4/14/2018

1

Role of next generation

sequencing in clinical care

Anne Slavotinek

Division of Genetics, Department of

Pediatrics, UCSF

Structure

1. Next generation technologies

2. Consent and secondary findings

3. Genetics of diabetes/clinical examples

4/14/2018

2

Sanger sequencing

National Human Genome Research Institute's Talking Glossary

Sanger sequencing

4/14/2018

3

Exome sequencing

Illumina

Exon Coverage

- exons are variably covered

- ‘fill in’ can improve coverage

Sarah Garcia, Personalis

4/14/2018

4

Whole exome sequencing (WES)What is an exome?

-protein encoding regions of genes

-1-2% of genome, but 85% mutations

-90-95% of exome ‘covered’

Uses of whole exome sequencing:

-non-specific phenotypes e.g. intellectual disability

-atypical presentations

-rare, novel phenotypes

-phenotypes difficult to confirm with clinical testing

-diseases where testing is expensive

-typically sent as a trio with both biological parents

Date of download: 9/6/2014Copyright © 2014 American Medical

Association. All rights reserved.

From: Korf and Rehm, New Approaches to Molecular Diagnosis

Figure Legend:

4/14/2018

5

What to expect?

Type of Mutation Novel Non-Novel Total

Missense 303 (AA) /192 (C) 10,828/9,319 11,131/9,511

Nonsense 5/5 98/89 103/93

Synonymous 209/109 12,567/10,536 12,776/10,645

Splice 2/2 36/32 38/34

Total 520/307 23,529/19,976 24,049/20,283

Bamshad et al., 2010AA = African American

C = Caucasian

3 general types of results:

- positive

- negative

- variant of uncertain significance (VUS)

2 general categories of results:

- primary findings

- secondary findings

Exome Sequencing

Sequence variants need to be sorted to find relevant ones:

1) Deleteriousness of variant

-frameshift, nonsense > missense

-sequence conservation; protein domain

-SIFT, PolyPhen-2, Mutation Taster, CADD

2) Existing biological/functional information

-segregation with disease

-variant not seen in unaffected individuals

-expression pattern

-predicted function; pathway/gene interactions

-animal models

Jessica van Ziffle

4/14/2018

6

Diagnostic Yield/Discovery

De Ligt et al,. NEJM, 2012

-100 patients with ID

-‘trio’ approach

-total yield 16%

Srivastava, Ann Neurol, 2014

-78 patients with neurodevelopmental disabilities

-total yield 41%

-19 AD, 11 AR, 1 X-linked, 1 AD and AR

-changed management in all situations

-yield depends on testing indication

Whole exome sequencing

- exome is being considered earlier

- ‘exotyping’ - phenotyping, genotyping, reinterpretation of phenotypic

data (Pinto et al., 2016)

- digenic diseases and blended phenotypes

Whole genome sequencing

- analysis of deep intronic regions, non-coding RNA, regulatory regions

Glissen et al., 2014

- 50 trios with severe ID; array and exome negative

- WGS had yield of 42% (13 SNVs and 8 CNVs)

- no mutations in regulatory regions

4/14/2018

7

Panel Sequencing

- panel targets <150, not 24,000 genes

- all exons are sequenced

- insertions/deletions detected; algorithm vs exon array

- use a panel when clinical evaluation suggests a diagnosis

- will not result in secondary/incidental findings

- may still result in variants of unknown significance

There are THREE outcomes from arrays/panel testing:

- we may find the cause of the condition in you and your family

- we may NOT find the cause of the condition in you and your family

- we may get a result that we cannot interpret

There are FIVE outcomes from WES testing:

The previous three PLUS

-we may find another change in the DNA of medical significance but not

for the condition we tested you for

-we may find family relationships to not be what you thought they were

(misattributed parentage)

Exome Sequencing

Bob Nussbaum

4/14/2018

8

Exome sequencing

Advantages of WES:

-syndromes with high genetic heterogeneity

-syndromes that are incompletely characterized

-Neveling et al., 2013

-WES covers 81% of known pathogenic mutations on gene sets

-improved diagnostic yield compensates for reduced sensitivity

-“even if all genes that could have been ordered by physicians had

been tested, the larger number of genes captured by the exome

would still have led to a clearly superior diagnostic yield at a fraction

of the cost”

Elements of Consent

Bick and Dimmock, 2011

-basic genetics (genes, mutations)

-inheritance patterns

-penetrance and expressivity

-types of DNA variants (pathogenic, benign, VUS)

-incidental findings

-false positives, false negatives

-scientific discoveries that may result from test results

-interaction of genes, environment

-information privacy

-non-paternity

-Genetic Information Nondiscrimination Act of 2008

4/14/2018

9

Limits to informed consent - “Can you really give informed consent when you look so widely [at the

genome]? Is that manageable for patients?... (patient representative)

- “In any case I think that it's very naïve to think that a patient is more able to choose [which results to receive] when he knows more. There are limits to what patients can comprehend. Decision-making in principle does not get easier, the more elaborately a patient is informed….the quality is important and also a discussion... (ethicist)”

- greater detail can lead to less understanding

- overwhelming; information overload

- thousands variants identified; need biological parents for interpretation

- only variants relevant to presentation reported

- if parental samples included, only one report generated

Elements of ConsentLimitations

-not every gene tested / not 100% coverage

-not all kind of DNA variants

-not all genetic changes in exome

-current genetic knowledge not comprehensive

Familial relationships/consanguinity

Protection against genetic discrimination

Secondary findings

-different depending on labs

-possible information on parents

4/14/2018

10

Secondary Findings Green et al., Genet Med, 2013“Results not related to the indication for ordering the test, but that may, nonetheless, be of medical value or utility to the ordering physician or patient”

ACMG recommendations:

-actively look for known pathogenic or expected pathogenic variants

-59 genes, 24 conditions

-prevention and/or treatment available

-may be asymptomatic for a long time

-excludes conditions screened by NBS

-in proband AND for family members (if WES performed), irrespective of proband results

-“..failure to report a laboratory test result conveying the near certainty of an adverse yet potentially preventable medical outcome would be unethical.”

Interpretation of Secondary Findings Goal: Maximize positive predictive value

LOW SENSITIVITY

High false negative rate

Data as generated by WES, not same standard as primary variant finding

-recommendations to be reviewed periodically

-system to submit new genes

Amendola et al., 2015

European ancestry

–112 genes medically actionable genes: 2.0%

–ACMG 56: 0.7%

African ancestry

–112 genes medically actionable genes: 1.1%

–ACMG 56: 0.5%

4/14/2018

11

Secondary Findings - Considerations

Burke et al., 2013:-screening results, not diagnostic test results

-ascertainment bias, as mutations identified in those with disease

-unknown natural history

-phenotypic spectrum and penetrance not known

-lack of controlled studies regarding interventions

-prior probability of disease is low

-costs should not be generated if patients do not wish for results

Shahmirzadi et al., 2014

-187/200 (93.5%) chose to receive ≥1 categories of IF

-manageable if consented well, understand implications

-conditions/sequence variants need to be curated

Importance of reinterpretation of

negative results

4/14/2018

12

Genetics of Familial Hyperinsulinism (FHI)

- hypoglycemia (neonatal onset, mild to severe) (Glaser, 2013)

Autosomal recessive ABCC8 (45%) /KCNJ11 (FHI-KATP) (5%)

- associated with large for gestational age infants

- severe refractory hypoglycemia with poor response to medical management

- may require pancreatic resection

Autosomal dominant ABCC8/KCNJ11 (FHI-KATP)

- normal for gestational age

- present around one year of age (2 d - 30 y)

- responds to diet, diazoxide

- other genes include GLUD1 (5%), HNF4A (5%), GCK, HADH, UCP2 (<1%)

Genetics of Familial Hyperinsulinism (FHI)

FHI - good situation for gene panel testing

-diagnosis relatively straightforward

-genetically heterogeneous

-known genes explain much of genetic variation

-panels vary in number of genes included and gene coverage

-want to do both sequencing analysis and del/dup testing

-Panel 1: ABCC8, AKT2, AKT3, GCK, GLUD1, HADH, HK1, HNF1A, HNF4A,

INS, INSR, KCNJ11, PDX1, PGM1, SLC16A1, UCP2

-coverage: 96% at 20x

-Panel 2: 50 genes; includes some metabolic conditions

-panel includes non-coding variants; omits some exons

4/14/2018

13

Genetics of MODY

Anik et al., 2015

-1-2% of DM in Europe; 21–45/1,000,000 children and 100/1,000,000 adults

-monogenic (AD) inheritance

-early onset of DM (<25 y)

-lack of autoimmune process/insulin resistance with lack of obesity

-endogenous insulin secretion is preserved

-80% are misdiagnosed as T1D, T2D

->10 known genes

-GCK, HNF1A, HNF4A, and HNF1B genes commonest causes in UK

-represent 32%, 52%, 10%, and 6% of MODY respectively

-another good situation for gene panel testing

Genetics of MODYExamples of available panels:

Panel 1: GCK, HNF1A, HNF1B, HNF4A, PDX1

-lists sensitivity

Panel 2: 13 gene panel that includes non-coding variants

-ABCC8, BLK, GCK, HNF1A, HNF1B, HNF4A, INS, KCNJ11, KLF11,

NEUROD1, PAX4, PDX1, RFX6

-lists non-coding variants

Limitations:

-complex inversions/balanced translocations

-gene conversions

-mitochondrial DNA variants/repeat expansion disorders

-single exon deletions/duplications

4/14/2018

14

Genetics of DM – type I

Type 1 DM:

-40-50% due to inherited factors

-HLA amongst first loci; >50 loci from GWAS

-T1DGC – aggregated studies for research

-41 loci; some well known (HLA, INS, PTPN22, CTLA4, IL2RA)

-27/41 were novel

-verified loci have candidate genes; allelic heterogeneity

Robertson and Rich, 2018

Genetics of DM – type I- research predominantly in Europeans

- prevalence increasing for other ethnic groups

- genetic risk prediction

- may help with treatment

- slice/exome?

Robertson and Rich, 2018

4/14/2018

15

Personalized Medicine

Whole Exome Sequencing

-testing with whole exome sequencing (WES) is effective

-variant return has implications for other family members

-variant interpretation is likely to change

-secondary findings can be medically actionable

-returning results in children for adult onset disorders

-how should care be delivered?

-genetics versus non-genetic professionals

-ad hoc versus a dedicated clinic

UCSF Personalized Genomics Clinic

- provides an identity for genomic services

- not a gateway

- interpretive and research function

- supportive role for clinical testing

- consent process, results provision/interpretation

- ‘registry’ function for annual review

- database for re-analysis

4/14/2018

16

UCSF Personalized Genomics Clinic

- started in 2013; was 2x per month with 2 physicians

- now weekly with 4 physicians

- >250 patients

- providers: GeneDx, UCLA, Baylor, Ambry, Personalis, Fulgent

- now to UCSF

- referrals: numerous testing providers from different specialties

Able to:

- facilitate re-analysis, longitudinal data collection

- aid to clinicians in variant interpretation

- dedicated clinic enables consistent provider care

- trainee education

Trios increase yield: 12/143 (8%) proband only

11/143 (8%) duo

120/143 (84%) trio

Lee et al., 2014: trio approach increases diagnostic yield

-proband only 74/338 (22%)

-trio 127/410 (31%)

Age group tested can influence yield: 15/143 (10.5%) ≤ 1 yr of age

60/143 (42%) 1-5 y

32/143 (22.5%) 6-10 y

20/143 (14%) 11-17 y

16/143 (11%) 18+ y

Posey et al., 2015: diagnostic rate: 85/486 17.5% in adults

-lower than for Pediatrics; 7% had blended phenotypes

4/14/2018

17

Patient examples

1. WES can result in unexpected findings that improve care

-11 yo male referred to Genetics

-sensorineural hearing loss at 2 yo

-loss is severe to profound

-diagnosed with type I diabetes after presenting with

several days of emesis and abdominal pain

-cytopenias, including anemia, also noted at presentation

-negative Otoscope gene panel (108 gene panel)

-chromosomal microarray arr(1-22)x2, (X,Y)x1

-MRI of inner ear normal

Patient examples-two pathogenic variants in SLC19A2

-maternally inherited variant: c.484C>T, p.Arg162Ter

-classified as pathogenic

-paternally inherited variant: c.515G>A, p.Gly172Asp

-previously reported variant, classified as pathogenic

-biallelic pathogenic variants in SLC19A2 are associated

with Thiamine-responsive megaloblastic anemia syndrome

4/14/2018

18

Patient examples

2. Results have implications for other family members:

-9 yo male with autism

-frameshift variant in TREX1; heterozygous; paternally inherited

-father had h/o iritis and fevers

-AR TREX1: Aicardi-Goutieres disease

-AD TREX1: chillblains; SLE

-51 yo male with retinal dystrophy

-deleterious variant in PRRT2; heterozygous; unknown inheritance

-paroxysmal kinesogenic dyskinesia

-benign seizures in grandchild

-importance of experienced counselors/genetic interpretation

Summary

-next-generation technologies are in common use

-panel and exome are most frequent tests

-differ in consent process, secondary findings

-testing has implications for other family members

-testing can yield complicated, unanticipated results

4/14/2018

19

Thank you!

UCSF Genomics Medicine Initiative - Pediatrics

Co-directors:

Neil Risch Pui-yan Kwok

Clinicians: Wet lab:

Anne Slavotinek Jessica van Ziffle

Joseph Shieh Heather Pua

Marta Sabbadini

Bioinformaticians: Pathology/Fellow:

Mark Kvale Jude Abadie

Ugur Hodoglogil

Ethics: Library/Scholarly Communication:

Barbara Koenig Megan Laurance