SG 102 Midterm

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DNA structure and organization

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1

DNA structure and organization

DNA is packaged into chromosomes, wrapped around histones

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Nuclear DNA

DNA that is present in the nucleus of a cell and that is inherited from both parents

Packaged into Chromosomes and Somatic Cells Have TWO Copies of the Entire Sequence

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mitochondrial DNA

DNA found in the mitochondria that is inherited only through mothers

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exon vs. intron

exon - protein coding intron - nonprotein coding

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central dogma

DNA-transcription-RNA-(introns removed through splicing) translation-protein

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types of mutations

point mutations: single nucleotide change

  • silent (no change), missense (amino acid substitution), nonsense (adds stop codon)

frameshift: Insertion/deletion of nucleotide(s) that shift reading frame or insert a stop codon

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monogenic conditions

conditions associated with single gene mutations (ex. Huntington's)

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most common health conditions made up of...

A combination of genetics and environment (not monogenic)

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examples of epigenetics

  1. Dutch Famine

  2. Barker's Fetal Origins of Adult Disease

Two different datasets that point to the importance of fetal and early life environment, not just genetics, for lifelong health

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meta ethics

the study of the origin and meaning of ethical concepts

What is morality/ethics? Where does it come from?

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normative ethics

How should we be? How should we act?

Examples: deontology, consequentialism, care and virtue ethics

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applied ethics

How do we put ethics into action?

Examples: bioethics, environmental ethics, medical ethics, legal ethics, business ethics, etc.

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Consequentialism

"ends justify the means"

  • Morality of an action depends on its consequences

  • Utilitarianism (Bentham): Goal should be to maximize benefit for the most people

  • would say to flip the switch in the trolley problem

criticism: prioritize individualism, abstract rules for right and wrong

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Deontology

Morality depends on the rightness of the action itself (outcome is irrelevant)

  • Rules determine morality: Do not lie, Do not kill

  • Kant's categorical imperative: don't treat people only as a means to an end (respect for persons)

  • would say not to flip the switch in the trolley problem; don't do direct harm

criticism: prioritize individualism, abstract rules for right and wrong

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virtue ethics

Good character traits (virtues) and bad character traits (vices)

  • Morality is less about what you do and more about how you are

  • Roots in Greek philosophy (Socrates)

  • Examples of virtues: Honesty, Patience, Wisdom

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ethics of care

  • Importance of relationships, benevolenceand empathy

  • Critiques individualistic, male-centered, hyper-rational (rules-based) deontology and consequentialism

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why did bioethics come about?

roots in in the 1960s/70s in the U.S.

Context:

  • Medical paternalism (doctor knows best)

  • Major technological and medical advancements (e.g., recombinant DNA, the atomic bomb, organ transplantation, contraception, life-extending care)

  • Unethical and horrific medical experiments coming to light: Nuremberg Trials, Tuskegee Syphilis Trials, Guatemala Syphilis Experiments

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Nuremberg Code

ethical code of conduct for research that uses human subjects (emphasizes autonomy and avoiding harm)

not legally-binding

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Declaration of Helsinki

The World Medical Association's international ethical guidelines for medical professionals researching human subjects

  • Builds on and clarifies items fromNuremberg Code

  • Directed towards physicians

  • Multiple revisions since first edition

  • Recent revisions address use of human samples/data, vulnerable populations

not legally-binding

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Tuskegee Syphilis Study

1932-1972: US Public Health Service & Tuskegee Institute study syphilis disease progression in Black men living in Alabama (399 with syphilis and 201without)

  • became public in 1972

  • No informed consent

  • Penicillin accepted treatment by 1945 but never offered to participants

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National Research Act of 1974

Authorized the creation of the Commission for the Protection of Human Subjects, which was charged with developing an ethical code and guidelines for researchers (informed consent & the role of assessment of risk-benefit criteria in the determination of the appropriateness of research involving human subjects)

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Belmont Report

made by the commission, summarizes the basic ethical principles and guidelines for the protection of human subjects of research (autonomy, justice, beneficence, nonmaleficence)

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four principles of bioethics

Respect for Autonomy - patients/participants have the right to make their own informed decisions, have a right to privacy and confidentiality

  1. Beneficence - obligation to provide services/treatment that are of benefit

  2. Non-maleficence - duty to avoid harm; harm should be outweighed by benefit

  3. Justice - duty to act fairly (everyone deserves what they're owed), balance risks and benefits

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complexities of distributive justice

when there's not enough resources for everyone

  1. To each person an equal share

  2. To each person according to need

  3. To each person according to effort

  4. To each person according to contribution

  5. To each person according to merit

  6. To each person according to free-market exchanges

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limitations of the four principles of bioethics

➔ Broad and/or vague in some scenarios - how do they guide action?

➔ May require weighting of different principles or specification (not absolute)

➔ What to do if 2 principles conflict with one another?

➔ Many other ethical decision-making paradigms that overlap or offer other guides

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public health/community ethics

contrast to clinical/medical ethics (individual)

  • Focus on preventing disease in communities and populations

  • Maximize community benefit and reduce community harm

  • Relational autonomy of interdependent citizens

  • Community consent through dialogue and community involvement

  • Concerned with equity and social justice, protection of vulnerable and/or marginalized groups

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feminist bioethics

Critiques: ➔ Under-representation of women and women's health issues in clinical trials and discussion ➔ Neglect of marginalized communities in mainstream bioethics ➔ Abstract, "one-size-fits all" sentiment in mainstream bioethics ➔ Narrow definition of autonomy

Emphasizes: ➔ Attention to unequal structures of power and privilege ➔ Social justice ➔ Attention to dependency, uncredited labor of caregiving, and vulnerability ➔ Relational autonomy - individual choices are constrained/informed by social and cultural context

More similar to public health ethics than traditional clinical ethics

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categories of genetic tests

preimplantation, prenatal, newborn screening, carrier screening, Pharmacogenetic and/or Tumor Testing, Predictive or Presymptomatic Testing, diagnostic testing

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two things that "keep Dr. Grody up at night"

variants of uncertain significance

incidental findings

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genomics

the study of: • the complete set of chromosomes characteristic of a species • the complete set of genes of an organism • the complete complement of DNA in the cell of an organism • The elucidation and use, for research and medical applications, of the complete DNA sequence on all the chromosomes of human cells

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Human Genome Project

An international collaborative effort to map and sequence the DNA of the entire human genome.

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Timeline of human genome project

1985 Exploratory conferences held at UC-Santa Cruz and Santa Fe

1986 Human Genome Initiative announced by DOE

1987 NIH funding commences; 15-year plan formulated (shortened due to next-gen sequencing)

1990 15-year NIH-DOE project formally begins; $3 billion in funding pledged

1991 Genome Database established

1992 Low-resolution linkage map of entire human genome published

1998 New 5-year plan announced for project completion by 2003]

1999 First human chromosome (#22) completely sequenced; Target date for draft sequence of entire human genome revised from 2001 to 2000

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Sanger sequencing

Dideoxynucleotides halt DNA polymerization at each base, generating sequences of various lengths that encompass the entire original sequence. Terminated fragments are electrophoresed and the original sequence can be deduced.

very slow

gives output in a graph where each color corresponds to a code

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Next generation sequencing

entire genomes sequenced using multiple parallel reactions to analyze short segments of DNA and compare the results to known sequences.

much faster

requires use of computers & bioinformatics

  • gives output in a "cluster array" no human can interpret

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massively parallel sequencing

uses laser excitation to get the identity of bases in clusters (3 days to run)

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progression from molecular to genomic medicine

from one gene/one disease to all genes/all diseases (many patients with undiagnosed disorders)

  • molecular = sanger

  • genomic = next-gen

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clinical applications of genome-level (next-gen) sequencing

• Diagnosis of rare or novel genetic disorders • Identification of "drug-able" mutation targets in malignant tumors • Noninvasive prenatal testing for aneuploidies and other genetic disorders (draw mother's blood) • Couple carrier screening for rare recessive disorders? • Prenatal testing? • Newborn screening? • Predictive risk assessment in healthy individuals?

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Whole exome sequencing

sequencing only the coding regions (exons) of DNA

  • first clinical grade genome sequencing

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variants of uncertain significance (VUS)

how do we interpret benign genetic variance?

a genetic sequence change whose association with disease risk is currently unknown

  • range from benign to pathogenic

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how are VUS's characterized?

• presumptive effect based on genetic code (ex. change in amino acid) • PolyPhen and other software tools • tolerance of missense and LoF variants • evolutionary conservation (usually based on Caucasians; but, extensive animal genome) • testing of other family members • population studies, databases • molecular or functional analysis

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discrepancies of VUS interpretation

37% discrepancy of VUS classification between labs

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Christian Millare (legal case) / Williams v. Quest Diagnostics

severe congenital epilepsy --> genetic test ordered --> clinicians assumed that VUS meant nothing --> variant was later re-classified as pathogenic --> mother sued, saying it should've been called likely pathogenic

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Case study of VUS pathogenic --> benign

BRCA VUS downgraded to benign after many women had major surgery

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classes of novel sequence variants identified by whole genome/exome sequencing

• Missense variants of uncertain significance in known gene (VUS)

• Variants and deleterious mutations in unknown gene(s) (GUS) -- often red herring, sometimes new disease gene

• Deleterious mutations in unintended target (e.g., BRCA mutations in a child) -- off-target finding: disclose or no?

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NIH task force on genetic testing conclusion

don't do predictive testing of children for adult-onset diseases (unless intervention can be done now)

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incidental findings

Undiagnosed conditions or other major findings that are discovered unintentionally during evaluation for another medical condition via genetic testing

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whole genome sequencing

sequencing ALL of the DNA (exons, introns, non-coding DNA)

  • now, patients have to say how much of their data they want to receive

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informed consent for whole genome sequencing (patient choices)

• Receive all information (CD, DVD?) • Receive relevant/targeted information • Receive medically actionable information for patient's age • Receive medically actionable information for future (ex. BRCA) • Receive medically actionable information for relatives (ex. inheriting BRCA from at-risk mother)

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recommendations of the ACMG incidental findings committee

• Mutations in a select list of high-penetrance, potentially lethal but actionable conditions must be sought and reported

• Same rules apply to sequencing of healthy parents in a "trio" or benign companion tissue when doing tumor sequencing

• Same rules apply whether the patient is an adult or a child

• These results are given to the ordering clinician who has responsibility for deciding which, when and how to convey to the patient

• The patient cannot opt out from receiving these incidental findings (controversial)

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reportable incidental findings by disease family

• Familial cancers (BRCA, Lynch, etc.) • Cardiomyopathies, hereditary arrhythmias • Connective tissue disorders (EDS, Marfan) • Neuroectodermal disorders (TS, NF2) • Miscellaneous (FH, MH)

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what field informed the controversial choice of the ACMG committee?

radiology precedent

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clinical genomics board

• Molecular/genomic laboratory directors • Laboratory technical staff • Genomic bioinformaticists • Clinical geneticists, paediatricians • Genetic counselors • Residents/fellows/student • Ordering clinicians (clinician who saw the patient)

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how does UCLA health resolve informed consent for genetic screening?

checkbox whether to get incidental findings or no

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clinical whole-exome sequencing at UCLA lessons

• Communication with the ordering clinician is essential • Diagnostic yield is better than expected (get an answer about 50% of the time) • Trio cases are much easier and more fruitful than singletons • Insurance coverage remains problematic

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ethical and legal dilemmas of clinical genomic sequencing

• Revelation of off-target/incidental findings • Many revealed disorders will have no prevention or treatment • Data storage and privacy • Costs of testing, genetic counseling and follow-up • Revelation of nonpaternity, consanguinity, incest • Risk of genetic discrimination

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UCLA ethical case study: pediatric cancer patient

trio sequencing for 18 month old cancer patient revealed nonpaternity of father

  • positive for p53 gene mutation (tumor suppressor)

  • concern about adverse outcome of finding in family

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reasons for not withholding the nonpaternity finding in the pediatric cancer case

• It affects our ability to determine whether or not thep53 mutation is de novo, and from that to counsel about recurrence risk

• >80% of p53 (LFS) mutations are inherited, not de novo; we need to test both parents in order to determine true recurrence risk

• Thus, it is likely the real father (and his siblings and other children) are at risk for malignant tumors

• The family might do Ancestry.com testing in the future and learn this information anyway, then sue us for not disclosing it

• We would not be telling "the truth, the whole truth, and nothing but the truth"

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ways pediatric cancer case could be resolved

• Inform the couple of the false paternity (and potentially cause breakup of the marriage)?

• Encourage the mother to contact the child's biological father to warn that he and his relatives may be at risk of LFS?

• Just say that the p53 mutation did not come from the mother, and don't say anything about the father?

• Simply identify the parental samples in the trio as "male adult comparator" and "female adult comparator"?

• Say that there was a technical problem in analyzing the father's DNA?

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Genetic Information Nondiscrimination Act (GINA) 2009

genetic information cannot be used by employers to determine job decisions or health insurers to determine coverage

  • Protects asymptomatic individuals only • Caveats: doesn't apply to life insurance, disability insurance, long term care insurance, US military, or employers with fewer than 15 employees

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WGS (vs. WES)

Advantages

  • Better overall coverage

  • Avoids the exon-capture step

  • Better detection of large deletions

  • Chance to identify mutations outside coding regions

Disadvantages

  • 10X more expensive

  • Difficult/impossible to interpret intergenic and non-coding regions

  • No insurance coverage

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advanced technology vs. expert clinical evaluation

clinical review uncovered the diagnosis at about the same rate as additional WGS

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should WGS be applied to...

Newborn screening? Prenatal diagnosis? Prenatal carrier screening? Population screening?

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issues with using genetic tests for wellness screenings

• Gene panel vs. whole exome vs. whole genome

  • the ACMG Incidental Findings panel?

  • a larger panel?

  • the whole genome?

• No a priori phenotype to guide interpretation

• Access only for the wealthy?

• Potential for insurance and employment discrimination

(ACMG already said it's not for general use)

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"next-next" generation sequencing

we are on the cusp of 3rd gen sequencing (even faster/cheaper)

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penetrance

the percent of individuals with a given genotype, who display the phenotype associated with that genotype"

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how to read a genetic report

BRCA2 Gene: c.7964A>G (p.Gln2655Arg)

mutation happens in the coding strand (a change in nucleotides)

  • in the protein product at the given position, the amino acid is changed

  • missense mutation

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how to determine effect of variants

● Population databases ● Family histories ● Conservation of amino acids ● Modeling of impact on protein ● Disease databases ● Other sci. lit.

(tolerance: some changes are know to be tolerated, ie. no effects)

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rules to protect healthcare and scientific workers from harm

HIPAA o Protects privacy and security of patient health information o Limits health insurers' restrictions based on pre-existing conditions o Ensures continuity of health insurance in case of job change or loss

  • doesn't cover DTC tests

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The Common Rule

● Federal guidelines for human participation in research (first published in 1991) ● Establishes ethics policies for Institutional Review Boards (IRB) and Informed Consent procedures ● Uses the Belmont Report as a framework ● Most recently revised in 2018

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informed consent

an instrument of mutual communication between doctor and patient with an expression of authorization/permission/choice by the latter for the doctor to act in a particular way

o Preserves patient autonomy: the ability to make one's own decisions

o Protects against paternalism: when an individual or group limits someone's autonomy forwhat is supposed to be "their own good"

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Medical Paternalism

when a doctor or healthcare provider makes choices for the patient without their consent or in opposition to it

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Informed Consent and the Common Rule key features

  1. Must disclose adequate information to make an informed decision

  2. Must ensure comprehension of the information

  3. Must ensure voluntary nature of decision (no coercion); murky with incentivizing

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All of Us

NIH program started under Obama to use as diverse genetic controls/references (and gene-environment interactions)

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expansion of genomics research

23andme also does expansive population-based genetic research

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idealized model of biobanking process

obtain informed consent --> collect biospecimens --> samples are processed & stored in biobank --> samples are made available for research

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ethical questions regarding biobank and genetic database use in research

  1. Return of Results → do researchers have an obligation to return clinically-significant results when participants give samples for research? Is this even possible?

  2. Informed Consent → are current methods adequate? Do they cover potential psychological risks in addition to physical risks of participation?

  3. Multiple Sample Usage and Right to Withdraw → what kind of informed consent is required? Is it possible to recontact participants to consent to re-use samples?

  4. Participant and Family Privacy & Protection from Genetic Discrimination → are procedures to anonymize biobanks and databases sufficient? What are the stakes if identification occurs?

  5. Commercialization of Data → is it okay for researchers to profit from research that relies on human samples?

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Havasupai Tribe vs Arizona State University

2004 Lawsuits: ASU researcher permitted to take blood samples for diabetes research

o Samples also used to study genetics of migration, inbreeding, and schizophrenia

o Settlement (2010): $700K, return blood samples (sacred), barred from reservation

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Moore v. UC Regents

Moore: patient at UCLA Medical Center starting in 1976: o hairy cell leukemia; spleen removal, collection of blood and tissue samples for multiple subsequent years

o Doctor and UCLA had patented a cell line derived from his white blood cells, which they profited from

o Moore sued based on property rights, saying he didn't give consent for the research

o Court's decision (1990): Cells had been altered in lab and were thus distinct from Moore's body; Concern about restriction of research and giving patients rights to sell body parts (as part of ownership); Acknowledgement of issues with consent

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Henrietta Lacks

Her cervical cancer cells (immortal --> kept replicating) were harvested without her or her next of kin's permission or consent. Millions of her cells have been used in research today with no financial compensation to her family.

issue: family shares her genetic information --> move to involve the family more

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how have contemporary guidelines aimed to address concerns of consent?

No consent necessary for future research if specimen is "de-identified"

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broad consent issue

The 2018 Common Rule allows for broad consent

  • give a bunch of ways it could be used and say yes or no

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where does biological ownership lie?

✔ Individuals don't have ownership rights over biological samples given for research purposes (Moore vs. UC Regents)

✔ Ownership lies with researchers and/or institutions (Washington v. Catalona)

✔ Informed Consent is Required

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patents

rights to a process or product that areexclusive to the patent holder

➔ Patent holder can prevent others from making the product ➔ Patent holder can charge others a licensing fee to use their design/make their product ➔ Rights are usually for 20 years (afterwards, generic versions can be made)

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Patent Criteria

It is new/novel

It is useful

It is non-obvious

It is not a product of nature

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why give patents?

Patents should benefit the inventor: o Rewards hard work and investment with rights to the invention o Can issue licenses to others (freely or for $$)

Patents should benefit society: o Fosters innovation, progress o Not forever - 20 years and then available to all

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history of US gene patenting

>6000 genes patented (20-25% of genes)

Celera submitted ~6500 gene patent applications during the race to sequence the human genome

Myriad Genetics patented BRCA1 & BRCA2 (1994-1995) - rights to all versions of the genes, diagnostic testing, and research

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Association for Molecular Pathology vs. Myriad Genetics

found that naturally-occurring DNA sequence can not be patented

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causes of tension between genomic testing, research, and the clinic

Autonomy & other ethicalconsiderations

Large-scale Genomics Research Requires Large Volumes of Data

Difficulty Re-establishing Consent to Use Stored Samples

Data Sharing for Research Advancement

Genetic Privacy and Genetic Discrimination

Unregulated genetic tests

Ownership, Profit, and Patents

Unintended and Uninterpretable Outcomes

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genetic counseling

Genetic counseling is the process of helping people understand and adapt to the medical, psychological and familial implications of genetic contributions to disease.

ex. • How inherited diseases and conditions might affect them or their families • How family and medical histories may impact the chance of disease occurrence or recurrence • Which genetic tests may or may not be right for them, and what those tests may or may not tell • How to make the most informed choices about healthcare conditions

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pre-test genetic counseling

Genetics team led by a genetic counselor o Sort out the motivations & assess the risks: ✔ Listen to and assess motivations ✔ Assess the situation (risk, family pedigree, medical history) ✔ Educate about inheritance ✔ Explain the procedure & consent ✔ Discuss the possible results

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post-test genetic counseling

Promote informed choices and adaptation to the risk or condition ✔ Review and explain the results ✔ Counseling on options ✔ Follow-up

Non-directiveness as a key goal (don't tell them what to do)

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some unintended outcomes of genetic testing that a genetic counselor could address

• Interpreting results: variants of uncertain significance (VUS), penetrance (high, low, moderate, etc.)

• Explaining potential unintended results (before and/or after test):

  • Misattributed parentage (trio testing)

  • Very close parentage (consistent with incest) (trio testing)

  • Information that impacts family members

  • Incidental findings (whole exome or genome sequencing)

• Discussing the possibility of diagnosis without therapy (before test)

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direct to consumer genetics

DNA tests consumers can buy; can even get WGS now

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23andme

most popular DTC, been around the longest --> microarray-based method

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microarray (chip-based) analysis

Advantages: o Can test many loci on plate o Can detect heterozygosity or homozygosity o Don't need to sequence entire gene (cheaper, faster)

Disadvantages: ● Only tests for specific sets of known variants (this could also be an advantage)

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benefits of DTC testing

• Find family members (ancestry testing) • Access for those without access to clinical genetics testing or with distrust of medical establishment • Ability to "take proactive steps in disease management or prevention" • Speed of results • Potential to keep results out of medical record - prevent possibility for genetic discrimination? • Potential to be more affordable than via clinic (e.g., insurance denials)

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concerns of DTC testing

protections of the medical field don't necessarily apply

● Accuracy of results/assay- do all labs interpret the results the same way and correctly? (40% false positives)

● Reliability & strength of association between genotype and phenotype - is there a strong, reliable link between the genotype and phenotype? How much does it elevate risk?

  • negative findings don't always mean no risk

● Importance of the results - do they merit follow-up with a healthcare provider? Are they clinically meaningful?

● Unanticipated Outcomes - will people be blindsided by health or ancestry results?; non-actionable

● Genetic privacy and potential for genetic discrimination - big datasets for research; law enforcement

● Commodification - big datasets for research

● Inconsistent regulation - "not for diagnostic purposes"

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23andme timeline

2010: FDA notifies 23andMe and other DTC companies that it considers their products "medical devices" and federal approval is required to market them.

o November 2013: FDA sends "cease and desist" orders to several companies -- 23andMe only authorized to offer ancestry tests and raw data

in 2015, 23andme starts adding back health info

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issues with ancestry testing

uses how similar your genetics are to other people in the world

  • can hurt families/communities

  • huge disparities in samples (some regions overrepresented)

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are public genetic databases really deidentified?

2013: Researcher took genetic data from a public database and combined with info on state and age → successfully identified 5 individuals and then their families based on Y chromosome data → 50 people total

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