Diagnostic Odysseys

Dusica Babovic-Vuksanovic, MD
Professor of Genetics and Pediatrics
Chair, Department of Medical Genetics
Center for Individualized Medicine, Mayo Clinic

GENETICSvery
simple!
Mendelian disorders
Autosomal dominant
Autosomal recessive
X-linked

Autosomal dominant
inheritance
Neurofibromatosis
Tuberous sclerosis
I

II

III

Marfan syndrome
Ehlers-Danlos
syndrome
Familial cancer
syndromes
Achondoplasia

Autosomal recessive
inheritance

Cystic fibrosis
Metabolic
disorders
Syndromes25%

Carrier

25%

25%

25%

Normal
Affected

X-linked
inheritance
Fragile X
Duchenne muscular dystrophy
Hemophilia A

Affected male
Normal male
Normal female
Carrier female

Risk for disease

CAD
70s

Colon Ca
80s

Lung Ca
75

55 y
Cleft
Palate
MR

45 y
BRCA

32 y

30 y

48 y
MI

20 y

Dementia

MVA
15 y

18 y

Dementia

Genetics of human disease

Is it so simple?
Mitochondrial disorders
Maternally inherited
mDNA

Epigenetics mechanisms
ANGELMAN SYNDROME PRADER WILLI SYNDROME
Paternal
Maternal
deletion/maternal UPD
deletion/paternal UPD
Hypotonia
Stiff ataxic gait
Developmental delay
Severe MR
Obesity
Absent speech
Hypogonadism
Almost never obese
OCD

Genetic
heterogeneity

Several genetic loci for

the
same phenotype
Neuropsychiatric
disorders
Autism
Schizophrenia
Hearing loss

Non-syndromic
>400 syndromes
Cell, Volume 141, Issue 2, 210-217, 16 April 2010

Allelic heterogeneity:
same gene-different
phenotypes
FGFR3
Achondroplasia
Crouzon syndrome/AN
Thanatophoric
dysplasia

LAMIN A
Progeria
ED Muscular
dystrophy
Cardiomyopathy
Lipodystrophy

Variable
penetrance
Incomplete penetrance
Deletion/duplication 22q
Age dependent penetrance
Alzheimer disease
Gender predisposition
X-linked disorders
Hip dysplasia

Anticipation
Trinucleotide repeat disorders
Huntington disease
Spinocerebellar ataxia
Myotonic Dystrophy
Fragile X syndrome

New Frontiers
Environment, including
microbiome

If the child looks like the father, its genetic

f the child looks like the neighbor, its environmenta

No rules? COMPLICATED
Non-Mendelian (common) disorders
Multifactorial disorders
Genetics/environment complex
interactions
Cleft lip/palate
Cancers
Diabetes
Hypertension
Aging..

DNA
(A, G, T, or C)
is 6 billion bits in
every cell, with a
total length of
about 6 feet of
DNA in every cell

From Nature, Feb 2001

Information
content of human

Human genome

~ 25,000 genes
Over 3,000 monogenic disorders
Human genome variations (Craig Venter)
3,213,401SNPs
53,823 block substitutions (2206 bp)
559,473 homozygous indels (182,711 bp)
62 CNVs
292,102 heterozygous insertion/deletion events
(indels)(1571 bp)
90 inversions
Levy et al., PLoS Biol. 2007 October; 5(10): e254

Personalized medicine: Era of

genomic sequencing
Risk for disease
Predict response to
drugs
Pre-symptomatic
diagnosis for
preventable and
treatable disease

Translation to clinical
medicine
Incidental findings
Ethical concerns
Emotional distress
Cost of medical care

Gene screening may refine

prediction of heart attack risk,
researchers say
Dec. 3, 2010 | This Week at Mayo Clinic | Volume 2, Number 1

Testing for 11 specific genetic variations in hundreds of people

with no history of heart disease provided information that led to
revision of their estimated heart attack risk, say Mayo Clinic
researchers.
Early findings suggest benefit may be most valuable in people thought to
be at intermediate risk using the current model
Dr. Kullo adds, however, that these findings need to be replicated and validated
in prospective studies before they are used in patient care.

What is a Diagnostic
Odyssey?
A protracted and arduous journey for
patients and families to find answers about
a rare medical condition they suffer from
A long sequence of medical evaluations
and referrals until patients have an
eventual diagnosis
Rare diseases affect about 25 million
people in the US
Cumulatively, rare diseases are not rare

Diagnostic Odyssey in the

Era of Precision Medicine
25% of patients on a diagnostic odyssey
identified a gap of 5 to 30 years between
getting their first symptoms and receiving
diagnosis
With advancements in genome sequencing
more patients are finding answers to their
diagnostic dilemmas
Some
are
treatments

even

finding

lifesaving

Genetic testing
Cytogenetic methods (karyotype, FISH etc)
Chromosome analysis
Chromosome microarray
Targeted microdeletion/duplication testing
Molecular methods (sequencing, UPD, methylation
assay etc)
Single gene analysis
Gene panels
Exome analysis
Genome analysis

21

From Gregory, Nature Reviews Genetics 6, 699-70

Genome sequencing

23

6 billion
bases!
lgvrdlmnqvtthequickababcmfxlqbrownfoxjulrcsmped
overthelazyyyzpolfdogjjirttiythedoglayhhbeldquetly
dreaminghwwiqldnsofdinnerplwosiucnd

25

Whole Exome Sequencing

(WES)
Analysis of all exons
180,000 exons arranged in ~22,000 genes
Portion of the human genome that contains proteincoding
sequences
2-3% of human genome

26

Whole Exome Sequencing

(WES)
Genes related to clinical phenotype
Medically actionable diagnosis (ACMGG guidelines)
Carrier status for AR conditions (CF, Tay-Sachs, Sickle
Cell, Familial Dysautonomia, Canavan disease, G6PD
deficiency)
Pharmacogenetic information (CYP2C9 and
CYP2C19), warfarin and Plavix metabolism
Not included adult onset neurodegenerative
disorders
Optional expended report includes deleterious
mutations and unclassified variants in genes
unrelated to disease phenotype and deleterious
mutations in genes with no currently known function
27

Whole exome sequencing

Need to have
parental samples
Complex test and
difficult interpretation
Variants of
unknown
significance (VUS)
Incidental findings

Were not all the same

28

Individualized Medicine (IM)

Clinic
IM Clinic Workgroup established in January
2012
Multidisciplinary, collaborative effort
Designed/implemented two IM Clinic service
lines

Sept 30th, 2012:

Advanced Cancer
Diagnostic Odyssey

IM Clinic: Diagnostic Odyssey

Undiagnosed patients with suspected
genetic component
Whole Exome Sequencing of patient
genomic DNA and affected or unaffected relatives - performed to find
genes contributing to disease
Dx provides:
Comfort, resolve, closure
Information for family planning
Possible therapy

Diagnostic Odyssey
IM Clinic
eConsult

Referral into
IM Clinic

Referral to
Mayo Clinic

Whole
Whole Exome
Exome Sequencing
Sequencing
of
of germ-line
germ-line

Genomic
Genomic counseling
counseling
IM
IM Clinic
Clinic Consultant
Consultant meets
meets patient
patient

Bioinformatics
Bioinformatics
analyzes,
analyzes, generates
generates
report
report
Results
Results presented
presented to
to
Genomic
Genomic Board
Board
Results
Results and
and possible
possible
treatment
treatment options
options
returned
returned to
to patient
patient

What is Standard Lab

Order?

IM Clinic
Genomic Odyssey Board (GOB)
Review new cases and test results.
Members include:

Diagnostic Odyssey
Case 1
Holoprosencephaly
Central incisor
Facial clefting
Diabetes insipidus
Ectrodactyly
Developmental delay

ECC (TP63 gene)

negative
Normal chromosome
array
Odyssey case!

Hartsfield syndrome

35

Hartsfield syndrome
Autosomal disorder
Only sporadic cases known
Recurrence risk low?

36

Second child
Ectrodactyly
Holoprosencephaly
Cleft lip and palate
Heart defect

Hartsfield syndrome
No other reported familial cases
WES: FGFR1 gene novel variant c.1880G>C
(p.R627T)
2 affected siblings with same mutation, parents
negative
Recurrence risk?

Third child
Ectrodactyly
Holoprosencephaly
Cleft palate
Encephalocele

3 affected siblings with same mutation, parents

negative, Odyssey continues!
Targeted testing of sperm: 23% of cells with the
mutation

Diagnostic Odyssey Case 2

Age : 26
Sex : Male
Progressive muscle weakness onset age 6 or 7
Difficulty sitting and standing up from sitting
position and difficulty climbing stairs
Difficulty swallowing solid foods
Mild form of ophthalmoplegia
Elevated CK (643)

Previous Genetic Testing

Prior muscle biopsies not specific
No genetic testing (insurance problems)
Referred for WES

IM evaluation
Personal history
Pedigree analysis
Physical examination
Review of previous evaluations
Differential diagnosis
Review of literature

44

Results

Progressive myopathy, suspicious family history,

Hutterite ancestry
Limb-girdle muscular dystrophy type 2H Sarcotubular myopathy
Autosomal recessive disorder previously described
in Hutterite population
Targeted gene testing

IM Clinic Diagnostic Odyssey

Consecutive Cases of 18 Months in
Operation

MedGen
Non
MedGen
Total

All Referrals
Declined
Proceeded with
WES
WES* testing
testing
42
23
8
50

Total
65

17

32

82

IM Clinic Diagnostic Odyssey

Consecutive Cases of 18 Months in Operation
Reason for Referral
Age
Neuro

5<5
18

>18

Total

All referred 17 26

50

All tested with WES 10 15

31

All positive 1

All referred 2

24

32

All tested with WES 1

14

19

All positive 0

Non-neuro

IM Clinic Diagnostic Odyssey

Consecutive Cases of 18 Months in
Operation
Cases
Total Complete

50

Positive

15

Negative

35

Success Rate

30%

IM Clinic Diagnostic Odyssey

Consecutive Cases of 18 Months in Operation
Results from Clinical Focused report
Tota
l

Range per
patient

22

0-3

Medically actionable*

0-1

Carrier status

0-1

76

0-4

223

0-16

5.6

Pathogenic/likely pathogenic
mutations

**PGX
***VUS possible related to
phenotype

* Medically actionable -- MUTYH (het), DSC2 Carrier - CFTR, G6PD, FANCC

** PGX: Pharmacogenomics
***Variants of unknown clinical significance

Averag
e

Cost for Evaluating a Patient on

a
Diagnostic Odyssey
Success
Rate

Cost

Conventional Genetic
Testing*

15%

~$25,0
00

Whole Exome Sequencing

30%

$8,000

Tests

* Shashi V et al., Genet Med 16;176-2;2014

IM Clinic Diagnostic Odyssey

Party Responsible for Payment
%

Category
Patients received complete or
31 partial commercial insurance
coverage
27

Medicaid

18 CIM
6

International patients

18 Other

WES and Still No Diagnosis:

What
is
Next
Associated
Variant Type
Phenotype(s)

Repetitive DNA, including

trinucleotide repeats

Fragile X syndrome
Huntingtons disease

Copy-number variants

DiGeorge syndrome
Charcot-Marie-Tooth disease type 1A

Long insertion-deletion variants*

Resistance to HIV infection

Structural variants

Chromosomal translocations
associated with spontaneous
abortions

Aneuploidy

Downs syndrome
Turners syndrome

Epigenetic alterations

Prader-Willi syndrome, BeckwithWiedemann syndrome

Acknowledgeme
nts
Center for Individualized Medicine
Leadership
Konstantinos N. Lazaridis, M.D.

Department of Medical Genetics

David R. Deyle, M.D.
Pavel Pichurin, M.D.
Ralitza Gavrilova, M.D.
Geoffrey Beek, CGC
Jennifer Kemppainen, CGC
Marine Murphree, CGC

Questions

Were not in the post-genome era yet!

Fromhttp://www.infosci.coh.org/corelab/genomics.htm