next generation sequencing in diagnosismedical applications biomedical research basic research...

ECS, UM, Braga Postgrad. Course on NGS 12/5/2013

Jorge Sequeiros 1

Next Generation Sequencing in Diagnosis:

do we need a next-generation ethics?

Jorge Sequeiros

ICBAS e IBMC, Univ. Porto

IV Jornada sobre

Enfermedades Genéticas y Discapicidad

Barcelona, 3 octubre 2013


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Centre for Predictive and Preventive Geneticswww.cgpp.eu

www.testegenetico.com

Genetic testing Genetic counselling

Education and training


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SUMMARY

1. Genomes in the news!

2. NGS in research

3. NGS in health care

4. Genetic testing and NGS

5. Ethical issues and dilemmas of NGS


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1. Genomes in the news


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Craig Venter

James Watson

Ozzy Ousbourne

Desmond Tutu


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"I was curious.

Given the swimming

pools of booze I've

guzzled over the years

– not to mention all of

the cocaine, morphine,

sleeping pills, cough

syrup, LSD,

Rohypnol…you name it

– there's really no

plausible medical

reason why I should

still be alive.

Maybe my DNA could

say why.”

Ozzy Osbourne


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The SOLiD™ System

Ion Torrent

Solexa Technology

454 GenomeSequencer

FLX System

NGS


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The SOLiD™ System Ion Torrent

HiSeq

NGS

Ion Proton

MiSeq

GS Junior System

454 Genome SequencerFLX System


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NGS


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Life

Technologies

Ion Torrent

Roche 454Illumina

Solexa

LT Applied

Biosystems

SOLiD

Sequencing

Chemistry

Ion semiconductor

sequencingPyrosequencing

Polymerase-based

sequence-by-

synthesis

Ligation-based

sequencing

Amplification

approachEmulsion PCR Emulsion PCR Bridge amplification Emulsion PCR

Mb per run 100 100 600,000 170,000

Time per run 1.5 hours 7 hours 9 days 9 days

Read length 200 bp 400 bp 2x100 bp 35x75 bp

Cost per run $ 350 USD $ 8,438 USD $ 20,000 USD $ 4,000 USD

Cost per Mb $ 5.00 USD $ 84.39 USD $ 0.03 USD $ 0.04 USD

Cost per

instrument$ 50,000 USD $ 500,000 USD $ 600,000 USD $ 595,000 USD

NGS - Next generation sequencing


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NGS IS HERE TO STAY AND IS ALREADY

ENTERING THE CLINICAL PRACTICE

Conclusion 1.


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2. NGS in research


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HeLa cells


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HeLa cells• Cervix cancer, Jan. 1951• Cells taken without her knowledge• 1st cell line immortalized in vitro• The most used ever• Initials of “Helen Lane” and “Helen Larson”• Name of Henrietta Lacks disclosed in 1971


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� More cells in circulation than during life� Many important discoveries:

• Anti-polio vaccine (Jonas Salk, 50’s)• Effects of zero gravity in space• Correct human chromosome number• >60,000 scientific articles• Contribution for ENCODE• Pharmacological assays

� Multimilionary businesses � Some protests from her family


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The director of the US National Institutes of Health

(NIH), Francis Collins met Lacks family members to

discuss what should be done with genome data

from their matriarch’s cell line.

On 8 August, Kate Hudson and F. Collins announced

that the family has endorsed case-by-case release of

the information, subject to approval by a committee

that will include family members.


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BLURRING OF THE DIFFERENCE

BETWEEN

RESEARCH AND HEALTH CARE

Conclusion 2.


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3. NGS in health care


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GWAs• GWA studies identify genetic variants with small effects• Need to validate findings and establish clinical relevance

• Insufficient evidence that genomic profiling is useful in measuring individual risks for common diseases, or developing personalised diet or lifestyle recommendations for disease prevention (Janssens et al., Am J Hum Genet 2008; 82: 593-599)

• Only 2 out of 5 genes in ‘osteogenomic profiles’ were significantly associated with some disease, but none with bone disease (Janssens et al., Am J Hum Genet 2008; 82: 593-599)

• This genetic astrology regarded as producing no more than entertaining horoscopes;potential for harm and need to ensure these tests are evaluated and used appropriately (Melzer et al., BMJ 2008; 336: 590-593)


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DTC

genetic testing

• direct-to-consumer (DTC) testing: • over the counter

• over the web

• marketing strategies (bypassing health professionals)

• override medical indications

• suppress prior information and genetic counselling

• publicity creates artificial needs (unsubstantiated claims)

• change patients into consumers


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Genetic horoscopes

Cassandra’s complex

“… a prophet of lies, a babbler from door to door?"

[Aeschylus, Agamemnon 1194]


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DTC testing(directly to the consumer)

• DTC often provides health-related information on genetic susceptibilities and recommendations for lifestyle changes

• Concerns include:(1) scientific and clinical validity of tests and their

clinical utility (transparency of claims)

(2) no medical indication (family history)

(3) quality of the testing services

(4) lack of appropriate genetic counselling (pre-test and interpretation of results)

(5) appropriate regulatory and policy response


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• at the reach of a sophisticated clairvoyant

• limited value (current state of knowledge / methods)

• may change in the future

“Genetic astrology”


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HYPE ON ANALYTICALLY AND CLINICALLY UNVALIDATED GENETIC VARIANTS AND DTC GT

Conclusion 3.


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25%

12.5%

25%

25%12.5%12.5%

50%50%

John

HD

? ?

50%

50% 50%

25%25% 25%

Sana

C

Mara


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25%

12.5%

25%

25%12.5%12.5%

50%50%

João

HD

? ?

50%

50% 50%

25%25% 25%

Sana

C

Luna

50% 50%50%

25% 25%

12,5% 12,5%12,5%

MaraVera


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Genetic information:

is it really different?

1. “Genetic exceptionalism” - all genetic information is special and needs exceptional means of protection

2. “Genetic inclusivism” - genetic information is not different from other medical information and should not be treated differently


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Table 2. Context definitions of human and medical applications of genetic testing, and of clinical genetics testing

NON-HUMAN � microbiology � plant � animal

HUMAN APPLICATIONS NON-MEDICAL

� forensic testing (civil, criminal) � ancestry testing

MEDICAL APPLICATIONS BIOMEDICAL RESEARCH � basic research

� clinical research

PUBLIC HEALTH GENETICS � population genetic screening

CLINICAL GENETICS TESTING Heritable Patient diagnosis: � diagnostic testing (confirmation, exclusion)

Genetic counselling: � presymptomatic testing (late-onset Mendelian diseases, non cancer)

� familial cancer testing (Mendelian cancers, with high or reduced penetrance)

� susceptibility testing (complex diseases/traits)

� prenatal diagnosis (Mendelian diseases, congenital malformations)

� pre-implantation genetic diagnosis (Mendelian diseases)

OTHER MEDICAL APPLICATIONS ART: � pre-implantation genetic screening (aneuploidy screening)

Drug treatment: � pharmacogenetics testing (adverse drug reactions, drug efficacy)

Somatic Cancer prognosis: � testing for somatic mutations in cancer

Modified from:Sequeiros J (2010): Regulating Genetic Testing: the relevance of appropriate definitions. Ch.3. In: Kristoffersson U, Schmidtke J, Cassiman JJ (Eds): Quality Issues in Clinical Genetic Services, Springer (ISBN: 978-90-481-3918-7).


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Table 3. Context definitions of “genetics laboratory-based testing” and “genetic information”

GENETIC INFORMATION

Genetics laboratory-based testing:

Cytogenetics tests (chromosomal-based) – identification of numerical or structural anomalies of individual chromosomes or chromosomal complement

Molecular genetics tests (DNA or RNA-based) – identification at the nucleic acid level of sequence alterations in the DNA molecule and its functional significance, and epigenetic (non-covalent) changes

Biochemical genetics tests (proteins, metabolites) – identification at the protein level of sequence alterations in the DNA molecule and its functional significance

Other sources of genetic information:

Family history

Personal history and physical examination

Other laboratory exams (haematological, biochemical, physiological, image, functional) exams

Sequeiros J (2010): Regulating Genetic Testing: the relevance of appropriate definitions. Ch.3. In: Kristoffersson U, Schmidtke J, Cassiman JJ (Eds): Quality Issues in Clinical Genetic Services, Springer (ISBN: 978-90-481-3918-7).


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(1) Analysis of genetic material

(2) Genetic information

Two consensus definitions?


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Ethical concerns

• counselling of persons at-risk for Huntington disease since the mid 80s

• fears of genetic determinism surrounding the human genome project and the ELSIdiscussion

• genetic antidiscrimination laws enacted in the USA, in the early 90s


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Genetic information

• Personal

• Familial

• Powerful

• Permanent

• Highly predictive

• Potentially discriminatory

unlike

a blood count

or glucose

unlike

cholesterol,

or “pedigree”

In the case of high penetrance

monogenic diseases

HGP


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Compelling to exorcize

genetic exceptionalism

• in these modern times of sophisticated, high-throughput technologies, many policy documents began by clearly dismissing it

• many came to embrace “genetic inclusivism”• some adding their rationale was that all medical

information needs much better protection


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25%

12.5%

25%

25%12.5%12.5%

50%50%

João

PKD

50%

50% 50%

25%25% 25%

Mara


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Specificity of genetic information

• The diagnosis of a genetic disease may change the risk for relatives (who are not examined or tested and may never want to be!)

• The major aim may be “non-medical”(diagnosis, treatment or prevention of disease), but

life decisions (e.g., reproductive choices, education, profession, change life styles)


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PKDpolycystic kidney disease

• fluid-filled cysts• blood in the urine• edoema• renal failure• dyalysis• transplant


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Not all human genetic information

is medical information

• paternity testing• twin-studies• civil identification• criminal investigation

• results from human genetics research

• predictive genetic information for severe diseases with no cure, effective treatment or prevention

forensic applications


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BLURRING OF DISTINCTION BETWEEN

GENETIC DATABASES AND BIOBANKS

Conclusion 4.


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END OF PERSONAL PRIVACY AND

CONFIDENTIALITY OF

“ANONYMOUS” GENETIC DATA

Conclusion 5.


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4. Types of genetic testing


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Not all health-related genetic information

is equally sensitive

• diagnostic genetic information is different from predictive information

• not all predictive information is equally sensitive (e.g. pharmacogenetics)

• presymptomatic information for Huntington disease is different from predisposition to hypertension

On the other hand,

• some non-genetic medical tests (even a physical exam) may provide sensitive genetic information


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Genetic Information:

is it special, is it really different?

3. some genetic informationis at least special and needs reinforced protection, as other sensitive medical information does,mainly in healthypersons


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Protection• Privacy• Confidenciality• Informed Consent

• Guidelines for testing and counselling• Quality assurance

• Data-protection• Antidiscrimination legislation

(insurance, employement, adoption)

• Clinical validity and utility of MGTs

• Verification• Sanctions


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Genetic testing

Components:

� Information, consent, preparation

� Laboratory assay

� Interpretation, support


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• Genetic counselling and psychosocial evaluation before and support after testing!

Presymptomatic testing

for late-onset incurable diseases

Candidates for

a presymptomatic test

register for counselling,

not for a mere blood test!


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PST Protocol

Written

Report

Neurological

Evaluation


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Children and adolescents

• Should not be tested for late-onset,

incurable diseases!

• Loss of autonomy

• Discrimination


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Susceptibility testing

• Common diseases - complex inheritance, i.e., mostly multifactorial, but also with a monogenic fraction (≈5%)

� many genes involved

� many non-genetic factors

• but, each with a very limited impact …!


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• professional enthusiasm (clinicians, scientists)

• urgent application to human health

• common diseases � commercial pressure(labs, biotechnology, insurance)



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Limitations:

• only small change in relative risk

• population diversity•

• analytical and clinical validity

• doubtful benefits for populationscreening


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Types of genetics tests

In the newborn, child, adult:

• Diagnostic testing• Presymptomatic testing (AD)

• Carrier testing (AR, XR)

• Susceptibility testing (multifactorial)

• Pharmacogenetics testing

In the foetus or embryo:

• Prenatal diagnosis• Pre-implantation diagnosis

In the population or populational group risk:

• Genetic screening

Predictivetesting


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BLURRING OF THE DIFFERENCE

BETWEEN

PREDICTIVE AND DIAGNOSTIC TESTING

Conclusion 6.


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5. Ethical issues and dilemmas

with NGS


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Applications of NGS

1. Whole-genome sequencing

2. Whole-exome sequencing

vs.

3. Panels of genes (heterogenous

disorders)


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1. NGS – ethical issues about

testing for health care purposes

(1) Sequence all genome/exome (if cheaper than

testing half a dozen of genes or even a single gene)?

• answer only the clinical question?

• what to do with all that information?

• keep rest of info. for future, if further requests?

• destroy and repeat the WGS (once it is cheap)?

(2) Do only gene panels for known diseases

(filtering the required information)?


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(1) What should/can be reported and what must not to requesting physicians?

• only disease-causing variants?

• variants of unknown clinical significance?

• normal variants?

(2) What to communicate to a patient/healthy person? • everything found?

• incl. susceptibility variants of low predictive value?

• incl. variants with no or still undetermined clinical validity and utility?

• all pathogenic variants of current or future clinical relevance?

• only mutations for diseases with treatment or preventive measures?

• information about children?

(3) What process of informed consent (opt-in, opt-out)?

2. NGS – ethical issues about

reporting results in health care


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“Incidental findings”

• Once you perform a WGS/WES, you will

necessarily have “incidental” findings

• We should talk instead of unsolicited or

unrequested findings


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CLINICAL APPLICATIONS OF NGS STILL A

MATTER OF HOT ETHICAL DEBATE

Conclusion 7.


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ABSTRACT

• In clinical exome/genome sequencing, there is potential

for the recognition and reporting of incidental or

secondary findings unrelated to the indication for

ordering the sequencing but of medical value for

patient care

• We recommend that laboratories performing clinical

sequencing seek and report mutations of the specified

classes or types in the genes listed

• ... irrespective of age, but excluding fetal samples.

• We recognize that there are insufficient data on clinical

utility to fully support these recommendations


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Primary Finding

This term is used to describe

pathogenic alterations in a gene or

genes that are relevant to the

diagnostic indication for which the

sequencing was ordered (e.g., a

mutation in MECP2 in a girl with loss of

developmental milestones).


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Incidental Finding

This term has been used in a variety of clinical andresearch contexts to indicate unexpected positive findings. Other terms have been used to describethese findings, particularly when they are soughtafter (rather than being unexpectedly discovered). These terms include “serendipitous and iatrogenic” findings, “non-incidental secondary findings”“unanticipated findings”, and “off-target results”. We use “incidental findings” in this paper to indicate the results of a deliberate search for pathogenic or likely pathogenic alterations in genes that are not apparently relevant to a diagnostic indication for which the sequencing testwas ordered.


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RECOMMENDATIONS

1. Constitutional mutations found in the genes on the minimum

list (see Table) should be reported by the laboratory, regardless

of the indication for which the clinical sequencing was ordered.

a. Additional genes may be analyzed for incidental (secondary)

variants, as deemed appropriate by the laboratory.

b. Incidental (secondary) variants should be reported

regardless of the age of the patient.

c. Incidental (secondary) variants should be reported for any

clinical sequencing conducted on a constitutional (but not

tumor) tissue. This includes the normal sample of a tumor-

normal sequenced dyad and unaffected members of a family

trio.


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RECOMMENDATIONS

2. The Working Group recommends that laboratories seek and

report only the types of variants within these genes that we

have delineated (see Table).

a. For most genes, only variants that have been previously

reported and are a recognized cause of the disorder or variants

that are previously unreported but are of the type which is

expected to cause the disorder, as defined by prior ACMG

guidelines, should be reported.

b. For some genes, predicted loss of function variants are not

relevant (e.g., COL3A1 and most hypertrophic cardiomyopathy

genes).

c. For some genes (e.g., APOB), laboratories should only report

variants for certain conditions.


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RECOMMENDATIONS3. It is the responsibility of the ordering clinician/team to

provide comprehensive pre- and posttest counseling to

the patient.

a. Clinicians should be familiar with the basic attributes

and limitations of clinical sequencing.

b. Clinicians should alert patients to the possibility that

clinical sequencing may generate incidental findings that

could require further evaluation.

c. Given the complexity of genomic information, the

clinical geneticist should be consulted at the appropriate

time that may include ordering, interpreting, and

communicating genomic testing.


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• WGA requires a justification in terms of necessity (theneed to solve a clinical problem) and proportionality(the balance of benefits and drawbacks for the patient).

• In the clinical setting, preferable to use a targetedapproach first, to avoid unsolicited findings or findingsthat cannot be interpreted (limit the analysis to specificsets of genes).

• Known genetic variants with limited or no clinicalutility should be filtered out (if possible, neitheranalyzed nor reported).

Recommendations


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Additional Protocol concerning

Genetic Testing for Health Purposes

• genetic tests for health purposes should

only be carried out once their clinical

utility has been proven, and under

individualised medical supervision

7 May 2008


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• Protocol should be in place for guidance on thereporting of unsolicited findings

• Guidelines for informed consent regardingdiagnostic testing need be developed

• Clinicians should be aware and safeguard thepatient’s position in the potential crossoverwith research

Recommendations


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• In case of testing minors, guidelines are needed as to what unsolicited information should be disclosed, to balance autonomy and interests of the child andparental rights and needs for information that may bein the interest of the family

• International collaboration to build sustainabledatabases on genotypic and phenotypic information ofvariants

• Genetic education of health-care professionals

• Inform the public and raise public awareness, enhancegenetic literacy in patients and the lay public will help to involve wider society in this debate

Recommendations


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Amb l'entrada de

força castellers

nous i d'una canalla

ben preparada ,…


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A “next generation” bioethics?


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► effective, timely and responsible

translation

for the befit of society!


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[email protected]


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“Henrietta Lacks”

Teresa Ricou

next generation sequencing in diagnosismedical applications biomedical research basic research...

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