GCD exam 3

Clinical Application of Pharmacogenomics

Pharmacogenetics-based testing can help determine which individuals may benefit from a specific drug and what is the most appropriate drug dosage. Unlike traditional Therapeutic Drug Monitoring (TDM), which is not performed until after a drug is administered, pharmacogenetics oriented TDM can be conducted even before treatment begins, decreasing the trial-and-error period or eliminating certain drugs from consideration. Genetic differences are explored for targeted drug therapy. Pharmacogenomics has a potential to reduce overall hospital cost. We need to determine the effects of genetic variation on drug interactions

possible CF treatments

The small molecule VRT-532 interacts directly with ΔF508 and the G551D mutations in the cystic fibrosis transmembrane regulator (CFTR) protein. Gentamicin An antibiotic that binds the 16S bacterial ribosomal RNA subunit. Thought gentamicin has a lower binding affinity for mammalian ribosomes, this binding results in ribosomes reading through nonsense mutations. For many diseases, a modest increase in normal protein can result in a normal or less severe phenotype. Gentamicin has been used in patients having cystic fibrosis and muscular dystrophy. Needs to be used in very high concentrations. Unfortunately, gentamicin can also result in hearing loss and kidney toxicity Read-through of premature stop codons by PTC124

Pharmacogenetics and pharmacogenomics

The study of how an individual's genetic inheritance affects the body's response to drugs (efficacy and/or toxicity) Goal: to select the drugs with the greatest potential for benefit along with the least likelihood of harm in an individual patient, based on their genetic make-up.

Mitochondrial donation for treatment of disease

The technique has only been legally approved in the UK, not the US, so Hope Fertility Center clinician (in New York City) John Zhang conducted the procedure in Mexico where, he claimed, "there are no rules". Previous children in this family had Leigh syndrome, a fatal disorder. He is adamant he made the right choice, saying, "To save lives is the ethical thing to do."

RNAi therapeutics

RNAi is a fundamental highly specific cellular mechanism for silencing gene expression triggered by double-stranded RNA (dsRNA). RNAi provides a possible mechanism for targeting (and eliminating) the cellular production or proteins associated with pathogenesis and disease. The synthesized siRNA is 21 nucleotides of duplex RNA (dsRNA) Then the siRNA enters the cytoplasm, it is recognized by the RNA Induced Silencing Complex Loading Complex (RLC). This then hands off the guide strand to the RNA Induced Silencing Complex (RISC). The active RISC with the RNA guide strand then binds to the target mRNA and cleaves the mRNA target by Ago2 RNaseIII RNAi therapeutics allows for selective RNA degradation

Dosing equation for warfarin using genomic data

SNP Allele Substitution Gene rs1799853 CYP2C9*2 Arg144Cys CYP2C9 rs1057910 CYP2C9*3 Ile359Leu CYP2C9 rs9923231 CàT in prom. VKORC1 Dose (mg/day) = 0.628 - 0.0135 (Age) - 0.240 (CYP2C9*2) - 0.370(CYP2C9*3) - 0.241 (VKORC1) + 0.0162 (height [in cm])

hallmarks of cancer cells

Self-sufficiency in growth signals Evade apoptosis Insensitivity to anti-growth signals Sustained angiogenesis Tissue invasion and metastasis Limitless replicative potential

Angelman Syndrome ("AS")

Severe mental retardation Seizures EEG abnormalities Hyperactivity Bouts of inappropriate laughter Mild hypotonia Sitting: 12-20 months (12 months in PWS) Walking: 3-5 years (2 years in PWS) Toilet training: 3-4 years Speech: Only a few single words or signs

Withholding information

Situations in which a professional must decide whether or not to share information with a patient. Case Examples 1. Unanticipated information (non-paternity) •55 year old woman presents with depression, chorea, cognitive decline. •Family history suspicious for Huntington disease. •Family counseled that a diagnosis of Huntington disease is likely. •Adult children present for appointment- considering predictive testing if diagnosis confirmed •Lab contacts clinician to discuss case prior to signout. •Physician asks whether standard report can be modified to indicate that result is "positive" without specifically indicating homozygosity. -Feels that she is protecting the patient's autonomy by only answering the question that was asked. •Clinical GC believes homozygosity must be reported- even though it answers questions that were not asked.

Confidentiality

Situations in which a professional must decide whether or not to share privileged information with family members, insurance companies, fellow professionals, or other third parties. Case Examples 1. Duty to warn family members about risk. •Sample received for "FAP" testing due to family history. •Documentation of familial mutation not received. •Lab GC "connects the dots" that wrong test is ordered. •Affected relative actually had MLH1 mutation •Patient is estranged from affected relative- can't reasonably obtain results.

Discrimination

Situations in which a professional or patient is concerned about unfair treatment by an insurance company or employer based upon the results of a genetic test. Case Examples 1. Loss of health insurance (MN statute protects in some circumstances). 2. Railroad requiring that some employees be screened for genetic susceptibility to carpal tunnel syndrome.

Informed consent

Situations in which a professional questions the patient's ability to make an autonomous decision due to a lack of information, misunderstanding of information, or situations in which patient cannot make a voluntary decision. Case Examples 1. Predictive testing for adult onset diseases 2. Testing minors

Diversity

Situations in which ethnicity, religion, socioeconomic status, language etc. present an obstacle in the genetic counseling process. Case Examples 1. Different cultural perceptions of the cause of disease. 2. Different cultural perceptions of importance of fertility. 3. The role of the family in making decisions (vs. the American system stressing patient autonomy)

Value conflicts

Situations in which there is a difference in personal/professional values between (a) professional and patient (b) professional and society (c) between two professionals. Case Examples 1. Sex selection 2.Patients selecting for such traits as deafness or dwarfism

Uncertainty

Situations in which there is ambiguity about how to incorporate genetic information into a patient's care. Case Examples 1.Variants of uncertain significance (mutation vs. benign variant) 2.Direct to consumer test results indicates a very modest increase in risk for a particular condition

Genetic mosaicism

Somatic mutations result in genetic mosaicism • Depending on the timing and cell of origin of a somatic mutation, its occurrence in the body may be very different • Common forms are somatic, germline, gonosomal, and in various patterns on the skin that follow from cell migration during development

Summary of Tumor Suppressor Gene (TSG) mutations and the two-hit hypothesis

Some TSGs can be involved in both familial and sporadic cancer. Familial: Inherit one mutant allele, Second mutant allele develops in somatic tissue Sporadic: Both mutant alleles develop in somatic tissue

Directiveness

To what extent should a professional influence a patient's decision making process. Case Examples 1. Situations in which a patient would clearly benefit from a particular course of action.

Problems with Gene Therapy

Transforming enough cells to allow for sufficient gene expression Immunogenic response to the virus vector Response to the expressed protein, especially in null mutations (May be best to do therapy on missense mutations with residual activity, protein that is present will produce immuno-tolerence.) Insertional mutagenesis

PKU treatment

Treatment of PKU requires a lifelong diet of reduced phenylalanine. Treatment of individuals with BH4-deficient forms of PKU requires a combined therapy, including Phenylalanine restriction, BH4 replacement and pharmacological doses of neurotransmitter precursors. "Phenylketonuria (PKU) is hypothesized to be a conformational disease, with loss of function due to protein destabilization, and the restoration of enzyme function that is observed in BH4 treatment might be transmitted by correction of protein misfolding." Addition of BH4 to misfolded protein may result in partially active holoenzyme, so it can still help treat PKU in the case of mutant enzyme

Unstable trinucleotide repeats

Triplet repeats are the sites of mutation in several heritable disorders, including Huntington's disease (HD), Spinobulbar muscular atrophy (SBMA), fragile X syndrome, spinocerebellar ataxia (SCA-1) and mytonic dystrophy (DM). These repeats are highly polymorphic in the normal population. Small or moderate increases in repeat number in Fragile X and DM are premutations with little or no associated disease. In each case, expansion of the trinucleotide motif to a "critical" number is associated with full expression of the disease.

tumor suppressor genes

Tumor suppressor genes are defined as genes whose reduced function can lead to neoplastic changes. To contribute to cancer, usually both copies in a cell have to be inactivated - TSGs are recessive at the level of the cell Many TSGs are check points of the cell cycle

Problems with the Cas9-mediated gene editing of HBB (but applies to other edits as well)

The efficiency of homology directed repair (HDR) of HBB was low. The edited embryos were mosaic. Off-target cleavage, as revealed by whole-exome sequencing. The endogenous delta-globin gene (HBD), which has similar sequence to HBB, competed with the intended exogenous donor DNA to act as the repair template, leading to unwanted recombination products.

DNA Editing using CRISPR

The gRNA can be synthesized to match DNA in virtually any gene Following Cas9/gRNA binding and target DNA cleavage, the double strand breaks are repaired by either of two processes: 1. non-homologous end joining (NHEJ), resulting in indels 2. homology directed repair (HDR), resulting in precise edits if a donor DNA molecule is present We can provide donor DNA ourselves, hoping that the cell will use it to precisely repair the double-strand break

treatment for complex diseases

The genetic contribution to a complex disease is usually stated as an increase in risk based on family history. Usually there is no specific treatment but environmental risk factors can be recognized. The following often help: Quit smoking (e.g. A1AT deficiency and emphysema) Exercise (e.g. cardiovascular disease) Reduce weight (e.g. diabetes) Intake of folic acid during pregnancy (e.g. neural tube defects) Eliminate ethanol during pregnancy (e.g. fetal alcohol synd.) Cigarette Smoking and Emphysema Cigarette smoke oxidizes the critical methionine residue at the active site of α1-antitrypsin (A1AT), reducing its ability to inhibit elastase by 2000-fold. Some individuals have only 40-60% of normal levels of A1AT (heterozygotes; ~10%). If these individuals smoke, a phenocopy of inherited A1AT deficiency is created.

Imprinting for complex traits

The human genome has about one hundred imprinted loci. Their effects on complex traits are subtle - just like of those common DNA variants.

liposome vectors

Typically no integration of the exogenous (foreign) DNA into the host genome

The international normalized ratio (INR)

The international normalized ratio (INR) measures blood coagulation relative to a standardized coagulation time. The INR target for warfarin therapy is between 2.0 and 3.0. The range of warfarin dosages given - to obtain an INR target between 2.0 and 3.0 - can range from dosages of <15 mg warfarin / week (7% of patients) to dosages of >60 mg warfarin / week (4% of patients). It was found that individuals with 1 or 2 allele variants of CYP2C9 were 5-6 times more likely to have an INR greater than the upper-limit of the therapeutic interval and up to 4 times more likely to have major bleeding complications compared to the general clinic population. Polymorphisms of VKORC1 and the INR

Delivery Methods of genes

Viral •Examples: Adenoviruses, adeno-associated viruses, lentiviruses •More efficient •Most studied/used nonviral •Examples: Stealth liposomes, polycationic particles, naked DNA •Easier to scale-up •Less likely to cause cancer. •Less likely to cause an immune response •Larger vector capacity Integrating vs non integrating integrating will be maintained over time, non integrating will be diluted by division

Vitamin K epoxide reductase complex

Vitamin K epoxide reductase complex subunit 1 (VKORC1), along with the cofactor vitamin K, is responsible for the activation of gamma-glutamyl carboxylase (GGCX) which activates the clotting factors.

Warfarin dosing needs to be carefully managed

Warfarin is a narrow therapeutic index agent. This means that a small change in systemic concentration of the drug may lead to significant changes in pharmacodynamic response. Careful clinical management is required to balance the risks of bleeding (overanticoagulation) with those of thrombosis (under-anticoagulation).

Genetic anticipation

The present concept of anticipation arises from the theory of degeneration by Benedict Auguste Morel in Traité des Dégénérescences (1857). It describes progressively worse illness from parent to child, becoming more severe form and occurring at an earlier age in succeeding generations, until the lineage is ultimately extinguished. The term genetic anticipation was introduced in 1911 to describe the progressively earlier appearance and increased severity of a genetic disease in successive generations. Initially thought to be a bias of ascertainment or an artifact of better diagnosis. Diseases are diagnosed at an earlier age.

Warfarin and anticoagulation

Warfarin is widely prescribed for the prevention and control of thrombo-embolism. That is, it's used for anticoagulation. Warfarin inhibits the vitamin K cycle, specifically vitamin K epoxide reductase complex subunit 1 (VKORC1). Reduced vitamin K is a required cofactor for gamma-glutamyl carboxylase (GGCX) that converts precursor forms of blood clotting factors VII, IX, X and prothrombin to active forms. Steady-state concentrations of warfarin are maintained through the balance of the dose administered, the level of cytochrome P4502C9 metabolism, and the renal elimination of the inactive hydroxy-metabolites and the active form.

Effect of CYP2D6 Polymorphisms

CYP2D6 deletions lead to poor metabolizer Duplications lead to extensive metabolizers It is estimated that 25% of all commonly used drugs are metabolized by CYP2D6

What is cancer

Cancer cells are defined by two heritable properties. They and their progeny: 1) divide in defiance of normal restraints (i.e. neoplasia) AND 2) invade and colonize territories normally reserved for other cells. We call such tumors "malignant" Cancer arises from a multistage process called "transformation" (normal -> proliferation -> local invasion -> invasion of other tissues -> distant metastases)

Summary: Cancer as a "genetic" disease

Cancer is a "genetic" disease in two senses: 1. Due to spontaneous mutations of one or multiple genes that regulate the growth of a cell 2. Some mutations are inherited in families, producing a genetic predisposition This inherited genetic cancer risk can be Mendelian (e.g. BRCA1) or genetically complex.

Final cancer summary

Cancer is caused by multiple "driver" mutations ("two-hit hypothesis") in oncogenes, tumor suppressor genes, and/ or DNA repair genes. Different tumors have different sets of mutations and are often composed of genetically distinct subpopulations. The multiple driver genes needed for cancer can be inherited, and/or can occur spontaneously Genomics is changing tumor classification and treatment

Cancer relevance

Cancer is the 2nd most frequent cause of death • 14 million new cases worldwide each year • 8 million deaths • $80 billion per year in the US • Always fatal if not treated • Vital to identify those at risk, and detect cancer early

Adeno-associated Virus (AAV) Gene Therapy

Cargo Capacity: ~4 kb - -Non-integrating -Infects quiescent cells - -Low oncogenic risk - -Lower immunogenic risk

Other Cas enzymes

Cas9 cannot target all bases in the genome, because Cas9 requires the presence of a PAM sequence "NGG" The PAM of Cpf1 is "NTT" (i.e. we can target different / more sites) Cpf1 guide RNA is half as long Cpf1 makes a staggered cut Cpf1 is a smaller protein (easier to deliver) There are other bacterial proteins that can be used for genome editing - the toolkit is constantly expanding

Components of today's commonly used CRISPR system

Cas9 protein: performs all CRISPR functions in a single protein. Cas9 makes double-stranded breaks in DNA "Protospacer adjacent motif" (PAM): required by Cas9 protein for gRNA binding Targeting sequence of the gRNA: tells Cas9 where to go by base-pairing with specific genome sequence Scaffold sequence of the gRNA: anchors the gRNA in the Cas9 protein guide RNA (gRNA): consists of scaffold and targeting sequence

Methylation of the Fragile-X gene

Cause of the symptoms is suppressed expression of FMR1 due to excessive DNA methylation of CGG repeats in the 5'UTR. Longer repeats are more unstable

Therapeutic options for genetic disorders

Change the environment: Dietary restrictions - PKU Provide the missing gene product: Protein replacement therapy Fix the mutated protein: Small molecules can sometimes prevent/correct protein mis-folding Eliminate gene expression or protein activity:Blocking antibodies, depletion, RNAi Replace the gene/tissue: Gene therapy - fix or supplement the gene Preventative care: Genetic counseling, prenatal diagnosis

Eugenics in MN

Charles Fremont Dight (1856-1938) Negative eugenics- Mandatory sterilization of the "feeble minded"- MN state Law enacted in 1925 and on the books until the 1970's.

Cystic Fibrosis (CF)

Clinical Problems 1. Chronic obstructive lung disease 2. Pancreatic insufficiency 3. Biliary obstruction and fibrosis 4. Infertility Physiological Problems 1. Abnormal mucous secretions 2. Abnormal sweat - increased Cl-, Na+ Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is mutated Experimental drugs have been designed to help mutated proteins fold. Most of these small molecules are not only protein specific but mutation specific as well. If an amino acid substitution (missense mutation) destabilizes the folding of the protein and resulting in a loss of function, the interaction of the small molecule with the protein while it is folding during translation and stabilize the structure providing (some) function. The small molecule VRT-532 interacts directly with ΔF508 and the G551D mutations in the cystic fibrosis transmembrane regulator (CFTR) protein.

CRISPR and Cas

Clustered regularly interspaced short palindromic repeat (CRISPR)- associated system (Cas) A weird signature in bacterial genomes, tandem repeats at fixed distances • Similar CRISPR ("clustered regularly interspaced short palindromic repeat") signatures were quickly found in many species of archaea and eubacteria • Often, they carried neighboring CRISPR-associated ("cas") genes in their vicinity CRISPR-spacers match phage DNA "There may be [...] a relationship between the presence of a CRISPR and the lack of a particular prophage. One possible explanation for that finding could be that CRISPRs are structures able to take up pieces of foreign DNA as part of a defence mechanism. In this view, it is tempting to further speculate that CRISPRs may represent a memory of past 'genetic aggressions'." An adaptive bacterial immune system

Epidemiologic studies of alcohol consumption & risk of CVD: Challenges to interpretation

Confounding Compared to abstainers and heavy drinkers, people who drink in moderation tend to be healthier, have more education, and have more favorable health habits Misclassification of exposure / information bias Current or past alcohol consumption is not reported accurately Alcohol consumption patterns change over time (one time measure may be inadequate to characterize a person Selection bias Studies of cardiovascular disease tend to select middle aged or older adults who started drinking as young adults. These studies likely disproportionately enroll moderate drinkers who are the most resilient or responsible, and have not experienced any adverse effects of drinking in young adulthood. Reverse causality Drinkers who develop cardiovascular disease may stop drinking, become part of the abstainer group In general, these things would tend exaggerate any possible benefits of moderate drinking

DNA repair genes

DNA repair genes are responsible for the repair of DNA damage. DNA repair genes are recessive at the level of the cell (like tumor suppressor genes). Heterozygous cells do not form tumors. Clinical features of DNA repair mutations Hypersensitivity to DNA damage Chromosome breakage Short Stature Increased rate of cancer (1,000 - 10,000X)

Methylation and Imprinting

Development involves dramatic shifts in DNA methylation, which involve both loss of methylation and de novo methylation. Imprinted genes somehow escape this demethylation. When an expressing allele at an imprintable locus passes through gametogenesis of the same sex from generation to generation, the expression and methylation state of the allele remains the same. However, when this allele passes through gametogenesis of the opposite sex, it becomes inactivated and is unable to be expressed (its methylation state is altered). This allele is now inactivated and has become "imprinted".

Organ Transplantation

Disease Gene Type of Transplant Polycystic Kidney Disease PK1, PK2 Kidney transplant Cystic Fibrosis CFTR Lung transplant Wilson disease Tyrosinemia ATP7B/TAT Liver transplant Type 1 Diabetes IDDM1 Pancreas transplant Hemoglobinopathies HHB Bone marrow transplant Hurler's syndrome MPS1 Bone marrow transplant

Factors that influence response to drugs

Dose ADME (Absorption, Distribution, Metabolism and Excretion) Age Environment Diet Lifestyle Gender Comorbid conditions e.g obesity and Genetic makeup

driver and passenger mutations

Driver mutations are the mutations that actually cause cancer. They lead to sub-clones whose size depends on the strength and timing of the driver. Passenger mutations do not cause cancer. They can expand in a sub-clone that is "driven" by a driver mutation. They can be used to trace the lineage of a given sub-clone.

Treatment for hunter and hurler syndrome

Enzyme replacement therapy (ERT) for Hurler and Hunter Syndromes Hurler syndrome (α-L-iduronidase) Hunter syndrome (iduronate-2-sulfatase)

Plasma Levels of Phenylalanine (μM) in PKU

Hyperphenylalaninemia (HPA) is defined as > 120 μM Phe non-PKU hyperphenylalaninemia (NPHPA) is 120-1000 μM Phe Phenylketonuria (PKU) is defined as > 1000 μM Phe

Important Points for Imprinting

Imprinting is reversible. There are differences in expression for some genes between the maternal and paternal chromosome. The paternal and maternal genomes are not equivalent. Some genetic disorders only arise when the mutant allele is on the maternal or paternal chromosome

In vivo vs. ex vivo

In vivo: direct inject of gene therapy vector Ex vivo: gene therapy targeting hematopoietic stem cells (remove bone marrow cells from patient, edit them, infuse them back into patient)

Polymorphisms Effect Drugs by...

Increasing or decreasing drug transport in and out of cells Increasing or decreasing metabolism Changes in binding to cellular target Increasing or decreasing the risk of toxicity or side-effects Increasing or decreasing efficacy

pharmacogenetics summary

Individuals respond differently to drugs. Genetics play a role in these differences. Identifying these differences will help us determine the right drug and the right dose to maximize efficacy while minimizing adverse outcomes.

Components of a Genetic Consult:

Information Gathering •Obtain a family history (pedigree) •Obtain medical history •Medical records from family members •Patient / family perception of the condition. Establish / Verify clinical diagnosis •Medical geneticist (M.D.) -Physical exam -Laboratory / radiology tests Risk assessment •Based upon known pattern of inheritance •Based upon empiric recurrence figures •Risks may be adjusted with mathematical calculations. Information giving •How was diagnosis / recurrence risk derived. •Description of condition, prognosis, medical management / intervention options. •Reproductive options •Support resources Psychosocial counseling •Direct impact for patient •Assessment of patient's support network •Discuss impact of information on relationships with family and friends. • Case example- 62 year old man tests positive for Huntington disease.

Gene Therapy

Insert a correct copy of a gene into the cell or inhibit the expression of a gene for purposes of correcting a disease producing error in the DNA sequence.

Knudson's 'two hit' hypothesis of carcinogenesis

Knudson reasoned that at least two mutations are necessary to create a retinoblastoma and that families segregating for the disease inherited one mutated allele with a second mutation occurring in a somatic cell.

Fragile X Mental Retardation Syndrome Clinical Characteristics

Mental retardation Males: Severe to moderate MR, Speech delay Females: Learning disability, Mild MR Characteristic face: Long, narrow face; Prominent jaw; Big ears Large testes after puberty Other Loose joints Seizures Autistic behavior

morphine metabolism

Morphine can also be metabolized by UGT2B7 to morphine-6-glucuronide (active) UGT2B7*2 p.His268Tyr (rs7439366) aka the "T" allele Adults who were homozygous for the UGT2B7*2 allele had higher M6G: morphine ratios than those who are homozygous for the wild-type allele

A proto-oncogene can be made oncogenic in several ways

Mutation of the coding sequence creates a hyperactive protein that is made in normal amounts. Gene amplification leading to too much production of a normal protein. Chromosomal rearrangements place proto-oncogene near a strong enhancer create an overproduced protein, or a fusion to an actively transcribed protein. (coding mutation that results in abnormal protein, regulatory mutation that results in excessive protein, translocation that results in novel protein, gene amplification that results in excessive protein)

precision medicine

Personalized medicine is the application of genomic and molecular data to better target the delivery of health care, facilitate the discovery and clinical testing of new products, and help determine a patient's predisposition to a particular disease or condition. Risk assessment Diagnosis Treatment Prognosis Pharmacogenomics

Opportunities and benefits of having healthcare providers have access to your genome sequence

Personalized medicine, reduced trial and error for treatment because you can make decisions based on genetics improved setting up of clinical studies by using genetics of the subjects Early preventative care measures could be taken if risk is known, would reduce cost of healthcare Access to drugs that were not previously available due to genotype dependent adverse affects Access to gene therapy due to identification of causal mutation Accurate diagnoses

positive selection and adaptation

• Positive, "Darwinian" selection is the only process in evolution that can result in adaptation: the development of traits that make a species better adapted to its environment • Certain mutations lead to higher fitness: the individuals that carry these mutations have more offspring • In a "hard sweep", a new, beneficial mutation occurs. • As the beneficial mutation rises to high frequency in the population: • variation around it is reduced • neighboring, neutral variants are brought along to high frequency • Positive selection produces distinctive patterns of genetic variation in a population, at a specific location in the genome • We can "scan" the genome for these patterns • Many cases of local, recent adaptation have been discovered in humans

Mendelian Randomization (MR)

• Refers to the random transmission (from parents to offspring) of genes influencing different traits or characteristics • Recognized as form of instrumental variable analysis (natural experiment) • First application in epidemiology is attributed to Katan (1986). He proposed to use genetics to help sort out whether or not low cholesterol was a cause of cancer.

Processes that can be altered in cancer

growth signals, receptors, signal transduction molecules, amplification cascade molecules, transcription regulators, cyclins, CDKs Everything involved in the cell cycle

Patterns of positive selection in genetic variation

lactase persistence, height, arctic environment, high fat diet, thick hair, starchy food, skin pigmentation, high altitude, trypanosome resistance, malaria, toxic arsenic rich environment, increased BMI

Can we make HIV resistant white blood cells

loss of CCR5 receptor in cells confers HIV immunity, performed by CRISPR We could also just eliminate the whole HIV genome from infected cells (cut out method of entry to prevent infection, cut out genome to cure infection)

What do epidemiology professionals do?

o Analyze public health trends o Design and implement research studies into causes of disease o Interpret study results for policy and program development o Translate health data into strategies to prevent disease and promote population health • Several UMN epidemiology faculty and students are part of the team building COVID 19 models for Governor Walz and other policymakers

three types of cancer genes

oncogenes, tumor suppressor genes, DNA repair genes

Risks of having genome sequence online/ accessible to others

potential for new eugenics: selecting partners based on genetics Insurance companies accessing sequence and leading to people who need care not being able to afford it Genome could be studied without consent possibility of identity theft if genome is used as ID Trail of data makes it hard for witness protection program efforts Use of data by investigators could expose a relative of a crime, or could be used to identify protesters, limit freedom etc Employers could discriminate Genetic screening for certain traits could pressure you to follow a certain path or prevent you from following a certain path Finding relatives you didn't know you had (half siblings) Could be tied to a crime scene Social discrimination Others could profit from your genetic data with no benefit to you DNA could be used to make people like you or to make weaponized biological warfare products to harm you

DTC genetic testing

primary clinical concerns •False reassurance "at least I don't have to worry about breast cancer." •Family history and past medical history may not be included in risk assessments. •Adverse reactions (anxiety, suicide) •Overuse of medical resources •Conflicting risk assessments •Data privacy -Patients don't want information in medical record, but are willing to submit it to a for-profit company. •Barriers to access will continue to fall •What will be the role of genetic counselors in this new world?

Directly fixing the mutation

Zinc finger proteins TALENS - Transcription activator-like effector nucleases CRISPR - clustered regularly interspaced short palindromic repeats • Zinc finger protein (DNA binding domain) fused with a catalytic nuclease domain • 1st engineered endonucleases used to edit genes • TALENS: DNA binding domain from Transcription Activator-Like Effectors produced by Xanthomonas plant pathogens CRISPR/Cas: Rapidly developing technology based on defense system that targets DNA invaders • Target recognition is RNA-based

Somatic mutation and disease

• Somatic mutations may underlie many syndromes that are especially severe, some with strong developmental effects, and with effects on the brain • If inherited as de novo mutations from the parents, the presence of the mutation in the entire body might not be compatible with life • The mosaic state allows patients to be born (and sometimes treated) • For example, Proteus Syndrome is due to mutations in AKT1, resulting in severe overgrowth of skin, bone, and connective tissue (the paralogous mutation in AKT3 causes HMG). • Proteus Syndrome has never been reported to run in a family. The associated lesions contain 1 - 50% of mutant cells • Somatic mutations can result in discordant monozygotic twins

A Brief History of Genetic Counseling:Genetic counseling emerges as a professional field. (modern times)

•1993 Gene for Huntington disease is cloned. For the first time, a precise molecular predictive test is available for an adult onset disorder. •1990's genetic basis of hereditary breast and colon cancers is elucidated. •Human genome project leads to discovery of subtle genetic changes that act as risk factors for disease.

Principle-based ethics- the four guiding lights

•Autonomy -Informed consent, confidentiality •Justice -Equitable treatment, equal distribution of risks/benefits •Beneficence •Non-maleficence

Competing ethical interests

•Beneficience/Non-maleficience -Doing the correct test could have life-saving benefit for patient -Doing the wrong test could do substantial harm. •Autonomy -Protecting the confidentiality of the proband and her right to protect or release her confidential medical information.

Viral Vectors

•Broad tropism • •High titers • •High and sustained expression levels • •Many active clinical trials

Coronary heart disease (CHD)

•Characterized by a build up of plaque in the heart's arteries that could lead to a heart attack • 33 50% lifetime risk • Leading cause of death in the U.S.

How to Create a Replication Incompetent Virus

•Create viruses that won't spread. • •Select tropism and pseudotype the viral vector to target specific cell types • •Insert gene into retroviral plasmid. • •Transfect plasmid into packaging cell line. • •Purify virions (active viruses)

ACMG standards and guidelines

•Define categories of variants (pathogenic, likely pathogenic, VUS, likely benign, benign) •Define the types of evidence used to make these categorizations. •Weight the strength of each type of evidence. •Define the level of evidence required for each level of variant classification. •Improve agreement/concordance amongst labs- standardize variant interpretation. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.

Ethical dilemmas

•Ethical dilemmas generally come into play when there are competing interests -Balancing different ethical obligations e.g. autonomy vs. justice -Balancing ethical obligations to different parties •Respecting the autonomy of parents vs. a child •Respecting the confidentiality of one family member when testing another

gene therapy summary

•Gene therapy is compelling vision, which has not been easy to realize •Gene editing approaches maximize safety •Gene editing also offers unparalleled opportunities for novel discovery and application •Gene therapy's unsolved problems means opportunities for you...

gene therapy vs gene editing

•Gene therapy: provision of full length, functional copies of a [defective] gene under the control of exogenous regulatory elements •Delivery: classified as viral or non-viral •Expression: classified as integrating or episomal •Gene editing: precision targeting at a genomic locus of interest that maintains endogenous gene regulation

Challenges of counseling for Multifactorial Conditions

•More than one risk factor may be segregating in a family. •Low penetrance (people who inherit genetic susceptibility, but do not manifest the disease). •Screening of asymptomatic family members-lack of guidelines.

How has history shaped genetic counseling, and how is genetic counseling changing?

•Recognition of past misuse of genetic information led to strong emphasis on informed consent. •Many guidelines in genetics are based upon highly-penetrant, single-gene conditions with a profound (often lethal) prognosis (i.e. Huntington disease). •We have typically worked with conditions in which the following is true: -The disease is caused by a single gene -All (or most) people who inherit the disease gene(s) develop symptoms. -If the disease gene is excluded, the individual will not develop the disease

Goals of the Eugenics movement

•Reduce social and/or economic burden of the genetically "unfit." •Preservation and/or purification of the "genetic stock" of a nation. •Prior to WW II, this was seen as a humane way to eliminate hunger, poverty, suffering, disease....very progressive idea.

Genetic variation and clinical molecular labs

•The reference human genome has one "correct" nucleotide at each position. •Every human being differs from this reference sequence at millions of positions •Scientists and informaticists are responsible for telling us where our patient is different from the reference genome •Our job is to answer a clinical question posed by a physician or genetic counselor -Is a particular variant(s) the cause of this patient's phenotype (diagnostic testing) -Is the variant associated with a significant reproductive risk (carrier testing) -Does the variant identify a future health risk (predictive testing) -Does the variant provide guidance for individualized medical management (?precision medicine)

Ethical professional domains

•Value conflicts •Confidentiality •Maintaining proficiency •Directiveness/non-directiveness •Determining primary patient •Withholding information •Emotional reactions (patient/professional) •Diversity •Discrimination •Colleague error •Documentation •Resource allocation (financial/time/organizational) •Facing uncertainty •Informed consent •Professional identity •Professional misconduct

issues to think about in genetic testing

•Will the sequencing and bioinformatics be so unwieldy that analysis will only occur in a handful of genome centers •Will analysis become so routine that the genetic analyzer sits next to the clinical chemistry machine in every clinic? •If there are no technical, cost, or interpretive barriers (i.e. no more VUS), will some genetic tests become as routine as a cholesterol test? -i.e. should everyone have a cancer risk assessment when they are born or turn 18? •Genetic counseling is a limited resource- how do we extend the benefits of genetic testing to as many patients as possible?

Neoplasia

"Neoplasia" is a disease process characterized by uncontrolled cellular proliferation leading to a mass or tumor ("neoplasm"). Neoplasia arises from an imbalance between the normal processes of cellular proliferation and attrition (e.g. programmed cell death or "apoptosis") Neoplasms are called "benign" if they do not invade surrounding tissue and do not spread around the body Their effects on the patient can be quite severe, even if they aren't technically "cancer"

Definition of Genetic Counseling:

(ASHG ad hoc committee on genetic counseling, 1975) Genetic Counseling: Genetic Counseling is a communication process which deals with the human problems associated with the occurrence or risk of occurrence of a genetic disorder in a family. This process involves an attempt by one or more appropriately trained persons to help the individual or family to: 1. Comprehend medical facts about a diagnosis. 2. Appreciate inheritance of disorder and risk to relatives. 3. Understand options for dealing with recurrence risk. 4. Choose most appropriate course of action. 5. Adjust to diagnosis or risk for recurrence.

ADME

(Absorption, Distribution, Metabolism and Excretion) elements of pharmacokinetics Absorption: Gut to liver / bloodstream Excretion: liver to kidney drug is transported from blood stream to target molecule Transporters Active transport of the drug into or out of the body or cells. Phase I Oxidation, reduction, hydrolysis, cyclization, and decyclization, addition of oxygen removal of hydrogen. Usually involves cytochrome P450 enzymes in the liver. Phase II Conjugation reactions such as with glucuronic acid, glutathione or amino acids. Targets Protein targeted by the drug. Examples of Phase I Drug Metabolism: Hydroxylation Examples of Phase II Drug Metabolism: Conjugation

Adenoviral Gene Therapy

- Cargo Capacity: 8-30 kb - -Non-integrating -Infects quiescent cells - -Low oncogenic risk - - Immunogenic risk

Lentiviral Gene Therapy

- Cargo Capacity: ~8 kb - - Integrating -Infects quiescent cells (+/-) - -Oncogenic risk

cause of fragile X

...is caused by a repeat of CGG in 5' untranslated region of first exon of FMR1 Repeat number is conserved in mammalian evolution. Most species have 10 - 12 repeats; Primates have around 30. Normal in humans: 10 - 50 "Premutation": 52 - 200 Mutation: 200 - 2000 Source of expansion is maternal Premutations sometimes shorten with paternal transmission The repeat forms a CpG island that becomes methylated with expansion: a potential promoter region that appears to be inactivated by DNA methylation.

Simultaneous delivery of Cas9 protein with sperm results in more efficient gene editing

72% of embryos with repaired allele Few mosaics No off-target effects detected • Most embryos did NOT use the provided template - they used the mother's DNA as the template to repair the DNA from the father's sperm • This allows repairing dominant mutations, but prevents addition of new DNA or alleles not present in either parent • It may only be a question of time for these technical issues to be resolved

genetics and tacrolimis

A single nucleotide variation in the CYP3A5 gene can lead to the use of a cryptic splice site and creation of a less functional of CYP3A5 rs776746 (aka ss1982931) G allele - CYP3A5(*3) - leads to reduced activity A allele - CYP3A5(*1) - gives 'wild type' activity of CYP3A5 Patients with CYP3A5(*1) activity have lower tacrolimus troughs, require higher doses and are at greater risk for transplant rejection

Huntington's Disease

Affects about 30,000 Americans. About 150,000 children have a parent with Huntington's disease. Autosomal dominant progressive neurodegenerative disorder characterized by personality changes and involuntary movements usually present in mid-life and progressive to death. Responsible gene on chromosome 4 has an expanded trinucleotide (CAG) repeat region The repeat encodes a polyglutamine tract Size of repeat region is correlated with age of onset. Alleles expand during paternal transmission Protein aggregation in Huntington's disease Nerve cells have died in the brain of a person with Huntington's disease, creating a large hole ("ventricle") in the center

all cancers are genetic

Alterations in genes that are important for control of cell proliferation Sequential mutations in cell regulatory factors lead to progressive loss of growth control

A Brief History of Genetic Counseling:

Ancient History (b.c - 1900) •Jewish law recognizes the inheritance pattern of hemophilia and exempts at-risk males from circumcision. •Societal taboos against marrying close relatives. Eugenics movement (1890-1930) Francis Galton (1822-1911) • Cousin of Charles Darwin • Coined the term "Eugenics" Positive eugenics--encourage "fit" families to have children Negative eugenics--mandatory sterilization. Recipients of "genetic counseling" identified by the state. Transition from social policy to medicine (1930-1970) •Following World War II, geneticists sought to distance their practices from those of Nazis. •Non-directive approach adopted. •Primary practitioners are PhD Geneticists in academic setting. •Sheldon Reed (U. of MN) coins the term "genetic counseling" in 1947. •Cytogenetic, molecular genetic tests to confirm diagnoses are not available. •Patients have only two options: have children or don't have children. Genetic counseling emerges as a professional field. (1970-present) •Cytogenetic tests become available that can precisely diagnose a limited number of conditions during pregnancy. •Abortion is legalized in the United States. •First genetic counseling training programs in the United States. •Formal definition of genetic counseling.

small molecules and genetic disease

BH4 cofactor Phenylalanine hydroxylase mutations PKU VX-770 CFTR - p.Gly511Asp mutation CF VX-809 CFTR - p.Phe508del mutation CF PTC124 Frameshift/nonsense mutations CF, DMD Hydroxyurea Sickle cell disease SCD Kuvan (BH4 cofactor) - $110,000 per year Ivacaftor/Kalydeco (VX-770) - $350,000 per year Ataluren or Translarna (PTC124) - $290,000 per year (England)

Tumor Suppressor Genes in Familial Breast Cancer

BRCA1 45% of families with an autosomal dominant pattern of breast cancer. 80% of families with early-onset breast cancer and ovarian cancer. BRCA2 45% of families with an autosomal dominant pattern of breast cancer. Associated with male breast cancer, but not ovarian cancer. P53 1% of families with an autosomal dominant pattern of breast cancer with onset before age 40 years.

Expected benefits of pharmacogenomics

Better and Safer Drugs Improved efficacy, speed recovery time and increase safety. More Accurate Method of Determining the Right Dose Decrease in the Cost of Healthcare Improvements in Drug Discovery and Approval Process Cost and risk of clinical trials can be reduced by targeting only individuals capable of responding to a drug. More Powerful Medicines Creation of new drugs that target a specific gene or gene defect.

Opportunities and benefits of comparing you genome sequence to millions of others

Better research -> better understanding of disease and what treatments are successful for certain genotypes Increased sample sizes, including people of different ethnic background More GWAS success and better trait prediction Better ability to identify common alleles with small effects due to increased statistical power that comes with larger sample size Connect with others who share similar variants or genetic signatures Discover forgotten ancestors or distant relatives Discover the phenotypic consequences of obscure variants Contribution of crowd sources research Better understanding of environmental contribution to disease Benefit from policy changes that emerge with universal genome sequencing and access Tools could be made to increase genetic literacy

uniparental disomy

Both chromosomes of a pair come from one parent Several potential mechanisms: Nondisjunction Duplication Frequency unknown In Prader-Willi syndrome, if both copies of chromosome 15 are from the mother (due to uniparental disomy) and none from the father, both chromosomes are genetically normal, but the genes relevant to Prader-Willi syndrome are transcriptionally silent. The same is true for Angelman syndrome when both copies of chromosome 15 are from the father.

Effects of Drug-Drug Interaction

Both drug A and drug B are metabolized by the same enzyme. If two drugs are metabolized by the same enzyme, it can result in less reduction of one drug in the body over time (because its metabolized less)

Different types of cancer can be treated by protein-specific monoclonal antibodies

Brain cancer Breast cancer Chronic lymphocytic leukemia Colorectal cancer Head and neck cancers Hodgkin's lymphoma Lung cancer Melanoma Non-Hodgkin's lymphoma Prostate cancer Stomach cancer Humanizing Mouse anti-Human Protein Antibodies Antibodies bind to and block growth receptors to inhibit cancer cells

Phenylketonuria (PKU) - Fölling's Disease

error in Phenylalanine hydroxylase (PAH) which converts phenylalanine to tyrosine, phenylalanine is instead converted to phenylpyruvic acid by transaminase. Symptoms of PKU caused by phenylpyrivic acid buildup Type II tyrosinemia: Tyrosine transaminase has an error, responsible for converting tyrosine to other product Phenylalanine hydrolase (PAH) requires a co-factor, tetrahydrobiopterin (BH4) so defects in the PAH gene aren't the only variants to lead to PKU

covers lectures 18-27

- Imprinting. Pay special attention to the mechanics of imprinting and inheritance, and how they can combine with genetic mutations to cause disease. - How "dynamic" mutations work in a pedigree and can result in disease - Characteristics that distinguish diseases exclusively caused by somatic mutations from diseases that can also be caused by germ line mutations - The three types of cancer genes - Roles of driver vs passenger mutations in cancer - The two-hit hypothesis in cancer - Genetic counseling: what is and is not part of this profession, and ethical considerations counselors have to weigh in their daily work - Technical challenges in engineering genomes with CRISPR - The phylogenetic tree of humans and how we know it - Evolution of the human compared to the chimpanzee genome - The concept of evolutionary conservation - Basic facts about what we know about Denisovans - Meaning of drug "ADME" - Codein metabolism and resulting risks for individuals with mutations in this pathway - Examples of diseases treated by various modes of therapy for genetic disease - Advantages and disadvantages of you and others knowing your genome sequence (see lecture next Tuesday)

Non-viral Gene Therapy

- Stealth liposomes (w/ polyethylene glycol [PEG]) - Higher nucleic acid cargo capacity - No viral proteins -Integrating/non-integrating -Delivery can be limiting

Mucopolysaccharidosis type I (MPS I) (Hurler syndrome)

- defect in alpha-L-iduronidase a-L iduronidase cleaves a terminal iduronic acid residue from dermatan sulfate and heparan sulfate (GAG) Virtually all tissues have some degree of storage and disease A lysosomal storage disorder • Deficiency of lysosomal enzyme a-L-iduronidase • Progressive accumulation of glycosaminoglycans (GAG) • Multi-systemic, heterogeneous • Severe morbidity and early mortality • Coarse features • Significant medical needs • Rare (est. incidence 1:100,000)

The "Two Hit Hypothesis" and retinoblastoma

1. 40% of the cases are inherited and 60% are sporadic. Inheritance is dominant. 2. Inherited cases have an earlier age of onset and are multifocal (multiple tumors) or bilateral (both eyes) in origin. Sporadic cases have a later age of onset and are usually unifocal (one tumor) and unilateral (one eye) in origin. 3. Some individuals with the dominant gene do not develop a tumor. Is retinoblastoma dominant or recessive? Tumor suppressor genes are defined as genes whose reduced function can lead to neoplastic changes. In families, tumor suppressor genes are inherited in a dominant fashion ...but they are recessive at the level of the cell. Recessive function with dominant inheritance! The RB1 gene encodes a phosphoprotein that normally regulates entry into S phase. It is a "checkpoint". Two deactivating mutations in a cell result in retinoblastoma

What constitutes a good candidate disease for gene therapy?

1. Diseases that have severe, lasting health issues 2. Diseases for which current available therapies do not exist or are insufficient 3. Diseases which can be corrected by manipulating a single gene target 4. Diseases for which potential patients are available

components of imprinting

1. Imprinting occurs before fertilization. 2. Imprinting confers transcriptional silencing. 3. Imprinting is stably transmitted through mitosis in somatic cells. 4. Imprinting is reversible on passage through the opposite parental germline (e.g. if an allele is maternally imprinted, this imprint is removed in the gametes of a male offspring).

Gene therapy strategies

1. Insertion of a functional copy of a gene to compensate for a nonfunctional gene 2. Replacement of a nonfunctional gene with a functional copy (dominant negative) homologous recombination 3. Gene addition to alter the expression of a disease-causing gene product (miRNA)

Presentation of untreated PKU

1. Unusual "musty" odor 2. Lighter pigmentation 3. Mental retardation 4. Behavioral changes 5. Seizures 6. Skin changes (eczema) Epilepsy 25% Profoundly retarded (IQ, < 35) ~50% Moderately retarded (IQ, 36 - 67) ~50% Slightly retarded (IQ, > 68) ~5%

Some oncogenes can be targeted by drugs

HERCEPTIN® (Trastuzumab) is a recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 protein, HER2.

Future challenges for genetic counselors

1.Making sense of genetic variation from exomes and genomes 2.What is the role of genetic counselors in a direct-to-consumer world

Progression of Research in CVD Genetic Epidemiology

Heritability: Does genetic variation contribute to risk of CVD? Discovery: Which genes and alleles are Translation: Can genetic discoveries lead to clinical or public health benefits?

Epidermolysis Bullosa

Epidermolysis Bullosa occurs approximately 1:50,000 and is due to mutations in the type VII collagen (C7) gene. The deficiency C7, can be ameliorated in both animal models of recessive dystrophic epidermolysis bullosa (RDEB) and in children with this rare genodermatosis. Healthy donor cells from the hematopoietic graft migrate to the injured skin, with a concomitant increase in the production of C7. This in turn is associated with increased basement membrane integrity and a reduction in skin blistering. (bone marrow transplant) Hematopoietic cell transplantation increases collagen 7 and improves skin blistering. Immunofluorescence staining and clinical photographs of the RDEB recipient of matched sibling HCT are shown. Antibody staining for C7 indicated faint, stippled labeling of C7 before HCT and continuous, bright, linear C7 labeling at 180 days after HCT

Some enzymes available for enzyme replacement therapy (ERT)

Gaucher Disease, Type I: Glucocerebrosidase Using the usually recommended dose of 60 units per kilogram of body weight every 2 weeks, Treatment for a 70-kg patient with Zavescais costs $350,200 per year. (2016) Mucopolysaccharidosis, Type I: Alpha-L-iduronidase Hurler Syndrome. Bone marrow transplantation Enzyme infusion BioMark-Genetics - Aldurazyme at $350,000 per year. (2016) Fabry Disease: Alpha-galactosidase A Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified alpha-galactosidase A in several cell types, suggesting diffuse uptake via the mannose 6-phosphate receptor. Mannose 6-phosphate is a lysosome directed protein tag. $300,000 per year. (2016) Glycogen Storage Disease II: Alpha-1,4-glucosidase Pompe Disease. Treatment with Lumizyme costs $630,000 per year. (2016) Mucopolysaccharidosis, Type VI: Galactosamine-6-sulfatase

Gene Therapy Adverse Events: Adenovirus 1999

Gelsinger received the highest dose of vector in the trial (3.8x1013 particles). A female patient who received a similar dose experienced no unexpected side effects. Gelsinger's precipitous and ultimately irreversible response to the experimental therapy included signs of disseminated intravascular coagulation (DIC), massive cytokine release, adult respiratory distress syndrome (ARDS), and multiple organ failure

Deletions at 15q11-13 underlie PWS & AS

Genes relevant to PWS are only expressed from the paternally inherited 15q11-q13 chromosome region. The maternally inherited genes are present and have normal sequence but are transcriptionally silent due to imprinting in the mother. Another gene in the 15q11-q13 region is responsible for AS. It is normally expressed by the maternally inherited chromosome. For this gene, the paternally inherited gene is imprinted and "turned off". About 70% of PWS and AS is caused by microdeletion, other 30% caused by uniparental disomy PWS = paternal deletion of maternal disomy AS = maternal deletion or paternal disomy

personalized medicine

Genetic variants associated with optimal... • disease diagnosis • drug usage • drug dosing • outcomes

Ways a gene is non-mendelian

Genomic imprinting Dynamic mutations and genetic instability (Fragile X) Somatic mutations

various classes of oncogenes

Growth factors PDGF, EGF Growth-factor receptors acting via tyrosine-specific protein-kinase activity PDGF receptor , EGF receptor (erbB) GTP-binding proteins Ras proteins [H-ras, N-ras, K-ras] Membrane/cytoskeleton-associated tyrosine-specific protein kinases Src protein kinase (src) Cytoplasmic tyrosine-specific protein kinases fes Steroid-type growth-factor receptors thyroid hormone receptor (erbA) Serine/threonine-specific protein kinases raf Nuclear proteins myc, fos, jun

oncogenes

Oncogenes [Greek onkos: mass, tumor] are genes whose expression can result in neoplastic transformation. They normally exist as proto-oncogenes that do not cause cancer. Function: Positive regulators of cell proliferation Mutation: One mutant allele -> gain of function -> unregulated cell proliferation Transformation to a tumor cell despite presence of the second, normal allele Oncogenes have dominant effects Clinical: Oncogene mutations found in most cancers.

Prader - Willi Syndrome ("PWS")

One of the most common micro-deletion syndromes (~1:10,000) in both sexes One of the most frequent genetic disorders seen in genetics clinics The most commonly recognized genetic form of obesity Hypotonia Failure to thrive in infancy Small hands and feet Hypogonadism Variable mental retardation Marked obesity Sitting: 12 months Walking: 24 months Adults: Adults remain mildly hypotonic with decreased muscle mass IQ: 60s to low 70s Growth hormone therapy helps in muscle mass

What makes us human?

Phenotypic differences between humans and other apes - Chimpanzees are NOT our ancestors • Instead, we share a common ancestor that was neither chimp nor human (~5-9 mya) • Their genomes have evolved just as much as ours The human and chimpanzee genomes • ...differ at ~1% of their single-copy (non-repeat) bases • This is 30 million differences! • 15 million each arose on the human and the chimpanzee lineage • 71% of protein coding genes carry at least one amino acid difference • ...but most of these (~90%) do not show evidence of positive selection • The 10% that have been under positive selection are enriched for genes with roles in immunity, reproduction, olfaction. These functions are enriched for fast evolution in all mammals! • Brain genes evolve more slowly than other genes. • To find the genes that "make us human", we cannot use the tools of GWAS, linkage, etc. because human-specific variants do not vary among us So we don't know

Humans as apes

Phylogeny based on anatomy: Elongated skull, brow ridge enlarged, shorter and stouter canines, front of upper jaw, fusion of certain bones in wrist, larger ovaries, reduced hairiness Larger brains, no tail, more erect, hips/ankles more flexible, changes in structure of arms/ shoulder Gene sequences clarified the position of humans within the great ape tree Mitochondrial cytochrome oxidase II alleles Fossils allow us to calibrate the molecular clock and obtain times for evolutionary events Human genetic diversity is low compared to the other great apes

What are the risks of having your genome sequenced?

Possible negative psychological effect of knowing risk for disease and passing that on to children Psychological stress of learning something you don't want to know such as mistaken paternity, infertility, genetic predisposition to disease, unexpected family links or ancestry, something that's untrue in the data or interpretation You could have to make challenging choices that you didn't have before: to screen partner or embryos for disease, to abort fetus likely to have genetic condition, to tell family, physicians, or insurers about findings

UBE3 may be the responsible gene in AS

Potential treatment for AS: Potential drugs are topotecan, irinotecan and etoposide. These are topoisomerase inhibitors used in chemotherapy. These drugs affect DNA replication. Drugs increase expression of Ube3a gene in the paternal chromosome in mouse neurons. "Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome."

Genetic counseling settings

Prenatal setting: •Carrier testing based upon ethnic background / family history. •Discuss screening and / or diagnostic tests during pregnancy. •Help patient understand a prenatal diagnosis. •Aid in the decision making process. •Non-invasive prenatal testing •Prenatal exome sequencing Pediatric setting: •Example: cystic fibrosis, fragile X, Down syndrome •Help families cope with diagnosis. •Help child understand their diagnosis. •Discuss risks for future children / other family members. Adult onset disorders: •Example: Huntington disease •Molecular genetic tests can predict who will be affected by autosomal dominant, adult-onset disorders. •Assessing pedigrees to differentiate Mendelian from multifactorial forms of common disease. Familial cancer clinics: •Example: BRCA1/2 •Evaluate family histories of cancer and determine most appropriate genetic testing options. •Discuss issues related to genetic testing •Cancer screening / prevention options Clinical laboratories: •Evaluate pedigrees and make recommendations to ordering physicians about most appropriate testing options. •Ensure that informed consent has been obtained from patients. •Variant interpretation/genome analyst •Report writing •Communication with ordering physicians about results and follow-up Research laboratories: •Coordinate the ascertainment of families for research. Facilitate ongoing contact with families. •Obtain informed consent from potential research subjects. •Writing grants and manuscripts. Public health department •Genetic counselors are often involved in coordinating newborn screening programs.

Tacrolimus dosing equation:

Tacrolimus TVCl/F (L/hr)=54.6 L/hr x (1.33, if days less than 9 post-transplant) x [(0.53, if 2 LoF alleles] x (0.85, if 1 LoF allele) x (1.23, if receiving a steroid) x (0.92, if receiving an antiviral drug) x (1.24, if recipient age 18-25 years) Daily dose (mg/day) = [TVCl/F x target tacrolimus trough concentration (ng/ml) x 24hrs]/1000

Tacrolimus

Tacrolimus is an important immunosuppressant used in solid organ transplantation. It inhibits the activation of T-cells by preventing the activation of nuclear factor of activated T-cells (NF-AT), which when activated moves to the nucleus of the T-cell and increases the activity of genes coding for IL-2 and related cytokines. The concentration of the drug in the blood is very important. If the drug level is too low, the transplant recipient is at risk for acute rejection and eventual graft loss. If the concentration is too high, the recipient is at risk for leukopenia (low white blood cell count) that puts them at risk for infection and cancer.

Codeine Metabolism

codeine (inactive) is converted to morphine (active) by CYP2D6 morphine is converted to morphine-3-glucuronide (inactive) by UGT1A Codeine is a prodrug with no analgesic activity. About 10% of a dose of codeine is converted in the body by CYP2D6 protein to morphine, an active metabolite. The morphine is glucuronidated to both active and inactive metabolites that are eliminated by the kidneys. The rest of the codeine is metabolized by glucuronidation and CYP3A4 to inactive metabolites. Thus, codeine metabolism by CYP2D6 is necessary for much of the analgesic action of codeine. Most Caucasians rapidly convert codeine to morphine via CYP2D6. Approximately 7% to 10% of Caucasians, however, have a genetic variant that produces limited CYP2D6 activity and slow metabolism of CYP2D6 substrates. In these patients, conversion of codeine to morphine is reduced, as is the analgesic efficacy. Administration of codeine to these patients will not provide the expected degree of analgesia.

retrovirus

contains reverse transcriptase and integrase

Combining crRNA and tracRNA into a single guide RNA (sgRNA) for in-vitro DNA editing

crRNA = target specific tracrRNA = sequence which binds to Cas protein

Hunter Syndrome (MPS II)

deficiency in Iduronate Sulfatase -heparan sulfate and dermatan sulfate accumulate -X-linked and generally milder than Hurler's Syndrome WITHOUT corneal clouding. It is associated with variable mental retardation (same pathway as Hurler's syndrome) Cells from an individual with Hurler syndrome can complement cells from an individual with Hunter syndrome (and vice versa)

metabolism of drugs

dose = drug increases, then decreases until next dose poor metabolizers have too high levels of the drug in their plasma -> levels above therapeutic range lead to adverse effects normal metabolizers: are generally within the therapeutic range ultrafast metabolizers: drug levels drop below therapeutic range quickly, no effects

X-linked SCID

severe immunodeficiency David Vetter, The "Bubble Boy" (1971-1984)

Origins of hominids

• "Hominids" or "hominins": species more closely related to human than chimp • Sahelanthropus tchadensis • Chad (Central Africa) • Dated to 6 - 7 million years ago • Posture uncertain, but slightly later hominids were bipedal Early hominid fossils were all found in Africa • Australopithecus afarensis ~3 - 4 million years ago in Africa • Homo erectus/ergaster ~1.9 million years ago in Africa • Use of stone tools • H. erectus in Java ~1.8 million years ago • First known Europeans date to ~800 KYA ("thousand years ago") • Ascribed to H. heidelbergensis • Homo sapiens ("wise man") • Anatomically modern humans in Africa ~130 KYA • In Israel by ~90 KYA • Initially did not spread around the globe • Modern human behavior starts to develop in Africa after ~80 KYA • By ~50 KYA, features such as complex tools and long-distance trading are established in Africa • Middle Stone Age technology in Australia ~50 KYA • Upper Palaeolithic technology in Israel ~47 KYA

Somatic mutation rates

• 2 - 10 mutations per diploid genome per cell division • Compared to 70 new germ line mutations per diploid genome per generation, the somatic mutation rate may be an order of magnitude higher. • During development, normal cells are expected to accumulate 100s to 1,000s of substitutions, depending on the organ and the number of cell divisions.

Selection and clonal expansions in normal cells

• 2 - 10 mutations per diploid genome per cell division • The human body has 10^14 cells, which arise from 10^15 divisions • This means that every cell in the body carries at least tens of thousands of mutations! Positive selection for precancerous driver mutations may be common even in non-cancer tissues • For example, by middle age, 25-30% of sun-exposed skin cells have accumulated a driver mutation • Many "normal" somatic cells have just as many mutations as a cancer cell!

The CCR5 protein is an entry point for HIV

• A chemokine receptor on white blood cells • Individuals who are homozygous, for the "delta32" mutation in CCR5 are protected against infection by some HIV strains • Other HIV strains use CXCR4 The researchers attempted to create CCR5 delta32 mutations to protect the twin babies from HIV infection Problems with this work: • No peer reviewed study; instead: press announcement • No independent verification of the genome (unknown if editing actually happened) • There are other ways to prevent and treat HIV; "No unmet medical need that [this] embryo editing addresses" • Mosaicism of the gene deletion means children may still get HIV; "risks of gene editing for no real necessary benefit" • One off-target edit identified before implantation; parents accepted this "glitch" • One edit only removed 5 amino acids from CCR5 - may have no benefit at all • Unknown side effects (e.g. normal CCR5 protects against other infections) • Informed consent obtained by investigator, not by a trained professional; possibly monetary coercion • Unclear if and where IRB approval was obtained; various hospitals denied knowledge of this work • The researcher has since been fired from his university and is under investigation for violating regulations

Gene editing in disease

• Because CRISPR can in principle create any change in a genome, it has huge potential for gene therapy • We can isolate cells from a patient, and turn them into iPS cells • Then make the desired edit to repair the disease mutation • ...and reinsert the repaired cells into the body • Alternatively, direct injection of CRISPR reagents into mice (without making cells first) has shown mutation correction in vivo

Somatic mutations in neurodevelopmental disease

• By contrast, somatic mutations arise in the patient during development • They may affect multiple (but usually not all) areas of the body • They may not be detectable in blood DNA, creating problems for diagnosis • For example, mutations in DCX result in "double cortex syndrome", in which neurons migrating to the cortex stop too early, creating extra gray matter. DCS mosaicism is further complicated by Xchromosome inactivation in females (where DCX resides) • Some somatic mutations may arise later in development, and are then present in just a single organ • For example, mutations in AKT3 result in hemimegalencephaly (HMG), which shows severe malformation and enlargement of one (not both) cerebral hemisphere • Neurons are born deep in the brain and then migrate to the cortex • Somatic mutations may alter proliferation or migration of brain (and other) cells • Neurons with somatic mutations may be dispersed widely across the brain, or remain in a focal area • Somatic mutations may also act to diversity neuron function in a "normal" brain

Recurrently mutated pathways & processes

• Cancer driver genes are often involved in signaling pathways, genome integrity, and chromatin regulation Classifying cancers based on molecular signatures is more precise than classification based on tissue

Some key points about cancer genetics

• Cancer is highly heterogeneous. It is not "one" disease. • Different sets of mutations can come together to result in cancer (but some genes are affected more often than others). • It takes multiple mutations to cause cancer. • Every individual cancer is genetically unique.

Ancient DNA

• DNA can persist for thousands of years • We can extract and sequence ancient DNA from human (and animal and plant) remains and study genetic variation in the past DNA preservation requires cold and dry climates We can now sequence entire ancient genomes

DNA as a record of the past

• DNA variants in a population affect phenotypes and disease. • But they also contain information about the evolutionary past. • We can read this information by sequencing & genotyping DNA, and then apply evolutionary mathematical models to the data. • Using DNA data from existing species and populations, we can: • Infer phylogenetic "trees" of species • Estimate past population movements & admixture • Search for the action of selection

De novo mutations in neurodevelopmental disease

• De novo mutations arise in the gametes of one the parents • For example, mutations in LIS1 cause lissencephaly, which results in a "smooth brain" phenotype • Such new mutations are not detectable in the parents, but are present in all cells of the patient

High altitude adaptation

• On the Tibetan plateau, oxygen levels are 40% lower than at sea level • When individuals from low altitude regions acclimate to high altitude, their hemoglobin levels increase • This may increase blood viscosity and increase the risk of heart problems • Tibetan populations have adapted to life at high altitude. Their hemoglobin is only slightly increased. Variants in EPAS1 contribute to high-altitude adaptation in Tibetan populations The high-altitude haplotype came from Denisovans

Duchenne muscular dystrophy

• Duchenne muscular dystrophy (DMD) is a lethal degeneration of cardiac and skeletal muscle • Affects ~1 / 5,000 males • More than 3,000 mutations in the X-linked dystrophin gene are known, many in "hotspot" regions of the gene • Survival to age 20 to 30s • No cure • If the dystrophin gene could be repaired, the missing muscle protein might be replaced in the body • DMD is often caused by frame shifts or premature stops in the spectrin-like repeats. These result in missing domains at the end of the protein, which are important for proper function. • The repeated domains in DMD suggest a gene therapy strategy: • Remove, or cause skipping of, the mutated exons • This removes one or a few exons from the middle of the cDNA, but restores the important binding domains at the end of the protein • The result is a much less severe phenotype called "Becker muscular dystrophy" Engineered skipping of the most frequently mutated exons (2 to 10 & 45 to 55) Repairing dystrophin in patient-derived heart cells

Illustrating Mendelian Randomization

• Exposure (X) is some modifiable factor of interest to us • Overall goal is to understand whether exposure (X) causes outcome (Y) • We need to identify one or more genotypes (Z) known to affect exposure (X). Genotype is our instrumental variable. (IV) • Such genotypes may have already been reported in the literature, especially in large genome wide association studies C = confounders

genetic epidemiology summary

• Family, twin, and adoption studies suggest moderate inherited genetic contributions to cardiovascular disease • Major advances in statistical methodology, genomic technologies, and computation have accelerated discovery of genes contributing to CVD • Translation of these discoveries into clinical and public health benefits is proceeding relatively slowly - To build better models to predict future risk of disease and identify best candidates for intervention (e.g. polygenic risk scores; epigenetic signatures - To clarify causal effects of possible modifiable risk factors (e.g. alcohol consumption) - To generate new targets for drug development (e.g., PCSK9 inhibitors ) or genetics guided approaches to prevention and treatment

Gene flow & admixture

• Gene flow describes the exchange of alleles between different populations • Gene flow can happen when individuals move and bring their alleles with them • Or when individuals in two populations have offspring, and those offspring spread alleles • Mating between historically separate populations results in genetic "admixture" • If a population has rare haplotypes that are common in another population, this suggests that there may have been gene flow and admixture in the past Ancient humans interbred with each other and with modern humans • In adaptive introgression, a beneficial variant is introduced from a different population There have been several adaptive introgressions from archaic populations into modern humans

Technical issues with editing embryonic stem cells

• Human cells tend to prefer NHEJ over HDR • In human embryonic stem cells: 2% efficiency • In human embryos: 14-25% efficiency

Imprinting and disease

• Imprinting is a normal element of human genetics & reproduction • But when paired with mutations on the nonimprinted parental allele, disease can result • A pair of syndromes, Prader-Willi Syndrome and Angelman Syndrome illustrate this.

Denisovans

• In 2010, Johannes Krause in Leipzig extracted DNA from a tiny piece of bone found in Denisova cave in Siberia, hoping to find a "good" Neanderthal bone with lots of ancient DNA • He found something quite different Denisovans are a human group more distantly related to us than Neanderthals • We now have the complete genome of this Denisovan female, but have essentially no other information about her, or other members of her population • She was more closely related to Neanderthals than us In 2019, a jaw bone found in Tibet in 1980 was confirmed to be from a Denisovan • Denisovans may have had big heads and weighed well over 200 pounds

Imprinting

• In imprinting, the two parental genomes do not play the same roles. • At an imprinted locus, only the maternal or the paternal allele is expressed • This happens irrespective of the alleles at these loci! • Females and males silence certain genes in their gametes • Mechanism not fully known, but DNA methylation is involved

consequences of DNA repair mutations

• Increased mutation rates • Sooner or later, these mutations will create an oncogene or destroy a tumor suppressor gene • DNA repair gene mutations do not themselves result in neoplastic transformation, but make other mutations that do much more likely

Many mutations do not affect fitness

• Many types of mutations have no, or only very small effects on the organism: • Mutations in many highly repetitive regions • Mutations in most noncoding DNA • Mutations in pseudogenes • Such regions are free to mutate • They will eventually change their sequence so much that we cannot recognize their shared ancestry anymore A consequence of the genetic code is that the third base in a codon can often mutate without changing the amino acid

DNA methylation

• Methylation is an example of EPIGENETIC modification • Mitotically heritable changes not coded in the DNA sequence • Associated with aging and may contribute to disease risk • 60-90% of CpG dinucleotides are methylated at cytosine carbon 5 • Methylation in promoters often suppresses gene expression • Methylation in gene body may regulate alternative promoter use and splicing

Mutations in some regions reduce fitness and are selected against

• Mutations that break important elements in a genome reduce fitness (for example, disease causing mutations) • Such mutations reduce the number of offspring an individual has • Therefore, these mutations are not passed on... • ...and the sequence of the genomic element does not change over time • Such elements show "evolutionary constraint" • And we call the region a "conserved element" Evolutionary conservation is visible by comparing species • About 10% - 15% of the human genome is conserved • Conservation scores reveal conserved elements • These elements often point to protein-coding exons • Conservation also indicates regulatory regions, such as enhancers • Conservation is a powerful to find functional regions in our genome Using evolutionary conservation to predict the severity of amino acid substitutions

epidemologist

• primarily focuses on environmental causes of disease, including behaviors and lifestyle D = E

geneticist

• primarily focuses on genetic causes of disease D = G

Fixation of drifting alleles produces a "molecular clock"

• Some DNA variants drift to high frequency, and eventually "fix" • "Fixation" means that the variant is homozygous in every individual in the species • (The time to fixation takes depends on population size) • Fixed mutations are called "substitutions" • The rate of substitutions is constant over time • After two populations have split (for example, due to a new spatial barrier (such as a river), they accumulate mutations independently • Over time, the populations become species that can no longer mate with each other • By counting the number of sequence differences, we can estimate how long ago two species split • Species that split from a common ancestor early on accumulate more independent mutations than more closely related species • We can count differences among genome sequences to reconstruct "phylogenetic trees"

Ancient human genomes

• Starting in 2010, complete genomes from ancient Homo specimen were published • They include Neanderthals, a human form that coexisted with modern humans for tens of thousands of years in Europe and Central Asia • We now have several complete genomes from individuals that lived tens of thousands of years ago

Discovery of CRISPR-Cas

• The CRISPR/Cas system was identified from basic research • This research did not have the goal of making a genome-editing tool • Curiosity-driven, fundamental research happened to find a tool with major consequences for research, disease, and - perhaps - human evolution

Cas9 PAM sequences correspond to splice sequences

• The Cas9 PAM is "NGG" • Some "NAG" sequences can also be used as a PAM • These two sequences often occur in the universal splice acceptor sequence (AG) and donor sequences (GG) • Splice sites should be easy to target by CRISPR • By destroying one splice site with NHEJ, a single gRNA can lead to exon skipping (i.e., there is no need to cleanly excise the exon from the genome with two gRNAs)

Gene editing in human embryos

• The applications we discussed so far all happen in cell lines that either cannot form a human embryo, or would need substantial further cell line work before they could be implanted (e.g. iPS cells) • By contrast, implanting gene edited embryos would result in a human being who carries the edit • The edit would also be included in the germ line, and might be passed on to future generations Started with mature fertilized human oocytes that had three pronuclei (fertilized with 2 sperm). Such embryos cannot develop into a human being. Created gRNA sequences that targeted the HBB gene (hemoglobin subunit beta). Because the sequences of the HBB and HBD genes are very similar, cells can use HBD as a template to repair breaks in HBB. Transfected 86 embryos with gRNA targeting HBB, Cas9 mRNA, GFP mRNA, and a ssDNA oligo donor template. Then wait 48 hours.

Somatic mutations

• Throughout our lifetimes, our somatic cells accumulate mutations • Most have no effects, but some can result in disease • One such disease is cancer

Gene-Environmental Impact on Cancer Risk

• UV light • Chemicals (smoking!) • Maybe some diets?

viral vs non viral vectors

• Viral vectors (transduction) most commonly used • Non-viral vectors (transfection) are safer but much less efficient at transferring genes Nonviral vectors: Plasmid DNA Liposome-entrapped DNA Protein-DNA conjugates Viral vectors: Retrovirus Adenovirus Adeno-associated virus Herpes simplex virus30

other CRISPR applications

• We can break the nuclease activity to make catalytically "dead" dCas9 • dCas9 can repress genes simply by being in the way of RNA polymerase • We call this "CRISPR inhibition", or CRISPRi • We can use this for programmable gene inactivation • dCas9 can be fused to many kinds of "effector domains" • Gene activation domains result in "CRISPR activation" (CRISPRa) • Chromatin modification: Writers, erasers • GFP

Base editors

• We can combine dCas9 with DNA modifying enzymes (e.g. APOBEC deaminases) • These can edit bases "directly", without a double-strand break, and without a repair template

Massively parallel, genome-wide screens

• We can synthesize pools of thousands of gRNAs • Each cell in a culture takes up one gRNA and deletes the given gene • In the whole cell culture, we knock out every gene in the genome • We can measure the consequences of each deletion by tracking gRNA frequencies over time • This allows us to probe the functional roles of thousands of genome regions in one experiment

Goals of The Cancer Genome Atlas (TGCA)

• Which genes are recurrently mutated? • Separate "driver" mutations from "passengers" • Which pathways are recurrently mutated? • Systematic patterns relative to body site? To cancer type? • Understand which cancers may respond to which treatment • From traditional to molecular classification for more precise treatment • For each gene, test if it has more somatic mutations in tumors than expected given the mutation rate • 299 cancer genes identified, 87 in multiple cancers: TP53 is the most studied gene in the human genome Most driver genes are now known • In big cancer genomics datasets, we do not find additional genes with systematic excesses of mutations • We find an average of 4-5 drivers per tumor

How to evaluate a gene therapy proposal or ongoing or completed study

• Why is gene therapy appropriate for the condition it is being applied to? Is there an obvious market for the therapy or innovation? • What is the relevant physiology of the condition the therapy is being directed toward? • What are the relevant genetics of the condition? • What organism and what genotype(s) is the therapy directed toward? Humans? Homozygous nulls? Hypomorphs? Heterozygotes? • What safety measures were taken to protect the patients/subjects? • What are the characteristics of the vector used? How was it constructed? Why was a vector with those features chosen? • What regulatory sequences were used in the expression/integration vector? Was a constitutive promoter used? Inducible? Tissue specific? Strong, medium, weak? • What was the transformation strategy? Why ex vivo or in vivo? How was it performed? • How was tissue specificity of the therapy determined? What about effective concentration of the gene/gene product? How was the cellular access determined? Physiological half-life? Longevity of the gene-expressing cells? • What about immunological responses? What if any measures were taken to avoid or reduce these? • How was the efficacy of the therapy determined and represented? How were the patients/subjects identified? How many individuals were studied? What controls groups were used? Were any other medications that might influence the recovery of the individuals taken concurrently? What were the measured variables? • When and how often was the efficacy of the treatment tested? What other parameters of the patients were assessed? How long did the results last? Must the gene therapy be given repeatedly over the years? • What level of variation in severity of their patients condition was permitted (or sought out) at the beginning of the therapy? • How many measurements were used to establish "baseline"? What other physiological parameters were measured? • What were the specific hypotheses tested in the study? Did the authors evaluate the potential of gene therapy or were they looking for the total solution to a given problem?

genetic epidemiologist

• focuses on both genetic and environmental causes of disease and their interactions D = G + E + GxE