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Sister Chromatids are 600 nanometers in width

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Nucleosomes are fundamental packaging: core of 8 histones; 146bp.

 2. Recognize normal and abnormal human karyotypes. SLIDES 7-9, 42

3. Define the following terms: Chromosome banding: staining of bands, or chromosome segments, allowing the precise identification of individual chromosomes or part of chromosomes. Centromere: mitotic spindle attachment. Found near the middle of the chromosome where the two identical sister chromatids come closest in contact. Telomere: region at the end of the chromosome that protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. p and q arms of chromosomes: long (q) and short (p) arms SLIDES 7 10 Autosome: any of the chromosomes other than the sex-determining chromosomes Sex chromosome: X and Y Metacentric: both arms are approximately equal in length Submetacentric: one arm is somewhat shorter than the other

Acrocentric chromosomes: one arm is much shorter than the other

Heterochromatin: Condensed (genetically inactive) form of chromosomal region is always condensed while facultative heterochromatin can become decondensed (genetically active form) in certain circumstances Euchromatin: Decondensed (genetically active) form of chromosomal region

Haploid: having one copy of each chromosome (N = 1); e.g. human gamete (Meiosis ) Genetic material is exchanged during meiosis through recombination. Diploid: having two copies of each chromosome (N = 2); e.g. human somatic cell (Mitosis) Aneuploid: having one or more extra or missing chromosomes (Trisomy 21)

Robertsonian translocation: joining of two acrocentric chromosomes at the centromeres during translocation (region on a chromosome that joins two sister chromatids, point of mitotic splindle attachement) with loss of their short arms (p-arms=Petit) to form a single abnormal chromosome.

Balanced and unbalanced chromosomal abnormalities

Down syndrome: 95% of cases due to trisomy 21 (47,XX,+21 and 47,XY,+21) Patau syndrome: trisomy 13 (47,XX,+13 or 47,XY,+13) Edwards syndrome: trisomy 18 (47,XX,+18 or 47,XY,+18)

4. Differentiate the following human cytogenetic abnormalities and list at least one Numerical abnormalities (i.e. abnormal number of chromosomes) Nondisjunction: the failure of the paired chromosomes to segregate properly during meiosis or mitosis, resulting in daughter cells with abnormal numbers of chromosomes (can occur during meiosis and mitosis) Turner syndrome (monosomy X): 45,X Klinefelter syndrome: 47,XXY Triple X syndrome: 47,XXX 47,XYY syndrome: 47,XYY Down Syndrome

Structural abnormalities (e.g. deletions, insertions, inversions, and translocations) Deletions/Insertions:

Smith-Magenis syndrome (SMS) Autosomal recessive 1/15,000 – 1/25,000, 17p11.2 deletions (RAI1 gene) Mostly caused by de novo mutations: during gamete formation in one parent, early fetal development Multi-systemic disorder: craniofacial abnormalities, intellectual disabilities, various organ systems •Autism Spectrum Disorders • Schizophrenia • Parkinson Disease • Alzheimer Disease • Emphysema Inversion: A segment of the chromosome is flipped around and the genetic material is inverted l Reciprocal translocation: segments from two different chromosomes have been exchanged CML >95% of chronic mylegenous leukemia patients have cancer cells with a ―Philadelphia chromosome,‖ reciprocal translocation between chromosomes 9 and 22. bcr-abl fusion gene with unregulated tyrosine kinase activity.

Robertsonian translocation: joining of two acrocentric chromosomes at the centromeres with loss of their short arms to form a single abnormal chromosome.

2-3% of Down syndrome cases, extra chromosome 21 may be due to a Robertsonian translocation that occurred in a parent. The long arm of chromosome 21 is attached to another chromosome in the healthy parent. This individual has a significant chance of generating a gamete with an extra copy of chromosome 21.

Ring chromosomes: usually occur when a chromosome breaks in two places and the ends of the chromosome arms fuse together to form a circular structure 3 Ring chromosome 14 syndrome, &RC 20(rare neurological disorders)

Human Inheritance I
Methods of Inheritance:

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Locus – genetic location on a chromosome Allele – each individual has two alleles at a locus, one copy from each parent Genotype – the record of an individual’s alleles, genetic information. Often written AA (homozygous dominant), Aa (heterozygous, sometimes called carrier), or aa (homozygous recessive) for a given trait. Autosomal – No effect of gender, males and females equally likely to be affected and to pass on trait. Sex-linked – Affected by gender, gene resides on one of the sex chromosomes (usually X). Dominant – One copy of gene confers trait. Affected seen in all generations, does not skip. About half of offspring of one affected and one non-affected parent will have the trait. Also called ―true dominant.‖ Co-dominant – In a heterozygote, both alleles are expressed as individuals. (ex: blood group AB) Semi-dominant – Subjects who are homozygous dominant more severely affected than heterozygotes. Recessive – Two copies of gene needed to confer trait. Often appears to skip generations, or a single generation may be affected. If both parents carriers, ¼ of offspring affected. X-linked – sex-linked trait on the X chromosome. More often expressed in men (only one X), will never see male-to-male transmission. Women are usually carriers (Xx) but could also show disease if trait is dominant, they are homozygous recessive, or there is skewed X-inactivation (Calico cats). Y-linked – sex-linked trait on the Y chromosome, very rare. Only male-tomale transmission, no skipped generations. Mitochondrial inheritance – all inheritance is passed from mother to child, regardless of child’s gender. No transmission from father to any offspring. Random (disassortative) mating – mate selection is not based on similarities or biased by background, physical ability, etc.

Founder effect – High frequency of a previously rare allele because population was started by small number of founders. Also called a ―bottleneck.‖ Mosaicism – Individual has more than one cell type due to an error in mitosis during development. Somatic mosaicism affects somatic tissues, sometimes only part of the body. Germline mosaicism shows normal parents but multiple affected offspring due to mutation in germline.

Relevant to dominant diseases: Expressivity – the amount that a trait is seen in an affected individual. ―Variable expressivity‖ refers to members of the same family showing striking variation in phenotype, even with same gene. Thought to depend on environmental factors. Penetrance – The frequency a specific genotype is expressed by those individuals that possess it, usually given as a percentage. With low penetrance, some genetically affected individuals may show no evidence of trait. (ex. skipping of generations) Age-related penetrance – refers to delayed age-of-onset, may be due to slow accumulation of toxins or inability to repair damage. NOTE: Penetrance is all-or-none, either the person does or does not express the disease gene. Expressivity refers to varying levels of severity of symptoms. Genetic anticipation – some dominant conditions become more severe and earlier-onset with successive generations (more repeats added or deleted) New mutations – sporadic and rare, usually dominant, should consider nonpaternity and reduced penetrance

Using a Punnett square: Both parents heterozygous: A A AA a Aa




Chance of affected child = ¼ , chance of carrier child = ½ One parent recessive, one homozygous dominant a A A Aa Aa

a Aa Aa

Chance of affected child = 0, chance of carrier child = 1

How to read pedigrees: Square is male (usually on left), circle is female, diamond is unspecified sex Black is affected, white is unaffected, dot in center is carrier, odd pattern of black (like one corner black) is affected and denotes different types of affected phenotypes. Arrow is proband – family member first brought to attention of clinician, often the person who sought medical advice. Not necessarily affected. Single lines show family tree relationships, diagonal line through symbol is deceased Double horizontal line is consanguineous mating by two related individuals. Be extra on the lookout for recessive traits appearing in their offspring! Draw oldest siblings on left, youngest on right

Human Inheritance II

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1. Understand the basis of multifactorial diseases and differences from Mendelian diseases. · Mendelian Diseases: o There is a single gene responsible for a disease (ex: his pea plants) o Mutations have a significant effect · Complex / Multifactorial Diseases: o Familial inheritance, but no clear segregation of phenotype into a defined pattern. o *They occur at a higher frequency in the population, and therefore are of greater public health concern (ex: Type II Diabetes). o Possibilities for causes: § Oligogenic: small # of loci § Polygenic: many loci, each has a small effect § One major locus w/ multifactorial background o Subtle mutations & environmental effects can aggregate to be pathogenic, but each alone is not.

2. Define the following genetic terms: Locus: location of a specific gene (Loci = multiple genes) Alleles: Different versions of a gene Genotype: genetic constitution Phenotype: physical appearance of trait Homozygous: dominant or recessive – same allele present on both chromosomes Heterozygous: Different alleles that are distinguishable Polymorphism: when an allele has a differing nucleotide sequence or repeated nucleotide units, compared to the population (>1% of chromosomes) that may or may not affect gene activity · SNPs (Single nucleotide polymorphisms): variation of a single nucleotide (differs between individuals or between chromosomes in same individual) · CNVs (Copy Number Variations) : insertion, deletion, or duplication of a large chromosome segment

· Microsatellite: repeating simple sequences Mutation: any event that changes genetic structure; any alteration in the inherited nucleic acid sequence of the genotype of an organism. Multifactorial Inheritance: trait depends on 2+ genes & environmental factors Polygenic Inheritance: a trait is dependent on multiple genes centiMorgan (cM): Represents 1% recombination frequency in linkage analysis LOD score: Logarithm of the odds which is used to determine linkage 3. Write and be able to apply the Hardy-Weinberg equation: p2 + 2pq + q2 = 1 when p+q=1

Conditions: randomly mating population with no outside influences, no new mutations, and there is no selective advantage to heterozygote (sickle cell, CF) Other Deviations: · Small population = genetic drift (one allele fixed, the other extinct) · Gene flow – migration/immigration of new population 4. Given a disease incidence, be able to calculate gene and carrier frequency EX: Disease incidence = 1/1,000 q2 = 1/1,000 q = .03 p = 1-.03 = .97 2pq = 2(.03)(.97) = .06 which is the carrier frequency Hardy-Weinberg can help determine likelihood of a child having a disease without doing a genetic test 5. Describe in general terms how genetic linkage analysis is done.

Linkage analysis: uses a known locus to estimate the location of another gene on the same chromosome (markers that cosegregated w disease) by calculating the recombination frequency (theta) · Linked: theta = 0 · Unlinked: theta = .50 · Can be evaluated with LOD Score: Logarithm of the Odds o log (Likelihood of observed data at theta / likelihood of observed data if Unlinked o >3 = linked (1/1000 chance that genes are unlinked) o <-2 = exclude linkage

Cancer Genetics

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1) Define genetic counseling and integration into the clinical setting. Genetic Counseling: the process of helping people understand and adapt to the medical, psychological and familial implications of genetics’ effect on disease. i) Interpretation of family and medical history to assess chance of disease/ reoccurrences ii) Education about inheritance, testing, management, prevention, resources, research iii) Counseling to promote informed choices about risks The Process: a) Determine patient’s understanding b) Gather family history c) Identify syndromes in family history d) Determine if testing is reasonable e) Discuss risks, benefits and limitations of testing f) Interpret test g) define risk and make recommendations h) provide counseling, updates on information i) manage psychosocial impact on family 2) Summarize the basics of cancer genetics and clinical contribution of hereditary cancer syndromes. - All Cancer is genetic: - 10% hereditary - 20% is shared familial risk factors Genetic (inherited) vs Somatic (developed/ acquired) Gatekeeper Genes: act directly to control cellular proliferation / differentiation Tumor Suppressor Genes: negative effect on cell cycle regulation - cancers occur when both TSGs fail - like brakes on a car Proto-oncogenes: turn on genes that would normally be turned off

- oncogene: mutated gene --> cancerous cell proliferation Caretaker Genes: genes that maintain integrity of genome DNA damage-response genes: repair mechanism for DNA during replication or from carcinogens. - cancer arises when both genes fail 3) Discuss the clinical impact of carrying a deleterious mutation in one of the BRCA genes. Both copies of a BRCA gene must be defective to get cancer (Knudson’s Two-Hit Hypothesis). If one gene is defective, the odds of acquiring a second hit are higher than with Sporadic (2 acquired mutations) cancer. Breast cancer will develop at a higher rate and earlier in life for this population. This population needs to: Increase Surveillance (check for lumps/ mammograms/ MRI after 25) Surveillance starting younger - Prevent Cancer through good lifestyle choices

4) Summarize the psychosocial considerations relevant to approaching the decision to undergo genetic testing for a hereditary cancer syndrome. Personal implications Familial implications Societal Implications Very emotional time - impact on family What if... positive? Negative? Who to tell? Why or why not? Family or cultural beliefs about cause of cancer Risk for depression and/or guilt

Other Terms:

Synthetic Lethality: when mutations in either of two genes individually has no effect, but the combined mutations leads to cell death (PARP & BRCA)

Genomic Medicine I
1. Define:

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Shotgun sequencing-DNA of interest is shredded into smaller, manageable fragments and then sequenced. Overlapping sequences are placed together in order to determine the genome  Hierarchical shotgun sequencing comes with higher accuracy and was used to initially sequence the human genome but is slower and labor-intensive  Whole genome-sequencing approach uses random fragmentation and computer algorithms to piece sequences together. It is faster and capable of sequencing individual genomes but requires the known sequence of the genome to be effective. Interspersed Repeats-Large sequences of repeats interspersed within the genome.  45% of our genome Segmental Duplications-Multiple copies of the same gene. De novo mutations-A sequence change that arises spontaneously as a result of a mutation in the germ cell, i.e. present in offspring but not in parents Copy number variants-A sequence that is at least one kilobase in length and polymorphic. GWAS (Genome-wide association study)-A study that looks at correlations between SNPs and a particular trait Epistatic Interactions-interactions between genes that modify a phenotype

2. Explain the genetic basis for CHARGE syndrome, age-related macular degeneration, and Type II diabetes mellitus CHARGE syndrome results almost entirely from de novo mutations arising in the CHD7 gene, validating the use of genomic studies on individuals in order to provide clinical correlations. The risk of age-related macular degeneration can be gauged by studying the sequences of a relatively small number of genes, showing the potential for genomic medicine in the clinical setting.

Studies on type II diabetes mellitus, however, have shown genomic studies to be as predictive as clinical correlations but there is the possibility that genomic medicine can eventually predict risk for type II diabetes.

3. Define the numbers of i) disease-associated sequence variants in a healthy person’s genome and ii) de novo mutations in a healthy person’s genome i) A person carries 50-100 sequence variants associated with disease.  250-300 LOF (Loss of Function) sequence variations ii) 70 de novo mutations arise per diploid genome 4. Explain the underlying motivation for GWAS studies:  GWAS studies address the hypothesis that common genetic variation is responsible for human disease.

5. Explain the risk factors common sequences confer in different common diseases Rare alleles tend to be associated with rare disease but have a very large effect size on phenotype, such as a single allele causing all of the symptoms seen in cystic fibrosis. Common alleles tend to be associated with common disease but have small effect sizes, such as in type II diabetes. Age-related macular degeneration is one of a handful of exceptions where common alleles confer a large effect.

Genomic Medicine II
1) Define:

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Silent mutation: a change in gene sequence that does not result in AA sequence change (sometimes activates cryptic splice sites) (10-12k in human genome) Missense mutation: 1bp change that alters AA sequence eg: CFTR G551D mutation loss of fxn for Cl channel, current Phase 3 drug tested increases Cl trafficking (does not help channel transport malfunction in ΔF508) Nonsense mutation: converts a normal codon into a stop codon (eg: animoglycosides, current treatment, have high toxicity) (drug PTC124 promotes read-through) Might be corrected by nonsense-mediated decay Might be in the noncoding region but might also interfere with folding Nonsense suppressor therapy: drugs that reverse the nonsense mutation (or nonsense mediated decay)  read-through promotion  Previously not tolerated well by the body: therapeutic window is small  basically replaces nonsense with missense mutation and applies to all stop codons  Challenges: stop sequence context and nonsense-mediated decay Driver mutation (20%): acquired changes that have functional significance Passenger mutation (80%): mutations that do not affect phenotype Pharmacokinetic effects: Genotype affects absorption, distribution, metabolism, and elimination of a drug (body’s effect on the drug) Pharmacodynamic effects: Genotype and physiology changes how active the drug is in the body (drug’s effect on the body) 2) Explain the basis for therapies based on the genotype of individuals with certain Mendelian disorders DMD: Duchenne muscular dystrophy – DMD exons deleted or duplicated (dystrophin stabilizes and links cytoskeleton of muscle fiber to the membrane and extracellular matrix – strengthens muscles)  

Exon skipping therapy: Antisense Oligonucleotide: binds and masks specific splice jxns on mRNA to skip exon readings and keep the right reading frame DMD is particularly good target because internal exons can be

deleted and still be left functional  Only works for a certain percentage of people CML: Chronic myelogenous leukemia – Philadelphia translocation between chromosome 9 and 22 (bcr-abl)  Unregulated tyrosine kinase acitivity  Treated by Gleevec: inhibits bcr-abl fusion protein activity and decreases cancer growth 3) Define the types of acquired loss of function and gain of function mutations often found in cancer cells and the genes affected by these mutations:  Loss of fxn mutations: knocks out tumor suppressors (eg: BRCA1)  Inactivating point mutations  Gene deletions  Epigenetic silencing  Gain of fxn mutations: Proto-oncogenes (eg: bcr-abl fusion protein)  Activating pt mutations  Gene amplification  Gene translocation 4) Explain the difference between passenger and driver mutations  Driver mutations are responsible for the development and progression of cancer  31% of driver mutations leading to cancer are mutations in protein kinases and therapy can be tailored accordingly  80% of mutations are passengers 5) Explain and give examples of pharmacokinetic and pharmacodynamic effects that relate to the genotype of an individual with cancer or a tumor sample  Cancer genome atlas: coordinated effort to understand the molecular basis of cancer through large-scale genomic databases and provide information for pharmacogenomic therapies See DMD example See CML example

Biomarkers on a person’s genome have pharmacogenomic consequences to taking certain drugs Pharmacokinetic effect: Genotype affects absorption, distribution, metabolism, and elimination of a drug Irinotecan is an anticancer prodrug which the body activates UGT1A1 gene enzyme attaches glucuronic acid to drugs to make it more soluble and allows easier processing through the body  Individuals with the 7TA repeats have less UGT1A1 enzyme and low glucuronidation levels which means that they metabolize the drug slower (toxicity, drug activity with same dosage)  Individuals with 6TA repeats have more enzyme and high glucuronidation levels which means that they excrete the drug faster (toxicity, drug activity with the same dosage)  Knowing genotype is essential for correct dosing Pharmacodynamic effect: Genotype and physiology changes how active the drug is in the body Gefitinib is a kinase inhibitor for non-small cell lung carcinoma  Individuals have mutations in the EGF (epidermal growth factor) receptor (tyrosine kinase always on)  Knowing mutation in the genotype of individuals allows targeted drug therapy 6) Explain and give examples of the use of gene expression data in the classification and prognosis of cancer:    Classification and prognosis of cancer on the individual level currently have little benefit 31% of cancer samples have driver mutations Eg: Diffuse Large B cell Lymphoma has high heterogeneity (different manifestations among different pts) o Following initial chemotherapy, <50% long-term remission Can subclassify cancers by how genes are turned on and off three subtypes of expression: Germinal Center B Cell Like, Subtype 3, and Activated B-Cell Like, each with a different probability of survival over 10 yrs Eg: Mantle cell lymphoma: ID small panel of gene expression profile and identified 4 quartiles o Tumors showing the most gene mutations for growth = worst prognosis

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Learning Objectives:

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1. Epigenetics vs. genetics: epigenetics refers to alterations that are meiotically or mitotically heritable changes but that are not based on DNA sequencing. Conversely, genetics is concerned with heritable alterations within the DNA sequence. a. Different cell types in the body are genetically identical but have different phenoytpes (e.g. liver cell v. fibroblast) b. Every cell: specific subset of required genes c. Epigenetic mechanisms: determine expression potential of genomic regions d. Examples: i. DNA methylation (add a –CH3 group) ii. Histone modifications iii. Chromatin structure/nucleosome positioning iv. Non-coding RNAs (coordinate transcription) v. Transcription factor regulatory networks Imprinted Regions: Genes where either only the paternal or maternal copy of a gene is expressed 2. a. b. c. d. e. 3. a. Epigenetic markers Writers: establish mark Readers: interpret mark Erasers: remove mark Remodelers: shift nucleosome about Insulators: segregate epigenetic domains Role of epigenetics in Mendelian disorders (w/ examples) Germline defects:   Inherited defects in epigenetic regulators (e.g. ICF, Rett, Kabuki syndromes --> mutations in methylation writers and readers) Uniparental disomy (UPD) of imprinted regions o Mono-allelic gene expression: only one of the two homologous alleles of a gene is expressed (e.g. X-inactivation in females, genomic imprinting—some genes activated by father, other by mother)

o UPD: person receives two copies of a chromosome (or part) from one parent and no copies from the other. Biallelic expression or no gene expression. (e.g. Angelman syndrome, Prader-Willi syndrome), Beckwith-Wiedemann syndrome) Systemic (inherited epigenetic defects) (rare) o Silencing of specific repair gene = predisposition to colorectal cancer (unsure…) o Transgenerational impact of environmental exposure (e.g. Dutch hunger winter phenomenon) Somatic defects:  Somatic mutations of epigenetic regulators in cancer o Many examples of somatic mutations in epigenetic writers, readers, erasers, remodelers and insulators  Somatic epigenetic defects in cancer (common) o Most human cancers have evidence of epigenetic alterations (e.g. silencing of tumor-suppressing genes) 4. a. Role of epigenetics in cancer (w/ examples) Epigenetic biomarkers: Diagnosis/Prognosis  i. Risk assessment ii. Disease detection iii. Disease classification iv. Prognosis v. Prediction of treatment response vi. Disease monitoring Epigentic Therapy: Treatment i. Inhibitors of Histone Acetylation Erasers (HDACs) ii. Inhibitors of DNA Methylation Writers (DNMTs)  E.g. MGMT methylation predicts response to alkylating therapy. MGMT enzyme repairs damage from chemotherapy. Epigenetic silencing of MGMT allows O6 methylguanine to persist in DNA, which interrupts DNA replication and causes apoptosis in tumor cells. 5. a. Role of epigenetics in other common diseases (w/ examples) common diseases? 



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