Jauch Symposium

Pamela Trapane, MD

Clinical Associate Professor, PediatricsMedical Director, Division of Medical GeneticsProgram Director, Medical Genetics Residency ProgramStead Family Department of Pediatrics, Division of Medical GeneticsUniversity of Iowa Hospitals and Clinics

Genetic Testing in the Genomic Era: What You Need to Know

Disclosures• I have no financial conflicts of interest or

connections/investments with any of the companies mentioned in this presentation

Objectives• Describe the technology and impact of genomic-scale testing in

research, clinical evaluation, and direct to consumer genetic testing

• Compare and contrast the strengths and weaknesses of various clinically available genomic-scale tests

• Given a clinical presentation, identify the most appropriate genomic-scale test

Genetics is Everywhere!!!FDA

Genetics/Genomics Saves Lives!!

Reardon S. Nature. 2014. Vol: 514: 13-14.

Park A. Time Magazine. Dec. 8th 2014.

http://raregenomics.org/

XIAP mutation discovered – cord blood transplant saved his life

Genetics/Genomics Makes Money!!

http://fastercures.tumblr.com/ Battelle Memorial Institute for United for Medical Research

…Or Does It??

Joyner, M. The New York Times. Jan. 29th 2015Graber C. The New Yorker. Feb. 5th 2015.

What About Actual Science?

Dewey FE, et. al. JAMA. 2014. Vol. 311: 1035-45.

What is Genomic-Scale?

• Genetics vs. Genomics

- Genetics: Single gene effects

- Genomics: Functions/interactions of all genes in the genome (or larger subsets of genes)

Genetics Genomics

The Genomic Era Begins…

…in the 1950s

• Each chromosome has a characteristic banding pattern– 1 band: ~8000 kb DNA (8 Mb)– ~50 genes

Fluorescent In Situ Hybridization = FISH• Fluorescent probes bind only to areas with high degree of

sequence similarity

• Detects submicroscopic chromosomal aberrations –100-300 kb (vs karyotype 1 band = 8Mb; visible deletions = 5Mb)–Microdeletion syndromes

• 22q11.2 deletion syndrome• Williams syndrome (del 7q11.2)

Chromosomal Microarray (CMA)• Whole genome view + increased resolution

• Detects copy number variants (CNVs)– Gains (duplications) and losses (deletions) – 1-5 kb– Will detect variants below the detection of both karyotype

(5000 kb) and FISH (100-300 kb)• ie, if you can see the CNV on a karyotype, you will see it on the CMA

– Does NOT detect re-arrangements or location of extra chromosomal material

• Need a karyotype and/or FISH for this

• First line test for patients with – intellectual disability and/or autism

AND– nondescript physical features

DNA from the patient and from a control (reference) is labeled with different colors and hybridized to a slide containing defined DNA probes

The fluorescence color is used to evaluate regions of DNA gain or loss.

http://www.genedx.com/index.php?PHPSESSID=582707572b20a30c56a4b6f7ef54fb12

http://www.genedx.com/index.php?PHPSESSID=582707572b20a30c56a4b6f7ef54fb12

Gene Sequencing – Sanger Method• The gold standard of sequencing

Gene Sequencing – Next Generation

Next Generation Sequencing (NGS)• High error rates

– 0.5 – 2% depending on platform

• Solution: Multi-Fold Coverage– Every genomic target (sequence target) is “covered”

by more than one sequencing read– No standardized requirement for fold coverage– Common suggestion: ~20x (10-50x fold coverage)

Multigene Panels• Covers multiple genes (coding regions) associated with a

phenotype– High sensitivity with very deep and uniform coverage of all

genes on panel with Sanger fill-in

• Examples– Thoracic aortic aneurysms– Noonan syndrome and associated conditions (RASopathies)– Intellectual disability

Whole Exome Sequencing (WES)• Moderate sensitivity

- coverage is not as deep and uniform as multi-gene panel

• Uncertainty- High rate Variants of Unknown Significance (VUS)

• Incidental DNA variations related to other genetic disorders

• Covers “all” genes- ~2% of the genome

What Does WES Miss?

Does NOT detect several clinically significant types of

genetic lesions

AND

Misses more than ~25% of coding exons

(~18% disease genes)

Santani, et. al. Medical exome: Towards achieving complete coverage of disease related genes. ASHG Meeting 2014.Biesecker, et. al. Diagnostic Clinical Genome and Exome Sequencing. NEJM. 2014. 370: 2418-25.

Incidental Findings• Reporting of “secondary variants” remains

controversial

• The “AMCG-56” includes many cancer syndrome genes– BRCA1 and BRCA2 are on this list

• Wellderly (80-104 y/o and healthy) study preliminary results– 6% have a HGMD/ClinVar pathogenic variant

present– 5% have a HGMD/ClinVar likely pathogenic

variant presentAriniello, et. al. Frequency of “ACMG-56” Variants in Whole Genomes of Healthy Elderly. ASHG Meeting 2014.

• Limited medical availability- Uses NGS technology to find single nucleotide polymorphisms (SNPs)- SNPs may be a/w increased or decreased risk of multifactorial diseases- Some SNPs are actual mutations a/w known genetic disease

• BRCA1 and BRCA2 but only the 3 most common mutations• This is NOT complete gene sequencing

• Family history still required for optimal interpretation

• Used for ancestry determination, identification of medical risks in adoptees, social/genetic networking

• Presents challenges for primary care doctors as data and risk magnitudes change almost daily and there is limited availability of genetic counseling

Direct to Consumer Genetic Testing

What does this look like in clinic?

• Pam is a healthy 48 year old who presents for her annual exam. – She reports that her mother’s first cousin recently died

from breast cancer.

• Pam has read the NY Times article by Angelina Jolie. – She is adamant that she would like to have BRCA

testing.– She also would like you to review her 23andMe results

and make a personalized health care plan for her.

• You review Pam’s chart: – previous mammograms demonstrated fibrocystic

breast tissue

• What should you do?

• Take a family history!• Pam is an only child• Family is mostly English and Irish. No known Ashkenazi ancestry.• Her mom’s first cousin died at age 55 from breast cancer

– She did not have genetic testing

family member with breast cancer: relative risk

first-degree relative: ~2first-degree relative <45: 2-5first-degree relative with bilateral disease: ~6two first-degree relatives: 2.5-9second degree relative: 1.4

• But Pam is part of a huge extended family with no other individuals with breast or other cancers

5 4 6 5

27

1 2

1530

N5

N N

2 N N N

astrocytoma

Breast Cancer: associated genetic syndromes

Ovarian

Desmoid

Breast Cancer

Colon

HNPCC (Lynch)

Mismatch repair genes

FAP (Gardner)APC

Hereditary Breast & Ovarian CancerBRCA1 & BRCA2

Thyroid

ProstateMultiple Endocrine

Neoplasias

Peutz-JeghersSTK11

Glioblastoma

Sarcoma

Li-FraumeniTP53

Hereditary Diffuse Gastric Cancer

CDH1

Gastric

CowdenPTEN

Pheo

• Pam is an only child

• Her mom’s first cousin died at age 55 from breast cancer

• But she is part of a huge extended family with no other affected individuals in her maternal lineage

• A newborn female has midface hypoplasia, upslanting palpebral fissures, low set small square ears, low tone, bilateral single palmar creases, and a heart murmur. You suspect Down syndrome.

• According to the AAP Health Supervision Guideline for Down syndrome, which of the following is the most sensitive test for Down syndrome in the first 24 hours of life?

A. Physical examB. KaryotypeC. Aneuploidy FISHD. Chromosomal microarrayE. Whole exome sequencing

Clinical Case

• A newborn female has midface hypoplasia, upslanting palpebral fissures, low set small square ears, low tone, bilateral single palmar creases, and a heart murmur. You suspect Down syndrome.

• Which of the following molecular genetic tests gives you the most information regarding Down syndrome?

A. Chromosomal microarrayB. Aneuploidy FISHC. KaryotypeD. Intellectual disability panelE. Whole exome sequencing

Clinical Case

• AS presents for her 9 month old well child visit. Her medical history is significant for:Hypotonia and global developmental delaysSeizures well controlled with KeppraShort stature and g-tube for poor weight gainHemangioma right nuchal foldChromosomal microarray by report was normal

• During your well child check, which of the following is the best first step in determining the etiology of the patient’s features?

A. Repeat chromosomal microarrayB. Epilepsy panelC. Intellectual disability panelD. Whole exome sequencingE. Consultation with genetics

Clinical Case

• AS presents to genetics clinic.

• Dysmorphic findings include: relative macrocephaly, frontal bossing, hypertelorism, sparse eyebrows, low nasal bridge with wide nasal tip, thick helices, cupped pinnae, fetal fingertips pads.

• Based on the clinical history and physical exam findings, which of the following is the best next step in determining the etiology of the patient’s features?

A. Repeat chromosomal microarrayB. Epilepsy panelC. Intellectual disability panelD. RASopathy panelE. Whole exome sequencing

RASopathy Gene Panel from GeneDx

• BL is a 37 year old who has been followed since age 15 for an unspecified Ehlers-Danlos syndrome.

• Infantile hypotonia led to muscular dystrophy evaluation and a normal muscle biopsy at 1 year of age (1978).

• Joint laxity, kyphoscoliosis, mild pectus carinatum, flat feet.

• No joint pain, subluxations or dislocations.

• Fragile skin splits easily. • Molluscoid pseudotumors removed 3-4 times. • Bilateral inguinal hernias repaired at age 11.

• Age 15, spontaneous rupture of the SMA successfully repaired.

Clinical Case

• Previous normal testing for EDS subtypes: Lysyl hydroxylase activity and urine pyridinium cross-links

- rules out kyphoscoliotic EDS (VI): PLOD1

Skin biopsy for Type I and III collagen- rules out arthrocalasia EDS (VIIa & VIIb): COL1A1 &

COL1A2- rules out vascular EDS (IV): COL3A1

Tenascin X gene testing-rules out autosomal recessive EDS: TNXB

• Which of the following is the best next step in determining the etiology of the patient’s features?

A. Chromosomal microarrayB. Muscular dystrophy panelC. Ehlers-Danlos syndrome panel (12 genes)D. Classic EDS panel (2 genes)E. Whole exome sequencing

Classic EDS panel from CTGT

Comprehensive EDS panel from CTGT

Comprehensive EDS panel from CTGT

Classic EDS panel from CTGT

Dermatosparaxis EDS (VIIc)

Occipital Horn EDS (IX)

Musculocontractural EDS (VIb)

Arthrocalasia EDS (VIIa/b)

Vascular EDS (IV)

Kyphoscoliotic EDS (VI)

Spondylocheirodysplastic EDS

Periventricular heterotopia EDS

EDS with progressive kyphoscoliosis, myopathy, and

HL

Mutation in type 5 collagen is consistent with the classic form of EDS. This mutation changes a conserved triple helical glycine to an arginine therefore changing the structure of the resulting protein.

Changes in COL5A1 and COL5A2 have been found in approximately 50% of individuals with the classic form of EDS

COL5A1 accounts for ~46% COL5A2 accounts for ~4%

• NC is a 38 year old who was clinically diagnosed with Marfan syndrome in childhood

Skeletal features (body habitus, tall stature, scoliosis)

High myopia (-11.5 diopters)

• Does NOT have ectopia lentis • Does NOT have aortic root dilation• NEGATIVE family history• Has NOT had FBN1 testing

• Scoliosis required fusion at 17 years of age• Does NOT have a chest wall deformity• Does NOT have flat feet• Left hip replacement in 2011• No history of pneumothoraces

Clinical Case

• Revised Ghent criteria (2010)– Major criteria:

•Ectopia lentis•Aortic dilation•Aortic dissection•Family Hx

– Need 2 or more major

– If only 1 major, need•≥ 7/20 systemic points (wrist/thumb, skeletal,

eye, pneumothorax, etc) or•FBN1 mutation

• Clinical diagnoses of Marfan syndrome in childhood based on skeletal features and high myopia.Does NOT have ectopia lentis or aortic root dilation/dissectionNEGATIVE family historyHas NOT had FBN1 testing

• Physical exam reveals a systemic score = 8Positive wrist and thumb signs (3)Hindfoot deformity (2)Myopia, scoliosis, facial features (1 each = total 3)

Which of the following is the best first step in determining the etiology of the patient’s features?

A. Chromosomal microarrayB. Plasma homocystine levelC. FBN1 gene testingD. Thoracic aortic aneurysm panelE. Whole exome sequencing

FBN1 testing from CTGT

FBN1 testing from CTGT

• Does NOT have a FBN1 mutation, deletion, or duplication

• Does NOT have ectopia lentis or aortic root dilation/dissection• NEGATIVE family history• Systemic score = 8

Positive wrist and thumb signs (3)Hindfoot deformity (2)Myopia, scoliosis, facial features (1 each = total 3)

Which of the following is a reasonable next step in determining the etiology of the patient’s features?

A. Chromosomal microarrayB. Plasma homocystine levelC. Thoracic aortic aneurysm panelD. Whole exome sequencingE. Watchful waiting

Whole exome sequencing from GeneDx

Which of the following is a reasonable next step in determining the etiology of the patient’s features?A. Chromosomal microarrayB. Thoracic aortic aneurysm panelC. Enroll in a research studyD. Review raw data from whole exome sequencing with molecular pathologistE. Watchful waiting

• GH is a 21 year old male with ID and autism spectrum disorderMyopia and congenital glaucoma (legally blind)Hearing loss requiring cochlear implants

Bifid uvula, high arched palate, micrognathiaShort stature, stiffing joints, flat feetType 1 diabetes diagnosed at age 15

• He does NOT have • a copy number variant on chromosomal microarray• a mutation of 7 genes on a Stickler panel: COL2A1, COL9A1,

COL9A2, COL9A3, COL11A1, COL11A2, VCAN

Which of the following is a reasonable next step in determining the etiology of the patient’s features?

A. Intellectual disability panelB. X-linked intellectual disability panelC. Hearing loss panelD. Whole exome sequencingE. Watchful waiting

Last Clinical Case

Whole exome sequencing from GeneDx

Wolfram syndrome-like diseaseautosomal dominantvariable optic atrophysensorineural hearing loss diabetes mellituspsychiatric illness

Wolfram syndromeautosomal recessiveoptic atrophy sensorineural hearing lossdiabetes mellitusprogressive neurodegenerative disorder

QUESTIONS?

Medical GeneticsUI Children’s Hospital

• Kim Turner, BSN (triage)(319) 356-2675

• Hannah Bombei, MS, CGC MJ Hajianpour, MD, PhD• Cathy Evers, BSN, MS Alvaro Serrano-Russi, MD• Kim Horton, BSN, MS, CGC Oleg Shchelchkov, MD• Jenny Marcy, MS, CGC Val Sheffield, MD, PhD• Ann Muilenburg, RN, MA Pamela Trapane, MD• Karin Panzer, MS, CGC

Joni Bosch, ARNP, PhD• Cheryl Stimson, MS, RD Myrl Holida, PA-C

Judy Miller, ARNP• Pam Deboer, RN Samantha Schutt, ARNP