1. Familial Disease
    • run in families; same disease is noticed in different generations of the same
    • genetically associated family members w/o a clear environmental factor
  2. Sporadic Diseases
    • appear in scattered or isolated instances w/o any genetic association to other
    • family members
  3. ABO Bloodtypes
    • gene to glycosyltransferase to putting on different patterns of carbohydrate
    • on cell surface
  4. Chromosome Organization?
    • the
    • shortening of chromatin results in small volume of each chromosome and a
    • reduction in exposed chromosome surface; least condensed = DNA double helix;
    • Chromatin visible = prophase; greatest condensation = metaphase
  5. Spectral karyotyping
    permit simultaneous visualization of all chromosomes in diff colors; chromosome-specific probes are fluorescently labeled
  6. Chromosome Abnormalities
    • Aneuploidy = change of total chromosome numbers
    • (gain/loss)
    • Structure alterations = terminal deletion, interstitial deletion,
    • insertion, inversion, reciprocal translocation
  7. Reciprocal Translocation
    • BCR-ABL
    • = fusion gene (aka Philadelphia chromosome)
    • Mys overexpression = overexpressing

    BCR-ABL = kinase; phosphoryltes other proteins to promoter cell proliferation and suvival
  8. Meiosis
    • - PROPHASE 1 (pachytene stage) CROSSOVER TAKES PLACE.
    • - crossing over creates new linear combinations of genes on a chromosome
  9. Law of segregation
    alternative alleles of genes account for variations in inherited characteristics
  10. Law of independent assortment
    inheritance pattern of one trait will not affect the inheritance patter of another
  11. Autosomal Dominant Inheritance
    • - one copy of disease allele to show phenotype
    • - affected person has at least one affected parent
    • - either sex
    • - children (with affected and unaffected parents) 50% to get it
  12. Autosomal Recessive Inheritance
    • - 2 copies of disease allele to show phenotype
    • - affected people are usually born to unaffected parents
    • - either sex
    • - child has 25% chance if both parents are carriers
  13. X-linked Dominant Inheritance
    • - one copy of disease allele on x-chromosome
    • - affects more female than male
    • - child has 50% chance of being affected
    • - for affected male, all daughters get it but none of the sons
    • TYPE1
  14. X-linked Recessive Inheritance
    • - affects mainly MALES
    • - affected males born to unaffected parents (mom is carrier)
    • - affected females if father affected and mother carrier (or result of
    • nonrandom x-inactivation)
    • - no male to male transmission
  15. Hemophilia
    • - inability to properly form blood clots because of lack of fibrin
    • Hemophilia A = deficiency of factor 8 (more common than B)
    • Hemophilia B = deficiency of factor 9 (aka Christmas disease)
    • - both prevent the making of thrombin, which activates fibrinogen to fibrin
  16. Von Willebrand Disease
    • - most common coagulation disorder
    • - vWF causes adhesion of platelets to collagen; synthesized in liver and in endothelial cells
  17. Management of Hemophilia
    • - standard care: replacement of deficient factor
    • - liver transplantation, blood transfusion
  18. Problems associated with unwanted blood clots: examples?
    • - deep vein thrombosis = blood clots in veins
    • - arterial fibrillation (stroke) = blood clots in arteries
    • - heart attack = blood clot in heart
  19. Male lethality may complicate X-linked pedigrees
    • - x-linked dominant inheritance: both males and females can be affected with only one copy.
    • - incontinentia pigmenti = affected males abort spontaneously
    • - frequent defective recessive allele can look like autosomal dominant pattern instead!
  20. Point Mutations (4 examples)
    • Silentmutation =same protein
    • Neutral mutation = change protein but has same properties to original
    • Missense Mutation = changes aa specified by DNA codon
    • Nonsense Mutation = changes it into premature stop codon
  21. Frameshift Mutations
    • Insertion
    • Deletion
  22. Sickle Cell Anemia
    • - RBCs (normally disk shape) contorted into rigid crescents
    • - pain = most frequent cause
    • - deaths from too much bacterial infections
    • - results from substitution of GLUTAMIC ACID to VALINE at aa#6 in beta-globin (missense mutation)
    • - AUTOSOMAL RECESSIVE! (one normal and one defective = carrier)
    • - in deoxygenated blood, HbSs aren’t as soluble as normal hemoglobin. They aggregate into rod-shaped fibers, which distort the RBC to a sickle shape;
    • these ones cant fit through capillaries, thus blocking blood flow and causing local ischemia (and extremely painful)
    • - vessel obstructive infarctions occur causing strokes….
    • - bone vaso-occulusion causes painful “crises”
    • - LOSS OF SPLEEN FUNCTION (immune system dysfunction, from infarction, increases susceptibility to bacterial infections
  23. Treatment of Sickle Cell Anemia
    • - oxygen inhalation
    • - transfusions
    • - antibiotics
  24. Thalassemia
    • - imbalance of globin-chain synthesis
    • - growth retardation, jaundice, bone marrow expansion
    • - 2 forms: alpha and beta (RBC are lighter colored and smaller)
    • - heterozygote advantage in providing some resistance to malaria
  25. Alpha Thalassemia
    • - reduced synthesis of alpha globin chains
    • - deletion of one to all 4 alpha globin genes on chr16 due to UNEQUAL CROSSING OVER and HOMOLOGOUS PAIRING
  26. Beta Thalassemia
    • - mutations that reduce beta-globin gene expression so unmatched alpha chains accumulate and precipitate as inclusion bodies (Heinz body) in RBC.
    • - These inclusions are removed by phagocytic cells, which cause premature destruction of the cell
  27. Treatment of Thalassemia
    • - blood transfusion + chelation with iron binding resin (iron overload due to breaking of RBCs)
    • - bone marrow transplantation ( too dangerous)
    • - gene therapy (technicaly immature)
    • - clinical trials – drugs that can increase the expression of fetal hemoglobin
  28. Epigenetics
    non-mendelian inheritance; study of heritable changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence
  29. Genomic Imprinting
    • expression of only one allele from a parent and inactivation of the counterpart allele from the other parent
    • Imprinted Gene = silenced allele
    • maternally imprinted = paternally expressed
    • paternally imprinted = maternally expressed
  30. Non-mendelian stuff..
    • Heritability = transmission of the gene imprint over rounds of cell division and generations
    • Reversibility = the imprinted locus can be erased and reset depending on the sex of the parent
  31. Genomic Imprinting: DNA Methylation
    • IMPRINTING = DNA methylation of cytosine at CpG island
    • Methyl donor: S-adenosylmethionine (SAM)
    • Enzyme: methyltransferase
  32. Genomic Imprinting: 2 types of methylation
    • 1) de novo methylation (DNMT 3)
    • 2) maintenance methylation (puts methyl group on other side w/ DNMT1)
    • - unmethylated new DNA strands to be methylated by maintenance methyltransferase
    • - DNMT1 recognizes hemimethylated site and add methyl group to cytosine
  33. How can the methylation status be experimentally
    • 1) distinction by the use of 2 restriction enzymes:
    • - Hpall (sensitive to methylation)
    • - Mspl (insensitive to methylation)
    • 2) Sequencing DNA after BISULFITE treatment
  34. Genomic Imprinting: Methylation status: BISULFITE TREATMENT
    • - methylated vs. unmethylated: bisulfate treatment = deaminates cytosine into uracil but not affect 5-methylated cytosine
    • - PCR and then DNA sequencing
  35. How does DNA methylation make gene locus inactive?
    DNA methylation induces histone H3 deacetylation and methylation at lysine 9. Histone H3 lysine methylation promotes the binding of HP1 (heterochromatin protein 1) and induces chromatin condensation (so gene expression in this locus is silenced…condensed chromatin = inactive)
  36. Genomic Imprinting : paternally imprinted and maternally expressed. Example of Disease?
    if it’s mutated or missing from the mother side, the offspring will have ANGELMAN SYNDROME!
  37. Genomic Imprinting : maternally imprinted and paternally expressed. Example of Disease?
    if it’s mutated or missing from father side, offspring will have PRADER WILLI SYNDROME
  38. Prader-Willi Syndrome
    1) symptoms?
    2) pathology?
    • -reduced muscle tone (hypotonia), lack proper suckling for milk in newborns depend on forced feeding
    • - age 1 -6: rapid weight gain, obesity, delayed development of motor skills
    • - adolescence and adulthood: dietary intake must be controlled to avoid excessive taking, infertility due to underdeveloped sex organs, mild to moderate learning disabilities
    • - early death by 30 years of age from obesity and related complications
    • - multiple genes in 15q11-q13
    • 1) large paternal deletion
    • 2) maternal uniparental disomy of chro15
    • 3) small paternal deletion
  39. Angelman Syndrome:
    1) symptoms?
    2) pathology?
    • - less common than prader-willi
    • - mental retardation, awkward gait, tremors, seizures, protruding tongue, absence of speech
    • - frequent uncontrolled outbursts of laughter and a happy disposition
    • - deleted UBE3A gene (ubiquitin protein ligase 3A) ; expressed in brain from maternal chromosome 15
  40. X-Chromosome inactivation
    • - leads to selective inactivation of gene expression on 1 of 2 X chromosomes in females
    • - provides a mechanism of “dosage compensation” which overcomes sex differences in the expected ratio of autosomal gene dosage to X chromosome gene dosage (male 1X; female 2X)
    • - choice of which of the 2 X chromosome is inactivated is RANDOM
    • - inactivated x-chromosomes forms Barr body
  41. X inactive specific transcript gene (XIST)
    • -XIST expressed from the inactive X (Xi) chromosome starting early around stage of 5- to 10-cell embryo
    • - product of XIST gene is 17kb non-coding, spliced, and polyadenylated RNA
    • - XIST RNA binds to X chromosome expressing XIST, but not to the other X chromosome and autosomes
  42. X-chromosome inactivation: after XIST RNA coating?
    • -upon XIST RNA coating, the painted chromosome is further modified by interacting with additional factors
    • - the CpG islands of Xi become highly methylated, bound histones and hypoacetylated
    • - DNA of Xi chromosome is condensed, replicates late during the S phase of the cell cycle
    • - Xi also associated w/ large amount of unique histone macroH2A1.2, which leads to further chromatin condensation
  43. How do you visualize Xist RNA?
    • - in situ hybridization used to visualize location of XIST RNA – only inactivated X-chromosome is marked
    • - the number of inactive X chromosome (Xi) is always one less than the total # of X chromosomes: barbody/No. Xi + 1 = number of x chromosomes
  44. Skewing of X chromosome Inactivation
    • - skewing defined as greater than or equal to 75% and greater than or = to 90% of cells expressing the same X chromosome
    • - mechanism is not fully clear; skewing of X chromosome inactivation occurs w/ various disorders
  45. Certain genes on Xi chromosomes are NEVER
    • - XIST gene is at Xq13.2
    • - long interspersed nuclear element, type 1 (LINE-1 or L1) is XIST RNA specific recognition site
    • - L1 elements are clustered near XIST gene and more in rest of Xq arm than in Xp arm
    • - most of genes escaped Xi are located in Xp; some of these genes have expressed loci on Y chromosome – no need for dosage compensation
    • - XIST RNA binding, DNA hypermethylation, histone hypoacetylation, macroH2A1.2
    • incorporation to the X chromo that produced XIST RNA lead to the inactivation of this X chromo
    • - RESULT: condense the inactivated X chromosome to form Barr body and silent most of genes on this inactivated X chromo
    • - XIST RNA only binds to the same X chromo that produces XIST RNA
    • - there are genes escape the inactivation due to the low level of XIST binding to their loci
  47. Environmental Influence on Epigenetics
    • - genomes of identical twins = same
    • - DNA methylation patterns are DIFFERENT!
    • - levels of difference isn’t only related to their age, but also w/ the time that the twins have spent apart from each other
    • - environmental factors can be permanently registered in our cells!
  48. Nondisjunction in Meiosis 1
    • - chromosomes not separated; homologous chromosome pairs go to the same daughter cell
    • - RESULTS: 2 of the gametes have an extra chromosome (trisomy), and the other 2 are missing a chromosome (monosomy)
  49. Nondisjunction in Meiosis 2
    • - sister chromatids segregate to the same daughter cell
    • - RESULTS: 2 of the gametes have normal #s of
    • chromosomes (Euploid), 1 has extra chromosome (trisomy), and other is missing one (monosomy)
  50. Consequence of Meiotic Nondisjunction
    • - autosomal monosomies are LETHAL
    • - trisomy 21 most common trisomy disease in humans
    • - people w/ monosomy or trisomy sex chromosomes are more likely to survive
  51. Down Syndrome: symptoms?
    (aka Trisomy 21)
    • -mental retardation, short stature flat facial profile, small ears, short and broad hands, a protruding tongue, and Brushfield spots (white/yellow spots seen on iris)
    • - probably not a disease of a single gene over dose
  52. Trisomy 13 (Patau Synrome)
    • - risks increase with older pregnant women
    • - severe mental retardation
    • - small eyes that may exhibit a split in the iris (coloboma)
    • - opening in the roof of the mouth (a cleft palate) and/or a cleft lip
    • - weak muscle tone (hypotonia)
    • - skeletal abnormalities & increase risk of heart defects
    • - rarely live past infancy
  53. Trisomy 18 (Edwards Syndrome)
    - heart defects, clenched fists, small eyes, rocker bottom feet, kidney problems…10% live over a year, a few last to 30’s
  54. Achondroplasia (Short Limb Dwarfism)
    • - most common form of dwarfism
    • - average height is 132 cm males, 125 cm females
    • - defective conversion of cartilage into bone
    • - AUTOSOMAL DOMINANT TRANSMISSION with complete penetrance
    • - mutations in fibroblast growth factor receptor 3 (FGFR3) gene (G1138A and G1138C)
    • - greater than 80% of individuals with achondroplasia have normal parents, de novo mutation occurs within a parental germ cell or very early in embryologic development
    • - FGFR3 de novo mutations have been associated w/ ADVANCED PATERNAL AGE
  55. XO or Turner syndrome, monosomy for the X
    • - some learning difficulties, but no mental retardation
    • - high incidence of congenital heart disease
    • - kidney abnormalities
    • - reduced life expectancy
    • - infertility
    • - 98% of all fetuses with turner syndrome spontaneously abort
  56. X and Y chromosomes pair at PseudoAutosomal
    Regions (PARs) during meiosis (possible crossover)
    -PARs of X and Y encode a same set of genes
  57. Turner syndrome results from haplo-insufficiency
    of certain genes on the X or Y chromosome
    • - Ex: SHOX gene is expressed from both X
    • chromosomes in female and both X and Y chromo in males
    • - SHOX mutation = individuals w/ idiopathic short
    • stature
  58. Short Stature Homeobox-containing gene (SHOX)
    • - SHOX gene located on PARs of the human X and Y chromo
    • - 2 functional copies of gene require for normal growth
    • - it codes for a key transcription factor that plays a critical role in controlling human stature primarily through regulation of chondrocyte/osteoblast development
  59. Treatment for Turner Syndrome:
    • - growth hormone therapy to improve growth
    • - estrogen replacement to improve development of secondary sex characteristics; estrogen therapy is impt for reducing risk of osteoporosis, which is common in Turner Syndrome
  60. SRY – Sex-determining region of the Y chromosome
    • - SRY is near but not in PAR; recombination may translocate this gene to the X chromosome
    • - PAR – pairs and recombines with X chromosome
  61. XX male
    • - translocation of SRY gene onto an X chromosome
    • - normal to short stature
    • - enter puberty spontaneously
    • - decreased testosterone, no sperm and sterile
    • - gynecomastia (abnormal enlargement of the breast in a male)
  62. XY Female
    • - mutation/deletion of SRY
    • - fetuses develop oocytes, but degenerate by birth, sterile
    • - taller than average for normal women
    • - not enter puberty spontaneously; need estrogen treatment
  63. Klinefelter Syndrome (XXY)
    • - performance: formation of peer relationships is difficult
    • - growth: long limbs, tall and slim stature
    • - hypogonadism and hypogenitalism: testes remain small; infertility
  64. Molecular Basis of Klinefelter Syndrome (XXY)
    • - SHOX gene in PARs of X and Y chromo isn’t inactivated and the increase # of SHOX alleles is associated w/ tallness
    • - testosterone replacement beginning in the early to the mid-adolescence years is recommended for Klinefelter syndrome; it will not reverse gynecomastia but will support secondary sexual characteristics
  65. XYY Syndrome
    • - unlike klinefelter males, these peeps have normal sexual behavior and normal secondary sexual characteristics
    • - 47, XYY males are TALLER and low weight; minor skeletal abnormalities; large hands and feet
    • - more aggressive and have behavioral and learning problems
  66. Triple –X female (XXX)
    • - no apparent physical abnormalities
    • - 47, XXX female discard additional X chromosomes by selective disjunction.
    • - children from XXX are usually normal
    • - social problems; tall for their age
    • - intelligence is normal
  67. What is nondisjunction?
    failure of paired chromosomes to separate in anaphase of meiosis 1; failure of sister chromatids to disjoin at meiosis 2.
  68. trisomy rescue
    occasionally, one of trisomy chromo is lost during the initial stage of embryo cell proliferation and development proceeds normally
  69. Uniparental Disomy
    when 2 rescued chromosomes are from the same parent; either heterodisomy (non-identical chromosome) or isodisomy (identical chromosome)
  70. Uniparental Disomy (UPD) and Genomic Imprinting
    • - UPD chromo carry silenced imprinted genes, then no functional gene product is made – developmental disorder
    • - if both UPD chromo carry active imprinted gene, the over production of these gene product may interfere normal devel
    • - UPD can lead to abnormal development and cancer
  71. Mitotic Nondisjunction
    • - sister chromatids not separated
    • - nondisjunction soon after fertilization leads to chromosomal mosaicism that can underlie some medical conditions, such as proportion of patients with Down Syndrome
  72. Independent Assortment
    • - occurs when genes affecting the phenotypes are found on diff chromo or separated by a great enough distance on the same chromosome that recombination
    • occurs at least half of the time
    • - the closer the 2 loci, the tighter the linkage
    • - genetic mapping can be conducted only with large numbers of readily identified polymorphic loci
  73. Polymorphic Locus
    2 or more alleles at a locus
  74. Cystic Fibrosis
    1) common mutation?
    2) why does mutation lead to disease?
    • - deletion of phenylalanine at positon 508 of CFTR gene
    • - F508 deletion mutant protein cannot reach the cell membrane and is degraded by proteasome after being modified by ubquitination
  75. Human Genome Project
    • - identify all genes in human DNA
    • - determine sequences of the 3 billion base pairs that make up human DNA
    • - store this info in databases; improve tools for data analysis
  76. Genetic Mapping
    relative distance (cM) between 2 loci based on the frequency of recombination
  77. Physical Mapping
    records actual distance (base pairs) between different DNA sequences
  78. Discovery Human Disease Genes:
    Functional-candidate gene cloning
    starts w/ either a known protein that is responsible for an inherited disorder or a protein that is considered a likely candidate based on the symptoms and biochem of the disease
  79. Discovery Human Disease Genes:
    Positional-candidate gene cloning
    when nothing is known about gene product; disease gene mapped to a chromosome location with polymorphic markers; a likely candidate is selected and a mutation detection assay is run with PCR probes based on gene sequence derived from the database
  80. Computer and Gene Discovery: Bioinformatics
    use of computers for storage an analysis of molecular data
  81. Computer and Gene Discovery : Genomics
    study of all features of genomes and individual genes at the level of DNA sequence: mutations polymorphisms, phylogenetic relationships
  82. Functional Genomics
    concerned with patterns of transcription (gene expression) as function of clinical conditions, in response to natural or toxic agents, or at different times during biological processes such as cell cycle
  83. SNP
    (Singe Nucleotide Polymorphism)
    • - single base pair difference at a specific location of the genome
    • -very useful as genetic markers
  84. SNP - recombination
    • - linkage studies for mendelian diseases based on idea that recombination is random
    • - this is true at the macroscopic level but zooming in on genome shows recombination hotspots that are more prone to crossover than the surrounding sequence
  85. Linkage Disequilibrium
    • - genomecontains recombination hotspots: this creates groups of variants that have an ancestral origin = Haplotype blocks
    • - variants seen together on the same haplotype block are said to be in linkage disequilibrium
    • - linkage diseq.= non-random association of 2 alleles; seen on the same chromosome more frequently than you would expect
    • - Haplotype Block = combination of variants which are transmitted together
    • - Recombination is not completely random; there are hot spots where recombination is more prone to occur
  86. Common disease-common variant hypothesis:
    - common heritable diseases (diabetes, cardiovascular, some cancers) are caused by many common alleles all with weak effects but act together to contribute to the phenotype
  87. Genome Wide Association Studies (GWAS)
    • - test each variant for an association to the disease
    • - bioinformatics needed to manipulate and handle big data
    • - 24 alleles showed strong association w/ one of the 7 diseases
  88. Wellcome Trust Case Control Consortium (WTCCC)
    • - 2 control groups – 3000 people total
    • - 7 primary diseases – 2000 people for each
  89. Hereditary Persistence of Fetal Hemoglobin (HPFH)
    • - continuously active gamma-globin gene expression leads to elevated fetal
    • hemoglobin (HbF) levels in adult life
    • - due to mutations of the beta-globin gene locus or the gamma-gene controlled region on other chromosomes
    • - patients with heterozygosity for sickle cell anemia (beta) and hereditary persistence of fetal hemoglobin (HPFH) mutations are largely asymptomatic
    • - Sardinians present relative genetic and environmental homogeneity
  90. 3 types of muscles
    • 1) skeletal = responsible for locomotion – contraction and relaxation controlled voluntarily
    • 2) smooth = propels material through internal passageways – functions involuntarily
    • 3) cardiac = pumps blood; functions involuntarily
    • - the elasticity of muscle tissue enables it to return to its original length after contracting or extending; nerve impulses stimulate voluntary and involuntary muscle contractions
  91. Muscle Disorders: mutations of genes that encode…
    • 1) enzymes that break down glycogen or fatty acids
    • for energy
    • 2) proteins that maintain the structural integrity and mechanical properties of a muscle cell
    • 3) proteins that either regulate or participate in the contractile process
  92. Terms used in Muscle Disorders:
    1) Atrophy
    2) Dystrophy
    3) Myopathy
    • -Atrophy = wasting away of the body or of an organ or part, as from defective nutrition or nerve damage
    • -Dystrophy = any of a # of disorders characterized by weakening, degeneration, or abnormal development of muscle
    • -Myopathy = any abnormality or disease of muscle tissue
  93. Recessive Muscle cell enzyme deficiency disorders
    • - McArdle disease
    • - Pompe disease
    • - Tarui disease
    • - Ceri or Forbes disease
  94. Duchenne Muscular Dystrophy
    • -EXTREMELY ENLARGED CALVES (pseudohypertrophy)
    • - muscular weakness of hips, inability to run, characteristic set of maneuver when rising from kneeling position
    • - confined to wheelchair by 10th year/ mean age of death is 20yr.
    • - gowers maneuver = uses his hands to walk up his legs to a standing position
    • - X-linked RECESSIVE! = mapped to X chromosome at Xp21.2
    • -2/3 cases are familial with other as carrier
    • - 1/3 cases are sporadic = new mutations
    • - most patients are males
    • - females who are heterozygous carriers for DMD gene usually show no muscle weakening (x-inactivation)
    • - some are found to have x/autosome translocations for females
    • - Males = DELETIONS of region containing Xp21.2
    • - females = TRANSLOCATION with an autosome
    • - DMD gene = DYSTROPHIN (one of the longest genes) ; so patients with DMD lack dystrophin
  95. Dystrophin Protein Schedmatic Structure
    - F-actin = filament actin, which forms microfilaments of cytoskeleton and thin filaments of muscle
  96. Becker Muscular Dystrophy
    • -X-LINKED RECESSIVE TOO!! (a mild form of DMD)
    • - muscle wasting becomes apparent later in life in DMD; life span is longer
  97. KNOW: DMD patients lack either entire dystrophin r
    missing the 2 critical C-terminal domains
    BMD patients have some deletion in the internal areas
  98. Explanation for high mutation frequency
    unequal crossing over b/w chromosomes containing tandem repeats
  99. 2 possible result from unequal crossovers:
    • 1) the reading frame is preserved = dystrophin is either longer or shorter, but may still be functional
    • 2) there is a frame-shift in the reading frame = results are truncated, useless protein
  100. -InDMD patients, along with dystrophin deficiency, there are reduced levels of the proteins of the sarcolemma (muscle cell membrane) associated with dystrophin
  101. in absence of these proteins, no bridge b/w
    internal contraction system and the extracellular matrix, which may lead to rupturing of the sarcolemma, muscle cell degeneration, and then deterioration of the whole muscle
  102. Limb-Girdle Muscular Dystrophy
    • - LGMD = group of disorders affecting voluntary muscles, mainly those around HIPS and shoulders
    • - patients have normal level of dystrophin
    • -LGMD1 = dominant inheritance
    • -LGMD2 = recessive inheritance
    • - all LGMD genes are located on autosomes
  103. Dilated Cardiomyopathy (DCM)
    • - enlargement of the ventricular chambers, esp the left chamber
    • - thinning of the vascular walls
    • - loss of contractility of the heart muscle
    • - fatal, with death occurring suddenly
    • - ~4% of population have DCM; leads to heart transplant
  104. DCM - symptoms and pathogenesis?
    • Symptoms
    • - sensation of rapid, fluttering, or pounding heartbeats
    • - fatigue and weakness
    • - shortness of breath (dyspnea) when active or lying down

    • Pathogenesis of DCM
    • - 25-35% are FAMILIAL.
    • - recessive, dominant, or X-linked
    • - obstruction of coronary arteries
    • - bacterial infections of heart
    • - nutritional deficiencies
    • - myocardial toxins (excessive alcohol)
  105. Primary cells
    cells taken from a living organism and NOT immortalized
  106. Established cell lines
    • established cell cultures that can proliferate indefinitely in supporting culture medium; IMMORTALIZED!
    • - provide large numbers of HOMOGENOUS cells for preparing protein, DNA, RNA; can be stored in liquid nitrogen for forever
  107. Common methods of gene transfer to mammalian
    cells in culture
    • Transfection = use of chemical /physical tricks to let cells take up DNA from culture medium
    • - transient transfection
    • - stable transfection
    • Transduction = virus mediated gene transfer
    • Microinjection = directly deliver DNA into individual cells
  108. Questions can be addressed using cell lines
    • -critical regulatory elements that control gene expression
    • - protein stability by stopping new protein synthesis
    • - function of interested proteins
    • --Immunostaining (using antibodies against a particular protein) to examine the protein location
  109. GFP as a reporter protein
    • - test the tissue-specific activity of a promoter
    • - determine when during development a gene is turned on
    • - fuse to another protein to make a chimeric protein that is still functional
  110. Transgenic Mice
    • -introduce DNA fragment into mouse GERMLINE cells
    • - have such genetic modification in ALL of their cells
    • - trans mice created by putting DNA fragment into specific location to remove or replace a gene function
    • - knock out/ knock in
  111. Classical Transgenic Mice
    • - created w/ DNA transfer approach
    • - location of DNA integration into genome is random
  112. Basic steps of generating Transgenic mice
    • - collect fertilized eggs
    • - inject DNA into the fertilized eggs at the pronuclei stage
    • - continue culture the cells in vitro for a while
    • - hopefully, the injected DNA fragments are integrated into mouse genome
    • - implant the embryos back into pseudopregnant mice
  113. How to identify Transgenic Mice?
    • - mark each mouse
    • - cut the tip of the tail
    • - extract DNA from the tip
    • - use the DNA to perform SOUTHERN BLOT or PCR for the detection of inserted DNA
  114. Application of transgenic mice
    • - study effect of overexpression of a gene product
    • - define critical regulatory elements of a gene expression
    • - express a particular protein in mice, such as Cre
  115. Knockout/ Knockin mice
    • Knockout mice = delete a specific area of a particular gene – LOSS FUNCTION
    • Knockin mice = create a specific mutation or insertion in a gene locus; expressed as wild type
  116. Neo & DTA
    • -Neo = makes the cells containing it resistant to
    • the antibiotic neomycin for a POSITIVE SELECTION; DNA frag gets in mice genome
    • -DTA (diphtheria toxin) = inactivates protein translation elongation factor 2 to stop cell growth for a negative selection
  117. Generate Knockout and Knock-in Mice
    • -embryonic stem cell lines originally obtained from inner cell mass of the blastocyst
    • - insert a DNA fragment into a specific locus of mouse genome via homologous recombination upon
    • transfection of DNA into ES cells
    • - select ES ells carrying correct insertion with drugs
    • - confirm the correct insertion by southern blotting or PCR
    • - inject these ES cells into isolated blastocysts
    • - reimplant these blastocysts into foster mothers
  118. Steps of chimeric ...
    • 1)isolate blastocyst from white mouse129
    • 2) isolate inner cell mass from blastocyst
    • 3) place in culture medium for cultured ES cells (+/+)
    • 4) target gene to get genetically modified ES cells (+/-)
    • 5) inject ES cells into cavity of blastocyst from BROWN mouse57
    • 6) reimplant blastocyst into foster mother
    • = it is expected that this chimeric (+/+, +/-) mouse
    • will have some genetically modified germ line cells, and produce some HAPLOID gametes with the modified gene
    • - Chimeric: brown C57 (+/+), white C129 (+/-)
    • 7) Mate chimeric mouse (+/+, +/-) with BROWN mouse57 (+/+)
    • 8) one result is (+/-) WHITE MOUSE!!!!
    • 9) another is (+/+) BROWN mouse…
    • 10) make crosses with heterozygotes (+/-) x (+/-) = (-/-) !!!!!
    • - may die as early embryo
    • - may die late in developmet or postnatally may be live-born and serve as model system for study
  119. Generate Conditional Knockout mice to avoid embryonic
    lethality or make cell type specific knockout mice
    - LoxP site can be recognized by Cre recombinase (cre = cause recombination)
  120. What is Diabetes?
    - a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin action, insulin secretion, or both
  121. Criteria for the Diagnosis of Diabetes
    • - symptoms of diabetes plus glucose concn checked at any time greater than
    • 200mg/dL
    • - fasting plasma glucose level greater than 126mg/dL
    • - 2 hr plasma glucose greater than 200mg/dL after drinking a standard glucose-containing drink (oral glucose tolerance test)
    • - if any one of these criteria are met, confirmation is required by any one of the 3 methods for the diagnosis to be made
  122. Hemoglobin A1C
    • - HgA1C reflects blood glucose over past 2-3 months
    • - hemoglobin that is irreversibly glycated at one or both N-terminal valines of the beta chains; doesn’t exclude hemoglobin that is additionally glycated at
    • other sites on the alpha or beta chains
  123. Diabetes: 2 underlying factors
    • 1) too little insulin produced
    • 2) how well the rest of the body responds to insulin
  124. Pancreatic islet Beta cells and insulin
    • -pancreas: 1-2% of mass is islets of Langerhans
    • Type 1: target of autoimmune attack
    • Type 2: impaired beta cell function;some beta cell loss/ cell death
    • - beta cells = source of hormone insulin
    • - increase in glucose = increase in ATP = increase in insulin
  125. Beta Cells
    - monitor and respond to blood glucose levels in very complex fashion
  126. Classification of Diabetes
    • -Type 1: immune mediated, beta cell depletion without evidence of autoimmunity
    • - type 2: obesity…
  127. Type 1 DIABETES: a disease of beta cell
    destruction and depletion
    • - usually before age 30
    • - insulin required for survival
    • - ketoacidosis
  128. Diabetes: Ketoacidosis
    • life-threatening syndrome in which lack of insulin causes the body to break down fat stores in an abnormal fashion and pH of the blood declines
    • - strong association with immune-related genes
  129. Human leukocyte antigen system
    • - name of major histocompatibility complex (MHC) in humans
    • - autoantibodies to islet proteins
    • - immune cell responses to islet proteins
    • - HLA association
    • - immunosuppressive drugs can ameliorate the disorder
  130. Islet autoimmunity
    • one or more autoantibody persistent for
    • at least 3-6 months
  131. Type 2 DIABETES:
    • 1) INSULIN RESISTANCE: normal amt of insulin produces a subnormal biological response
    • 2) Beta cell dysfunction leads to insulin deficiency
    • - a condition characterized by both insulin resistance AND insulin deficiency
  132. Key sites of insulin action in DIABETES 2
    • 1) tissues
    • 2) liver (glucose production and lipid metabolism)
    • 3) fat tissue
    • - beta cell failure, insulin resistance
  133. Diabetes Type 2: beta cell defect
    • - no islet autoimmunity
    • - relative reduction of beta-cell mass
    • - relative insulin deficiency: beta-cell functional impairment
    • - risk is largely determined by age, OBSEITY, family history, and ethnicity
    • - EPIDEMIC!!
  134. Terms:
    1) Microvascular
    2) Retinopathy
    3) Neuropathy
    4) Microalbuminuria
    • -Microvascular = affecting small blood vessels, such as those in the skin that go to the nerves or in the back of the eye
    • -Retinopathy = damage to the retina
    • -Neuropathy = disease affecting the nerves
    • -Microalbuminuria = leakage of protein into the urine that can be caused by diabetes; early sign of kidney disease
  135. Monitoring of blood glucose
    • -SMBG (self-monitoring blood glucose): finger sticks, capillary blood, glucometer
    • - HgA1C (hemoglobin A1C) reflects blood glucose over past 3months
  136. Major Classes of Medications for Diabetes
    • 1) drugs that sensitize the body to insulin and/or control hepatic glucose production: thiazolidinediones, metformin
    • 2) drugs that stimulate the pancreas to make more insulin: sulfonylureas, meglitinides
    • 3) GLP-1 (glucagon-like hormone 1) – related drugs
    • - forms inhibitors of the enzyme that breaks down GLP-1
  137. Diabetes: Glucagon-like Peptide 1
    • - beta cell: stimulates glucose-dependent insulin secretion increases beta cell mass
    • - liver: reduces hepatic glucose output by inhibiting glucagon release
    • - alpha cell: inhibits glucagon secretion in a glucose dependent fashion
  138. SLGT2 Inhibitors
    • - block kidney’s ablity to recover glucose from the urine; glucose loss in urine
    • - renal sodium glucose transporters (SGLT2) reabsorb glucose from the filtrate preventing loss of glucose in the urine; SGLTs use the energy from a + sodium gradient to transport glucose across the membrane
    • - SGLT2 inhibitor: dapafliglozin
  139. What is Ectopic Lens?
    • - autosomal dominant trait
    • - mutations in Fibrillin-1
    • - aneurysm (bulging of blood vessels) and dissection of aorta
  140. What is Retinoblastoma?
    • - most frequent childhood eye cancer
    • - recessive oncogene (tumor suppressor gene)
    • - presence of leucokoria
    • - gene : Rb
  141. What is Retinitis Pigmentosa?
    • - genes involved in phototransduction
    • - mutations in genes in vitamin A metabolism
    • - night blindness
    • - constrictions of visual field
  142. Major Causes of Blindness in elderly population?
    • - cataract
    • - AMD
    • - Diabetic Retinopathy
    • - Glaucoma
  143. What are Cataracts?
    • - cloudy lens
    • - use ultrasound to break lens and suck out cataract
  144. What is Glaucoma?
    • - optic nerve cupping/ atrophy and ganglion cell death
    • - visual field loss
    • - associated w/ elevated intraocular pressure (IOP)
    • - strong genetic predisposition
  145. What is Primary Open-Angle Glaucoma?
    • - loss of retinal ganglion cells and axons
    • - associated with increased IOP
  146. What are Diabetic Microvascular Complications?
    • - diabetic retinopathy (leading cause of blindness)
    • --> proliferative diabetic retinopathy (PDR) - most severe form
    • - diabetic nephropathy (leading ause of end stage renal disease)
    • --> requires dialysis and kidney transplant

  147. What is AMD? Soft Drusen?
    • - soft drusen = debris that collects in retina and macula
    • 1) Dry (geographic atrophy)
    • 2) Wet (choroidal neovascularization); abnormal blood vessels form
    • - development multifactorial:
    • --> environmental factors: smoking, obesity, nutrition
    • --> familial risk factors
  148. AMD: Gene Loci?
    • complement factor H (CFH) region and chromosome 10q locus
    • - CFH has single-nucleotide polymorphism (SNP) coding (Tyr402His) and non-coding polymorphisms in complement factor H
    • - increased HTRA1 expression in AMD with wet AMD in chinese/whites
  149. Dry AMD: geographic Atrophy
    what are specific genes?
    • - CFH
    • - HTRA1
    • - Toll-like receptor 3 (TLR3)
  150. In dry AMD, what does the gene TLR3 do?
    viral dsRNA sensor
  151. TLR3-/TLR3- mice....
    are resistant to dsRNA induced RPE and retinal cell death
  152. How will genetics help our patient care?
    • - classification of disease according to genotypes
    • --> risk levels and prognosis due to gene/risk allele combos
    • - rational drug design to target specific defect or pathway: increase specificity and redue side effect
    • - animal models for new drug screening
    • - customized medicine
  153. Treatments of WET AMD:
    • -targeting VEGF pathway; trying to block VEGF from binding to receptor by using antibodies
    • - Lucentis: ranibizumab= mAb binds VEGF and decreases VEGF protein
  154. What is the Anti-VEGF treatment Response?
    • - hypothesis: amd genetic factors may influence response to anti-VEGF therapy
    • - VEGF = vascoular endothelial growth factor which promotes angiogenesis (blood vessel growth)
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