • Tissue specific expression - function in patterning the body axis. They provide identity to particular body regions

Linkage Analysis

- gives probability of family member with high probability of developing disease phenotype - done when genes in particular disorders were mapped but not identified - done when gene unknown OR large genes with high ALLELIC heterogeneity - becoming less important as sequencing data accumulates - but it is still used Genetic Principle: recombination frequency of linked loci is low ***Identification of the actual disease causing gene is accomplished by sequencing nearby regions of the genome (in the past), or by use of the human genome databases (current)

Syndromes

- groups of anomalies that consistently occur together due to a single underlying cause, e.g. Down syndrome dysmorphologies due to trisomy 21. - type of Multiple Abnormality Classification of Developmental Defects

Huntington Disease

- high penetrance but delayed onset neurodegenerative disorder --> Manifests at approx 40 years - Genetic Fitness is high - Cause of death is not necessarily caused by the disease due to late onset - Progressive dementia, loss of motor control - CAG (Glutamate) repeat sequence on 4p - Normal repeat: 9-35 - Pathogenic Repeat: 36-100 - Autosomal dominant -Paternal anticipation (age of onset and rate of decline) -Polyglutamine disease

CYP2C9

- highly polymorphic - metabolizes warfarin to inactivate form - low activity (*2,*3) => decreased inactivation of warfarin => increases hemorrhaging **also metabolizes NSAIDs and Hypoglycemic Drugs

b-thalassemia

- is caused by reduced b chain synthesis -AR with high carrier frequencies in the Mediterranean and in North Africa and the middle east. -20-25% carrier frequencies have been observed in Rhodes and Cyprus. - Un paired a-globin forms precipitates which inhibit red blood cell formation and result in severe haemolytic anaemia. -• Bone marrow tries to compensate and expands to perform erythropoiesis and this lead to bone deformity and fractures (as also occurs in a-thalassaemia) -• There are many mutations that cause b Thalassaemia. ---> b+ type => reduces gene expression and --->b0 type completely suppresses it -• The net effect is the reduction or absence of synthesis of the b-globin chains of Haemoglobin.

CYP2D6

- member of Cytochrome P450 superfamily - major drug metabolizing enzymes - metabolizes a large range of drugs - antidepressants, antiarrhythmics, analgesics -polymorphisms originally suspected by using phenotypic assays with probe drugs e.g. Debrisoquine --[CYP2D6]--> 4-Hydroxydebrisoquine Poor Metabolizers - homozygous recessive for defect allele Extensive metabolizers - heterozygous/homozygous for wild type Ultrametabolizers - gene duplication

CYP2D6

- member of the cytochrome P450 enzyme superfamily, - the major phase I drug metabolizing enzymes. - Enzyme metabolizes a large range of drugs including antidepressants, antirrythmatics and analgesics. - Polymorphisms in the gene originally suspected based on phenotypic assays using probe drugs e.g. debrisoquine. - Important clinically used drug metabolised by CYP2D6 include: 1. Adrenergic blocker -metoprolol 2. Antipsychotics - haloperidol 3. Antidepressants - fluoxetine, imipramine, desipramine 4. Opioids - codeine(--> morphinr) and dextramethorphan.

Velocardiofacial Syndrome (DiGeorge Syndrome)

- microdeletion in chromosome 22q (del(22)(q11.2q11.2) • Cleft palate and speech difficulty • Cardiac anomalies • Facial anomalies include narrow palpebral fissures and prominent nasal root • Immunological problems due to underdevelopment of the thymus • Population prevalence of 1:4,000 to 1:6,000 DIAGNOSIS: FISH, CGH Array

HbC

- missense point mutation at the 6th position of the β-globin gene (Glu→Lys) - homozygotes have mild hemolysis - this Hb has lower solubility than HbA and tends to crystallize in RBCs - DOES NOT form fiber, just crystalize - compound heterozygotes for HbSC, may have episodes of sickling that are similar to sickle cell disease

DNMT1

- most abundant methyltransferase in somatic cells - essential for proper embryonic development, imprinting and X-inactivation • a maintenance methylase that can methylate heme-methylated DNA • Important in maintaining transcriptional repression following cell division i.e. NOT De novo!!!

Depurination

- most common form of spontaneous lesion ~10,000/day/cell - breaking of glycosidic bond between base and sugar in purine nucleotides - sugar-phosphate backbone remains but base is lost - if it persists through replication, then mutation can occur - occurs with A or G -spontaneous endogenous lesion

b-thalassemia intermedia

- mostly homozygotes or compound heterozygotes (different mutations on the b-globin genes) - Low HbA levels - but always β+β+

HoxA13

- mutation in this hox gene causes shorter thumbs shifted down the hand, and similar abnormalities in big toes - mutation have been found in a Michigan family - HEREDITARY abnormalities of the limb do not cause any apparent hardship. - Three women in the family also have uterine abnormalities that led one to be infertile and the others to have difficulty conceiving

Molecular Mechanism of Action of Sonic hedgehog Protein

- normal action of Ptch is to inhibit Smo, but when Ptch is bound by Shh this inhibition is removed activating downstream signaling - Gli protein (activated by Smo) and CREBBP interact to activate gene expression by binding to DNA regulatory sequences NOTE: Some of these genes are Hox Genes

Human Epidermal growth factor Receptor 2 overexpression

- occurs in sporadic breast cancer ~30% of breast cancers have HER-2 amplified

OI type II

- perinatal lethal OI due to severe defects in collagen formation - dominant negative => destabilize the collagen triple helix. - point mutation - most severe (perinatal lethality) - more severe than loss of function - 25% of the collagen that is made is normal, 75% is created with an abnormal protein which destabilizes the final collagen structure (this is dominant negative).

Sickle cell trait

- produce both normal and abnormal hemoglobin. - Hb genotype AS - benign condition - may have rare complications - can develop clinical problems of sickle crisis in low oxygen saturation --->Deep sea diving and unpressurised aircraft. --->Also extreme exercise such as army training. Which accounts for problems of some black males during army induction.

OI type III

- progressively deforming - dominant negative

A1AT

- protease inhibitor family (serine protease inhibitors or SERPIN). - Expressed in the liver and secreted into plasma Function: 1. bind to and inhibit elastase particularly elastase released from neutrophils in the lower respiratory tract. 2. inhabit other proteases

Osteogenesis Imperfecta

- rare condition - most common genes associated with OI are the COL1A1 and COL1A2 - Genes are typically inherited in an autosomal dominant fashion

a-thalassemia

- reduced a-chain synthesis - major cause of ill-health in South East Asia - Carrier frequency may go as high as 1 in 5 in some communities. -Also common in Africa and the Mediterranean -a-globin gene DELETION can be caused by UNEQUAL CROSSING OVER during homologous recombination

Myc

- regulates 15% of all genes - overexpression dramatically disrupts the equilibrium between activation and repression of genes

ascorbic acid (vitamin C)

- required for hydroxylation of proline and lysine in procollagen.

FIX

- small gene - codes for contains 415 amino acids - Point mutations and deletions in the gene are the most common causes of hemophilia B

Reasons for no manifestations of the disease in dominant disease heterozygote

- the phenomenon of skewed X chromosome inactivation OR - the disorder may be due to a germline mutation in the mother. The mother is a germline mosaic

Tumor suppressor genes

- these are genes that produce proteins that inhibit the cell cycle preventing cell proliferation - Usually a loss of function mutation results in cancer - requires loss of both alleles. - Functions like the 'brakes' of a car -Genes that cause cancer when they are lost -normal function is to control and regulate growth (i.e. stop cancer) 1) Cell cycle control genes 2) apoptosis promoting genes 3) DNA repair genes.

Purpose of Human Genome Project

- to determine the entire sequence of the human genome - to identify and map all of the genes within that genome - to analyze single nucleotide polymorphisms - to deal with ethical issues as they arose

Associations

- traits coincide more often than expected by chance - type of Multiple Abnormality Classification of Developmental Defects

Nonsense Mutation

- type of substitution mutation - change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

Silent Mutation

- type of substitution mutation -have no effect on the amino acid produced by a codon because of redundancy in the genetic code

Missense Mutation

- type of substitution mutation -still code for an amino acid, but not the correct amino acid

F8C

- unusually large gene - codes for 2332 amino acids - Mutation leads to Hemophilia A of which: --> Approximately 40% of cases of severe arise from a large inversion that disrupts the gene. Deletions, insertions, and point mutations account for the remaining 50- 60% of hemophilia A defects

E. Coli

- uses glucose as its energy source - Glucose is the preferred energy source - In the absence of glucose they can metabolise lactose as an energy source. - In media with both glucose and lactose, only glucose is used as an energy source. Genome: 4.6 million base pairs ~4300 codes for proteins

OI type IV

- variable phenotype OI, but with normal (not blue) sclera - dominant negative

Cellular Effect of the dF508 mutation on CFTR protein

--> CFTR is a glycosylated membrane spanning protein which is processed in the ER. --> DF508 mutation (class 2 defect in CFTR gene i.e. defective protein processing due to misfolding of protein) =>Mutant protein does not fold normally enough to allow exit from the ER => undergo a ubiquitin/proteasome dependent degradation in the ER => CFTR protein in the plasma membrane.

Loss of Heterozygosity (LOH)

-2nd hit/somatic mutation usually caused by: 1) Non-disjunction 2) Mitotic Recombination 3) Gene deletion 4) Point Mutation 5) Aberrant Methylation

Butyrylcholinesterase Deficiency

-Autosomal Recessive - Genetic variation leads to a decreased rate of succinylcholine metabolism => prolonged drug action => prolonged paralysis NOTE: defect is a structurally altered enzyme and not from an absence of the enzyme - pseudocholinesterase Condition is rare - not clinically screened Second isoform exists with increased activity

Spontaneous lesions

-Changes that occur in a resting cell due to the chemical nature of the DNA -chemical changes that occur spontaneously -converted to mutations upon DNA replication -only a problem during S phase of cell division -Extremely common = tens of thousands of DNA damage events/cell/day

Procollagen Amino Acid Sequence

-Composed of the three amino acids [glycine-x-y]. ---> The presence of glycine, which holds the smallest R-group of all of the amino acids is significant as it allows tight packing of the triple helix. ---> The -x and -y amino acids have a high proportion of proline and lysine, many are post-translationally hydroxylated (facilitates cross-linking of collagen => increase stability of mature protein)

Wilms' Tumor

-Example of Autosomal Dominant Inheritance - Results from loss-of-function in the WT1 gene on Chromosome 11, which encodes a transcription factor important in the control of cell growth and differentiation.

Hemophilia B

-Gene FIX -factor IX deficiency -X linked -very similar to another Hemophilia and can only be distinguished by assays of factor VIII and factor IX activity -rare - Incidence: 1 in 50,000 births (1 in 25,000 males)

T tract polymorphism

-Located in intron between Exon 8 and 9 -Usually cis with less severe mutation e.g. R117H mutation

Clinical Presentation of DM1

-Myotonia and weakness in distal muscle -Cognitive problems -Weakness in face and jaw muscle -Drooping eyelids (Most Common Myotonic Dystrophy)

Hemoglobin H (HbH) disease

-Should be suspected in an infant or child with a mild-to-moderate (rarely severe) microcytichypochromichemolytic anemia and hepatosplenomegaly. -Mild thalassemia-like bone changes are present in approximately one-third of affected individuals. -Unlike HbBart syndrome, HbH disease is compatible with survival into adulthood. -Frontal bossing, molar prominence -indicative of extramedullary hematopoiesis ( the marrow is trying so hard to make blood it expands and deforms bones) -'Hair-on-end" skull radiograph

Family studies

-Similar to twin studies but not so precise -More genetic variability

Hereditary Nonpolyposis Colon Cancer

-a result of mutations in mismatch repair genes MSH2*, MLH1*, PMS1, PMS2, or MSH6 -results in microsatellite instability= HYPERMUTABLE PHENOTYPE

Oxidative damage

-a result of the production of reactive oxidative compounds due to oxidative metabolism -superoxides, peroxides, etc. -causes oxidative damage to many parts of cell : adds oxygen groups to nucleotides e.g. 8-oxo-7-hydroxyguanosine -results in MISPAIRING with A and potential TRANSVERSION Repair Method: Base Excision Repair -spontaneous endogenous lesion

Inherited Autosomal Dominant Cancer Syndromes

-actually recessive at the cellular level, but with guaranteed loss of second copy e.g. retinoblastoma -associated with a single, susceptibility gene

genes implicated in AD

-amyloid precursor protein (APP) -apolipoprotein E -presenilin 1 (PS1) -presenilin 2 (PS2)

b-thalassemia minor

-are also called "b-thalassemia carrier", "b-thalassemia trait" or "heterozygous b- thalassemia" - •are mostly heterozygotes (one normal and one mutant b-globin gene) • Almost normal HbA levels

Mechanism of Friedrich ataxia

-autosomal recessive -possible paternal anticipation - GAA trinucleotide repeat expansion in intron 1 of the frataxin gene. - repeat expansion alters chromatin through DNA methylation and then histone modification => silencing of frataxin gene. - Frataxin appears to be involved in mitochondrial iron metabolism

Mutagens

-cause mutations by increasing the frequency of damage and mutations.• ultraviolet (UV) light. E.g. • ultraviolet (UV) light. • ionizing radiation • aflatoxin (from moldy grain) • benzene • Formaldehyde • mustard gases • Alcohol • cigarettes • barbequed hamburgers

Balanced Chromosome Structural Abnormalities

-chromosomes are altered but all the genetic information is present (no loss or gain) -usually phenotypically normal = inversion, reciprocal and Robertsonian translocations

Apoptosis in Alzheimer's Disease

-considerable evidence for DNA fragmentation in the brain cells of AD patients (also seen in schizophrenia) In AD brain -pro-apoptotic Bak and Bad protein levels increased (anti-apoptotic proteins Bcl-2 and Bcl-xL also increased, perhaps compensatory response in surviving neurons) -APP, PS1 and PS2 proteins are cleaved by caspase-3 -neuronal caspase-3 immunoreactivity increased

recombinational repair

-does use homologous chromosome -less error prone than non-homologous end joining (NHEJ) - one of the two mechanisms for Double stranded break repair - used in G2

UV light

-generates several deleterious photoproducts -such as cyclobutane pyrimidine dimers or 6-4 photoproducts -covalent linkages between bases on the same strand => Bulky Adduct => Nucleotide Excision Repair -interfere with normal pairing and block replication

Ionizing Radiation

-include X-rays, γ-rays and radioactive particles (α, β, γ) --> also causes extensive damage to DNA (base damage and strand breaks) leading to heritable mutations - most severe damage = double stranded DNA break = most time lethal

breast cancer type 1 and 2 susceptibility protein

-involved in repair of DOUBLE STRAND BREAKS = Defective Homologous Recombination Repair -contributes to apoptosis when DNA can't be repaired. -women with BRCA1 or BRCA2 mutations have up to an 85% risk of developing breast cancer by age 70 -and the risk of developing ovarian cancer is about 55% for BRCA1 and 25% for BRCA2 mutations -hundreds of mutations have been identified in the BRCA1 gene, most associated with an increased risk of cancer (= allelic heterogeneity)

Liability threshold model for Pyloris Stenosis

-males are more prone to get the condition (i.e. have a lower liability threshold) -therefore, for a female to be affected, she must be at the high end of the curve (has more 'contributing' genes) -therefore, the children (or siblings) of an affected female are more at risk of having the condition than if she was male -and her sons (or brothers) are more at risk than her daughters (or sisters)

Errors of replication

-mistakes in base-pairing made during replication -insertions/deletions -only relevant if lesion is present during S phase of cell division - Repair Method: Mismatch Repair

Ryanodine Receptor

-muscle membrane ion channel located on Chromosome 19 -Associated with DHPR (dihydropyridine receptor) - Functions to release Ca from sarcoplasmic reticulum

Clinical Presentation of DM2

-muscle pain, stiffness, fatigue -weakness in proximal lower extremities such as neck shoulders, hips and upper legs

Pallister-Hall Syndrome (extremely rare)

-mutation in Gli genes -brain tumors, polydactyl -Extremely rare!

Gorlin Syndrome (Nevoid basal cell carcinoma)

-mutation in Patched (Ptc) -early age basal cell carcinoma (<20 years) -rib defects (1/56,000 to 1/164,000)

Rubinstein-Taybi Syndrome

-mutation in the CREBBP gene which interacts with the Gli gene. -broad thumbs and toes, mental disability, short stature, small head, facial features -1/125,000 births

Thanatophoric Dysplasia (TD)

-mutation in the extracellular domain and distal tyrosine kinase domain -severe (lethal): similar to homozygous achondroplasia -short limbs, narrow chest, small ribs, undeveloped lungs, enlarged head -usually still born or death results shortly after birth from respiratory failure

Hypochondroplasia

-mutations in the proximal (most common) and distal tyrosine kinase domain -mild severity -Normal appearance at birth -as child grows the arms and legs do not develop properly and proportionately. -individuals live a normal life span

Nucleotide Excision Repair

-removes a few to more than a few (up to 29) bases around a damaged site involving a bulky non-coding lesion (UV damage) - Ability to recognise and remove bulky distortions/adducts in DNA NER involves co-ordinated action of 11 enzymes including ERCC-1, to remove, resynthesise and religate short oligo (25-30 mer)

Base Excision Repair

-repairs a single damaged base by removing it (damage by methyltion, oxidatio, deamination). -Uracil glycosylases recognize U :: G mismatches resulting from spontaneous C deamination to U in DNA. Remove U. -Specific Glycosylases that recognize T :: G mismatches (e.g. from deamination of 5-mC to T). Remove the T -Specific Glycosylases remove oxidized bases - (e.g.recognize 8-oxoG :: A mispairing).

Gene Annotation

-requires powerful computers to search for all of the known sequences that would define "a gene" based on a large number of criteria including; 1) ORFs (open reading frame), has a sequence that might make a protein, eg it has start site, stop site in same frame and associated splicing sequences 2) tailing sequences 3) regulatory domains (e.g. TATA boxes, GC-rich regions) - etc. Method misses any gene not translated into protein (rRNA, tRNA, etc.) And still mistakes made and things missed FORMS A DRAFT!

Unbalanced Chromosome Structural Abnormalities

-structural alterations result in the loss of gain of genetic information -therefore generally much more serious clinical ramifications = deletion, duplication, ring, isochromosome

Frameshift mutations

-tend to occur at positions where there are base repeats (e.g. GTCGAAAAACTCA) -thought to result from 'slipping' of DNA polymerases during replication of these repeats -DNA loops or kinks at these points and one or more bases are not copied or are copied twice - caused by insertions and deletions

non-homologous end joining (NHEJ)

-the more common of the two methods used for Double stranded breaks -does not use homologous chromosome to repair the break - Occurs in G1

variomics

-the study of all the variation that exists in the human genome

epigenomics

-the study of the epigenetic component of cell (DNA methylation)

splicosomics

-the study of the total alternatively spliced products of a cell/tissue

lipidomics

-the study of the total lipid component of a cell

transcriptomics

-the study of the total mRNA component of cells/tissues

metabolomics

-the study of the total metabolites in a cell/tissue

proteomics

-the study of the total protein component of the cell • Attempts to look at the protein fingerprint of a disease state

Somatic errors

-these types of replication errors can result in cancer -does result in aging of the person

Germline errors

-these types of replication errors can result in genetic diseases visited on your descendants -bad for the person's kids!

digynic

-two sets of maternal chromosomes -usually due to fertilization of a diploid ovum - small, non-molar placenta - one of the two types of triploidy

diandric

-two sets of paternal chromosomes -due to fertilization of a normal ovum by a diploid sperm or by fertilization by two sperm (dispermy) -produces a molar pregnancy (abnormal placenta with little embryonic tissue) and spontaneous abortion

DNA Microarrays

1) CGH arrays 2) SNP chips -single base pair changes, polymorphisms, and point mutations 3) Expression Arrays (cDNA) -gene expression

Control of Gene Transcription in Eukaryotes

1) Cis regulatory sequence of DNA 2) Transcripion factor complexes (=Trans acting factors) 3) Epigenetics 4) Small nuclear RNAs

Logic of Human Genome Sequencing

1) Clone large piece (200,000 bp) into a BAC clone (or YAC) 2) Cut BAC clone with a variety of enzymes 3) Sequence all fragments 4) Line them up and generate a "contig" map (continuous mapping) => Find the overlap between pieces => Put it all back together like a giant jigsaw puzzle 5) Repeat for each BAC clone

Heavy chain recombination

1) D + J recombination will join together in the DNA one version of D and one version of J. 2) DJ + V recombination joins the previously joined DJ exons with a version of the V region. The recombined gene will be transcribed into mRNA. 3) RNA processing will remove introns and alternative splicing with include the C region Cμ or Cδ. 4) Translation yields Ab peptide 5) Heavy & Light chains associate by disulphide bonds

appropriate disorders for gene therapy

1) Disorders that are a result of a single gene defect 2) Disorders for which we have the gene identified and cloned in its functional form (complete) 3) Disorders in which the affected tissue is accessible for gene delivery

Methylation Inheritance and transcriptional repression

1) Dnmt3 methylates unmethylated DNA (De novo methylation) 2) MeCP2 recruites either HDAC1 or Dnmt1 3) HDAC1 deacetylates histones 4) Dnmt1 methylates hemimethylated DNA (inherited methylation)

Gene Duplication

1) Functional Redundancy - second copy of the gene is often free from selective pressure — so mutations can accumulate without losing gene function 2) Genes often evolve subtle differences in function. 3) can evolve faster than a functional single-copy gene, over generations of human evolution. 4) Can occur through: - error in homologous recombination, - a retrotransposition event, or - duplication of an entire chromosome Notable Example = Globin Gene

Mechanisms producing 2nd hit

1) Loss through Non-disjunction 2) Mitotic Recombination 3) Gene Deletion 4) Point Mutation 5) Epigenetic mechanism

Limitations of twin studies:

1) May underestimate heritability (only addresses differences between 100% and 50% identical genomes) 2) MZs do have some different genes (mitochondrial genes) and epigenetic differences 3) Different environmental exposures (in utero and outside) 4) Different genes in different twin pairs (studies between different sets of twins may point to different contributors for same phenotype) 5) Ascertainment bias 6) Most studies do NOT specify the loci and alleles

Restriction Endonuclease Requirements

1) Only Recognize Palindromes!!!! => Must read the same in 5' -> 3' direction 2) Recognise a short DNA sequence (4, 6 or 8 bp) 3) Cleave double-stranded DNA on both strands creating a 5' phosphate and 3'OH

Genetic variation effecting drug response

1) Pharmacokinetic variation - Variation in proteins involved in drug metabolism and transport. 2) Pharmacodynamic variation - Variation in drug targets or pathways associated with these targets 3) Variation associated with idiosyncratic adverse drug effects.

Genes that regulate phosphorylation of Rb

1) RB, 2) CDK4, 3) cyclin D gene (ClnD) 4) CDKN2A (p16).

characteristics of an effective gene delivery system

1) able to target the correct cell type (where the gene needs to be expressed) 2) effective at getting the gene into a number of cells 3) able to get the gene into the nucleus (intact) where it can be expressed

Types of Spontaneous Lesions

1) deprivation 2) deamination 3) oxidative damage

Apoptosis in Disease

1) excessive loss of cells and stimulation of apoptosis e.g. Alzheimer's diseases and schizophrenia 2) viral infection e.g. HIV-1/AIDs 3) excessive growth of cells and inhibition of apoptosis e.g. cancer

low-frequency variants

1) recent, and 2) enriched for changes that alter protein sequence and function and, hence, are potentially involved in disease.

Vectors for Gene therapy

1. Adenovirus 2. Retrovirus 3. DNA approaches such as liposomes

Long Term Consequences of DNA Damage

1. Aging 2. Diseases, especially cancer

Targets of Caspases

1. Apoptosis regulators - Bcl-2, Bax - Procaspases - Inhibitors of apoptosis Cell 2. Adhesion Molecules - catenins, cadherins 3. Cytoskeletal Proteins - actin, keratins, tubular 4. Nuclear Structural Proteins - lamina Many others: ER and Golgi proteins, cell-cycle proteins, proteins involved in DNA synthesis and repair proteins, transcription and translation proteins, cell receptors, kinases, phosphatases, signalling molecules, etc.

Short Term Consequences of DNA Damage

1. Cell cycle arrest 2. Diminished transcription 3. Mutations 4. cell death (apoptosis)

Transcription factor Domains (Trans Element Domains)

1. DNA Binding Domain --Transcription factors have DNA binding domains that only bind to certain DNA sequences 2. Dimerisation Domain --Two transcription factors bind together to form a functional DNA binding unit (Dimer). Dimer formation adds an extra element of complexity and versatility. 3. Activation/repression domain --Binds to the enhancer binding proteins or other transcription factors and modulates their function

Mechanism of Translational regulation by siRNA

1. DNA of Engineered Gene delivery vehicle (i.e. stripped out virus ) --Double stranded RNA Often transcribed of two opposable promotors in a gene delivery vector engineered by a molecular biologist 2. Processed by dicer enzyme 3. Regulates expression through mRNA degradation • Associates with RNA Interference silencing complex. This mechanism takes advantage of the innate machinery used to destroy Viral dsRNA

X - Linked Recessive Disorders

1. Dystrophin-associated muscular Dystrophy - Duchenne muscular dystrophy (severe) - Becker muscular dystrophy (milder form) - both are due to mutations on the dystrophin gene 2. Glucose 6 phosphate dehydrogenase (G6PD) deficiency - hemolytic anemia on ingestion of primaquine, sulfa drugs 3. Hemophilia A and B result in bleeding tendencies 4. Lesch-Nyhan syndrome [Hypoxanthine Guanine Phospho ribosyl transferase (HGPRT) deficiency] - causes hyperuricemia, gout, & self mutilation Red-green color blindness (non-lethal) X-linked SCID (defect in the SCIDX1 gene)

Disadvantages of Array CGH

1. Finding a Baseline normal CNV (copy number variants) is difficult 2. Hard to determine significance of variations 3. Diagnostic tool does not have high dynamic range e.g. 8 or 10 extra copies looks the same as 50

Neurological Function of the Dystrophin Protein

1. Functions of the dystrophin complex is its involvement in the positioning of proteins in the complex so that they can function correctly. e.g. dystrophin complex is required at the neuromuscular junction for proper acetylcholine clustering during development.

Problems with Gene therapy

1. Immune response • one gene therapy volunteer with a non-life threatening condition died after receiving the ornithine transcarbamylase (OTC) gene 2. Tumor formation • several patients developed leukemia after being given gene therapy for X- SCIDs • Insertional mutagenesis induces a tumor - If the DNA is integrated in the wrong place in the genome: a) May destroy a tumor suppressor gene b) May activate a protooncogene 3. Gene therapy was successful but does not fix the problem examples: temporary fixes

Cellular Growth Control

1. Instructions come from outside the cell (growth factors) 2. Bind to GF Receptor Tyrosine Kinases on cytoplasmic membrane 3. functions via cell surface receptors triggering signal transduction pathways by: - activating cytoplasmic tyrosine kinases ==> Proteins with GTPase activity are activated ==> Activation of DNA-binding nuclear protein transcription factors 4. Activate/Deactivate gene expression to drive DNA replication and other aspects of cell division.

Cell treatment for preparation of a standard G-banding karyotype analysis or FISH

1. Isolation of cells from peripheral blood sample. 2. Culture of cells in the presence of phytohemagglutinin to stimulate cell division. 3. Arrest of cells at metaphase by treatment with colchicine or colcemid. 4. Cells are swelled by hypotonic solution, and burst onto a microscope slide. 5. Chromosomes are fixed and prepared for staining and microscopic visualization.

dystrophin protein

1. Links actin cytoskeleton with sarcolemma membrane (SM) protein complex 2. Required to position other proteins in the SM complex for correct function => e.g. In NMJ for proper acetylcholine clustering during development. 3.In absence, cell membrane becomes permeable, leads to damage to the muscle fibers =>sarcolemma breaks down => calcium influx => phospholipase activation => oxidative cell injury => Serum Creatine kinase leaks out of the muscle (=>myonecrosis).

Mechanism of Translational regulation by miRNA

1. MiRNAs bind to sequence elements in the 3' UTR of a specific mRNA and base pairing with it. 2.This prevents the interaction of the translational machinery with the 5' cap structure. THE COMBINATION OF TRANSCRIPTION FACTORS AND miRNAs EXPRESSED BY A CELL DEFINE THE CELL

Reasons for different severity in exact same CFTR mutation

1. Patient has less reactive inflammation (TNF, IL-1) response to bacterial infection => less lung damage 2. Something else in genetic b/g protects patient 3. environmental factors

Common Alleles of SERPINA1 Gene that encodes A1AT

1. Pi M 2. Pi Z (Glu342Lys) 3. Pi S (Glu264Val):

Reasons for Spontaneous Mutation

1. Random single mutation 2. Random ovum mutation 3. Random fertilized egg mutation 4. Germline Mosaicism (with/without somatic mosaicism

somatic cell nuclear transfer

1. Skin cells from patient with a monogenic disorder are cultured and the mutation corrected by recombinant technology in vitro. 2. The nucleus from the corrected cell is transferred to an enucleated egg or Embroyonic stem cell.

Philadelphia chromosome

1. This is a reciprocal translocation between chromosome 9 and chromosome 22, (specifically designated t(9;22)(q34;q11) 2. Creates the BCR-ABL oncogene 3. Causes chronic myeloid leukemia 4. NOT detected by array CGH; detected by SKY-FISH

Glucocorticoid-induced GR Activation

1. When glucocorticoid (cortisol) binds to GR, it dissociates from regulatory complex 2. The GR dimerizes 3. Then the GR moves to the nucleus 4. In the nucleus, GR may induce or repress target genes 5. GR binds response element DNA in the promoter regions of glucocorticoid-responsive genes

Morphological Progression of Apoptosis

1. cell shrinkage and rounding (breakdown of the cytoskeleton) 2. chromatin undergoes condensation forming patches against the nuclear envelope (called pyknosis) 3. nuclear envelope becomes discontinuous and the DNA is fragmented (called karyorrhexis) 4. nucleus breaks into discrete chromatin bodies due to the degradation of DNA 5. the cell membrane shows irregular buds known as blebs 6. cell breaks apart into several vesicles called apoptotic bodies 7. phagocytosed by neighboring cells

Reason for Female Carriers presenting with clinical manifestations of DMD

1. unequal (skewed) X-inactivation: => normal DMD X chromosome inactivated in higher % of cells (higher the % worse the symptoms) 2. (rare) X; autosome translocations 3. (rare) turner syndrome (XO) 4. . (rare) Heterozygous monozygotic twins (one gets mutant X)

Mendelian Retinoblastoma

30-40% of retinoblastoma cases Autosomal Dominant Cancer Syndrome Germaine Mutation + Sporadic Mutation --> Multiple Tumors, Bilateral, Early Onset

Glucocorticoid Response Element DNA Sequence

5' AGAACAnnnTGTTCT 3' 3' TCTTGTnnnACAAGA 5' where n represents any nucleotide. (Note the inverted repeats.)

Sporadic Retinoblastoma

60-70% of retinoblastoma cases Normal Gene + 1st Somatic Mutation + 2nd Somatic Mutation --> Single Tumors, Unilateral, Later Onset

Caspase

= Cysteinyl aspartic acid-proteases which cleave proteins at aspartic acid residues - Once apoptotic signals are received by the cell, the final stages that lead to the breakdown of the cell is carried out by a sub-family of enzymes - highly selective enzymes that break down proteins

premutational

= Patients with Repeat numbers that are between normal and pathogenic numbers. - sometimes have a mild, late onset phenotype

Ras

= a GTPase involved in the major cell proliferative pathway -Ras activated by binding GTP -initiates a phosphorylation cascade that activates cellular proliferation -quickly inactivated by intrinsic GTPase activity (GTP→GDP) = self regulation ---> Ras-GTP activates Growth pathway ---> Ras-GDP inactive (No growth) -therefore, mutation of GTPase => constant stimulation required to continue to grow

Karyotype analysis

= a detailed description of the chromosomal content of an organism -Includes; 1) number of chromosomes 2) size of chromosomes 3) position of centromeres (=length of chromosome arms) 4) banding pattern -karyotype analysis can identify chromosomal abnormalities by deviations from the norm

Double stranded breaks

= a difficult type of damage to repair particularly dangerous to dividing cells, = high probability of loss of genetic material

Chromosome Structural Abnormalities

= breakage and rejoining of chromosomes producing abnormal forms CAUSE: -chromosomal rearrangements can be due to simple breakage and loss or breakage and incorrect rejoining -More often due to recombination between similar sequences (e.g. LCRs) Therefore, often the same rearrangement event is seen in multiple patients. This occurs much more often than expected by random chance

Monozygotic (MZ) twins

= derived from a single ovum -genetically identical (with potential exception of mitochondrial DNA)

Dizygotic (DZ) twins

= derived from two separate ova but share intrauterine environment -just siblings (~50% identical)

individualized medicine

= determining your genetic makeup and revising drug treatments accordingly EXAMPLES: 1. Screening for Cytochrome P450 variants before warfarin therapy - to prevent bleeding complications 2. Screening for Factor V Leiden mutation before oral contraceptive treatment - to reduce risk of thrombosis 3. screening of HER2 receptor in metastatic breast cancer - to determine if Herceptin is useful

telomere

= ends of chromosomes also repetitive DNA

Xeroderma pigmentosum

= extreme sun sensitivity - AR - mutations in 9 Nuclear Excision Repair genes (=locus heterogeneity) -clinical sun sensitivity, = sunburn, blistering, freckled with hyperpigmented skin lesions -ocular involvement = conjunctivitis, ocular tumors > 1000-fold increase in skin cancer including melanomas -20% have progressive neurologic degeneration -DNA damage is CUMULATIVE & IRREVERSIBLE

Pharmacodynamic Variation

= genetic variation in target or associated component of signaling pathway

Population/migration studies

= indicative of environmental factors • Disease incidence differences between populations suggest a genetic basis for that disease • However, could also be due to cultural/lifestyle differences e.g. Incidence of breast cancer is lowest in Asian women After immigration to the U.S. the incidence increased to near American levels within one to two generations Therefore, genetic contribution is the same, but lifestyle (environment) is different

Genomic Instability

= mutations in gene involved in resting DNA repair and chromosome break repair will de-stabilize the genome e.g. Bloom Syndrome

Mutations in DNA repair genes

= mutator genes - Increase the frequency of mutations in cells due poor repair

sister chromatids

= pair of replicated chromosomes called

Mismatch Repair

= post-replicative (G2 Phase) repair mechanism -corrects errors in DNA arising during replication, e.g. insertion -Recognises strand discontinuities e.g. parent strand is methylated and daughter is unmethylated -mismatched bases are recognized and excised -also very important in relation to removing small repeats that tend to expand (e.g. triplet expansion disorders)

centromere

= several hundred Kb of repetitive DNA - required for spindle fibre attachment during mitosis - NO function significance known, but can be used to produce a centromeric index that helps to identify chromosomes

Microsatellite instability

= simple repetitive DNA sequences show size variability due to inaccurate replication HYPERMUTABLE PHENOTYPE

Chromosome Staining

= staining with DNA stains to generate a reproducible banding pattern Examples: 1)G-banding (giemsa stain) -- light and dark regions due to differences in sequence (AT rich = dark 2)R-banding (reverse stain) 3)Fluorescent stain (SKY-FISH)

trisomy

=2n+1 Gain of one chromosome Compatible with Life: • Down syndrome (21) • Edward syndrome (18) • Patau syndrome (13)

tertrasomy

=2n+2 Gain of two chromosomes

monosomy

=2n-1 Loss of one chromosome (only viable one is Turner's Syndrome)

Turner Syndrome (45,X)

=> nondisjunction during meiosis • X chromosome monosomy • Short stature • Webbed neck, Cystic hygroma at birth (neck swelling) • Primary amenorrhea • Gonadal dysgenesis • 'Streak ovaries' Diagnosis: Cells with NO BARR BODY Many patients are mosaics • Some of their cells are 45,X • Other cells are 46,XX and 47,XXX (indicate mitotic nondisjunction during embryogenesis)

Klinefelter syndrome (47,XXY)

=>nondisjunction during meiosis • Testicularatrophy • Gynecomastia • Female distribution of hair • Infertility Presence of a Barr body (X chromosome inactivation) in the buccal mucosal cells in MALE CAUSE: nondisjunction during meiosis I or II in the mom, or nondisjunction during meiosis I in dad (meiosis II nondisjunction in dad cannot result in this syndrome!)

25%

A 23-year-old female is pregnant and has come for genetic counseling. Her father (50 years old) is diagnosed to have Huntington disease. What is her risk of transmitting the mutant gene to her child?

Check for mutation in the parents

A couple who have a child with classic symptoms of OI seek advice regarding recurrence risk as they are expecting a second child. The COL1A1 gene in their affected child has been sequenced, and a known mutation has been identified. What is the next step that needs to be taken to address the question of recurrence risk?

Dominant-negative mutations (dominant negativity)

A mutant gene product interferes with the function of the normal gene product - Examples include: --> collagenopathies such as OI Type II, III, or IV --> Marfan syndrome (defect in fibrillin)

Silent Carrier (Heterozygote)

A person who has 3 total functional a-globin genes

residual risk

A person who has been identified as a "non-carrier" - may be a carrier of an infrequent mutant allele, that is not included in the panel

Centromeric Index

A ratio usually expressed in % = short arm / total chromosome -metacentric = highest = 48% (Chromosome 1)

Ring chromosomes

A ring chromosome forms when a chromosome loses genetic material at the terminal portions & the ends fuse to form a ring like structure

Immunodeficiency centromeric instability-facial anomalies syndrome (ICF)

A) Mutation of DNMT3B, results in loss of methylation from repeat sequences adjacent to centromeres. B) Hypomethylation leads to decondensation of heterochromatic regions surrounding the centromere. C) Results in 'sticky' centromeres that lead to the formation of multiradiate chromosomes. • Hypomethylation of CpG sites on chr. 1,9 and 16 lead to centromeric instability

Categories of caspases

A. Initiators (caspase-2,-8,-9,-10) B. Effectors or executioners (caspase-3,-6,-7) C. Inflammatory caspases (caspase-1,-4,-5)

Catabolite gene Activator Protein (CAP)

ALSO CALLED cAMP Regulatory Protein (CRP) - Active cAMP-CAP binds lacP and helps activate transcription in the presence of lactose

APC Pathway

APC is a component of the WNT signaling pathway

Progressive/Cumulative Nature of Cancer in FAP

APC mutation is the "Gatekeeper" event. However, Loss of p53 is the late even that directly sends cell into carcinoma state.

Amplified Oncogenes

Abnormal Homogeneously Staining Regions of Chromosomes in Cancers Often Contain ........[fill in the blank].... e.g. N-MYC amplification in neuroblastomas (NB)

Zero genetic fitness (genetic lethal)

Affected individual is unable to reproduce. e.g. DMD; Tay Sachs disease.

Liability

All factors that contribute to the disease => produces normal distribution

Pi S

Allele most often occurs with Pi M i.e. Pi MS in about 8% of caucasian population - only clinically significant when deficient variant reduces A1AT to 40% deficiency

Regulatory mutation

Alteration of a promoter sequence

cDNA Microarray

Analysis tool May help to molecularly characterize differences between the genes expressed in cancer tissues compared to non- cancerous tissues. 1) Isolation of two tissues 2) Isolation of RNA 3) Production of labeled cDNA 4) Hybridization of labeled cDNA to microarray 5) Visualization of hybridization to compare 85

CLIA (Clinical Laboratory Improvement Amendments)

Any lab that provides a genetic test for diagnostic purposes and charges a fee for that service must be certified under this organization. Ensure: - All tests are validated - No patent infringements

HIV-1

Apoptosis can be facilitated by this virus at several points in its life cycle: 1) Infection can lead to inactivation of anti-apoptotic protein Bcl-2 and activate procaspases priming the cell for entry into the apoptotic pathway. 2)Appears to promote the extrinsic Fas-mediated apoptosis pathway in CD4+ cells. 3)HIV proteins such as tat, nef and vpr may induce apoptosis by interacting with p53 or other pro-apoptotic transcription factors. 4)Virus' tat protein secreted by infected cell may taken up by other cells inducing apoptosis.

De novo mutation occurence

As an autosomal dominant disorder exhibits lower genetic fitness, the probability that the disorder is caused by a de novo mutation increases. [NOTE: This is not the case for autosomal recessive disorders since most disease alleles are masked by the functional allele. Another way to put this is that for AR disorders, most disease alleles are hidden in heterozygotes.]

G1/S

At what stage in the cell cycle does cyclin/Cdk hyperphosphorylate Rb => Rb no longer represses E2Fs => E2Fs activate S-phase genes => No G1 arrest => cell cycle progresses

Complementation

Autosomal Recessive Inheritance & Locus Heterogeneity usually help mask deleterious alleles because aaBB x AAbb give rise to AaBb offspring.

pro-apoptotic Bcl-2 family members

BID, BAX and BAK

Pro-apoptotic proteins

Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk. -upregulation of these proteins or their increased activation may offer an approach for cancer therapy -p53 upregulats these proteins NOTE: these proteins are part of the Blc-2 FAMILY of proteins BUT the Blc-2 protein itself actually inhibits their action.

Anti-apoptotic proteins

Bcl-2, Bcl-x, Bcl-xL, Bcl-XS, Bcl-w, and BAG. -some of these proteins are currently under investigation as potential targets for anticancer therapy

CFTR protein

Belongs to ABC family of transport proteins Function: • CFTR controls Cl-movement in and out of the cell (active transport) =>water follows hence thickened mucus • Regulatory domain controls opening of the channel and ion movement

Sporadic Cancers

Below is the profile of what kind of cancer: Age of onset: Later Multiplicity (multifocal): few Paired organs: unilateral Types of tumors: 1 Family pattern: None (no clustering, no inheritance) Marker: No Degree of risk: much lower

Inherited Cancers

Below is the profile of what kind of cancer: Age of onset: earlier Multiplicity (multifocal): multiple Paired organs: bilateral Types of tumors: >1 Family pattern: Monogenic = Mendelian Multifactorial = cluster Marker: Monogenic = yes Multifactorial = no Degree of risk: Monogenic = high Multifactorial =lower

Enhancer binding proteins

Bending of DNA so that the enhancer binding proteins can interact with the transcription factors and enhance transcription by RNA Polymerase II ; high levels of transcription = UPREGULATION • Bending of DNA allows formation of a 3D shape • => can be located before (UPSTREAM) or after a gene (DOWNSTREAM) or even in an intron! • => can be quite distant from the gene (thousands of base pairs)

SKY FISH (Chromosome Painting)

Best method for viewing translocations

Trisomy rescue

By chance the problematic allele is ejected e.g. in XXY ejects the extra X or YYX ejects the extra Y.

Pure Gonadal Dysgenesis (46, XY Swyer Syndrome)

CAUSED BY A LOSS OF FUNCTION MUTATION OF THE SRY GENE (CODING SEQUENCE MUTATION OR REGULATORY SEQUENCE) Incidence: 1/80,000 births Characteristic Features: •Normal female genitalia •Well-developed mullerian structures •Bilateral streak gonads instead of ovaries or testes •Teens show delay in the onset of puberty •Elevated luteinizing hormone (LH) causes clitoromegaly Treatment: •Due to increased chance of germ line tumors (30% risk by 30 years old) streak gonads are removed •Hormone replacement with estrogen and progesterone ***not to be confused with the features of androgen insensitivity syndrome

p16

CDK inhibitor - involved in G1/S checkpoint regulation

Spinocerebellar ataxia 8

CTG repeat expansion in the 3' terminal exon of a non-protein coding RNA from the SCA8 gene. The function of this non-coding RNA is unknown but it has been shown that the repeat expansion alters protein binding to the RNA.

Codeine

CYP2D6 converts this drug to morphine

G-banded karyotypes

Can detect insertions, deletions, and rearrangements on the several or many mega-base (Mb) level

natural selection that gives competitive survival advantage

Cancer is an unusual disease because of its tendency to become progressively more aggressive over time. This is a consequence of:

Inherited Autosomal Recessive Cancer Syndromes

Cancer syndromes of Defective DNA Repair e.g. Xeroderma pigmentosum; Ataxia-telangiectasia; Bloom syndrome; Falcon anemia -associated with a single, susceptibility gene

Familial Cancers

Cancer syndromes of multifactorial cause -Familial clustering of cases, but role of inherited predisposition not clear for each individual: Breast cancer; Ovarian cancer; Pancreatic cancer

WAGR syndrome

Caused by small microdeletions on chromsome 11 1. Wilms' tumor, 2. Aniridia, 3. Genitourinary malformations, and 4. Retardation of growth and development

de la Chapelle syndrome (46, XX Male Syndrome)

Characteristic Features: •Are typical boys and men •Slightly shorter than normal stature •Normal male external genitalia •10% show hypospadia •Urine comes out of wrong part of penis •Mullerian tissue absent •Infertility CAUSED BY A TRANSLOCATION OF THE SRY GENE FROM THE Y TO THE X CHROMOSOME AS A RESULT OF UNEQUAL CROSSING OVER DURING MEIOSIS. Incidence: 1/20,000 males

Clinical Features of Fragile X syndrome

Characteristic features: -Mental retardation, learning difficulties, - prominent ears, - elongated face, - macro orchidism (enlarged testis) • Analysis of the mother's X chromosome usually demonstrates a premutation Diagnostic tests: • Southern blot analysis of the triplet repeats gives an indication of the number of triplet repeat sequences • Cytogenetic test- the X chromosomes show breakage (fragile X) in a folate deficient medium - req full mutation to test positive

Nucleic acid hybridization

Characteristic of Nucleic Acid structure that is important for most diagnostic methods

sodium benzoate

Chemical that diverts ammonia to glycine synthesis, and the nitrogen moiety is subsequently excreted as hippurate. - Administered in Patients with a Urea cycle disorder

Chromosome 16

Chromosome containing alpha-like genes in humans

Chromosome 11

Chromosome containing b-like genes in humans

Severe Classification in Hemophilia

Classification is by Clinical bleeding symptoms or by Plasma procoagulant levels < 1% normal factor activity= Severe 1 - 5% normal factor activity =Moderately severe 5 - 40% normal factor activity = Mild

A1AT Clinical Symptoms

Clinical Symptoms: - COPD specifically emphysema (most common) => smoking major factor influencing course of COPD -->Smokers present (40-50yrs) -->Non-smokers (6th decade)-approach normal life span -Liver disease may occur in some (but not all) children and adults

Beckwith-Wiedemann Syndrome

Common Features: -birth weight and length greater than 90 percentile -ear creases or pits -neonatal hypoglycemia -increased risk of cancer Genetic Cause: -typically sporadic -maternal chromosomal rearrangements of 11p15 (imprinted region) -paternal uniparental disomy (UPD) -abnormal methylation at 11p15 ***children of IVF have 4-9 fold higher chance of BWS (1/4000 IVFs). In vitro fertilization causes abnormal DNA methylation of region 11p15. Angelman syndrome also increased in IVF

Meiosis 1 nondisjunction

Copies of different maternal chromosomes

Cholera

Cystic Fibrosis gives resistance or advantage to this disease.

1 in 25

Cystic fibrosis incidence = 1 in 2500 children. What is the carrier frequency? NOTE: for q<5% p ~ 1 => Carrier Frequency = 2 x the root of the disease incidence, I => Answer displays how a rare condition has a relatively high number of carriers

DMD vs Becker

DMD is mostly caused by deletions that lead to loss of proper reading frame - causes very severe disease, whereas in BMD, the deletion or duplication often maintains the reading (reading frame not affected). But in BMD, there is some deleterious effect due to the mutation, it is just not as drastic as in DMD.

distinction between DNA damage and mutation

DNA Damage is recognized and efficiently repaired vs Mutation, which occurs when there is replication across a damaged region that has not yet been repaired will create mutations by introducing incorrect bases.

exogenous DNA Damage factors

DNA damage from e.g. UV light, chemicals, radiation, pH, smoking

endogenous DNA Damage factors

DNA damage from e.g. cellular metabolism

Bloom Syndrome

Defect in BLM gene =RecQL3 DNA helicase, required for replication repair, recombination Characteristics: -smaller than average -narrow chin, prominent nose and ears -facial rash (pigment and dilated blood vessels) upon exposure to sun -often get diabetes and have neurological, lung and immune system deficiencies -Chromosomal instability resulting in many chromosomal breaks and sister chromatid exchanges -High incidence of cancer of a broad range of types

Phenylalanine Hydroxylase

Deficiency in PKU-I

BH4

Deficiency in PKU-II

Clinical Utility

Degree to which the test result informs clinical decision making. ---> These questions need to be considered before embarking on testing (there may be tangible benefits/ risks to both the individual being tested and/or their family)

age dependent penetrance

Delayed age of onset of disease manifestations e.g. Huntington disease

Compound Heterozygotes

Describes an affected individual where there are two different (or non-identical alleles) of the same gene that are inherited to cause the disorder e.g. 1. D508 and R117H mutant alleles which together may cause CF 2. Hb S and Hb C mutant alleles which together cause Hb (Beta-Globin) disease

Hardy Weinberg equilibrium

Describes the relationship between gene frequencies and genotype frequencies - Concludes that in a model population the relative proportions of the different genotypes remain constant from one generation to another. - If the population get large the relative proportions of genotypes remain constant.

Multiplex PCR

Diagnostic tool that uses many PCR primers in one reaction tube e.g. PCR primers for commonly deleted Exons in DMD to compare normal vs affected.

11nm

Diameter of "Beads on a String" form of chromatin

700nm

Diameter of Condensed section of chromosome

2nm

Diameter of DNA double Helix

300nm

Diameter of Extended section of Chromosome

1400nm

Diameter of Metaphase chromosome

30nm

Diameter of chromatin packed nucleosomes

alleles

Different forms (versions) of a gene

5' - 3' direction

Direction in which DNA polymerases synthesise new DNA Following synthesis of Okazaki fragments:- • Ribonuclease removes the primer • Gap filled by a DNA Pol • Ligase joins the adjacent fragments

Disadvantages of siRNA treatment

Disadvantages: 1. OFF TARGET effects due to non-specificity of to specific mRNA 2. Too much siRNA can result in non-specific events due to activation of innate immune response - cells detect RNA and use viral defense mechanisms against "viral attack"

Clinical Manifestations of BMD

Disease Severity: - May have same features as DMD but less severe - Age of onset usually after 7 years old Genetic Fitness: - Relatively high - Up to 70% of normal

Chronic myeloid leukemia

Disease cause by Reciprocal Translocation resulting in the Philadelphia chromosome → Activation of abl proto-oncogene in hematopoietic cells =>The bcr-abl abnormal fusion gene is produce = an Unregulated cytosolic tyrosine kinase Abl is stuck in its active form fusion- tyrosine kinase that stimulates uncontrolled division

Maleria

Disorders below give resistance or advantage to this disease. 1. Sickle-cell 2. a & b-thalassemia 3. G6PD deficiency

HoxD13

Disruption of this hox gene form Polydactyly

Pachytene quadrivalent

Due to Reciprocal translocation occurs in gamete progenitor cells The chromosomes have a problem lining up during gametogenesis and create this structure ALTERNATE segregation = balanced ADJACENT segregation = unbalanced (contains duplicated and missing regions) => most lead to spontaneous abortions

3-6 months

Duration of Embryonic Hb

Transacetylase

E.coli protein of unknown function but may help in detoxification of b- galactosides

B-galactosidase

E.coli protein that breaks 1- 4 glycosidic link in the lactose disacharide

Permease

E.coli protein that helps lactose move through the cell membrane

Dual System of Negative AND Positive Regulation of Same Operon

Enables Bacteria to make choices e.g. When there is glucose AND lactose present E Coli can still choose to inhibit Lac Operon expression in favor of metabolizing glucose

Normal Genetic Fitness

Examples include Huntington disease (because onset typically occurs after child bearing years), and adult onset polycystic kidney disease.

Direct detection Molecular Diagnosis that query entire genome

Examples: - Karyotypes (G-banding) - Array CGH, SNP chip, expression arrays - Spectral karyotype - Next gen sequencing

Transcriptional control (DNA Gene Regulation)

Examples: - Nuclear compartmentalisation, - chromatin remodelling, - recruitment of transcription machinery, - transcription initiation, - transcription elongation, - transcription termination

Direct detection Molecular Diagnosis that query specific gene/ sequence

Examples: - Southern-RFLP, PCR-RFLP, PCR sizing, ASO blot - FISH - Northern, Western - Sanger sequencing

Examples of single gene analysis

Examples: • Southern blot • Northern blot • Western blot • Sequencing of a PCR product • ASO detection on DNA • Allele specific primers for PCR

Ways DNA may be altered

Examples: •Amplification of repeat sequence - Genomic instability - A transposon may disrupt a gene • Regulatory mutation - Alteration of a promoter sequence • Triplet repeat expansion - May lead to gain of function, other effects • New splice donor in exon or intron • New splice acceptor in exon or intro

Liability threshold model

Explains the pattern of recurrence risk in families: 1) Risk of recurrence is higher in relatives of severely affected individuals (severely affected individuals implies more contributing genes in the family) 2) Recurrence risk is high in close relatives and decreases rapidly in more distant relations 3) Greater risk if more than one close relative is affected (multiple affected individuals implies more contributing genes in the family) **This is unlike Mendelian genetics where risk does not change, e.g. for autosomal recessive, all offspring of heterozygous parents have 1⁄4 risk

46, XX, t(2;4)(p12;q13)

Express in ISCN for Chromosomal Abnormality: Balanced translocation between the short arm of chromosome 2 and the long of chromosome 4 (female)

46, XY, del (1)(p21)

Express in ISCN for Chromosomal Abnormality: Deletion on chromosome 1 (male) short arm in the region 2-1

BID

Extrinsic pathway can also act through mitochondria due to the action of the this Bcl-2 family member protein which is activated by caspase 8

Achondroplasia Symptoms

Facial Features: -large head -flat nose at bridge Hand Features: -shortened and stubby fingers -trident hand (separation between middle and adjacent fingers) -individuals usually live a normal life span

NF-2

Features: - Bilateral vestibular schwannomas - Cranial and peripheral nerve schwannomas - Meningiomas - Ependymomas Gene: - NF2: chromosome 22 - Encodes merlin - cytoskeletal protein Frequency: - 1:30,00

Schwannomatosis

Features: - Schwannomas - Pain Gene: - SMARCB1: chromosome 22 - Encodes chromatin remodeling complex protein Frequency: -1:40,000

NF-1

Features: - Café-au-lait macules - Skinfold freckles - Neurofibromas - Optic gliomas - Lisch nodules Skeletal dysplasia - Malignancy Learning disabilities Gene: - NF1: chromosome 17 - Encodes neurofibromin - GTPase-activating protein Frequency: - 1:3000

Fluorescent in situ hybridization (FISH)

Fluorescently labeled probes can be used to hybridize partially denatured chromosome spreads ***Cells do not need to be in metaphase*** • Probes are 10 - 100 kb (or more) long so that many fluorescent tags can be packed onto the probe to allow visualization • Key Point: Resolution is improved over G-banding •Advantage: - Better resolution compared to G-banding e.g 22q11.2 deletion (DiGeorge) syndrome •Disadvantage: - The geneticist must "know what to look for" NOTE: similar to ASO in specificity however ASO is only 15b whereas FISH is 10,000-100,000b!!!!!!!! Also in ASO you fully digest DNA with endnucleases first.

Hardy Weinberg Principles

Frequency of an allele or a genotype is constant in a population over time if: 1) Population is large 2) Mating is random (non selective) 3) There are no new mutations 4) There is no selection/discrimination to affect allele frequencies 5) There is no migration (gene flow) to change allele frequencies

Rb protein

Functions as a regulator of G1/S phase transition

Molecular Defect in Sickle Cell Disease

GAG-->GTG single nucleotide change (point mutation) resulting Glu6Val (glutamic acid to valine) at the sixth position of the b-globin polypeptide => missense mutation

Effects of reciprocal translocation during gametogenesis

Gamete that has a reciprocal translocation just like the parent (balanced genetic material). On fertilization results in a translocation carrier (similar to the parent) Translocation carriers can thus have - normal children (fertilization of gamete 1) or - translocation carriers (fertilization of gamete 2)or - spontaneous abortions (fertilization of gametes 3, 4)

shotgun sequencing

Gene Sequencing Method used by Venter/Celera (MODERN METHOD) - cheaper and faster than Government Method (evolved out of lessons learned from government so Government still important)

APC

Gene assisted with: SYNDROME: Familial adenomatous polyposis MAJOR TUMORS: Bowel carcinoma ( A Colorectal Cancer)

MSH1/2,PMS1/2

Gene assisted with: SYNDROME: Hereditary nonpolyposis colon cancer MAJOR TUMORS:Colorectal carcinoma, endometrial carcinoma

TP53

Gene assisted with: SYNDROME: Li-Fraumeni Syndrome MAJOR TUMORS: Soft-tissue sarcoma, glioma, leukemia

MEN1

Gene assisted with: SYNDROME: Multiple endocrine neoplasia 1 MAJOR TUMORS: Islet cell adenoma, pituitary adenoma, parathyroid adenoma

RB

Gene assisted with: SYNDROME: Retinoblastoma MAJOR TUMORS: Retinoblastoma, osteosarcoma

TSC1/2

Gene assisted with: SYNDROME: Tuberous sclerosis complex MAJOR TUMORS: Cortical dysplasia, renal angiomyolipoma

WT1

Gene assisted with: SYNDROME: WAGR (Wilm's tumour, aniridia, genitourinary anomalies, growth retardation MAJOR TUMORS: Wilm's tumour

Rett Syndrome

Gene responsible: MECP2 (MIM 300005) - X-linked dominant • MeCP2 remodels chromatin and linked to specific defects in 3D folding of chromatin • MeCP2 protein represses genes - Males with the mutant X-gene, usually die in utero or soon after birth • Proposed mechanism; defective or absent MeCP2 protein might cause Retts Syndrome by failing to attach to methylated CpG dinucleotides and fail to repress inappropriate gene expression in the brai SYMPTOMS: - Compulsive hand wringing - Decrease in head growth rate. - Normal early development, followed by developmental regression.

CFTR gene

Gene showing Allelic Heterogeneity in Cystic Fibrosis

HFE Gene

Gene showing Allelic Heterogeneity in Hemochromatosis

Regulated Genes

Genes are required only under certain conditions - To reduce wasted effort these gene are regulated i.e. they are only expressed under certain conditions.

Constitutive Genes

Genes that are always expressed - often referred to as house keeping genes

Repair genes

Genes that normally limit mutations

Trisomy 13 (Patau syndrome)

Genetic mechanism is nondisjunction during oogenesis Features: • Polydactyly • Cleft lip and palate • Microphthalmia • Microcephaly • Mentalretardation • Cardiac anomalies

Trisomy 18 (Edward syndrome)

Genetic mechanism is nondisjunction during oogenesis Features: • Clenched fist, overlapping of fingers • Rocker bottom feet • Congenital heart defects • Low-set ears, small lower jaw (micrognathia) • Mental retardation

2pq

Genotype frequency of carriers (heterozygotes) in Hardy Weinberg

q^2

Genotype frequency of homozygous affected in Hardy Weinberg

p^2

Genotype frequency of homozygous normal in Hardy Weinberg

Transcriptional Control

How are genes regulated in prokaryotes? - Bacteria Mainly use this regulation method

Zone of polarizing activity (ZPA)

Hox genes are sequentially expressed in this region of cells to specify LIMB PATTERNING. e.g. - Cells in the early limb are only exposed to Hox 9 and d10 so become humerus later in development - Cells in the late limb are exposed to Hox A13 HoxD13, etc and exposure to these dictates which cells form which digits later in development

opsin gene duplication

Humans have three opsin genes. This is a recent evolutionary event as the first simians (prosimians) are likely to have only had two. Gene duplication on X chromosome must have allowed divergence in opsin gene function to give us good colour vision.

heritability of a disease

Identifying the genetic component of a complex disease

US government organized consortium

In Sequencing the Humans Genome Draft this group: -used "several" volunteers recruited from population nearby the labs involved - identity purposely hidden (not even the volunteers know as not all who donated were used) - claims to be from a "diverse" group Included: University and government labs from around the world including the U.S., U.K., Japan, France, Germany, China

Celera

In Sequencing the Humans Genome Draft this group: -used 5 individuals, - two males and three females -including Caucasian, African-American, Hispanic and Asian -first to report the completion of a draft of the human genome but the government group followed close behind.

Perforin/Granzyme Pathway

In response to virus-infected cells 1. Cytotoxic T-cell secretes perforin and granzymes. 2. Perforin forms pore in target cell. 3. Granzyme B is a protease that enters cell and activates caspases 8 and 10 by cleavage resulting in the inactivation of apoptotic inhibitors. 4. Also activates caspase 3 = execution of apoptosis

Use of PCR for the detection of triplet repeat expansion disorders

In this application, the size of the PCR product itself may be diagnostic for the disease. -->Primers flanking the repeat region will create different sized PRC products Used in: • Huntington's disease (AD) • Myotonic dystrophy (AD) • Fragile X syndrome (X-linked)

No WNT Signal

In what context does the following occur? 1) APC interacts with β- catenin 2) triggers phosphorylation 3) ubiquitination and β- catenin degradation 4) therefore, low β-catenin levels

WNT signal present

In what context does the following occur? 1) destruction complex inactivated 2) β-catenin not degraded 3) β-catenin moves to nucleus, forms complex with TCF-4 4) activates growth promoting genes

Absent cyclinD/Cdk4

In what context is: Rb hypo- phosphorylated => Rb/E2F complex binds DNA => recruits histone methyl transferase and histone deacetylase => transcriptional block => G1 arrest (cell cycle arrest) => NO Cell Division

I in 1000

Incidence of nucleotide variation (genetic variation) between two individuals in a population. Most of these are SNPs (total = 3 million differences in entire genome)

Example of Female mosaic

Incontinentia pigmenti causes patchy, darker pigmentation where the normal X has been inactivated (mutant X is active) • Areas of normal pigmentation indicate the areas where the normal X is active

Anticipation

Individuals in the recent generations of a pedigree develop disease at an earlier age and with greater severity e.g. in Huntington Disease triplet repeat expansion of CAG causes earlier age of onset - The larger the repeat, the greater is the chance for it to be unstable and it tends to expand from one generation to the next = increase in severity and decrease in age of onset in successive generations - paternal or maternal anticipation is the result of meiotic drive.

Rb

Inhibitor of E2F - involved in G1/S checkpoint regulation

Liver

Initial Location of Fetal Hb Synthesis

Ecogenetics

Interaction between environmental factors and different human genotypes e.g. Early Onset A1AT <= Smoking

LINE and the SINE

Interspersed Nuclear Elements (Long and Short, respectively). These are highly repetitive DNA sequences that are found interspersed throughout the genome. The long ones can create their own reverse transcriptase so that they can make copies of themselves, and propagate in the genome. The short ones have the ability to hijack reverse transcriptase from some other source, and they are also able to propagate in the genome.

Succinylcholine

It is a fast acting drug (approx 1min) but is rapidly metabolized by plasma butyrylcholinesterase resulting in neuromuscular blockade for 5- 10min. Genetic variation in the enzyme leads to a decreased rate of succinylcholine metabolism and hence prolonged drug action => prolonged paralysis

lac operon Regulatory protein

Lac I- Lac Repressor - turns gene off

Constituitive expression of genes (System always ON)

LacI- :Non-functional repressor, unable to bind the operator to shut off transcription.

Lactose cannot be metabolized (Lac Operon system always OFF)

LacIs: Super-repressor, will not dissociate from operator

Constituitive expression of genes (System always ON)

LacOc: Non functional operator, repressor cannot bind. Cannot be shut off

Lactose cannot be metabolised

LacP- :non functional promoter. RNA pol cannot bind - genes cannot be transcribed

Lactose cannot be metabolised

LacZ- lacY- and lacA- are mutations in the genes = Non functional proteins

Spleen

Later Location of Fetal Hb synthesis

Penitrance

Liklihood of physician diagnosing/seeing the mutation

Bone Marrow

Location of Adult Hb synthesis

Yolk sac

Location of Embryonic Hb synthesis

reason for the high mutation rate for DMD

Locus is a large target for mutation due to Largest Gene in human genome (2.5-2.7Mb).

Haploinsufficiency

Loss-of-function/expression mutations in which half normal levels of the gene product result in phenotypic effects - Examples include: --> cell membrane receptors (familial hypercholesterolemia) -->AIP (enzyme deficiency, heme can't be produced fast enough) -->OI type I

Read depth in NGS

MUST read the DNA through NGS hundreds of more times. If 50/50 distribution of nucleotide at a specific position then we can conclude polymorphism/ possible mutation at that sire.

Allelic Heterogeneity

Many allele variations of the mutation of SAME Gene e.g. Hemophilia A, Hemochromatosis (HFE Gene), CF (CFTR Gene), NF (NF-1 Gene) - Gives rise to the possibility of a compound heterozygote of a recessive mutant allele having disease phenotype

androgen insensitivity syndrome

May be caused by loss of function mutations in the gene encoding the androgen receptor - The AR binds dihydrotestosterone. - These XY males have female genitalia, breast and a blind vagina, absent uterus and abdominal testis

Slippage

Mechanism for triplet repeat expansion in which: 1) DNA polymerase replicates a repeated DNA sequence (eg. Trinucleotide repeat). 2) DNA polymerase is arrested and dissociates from the newly synthesize strand. 3) The newly synthesized strand separates from the template and realigns with another repeated sequence. 4) DNA polymerase reloads and replication resumes. NOTE: repair proteins may misrepair the repeat by adding to the parent strand.

unequal crossing over

Mechanism for triplet repeat expansion: 1) During meiosis two pairs of sister chromatids line up. In the case of repetitive sequences, a repetitive region of one chromatid does not line up exactly with its corresponding repetitive region in the other chromatids. 2) This will result in two germ cells: one with a shortened repetitive region and another with an expanded repetitive region.

Coenzyme supplementation

Mechanism of mutant apoenzyme response to administration of its cofactor at high doses. - Vitamin-responsive enzyme defects are sometimes due to mutations that reduce the normal affinity of the enzyme protein (apoenzyme) for the cofactor needed to activate it. - In the presence of high cofactor concentrations that result from the administration of up to 500 times the normal daily requirement, the mutant enzyme may acquire a small % of activity (often enough to restore normalcy)

BRCA1 protein function

Mediates: - Damage Sensor - Chromatin remolding - Bloom Helices - RECOMBINATION REPAIR System (Homologous recombination repair) - Cell Cycle Control - Regulation of Apoptosis

Generation of SKY FISH probes for whole chromosome analysis

Method 1) Sequences unique to each chromosome are created and pooled into individual tubes. 2)Probes unique to each chromosome are created by labeling with a different colored fluorescent molecule. 3)These labeled DNA probes are combined (mixed), and hybridized to chromosome spreads. 4)Labeled chromosome spreads are analyzed by fluorescence microscopy either manually or using automated computer systems.

LOD (Logarithm of Odds) score

Method of Scoring: • Data from multiple families is combined to get better statistical significance - Attempt to demonstrate or disprove linkage Pitfall: Locus heterogeneity will confound results

SNP microarray (SNP chips) Analysis

Method: 1. DNA obtained from an individual, ssDNA prepared, shredded into small lengths 2. This ssDNA is labeled with fluorescent tag to form ssDNA* 3. ssDNA* is used to find the immobilized probe on the chip 1. A positive signal indicates the presence of a particular SNP within the genome of that particular individual 2. Advantage: thousands of probes (spots on the array) may be searched in a single experiment

PCR-Agarose gel electrophoresis of a patient with Huntington's disease

Method: 1. DNA sample from the patient is obtained 2. PCR amplification of the locus containing the CAG repeat 3. Agarose gel electrophoresis to separate the DNA fragments based on the size of the fragment 4. After staining with ethidium bromide DNA will fluoresce under UV light; can see that shorter fragments move faster than the larger fragments. Can diagnose HD presymptomatically

Western Blot

Molecular Analysis tool used to detect and quantify the amount of a particular protein: Method: 1. -electrophoresis of protein 2. -transfer nylon membrane 3. -detection using a labeled antibody system (typically an enzyme label and a chemiluminescent substrate)

RFLP (Restriction Fragment Length Polymorphism)

Molecular Diagnosis tool allows for DIRECT detection when mutation interferes with restriction site. NOTE: mutation may be from: 1. Creation of new site (extra bands, smaller segments) 2. Loss of normal site i.e. loss of recognized palindrome sequence (less bands, larger segments)

Southern blot

Molecular Diagnosis tool used to detect insertions or deletions because size of fragments may change Principle: - DNA is digested with a RE & the fragments separated on the basis of size (agarose gel) - DNA is then transferred onto a membrane by blotting (invented by Ed Southern). - Sequence of interest is analyzed using a labelled probe.

PCR-RFLP

Molecular Diagnostic PCR tool used in Sickle Cell Anemia Diagnosis

Sizing of PCR Products

Molecular Diagnostic PCR tool used to determine - HD severity - Forensics/ Paternity testing using Simple Sequence Repeats (SSR)

DNA sequencing of PCR products

Molecular Diagnostic PCR tool used to determine the sequence of PCR products

ASO PCR

Molecular Diagnostic PCR tool used to determine: - Rare familial mutations, - Research tool, - Difference between genotype and gene expression; epigenetics, - X-inactivation

Tyrosine Kinase Inhibitor

Molecular Mechanism of Action of Imanitib mesylate in management of Chronic Myeloid Leukemia.

demethylating agent

Molecular Mechanism of action of 5-azacytidine

inhibit histone deacetylation

Molecular Mechanism of action of Butyrate Compounds

Euploidy

Monopoloidy in humans = germ cells no known examples of parthenogenetic development in humans •triploidies occur in ~ 6% of spontaneous abortions •triploidy is incompatable with life (pregnancy usually terminate in 1st trimester) •shows severe intrauterine growth restriction particularly of the trunk region • triploidy is an accident and having one triploid pregnacy does NOT mean your risk of having another is increased • there is no increase in frequency associated with maternal age • If the pregnancy is a partial molar one there is a small risk of cancer due to molar cells left behind but the prognosis for this type of cancer is good

age dependency of cancer

Most cancers occur with the 5th to 6th power of age, suggesting that they result as a consequence of multiple independent events <50yrs suggests genetic inheritance

Pi M

Most common SEPINA1 (A1AT) Gene allele in normal population

Alu

Most common SINE in humans -named after restriction enzyme that recognizes the sequence

Pi Z

Most common allele deficiency in A1AT deficiency (95% of cases)

CMT1A

Most common mutation in Charcot-Marie Tooth Disease - Autosomal Dominant - duplication in PMP22

Genes are hot spot for mutation

Mostly due to large size of gene Examples: - NF1 - FGFR3 - Dystrophin Gene

Polymorphism

Multiple forms (alleles) of a gene in population

Cystathione B-synthase

Mutant apoenzyme with defective cofactor binding site in homocystinuria

De-novo point mutations in DMD & BMD

Mutation Formed during spermatogenesis. -If this sperm fertilizes a normal ovum, it will result in a carrier female.

VHL

Mutation in this Gene causes Von Hippel-Lindau Syndrome Major Tumor: Hemangioblastoma, pheochromcytoma, renal cell carcinoma

5a-reductase type II Mutation

Mutation in this protein's gene causes: - child has ambiguous genitalia at birth because: - Inability to convert testosterone to dihydrotestosterone - However during puberty the 5a-reductase type I is synthesised so dihydrotestosterone may be formed • These intersex boys then masculinize during puberty • An important consideration when deciding to rear a child as a boy or a girl if they have ambiguous genitalia

Mutation in Splice Acceptor Site

Mutation may: - Lead to reading frame change - Lead to loss of an exon

Retinoblastoma

Mutation of Rb gene on chromosome 13 -a childhood cancer - Incidence: 1 in 20,000 births - high probability of LOH Mutation => other tumors after on in life

Germline mosaicism

Mutation on found in gametes NOT somatic cells Pedigree: 1. presence of two affected children 2. There is NO family history of the disease

Mutation in Splice Donor Site

Mutation: - Always cause an intron to be included in the mRNA (RNA looks bigger by northern) - May shorten protein product if stop codon is in the intron (in this case the protein is smaller or not detected by western)

De-novo large deletion mutations in DMD & BMD

Mutations Occur during oogenesis. - If the mutant ovum is fertilized by a normal 23X sperm, the female will be a carrier, unless skewed X-inactivation occurs.

Locus Heterogeneity

Mutations in two different genes, at two distinct chromosomal locations contribute to the same phenotype e.g. COL1A1/2 related OI

Digenic disorders

Mutations in two genes (independent genetic loci) are additive and necessary to produce the disorder NOTE: DO NOT confuse this with Locus heterogeneity where you only need one mutation to express disease phenotype

Four classes of triplet repeat disorders

NOTE: Repeats are unstable, and repeat size of influences disease severity - explains phenomena called anticipation

Tyrosine Kinase Receptor Activation

NOTE: Truncations or point mutations can render a receptor constitutively active e.g. mutation in the extracellular domain and distal tyrosine kinase domain of FGFR3 => constitutive activation => Thanatophoric Dysplasia

Oncogene-induced senescence (OIS)

Negative feedback loops abolish the increased mitogenic signaling resulting from the oncogenic mutations -cells that gain oncogene signalling without loss of p53 eventually enter oncogene-induced senescence

Sulfadimidime

Non-toxic Drug used to Test for Acetylator Status of Patient

Tumor Progression

Normal --> Hyperplastic --> Dysplastic --> Neoplastic --> Metastatic - results from waves of mutation followed by clonal expansion.

Her2 in breast cancer

Normal Her2 but over-expressed.

thalassemias

Nucleated red blood cells are one of the diagnostic features of these types of blood diseases in general

Burkitt lymphoma

Oncogenic activation by translocations (a.k.a. illegitimate recombination) leading to activation of myc - Myc oncogene is fused to immunoglobulin locus (t 8:14) - Myc is a transcription factor important for G1/S transition -Level of oncogene expression is increased as myc is now under the regulation of IgH promoter ( -Increased myc production -Lymphocytes fail to differentiate (undergo rapid cell divisions) = cancer

Chromosome-specific unique sequence probes OR Gene specific probes

One of the two types of FISH Analysis. Characteristics: - Hybridizes to a specific locus on a chromosome (only one locus) - These probes are useful for identifying: 1. submicroscopic deletions, 2. translocations and 3. duplications of genes

Chromosome painting - or SKY (Spectral karyotyping)(SKY FISH)

One of the two types of FISH Analysis. Characteristics: - Uses whole chromosome specific probes to paint each chromosome a different (unique) fluorescent color - These probes are useful for characterizing: 1. complex chromosomal rearrangements and 2. identifying the chromosomal origin of rearranged genetic material e.g. A highly aberrant karyotype associated with a highly aggressive primary ovarian carcinoma.

Sanger sequencing

Original method: X-ray film Modern Method: Sequencing using labeled dideoxynucleotide and computer analysis

Primaquine

Oxidant Anti-malerial Drug

Acetanilide

Oxidant Antipyretic/Anelgesic Drug

Sulfamethoxazole

Oxidant Sulfonamide Drug

Friedreich Axatia

PHENOTYPE: - Onset typically occurs between 5 to 15 years of age with late onset occurring between between 20 to 35 years. - the first symptoms are difficulty walking and loss of tendon reflexes in the ankles and knees - Later slurring of speech, and vision and hearing loss. - Associated heart disease is the most common cause of death - Generally 10 to 20 years after onset the individual is confined to a wheelchair and may be completely incapacitated. -ataxia and muscle weakness -vision and hearing impairment -scoliosis of the spine -diabetes

Spinocerebellar ataxia

PHENOTYPE: highly variable symptoms include: -Lack of muscle coordination -Lack of coordination of hands, eyes and speech -Generally full mental capacity is retained

Variable Expressivity

Patients dont express with same severity

Autosomal Dominant

Pedigree exhibits -->VERTICAL INHERITANCE (skipped generations not common) -->Males and females affected with equal frequency -->male to male transmutation is seen

Autosomal Recessive

Pedigree exhibits: --HORIZONTAL INHERITANCE (seen in only one generation) ---Siblings more commonly affected. -Only expressed (phenotype) in the homozygous state. -Both parents are usually carries -Carriers are phenotypically normal

Assortative mating

People associate and marry with "like" e.g Hearing or visually impaired individuals, or couples with phenylketonuria marry usually each other

99.9%

Percentage of identical nucleotide sequences in all humans found in the Human Genome Project using representative human genome

N-acetyltransferase 2 (NAT2)

Phase II drug metabolizing enzyme that metabolizes the anti- mycobacterial agent (tuberculosis), isoniazid and other drugs via acetylation. Slow acetylators (metabolize the drug slowly and have high blood conc.) or fast acetylators (metabolize the drug quickly and have low blood conc.).

Pleiotropy

Phenomenon of a disease causing mutation affects multiple organs e.g. Marfan syndrome; Osteogenesis Imperfecta

Variable Expression

Phenomenon where in Individuals who have inherited the same mutant allele, only some individuals are severely affected and others are mildly affected • Three reasons: - random chance - other genetic factors (modifier loci) e.g. bad poly T tract - environmental exposure e.g. smoking in A1AT NOTE: In pedigree, less severe and more severe expression look the same = shaded for having disease

Neu Oncoprotein

Point Mutation of Her2 Receptor (Val-Gln) - The abnormal protein is active even in the absence of a signal (constitutively active)

Heterozygote

Possess different alleles at a specific locus

Homozygote

Possess the same alleles at a specific locus

Stem cell therapy

Post-somatic cell nuclear transfer to enucleated egg: - The egg, now containing the genetically corrected genome of the patient, is activated to develop into a blastocyst in vitro, and corrected autologous stem cells are derived from the inner cell mass. - The stem cells are then directed to differentiate into a specific cell type and transferred to the patient, thereby correcting the disorder. => Nuclear reprogramming by factors in the cytoplasm of the ova make the nuclei "reset" to behave as embryonic tissue

Germ Line Mosaicism

Presence of more than one cell line in the gametes in an otherwise normal parent and is the result of a mutation during EMBRYONIC development of that PARENT =>mutation occurs during germ cell development and may involve multiple sperms and egg cells =>increased risk of having more than one affected child in spite of not being affected with the trait.

Recurrence Risk

Probability that the offspring of a couple will express the genetic disease. For single gene disorders, it does not depend on the number of previously affected/unaffected offspring. Depends on mode of inheritance of a disease

Drosha

Processes long primary- miRNAs to pre-miRNA hairpin structures

A1AT Deficiency

Progressive degradation of the elastin of alveolar walls. This results in decreased elasticity of lung alveoli. Inheritance: autosomal recessive 1. Severe Lung Damage (assoc. Z allele of A1AT) - alteration in balance of elastase: A1AT = Loss of Function Mutation Z/Z - 15% normal concentration of A1AT; 20% of ability to inhibit neutrophil elastase

Multiple myelomas produce a monoclonal Ig

Proof of monoclonal nature of tumors seen in Multiple myeloma (a malignancy of B-cell (precursors of antibody- producing plasma cells). NOTE: normal tissue has is polyclonal Ig

all cells in the tumor contain chromosomal aberration

Proof of monoclonal nature of tumors using Chromosomal abnormalities.

all cells in tumor have the same copy of the X chromosome inactivated

Proof of monoclonal nature of tumors. NOTE: All tissues are mosaic for X- inactivation

Doubles every 17min

Rate of cell division in E. coli

allelic heterogeneity

Reason for compound heterozygotes in thalassemias: Gene deletions, point mutations, splicing site changes, removal of regulatory sequences outside of b-globin genes all cause the clinical manifestations of b-Thalessaemia.

low recurrence risk

Recurrence risk for new mutation

Vitamin K Epoxide Reductase

Reduced affinity of this enzyme for warfarin contributes to pharmacodynamic variation in warfarin efficacy. - Encoded for by the Vitamin K epoxide Reductase Complex 1 (VKORC1) - VKORC1, polymorphic gene - dose requirement may be 2-fold.

Treatment of b-Thalassemia manifestations

Regular transfusions correct the anaemia and suppress erythropoiesis, but lead to iron deposition . -->Iron chelation and dietary control can manage these complications • The only available definitive cure is bone marrow transplantation from an HLA-identical sibling. - EXPENSIVE!!!!

p21

Regulated by p53 (p53 is the transcription factor for this protein's gene) - similar to p16 in action i.e. inhibits CDK4 => hypomethylated Rb => Rb-E2F complex => recruiting of Histone MT and HDAC => G1 arrest

Induction of the Lac operon

Regulation Stage of Lac Operon when Lactose Present Mechanism: 1. Lactose always contains a little of its isomer allolactose (1,6 Glycosidic Bond) 2. Allolactose binds to the repressor protein 3. repressor undergoes a conformational change and dissociates from the operator sequence 4. RNA polymerase is then free to initiate transcription and make the poly cistronic mRNA (LacZ LacY LacA). RNA. HOWEVER the RNA polymerase needs activating (by catabolite activator protein or CAP) to make lots of the polycistronic RNA for the three enzymes to be manufactured

Epigenetics

Regulation of chromatin conformation to alter transcription 1) Gene methylation 2) Histone acetylation

Positive regulation of Lac Operon

Regulation when Glucose is ABSENT - Lac Operon System Turned ON 1. When glucose runs out a cAMP is produced by active adenyl cyclase (converts ATP to cAMP) 2. cAMP binds CAP, activating it. 3. Active CAP (cAMP-CAP) binds the lacP sequence. 4. Active CAP allows RNA polymerase to more effectively initiate transcription. 5. More mRNA more protein Lac Operon Most Active when Glucose absent, lactose present

Cytochrome c

Release from the mitochondria is the result of channels formed in the membrane by Bax and Bak - release leads to the activation of caspase 9 - forms the apoptosome with Apaf-1 - Can be often used as a measure experimentally to indicate apoptosis (rather than necrosis)

AP endonucleases

Repairs Apurinic and apyrimidinic (AP) sites that result from the action of enzymatic base removal or spontaneous deamination - Cleaves the phosphodiester backbone 5' and 3' to the AP site - a form of nucleotide excision repair. 28

premutational Triplet Repeat

Repeat numbers that are between normal and pathogenic numbers and sometimes have a mild, late onset phenotype

Dominant

Require only one copy of the mutation to produce disease

requirements for linkage mapping

Requirements: - Large family with several affected individuals - Linkage map (markers near the region of interest)

Recessive

Requires two copies of the mutation to produce disease

Hypoxia Response Element

Response Element found associated with the following genes: 1. VEGF => Angiogenesis 2. Glycolysis Genes => Anaerobic Respiration 3. iNOS/HO-1 => vasodialation, respiration 4. EPO => Erythropoiesis 5. BNIP3 => Apoptosis

HbBart

Result of a having a deletion/mutation in both a-globin genes on both chromosomes (i.e. deletion of all 4 a-globin genes) - 4 gamma chains come together -lethal in fetus -hydrops fetalis (better name)

Mosaic

Result of a nondisjunction in Embryogenesis (not seen when occurs in oogenesis)

Heterozygote

Result of having 2 or 3 total a-globin genes normal (can be on the same chromosome and/or shared between the two chromosomes

HbH

Result of having one total normal a-globin gene out of the 4 possible genes normal - Beta-chain tetramer so instead of HbA(a2b2) they are b4. (at birth you may see HbBart, but is a minor compartments and disappears to trace amounts)

Aerosol mediated gene therapy

Results of this kind of vector delivery method: -low uptake of virus -transient results -inflammatory response clears gene therapy delivery virus before cell infection & may result in additional tissue damage => It works, but only transiently and not efficiently , and it can't be done multiple times - NOT effective (failure) NOTE: BOTH adenovirus or with a liposomal DNA approach FAIL in treatment of Cystic Fibrosis

Thiopurine-S-methyltransferase (TPMT)

S-methylation of anticancer drugs - mercaptopurine and azopurine but also thioguanine - treatment of leukemia, thematic disease & inflammatory bowel disease - NARROW THERAPEUTIC WINDOW -> mylosuppression - Risk increased by low enzymatic activity - 1:300 possess low enzyme activity - 3 alleles: (i) TMPT*2 (ii). TPMT*3A (iii) TPMT*3C - Low activity patients given 1/10 std dose - Efficacy of enzyme also affect by GSTM1 & thymidylate synthetase

Thiopurine S-methyltransferase (TPMT)

S-methylation of anticancer drugs - mercaptopurine, azothiopurine, thioguanine - treatment of leukemia, rheumatic disease and inflammatory bowl disease - NARROW THERAPEUTIC WINDOW - mylosupression --> risk increased by low enzyme activity 3 Alleles: - TPMT*2, - TPMT*3A, - TPMT*3C -Polymorphisms least to increased mylosupression at standard dose -Low Activity patients given 1/10 std dose

Array technology Vs. single gene analysis

Single gene analysis gives a result for one item. Array methods may test 1000's The single analysis will always be useful for confirmation. If a gene is shown to be overexpressed by a cDNA microarry, the northern blot may be used to confirm it.

Reduced genetic fitness

Some Individuals who have the disorder are still able to reproduce e.g. any disorder which has a negative impact on a person's ability to have a child.

Mutational Hotspots

Some sites are mutated at a much higher frequency than other sites. -Not all positions are equally mutable, hotspots include things like 5-methyl cytosine or repeated bases (AAAAA)

Population Genetics

Study of genetic variations in populations

Caffeine-Halothane Muscle Contracture Test

TEST FOR MALIGNANT HYPERTHERMIA 1. Take Muscle Biopsy 2. Split into Strips 3. Muscle placed in a device to measure force of contraction and then expose to caffeine or halothane Abnormal Muscle produces >0.2g tension to 2mM Caffeine Response of 0.6g tension to 1mM caffeine, 1.6g tension to 2mM caffeine Halothane response of 3% dose (Abnormal >0.5g tension) Normal muscle will not change baseline by more than 0.5g tension

Tuberculosis

Tay-Sacks Disease gives resistance or advantage to this disease.

Twin Studies

Test Group: Monozygotic Twins Control Group: Dizygotic Twins => Test Disease concordance in MZ twins concordance = 100% genetically determined concordance <100% non-genetic factors involved =>The greater discordance = the greater environmental input e.g. Female Alcoholism: MZ Twins = DZ twins => not genetic; environmental Male Alcoholism: MZ>DZ => GENETIC contribution Autism: MZ>>DZ => MOSTLY GENTIC

Analytical Validity

Test Result Accuracy. --> high for DNA-based tests

CpG Islands

The "p" notation refers to the phosphodiester bond between the cytosine and the guanine. - 300-3,000bp in length - Found in about 70% of human promoters - Where Genes expressed islands of promoters are unmethylated - Where Genes silenced Methylated promoters tend to be shut down

two hit hypothesis

The Hypothesis that for tumor suppressor genes, two mutations are needed for tumor initiation i.e all loss of tumor suppressor activity must be lost for cancer to ensue. 1st "hit" = spontaneous or familiar (inherited) 2nd "hit" = loss of heterozygosity

SSR (Simple Sequence Repeat)

The SSR is a highly polymorphic sequence that consists of 2, 3 or 4 bp repeated tandemly in the genome. (sometimes called simple tandem repeats (STR) but this may lead to confusion with VNTR - Used in FORENSICS e.g. Mother has 3 repeats of C-A (2 bp) and Father has 5 repeats of C-A (same di nucleotide being compared), Child should have contributing bands from both.

Proband

The affected individual in the family who gains the attention of the physician due to a genetic condition

Reason for Increased risk of Cardiopathy in BMD

The cardiac muscle cells in his heart express only little amount of dystrophin protein => increased risk of having a cardiac problem in his lifetime

familial relative risk

The more closely related you are to someone with a complex disorder, the more likely you are to have some of the same allele

Consultand

The person who approaches a physician or geneticist for a consultation - may or may not be affected

Non-directive counseling

The point of this kind of counseling (used in genetic counseling) is NOT to tell the patients what to do rather it is to give them enough information (empower) so that they can make the best decision for them and their families

VNTR (Variable Number Tandem Repeat)

These are tandem repeats that are bigger than the SSR, 5bp ,10bp or 100s. These are polymorphic, but not as variable as the SSR.

Basal promoter sequence

These bind the general transcription factor proteins which are associated with RNA Pol

Proximal control regions

These bind transcription factor proteins, and are found near the promoter

LCR (Low Copy Repeat)

These can be quite large, (1,000 of bp, and bigger), and are found in only a few places in the genome, and they are usually very similar (or the same) between individuals. As a general rule, the shorter the repeat sequence, the more variability it is likely to have. - May cause mis-pairing during meiosis or mitosis - because you have introduced abnormal alignment site.

Insertions and Deletions (InDel)

These mutations have a disastrous effect on the resulting protein more often than substitutions do -may alter the reading frame, producing a frameshift mutation -Spontaneous mutations can occur during DNA replication, recombination, or repair

Down's syndrome due to Robertsonian translocation

This 5% is usually of all cases due to t(14q;21q) Trait is inherited through gametes that underwent ADJACENT segregation in Meiosis Karyotype: 46, XX, +t(14;21). Two copies of Chromosome 21; Derivative chromosome 14 has the long arm of chromosome 21

Intrinsic Pathway

This apoptotic pathway is used to eliminate cells in response to genotoxic damage, mitochondrial damage, absence of growth factors, loss of substrate adhesion, etc.: 1. Bax or Bak proteins insert into the mitochondrial membrane and causes the release of cytochrome C from mitochondria. 2. Cytochrome C binds to the adaptor protein Apaf-1 causing its aggregration and binding to procaspase (APOPTOSOME complex). 3. Procaspases are cleaved to form active caspases.

CNV (Copy Number Variant)

This describes changes in copy number but does not apply to polymorphic sites such as the SSR, and the VNTR.

p53 influence on apoptosis

This protein can influence apoptosis in several ways by increasing expression of: 1) pro-apoptotic Bcl-2 family members 2) Fas receptor (CD95) 3) IGFBP-3 (sequesters cell survival proteins like IGF1/2 away from receptors)

Genetic Fitness

This refers to the ability to pass on ones genes to offspring, the ability to reproduce NOTE: Genetic fitness can change depending on treatment available. e.g. appropriate dietary treatment for PKU will lead a normal life, and have normal genetic fitness. vs. untreated PKU patient will have severe intellectual impairment, and is unlikely to reproduce - very low genetic fitness.

mannose

This residue targets proteins to lysosomes

0%

Thomas is seen for genetic counseling because a diagnosis of MELAS has been made in his sister and brother. What is the risk of the disorder being present in Thomas's children?

Treatment of AR-SCID (ADA Deficiency)

Three possible treatments: 1. Bone marrow transplant (HLA matched patient) 2. Protein replacement therapy --> NOTE: does not completely correct immune function, but it helps PEGylated version superior to unmodified protein) 3. Gene therapy

Hypoxia Inducible Factor (HIF)

Transcription Factor used as a Therapeutic Target for: • Anemia • Inflammation • Cancer

Foetal haemoglobin inducers

Treatment for Sickle Cell

PKU-I

Treatment: - Low Phenylalanine diet (also supplement with Tyrosine) - Pharmacological doses of BH4 (sapropterin) in some patients - PAH enzyme replacement therapy is being tested

ErbB Oncoprotein

Truncation/ deletion of EGF receptor - The abnormal protein is active even in the absence of a signal (constitutively active)

Molecular Mechanism of Huntington's Disease

Two mechanisms have been proposed to account for the development of Huntington's disease: 1)Increase in polyglutamine tract length causes Huntington protein to aggregate, form inclusion bodies, and behave as a toxin. 2)Abnormal Huntington protein has been shown to interact with a number of different transcription factor proteins causes dyregulation of gene expression.

Periodic Starvation

Type 2 Diabetes gives resistance or advantage to this.

Pharmacodynamic variation

Variation in drug targets or pathways associated with these targets

Pharmacokinetic variation

Variation in proteins involved in drug metabolism and transport. Examples include: 1) CYP2D6, 2) butyrylcholinesterase 3) N-acetyltransferase 2 4) thiopurine-S- methyltransferase. NOTE: drug metabolism is not only a detoxification process but also may be an activation process e.g. cyclophosphamide --[CYP2B6]--> cytotoxic phophomustard derivative

Lipoprotein Lipase Gene Therapy

What Gene is targeted in Glybera Gene Therapy? NOTE: GLybera is the 1st Gene Therapy Drug in European Union

mismatch

Wrong base is incorporated by DNA polymerase --> due to chemistry of the nucleotides

Polydactyly

a condition of having more than the normal number of fingers. The result of disruption of the HoxD13 gene, over expression of SHH, Gli3 mutations

5-azacytidine

a demethylating agent, can elevate hemoglobin F - not as safe as Hydroxyurea -alter epigenetic gene regulatory mechanisms and may change the acetylation of chromatin proteins. Altering gene silencing patterns. Leading to more HbF - NOTE: cannot get rid of HbS completely e.g. HbS = 75% HbF = 25%

Hydroxyurea

a demethylating agent, can elevate hemoglobin F - safer than 5-azacytidine -alter epigenetic gene regulatory mechanisms and may change the acetylation of chromatin proteins. Altering gene silencing patterns. Leading to more HbF - NOTE: cannot get rid of HbS completely e.g. HbS = 75% HbF = 25%

morphogen

a molecule that diffuses to form a concentration gradient with protein abundance levels dictating cell fate

rapid technological advances

accelerated the Human Genome Project completion date to 2003 (originally plan was 15 years but ended up being a 13-year effort coordinated by the U.S. Department of Energy and the National Institutes of Health)

induced pluripotent stem cells (iPSCs)

adult cells that have been genetically reprogrammed to an embryonic stem cell-like state.

Complete Penetrance

all the people carrying the mutation, express the manifestations of the disease e.g. NF-1

Net Cell Growth

balance of Cell Cycle Control (cell birth) and Apoptosis Control (cell death)

euploidy

changes in copies of the entire genome

aneuploidy

changes in the number of individual chromosomes -most common cause is nondisjunction during meiosis = sister chromatids do not separate during anaphase, one cell gets two and the other gets none -in Meiosis I = cell with two different homologues -in Meiosis II = cell with two copies of the same homologue Result: -usually non-viable and creates a spontaneous abortion. Only a few get to term

Pyridoxine (Vitamin-B-6)

coenzyme supplementation in homocystinuria

genetic determinism

concept that genetic factors are completely determinant of phenotypic characteristics -is a misconception that can be damaging to public perception and trust of the role of genetics in medicine. It may set up unrealistic expecta- tions for the power of the genetic approach to prevent, diagnose, and treat disease. It also may discourage efforts to reduce exposure to environmental factors that contribute to disease and even create a sense of futility

Increased error rate

consequence of DNA repair gene mutations that: • produces mutator phenotype - particularly true for mutations in proofreading and MMR proteins => damaged bases are not repaired efficiently NOTE: DNA repair genes are also vulnerable to mutation

cDNA

derived from mRNA using reverse transcriptase - gives information about gene expression i.e. phenotype NOTE: DNA gives information about genotype (e.g. heterozygous, T-tract polymorphisms, intron information etc) E.g. in X inactivation of heterozygote of p55 gene, DNA shows both alleles but cDNA only shows allele on activated x chromosome

Incomplete Penetrance (Non-penetrance)

disease genotype but does not display the disease phenotype e.g. hemochromatosis (autosomal recessive), Van der Woude (autosomal dominant) - An autosomal Dominant pedigree may be complicated by this phenomenon because individual appears non-diseased

Herceptin

drug (antibody) that binds to HER2 and prevents binding of EGF to HER2 •Addition of Herceptin to the treatment results in decreased tumor cell proliferation NOTE: Very effective for Her2+ tumors but not effective for treatment of Her2- tumors

Toxins

eg. Arsenic and nickel cause hypomethylation in cell cultures; Bisphenol A found in plastics causes hypomethylation.

Temperature

eg. DNA methylation of zebrafish transgenes (Environmental Factors that have been show to influence or disrupt epigenetic gene silencing)

Hypoxia

eg. Demethylated HRE in the erythropoietin gene required for activation (Environmental Factors that have been show to influence or disrupt epigenetic gene silencing)

Diet

eg. Folate and homocysteine deficient diets alter DNA methylation (Environmental Factors that have been show to influence or disrupt epigenetic gene silencing)

Maternal care

eg. Modification of glucocorticoid receptor methylation Environmental Factors that have been show to influence or disrupt epigenetic gene silencing: Methylation patterns of this gene are established during the first week of life and maintained in the adult Reduced methylation results in higher levels of glucocorticoid receptor and an ability to handle stress better. Methylation of the promoter of the Glucocorticoid Receptor gene in Mouse pups with high and low maternal care (grooming and licking!) during the first week of birth.

Butyrate compounds

elevated in the plasma of mothers with diabetes whose fetuses have delayed "switching," - alter epigenetic gene regulatory mechanisms and may change the acetylation of chromatin proteins. Altering gene silencing patterns. Leading to more HbF - NOTE: cannot get rid of HbS completely e.g. HbS = 75% HbF = 25%

gene duplication

engine of evolution found discovered through human genome project

Genomic imprinting

epigenetic process that involves methylation and histone modifications in order to achieve monoallelic gene expression without altering the genetic sequence" • gamete-of-origin dependent modification of phenotype. • In imprinted genes, only one parental copy is active; the other copy is silent • Methylation states are 'passed on' (inherited) i.e. Maternal Expressed => only egg-chromosomes express Paternal Expressed => only sperm-chromosomes express

Double minutes

extrachromosomal fragments of DNA containing an amplified region of the chromosome -often seen in tumors where the amplified region includes an oncogene e.g. -EGFR in advanced gliomas

Dicer

further processes pre-miRNA hairpin structures to single stranded RNA and initiates the formation of the RNA-induced silencing complex (RISC).

OPN1SW

gene for blue/violet opsin

OPN1MW

gene for yellowandgreen opsin

FMR1

gene involved in Fragile X syndrome (triplet repeat expansion, X-linked)

OPN1LW

gene makes a pigment that is more sensitive to red light and mutations are responsible for the majority of red/green color blindness - 8% of males and .5% of females in some Northern European countries (x-linked)

Disease Modifying Genes

genes which contribute to severity of expression e.g. in SHH mutations where there is wide clinical variability, with family members presenting with holoprosencephaly, facial abnormalities to mild microcephaly alone

Myc-Max

heterodimer can bind DNA and transactivate homodimer cannot activate transcription!

Pyloric stenosis

hyperplasia of the muscle (pylorus) between stomach and intestines, causing it to narrow (stenosis) impeding gastric emptying -can cause severe vomiting in babies -palpation of the abdomen may reveal a mass in the epigastrium -may cause other problems such as dehydration and salt and fluid imbalances

"common-disease rare-variant" hypothesis

hypothesis posits that variants affecting health are under purifying selection and thus should be found only at low frequencies in human populations.

cyclinD/CDK4

inactivates Rb by phosphorylation - involved in G1/S checkpoint regulation

SNP (Single Nucleotide Polymorphism)

is a relatively common single base pair change which is found in approximately one in a thousand base pairs. The term polymorphism is reserved for those DNA changes that are relatively frequent in populations (most experts agree that this is ~1% of the population).

Microdeletion syndromes

large deletions but but too small to see under a microscope <5Mb Are detected by high resolution prometaphase banding, fluorescent in situ hybridization (FISH) or comparative genomic hybridization (CGH), chromosome-CGH or (microarray-CGH)

Extracellular Matrix

location of pro collagen assembly to collagen - assembly begins from C-terminus

cytoplasm of fibroblast

location of procollagen synthesis and post-translational hydroxylation

Basal transcription machinery

maintains a low level of expression

CYP450 array

may genotype a patient's CYP450 alleles Importance: Variation in the DNA sequences of members of the Cytochrome P450 gene family may affect the ability to metabolize many different drugs

Brachycephaly

meaning wide head

Concordance

means in a twin study both MZ twins have the same disease

Discordance

means in a twin study one of the MZ twins has it but the other does not

Familial aggregation

measured by frequency (prevalence) of the disease in the relatives of an affected proband versus frequency in the general population. The larger λr, the greater is the familial aggregation λr = 1 relatives have no greater risk than any individual in the population Measure λr for a particular class of relatives (e.g., r = s for sibs, r = p for parents) Example disease for relative risk calculations. Polygenic Diseases have different Relative Risks NOTE: Relative Risk is always MUCH higher for Mendelian Inheritance than Multifactorial Inheritance!

hypertelorism

mediolateral expansion of the face and ventral forebrain - occurs due to Increasing SHH in the frontal nasal prominence NOTE: True facial duplications are extremely rare in humans suggesting that they may be associated with other developmental disturbances that are incompatible with life.

CYP2C

members of this cytochrome P450 family metabolize 20% of clinically used drugs

Small nuclear RNAs

miRNA, siRNA. Never leave nucleous or get translated but regulate transcription e.g. Xist (X-inactive specific transcript) (siRNA is something we make that modifies transcription)

Consanguinity

might be a finding in an autosomal recessive pedigree (usually NOT in western nations)

Competition

mode of action of transcription factor acting as a repressor in which the repressor binds enhancer sequence on DNA and competes with enhancer proteins - reduces transcription by preventing enhancer protein binding

Quenching

mode of action of transcription factor acting as a repressor in which the repressor binds to the activator protein and blocks its ability to bind the DNA site - Repressor soaks up the activator protein - Activator protein is not able to bind to the DNA

Blocking

mode of action of transcription factor acting as a repressor in which the repressor binds to the activator protein's activating domain and prevents it from interacting with general transcription factors - The activator protein still binds to DNA, but it is not functional as it cannot interact with the rest of the transcriptional machinery

Array comparative genomic hybridization (array CGH)

molecular cytogenetic technique that allows detection of copy number variants (CNV) i.e. amplifications or deletions BUT CANNOT detect balanced rearrangements Advantages: Useful in Tumor genetics - As cancer progresses, the extent of chromosomal abnormalities increases -CGH can quickly analyze these changes where using single probes or FISH staining may take a long time to detect all the changes.

Oncogenes

mutant or misrelated (usually upregulated) forms of porto-oncogenes e.g - Anti-apoptotic: BCL2 mutated to be upregulated - GF Receptor: c-erb (epidermal growth factor receptor kinase) e.g. HER-2 in breast cancer - G protein/ signal transduction: ras -Intracellular Tyrosine Kinase: abl - Transcription Factors: c-myc, c-fos, c-jun

Transcripion factor complexes (Trans acting factors)

nuclear proteins bind to promoter or enhancer sequences of genes and modify transcription

acrocentric

only chromosomes are 13, 14, 15, 21, 22 - Involved in Robertsonian translocation

apoptosome

only ever formed with the intrinsic pathway Protein complex made up of Apaf-1 + CytC + Caspase 9

Angelman Syndrome

prevalence of 1/12,000 also called "happy puppet" syndrome - holding hands in funny puppet way, always smiling, always happy. Severe developmental delay, little or no speech, movement or balance disorder, ataxia, most have seizures, hyperactive, wide mouth, usually happy. Often use augmentative communication device.

Prader-Willi syndrome

prevalence ~ 1/10,000 Hypotonia and failure to thrive in infancy, rapid weight gain 1 to 6 years, round face, almond shaped eyes, hypogonadism, 40% borderline and 40% mild MR Behavior problems including voracious appetite (elevated ghrelin), food hoarding and other OCD behavior, skin picking, sleep disturbance/sleep apnea, short stature, hypopigmentation, hypofunctioning of the hypothalamus

PKU-II

prognosis is always worse - Blood-brain barrier keeps BH4 supplements out.

50%

recurrence risk for every new child born to couple with autosomal dominant disease (with 100% penetrance) heterozygote parent

37.5%

recurrence risk for every new child born to couple with autosomal dominant disease (with 75% penetrance) heterozygote parent

mRNA Stability regulation (method of gene regulation)

regulation of Localisation and/or Degradation of mRNA.

mRNA processing regulation (method of gene regulation)

regulation of polyadenylation Splicing of mRNA

mRNA Transport regulation (method of gene regulation)

regulation of: - Nucleus to Cytoplasm transport; mRNA - storage and choosing when to translate

Gene repression

results when only the Max polypeptide is made in the cell

International System for Human Cytogenetic Nomenclature (ISCN)

short arm = p long arm = q centromere = 10 (one-zero) loci numbered from centromere to telomere e.g. 1p31.1 1 = Chromosome 1 p = Short Arm 31 (three-one) = Region 3, Band 1 .1 = Sub-band 1

ASO (Allele Specific Oligonucleotide) probes

short oligonucleotide sequences (~15 bp long) that specifically bind to a single allele of a gene (possible only if the exact mutation in the gene has been identified) - less useful in disorders with increased allelic OR genetic heterogeneity e.g. CF and hemochromatosis

Response Element

short sequences of DNA within a gene promoter region that are able to bind a specific transcription factor and regulate transcription of genes"

Beta-Globin chain

site of the sickle mutations

Cis regulatory sequence (Cis regulatory elements)

small DNA sequences near promoter (eg. enhancer sequences) -- DNA motifs such as transcription factor binding sites or enhancer binding sites

Genetic Basis for Paternal Age effect on ccurrence of new Autosomal dominant mutants

spermatogonia continually divide & sperm cells from older fathers may contain replication error mutations (point mutations)

Burkitt's lymphoma

t(8;14) => What Disease?

Rare variants

tend not to be shared between these populations (are private), meaning that they arose more recently.

Common variants

tend to be shared between the African-American and the European-American populations, meaning that they are ancient.

Tautomerism

the ability of certain chemicals to exist as a mixture of two interconvertible isomers, leading to mismatch

Clinical validity

the likelihood that the presence of a sequence variant correctly diagnoses a condition, or its absence indicates that the condition will not occur i.e. diseases with 100% Penetrance e.g. NF1

pyknosis

the process of chromatin condensation forming patches against the nuclear envelope

Genetic counseling

the process of helping people understand and adapt to the medical, psychological and familial implications of genetic contributions to disease.

karyorrhexis

the process of nuclear envelope becoming discontinuous and the DNA is fragmented

Pharmacogenetics

the study of how genetic variation affects drug response

Regulators

these proteins act to either inhibit apoptosis (eg. Bcl-2 and Bcl-xL) or initiate the apoptotic pathway (eg. Bak and Bax)

Retroviruses

this gene therapy vector can insert the genetic material into the genome

Adenoviruses

this gene therapy vector is able to get to the nucleus and the genetic material exists as a separate entity (extrachromosomal)

Methylated cytosines

this type of nucleotide base produces a mutational hotspot

E2F

transcription factor required for G1/S transition (=> transcription of S-phase genes)

pancreatic enzyme supplements

treatment for fat malabsorption in CFTR patients (90% have pancreatic insufficiency)

p53

tumor suppressor that controls both cell birth and cell death = transcription factor activated by cell stress, particularly DNA damage and it impinges on the G1/S checkpoint. = "guardian of the genome" Function: 1) G1 phase arrest - a) arrests cell cycle, allows time to do DNA repair b) increases DNA repair capabilities 2) Activate Cell Death- if damage is too great => accumulation of high levels of p53 => initiates apoptosis

Adapters

typically these proteins act by binding to procaspases (inactive caspase) and either facilitate proteolysis of procaspases to active caspases directly or cause the aggregation of procaspase resulting in self activation. eg. Apaf-1

Lac Operon

~6000bp long collection of genes involved in sugar metabolism •Four proteins are coded in the operon (polycistronic) •Metabolise the sugar "lactose" if there are no other energy source • All three genes for the proteins (b-galactosidase, Permease, Transacetylase ) share the same promoter (LacP),the same operator (LacO) and are transcribed as a single mRNA (polycistronic). • The gene for the regulatory protein (LacI) is found near the operon.

Molecular Basis of F508del mutation

ΔF508 is the predominant mutation in Caucasian CF patients - Detected in ~66% of all CF patients as at least one disease allele. - Prevalence varies between populations - It results from 3bp (AGA) deletion of a phenylalanine residue of CFTR at position 508 on the protein. - CFTR never gets to plasma membrane. The CFTR protein never makes it through the ER, it is recognized as a misfolded protein, and is targeted for degradation by proteasome

Angelman syndrome

• "Happy Puppet syndrome" • Happy disposition, laugh inappropriately • Severe mental retardation, seizures • Puppet like posture of limbs Cause: 1. Deletion of maternal 15q11-13 (absence of active UBE3A gene) 2. Uniparental disomy of paternal chromosome 15

Chromosomal Microarray Analysis is very useful in diagnosis of birth defects

• A baby is born with birth defects: 1. G-band karyotype looks normal 2. Next do chromosomal microarray analysis (Array CGH and a SNP chip) - This test is more likely to detect an abnormality

Mitochondrial Inheritance

• ALL offspring of an affected female are affected • Only females transmit the disease • Affected males do NOT transmit the disease • Both male and female children of an affected female are affected

Histone modification

• Alters the interaction of the DNA and the histone tails or allow access to other factors • Histone tails particularly highly modified: • Modifications - Methylation - (De)acetylation - Phosphorylation - Uitquitination - Sumoylation

Gaucher disease

• Bone marrow becomes damaged • TREATMENT: enzyme replacement therapy PEGylation of recombinant β-glucocerebrosidase protein --> RESULTS OF THERAPY: improves Hb levels ↑platelets, ↑growth ↓liver/spleen enlargement ↓skeletal abnormalities

Prader Willi Syndrome

• Caused by: - Microdeletion of paternal chromosome 15 (70% of patients) - Maternal uniparental disomy of chromosome 15 • Genetics: 1. micro deletion of Paternal 15q11-13 (absence of SNRPN) - Microdeletion of this region in paternal chromosome → this syndrome (about 70% of cases) - Can be detected by FISH using specific probes against the region 2. Uniparental disomy - two copies of the maternal chromosome 15 (absence of paternal chromosome 15)

Modes of management of genetic disorders

• Coenzyme supplementation e.g. Vitamin B-6 in Homocystinuria • Dietary restriction of substrate e.g. Phenylalanine restriction in PKU • Chemical diversion e.g. Sodium Benzoate mediates diversion of NH3 to glycine synthesis in Urea Cycle Disorders • Protein/ enzyme replacement e.g Factor VIII replacement therapy in Hemophilia A • Targeting signal transduction pathways: - Enzyme inhibition (Gleevec for Ph+ CML) - Specific antibodies (HER-2 antibodies) • Epigenetic modification e.g. hydroxyurea increasing HbF in Sickle Cell Anemia • Gene therapy e.g. Glybera for lipoprotein lipase • Stem cell therapy • RNA interference (miRNA and siRNA)

Autosomal Recessive disorders

• Cystic fibrosis • Sickle cell anemia • Phenylketonuria • Tay-Sachs disease (Hexosaminidase A deficiency) • Congenital deafness (autosomal recessive) • Hemochromatosis (delayed age of onset) • Alkaptonuria (delayed age of onset) • Homocystinuria • Galactosemia - a1-antitrypsin deficiency • SCID due to adenosine deaminase deficiency - Severe combined immune deficiency • Most enzyme deficiencies are AR - Beware those that are X-linked, and IAP (Intermittent acute porphyria)

Wolf- Hirschhorn syndrome

• Deletion of ch 4p • Facial anomalies widely spaced eyes, prominent nose, abnormal iris • Cardiac anomalies • Mental and developmental delay

Disorders with non-Mendelian (unexpected) inheritance patterns

• Digenic disorders - Retinitis pigments • Imprinting - Prader Willi syndrome - Angelman syndrome • Triplet repeat disorders - Huntington disease - Myotonic dystrophy - Fragile X syndrome (X-linked)

Achondroplasia Biochemistry

• FGFR3 mutations • FGFR3 codes for a transmembrane receptor that is involved in differentiation of cartilage to bone • 80% De novo mutation • G-C transversion at nucleotide 1138 - this site is considered to have one of the highest mutation rates in the human genome (located at a CpG site). - However, the hot spot maybe be an artifact of sperm with FGF3R mutations having a selective advantage. • Mutations in FGFR3 result in severe stunting of growth - the mutated form of the receptor is constitutively active always signals to stop chondrocyte production - preferentially affects long bones (limbs) • Dominant Negative • Gain-of-function mutation - Heterozygote has short stature, homozygote is lethal

In vivo Gene therapy

• Gene therapy attempts to get genetically modified cells directly into the patient -e.g. X-SCID • Gene therapy is not limited to stem cells: -e.g. CFTR gene therapy using liposomes or adenovirus via nasal sprays

Ex vivo gene therapy

• Gene therapy involves removal of cells from the patient, modification in vitro and then return to the patient • Gene therapy may use stem cells or differentiated cells - Treatment of fibroblasts from patients with hemophilia B by the addition of the factor IX gene. Modified fibroblasts are then injected into the stomach cavity (differentiated cell example)

Deletions in chromosome 15

• Genetic Basis of Angelman & Prader Willi syndrome • Angelman syndrome is due to deletion of maternal chromosome, whereas Prader Willi syndrome is due to deletion of paternal chromosome

Max

• Homodimerises when there is no mic available => gene repression

Diseases displaying Delayed age of onset

• Huntington disease (AD) • Hemochromatosis (AR) • Familial breast cancer

Down Syndrome dysmorphology

• Hypotonia, • Brachycephaly (wide head) • Upslanting palpebral fissures • Brushfield spots of iris • Adult cataracts, • Clinodactyly • Single palmar crease

Bcl-2

• In healthy cells there is a fine balance between p53 [proapoptotic] and this anti-apoptotic protein. • p53 can directly bind to to this protein and inhibit its activity • Over expression of this protein can prevent apoptosis in cells that are damaged • Up regulation of this protein is common to many human tumors NOTE: over expression does not result in increased cell division. It inhibits cell death by BLOCKING release of CYTOCHROME c

Mitochondrial diseases

• Leber hereditary optic neuropathy - Manifests as progressive blindness around 20-30 years • MELAS: Mitochondrial encephalopathy, lactic acidosis, and stroke like episodes • Myoclonic epilepsy with ragged red muscle fibres (MERRF)

Tumor cells Apoptosis Evasion Strategies

• Loss of p53 • Downregulation of BAX, BAK • Upregulationofantipoptoticproteins e.g. BCL2 • Upregulation of IAPs (inhibitor of apoptosis proteins) e.g. Survivin

Reifenstein syndrome

• Lower androgen receptor levels • have micropenis, gynecomastia and azoospermia.

DNA methylation

• Marks CpG bases and repress gene activity - one of the Two main components of epigenetic regulation - Requires Folic Acid and SAM as cofactors!!!!

Inversions

• May be pericentric (involves the centromere) or paracentric (does not involve the centromere) • Usually balanced & no clinical problems in carriers • Inversions usually result in a change in the banding pattern of the chromosome and can be identified by karyotype analysis

Methylation analysis

• Methylation sensitive restriction enzyme analysis • The restriction enzyme does not cleave methylated DNA • Paternal chromosome 15 at this region is cleaved by the enzyme • May be exploited by Southern blot

Heteroplasmy

• Mitochondrial DNA segregates passively when a cell divides. • Unequal distribution of mutant and non-mutant mitochondrial DNA , results in the phenomenon of heteroplasmy in mitochondrial disorders. • Explains the variable expression of the disease in the offspring.

Trisomy 21 (Down syndrome)

• Most common autosomal trisomy • Risk factor: Increased maternal age => increases risk of meiotic nondisjunction during oogenesis (most common is meiotic I nondisjunction) -Features: •Mental retardation •Short stature •Congenital heart defects Facial features •Depressed nasal bridge •Upslanting palpebral fissures •Epicanthal fold Hand feature •Single palmar crease • Develop changes similar to Alzheimer disease at a relatively young age. One of the genetic factors responsible for Alzheimer is localized to chromosome 21

Myotonic dystrophy

• Mutation in the DMPK gene • Most pleiotropic phenotype of all unstable triplet repeat disorders • Characterized by wasting of the muscles, cataracts, heart conduction defects, endocrine changes, and myotonia -Autosomal dominant inheritance - Maternal anticipation (variable age of onset). - Repeat sequence: CTG at 3' UTR - Normal Repeat #: 5-35 - Pathogenic Repeat #: 75-11,000 - RNA binding proteins bind to CTG repeat motif => accumulate => sequestered from normal functioning => => mis-regulation of these RNA binding proteins => global splicing alterations

Sonic hedgehog (SHH) mutations

• Mutations in this gene lead to: - Infant with classical holoprosencephaly. - Unusual eyes - Flattened nose and midline cleft lip and palate. - There is an underlying brain abnormality with lack of development of midline cerebral structures. - Death usually occurs in the neonatal period. • Mutations can run in families or be sporadic. • In humans, Heterozygous mutations of this gene result in one form of autosomal dominant holoprosencephaly NOTE: VARIABLE EXPRESSION - There are DISEASE MODIFYING genes that contribute to severity of expression

main types of regulatory proteins in bacteria

• Negative regulation through Repressors which bind to an operator and prevent RNA polymerase initiating transcription. • Positive regulation through Activators which bind to an operator and allow RNA polymerase to initiate transcription. A gene can use both types of regulation e.g. Lac Operon

Robertsonian translocation

• Occurs in acrocentric chromosomes • Chromosome 13, 14, 15, 21, 22 • There is loss of the short arms of the two chromosomes • The satellite structures in the acrocentric chromosomes contain genetic material for RNA • Fusion of the long arms of the two chromosome

Polyethylene glycol derivatization (PEGylation)

• Overcomes Protein stability problems encountered in protein replacement therapy - May protect protein from rapid degradation --> Carbohydrate moiety - shields the protein, but does not interfere with enzyme active site - May reduce clearance by the kidney - May reduce the chance of an immune response • Allows greater potential for therapeutic use of protein/ enzyme replacement therapy

PCR Applications in Medical Diagnostics

• PCR - RFLP • To determine if there is a deletion - Multiplex PCR in Duchenne Musc Dystrophy • Sizing of PCR products • DNA sequencing of PCR products • ASO PCR of genomic DNA or cDNA

Tools for determining genetics vs. environment

• Population/migration studies • Family studies • Twin studies • Adoption studies • Association studies

Allele frequency

• Proportion of chromosomes that contain a specific allele -->Different alleles at a locus can be determined by RFLP NOTE: -homozygous aa has 2 chromosomes with a -heterozygous aa has 2 chromosomes with a i.e. in this population of 2 people (aa and Aa) => 4 chromosomes total, the chromosome population containing a allele = (2+1)/4

Parts of the Lac Operon

• Proteins coded - Lac Z - b- galactosidase - Lac Y - lactose permease - Lac A - Transacetylase • Control regions - Lac O - Operator - main 'switch' - binds repressor protein - Lac P - Promoter - binds RNA polymerase

Transition mutation

• Purine ->Purine (A to G or G to A) OR • Pyrimide ->Pyrimidine (T to C or C to T). e.g. most common mutation that leads to hemochromatosis (G--> A) C282Y (cytosine to tyrosine at position 282)

Transversion mutation

• Purine is replaced by a Pyrimidine OR • pyrmidine is replaced by a purine -less common then transition mutations e.g Mutation in HbS is a transversion mutation (A→T) GLutamine to Valine at position 6

Hallmarks of Cancer

• Self-sufficiency in growth signals • Insensitivitytogrowth-inhibitorysignals • Evasionofapoptosis • Limitlessreplicativepotential • Sustainedangiogenesis • Ability to invade and metastasize • Loss of contact inhibition of growth • Loss of cell-cell adhesion • Escape from immune attack • Genomic instability: Breakdown in DNA repair

Pseudogenes

• Sequences that look like real genes but are not functional (no protein product).

CIS factors

• Serve as binding sites for general transcription factors recognized by RNA Polymerase II • Consensus sequence of a TATA (hogness box) ~25bp upstream of start codon. • CAAT box: - Often 70—80bp upstream of start codon is a second cis element a CAAT box. • GC box (GGGCGG) often used in gene

HDAC

• Sin 3a/histone deacetylase 1⁄2 complex • A chromatin remodelling complex that is recruited to the DNA by MeCP2 • Deactylates lysine residues on the histone tails and causes gene inactivation - Methylation player

X-Linked Dominant Disorders

• Skipping of generations not common • Preponderance of females compared to males • No male to male transmission • Affected male transmits the disease to all his daughters, but none of his sons would be affected

Aspects of the Genome missed by Gene Annotation Method

• Small ORFs (open reading frames) • Genes within genes • Gene that are not translated into protein - (rRNA, tRNA, other RNA.) • Other unknown RNA species • And still mistakes made and things missed

X-Isochromosome i(X)

• Some children with Turner syndrome have an isochromosome of one of the X chromosomes • They have only one copy of the short arm of X chromosome = 46,X, i(X)(q10)

Imprinting (Parent of origin effects)

• Some genes are active only when transmitted by mother or father e.g. UBE3A expressed by maternal chromosome 15, SNRPN always expressed by paternal chromosome 15 • Imprinting involves methylation of specific loci (epigenetic change) & silencing of the gene • Normal imprinting pattern on chromosome 15: SNRPN gene is silenced by methylation on maternal ch15, but UBE3A is active. On paternal ch15, SNRPN is active and UBE3A is inactive. (silenced by methylation). - UBE3A is active on the maternal chromosome 15 - SNRPN is active on the paternal chromosome 15

Giemsa staining (G-Banding)

• Standard for karyotype analysis. - Became a test for many major chromosomal abnormalities such as aneuploidy and large translocations, insertions, deletions. G-banding is imaged after binding of Giemsa stain to A-T rich regions of the genome (dark bands are A-T regions, and light bands are G-C rich regions). Disadvantages: -low resolution - insertion/deletions (InDel) must be >5million bases (5Mb)

Isochromosomes

• There is loss of one arm of a chromosome & duplication of the other arm • X isochromosome: long arms of the X chromosome join to form an isochromosome

Intron polymorphisms that affect splicing

• These sequences are found in specific introns of the CFTR gene • Bad poly T-tract alleles (or TG tract) cause a % of the mRNA to be spliced incorrectly =>Less protein is able to be made • If a mild mutation is found in cis with a severe T-tract (or TG tract) polymorphism then the effects of that allele would be worsened. => bad poly T

HapMap Project

• This will describe the common patterns of human genetic variation. • expected to be a key resource for researchers to find genetic variants affecting health, disease and responses to drugs and environmental factors. • information produced by the project is made freely available to researchers around the world. • The variants found may be disease causing mutations or may be in linkage with disease causing mutations and are therefore useful.

The transcriptome

• Use of cDNA microarrays to determine the genes expressed - Various tissues - Various pathological conditions - Other physiological conditions • Allows comparison of expressed genes between normal and diseased states Uses the principal of cDNA microarray assay gene expression

Examples of X-linked dominant disorders

• Vitamin D resistant Rickets (hypophosphatemic rickets) • Rett syndrome (lethal in males) • Incontinentia pigmenti (lethal in males)

MeCP2

• a protein that binds to methylated cytosines - Methylation player

Hemophilia C

• gene for factor XI is on the distal arm of chromosome 4 (4q35) • mutations include missense mutations, nonsense mutations, deletions and/or insertions, and splice-site mutations • Mutations described so far are associated with mainly failed or reduced production of the active protein, and only a few are related to the production of a dysfunctional molecule. • Incidence: 1 in 100,000 population

Spina Bifida

• incomplete closure of the spine • can be quite variable in severity • repair may be done in utero or postnatally but success treatment is variable - Incidence 1-2:1000 - Evidence for GENETIC: =>varying prevalence in different human populations => a woman who has had one child with a neural tube defect such as spina bifida, has ~3% risk of having another (much higher than population risk) - Evidence for ENVIRONMENT: => supplementation of the mother's diet with folate (400mg per day) can reduce the incidence of neural tube defects by about 70% => the 3% risk that a woman will have a second child with a neural tube defects can be reduced to 1% with high doses of folic acid

Sonic Hedgehog (SHH)

• involved in forming and regulating the Zone of Polarizing Activity (ZPA)LIMB PATTERNING • plays a key role in regulating vertebrate organogenesis •Growth of digits on limbs and organization of the brain are controlled by concentration gradients of Sonic hedgehog • is a morphogen — a molecule that diffuses to form a concentration gradient with protein abundance levels dictating cell fate. •In adult tissues controls cell division of adult stem cells and has been implicated in development of some CANCERS

MicroRNA in Tumorigenesis

• miRNAs act to reduce the expression of genes by targeting specific mRNAs - e.g. Down- regulation of miRNAs in leukemias and lymphomas results in increased BCL2 - e.g. reduced miRNA-mediated upregulation of RAS and MYC detected in lung tumors and B- cell leukemias, respectively. • Aberrant expression/activity of miRNAs could be tumorigenic • miRNAs have been shown to undergo changes in expression in cancer cells with frequent amplifications and deletions of miRNA loci

Epigenetics in Tumorigenesis

• miRNAs have been shown to undergo changes in expression in cancer cells with frequent amplifications and deletions of miRNA loci 1) Reduction of miRNA that inhibits oncogene RNA e.g. miRNA-mediated upregulation of RAS and MYC detected in lung tumors and B- cell leukemias, respectively. 2) Increase in miRNA that inhibits tumor suppressor RNA -DNA methylation and histone modifications are frequently altered in tumor cells -tumor suppressor loci are frequently hypermethylated in cancer cells (silencing of tumor suppressor genes e.g. 1) Loss of imprinting causing activation of growth associated genes (e.g. de-silencing of maternal copy of IGF2) 2)Silencing of tumor suppressor loci causing cell overgrowth

Explanation of discrepancy between protein and gene numbers

• potential for dozens of different proteins from one gene •It is estimated that 40-60% of human genes produce more than one protein) • With all of these potential variations the human genome with its 20,000-25,000 genes has the capacity to produce several hundreds of thousands of protein products • 1) multiple start sites • 2) alternate splicing • 3) RNA editing same protein, different function in different contexts 4) also many proteins work as complexes that can be put together in many ways

b-thalassemia major

• subjects are homozygotes OR compound heterozygotesf or β0 or β+ genes • Mutations in both the b-globin genes • Very low or absent HbA levels; High HbA2 and HbF levels a.ka. 'Cooley's Anemia' and 'Mediterranean Anemia'

Goals of Genetic Counseling

• the characteristics of the disorder • the probability of developing the disorder • the risk of passing the disorder on to their children • about the options to prevent or ameliorate the disorder

General transcription factors

• trans acting factors (ie they are transcribed at a different location on the genome are translated in the cytosol and then bind DNA at the cis elements) • General transcription factor bind cis elements • Bind RNA polymerase II and can activate transcription.

α-thalassemia carrier/trait

•Can result from deletion or inactivation of two α-globin genes (--/αα in cis configuration or α-/-α in trans configuration). -may have normal hemoglobin levels or slightly low hemoglobin levels

Incontinentia pigmenti

•Caused by a mutation of the X-linked IKBKG gene •inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma - Males with the disorder die in utero - only survived in females that have skewed X- inactivation in favor of the normal IKBKG allele - Manifests as rashes & blisters in early life - Later, patches of hyperpigmentation, 'Marble cake appearance' of skin - Intellectual and learning disability (in some patients) - Retinal detachment (in some patients) - Variable expressivity in females: Due to the phenomena of X- inactivation

Transcription and translation in bacteria

•DNA free floating in cytoplasm •Ribosomes bind and make many copies •Geared for speed Transcription and Translation occur simultaneously

Autosomal Dominant disorders

•Familial hypercholesterolemia (LDLreceptor deficiency) • Huntington disease • Myotonicdystrophy • Marfansyndrome • Osteogenesisimperfecta • Achondroplasia • NeurofibromatosistypeI • Acute intermittent porphyria

concerns with gene therapy

•Phenotoxicity: complications arising from overexpression or ectopic expression of the transgene •Immunotoxicity: harmful immune response to either the vector or transgene (or the combination) => mule-organ failure •Risks of horizontal transmission: - Somehow the vector becomes infectious and it enters environment •Risks of vertical transmission: - germline transmission of donated DNA

Characteristics of multifactorial inheritance

•Trait does not demonstrate a simple Mendelian pattern of inheritance •Familial aggregation •More common among the close relatives of the proband and less common in relatives who are less closely related •Environment also interacts with genotype to produce the final phenotype

Anencephaly

•is the most severe of the neural tube defects • is rare and it is fatal • underdeveloped brains and incomplete skulls •may have brain stem function •most do not survive more than a few hours after birth -Incidence = 1:5000

lymphoma

•miRNAs found commonly in this cancer

Smith-Lemi-Opitz Syndrome

•mutation in 7-dehydrocholestrol reductase which block cholesterol metabolism so cannot bind PTCH •Consequence is SHH binding to cholesterol is disturbed •Limb abnormalities, holoprosencephaly, growth retardation and limb abnormalities. - (1/20,000 to 1/40,000 births) -If a fetus is exposed to drugs that block cholesterol biosynthesis similar problems can occur!!!!!!!!!