Genetics Exam 2b- Study Guide-KHA 11-30-23 copy.pdf

Class of 2027 Genetics Exam 2: Review Sheet Exam on Monday October 30, 2023. Exam Two will be around 50-60 multiple choice questions and you will have two hours to answer the questions. The exam will cover topics 7 – 15 and topic 17 (NOT TOPIC 16). The follow topics below will be the high yield topi...

Class of 2027 Genetics Exam 2: Review Sheet Exam on Monday October 30, 2023. Exam Two will be around 50-60 multiple choice questions and you will have two hours to answer the questions. The exam will cover topics 7 – 15 and topic 17 (NOT TOPIC 16). The follow topics below will be the high yield topics on the exam, in addition to the topics below, it would be helpful to know the “Concept/Terms to Know” slide at the end of each lecture. Also read the papers mentioned in this review sheet. ONLY ONE PAPER TO READ FOR EXAM in Topic 15 “MGM researchers identify a potential method for treating fragile X syndrome” For any questions, email us or Professor Astrin himself at [email protected] Topic 7: Genetic Variation - Definition of polymorphism (minor allele must be 1% or more in a population) Inheritance of a trait controlled by a single locus w/ at least 2 alleles, where the least common allele has a frequency of at least 1%. (If it is less than 1% would be considered a variant or mutation.) Can be studied in both DNA and protein. Most variants that become polymorphisms are neutral or do not really have an effect. Although, some affect disease susceptibility and drug responses. Remember, that a rare disease allele in one population can be considered a polymorphism in another, such as the sickle cell disease gene mutation, endemic to certain regions. - ABO Blood Groups ABO blood groups is a classification of blood based on presence of antigens on surface of RBCs. These antigens can be proteins, carbs, glycoproteins, and/or glycolipids. There are 30 known antigens for blood typing even thought we mostly just use ABO and Rh. The ABO antigens are integral to all cells and are also present in plasma. Someone who is type A will have antigen A on surface of cells and Anti-B antibodies in plasma. Type B will have anti-A antibodies and B antigens. Type AB will have neither of these antibodies, but both A and b antigens. Type O will have no antigens, but both Anti-A and Anti-B antibodies. Type O is the most common blood type in the world. AB being the rarest. - ABO; one gene- 3 alleles Know that there is 1 gene bc the ABO gene is polymorphic, with 3 alleles. Each allele codes for a different blood type. Inherited in Mendelian fashion. Autosomal co-dominant (how people can be AB, not just A or B). Phenotypes possible are A, B, AB, and O. Genotypes can be AA or AO (type A), BB or BO (type B), AB (type AB), and OO (type O). - How do the ABO proteins differ? 1 This ABO gene codes for a glycosyltransferase that transfers specific sugar residues to a protein precursor H antigen. The A allele codes for transferase A (alpha 1-3-n-acetylgalactosaminyltransferase) that recognizes N-acetylgalactosamine and adds this to the H antigen. The B allele codes for transferase B, alpha-1-3-galactosyltransferase that recognizes D-galactose and adds it to the H antigen. The O allele does not have such enzymatic activity and therefore leaves the H antigen alone. Alleles coding for enzymes that form A and B blood groups have SEVEN SNPs resulting in AA changes. The cDNA for O type has a 1 base deletion at cDNA 261 (G) which results in a frameshift and early termination resulting in no enzyme activity. The H antigen is the essential precursor for ABO antigens. The H locus is located on chromosome 19 and it encodes an enzyme that adds L-Fucose to the H precursor. This results in the H antigen having a carb chain at its ends of N-acetylglucosamine, galactose, and fucose. - Blood transfusion with washed RBCs and plasma - know which transfusions are possible 2 If this image is confusing, the top refers to RBC transfusions and the bottom to platelet/plasma. Remember, that RBC has the antigens and that plasma has the antibodies that clump against their respective antigens. Think about which antigen and antibodies are found in what blood type and this image should begin to make sense. Also, from what I found online, platelets and plasma do not contain cells, so antigens cannot be present in these transfusions. - No HLA on exam - How to use repetitive DNA for forensics and paternity tests Be able to figure out how to determine to whom a sample DNA belongs to VNTRs are used to identify matches in DNA based on how many repeats are found for various markers. - Concept to know: RFLP RFLPs are due to single base changes. They are usually of a 2 or 3 allele system and require restriction enzyme digestion. Restriction enzymes recognize a specific nucleotide sequence and make a cut between 2 specific nucleotides in that 3 sequence. If there was a change in this sequence, they would not be able to recognize and make the cut. Example below. Topic 8: Pedigree App − There will be pedigrees on the exam Know symbols Know how to read a pedigree Pedigrees are used to visualize of family medical history using symbols. Shows inheritance of genetic conditions within a family. Helps determine mode of inheritance. 4 Topic 9: Autosomal Recessive Inheritance - Know pedigree notations (A = wild type and a = mutant allele) Every generation on pedigree will have a Roman numeral notation. Arabic numbers are used to identify individuals of a generation. For example, III-4 would be individual 4 or generation III. Affected individuals will have symbol filled in and carriers are not usually indicated but if they are there will be a dot in the middle of their symbol. Proband arrow indicates who is being consulted for family history. - Use of punnett square Slides 19-20… Rules of autosomal recessive Affected Individuals: Phenotype expressed only when individual has 2 mutant alleles. They will have clinical symptoms and are rarer than carriers. Usually there will be little to no protein function coded for by the mutant genes. Most auto rec diseases are loss of function diseases. Carriers/Heterozygotes: Will have only one mutant allele. Typically will not have clinical symptoms and are more common than affected homozygotes. It is usually not possible to tell if someone is a carrier w/o biochemical/DNA testing. These individuals will usually have about 50% normal protein function. - Know what an autosomal recessive pedigree looks like A auto rec. pedigree can be identified as affected individuals may be siblings, but their parents or offspring will typically not be affected. Parents of affected children may be related. The trait will also appear sporadically in small families. There will also be complete penetrance usually with auto. rec. diseases. Heterozygotes will be identified by half of their symbol being colored in. - Know clinical symptoms of Cystic fibrosis 5 Most common auto rec disease amongst Caucasians. Resp and GI problems and elevated sweat electrolyte concentrations. Most characteristic symptom is excessive production of thick, sticky mucus in lungs’ airways. - Do not need to know the frequencies Know there are: large number of mutations but Δf508del is the most common mutation (only need to know deltaf508) CF (auto rec) is caused by mutations in CFTR gene. The phenylalanine deletion at locus 508 accounts for 70% of CF patients worldwide. Although, there are almost 2000 mutations that can cause CF. With the CFTR delta 508 mutation, the CFTR protein is made, but does not get to the cell surface and instead just floats around in the cell. It is a misfolded protein and is eventually degraded in the ER. The lack of the CFTR protein channel being at the cell surface prevents Cl- from efflux into ECF. This prevents mucus from being fluid and makes it so that it is thick and sticky. In delta 508, the PT can be given a corrector drug to boost the CFTR protein to the surface and another doorman drug to open the channel so that the Cl- can pass. - Know: Characteristics of Autosomal Recessive Inheritance - AR phenotype usually seen in sibs of proband, not in parents, offspring, or other relatives. - Recurrence risk for offspring of 2 carrier parents is ¼. - Parents of affected person may be consanguineous. Especially if gene responsible for condition is rare in population. - Parents of affected children are known as heterozygotes. - New mutations in AR disorders are rare. - If one parent is carrier, affected offspring are rare because risk of new mutation by spontaneous mutagenesis ranges from 1 in 100,000 to 1,000,000. - For most AR diseases, males and females equally likely to be affected. - Almost always complete penetrance in AR whereas can be incomplete in AD - AR inheritance more common than AD inheritance (maybe due to penetrance) - Concepts to know: - Examples of AR diseases: Gaucher’s, CF, Tay-Sachs, thalassemias, sickle cell Topic 10: Hb & SCD - Sickle Cell Follows autosomal recessive inheritance pattern and is caused by a single mutation. About 2.5 million in the US have sickle cell trait and 100,000 suffer from the disease. Worldwide there are 3.2 million with the disease and 4.3 million carriers. 1 in 365 black - 6 newborns will have SCD. 1 in 13 will have the trait. 1 in 16,300 hispanic american newborns will have SCD. Normals RBCs turnover every 120 days, but sickle cells turnover every 10-20 days. - Know mutation in which gene - alpha or beta Alpha globin gene is normal. Beta globin gene is mutated with a change in 1 amino acid. The mutation is a change from glutamate (GAG) to valine (GTG) at position 6 (E6V) in beta globin. - How the mutation causing sickling Glutamate has an acidic side chain containing a negatively charged carboxylate (COO-). Valine has a non-polar side chain. The change in amino acids results in valine being on surface of protein although it is hydrophobic. There are 2 valine substitutions per Hb tetramer since there are 2 beta chains per Hb. In the deoxy conformation, the valine side chain of a beta-chain in one HbS binds to the hydrophobic pocket on surface of beta-chain of another Hb tetramer. - When does sickling; occur in the T or R forms? Cells are in the sickled form only in a low oxygen state. In oxygenated conditions, you may not see sickled cells. The complementary binding site of the valine is formed by phenylalanine beta85 and leucine beta88 and is exposed in the deoxygenated state but not when oxygenated. The deoxygenated Hb units polymerize, especially at low oxygen tension. As they polymerize they form long, rigid fibers which ultimately results in the RBCs sickling. - - Consequences of sickling Normal RBCs are pliable and can squeeze thru. Sickle cells are rigid and cannot deform to get thru leading to restricted or totally blocked blood flow thru capillaries. Organ damage, blindness (sickle cells occlude small vessels to eyes), skin ulcers, gallstones, and priapism (painful erections). RBCs are larger in diameter than capillaries. - Symptoms of SCD Anemia, episodes of pain (aka crises), hand-foot syndrome aka dactylitis (swelling in these extremities), jaundice, splenic problems, frequent infections, delayed growth, and vision problems (vessels to eye blocked eventually leading to blindness). What is an acute crisis in SCD and factors that can cause SCD acute crisis Acute pain in SCD pts is caused by ischemic tissue injury resulting from occlusion (clumping of sickle cells) of microvascular beds by sickled erythrocytes 7 - - - during an acute crisis. Usually, a crisis resolves in 5 to 7 days, however a severe one can last weeks to months. Chronic pain can result from destruction of bones, joints, and visceral organs. A sickle cell crisis can be brought on by infections, low oxygen tension, other medical conditions (diabetes), dehydration, acidosis, extreme physical exertion, stress (physical or mental), alcohol, pregnancy, and cold weather. Inheritance Follows autosomal recessive mode of inheritance. If one parent has SCD and other parent is unaffected, then all offspring will have sickle cell trait (carriers). If one parent has trait and other has SCD then 50% that offspring will have SCD and 50% that they will be carriers and have sickle cell trait. If one parent has trait and other is unaffected then 50% that offspring will be carrier and 50% for being unaffected. Population screening in black community to identify carriers as frequency of carriers is about 1 in 12 and frequency of those with disease is about 1 in 500. Treatment – present and experimental Treatment is constant in SCD patients. Have to ensure adequate fluids, folic acid, analgesics for pain, antibiotic therapy from ages 2-5 to prevent infections, aggressive antibiotic therapy for adults, blood transfusions, hydroxyurea, and bone marrow (stem cell) transplants. Standard Treatment: Hydroxyurea mechanism of action unknown, however it increase HbF concentration of RBCs. Given by IV infusion over several hours. Fetal Hb cannot sickle and so if its concentration is incr. in RBC, it could prevent symptoms of SCD. Experimental Treatment: Gene therapy to delete/alter HbF repressors or replace SC mutation with WT - Gene therapy involves taking stem cells from pt, growing them in tissue culture, doing gene editing to cells, and then infusing these cells back into pt. - Infusing CTX001 (experimental gene-editing cell therapy) results in increased HbF - Two ways CRISP/CAS can treat SCD - CRSPR reactivates HbF gene by turning of BCL11A gene - KLF1 expression increases BCL11A and beta-globin expression. BCL11A inhibits gamma-globin production which is essential in HbF. Knocking out the BCL11A gene allows reactivation of HbF production. Thus, KLF1 and BCL11A are both therapeutic targets for SCD. - CRSPR and DNA template fix the mutation in HbA gene - Potential Risks of gene therapy - Insertional oncogenesis (chance that alteration of DNA in stem cell leads to change in genes nearby potentially repressing tumor suppressors or activating oncogenes) - Off-targeting (gene changes made to incorrect target) 8 - - - Unintentional gene activation (risk of complications that unintentionally prevent function of another important gene) Butyric acid to turn on HbF gene Clotrimazole to prevent water loss in RBCs Nitric Oxide (NO) which is usually lower in SCD pts is given to keep blood vessels dilated and reduce crises Fetal hemoglobin: regulation of synthesis by BCL11A Already included above Know first gene therapy treatment using CRISP/Cas9 to delete BCL11A Already included above Concepts to know - Location of SCD - Origins of Saudi Arabia, central India, and Africa - Screening for SCD - DNA gel and protein gel. (The leftmost image is protein analysis while the other 2 are DNA). Lecture 11: Structural Hb Variants, Thalassemias and HPFH - Structural Variants: only need to know HbC variant SVs are alterations in globin peptide sequence w/o affecting rate of synthesis (usually resulting from SNPs). HbC is due to substitution at 6th amino acid of beta-chain. Mutation is glutamate to lysine at position 6 (E6K). This does NOT cause RBCs to sickle, but instead results in a milder hemolytic disorder. Common in west Africa. With homozygous state of HbC disease, can expect mild anemia and splenomegaly presence. - Alpha and Beta Thalassemias Thalassemias are disease in which there is reduced synthesis of either the alpha or beta chains leading to distortion of alpha:beta chain ration. - Know the difference between α and β thalassemia Alpha is where alpha-chain synthesis is reduced or absent and there is excess of beta chains. Common mutations are deletions. Beta is where beta-chain synthesis is impaired and there is excess alpha-chain. Common mutations are of point mutations. - α-Thalassemia - How many α-globin genes in a cell = 4 (2 on each chromosome 16) 9 - Know different clinical symptoms of α thal depending on number of alpha genes mutated WT (normal): M: 14.5-16.5 F: 13-15 alpha:alpha/alpha:alpha Carrier (asymptomatic): M: 13-15.5 F: 11.5-13.5 alpha:——/alpha:alpha Alpha thal minor (asymptomatic): M: 12-14 F: 10.5-12.5 ——alpha/——alpha OR ——:——/alpha:alpha HbH Disease (symptomatic): M: 10-12 F: 8.5-10.5 ——:——/——:alpha Hydrops Fetalis (incompatible w/ life): Severe anemia of fetus ——:——/——:—— - Symptoms of HbH Disease 3 of 4 alpha genes deleted; Hemolytic & microcytic anemia and splenomegaly. Many pts w/ HbH are healthy but at risk for hemolytic episodes, aplastic crises, iron overload, hypersplenism, and endocrine disease. PTs may have mild jaundice, gallstones, pallor, changes in pigmentation of skin (due to iron overload), skeletal deformity, and potentially slow growth in children. HbH is unstable and will lead to precipitation of Heinz (inclusion) bodies forming overtime. HbH has 4 beta-globin chains in tetramer form. The beta-4 tetramers damage RBC membrane and will induce hemolysis. - Symptoms of HbBarts-hydrops fetalis All alpha genes deleted; severe anemia in fetus and incompatible w/ life. Only common in Southeast Asia. Genetics of alpha-thal - Common type of mutations causing alpha-Thal - deletions - 10 - - Summary slides β-Thalassemia - How many β thal genes in a cell = 2 (one on each chromosome 11) - Different types of beta-Thal depending on number of genes mutated and type of mutations: point mutations - What are beta+ and beta0 mutations? Know that the there are fewer types of β-thalassemia but the types of mutations are more significant Clinical symptoms of different β-thal B-thal trait or Minor: PT will have at most slight lowering of Hb level in blood. Condition will closely resemble mild-iron deficiency, however usually pt w/ minor b-thal will have normal iron. No treatment necessary. B-thal intermedia: Globin-chain production will be slightly impaired. PT will have mild anemia. Occasional blood transfusions may be needed depending on severity. B-thal major (Cooley’s): Abdominal swelling, growth slowed, irritability, jaundice, pallor, skeletal abnormalities, splenomegaly, and will require lifelong transfusions. Will also have poor appetite, dark urine, leg ulcers, 11 - gallstones, bone problems, liver failure, endocrine disorders, extramedullary erythropoiesis and frequency congestive heart failure. Will have Hb value lower than 7 g/dL. Why beta thal symptoms more severe than alpha thal symptoms Alpha-chains dissociate more readily into monomers than beta-chains. Beta-chains form hemichromes at a faster rate, making beta-thal more severe. In beta-thal, accumulation of unstable alpha globin chain tetramers leads to premature cell death in bone marrow. In alpha-thal, beta-chain accumulation leads to precipitation of beta tetramers only damaging the cell membrane and not really affecting erythropoiesis in bone marrow. Summary of beta-thal symptoms Mentioned above Inheritance slide - Hereditary persistence of fetal hemoglobin: know consequences - - - Benign condition in which there is significant HbF production that persists in adulthood. Mutations such as large deletions that remove whole betaglobin locus or other genes of beta-globin cluster result in this condition. Loss of locus control region is often seen in HPFH. No clinical symptoms. Treatment slides Blood transfusions and chelation therapy for excess iron required for those with a sever thalassemia. ZYNTELGO is a one time gene therapy for beta thal. It is made specifically for each pt using their own blood’s stem cells and adding functinal copies of beta-globin genes to these cells. May allow pt to thrive w/o need of repeat transfusions. 12 Hydroxyurea helps by stimulating HbF production. Also two methods of using CRISPR: One to repair defect in beta-globin gene and another to upregulate HbF production. Lecture 12: Autosomal Dominant Inheritance - Know definition of autosomal dominant (AD) Clinical phenotype is expressed even when just ONE mutant allele is present. Many pts w/ AD diseases have de novo mutations. Mutations are often in genes coding for nonenzymatic structural proteins and protein components of membranes or receptors. Remember AR disorders will typically result in enzyme defects. AD disease seen in about 1/200 individuals. - Know the rules of autosomal dominant - **Know that the vast majority of those with Autosomal dominant diseases are heterozygous (consider affected Aa in problems on test unless told otherwise 98% of the time one w/ AD disorder inherits due to having one affected parent and one unaffected. VERY RARE to see homozygous in AD. - Genetics - Know difference between complete and incomplete AD inheritance There is complete AD inheritance and incomplete. Complete is where the clinical symptoms of a heterozygote and homozygote are identical. In incomplete, the symptoms will be lesser in heterozygotes. Phenotype will be seen in every generation EXCEPT if there is a new mutation or if there is reduced penetrance. PTs w/ severe diseases do not or may not reproduce therefore, all newborns w/ the disease are due to de novo mutations. - Characteristics of AD inheritance AD diseases are common in certain populations. For example, familial hypercholesteremia is 1 in 100 in South African population. Risk and severity of 13 AD disease in offspring is dependent on is trait has variable expression and if expression of symptoms are complete or incomplete. In mating of affected w/ WT, there is always 50% chance of offspring being affected. - - - Know what autosomal dominant pedigree looks like Generations are not skipped in AD pedigrees. Disorder will be present in all generations. It is equally likely to see disorder in males and females. You will also see male to male (father to son) transmission which is NOT seen in Xlinked. Familial hypercholesterolemia Most common AD disorder. Most cases have one mutation allele and are called heterozygous FH (HeFH). Since FH is so common though, there are those w/ homozygous FH (HoFH). HoFH involves more sever clinical symptoms. Mutations in the LDL receptor are the most common cause of FH (>90% cases). Know the difference between the clinical symptoms of HOF and HEF and the frequency of both HeFH: one mutated allele - Characterized by incr. levels of LDL chol. - Causes premature cardiovascular disease - 1.3 million people in US w/ HeFH - About 90% of those w/ HeFH are undiagnosed - Chol. Levels are greater than 300 mg/dL in adults, 250 in children; normal is 200 or less in adults - LDL levels are greater than 170-200 in children, 220 in adults; normal is about 120 in adults - Triglycerides will be normal in those w/ HeFH - Xanthomas, Xanthelasmas, and Corneal arcus (waxy chol. Deposits on skin, tendons, eyelids and around cornea) - Angina (chest pain) which may also signify heart disease presence - Men w/ HeFH by age 60: - 75% develop CAD and 50% had fatal MI - Women w/ HeFH by age 60: - 45% develop CAD and 15% fatal MI - Prevalence is 1/244 HoFH: two mutant alleles - Prevalence is 1 in 160,000-300,000 - Total chol. and LDL levels over 600 mg/dL - Symptoms consistent with heart disease, PVD, cerebrovascular disease, or aortic stenosis - Articular symptoms like tendonitis or arthralgias - Unusual skin lesions like xanthomas at birth or very early childhood - Corneal arcus - Murmur of aortic stenosis 14 - - - - - Usually will not survive adulthood beyond age 30 unless treated w/ unusual methods Which gene is mutated) is the most common cause of FH Most common cause is mutations in LDL receptors (AD,~93%). Mutations decr. number of functional receptors on cell membrane, reducing LDL uptake by liver and reducing clearance of LDL from blood. Cholesterol synthesis is not repressed and elevated plasma levels of LDL results in atherosclerosis. LDL receptor gene located on chromosome 19. Which gene mutations are rare causes of FH - Apoprotein b-100 (AD, Het. 1/1000, ~5%) - ARH adaptor protein (AR, very rare) - PCSK9 (AD, very rare, ~2%) Treatments HeFH: Statins (inhibit HMG-CoA reductase resulting in lowered cholesterol in cells, triggering upregulation of LDL receptors, promoting uptake of LDL from blood), ezetimibe (decr. chol absorption in small intestine), antibodies against PCSK9 (hepatic protease that attaches and internalizes LDL receptors into lysosomes, destroying them), and treatments to decrease LDL levels to near normal HoFH: More difficult to treat than HeFH. High levels of statins, lomatipide (inhibits microsomal TAG transfer protein which is needed for VLDL assembly), LDL apheresis (like kidney dialysis but involves filtering LDL from blood), and liver transplant in rare cases. Marfan syndrome know mutated gene and clinical symptoms Affects connective tissue. Since CT is found all over body and in multiple organ systems, there are a host of problems that can present. Follow AD inheritance. Incidence is 1 in 5 to 10 thousand live births. Is caused by mutation in fibrillin-1 (FBN1) gene. Fibrillin-1 is component of microfibrils, which are structures in ECM. About 25% of pts have de novo mutations. Clinical symptoms are skeletal, optical, and cardiovascular. Skeletal involves long fingers and toes, extreme lengthening of long bones, scoliosis, rib and sternum abnormalities, pectus excavatum (pitting of sternum), etc. Optical involves ectopia lentis where lens are dislocated into anterior chamber of eye. Cardio problems may include enlarged aortic root (widening), valve issues, and dissecting aneurysms, largely responsible for shortened lifespans of pts. - Has variable expressivity 15 - - Symptoms can vary from presumed normal to things like: eye lens displacement, congential heart disease, and long digits and toes (or very long). - Huntington's disease: Caused by mutation in huntingtin gene (HTT). Follows AD inheritance. Caused by same mutation in ALL pts. Is a progressive neurodegenerative disorder. - Clinical stages Age of onset is late childhood to late adulthood, thought usually in 30s and 40s. Symptoms become more severe as disease progresses over time. Average length of survival after clinical diagnosis is usually 10-20 years. Early stage: Symptoms may affect cognition, mobility, depression, mood swings, memory issues, clumsiness, involuntary twitching, and lack of coordination. MidStage: Concentration and short term memory diminish, involuntary movements of head trunk and limbs will increase. Most common invol. Movement is chorea which involves abnormal invol. movement that is brief and abrupt. Walking, speaking, and swallowing become more difficult. Late stage: PT unable to care for self. Death follows complications such as choking, infection, or heart failure. Know single mutation is in Huntingtin gene and the mutation is expansion of a CAG repeat in front of the gene Relationship between number of CAG repeats and age of symptoms and risk for HD All alleles of 27 repeats and higher are unstable and prone to expand in future generations, particularly when transmitted by male parent. Although most pts w/ HD have affected parent, about 3% of cases result from new expansions into the disease range. Pedigrees … Effect of increased CAGs Mentioned above Summary: Rules for AD inheritance slides 1) Phenotype appears in every generation; each affected person has one affected parent unless new mutation or reduced penetrance occurs 2) Any child of an affected parent has a 50% risk of inheriting mutant gene 3) Phenotypically normal family members usually do not transmit the phenotype to offspring EXCEPT in cases where there is reduced penetrance 16 - 4) Males and females equally likely to transmit phenotype to child of either sex Comparison of AR and AD inheritance slide Topic 13: Factors Affects Inheritance - Know factors that may complicate Inheritance Patterns New mutations, germline mosaicism, delayed age of onset, reduced penetrance, variable expression, pleiotrophy, and locus/allelic/clinical heterogeneity - New mutations: major cause most cases of achachondroplasia Case in which a child with a genetic disease is born into a family w/ no history of it. Usually this mutation will have occurred in the germ cells of one (rarely both) of the parents. 80% of cases of Achondroplasia are due to new mutations. This is due to the fact that usually one w/ this disease does not reproduce due to the severity of the disease. However, if inherited the disease is AD and has complete penetrance. It is a disorder of bone growth and frequency is 1 in 25 to 30 thousand live births. This disease is caused by 2 specific mutations of the FGFR3 gene: 1138G>A in 98% of cases and 1138G>C in 1-2%. FGFR3 is a transmembrane tyrosine kinase receptor that binds to fibroblast growth factor. New mutations occur only in males during sperm formation and likelihood of mutation increases with age of father rising. - Know what is germline mosaicism Occurs when all or part of parents’ germline cells have disease mutation but somatic cells do not. This mutation in germline occurs during embryonic development. It is suspected in cases where parents are unaffected but have 2 or more children w/ AD disease. - Know definition of penetrance Penetrance is probability that mutant allele will have any phenotypic expression in individual w/ mutation (mostly refers to AD diseases). Penetrance is ALL OR NONE. In reduced or incomplete penetrance, person does not show any disease phenotype. Possible causes of reduced penetrance 17 Expression could be age-related, environmental modifiers, genetic modifiers, complex genetic and environmental interactions, and epigenetic regulation. - - Remember that reduced penetrance not only explains why an offspring does not show phenotype but can ALSO explain why seemingly unaffected parents have children w/ phenotype. Polydactyly and osteogenesis imperfecta are good examples of reduced penetrance conditions. Definition of variable expressivity Expressivity is degree to which trait expression of disease phenotype differs among individuals with same genotype. DO NOT MIX THIS UP WITH PENETRANCE! Variable expression leads to some people showing milder symptoms and others showing more severe conditions of same disease. This may be caused by environmental factors, modifier genes, allelic heterogeneity (diff mutations in same gene cause diff phenotypes). However, ALL affected individuals will have AT LEAST one symptom. Example: Neurofibromatosis type 1 (NF1) Disorder of nervous system, eyes, and skin. Frequency is 1 in 3500 births. This disease has complete penetrance (varies w/ age) BUT variable expressivity. Is caused by mutations in NF1 gene resulting in loss of function of neurofibromin (tumor suppressor gene). Follows AD inheritance and 50% of cases are due to new mutations. Diagnostic criteria for NF1: Pt has 2 or more of the following: - Flat, light brown spots on skin - Freckling in armpits or groin - Tiny bumps on iris (Lisch nodules) - Soft bumps on or under the skin (neurofibromas) - Bone deformities - Tumor on optic nerve (optic glioma) - Learning disabilities - 1st degree relative w/ NF1 KNOW DIFFERENCE BETWEEN REDUCED PENETRANCE AND VARIABLE EXPRESSIVITY Diseases in the same family can have both together reduced penetrance and variable expressivity 18 - - Example disease is Camptodactyly in which pt has immobile, bent pinky fingers. Variable expressivity in this disease comprises of phenotypic expression in no hands, one hand, or both hands. Know definition of Pleiotropy Occurs when mutation in one gene influences 2 or more seemingly unrelated phenotypic traits. For example, CF affects lungs and pancreas and Marfan’s affects eye, skeleton, and cardiovascular system. Basically, you need to see effects in more than one organ system to identify a mutation as pleiotropic. Know definitions of locus and allelic heterogeneity and the difference between them Table LOCUS: How the same or similar phenotype can be caused by mutations in different genes. Examples mentioned by Astrin: Cornelia de Lange syndrome (developmental disorder) can be caused by mutations in at least 3 diff genes. Retinitis pigmentosa is another example in which there are at least 43 different loci w/ 5 X-linked forms, 14 AD forms, and 24 AR forms. ALLELIC: When diff mutations at same locus lead to same or very similar phenotypes. (Diff mutations in HGO gene all cause alkaptonuria). How CFTR mutations can cause phenotypic traits in lungs, pancreas, and sperm cells. Tay-Sachs: AR lysosomal storage disease caused by mutations in HEXA gene which causes deficiency of enzyme hexosaminidase A. Exists in both infantile and adult forms. Infantile involves neuro degen beginning b/w ages of 3-6 mo. and progressing to death at 2-4 years of age. Adult form involves unsteady gait, progressive neurodegen. Symptoms present in adolescence or early adulthood, including speech and swallowing difficulty, gait issues, spasticity, cognitive decline, and psych illness. However, they tend to live into their 60s. Summary slides Lecture 14: X-Chromosome Inheritance 19 - - - - Dosage compensation - Evidence for it: cytogenetic and biochemical - Single isozyme in single hair follicle - Barr body: Lyon hypothesis: know details Mechanism of X inactivation - Inactivation center XIC - Tsix Gene and Xist gene: - Choosing which chromosome is Xactive and X inactive: Pairing? - Spreading - Maintenance of inactivation - Not all the genes are actually inactivated – around 15%-25% are not inactivated (mainly on short arm) Comparison of Xinactive and Xactive Features (Table) X chromosome inactivation in women results in women being mosaics X chromosome inheritance of mutant alleles - Males cannot inherit from their fathers - Females always inherit mutation X from affected father - Offspring of carrier mother x normal father can be wild type, affected or carriers - Summary of inheritance Know what X-linked pedigree looks like Lecture 14: X-Chromosome Inheritance (65 slides) - Kyle. Orange text for the season. 🎃🦇👻 − Dosage compensation: Think of it like a mechanism for an OD. You took one to many Xanax for the biochem 4 exam? No? Just me? Anyway, in genetics we’re talking about X chromosomes. Females inherit two X chromosomes (one paternal, one maternal) but only need to express one. So to compensate for this additional dosage, one is inactivated (Xinactivation). − Evidence for it: cytogenetic and biochemical − Single isozyme in single hair follicle: − From the slides: “Females who are heterozygous for G6PD and/or PGK isozymes expressed only one isozyme in each cell and also only have one barr body… Therefore there must be a mechanism for dosage compensation.” − I am taking this as “Basically here’s two examples of how we know that this goes on. Female inherits two copies of slightly different isozymes, one on each X chromosome.. but instead of making both isozymes they only express one: because one chromosome is inactivated. − Barr body: a small chromatin body found in the cells of normal females but not in males. The chromatin is composed of inactive X chromosome material that has been condensed and is heterochromatic. (Recall, heterochromatin and Euchromatin) Heterochromatin can’t be transcribed because there isn’t any physical space for the transcriptional enzymes, and therefore it is inactivated. It gets replicated later in the cell cycle than all the other 20 − − − − − − − − − chromosomes, which I assume helps the cell not to F up the whole inactive thing. You can see them in cells if you stain them right. Here’s a picture. ← that is a hyperlink. He made a slide about there always being one fewer barr bodies than x chromosomes a person has. So if you inherit 3 because of a mistake in gamete production you aren’t necessarily worse off: your body will just inactivate 2 instead of 1. Lyon hypothesis: know details Named after the scientist, Mary Lyon. It is a hypothesis for the dosage compensation mechanism. Inactivation occurs EARLY in embryonic development, like when the embryo is only 8-32 cells in total. (Don't memorize the number I’m just giving a size reference) Because it occurs when there are more than 1 cell, the end result is a mosaic or mixture of x chromosome expression in female somatic cells. Which X chromosome gets inactivated is random. Once it’s inactivated it remains inactivated for all descendant cells. Mechanism of X inactivation (Exactly exactly isn’t completely understood.. but see below anyway. There are 3 steps: Initiation (transcription), Propagation (spreading), and Maintenance. − Inactivation center XIC − This is a locus, called Xic. It governs inactivation. Has several genes which are nonproteincoding genes that include Xist, Tsix, Jpx, and Xite. − It’s about in the center of the chromosome − Tsix Gene and Xist gene − Xist is transcribed, producing RNA. − Tsix plays a role in pairing. (see below) AND is an inhibitor of Xist transcription. − Choosing which chromosome is Xactive (Xa) and X inactive (Xi): Pairing. 21 − It’s how the cell knows how many X chromosomes to inactivate. We don’t know the mechanism for sure. The pairing of X chromosomes is called “X pairing.” Shocker. − Tsix makes Rna. So does Xist. This RNA and the lining up of inactivation loci (XIC) of both chromosomes (and I kid you not this is exactly what the slide says) “generate the epigenetic asymmetry that enables one X to become the Xi and the other to remain the Xa.” − The chromosome that makes more Xist RNA will become the inactive chromosome. The other chromosome is expressing Tsix, which silences Xist and makes that chromosome active. − − − Spreading of Xist RNA 22 − Makes a 17kb functional RNA molecule (So it is NON CODING RNA) − Not expressed in males (we dont inactivate our X chromosomes or we die) − Xist RNA spreads over the chromosome and it attracts protein factors that lead to modification of the histone tails, thereby inactivating the chromosome. − Maintenance of inactivation − Includes stuff like methylation, histone hypoacetylation, and some other stuff he didn’t explain at all in class so I am skipping it (But it is slide 39 if anybody is feeling feisty. − Not all the genes are actually inactivated – around 15%-25% are not inactivated (mainly on short arm). So in the real world if a female has a gene that falls into that 15-25%, she will have two copies of that gene and potentially express something like isozymes due to this. − Comparison of Xinactive and Xactive Features (Table) − − Also, Xi is found in the periphery of the nucleus while Xa is found interiorly. − Note from Kyle… not listed on review sheet but he talks about it a lot and it has to do with above concepts. − He also talks about how these things are important clinically. For example if a female inherits a mutant allele on one X chromosome, due to dosage compensation and the random nature of how which cell is chosen, the symptoms from one person to the next can vary. If you get really unlucky and all your liver cells express the mutant alleles? RIP your liver. Your mom was just luckier than you because instead of the mutant allele being expressed in majority in her liver, it was a minority so she didn't ever suffer from any major symptoms. − Also he notes that if a dude inherits an x chromosome mutation? That's it, we get the mutation. Maybe that's part of why there are very slightly more women born every year than men? He talked about that one time. 23 − Vocab: Manifesting heterozygote: A female who shows the phenotype due to “unfavorable lyonization.” (Bad dice roll. Nat 1. Mosaic isn’t 50/50 or it is but there’s a large number of bad ones in a specific organ..etc. − We can’t tell what the phenotype will be. We can only know what the genotype is, for these kinds of mutations and inheritance patterns. − − − − − − − − − − − X chromosome inheritance of mutant alleles Males cannot inherit from their fathers Females always inherit mutation X from affected father Offspring of carrier mother x normal father can be wild type, affected or carriers Summary of inheritance There kind of isn’t such a thing as X linked dominant or recessive. There was, but because of inactivation the scientific community is moving away from those terms. It is simply “X linked.” Know what X-linked pedigree looks like Males are much more affected than females. There is NO MALE TO MALE transmission (affected fathers will NOT have affected sons, if the mother is not a carrier.) Pedigrees included on next page. Know how to do a punnett square. He does a few of them in the lecture, examples include: WT mother x Affected Father Carrier mother x WT father Let me know if I can explain anything from this section! 24 25 Lecture 15: Fabry & Fragile X Diseases - Fabry disease Lysosomal storage disease with deficiency of alpha-galactosidase enzyme. Frequency is 1 in 30 to 40 thousand males. Primary pathologic site of accumulation is in small vessel walls or kidney, heart, skin, etc. Disease leads to premature death from renal failure, cardiac issues, and cerebrovascular disease. KNOW THAT if father is affected, then all daughters are heterozygotes and all sons unaffected. If mother is heterozygous then 50% sons affected and 50% daughters heterozygotes. - Clinical symptoms - - Mutated gene in Fabry disease and metabolic defect Mutation in alpha-Gal A leads to decrease enzyme activity and thus build up of Gb3 (GL-3) in lysosomes and lysosomal inclusions will be present. A carrier will have some accumulation, affected male will have accumulation in all cells, and normal individual will have very little to no accumulation. - Variable expression in females in families with same mutation Can range from no symptoms for females to stroke/TIA, cardiac issues, pain, proteinuria from renal dysfunction, etc. However, ALL males express symptoms. - Identification of female carriers. Need to use DNA mutation analysis DNA testing is needed instead of enzyme activity analysis for females as control for normal alpha-Gal activity and that of female heterozygotes may overlap and make it hard to distinguish. Most accurate diagnosis can be made from testing for mutation in GLA gene. Fragile X Caused by increase in CGG repeats. Up to 44 repeats is normal and then disease manifestation can be seen in higher repeats. This disease is leading known cause of intellectual disability and most widespread single-gene cause of autism. Seen in 1 of 3 to 4 thousand males and 1 in 8,000 females. Age of onset is childhood. There is a strong association b/w FXS and autism as 50% people w/ FXS and 2 of 3 males w/ FXS meet criteria for autism. - Symptoms 26 - Common physical features include: elongated face and broad forehead, large prominent ears, high arched palate, prominent jaw and dental crowding, macroorchidism, squint, murmur, mitral valve prolapse, cardiomegaly, dilation of aorta, hypotonia, joint laxity, flat feet, hollow chest, and scoliosis. Note that nose will be of normal shape. Behavioral symptoms include: Developmental and behavioral issues, attention deficits, autistic behaviors, aggression, anxiety and hyperactivity, sleep disorder, seizures, hypersensitivity to sensory stimulation (noise and touch), and deficits in social-personal skills. - Location of FXS gene on X Chromosome FXS is caused by gene FMR1. In 5’ UTR region of first exon of FMR1 there is tandem repeat of CGG. Normal individuals have small number of repeats, but this is expanded in pts w/ FXS. Specific location on X chromosome is Xq27.3. 30-50 repeats is normal and unmethylated. 96 repeats is premutation but still unmethylated. Over 200 repeats is full mutation and there is methylation. - Mutation in fragile X gene (expansion CGG in first exon) Mentioned above - When and in whom does triplet expansion occur The CGG repeat is unstable and therefore length in normal population is variable ranging from 6-55 repeats. Repeat can become unstable upon maternal transmisison and number of repeats are ONLY expanded during formation of eggs in females who are known to be premutation carriers. Premutation can lead to full mutation in subsequent generations when transmitted thru a female carrier. Also, males with premutation are at risk of developing FXTAS. Premutation protein w/ 55-200 repeats causes Fragile X Associated Tremor/Ataxia syndrome (FXTAS). The premutation protein may also cause in women Fragile X Associated Primary Ovarian Insufficiency (FXPOI). The FMR1 gene w/ over 200 repeats causes methylation of the gene and thus lack of transcription. Other triplet repeat diseases. Don't need to know names of triple repeat diseases (just know they exist) 16 disease have been identified caused by triplet repeats. Many of these have smaller degree of expansion than FXS. Triple expansion is caused by slippage during DNA replication. Repeats can be found in UTR, intron, or exon. 27 Topic 16 is NOT on Exam Two Topic 17: Pedigree and Probability Problems Know what probability is. Rules of probability Multiplication rule -if events are independent Addition rule – if events are related How to use probability in pedigrees Topic 17: Pedigree and Probability Problems (17 slides) – Sneha Know what probability is: Probability refers to the chance or likelihood that a specific outcome or form of an event will occur in a particular situation (relative to all possible outcomes or forms of the events) Rules of probability: Multiplication rule -if events are independent (this AND this) − If the outcome of each event has no effect on subsequent outcomes, the events are said to be independent. − If two or more events are independent, the chance that they will occur together is the product of their separate probabilities. − Note: Sex of child at each birth is independent of sex of previous child. Examples: 5) Two female children in a row would be? − ½ X½ =¼ 6) What is the probability of having three girls in a row? 28 − Probability of having a girl at each birth = ½, Since reproductive events are independent of each of other, the probability is: − ½ X½ X½ =⅛ 7) What is the probability of having three girls in a row if a couple has two daughters already? − Probability of having a girl at each birth = ½ − Since the first two girls are already born, these events are no longer probabilities and only the birth of the third girl is a probability which is ½ Addition rule – if events are related (this OR this) − we want to know the probability of either one outcome OR another, one adds the respective probabilities together. − If either one or the other event must occur, the chance that they will occur together is the sum of their separate probabilities. Examples: 8) The probability of getting two heads in a row (1/2 X 1/2) OR the probability of getting two tails in a row (1/2 X1/2) is the sum 1/4 + 1/4 = ½ 9) The probability of having two girls OR two boys in a row 1/2 X 1/2 = 1/4 1/4 + 1/4 = 2/4 =½ (ignoring order of births) 10) If a couple is planning to have only three children and don’t want them to all be of the same sex, what is the probability of having both sons and daughters? − As we said before, the probability of having three daughters or three sons is each ⅛. Probability of producing 3 girls OR 3 boys is 1/8 + 1/8 = ¼ − Therefore the probability is having some combination of boys and girls is ¾ How to use probability in pedigrees − Important note: remember the probability that a parent will transmit one of the two alleles at a locus is: Independent from one birth to the next. − If a person is a carrier of a recessive disease, the person’s genotype is Aa (a = disease allele). − The carrier’s eggs or sperm will have either the A allele or the a allele with a 1/2 or 50% chance of having either one or the other. − Therefore a child has a 50% chance of inheriting the mutant allele a from one parent. 29 NOTE: PRACTICE Pedigrees: − @Ch17 from Slide 20, Answers on Canvas − @Halloween Pedigree analysis Extra Resource (NOT FROM SLIDES): Modes of Inheritance: Autosomal Dominant − Aa and AA will have disease − aa will not Autosomal Dominant with reduced Penetrance: − In incomplete or reduced penetrance, some individuals will not express the trait even though they carry the allele. Autosomal Recessive − Aa, AA will not have disease − aa will have disease − Probability of being a carrier = ⅔ X linked Dominant − Affected male with unaffected female will have 100% affected daughters X linked Recessive − Female carrier and normal male will have high rates of affected sons − Example: hemophilia Y linked − Only males are affected, this inheritance never skips generations Important Terminology: Allelic Heterogeneity: Allelic heterogeneity occurs when two or more alleles of a single locus are independently associated with the same trait “Different trait, same location” Locus Heterogeneity: locus heterogeneity occurs when two or more DNA sequence variations at distinct loci are independently associated with the same trait. “Same trait , different location” 11. PAPER: Topic 15 “MGM researchers identify a potential method for treating fragile X syndrome” – Sneha Summary: Researchers from Massachusetts General Hospital (MGH) have identified a potential treatment approach for fragile X syndrome (FXS), a leading cause of autism spectrum disorders. FXS is 30 due to an expansion of the CGG trinucleotide repeat within the FMR1 gene, leading to reduced expression of the FMRP protein which is vital for brain development. This reduced expression results in developmental delays, learning disabilities, and various social and behavioral problems.The disorder affects 1 in 3,000 boys and 1 in 6,000 girls. Instead of employing gene therapy or editing to restore FMRP expression, the MGH team aimed to contract the expanded CGG repeat by leveraging the body's natural DNA repair mechanisms. By using models derived from FXS patient cells, they found that inhibiting two kinases, MEK and BRAF, led to increased production of "R-loops" (nucleic acid structures) that the cells interpret as DNA damage. The cells then initiate repair mechanisms which remove the expanded CGG repeats, thereby normalizing CGG levels and reactivating the FMR1 gene. Lee suggests that this method of contracting the CGG repeat might serve as a one-time treatment for FXS. Current efforts are focusing on extending this technology to patient neurons and testing in animal brain models. 31