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Gene mapping | Biomolecules | MCAT | Khan Academy

Lecture 18: 3 Point Gene Mapping

Lecture 1 Meiosis and Genetic Recombination Meiosis: Two nuclear divisions that result in haploid gametes from diploid cells.k Involves production of tetrads, which contribute to genetic diversity through recombination. One round of DNA replication followed by two cell divisions. Homologous chromosomes pair up, allowing for genetic exchange through crossover. Homologous Recombination: Exchange of DNA between non-sister chromatids during meiosis. Breakage and reunion at crossover points; enhances genetic variation. Breakdown classifications: recombination event (crossovers and non-crossovers). Resolution of Holliday Structures: Holliday junction is formed during recombination. Resolved via either horizontal or vertical breakage, resulting in crossover or non-crossover outcomes. Illegitimate Recombination: Non-homologous end joining, less fidelity, can lead to genomic instability. Genetic Disease Patterns Types of Inheritance: Autosomal Dominant: Only one copy of mutant allele needed. Autosomal Recessive: Two copies of the allele are necessary to exhibit the trait. X-Linked Inheritance: Alleles located on the X chromosome; males more affected. Important Terms: Penetrance: Proportion of individuals with a mutation who exhibit symptoms. Polygenic Traits: Traits influenced by multiple genes. Genetic Imprinting: Differential expression of genes depending on the parent of origin. Population Genetics Genetic Variation: Differences in alleles among individuals. Hardy-Weinberg Equilibrium: Model that describes frequency of alleles in a non-evolving population. Conditions for equilibrium include large population size, random mating, no mutations, no migrations, and no natural selection. Factors Affecting Allele Frequencies: Mutation, migration, genetic drift, and natural selection. Quantitative Traits & Heritability Twin Studies: Used to estimate heritability by comparing traits in identical vs. fraternal twins. Heritability: Proportion of variance in a trait attributed to genetic factors. Cytogenetics Analysis Identify chromosome abnormalities: Involves karyotyping and molecular techniques to analyze structure. Useful in detecting genetic diseases and cancers. Molecular Markers in Genetics Types: RFLPs (Restriction Fragment Length Polymorphisms): Variations in DNA sequences detected by restriction enzyme digestion. VNTRs (Variable Number Tandem Repeats) and Micro-satellites: Repeated sequences of DNA used in mapping and fingerprinting. Genetic Mapping and Association Studies Genetic Mapping: Determining the location of genes on chromosomes. Positional Cloning: Finding disease-related genes based on their location in the genome. Genetic Association Studies: Investigate the relationship between genetics and disease. Cancer Genetics Understanding genetic mutations that lead to cancer. Involves studying tumor suppressor genes and oncogenes. Mouse Mutants and Gene Therapy Production of Mouse Mutants: Model organisms for studying gene function and disease models through targeted genetic modifications. Human Gene Therapy: Experimental technique to treat genetic disorders by correcting defective genes. Recommended Texts for BC3007 Human Molecular Genetics III or IV (Strachan & Read) Genes X or XI (Lewin) Reliable online resources through reputable websites. Historical Context in Genetics Thomas Hunt Morgan (1910): Evidence for genes located on chromosomes using fruit fly mutations. Alfred Sturtevant (1913): Proposed linear arrangement of genes based on genetic linkage phenomena. Lecture 2 Genetic Disease Definition: A condition with an observable/detectable genetic component. Factors: Non-genetic diseases may also be influenced by genetic susceptibility loci. Example: Cystic fibrosis, resulting from mutations in a chloride ion channel. Considerations: Secondary infections associated with CF: Could CF be seen as an infectious disease? Genetic component in road traffic accidents: Could injuries have a genetic basis? Genetic Contributions to Disease Types of diseases: Simple, monogenic, Mendelian diseases (e.g., cystic fibrosis). Several thousand such diseases identified in humans. Notable: Different genes can lead to similar phenotypes (e.g., epilepsy and autism). Types of genetic disorders: Single gene disorders. Complex oligogenic or multigenic disorders (e.g., hypertension, diabetes). Involvement of multiple loci and interactions between them. Key Question: Are rare de novo mutations or common variants responsible for these diseases? Types of Chromosomes Metacentric: Centromere is median or near median. Two well-defined arms with a ratio from 1:1 to 2.5:1. Acrocentric: Centromere is close to one end. One arm significantly smaller, arm ratio from 3:1 to 10:1. Telocentric: Centromere is terminal, presenting a one-armed structure. Importance of centromere position and arm length ratio, and observation of secondary constrictions (nucleolar organizers). Karyotype Definition: The number, size, and shape of chromosomes in a somatic cell arranged systematically. Human karyotype: 46 chromosomes total (23 from each parent). Sex determination: Males: XY Females: XX Group classifications: Group A: Large metacentric chromosomes (1-3). Group B: Large submetacentric (4-5). Group C: Medium-sized submetacentric (6-12, X). Group D: Medium-sized acrocentric (13-15). Group E: Short metacentric (16-18). Group F: Short metacentrics (19-20). Group G: Short acrocentrics (21, 22, Y). Sex Chromosomes Mapping of homologous genes in non-recombining regions of X and Y chromosomes. Pseudoautosomal regions of X and Y are marked (black) as these regions pair/recombine during male meiosis. Notable features: Heterochromatic region of Y (gray). Centromeres denoted by clear ovals. Gene clusters on both X and Y chromosomes indicated (labeled a, b, c). Chromosome Substructure Distinction between chromosomes is achieved through banding techniques. Tissue sources for karyotype: blood lymphocytes, amniotic fluid, bone marrow, skin. Karyotype analysis involves: Blocking cells in mitosis with colchicine. Digestion with trypsin. Staining condensed chromosomes (Giemsa dye). Banding results: Dark bands: Heterochromatic, gene-poor, late replicating, AT-rich. Light bands: Gene-rich (R bands). Specific stains can reveal centromeric and nucleolus organizer regions. Chromosome Paints Combinatorial FISH technique used. Example from a CML patient showing a complex Philadelphia Translocation between chromosomes 8, 9, and 22. Use of chromosome-specific DNA probes labeled with fluorescent dyes, allowing for multiplexing. FISH (Fluorescent In Situ Hybridisation) Applications include prenatal diagnosis to identify numeric chromosome abnormalities. A numerically normal result does not confirm a chromosomally normal fetus; FISH can identify abnormalities in pregnancies (e.g., Down Syndrome risk). Chromosome Nomenclature Short arm labeled P; long arm labeled Q. Standard Paris nomenclature developed: total chromosomes and sex chromosome constitution depicted. Example: 46,XX indicates a normal female; 46,XY indicates a normal male. Chromosomal Abnormalities Definition: Changes in number (numerical) or structure (structural) of chromosomes. Types of abnormalities: Numerical: Loss or gain of chromosomes or segments. Structural: Leads to genetic mosaicism- cells having different genotypes. Numerical Chromosome Abnormalities Types: Polyploidy: Abnormality of entire chromosome set (e.g., triploidy, tetraploidy). Aneuploidy: Abnormal number of a specific chromosome (e.g., monosomy, trisomy). Aneuploid cells arise through nondisjunction or anaphase lag during cell division. Mixoploidy Occurs when individuals have differing chromosomal compositions. Related to mosaicism (from a single zygote) or chimerism (from different zygotes). Aneuploidy mosaics are common due to nondisjunction in early embryonic cells. Structural Abnormalities Results from chromosome breakage: Single break (repaired or degraded). Multiple breaks lead to structural aberrations (balanced/unbalanced). Types of structural change: chromosomal inversion, interstitial deletion, ring chromosome. Chromosomal Translocations Defined as joining segments from different chromosomes. Types include: Balanced reciprocal: material exchanged between chromosomes, often asymptomatic. Robertsonian fusion: acrocentric chromosomes fuse, potentially causing syndromes in offspring. Insertional translocation: a segment excised and inserted elsewhere, presenting risks of monosomy or trisomy in progeny. Types of Mutation Types of chromosomal mutations: deletion, insertion, duplication, translocation, and inversion. Balanced reciprocal and Robertsonian translocations can lead to conditions like Down syndrome (47,XX,+21). Uniparental Diploidy & Disomy Results in imbalance in maternal/paternal contributions to karyotype. Uniparental diploidy: all chromosomes from one parent, could lead to abnormalities due to genomic imprinting. Uniparental disomy: both homologues of a specific chromosome from one parent, implications for genomic function. Nomenclature of Chromosomal Abnormalities Types of numerical abnormalities: Triploidy: e.g., 69,XXX; 69,XXY. Trisomy: e.g., 47,XX,+21. Monosomy: e.g., 45,X. Mosaicism: e.g., 47,XXX/46,XX. Structured description includes indicators for types of structural abnormalities (deletion, inversion, etc.). Example of CFTR gene location on chromosome: 7q31.2. Lecture 3 Diseases Caused by Chromosomal Abnormalities Examples of Diseases Down syndrome & other trisomies Turner syndrome Prader-Willi syndrome Burkitt’s lymphoma Chronic myelogenous leukemia Down Syndrome (Trisomy 21) Incidence: 1 in 800 live birthsCharacteristics: Growth retardation Mental retardation Distinctive abnormal facial features (e.g., flat facial profile, slanted eyes, protruding tongue) Congenital heart defects (approximately 40-50% of cases) Higher incidence of leukemia (10-20 times more likely) Signs of premature aging, including issues with vision, hearing loss, and cognitive decline with age Genetic Causes: 95% of cases due to trisomy 21, primarily caused by nondisjunction during meiosis. Majority (approximately 80%) involves maternal nondisjunction at meiosis I. About 5% caused by unbalanced translocation involving chromosome 21, which leads to a partial trisomy. Genomic Details: Chromosome 21 is small (approximately 1.7% of total DNA, containing about 1500 genes). Important gene regions include q21.2-q22.3, containing critical genes such as amyloid precursor protein, superoxide dismutase-1, and alpha-A crystalline, which are believed to play roles in the cognitive decline associated with aging and the pathology of Alzheimer's disease. Maternal Age Factor: The risk of Down syndrome significantly increases with maternal age, particularly after age 35, where the risk escalates to approximately 1 in 270 at age 35 and rises even more dramatically afterward. Other Chromosomal Abnormalities Patau Syndrome (47, +13) Incidence: 1 in 19,000 live birthsPrognosis: Poor survival; many affected infants die within the first year of life.History: Observed increase in incidence in Pacific Islands post-atomic bomb tests, suggesting environmental factors may interact with genetic predispositions. Edwards Syndrome (47, +18) Incidence: 1 in 8,000 live births.Survival Rates: 95% die in utero; less than 10% of live births survive the first year.Mosaic Cases: Some may survive into adolescence, characterized by notable physical and developmental challenges, but overall health outcomes are severe. Turner Syndrome (45, X) Characteristics: Phenotypic female with gonadal dysgenesis Symptoms include sexual immaturity, primary amenorrhea, short stature (typically under 5 feet), webbed neck, and various cardiovascular and renal anomalies. Genetics: The 45,X karyotype is found in 50% of cases, often resulting from nondisjunction during male meiosis. Abnormal X chromosomes present in 15% of cases, including deletions, rings, and isochromosomes. Mosaics like 45,X/46,XX found in 15% of cases, suggesting early mitotic nondisjunction, leading to a variation in phenotype severity. Angelman & Prader-Willi Syndromes Illustrative Genetics: Both syndromes are associated with deletions in region 15q11-q13 and exemplify genomic imprinting. Prader-Willi Syndrome (PWS): Result of loss of paternal genes, leading to symptoms such as short stature, hypotonia (poor muscle tone), poor feeding in infancy, transition to hyperphagia (insatiable appetite) and obesity later in life as a result. Incidence: 1 in 10,000 live births. Angelman Syndrome (AS): Result of loss of maternal genes, characterized by severe mental retardation, absence of speech, seizures, and inappropriate laughter. Incidence: about 1 in 15,000 live births. Genetic Mechanism: The presence of genomic imprinting critical for gene expression linked to these syndromes underlines the importance of parental origin of alleles in regulating genetic disorders. Burkitt's Lymphoma Genetic Cause: Associated with a translocation between chromosomes 8 and 14, causing constitutive activation of the c-Myconcogene. Epidemiology: Commonly found in parts of Africa, often in conjunction with Epstein-Barr virus infection, which is thought to contribute to lymphomagenesis. Mechanism: The translocation positions the MYC oncogene under the influence of the immunoglobulin heavy-chain locus, resulting in continuous overexpression, leading to uncontrolled proliferation of B cells and consequently, lymphoma. Chronic Myelogenous Leukemia (CML) Philadelphia Chromosome: Found in 90% of CML cases; characterized by the translocation t(9;22), which leads to a shortened chromosome 22, known as the Philadelphia (Ph) chromosome. Genetic Composition: The ABL proto-oncogene located on chromosome 9q and the BCR gene on 22q produce a BCR-ABL fusion protein that has inappropriate kinase activity driven by the BCR promoter, causing uncontrolled cell division and survival of myeloid cells, characteristic of CML. Lecture 4 Mutation & Polymorphism Definition: Allelic sequence variations occurring at a frequency > 0.01 (1%) are termed DNA polymorphisms. Mean Heterozygosity: Human DNA has a mean heterozygosity of ~0.004, meaning ~1 in 250 bases differ between allelic sequences (HLA has higher variation). De Novo Mutations: Occur at a frequency of ~10⁻⁶ to 10⁻⁸ per nucleotide base per generation; roughly equivalent to 10⁻⁵ per gene per generation. New studies may update this data. Inheritance: Most human allelic differences are inherited, not due to new mutations. Types of Mutations Base Substitutions (Point Mutations): Common in coding and non-coding DNA. Types: Transitions & Transversions Synonymous & Nonsynonymous Conservative & Nonconservative Gene conversion-like events (multiple base substitutions) Insertions: One or a few bases can lead to frameshifts; common in non-coding DNA. Triplet repeat expansions and large insertions (e.g., transposons). Deletions: One or a few bases can lead to frameshifts; common in non-coding DNA. Large deletions, typically at regions with tandem repeats or interspersed repeats. Copy Number Variations (CNV): Evidence suggests increasing submicroscopic variations. Chromosomal Abnormalities: Refer to earlier lectures for details. Epimutation: Alteration in gene function without changing the DNA sequence, e.g., altered DNA methylation. Sources of Mutations Environmental Mutagens: Sources include ionizing radiation, chemicals, and ultraviolet light. Reactive Metabolites: E.g., reactive oxygen species. Endogenous Mutation: Most common causes include: Errors in DNA replication/repair. Spontaneous Chemical Reactions: Depurination: Loss of purines (A or G) from DNA; ~5,000 purines lost/day per human cell. Deamination: Conversion of cytosine to thymine (~100 bases lost per cell/day). Endogenous Somatic Cell Mutation Rate Cell Count: ~10¹⁴ cells in an adult human (100 million million cells). Cell Divisions: ~10¹⁷ cell divisions occur in a human lifespan. Nucleotide Incorporation: Each cell division incorporates ~6 x 10⁹ new nucleotides. Total Nucleotides: ~10²⁶ nucleotides incorporated in a human lifespan. Replication Errors: Uncorrected replication errors occur at ~10⁻⁸ per incorporated nucleotide. For a 1.5 kb coding sequence (CDS), mutation risk is 1.5x10⁻⁵ per CDS per cell division, leading to ~10¹² ‘hits’ across a human lifespan. Base Substitutions are Non-Random Transition vs. Transversion: Transitions are twice as common as transversions in both coding and non-coding DNA. This pattern arises due to multiple factors: Polypeptide sequence conservation. High frequency of C to T transitions (5mCpG to TpG). Differential repair efficiency of mispaired bases. Coding vs. Non-Coding DNA Mutation Spectra: Similar, but viability is compromised in the 3% of DNA that is coding. Synonymous Mutation: Does not change gene product (commonly silent). Nonsynonymous Mutation: Alters protein or functional RNA, potentially harmful, beneficial, or silent. Selection Influence: Frequency of nonsynonymous mutations is strongly influenced by natural selection, while non-coding DNA exhibits a higher mutation rate due to reduced selection pressure. Base Substitutions in Coding DNA Non-Random Patterns: Reflect requirements to preserve gene protein-coding function. First, second, and third base positions of codons exhibit different degrees of degeneracy: Non-degenerate Sites: All substitutions are nonsynonymous (e.g., 2nd base position of all codons). Very low substitution rate. Twofold Degenerate Sites: One of the three substitutions is synonymous, often found at 3rd positions. Intermediate substitution rate. Fourfold Degenerate Sites: All substitutions are synonymous, found at specific positions. Substitution rate resembles introns and pseudogenes. Amino Acid Function / Chemistry Influences Substitution Rate Cysteine: Unique for its sulfhydryl group; highly conserved due to its role in forming disulfide bridges. Serine and Threonine: Similar side chains, leading to mutable substitutions at codon positions. Classes of Base Substitutions in Coding DNA Synonymous: No change in the amino acid; common due to third base wobble. Some synonymous mutations can be pathogenic. Nonsynonymous: Includes: Nonsense Mutation: Replaces a codon with a stop codon (very rare). Missense Mutation: Encodes a different amino acid. Conservative Substitution: Amino acid replaced by a chemically similar one; minimal effect on protein function. Nonconservative Substitution: Replaces amino acid with dissimilar one, may affect protein function. Nomenclature for Describing Allele Effects Null Allele / Amorph: Produces no product. Hypomorph: Produces a reduced amount of product. Hypermorph: Produces an increased amount of product. Neomorph: Allele with a novel activity/product. Antimorph: Activity/product antagonizes normal product. Frequencies of AGXT Mutations in Italian Patients Mutation of AGXT gene in patients diagnosed with Primary hyperoxaluria type 1 (PH1): Various mutations with respective gene frequencies listed, indicating prevalence. Mechanisms to Reduce/Abolish Gene Function Deletion: Can be of the entire gene or part thereof (e.g., α-thalassemia, Duchenne muscular dystrophy). Insertion: Sequence inserted into the gene (e.g., LINE-1 into F8C gene in hemophilia A). Gene Structure Disruption: Achieved via translocation, inversion, or preventing promoter function (examples provided). mRNA Destabilization: Caused by mutations impacting polyadenylation sites or nonsense-mediated RNA decay. Splicing Issues: Prevented by inactivating donor/acceptor splice sites or activating cryptic splice sites. Frameshift: Introduction alters translation, impacting gene function. Factors Affecting Allele Frequency in Populations Natural Selection: Acts on phenotype encoded by genotype affecting fitness (survival & reproduction). Nonsynonymous Mutations: Typically reduce fitness and are subject to negative/purifying selection. Neutral Alleles: Fate determined by random genetic drift in small populations. Advantageous Mutations: Undergo positive directional selection. Codominant Selection: Heterozygote fitness may be intermediate compared to homozygotes; may lead to balancing selection. Rate of Nucleotide Substitution Varies across different gene components and associated sequences. Non-degenerate sites, twofold degenerate sites, and fourfold degenerate sites have differing substitution rates. Ka / Ks Ratio dN/dS Ratio: Infers natural selection on protein coding genes. 1 indicates positive selection; <1 implies purifying selection; 1 suggests neutral selection. Potential combination of positive and purifying selection observed throughout gene evolution. Comparative Genome Hybridization Conventional Setup: Label patient & control DNA with different fluorescent dyes. Analyze hybridization to detect duplications or deletions. Genome-wide Screening for Chromosomal Imbalances Matrix-CGH: Used for detecting chromosomal gains/losses in cell lines (represented in fluorescence signal ratios). Copy Number Variations (CNV) Refers to large DNA segments (10,000-5 million letters) variations, often overlooked in genetic disease. CNVs linked to various diseases and conditions; the role in common diseases is currently under investigation. Flavors of Sequencing Whole Genome Sequencing (WGS): Sequence entire genome. Whole Exome Sequencing (WES): Focuses on protein-coding regions; cost-effective and reduces analysis time. Custom Capture: Targets specific DNA sequences. Amplicon Sequencing: Amplifies and sequences target DNA. Whole Genome vs. Exome Sequencing Tables comparing features, costs, success rates, and types of sequences included. Next Generation Sequencing (NGS) Overview: Generates a larger number of shorter reads through parallel reactions, with key applications described. Single Molecule (3rd Gen) Sequencing Techniques like Single Molecule Real Time (SMRT) and Nanopore sequencing, highlighting novel detection methodologies. Lecture5 Inheritance Patterns of Genetic Disease Mendelian Characters Depend on the genotype at a single locus. Phenotypic character can be either dominant or recessive. Dominance/recessiveness pertains to the trait, not necessarily to specific genes. Example: Sickle cell anemia shows this dominance pattern. Semi-dominant Traits: Heterozygote presents with an intermediate (milder) phenotype compared to homozygote. Gene Complexity: Classical genes are not always single transcription units (may include deletions, insertions, or rearrangements). Locus Heterogeneity: Certain phenotypes (e.g., mental retardation) can arise from mutations in different genes. Allelic Series / Clinical Heterogeneity: Variations in alleles of a single locus can lead to different severities or diseases (e.g., dystrophin gene abnormalities). Complementation Test: Used to determine if characters are from one locus or two; conducted in model organisms like mice, Drosophila, and C. elegans. Sickle Cell Anemia Genotypes and Phenotypes: Genotype: +/+ (normal) , +/HbS (sickling trait), HbS/HbS (sickle cell anemia) Inheritance Mode: Dominant and recessive scenarios. Prevalence of Single Gene Disorders Autosomal Dominant Disorders: Familial hypercholesterolemia: 1 in 500 Polycystic kidney disease: 1 in 1250 Huntington disease: 1 in 2500 Hereditary spherocytosis: 1 in 5000 Marfan syndrome: 1 in 20000 Autosomal Recessive Disorders: Sickle cell anemia: 1 in 625 (common in African Americans) Cystic fibrosis: 1 in 2000 (common in Caucasians) Tay-Sachs disease: 1 in 3000 (common in American Jews) Phenylketonuria: 1 in 12000 Mucopolysaccharidoses: 1 in 25000 Glycogen storage diseases: 1 in 50000 Galactosemia: 1 in 57000 X-linked Disorders: Duchenne muscular dystrophy: 1 in 7000 Hemophilia: 1 in 10000 Genetic Complementation Test Used in model organisms to determine if two recessive mutations are allelic (e.g., Uncoordinated mutations in C. elegans). Unusual (Non-Mendelian) Inheritance Types Mitochondrially Encoded Diseases: Features matrilineal inheritance and heteroplasmy (mixture of normal and mutated mitochondria). Selection for 'fitter' mitochondria (germline bottleneck). Mitochondrial genome: encodes essential components for mitochondrial function. Some mtDNA mutations can cause severe conditions in high-energy tissues due to variable penetrance. Leber Hereditary Optic Neuropathy (LHON) Symptoms: Mid-life central vision loss, leading to central scotoma and blindness. All mutations (~15 identified) are missense mutations in mitochondrial genes related to respiratory chain complex. Variable penetrance and often occurring as de novo mutations. Males are at a higher risk. Rodent models used to study the effects of mitochondrial dysfunction on disease progression. Genomic (Parental) Imprinting Certain disease genes are expressed differently based on parental origin (e.g., Prader-Willi syndrome). Autosomal dominant conditions may involve reactivation of silenced genes depending on maternal/paternal inheritance. Non or Incomplete Penetrance Penetrance: Probability that the genotype expresses the phenotype; variation seen largely in dominant traits. Example: Huntingtons can show incomplete penetrance, leading to skipped generations in inheritance. Variable Penetrance and Expressivity Penetrance: Complete (100%) or incomplete (e.g., 50% for some traits). Expressivity: Variability in how traits manifest; influenced by genetic makeup and environmental factors. Anticipation Certain dominant diseases become more severe across generations due to unstable trinucleotide repeats (e.g., Huntington disease). Mosaicism / Chimerism Germline mosaicism: Found in autosomal dominant and X-linked diseases influencing reproduction. Microchimerism: Exchange of cells between mother and fetus during pregnancy can lead to autoimmune diseases. Somatic Cell Mosaicism: Observed in conditions like anhidrotic ectodermal dysplasia; affects skin and muscle proportions based on active X chromosome. Hypomelanosis of Ito Characterized by white patchy skin, possible intellectual and developmental disabilities, seizures, and skeletal issues; linked to chromosomal mosaicism. X Chromosome Inactivation (XCI) Females may show milder phenotypes due to random XCI resulting in mosaic cell populations for maternal and paternal X chromosomes. Skewed XCI can cause differing clinical expressions between genders, impacting the severity of X-linked diseases. Quantification of Skewed X Inactivation Involves assessing both random and nonrandom patterns during XCI, understanding the effects on health conditions. X-linked Conditions Lethal in Males Many conditions lethal in utero present significant phenomena in females, like Incontinentia Pigmenti. New Mutations Severe genetic diseases often arise from new dominant or recessive mutations in unaffected parents, without a family history. Y-linked Diseases Very few genes lead to few associated diseases, primarily related to male infertility and possibly Y-linked deafness. Increased Y chromosome counts (XYYY, etc.) can lead to various health issues, though conditions are often variable in severity. Lecture 6 Human Pedigree Analysis Symbols in Human Pedigree Charts Unaffected male: represented by a square. Affected male: represented by a shaded square. Unaffected female: represented by a circle. Affected female: represented by a shaded circle. Person whose sex is not known: represented by a diamond. Identical twins: represented by connected circles or squares. Non-identical twins: represented by two distinct circles or squares. Marriage (mating): represented by a horizontal line connecting two individuals. Vertical line: indicates the offspring of the parents, branching downward. Carrier (optional): often indicated by a half-shaded circle or square. Consanguineous marriage: indicated by a double line connecting partners. Dead: represented by a line through the symbol. Punnett Square Example of Punnett Square with Genotypes: AA, Aa, Aa, aa Proband (II2): Genotype: aa Phenotype: affected Autosomal Dominant Inheritance Characteristics: Affected individuals typically have an affected parent. The condition can affect individuals of either sex and can be transmitted by either sex. Typical mating scenario: M/m (heterozygous) x m/m (homozygous recessive) results in a 50% chance of offspring being affected. Examples of Conditions: Tuberous sclerosis: characterized by benign tumors in multiple organs. Neurofibromatosis: a genetic disorder causing tumor growth on nerves. Retinoblastoma: a rare form of eye cancer affecting young children. Autosomal Recessive Inheritance Characteristics: Trait manifests only when an individual is homozygous for the gene variant. Severe conditions may prevent homozygotes from reproducing. Condition often arises from matings between asymptomatic heterozygous carriers. There is a ¼ chance of any child being affected with such matings. Inbreeding and Its Risks Inbreeding: Occurs when parents are closely related (e.g., cousins), increasing the risk of offspring inheriting identical alleles. F (coefficient of inbreeding): measures inbreeding risk, calculated based on lineage. Example: Risk calculation for first cousin mating: 1/16 probability of an affected child. Pedigree Analysis and Carrier Detection Investigation of a pedigree can reveal insights regarding inheritance patterns: If dominant: low risk of affected children for individual III6. If recessive: obligate carriers include individuals I2, II1, II5, II6, II8, III1, III2, III4, III5, III6, III7, IV1. Carriers have a 50% risk of passing on the trait (e.g., in individuals III10, III11, III12). X-Linked Recessive Traits Characteristics: Predominantly affects males due to the presence of only one X chromosome. Affected females pass the trait to all sons, whereas affected males do not transmit the trait to their sons. Mitochondrial Inheritance Characteristics: Inherited exclusively through the egg; sperm do not contribute to the mitochondrial genome. Few diseases are caused by mutations in the mitochondrial DNA. Inheritance of Specific Traits Red-Green Color Blindness: an X-linked recessive trait. A male with this condition likely has a maternal grandmother who is affected. Tay Sachs Disease: occurs when an affected child inherits the condition as an autosomal recessive disease from unaffected parents. Predicting Inheritance Patterns Example: For a purple ears trait, observations suggest that autosomal dominant inheritance is likely since the trait appears in both genders. Diseases and Mechanisms of Inheritance X-linked Dominant Disease: Example: Hypophosphatemia (Vitamin D-resistant rickets). This condition leads to bone deformities, where ingestion of vitamin D is ineffective. It is associated with mutations in the PHEX gene located on chromosome X. Inheritance Pattern Identification Common Mechanisms: Autosomal recessive Autosomal dominant X-linked dominant X-linked recessive Y-linked recessive Mitochondrial Summary of Mechanism Identification Determine the probable mechanism of inheritance based on pedigree observations and genetic traits, which can inform medical decisions and genetic counseling. Lecture 7 Page 1: Introduction to Genetic Markers Objective: To associate genetic markers with phenotypes or other genetic markers. Definition of Genetic Marker: A polymorphic allelic variant detected directly or indirectly through its encoded phenotype. Page 2: Types of Genetic Markers Morphological Markers: Physical characteristics for identification. Molecular Markers: Variations at the molecular level. Biochemical Markers: Variations evidenced through biochemical properties. Classification: Non-PCR Based Based on PCR Isoenzymes AFLP (Amplified Fragment Length Polymorphism) RAPD (Random Amplified Polymorphic DNA) SSR (Simple Sequence Repeat) STS (Sequence Tagged Site) ISSR (Inter Simple Sequence Repeat) SCAR (Specific DNA Amplified Region) SNP (Single Nucleotide Polymorphism) Page 3: Detection of Protein Variants Variability Factors: Size, charge, and shape variations due to: Alternative splicing Mutations Post-translation modifications. Detection Techniques: Gel Electrophoresis/Iso-electric focusing Chromatography Immunological Methods Focus: Detection of variants between different individuals/alleles, not tissue-specific changes within an individual. Page 4: Protein Variability Observations: Some proteins show no detectable variability, dependent on detection methods. Molecular Weight Range: Common molecular weights, e.g., kDa. Page 5: Detecting Polymorphism in Proteins Polymorphism in Proteins: ~25% do show polymorphism. Historical Context: Initial studies in the 1960s-70s focused on soluble enzymes and serum proteins detectable through electrophoresis. Detection Methods: Use of ‘in situ’ assays Antibody binding Detection of size variations through denaturing SDS-PAGE. Page 6: Isozymes of Phosphoglucomutase Isozyme Patterns: Multiple isozymes result from gene expression, allelic variation, and post-translational modifications. Focus on phosphoglucomutase enzyme in red blood cells. Page 7: Glucose-6-Phosphate Isomerase Isozymes in Mice Enzyme Function: Converts fructose-6-phosphate to glucose-6-phosphate. Detection: Use of various visualization methods and staining solutions. Page 8: Basics of Electrophoresis Key Tool: Electrophoresis is vital for analysis of DNA and proteins. Mobility Equation: v = q/f (where q is charge and f is frictional resistance). Separation Variables: Molecules separated based on charge and size. Page 9: Immunological Detection Blood Typing: As an example of genetically encoded phenotype differences. Genotype Inference: Inferring genotype from observable phenotype. Page 10: DNA Variants Detection Methods Common Techniques: Southern blotting RFLPs (Restriction Fragment Length Polymorphisms) PCR methods Single nucleotide polymorphism detection. Page 11: Electrophoresis for DNA Polymorphism Detection Separation Basis: DNA separates based on charge/mass ratio, allowing detection of single nucleotide changes. RFLP Detection: Focuses on restriction enzyme sites. Page 12: Example of Allele Variants Allelic Variants Shown: Differences between alleles based on EcoRI cutting activity. Page 13: Repetitive DNA in Gene Mapping Mechanisms: Minisatellites/VNTRs: Unique to individuals, except monozygotic twins. Microsatellites/SSRs: Numerous in the genome, applicable for PCR. Page 14: Size Difference Detection in DNA Techniques: Use of electrophoresis for insertions, deletions, and repeats. Page 15: Initial DNA Fingerprinting Technique: Examined multiple loci for DNA fingerprinting; provides insight into genetic relationships. Page 16: VNTR Polymorphism Analysis Technique: Variable number of tandem repeats can be visualized using Southern blot. Importance: Offers genetic complexity and variability. Page 17: Applications of DNA Analysis Examples: Paternity testing and forensics, including sexual assault cases. Page 18: Replication Slippage and STR Alleles Mechanism: Leads to new STR alleles through misalignment during DNA replication. Page 19: Understanding SNPs Definition: Single nucleotide polymorphisms, frequent in non-coding areas. Detection: Utilizes fluorescent labeling post-PCR. Page 20: Techniques for Polymorphism Detection Methods such as SSCP, heteroduplex analysis, and SNuPE for detecting nucleotide changes without sequencing. Page 21: SNP Genotyping Arrays Commercial Implementation: High-throughput analysis for whole genomes. Page 22: Developments in Whole Genome Sequencing Cost Evolution: Significant reductions over time, e.g., from hundreds of millions to nearly $1,000. Page 23: Methylation Analysis Techniques Process: Involves cutting DNA, linking, and sequencing with bioinformatics applications. Page 24: Bisulfite Treatment for Methylation Analysis Method: Treating DNA with sodium bisulfite to detect methylation patterns. Page 25: RNA Sequencing Overview Steps for RNA-Seq Library Preparation: Purify RNA Bind polyA fraction (mRNA) Fragment RNA Convert to cDNA Apply adaptors and sequence Analyze the sequence data. Lecture 8 .Trinucleotide Repeat Disorders Discovered in 1991, adjacent to genetic understanding and evolution of diseases. Triplet Repeats: Occur throughout the genome, including regulatory regions and reading frames. Expansion Dynamics: Expansions can be stable or unstable, categorized as dynamic mutations. Disease Association: Repeat expansions contribute to various diseases, which may be dominant, X-linked, or recessive. Theoretical vs. Practical: Theoretically 64 triplets (4x4x4), but practical applications show only about 10 distinct types (e.g., CAG, AGC, GCA). Anticipation in Disorders Genetic Anticipation: Severity tends to increase with successive generations inheriting triplet repeat disorders. Triplet Expansion: Investigates why three nucleotides are significant in expansion likelihood versus other repeat sizes (e.g., dinucleotides). Frameshifts: Non-multiples of three lead to frameshift mutations, but issues persist even in non-coding regions. Types of CAG Repeat Problems Polyglutamine Tract Expansion: Modest expansions (10-30 repeats): Non-pathogenic. Larger expansions (40-200 repeats): Pathogenic leading to protein aggregation and cell death. Non-Coding Repeat Expansions: Types: CGG, CCG, CTG, GAA. Non-pathogenic <50 repeats; significant pathogenicity generally at 100's to 1000's, can create fragile sites. Mechanism of Repeat Expansion Unstable Above Threshold: Below a certain length, repeats are stable during cellular replication; above, they tend to either contract or expand with a bias towards expansion. Molecular Mechanism: Slipped strand mispairing is theorized to cause instability in expansions. Disease Mechanisms Anticipation Role: Age of onset lowers, with severity increasing in successive generations. Non-Coding DNA Expansions: E.g., Fragile-X leads to transcriptional silencing and dysfunction in neighboring gene expressions. Coding Regions Expansions: Contributes to multiple late-onset neurodegenerative diseases, correlating with repeat length and age of onset. Diagnosed using PCR and Southern blotting techniques. Non-Polyglutamine Disease Examples Type Gene Codon Normal/Wildtype Pathogenic FRAXA FMR1 (X-linked) CGG 6 - 53 230+ FRDA FXN GAA 7 - 34 100+ DM DMPK CTG 5 - 37 50+ SCA8 OSCA CTG 16 - 37 110 - 250 SCA12 PPP2R2B CAG 7 - 28 66 - 78 Polyglutamine Diseases Type Gene Normal/ Wildtype Pathogenic HD HTT 10-35 35+ SCA1 ATXN1 6-35 49-88 Disease Overview & Mechanism Huntington Disease: Associated with increase in CAG repeats in the huntingtin gene (autosomal dominant inheritance). Symptoms progress through motor, cognitive and psychiatric changes, with average onset in mid-adulthood. Polyglutamine Pathology: Inherent common mechanisms across polyglutamine diseases, including protein aggregation and altered cellular processes leading to neurodegeneration. Fragile X Syndrome Inheritance: Unusual X-linked dominant; affected males tend to show various symptoms including mental retardation and distinct physical features. FMR1 Gene: Encodes an RNA-binding protein; expansions cause hypermethylation leading to gene silencing. The Sherman Paradox indicates that the severity of symptoms can skip generations. Myotonic Dystrophy The most common inherited muscle disease, characterized by weakness and myotonia, showing variable severity. DMPK gene repeat sizing correlates with the form of the disease - classical vs. congenital, with implications on symptom severity. Southern Blotting Techniques Used for diagnosing repeat expansions in various diseases, determining genetic traits and potential pathogenic mutations through analysis of sized bands corresponding to repeat numbers. Lecture 9 Population Genetics of Disease Focuses on the frequency and distribution of alleles related to genetic disease susceptibility. Investigates various factors impacting allele frequencies. Factors Affecting Allele Frequencies in Populations Natural Selection: Acts on phenotype, encoded by genotype; fitness is a measure of survival (viability) and reproduction (fecundity). Nonsynonymous mutations typically reduce fitness and are purged through negative selection. New mutations can have equal fitness as existing alleles, leading to random drift in smaller populations. Positive Directional Selection: Favors advantageous new mutations. Codominant Selection: Heterozygote shows intermediate fitness. Heterozygote Advantage: Balancing selection occurs where heterozygotes are favored. Types of Natural Selection Directional Selection: Favors one extreme form of a trait with highest fitness. Stabilizing Selection: Favors the average form of a trait over extremes. Disruptive Selection: Favors both extreme forms over the average. Hardy-Weinberg Principle (HW) Without Selection: Allele and genotype frequencies remain constant if assumptions hold. Example Population: 50% AA and 50% aa (p = q = 0.5) results in specific genotype ratios after random mating: AA (0.25), Aa (0.50), aa (0.25). Frequencies remain fixed over generations. Hardy-Weinberg Equilibrium Assumptions Organism is diploid. Reproduction is sexual. Non-overlapping generations. Random mating. Large population size. Zero migration. Zero mutation. Natural selection does not affect the gene. Basic Hardy-Weinberg Calculations In a study of 5000 individuals: Genotypes: A1A1: 2592, A1A2: 2016, A2A2: 392. Genotype Frequencies: p² = 0.518, 2pq = 0.403, q² = 0.078. p = 0.719, q = 0.279. Autosomal vs. X-linked Loci Autosomal Locus: Genotype frequencies same as x-linked under random mating. X-linked Locus: Different implications for male and female offspring. Incorporating Selection into Hardy-Weinberg Use selection coefficient (s) to gauge influence on genotype frequencies. At equilibrium, loss through lethality equals gain through new mutations. Cystic Fibrosis Example Autosomal recessive inheritance: Mutation-Selection Balance: sq² = m(1 − q²) approximates m if q is small. Mutation rate.M = 5 x 10^-4 is notably high for cystic fibrosis. Heterozygote advantage may explain persistence despite deleterious alleles due to protection against cholera. Magnitude of Selection Favoring Cystic Fibrosis Heterozygotes Disease frequency: ~1 in 2000 births; frequencies computed yield q = 0.022 and p = 0.978. Maintenance of allele frequency if heterozygotes have an average of 2.3% more surviving children than homozygotes. Genetic Counseling in At-Risk Families Autosomal recessive condition: 1 in 10,000 frequency. Expected carrier frequency calculated to be approximately 1 in 50. Risks of Producing Affected Children in New Marriages Risk of 1 in 200 for two carrier parents based on carrier frequencies. X-linked Red-Green Color Blindness Affects 1 in 12 males: Proportion of females who will be carriers and affected calculated, yielding a carrier rate of ~15% and affected rate of ~0.7%. Multifactorial Characters Mendelian Characters: Dichotomous traits; present or absent. Non-Mendelian/Quantitative Characters: Polygenic traits, often showing continuous variation (e.g., size, strength). Characters tend to follow a Gaussian distribution driven by the additive effect of multiple genes. Heritability and Regression to the Mean Heritability (h^2): Proportion of variance in a trait due to genetic effects. Formula for genetic variance and assumptions of random mating evaluated. Regression to Mean: Child’s traits are influenced by parental traits assuming no dominance. Polygenic Threshold Theory Extended theory on traits leading to discontinuous traits expressed when exceeding a threshold. Traits tend to cluster in families, indicating shared genetic risk factors. Sex-specific Thresholds in Disease Susceptibility Observed in certain congenital conditions; typically, females exhibit higher thresholds. Summary of Genetic Architectures of Disease Trait Type Mode of Inheritance Environmental Effects Mendelian Simple (e.g. enzyme defects) Little Complex Quantitative (e.g. height) Moderate to Great Polygenic Threshold (e.g. pyloric stenosis) Great Summary of Genetic Architectures of Disease lecture 10 Identification of Disease/Susceptibility Genes Single gene: Refers to conditions caused by a mutation in a single gene. Mendelian inheritance: Disease caused by a single gene, following Mendel’s laws, which usually exhibit clear inheritance patterns. Reduced penetrance: Not all individuals with the genotype express the phenotype. Multifactorial inheritance: Involves multiple factors determining traits or conditions, often includes a single major gene and polygenic influences. Polygenic: Trait influenced by multiple genes. Environmental factors: External factors interacting with genetic predispositions. Box 2: Human characters typically do not fall purely under Mendelian, polygenic, or environmental categories; rather, they exist in a spectrum combining these influences. Gene-Hunting Strategies Functional Cloning: Requires knowledge of the normal function of the disease gene. Involves antibody production or functional assay. Techniques: Screen expression library or use PCR to clone cDNA. Positional Cloning: Does not require prior knowledge of gene function. Constructs genetic and physical maps from pedigrees and cytogenetic data. Tests candidate genes in specific chromosomal regions. Position-independent candidate genes: Suggest identification based on similar diseases in animal species or molecular pathology. Systems biology allows utilizing vast data from animal models and genome sequences for identification. Positional Cloning Methods Initial information sources for gene location: Linkage studies in pedigrees. Population or family-based association studies. Loss of heterozygosity screening (e.g., in tumors). Chromosomal abnormalities (e.g., translocation breakpoints). Linkage Analysis Overview Disease definition: Accurate identification is crucial for successful analysis. Large, multi-generation pedigrees are used to track inheritance and genetic markers. Linkage analysis goal: Determining marker loci near the disease-causing locus to identify genetic factors contributing to disease. Independent Segregation in Linkage Analysis Unlinked disease and marker loci segregation: On different chromosomes: Independent assortment occurs. On the same chromosome: Markers exhibit different segregation patterns. Inheritance patterns in disease pedigrees illustrate genetic recombination possibilities. Co-segregation and Recombinants Co-segregation: Disease and marker loci that are physically linked remain together during meiosis. Recombinants: Offspring that inherit different combinations of alleles due to crossing over during meiosis. Meiosis Complexity and Recombination Meiosis: Complex process involving single and double crossovers. Recombinants: Typically 50% of offspring due to independent assortment and crossover events. Figures detail the result of single and double crossovers involving chromatids. Phase of Disease and Marker Alleles Phase: Relationship between alleles at a marker locus in relation to the disease locus. Identification of alleles linked to disease is essential for informative meioses. Linkage disequilibrium may occur when marker loci are very close to the disease locus. Informative and Uninformative Meioses Informative meiosis: Allows differentiation between recombinant and non-recombinant gametes. Examples of uninformative meioses: Cases where allele combinations do not provide linkage information due to homozygosity or ambiguous inheritance. Significance of Linkage Analysis Linking analysis challenges with small family sizes: Relies on counting recombination events among individuals. Baseline probabilities and odds ratios: Essential for establishing statistical significance of linkage results. Traditional threshold for significance is a odds ratio of 1000:1 with a LOD score of +3. Calculating LOD Scores LOD score (Z): Evaluates significance by comparing odds of obtaining data through linkage versus chance. Calculated using the ratio of likelihoods based on observed recombination events. A LOD score of +3 indicates strong evidence of linkage, while -2 means exclusion of linkage. Example Case Studies Linkage analysis for NF1: Evaluates potential allele associations and recombinants in familial cases. Pedigree analysis: Used with Southern blot data to assess RFLP loci for evidence of linkage in autosomal recessive conditions. Evidence for linkage is assessed through recombination data and allele inheritance. LOD Score Curves and Calculations Graphs demonstrating LOD scores provide visual representation of data support. Computational methods enhance accuracy in LOD score calculation, using logistic regression and other statistical models for analysis. Conclusion Understanding genetic linkage and methodologies for identifying disease susceptibility genes is crucial for disease mapping and genetic counseling. Lecture 11 Linkage versus Association Linkage: Property of relative position of loci, not their alleles. Co-segregation of a disease or trait with a specific genomic region in multiple families. Involves any allele at the marker locus in a given family. Association: Property of alleles. A specific allele of a gene or marker found to co-segregate with a disease or trait in a population. Linkage Disequilibrium: Presence of linkage and association. Page 2: Co-segregation in Pedigrees Co-segregation of disease and tightly linked marker allele indicates: Linkage and association. Page 3: Complexity of Association Association is a statistical concept rather than strictly genetic. Defined as the co-occurrence of alleles or phenotypes. Example: Allele A associated with disease D if its frequency in affected individuals is greater than expected based on population frequencies. Causes of Association: Direct causation. Linkage disequilibrium. Natural selection affecting phenotype at unlinked loci. Population stratification. Type 1 error. Page 4: Finding Disease Genes with Association Tests Conducting Case–Control Studies: Collect diseased and normal individuals, no familial relationships needed. Genotype all individuals for numerous genetic markers. Compare allele frequency in diseased versus normal populations. Identify alleles with differing frequencies, potential causative variants, or false positives. Page 5: Variants and Their Use Common, Low Penetrance Variants: Utilize association methods. Rare, High Penetrance Mutations: Engage linkage methods. Considerations: Frequency in population, magnitude of effect. Page 6: Importance of Linkage Disequilibrium If all polymorphisms were independent, studies would require examining over 10 million cataloged variants. Linkage disequilibrium reduces costs significantly by making tightly linked variants correlated, but whole genome sequencing may replace these methods. Page 7: Mosaics of Linkage Disequilibrium Chromosomes display mosaicism influenced by: Recombination rate. Mutation rate. Natural selection. Population size effects: bottlenecks and founder effects. Page 8: Haplotypes and SNPs Combinations of alleles at proximally located markers reflect ancestral haplotypes. SNPs (Single Nucleotide Polymorphisms) example: DNA sequences representing variants. Haplotypes: A set of closely linked genetic markers on one chromosome inherited together. Page 9: Linkage Disequilibrium Measures Perfect and Complete LD: No LD due to recombination. Perfect LD if r2=1 (Ratio A/C = B/D). Complete LD if D’=1 (Ratio A/C ≠ E/F). Page 10: The HapMap Project Structure showing blocks of haplotype segments (20 kb, 25 kb, etc.) Page 11: Tagging SNPs Within short chromosome segments, only a few distinct haplotypes exist. Carefully selected SNPs can infer the status of other SNPs associated with haplotypes. Page 12: Challenges in Case-Control Studies Common Issues: Inconsistent results and lack of replication. Population stratification, phenotypic heterogeneity, genetic heterogeneity, random errors. Problems related to study design, including failure to correct for multiple comparisons and poor control group selection. Page 13: Avoiding Population Stratification Methods to reduce bias: Careful matching of case and control origins. Genotyping unlinked markers. Employ family-based methodologies like the Transmission Disequilibrium Test (TDT). Page 14: Transmission Disequilibrium Test (TDT) Involves both parents and at least one affected sibling. Tests whether marker allele M1 is over-transmitted to affected offspring. Test statistic: (a-b)² / (a+b) where: a = number of M1 transmissions. b = number of M2 transmissions. Software examples: GENEHUNTER, TRANSMIT. Page 15: TDT Analysis for Non-LD Situations Understanding marker distribution under no LD with causative variant. Page 16: TDT Analysis for LD Situations Assessment where marker is affected by LD with causative variant. Page 17: Sib Pair Analysis Sib pairs may share: 0, 1, or 2 parental haplotypes based on random segregation. Affected sib pairs share haplotypes indicating condition specifics (dominant/recessive). Page 18: Multiple Testing Problem 1 in 20 tests expected to yield significant results purely by chance at α = 0.05. False positive rates increase with multiple independent tests (each marker as a separate hypothesis). Page 19: Solutions to Multiple Testing Issues Bonferroni correction: Adjust significance level per number of tests. Permutation Tests: Use reference distributions from data rearrangements. Monte Carlo Sampling: Sample possible replicates repeatedly. Replication: Ensure findings are replicable. Page 20: Linkage vs. Linkage-Disequilibrium Similarities in measuring correlation but different focuses (locus vs. allele). Differentiation based on timeframes of recombination events and detection methodologies. Linkage detected for markers close to disease genes; LD detected for markers even closer. Page 21: Disease Gene Identification Workflow Steps include: Population resource collection (trios or case-control). Whole-genome genotyping. Genome-wide association and fine mapping. Gene mining and sequencing for polymorphisms. Identification of causative SNPs and pathway analysis. Lecture 12 GENE INTERACTIONS Pleiotropy: A single gene affects multiple traits. Epistasis: Multiple genes interact to affect a trait. Interrelation between multiple traits impacts fitness, leading to natural selection for gene combinations. DISORDERS OF COMPLEX INHERITANCE The “curse of dimensionality”: Challenges in calculating numerical solutions when models have numerous parameters. With increasing factors (e.g., genetic or environmental) behind a disease, the combinations (alleles) grow exponentially. LEVELS OF ANALYSIS Ranges from species-level differences to individual variation. Severe disorders: Often caused by a single gene and are sufficient for the disorder. Common mild disorders: Known as 'complex disorders' due to their influence by multiple genes and environmental factors. Some researchers argue that common mild disorders might be just extreme variations of normal traits, not distinct disorders. Quantitative Trait Loci (QTLs): Genes in polygenic systems likely lead to quantitative (dimensional) outcomes instead of qualitative (categorical) disorders. Emphasis on the spectrum of genetic variation influencing phenotypic variation. EPILEPSY Types of seizures: Partial Seizures: Can be simple partial (consciousness intact) or complex partial (consciousness impaired). Generalized Seizures: Absence seizures (consciousness not impaired), tonic-clonic (grand mal), myoclonic, atonic. Partial seizures can evolve into generalized tonic-clonic seizures. Environmental causes (e.g., neonatal hypoxia) may contribute alongside genetic factors. GENE-ENVIRONMENT INTERACTIONS (GxE) Rare diseases like Huntington's or Tay Sachs are often linked to single gene deficiencies but are outnumbered by common diseases resulting from genetic and environmental interplay. Phenylketonuria (PKU) requires both genetic variant (metabolism deficiency) and environmental factor (dietary phenylalanine) for manifestation. Genetic variations contribute to disease susceptibility rather than directly causing diseases. Individual responses to the same environmental factors can vary significantly due to genetic differences. ENVIRONMENTAL PROJECTS AND EPIDEMIOLOGY Focus on hypothesis development regarding exposure-disease relationships and methods for prevention/intervention. COAL WORKER'S PNEUMOCONIOSIS Interaction between oxidant exposure and polymorphisms in antioxidant and pro-inflammatory genes influences disease development. Factors include: Exposure: Coal dust and cigarette smoke. Genetic susceptibility: Various gene polymorphisms (e.g., TNF-a, GST). Disease factors: Changes in lung function, nodule development, inflammation. SMOKING-RELATED LUNG DISEASE Factors influencing lung disease risk: Amount of cigarettes smoked, reaction to smoke, novelty seeking behavior. Genetic components alter nicotine metabolism (e.g., CYP2A6). Effects on lung structure: Destruction of alveolar attachments, lumen obstruction, inflammation effects on airway narrowing. TYPE 2 DIABETES Multiple interacting factors: Genetic Factors: Genes like PPARG, FTO. Environmental Factors: Obesity, exercise, diet, smoking, and drugs. Pathophysiology demonstrates insulin resistance and defects leading to hyperglycemia. ENDOPHENOTYPES AND PATHOPHENOTYPES Endophenotypes: Intermediate phenotypes influencing clinical presentations and disease expressions based on genetic/environmental factors. Relationship of these variables to the pathophysiological states leading to distinct disease outcomes. DISEASE NETWORK AND GENES Disease gene network: Represents genes linked to the same disorders; node size indicates the number of associated disorders. GENE EXPRESSION PROFILING Comparison of gene expression between obese and non-obese individuals aids in identifying disease-related genes. Aims to lead to new diagnostic tests and drug targets based on differences observed in expression profiles. PHARMACOGENETICS / PHARMACOGENOMICS Individual responses to drugs show significant genetic variation through metabolic pathways (e.g., cytochrome P450). Genetic factors explain 20-40% of differences in drug efficacy and adverse effects. Adverse drug reactions account for significant health risks, emphasizing the importance of personalized medicine. CASE STUDY: PHARMACOGENETICS 6-mercaptopurine: Varying response due to the TPMT enzyme's genetic variations. Blood tests allow for tailored medication dosing based on metabolism rates, crucial for effectively treating conditions like leukemia. Lecture 13 Page 1: Introduction Title: Cancer prevalence and survival rates Key Author: Steven A. Belinsky Publication: Nature Reviews Cancer (September 2004) Focus: Gene-promoter hypermethylation as a biomarker in lung cancer. Page 2: Carcinogens Definition: Carcinogens are substances capable of causing cancer in living tissue. Occupational Carcinogens: Certain occupations expose workers to carcinogens, leading to increased cancer risk. Key examples include: 4-aminobiphenyl: Chemical and dye workers → Bladder cancer. Asbestos: Construction workers → Lung cancer. Benzene: Leather and petroleum workers → Leukemia. Vinyl chloride: Rubber workers → Liver cancer. Radon: Underground mining → Lung cancer. Page 3: Preventable Cancers Some cancers are preventable through the understanding of factors like: Inflammatory Cells: Asbestos fibers interact with macrophages and cytokines, leading to cellular changes. Direct Interaction: Asbestos fibers cause free radicals and reactive oxygen species, damaging target cells (e.g., bronchial epithelium). Types of Tumors: Mesothelioma Bronchogenic Carcinoma Page 4: Cancer Genetics Process: Cancer arises from natural selection on cell phenotypes enhancing division. Mutations: Predominantly somatic but some inherited. Terminology: Cancer: Systemic disease. Tumor: Physical entity. Adenoma: Benign glandular tumor. Carcinoma: Malignant glandular tumor. Sarcoma: Malignant connective tissue tumor. Leukemia: Blood cell cancer. Page 5: Complexity of Cancer Key Factors: Cancer is highly complex; influenced by causes, histology, gene expression, and prognosis. Examples of common cancers: Gastric and breast cancer. Page 6: Development of Cancer Process: Generally a multi-step progression: Normal epithelium → Dysplasia/Hyperplasia/Metaplasia → Tumor Development of primary tumors can lead to secondary metastasis. Page 7: Characteristics of Tumors Successful Tumors Must achieve: Independence from growth signals. Insensitivity to growth suppressors. Avoid apoptosis. Achieve indefinite replication. Sustain angiogenesis. Capable tissue invasion and metastasis. Page 8: Oncogenes Definition: Mutated genes that promote cell division (gain-of-function). Examples: MYC, RAS. Activation Methods: Gene amplification, point mutation, translocations, etc. Page 9: Tumor Suppressors Function: Inhibit inappropriate cell division, ensure apoptosis, and maintain genome stability. Require inactivation of both copies (e.g., p53, Rb). Knudson's Two-Hit Hypothesis: In retinoblastoma, one mutation is inherited; the second occurs somatically. Page 10: Knudson's Two-Hit Model Mechanism: Development of cancer requires mutations in both copies of tumor suppressor genes. A) Sporadic case with two somatic mutations. B) Hereditary case with independent tumor development. Page 11: Loss of Heterozygosity (LOH) Mechanisms: Include deletion, recombination, chromosome breakage/loss, nondisjunction. LOH screens are vital for identifying tumor suppressor loci. Page 12: DNA Repair Defects Importance: Cells constantly repair damaged DNA to prevent cancer. Mechanisms They Address: Nucleotide excision repair: Errors from UV light (e.g., xeroderma pigmentosum). Double-stranded break repair: Involves BRCA1 and BRCA2 for breast cancer risk. Page 13: Genome Instability Feature of Cancer: Chromosomal instability manifests as loss or gain of chromosomes. Mechanisms: Loss of spindle checkpoint, aberrant DNA replication, loss of telomeres. Page 14: DNA Double-Strand Break Repair Pathways Involved: Non-homologous end joining (NHEJ) Homologous recombination repair (HRR) Signaling Response: Activates key proteins like ATM and MRN complex which influence cell cycle arrest and apoptosis. Page 15: Control of the Cell Cycle Cell Fate Decisions: Cells can remain static, divide, die (apoptosis), or differentiate. Checkpoints: DNA damage checkpoints arrest progression for repairs, critical to tumor suppression. Page 16: RB Protein and Cell Cycle Regulation Role: RB inhibits E2F transcription factors necessary for S-phase progression. Mitogenic Signals: Cyclins and CDKs phosphorylate RB, leading to cell cycle progression. Page 17: PI3K Signaling Pathway Function: Growth factors activate this pathway via receptor tyrosine kinases. PTEN Role: Opposes PI3K activity, crucial for regulating cancer growth. Page 18: P53 and Apoptosis Function: p53 monitors DNA integrity; initiates cell cycle arrest or apoptosis in damage cases. Loss / Mutation Implications: Common in many tumors, leads to various cancer types, including Li-Fraumeni syndrome. Page 19: Therapeutic Strategies Targeting p53 Aim: Reactivate p53 pathways to induce apoptosis in tumor cells. Compounds: Target different aspects of p53 activity. Page 20: Genetic Predisposition to Cancer Indicators: Multiple cancers in families, early onset cancers, rare cancers, etc. Page 21: Common Cancers with Inherited Components Examples: BRCA1 & BRCA2: Linked to breast and ovarian cancer. APC: Associated with familial adenomatous polyposis. Management Strategies: Prophylactic surgeries and regular screenings. Page 22: Evolutionary Concepts in Cancer Somatic Mutations: Enhance survival and are selected over generations. Cancer Prevalence Post-Reproductive Age: Limited selection pressure for cellular integrity after reproduction, leading to increased cancer risk in older populations. Lecture 14 Co-operative and Self-reinforcing Organization of Chromatin Histone Modifications: Includes lysine (K) acetylation (Ac) and lysine methylation (Me). Lysines at various positions can also be modified, playing a critical role in gene silencing. HP1 Protein: Recognizes MeK9 modifications. Binds histone methyltransferase (HMT), facilitating heterochromatin spread. Histone Deacetylases (HDAC): Deacetylate lysine residues which is essential for subsequent methylation processes. DNA Methyltransferases (DNMT): Participate in multiprotein complexes with HDACs and HMTs. Methyl-C binding proteins (MBD) bind to methylated DNA through interactions with HDACs and HMTs. Evidence primarily from studies on constitutive heterochromatin, with implications for genes silenced in cancer cells. Dynamic Nature of Epigenetic Modifications Epigenetic Modifications: These changes on histone N-terminal tails and DNA are dynamic. Enzymes responsible for depositing and removing these marks are crucial. Any deregulation of these enzymes can potentially lead to oncogenesis. DNA Methylation DNA Methylation Reactions: Methylation is catalyzed by DNA Methyltransferase (DNMT). The reaction converts deoxycytidine and S-adenosyl-L-methionine (SAM) into 5-Methyl-cytidine and S-adenosyl homocysteine (SAH). Roles of DNA Methylation in Mammals Key Functions: Transcriptional gene silencing Chromatin compaction Genome stability Suppression of homologous recombination Genome defense mechanisms X chromosome inactivation in females Study Techniques: Methylation-sensitive restriction enzymes (REs), bisulfite treatment combined with PCR/sequencing, and gene arrays. DNA Methyltransferase Family in Mammals Current Members: Five known mammalian DNMT family members. All proteins have a C-terminal catalytic domain and an N-terminal regulatory domain (except DNMT2). N-terminal region mediates most protein-protein interactions. Interactions with Other Proteins Methyltransferases interact with: Tumour-associated oncogenes and tumour suppressors. Proteins that modulate chromatin structure including pRb, HDACs, and HP1. Epigenetic Changes and Cancer Initiation Mechanism of Mutations: Epigenetic changes such as gene silencing may occur prior to DNA mutations, initiating the cancer process. Sequence of events from epigenetic alteration to cancer progression. Tumor Suppressor Gene Silencing Hypermethylation: Frequently silences tumor suppressor genes in cancer, contrasting the overall hypomethylated state of tumors. Some genes like HIC1 on 17p13.3 may be solely disabled by hypermethylation mechanisms. Diet may influence DNA methylation levels. Methylation Alterations in Cancer Table of Genes Altered by Methylation: CDKN2A, PAX5, MGMT, among others, varying prevalence across different tumour types (e.g., NSCLC, AdC, SCC). Notable methylation rates are reported for gene alterations related to the cell cycle and apoptosis. Beckwith-Wiedemann Syndrome (BWS) Clinical Signs: Associated with deregulation of IGF2 imprinting leading to increased expression. Examples of clinical manifestations include hypoglycemia and hemihyperplasia. Molecular Events in Imprinting and Cancer Events Leading to Abnormal Expression: Include altered growth and survival pathways, potentially leading to developmental abnormalities and cancer. Loss of Imprinting (LOI) in Tumours Mechanisms of LOI: Can result from sporadic somatic mosaic alterations or early developmental events. Linked to overgrowth conditions and tumour formation, particularly in Wilms tumour. Wilms Tumor - Characteristics and Genetics Definition and Incidence: Pediatric kidney cancer with a complex genetic basis, generally arising from embryonic renal precursors. High cure rates (~80%) with appropriate treatment. Genetic Associations: Mutations in genes such as WT1 and alterations on chromosome 11p. Types of Cancer Associated with IM and LOI Not only Wilms tumor but also various other childhood and adult malignancies such as leukemia, hepatoblastoma, and colorectal cancer. Cancer as a Multistep Process Pathway: Involves a series of mutations leading from normal cells to metastatic tumors. Describes stages from mutation to hyperproliferation. Colon Cancer Types Distribution: Most commonly sporadic (60-85%), familial (10-30%), and hereditary non-polyposis directly referenced as HNPCC. Evolution of Colon Cancer Genetic Alterations: Key mutations involve MLH1, MSH2, APC gene, and K-ras among growth control genes. Distinct pathways leading to chromosomal instability relevant to cancer progression. Familial Adenomatous Polyposis (FAP) Characteristics: Defined by the presence of >100 adenomatous colon polyps. High penetrance (100%) with surgery necessary. Probability of Cancer with FAP Without intervention, colorectal cancer risk approaches 100% with age. The Angiogenic Switch and Tumor Growth Activators and Inhibitors: Involves complex interactions with VEGF and its receptors activating tumor angiogenesis. Tumor hypoxia and other factors play a pivotal role in promoting vascular growth. Tumor-associated Inflammatory Cells Contributions to outcomes: Immune cells can be pro or anti-tumourigenic. Potential for therapeutic modulation through cancer vaccines. Emerging Immune Therapies Combination Strategies: Integrative approaches utilizing classical tumor therapy alongside novel immunotherapeutics addressing immunosuppressive networks are being explored Lecture 15 Genetic Diagnosis and Gene & Stem Cell Therapies Repairing faulty genes or replacing damaged cells could significantly improve treatments for various genetic conditions. Prenatal Diagnosis Maternal Serum Markers (15-19 weeks) Used to screen for genetic disorders. Down’s Syndrome: markers: hCG fetal cells, Y-chromosome, other DNA markers. Neural Tube Defects: marker: α-fetoprotein. Preimplantation Diagnosis: Applied in IVF/fertility clinics. Zona drilling: Techniques: acid tyrodes or laser to remove blastomere (4-8 cell stage) or polar body (checks maternal genes only). Ethical issues arise due to difficulties in growing cells and managing embryos. Amniocentesis (15 weeks onward): Removal of 20 ml of amniotic fluid containing fetal cells. Cells cultured to expand population; 1% risk of miscarriage. Chorionic Villus Sampling (CVS): Earlier method than amniocentesis, biopsy via laparoscopy & ultrasound for direct testing. Fetal Blood Sampling: Conducted late in pregnancy but well tolerated; detects fetal karyotypes, hemoglobinopathies, and infections. Ultrasound: Non-invasive; detects gross abnormalities during the 1st or 2nd trimester (e.g., spina bifida). Gene Therapy Definition: Treatment via genetic modification of patient's cells. Methodology: Direct transfer of therapeutic genes in vivo or modify & reinsert cells ex vivo. Targets: Infectious diseases, cancers, inherited disorders, and immune system disorders. Strategies: Produce absent products. Generate toxins to kill diseased cells (prodrugs). Activate immune cells via cytokine transgenes. Inhibit harmful gene activity (using ribozymes, antisense, shRNA, CRISPR). Correct genetic defects through homologous recombination. Expression Cloning and Recombinant Proteins Techniques in mammalian cells and transgenic livestock to produce modified proteins, including: Blood clotting factor 8 (hemophilia A). Erythropoietin (anemia treatment). Insulin (for diabetes). Recombinant proteins: Cultured for clinical trials and drug development, but transgenic livestock have faced commercial challenges. Genetically Engineered Antibodies & Vaccines Potential in therapeutic areas (notably spearheaded by Greg Winter / Cambridge Antibody Technologies). Monoclonal Antibodies: Produced by hybridomas (fusion of B lymphocytes and lymphoma cells). Challenges: rodent antibodies cause generic immune responses. Solutions: Humanized Antibodies: engineered antibodies combining rodent variable regions with human constant regions. Fully Human Antibodies: derived from phage display systems or transgenic mice. Genetically Engineered Vaccines Types: DNA vaccines using plasmids encoding antigens and cytokine genes. Engineered antigens fused with cytokines. Modified viruses for delivering genes from various pathogens. Cancer vaccines targeting tumor cells. Gene Therapy Techniques Efficiency in Gene Transfer: Must efficiently affect a sufficient number of cells, especially for tumor treatments. Types of Gene Transfer: Minigenes commonly transferred with strong viral promoters for expression. May result in chromosomal integration or transient expression as episomes. Challenges: Integration site randomness can lead to silencing and potential tumorigenesis. Viral Vectors in Gene Therapy Types of Vectors: Oncoretroviruses: infect dividing cells; inserting RNA virus genomes into chromosomes. Adenovirus: high transduction efficiency, utilizes receptor-mediated endocytosis. Adeno-associated Virus (AAV): integrates at specific sites, providing safety and long-term expression. Lentiviruses: transduce non-dividing cells and integrate into chromosomes; potential for widespread applications. Non-Viral Delivery Systems Methods: Liposomes: facilitate endocytosis for DNA delivery but offer low expression efficiency. Direct DNA Injection and Gene Guns: deliver DNA for muscle gene therapeutic applications. Receptor-mediated endocytosis: enhancing targeting to specific cell receptors. Direct Delivery of Therapeutic Genes The therapeutic gene can be packaged into various delivery vehicles. Cell-based Delivery: uses genetically modified embryonic stem (ES) cells to block immune rejection. Chimeric Antigen Receptor (CAR) T Cell Therapy Processes: Leukapheresis: collecting T-cells from the patient. Cell Modification: using viral or non-viral methods to insert genes into T-cells. Expansion: growing modified T-cells. Infusion: reintegrating modified T-cells into the patient post-chemotherapy. Gene Therapy for Inherited Disorders Initial optimism in the 1990s saw few successes and some disasters, particularly in complex diseases. Best Candidates for Success: Recessively inherited disorders, requiring precise regulation of transgene expression. Adenosine Deaminase (ADA) Deficiency Overview: A significant condition leading to severe immunodeficiency; potential for treatment via gene therapy. Potential Gene Therapy Steps: Clone ADA gene into a retroviral vector. Transfect patient’s T-cells and expand resulting ADA+ cells. Re-insert engineered cells into the patient. Safety Concerns in Gene Therapy Risks include potential diseases caused by the vector, randomness in DNA insertion, and associated complications leading to adverse events observed in clinical trials. Advancements in Cancer & Infectious Disease Therapies Many trials focus on enhancing immune responses to cancer or targeting specific infected cells. Challenges in Gene Therapy Implementation Short-lived Nature: Therapeutic DNA must remain functional and stable in host cells. Immune Response: Introduction of foreign DNA often activates the immune system, reducing effectiveness. Viral Vector Problems: Include immunogenicity, targeting issues, and potential disease recovery. Multigene Disorders: More complex disorders pose greater challenges for effective gene therapy solutions. Sources of Stem Cells Types: Embryonic stem cells (blastocyst stage). Umbilical cord blood. Adult bone marrow. Tooth and hair follicle stem cells. Induced pluripotent stem cells (iPSCs) Stem Cell Development and Applications Key Stages: Starting from fertilized eggs, developing into morula and blastocyst stages to produce various cell types (e.g., hepatocytes, neurons). Legacy of Cloning Technologies** SCNT (Somatic Cell Nuclear Transfer): Techniques for reproductive and therapeutic cloning, allows derivation of pluripotent stem cells from adult cells. Lecture 16 Animal Models of Human Disease Animal husbandry is a highly technical and expensive field utilized in research. Animal Care & Handling Factors Type of Caging: Various types utilized depending on species and research needs. Room Temperature: Critical for maintaining animal health. Relative Humidity: Important for comfort and health of animals. Ventilation: Necessary to ensure a healthy environment. Physical & Environmental Factors: Include impacts of room conditions on animal welfare. Animal Factors Age, Sex, Reproductive State: Influences research outcomes and results. Diet & Water: Quality and quantity affect animal health and study validity. Daily Routines: Regular schedules minimize stress. Gentle Handling: Reduces stress and trauma to animals. Metabolic State: Considered to ensure accurate data. Biological Rhythms: Affects behaviors and reactions. Light Cycle & Quality: Impacts animal physiology and behavior. Research Manipulation Factors Time of Manipulation(s): Duration and timing are crucial for study conditions. Number of Animals per Cage: Affects social dynamics and stress. Experimental Stressors: Must be managed to avoid skewing results. Pain and Distress: Ethical considerations must be taken into account. Commonly Used Animal Models Mammals and Birds: Mouse, Rat, Rabbit, Hamster, Guinea Pig, Ferret, Pig, Sheep, Goat, Cattle, Horse, Cat, Dog, Primates (chimp, macaque, marmoset, baboon). Non-Mammalians: Birds, Frogs (Xenopus laevis), Roundworm (Caenorhabditis elegans), Fruitfly (Drosophila melanogaster), Zebrafish (Danio rerio). Ethical Issues Key Questions: Balance of harm versus benefit in using animal models. Assessment of harm to whom and benefit to whom? Measuring 'suffering' in models; appearances can be misleading. Mouse Mutants & Strains Thousands of mouse mutants and strains used in research, including: Specific Mutants: e.g., nude (nu), obese (ob). Inbred Strains: e.g., C57Bl/6 (Bl/6), CBA, DBA/2. Outbred/Randombred Strains and Congenic Strains. Recombinant Inbred Strains for specific studies. Customised Genome Modifications Routine processes implemented in current research practices. Transgenesis Mechanisms: Usage of plasmids, BACs, YACs, retroviral vectors, episomal MACs to insert genetic material. Insertion Method: Non-homologous integration at double-strand breaks leading to random insertion points and variable expression. Gene Targeting in ES Cells Process Overview Introduction of a targeting vector via electroporation into embryonic stem (ES) cells. Targeted ES cells injected into mouse blastocysts for creating chimeric mice. Mating between chimeric and normal mice produces gene targeted offspring. Mutagenesis Techniques Insertion Mutagenesis Vector Elements: Left and right homology arms, Neor selection. Results in interrupted open reading frame (ORF) in one gene. Deletion Mutagenesis Similar vector setup as insertion mutagenesis, results in exon deletion. Negative Selection Screens Utilizes Neor and HSV-tk for selecting against non-homologous integrations. Targeting Vector Components Contains homology arms, neor (antibiotic inactivation gene), and tk (thymidine kinase gene). Negative Selection: Optional, depends on targeting efficiency. Screening ES Cell Colonies Utilizes probes to distinguish between wild-type and targeted alleles in ES cell colonies. Site-Specific Recombination Systems Components: Recognition sites, Cre-loxP (phage P1), Flp-FRT (S. cerevisiae). Tissue and Stage Specific Mutagenesis Understanding the Cre-loxP system for tissue-specific gene targeting. RNAi Screening Techniques High content screens for target discovery, utilizing libraries of siRNA or dsRNA in various cell types. Techniques maximize throughput and data depth for functional studies. Gene Trap Libraries & Mutagenesis Gene Trap Principle Random integration of selector reporter cassettes leads to trapped genes for downstream studies. ENU Mutagenesis in Mice Implementation of N-ethyl-N-nitrosourea as a potent mutagen; screens for heritable phenotypic abnormalities. Distinguished from gene targeting, often results in point mutations. CRISPR Technology Overview CRISPR/Cas systems harnessed for precise genome editing. Three CRISPR/Cas systems, with Type II utilizing Cas9 for cleaving DNA at specified sequences. Recent developments include: base editing, prime editing, and epigenetic modifications. n

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