Download Genetic Testing in Clinical Neurology

Presented at the 58th Annual Meeting of the American Academy of Neurology,
San Diego, California in April, 2006
NEUROGENETIC TESTING:
A CASE-BASED APPROACH
Thomas D. Bird, MD
Roberta A. Pagon, MD
Departments of Neurology and Pediatrics
University of Washington
VA Medical Center / Children’s Hospital &
Regional Medical Center
Seattle, WA
Clinical Implications of Genetic Testing
• Ten vignettes with questions
• Focus on understanding
 Test results
 Common test methods
 Testing strategy when
• More than one test method is available
• More than one gene is associated with a phenotype
• Testing at-risk relatives
 Commonly used and abused genetic terms
 Uses of genetic testing
Case Vignette 1
• Three siblings all in their 30’s are at
50% risk to inherit Huntington disease
from their father.
• All 3 siblings have DNA testing of the
HD gene.
 One has 42 CAG repeats
 One has 38 CAG repeats
 One has 35 CAG repeats
Case 1: Question
Do the siblings have different risks for
developing Huntington Disease?
A. Yes
B. No
www.genetests.org
Located at
University of Washington
Seattle, WA
Funded by
National Institutes of Health
Huntington
testing
Trinucleotide repeat: Sequences of three nucleotides repeated a number of times in tandem within a
gene. Normal polymorphic variation in repeat number with no clinical significance commonly occurs
between individuals. Abnormally large alleles are classified in increasing order of size as mutable normal
alleles, reduced penetrance alleles, and full penetrance alleles, respectively.
Trinucleotide Repeat Diseases
Mode of
Inheritance
Trinucleotide
Repeat
Huntington disease
AD
CAG
Myotonic dystrophy 1
AD
CTG
Spinocerebellar ataxia 1
(SCA1)
AD
CAG
DentatorubralPallidoluysian atrophy
(DRPLA)
AD
CAG
Fragile X syndrome
XL
CGG
Oculopharyngeal
muscular dystrophy
AD and AR
GCG
AR
GAA
Disease
Friedreich ataxia
Case 1: Question
Which of the following is true for the sister
with 42 CAG repeats?
A. She will eventually develop HD.
B. She will not develop HD, but her children are
at risk.
C. She may or may not develop HD.
Huntington Disease
Molecular Genetic Testing
Allele sizes
• Full penetrance alleles: 40 or more CAG
repeats. Alleles of this size are associated
with development of HD.
Case 1: Question
Which of the following is true for the sister
with 38 CAG repeats?
A. She will eventually develop HD.
B. She will not develop HD, but her children
are at risk.
C. She may or may not develop HD.
Huntington Disease
Molecular Genetic Testing
Allele sizes
• Reduced penetrance alleles: 36-39 CAG
repeats. An individual with an allele in this
range may or may not develop symptoms of
HD in his/her lifetime.
Case 1: Question
Which of the following is true for the brother
with 35 CAG repeats?
A. He will eventually develop HD.
B. He will not develop HD, but his children are
at risk.
C. He may or may not develop HD.
Huntington Disease
Molecular Genetic Testing
Allele sizes
• Intermediate alleles: 27-35 CAG repeats. An
individual with an allele in this range is not at
risk of developing symptoms of HD but may be
at risk of having a child with an allele in the
abnormal range. Alleles in this range have
also been described as "mutable normal
alleles“.
Focus
Test results
 Allele sizes in trinucleotide repeat disorders
vary by disease.
 The GeneTests Web site is a resource for
test result interpretation.
 Repeat size may influence phenotype.
Uses of genetic testing
 Presymptomatic testing
Case Vignette 2
A 45 year old man has had a slowly
progressive, symmetrical, peripheral
neuropathy for 20 years.
 NCV are slow.
 He has no family history of neuropathy.
 His two sons and one daughter are young
adults.
Case 2: Questions
Which of the following is the most likely
cause of this patient’s neuropathy?
A.
B.
C.
D.
E.
Autosomal dominant inheritance
Autosomal recessive inheritance
X-linked inheritance
Not genetic
Cannot determine inheritance pattern at
present
Case 2: Simplex vs sporadic
• Simplex: A single occurrence of a
disorder in a family
• Sporadic: The chance occurrence of a
disorder or abnormality that is not likely
to recur in a family
Case 2: Simplex
Possible explanations for a single
occurrence of an inherited disorder in a
family:
 One offspring with an autosomal recessive
disorder or an X-linked disorder
 One person with a new mutation for an autosomal
dominant disorder or an X-linked disorder
 Decreased penetrance for an autosomal dominant
disorder
 Alternate paternity
Case 2: Sporadic
Non-genetic causes of neuropathy
 Alcohol/nutritional neuropathy
 Diabetic neuropathy
Case 2: Question
Should this man have DNA testing for CMT?
A. Yes
B. No
Case 2: Question
Which of the following tests for CMT
should be ordered?
A. PMP22 duplication testing
B. Myelin P zero (MPZ) sequencing
C. Connexin 32 (GJB1) sequencing
Case 2
Possible testing strategies
• MD: Single sequential testing
• Lab: Batching
• Lab: Tiered approach
Issues to consider
• Subtype prevalence
• Time
• Cost
Prevalence of CMT Subtypes
Strategy
Single
sequential
Batched
Gene
Proportion
of all CMT
Cost
Time
PMP22
(CMT1A)
~60-70%
~ $800
1 month
MPZ
(CMT1B)
~5%
~ $800
1 month
Connexin 32
(CMTX)
~10%
~ $800
1 month
All 3
~ 75-85%
$2,000
1 month
Case 2: Test Results
• The patient has a mutation in GJB1,
the gene encoding connexin 32. The
mutation is T>C at nucleotide 597
(Cys179Arg)
• This mutation confirms the diagnosis
of CMT X
Case 2: Question
What are the risks of CMT X to his children?
A.
B.
C.
D.
No risk
50% risk to each child
25% risk to each child
No risk to sons / All daughters are carriers
Case 2
X-linked Inheritance
X Y
X X
X X
X Y
X Y
Focus
• Commonly used and abused genetic terms
• Testing strategy when more than one
gene is associated with a phenotype
• Uses of genetic testing
 Diagnosis
 Genetic Counseling
Case Vignette 3
Newborn male with:
• Profound hypotonia
• No respiratory distress
• Lethargy
• Poor feeding
• Cryptorchidism
Case 3: Question
Prader-Willi syndrome is suspected. Which
of the following genetic tests would you
order?
A.
B.
C.
D.
Karyotype
FISH analysis
Uniparental disomy testing
Methylation analysis
Routine banded
karyotype
Reprinted from Thompson & Thompson Genetics in Medicine, 6th Edition; RL Nussbaum, RR McInnes, HF
Willard, Chromosomal Basis of Heredity, pg 8, Copyright 2001, with permission from Elsevier.
Routine banded karyotype detects
 Abnormal number of chromosomes
 Large (visible) duplications and deletions
 Balanced rearrangements
(translocations, inversions)
FISH
fluorescent in situ hybridization: (FISH) A technique used to identify
the presence of specific chromosomes or chromosomal regions
through hybridization (attachment) of fluorescently-labeled DNA
probes to denatured chromosomal DNA.
Step 1. Preparation of probe. A probe is a fluorescently-labeled segment of
DNA complementary to a chromosomal region of interest.
Step 2. Hybridization. Denatured chromosomes fixed on a microscope slide are
exposed to the fluorescently-labeled probe. Hybridization (attachment) occurs between
the probe and complementary (i.e., matching)
chromosomal DNA.
FISH
Step 3. Visualization. Following hybridization, the slide is examined
under a microscope using fluorescent lighting. Fluorescent signals
indicate the presence of complementary chromosomal DNA; absence
of fluorescent signals indicate absence of complementary
chromosomal DNA.
Green signal =
Normal control
Pink signal =
Chromosome region of interest
Normal control:
Two green signals
Two pink signals
Patient with deletion:
Two green signals
One pink signal
FISH detects small (submicroscopic)
chromosome
 Deletions
 15q11.2 deletion in Prader-Willi syndrome
and Angelman syndrome
 22q11.2 deletion in velocardiofacial syndrome
 Duplications
 PMP22 – CMT1A
 PLP1 – Pelizeus-Merzbacher leukodystrophy
 -synuclein familial Parkinson disease
Karyotype
FISH
Intragenic deletion
Base pair deletion
Case 3
uniparental disomy study: (synonyms: UPD analysis, UPD study) Testing used to identify
if specific chromosomes or chromosomal segments are maternally or paternally derived;
can aid in confirming the clinical diagnosis of certain disorders for which UPD is a possible
underlying etiology
I
K
M
O
J
L
N
P
Father
J
Mother K
N
O
Patient
J I
K L
N M
O P
I
L
M
P
Case 3
methylation: The attachment of methyl groups to
DNA at cytosine bases; correlated with reduced
transcription of the gene and thought to be the
principal mechanism in imprinting
Prader-Willi Syndrome
Molecular Genetic Testing
Test Methods
Mutations
Detected
Percent of
Individuals
Methylation analysis
Methylation
abnormality
99%
FISH /
Quantitative PCR
Deletion of PWCR
70%
Uniparental disomy
studies
UPD of PWCR
25%
Sequence analysis
Imprinting center
defect
<1%
Case 3: Test Results
Methylation testing is consistent with expression
of maternal genes only in the PWS/AS critical
region of 15q11.2
Conclusion: Patient has Prader-Willi syndrome
Next step for genetic counseling: Determine if
he has a deletion, UPD, or an imprinting center
mutation
Focus
Test methods
• Often one method has a higher sensitivity
than others
• Sometimes the laboratory will not tell you this
until it reports the result of the test that was
ordered.
Case Vignette 4
40 year old man
• Muscle weakness since age 30
• Slowly progressive
• Very large calves
• CK 4,000 u
• Negative family history
• 7 sisters, 2 brothers
• 2 daughters, 2 sons
Case Vignette 4
In 1998 at age 33 years
• DMD (dystrophin) gene analysis on blood
• “Multiplex PCR gene amplification”
(deletion screening)
• Result: No deletion in the dystrophin
gene
Case 4: Questions
• In 2005 at age 40 years
• Is there additional testing for
mutations in DMD?
A. Yes
B. No
Dystrophinopathy
Molecular Genetic Testing
Test
Method
Deletion /
duplication
testing
Sequence
analysis
Mutations Detected
% of Males
with DMD
% of Males
with BMD
% of Males
with XLDCM
Deletion of one or more
exons of DMD gene
~65%
~85%
Unknown
Duplication of one or
more exons of DMD
gene
~6-10%
~6-10%
Unknown
Small insertions/
deletions/point
mutations/splicing
mutations of DMD gene
~25-30%
~5-10%
Unknown
Case 4: Test Results
DMD sequence analysis
“A to T change at nt 435 in exon 3 resulting
in asp46val in actin binding domain.”
Focus
Test methods
• VERY likely to change over time
• Especially:
• CMTs
• Muscular dystrophies
• Hereditary ataxias
• Check to see what methods are
currently available
Case Vignette 5
A 40 year old woman has a long history of
peripheral neuropathy.
• Three other family members in two
generations are also affected.
• Her DNA test for CMT shows a G>C at
nt 1064 producing a glycine for arginine
substitution at codon 163 (G163R) of MPZ,
the gene encoding myelin P zero.
MPZ Gene
Mutation = G163R
Codon 163
G* G U
Amino Acid
Gly (G)

C G U
*Nucleotide 1064
Arg (R)
Case 5: Test Results
The laboratory says this is an
“indeterminate result” because it has
not been previously reported.
Sequence analysis
Types of sequence alterations that may be detected
◦ Pathogenic sequence alteration reported in the literature
◦ Sequence alteration predicted to be pathogenic but not
reported in the literature
◦ Unknown sequence alteration of unpredictable clinical
significance
◦ Sequence alteration predicted to be benign
(polymorphism) but not reported in the literature
◦ Benign sequence alteration (polymorphism) reported in the
literature
Case 5: Questions
What is the best interpretation of this result?
A. Probably a benign polymorphism
B. Definitely a causative mutation
C. Could be a causative mutation, but
cannot be certain
Case 5: Questions
Will any further testing help?
A. Yes
B. No
Case 5: Interpretation of test results
Determine how the mutation segregates with the phenotype in the family
1
?
2
66
1
2
?
68
+
3
63
1 +
44
+ = Arg163Gly
= Affected with CMT
2
4
42
+
42
3 +
40
53
4
38
Focus
Test results
The laboratory may be able to offer additional
testing to clarify indeterminant results.
Case Vignette 6
35 year old man
• Onset ataxia age 22
• Slowly progressive
• Depressed DTR’s
• Dysarthria
• ↓ Vibration in feet
• ↓ Plantar reflexes
• Normal MRI
• Negative family history
Case 6: Questions
Would you test him for Friedreich ataxia?
A. Yes
B. No
Case 6: Test Result
Friedreich ataxia molecular genetic test
One normal allele
One allele with 120 GAA repeats
What is the best interpretation of this result?
A. Normal
B. Abnormal
C. Indeterminant
Friedreich Ataxia
Molecular Genetic Testing
Allele sizes. Three classes of alleles are recognized for the
GAA triplet repeat sequence in intron 1 of the FRDA gene:
• Normal alleles: 5 to 33 GAA repeats.
• Premutation alleles (mutable normal alleles):
34 to 65 pure (uninterrupted) GAA repeats
• Disease-causing expanded alleles (full
penetrance alleles): 66 to 1700 GAA repeats.
Case 6: Question
FA is an autosomal recessive disorder
– diagnosis requires presence of two
abnormal alleles
Can any other test be done to confirm or
exclude FA?
A. Yes
B. No
Friedreich Ataxia
Molecular Genetic Testing
Test Method
GAA repeat
analysis
Sequence
analysis
Mutations
Detected
Prevalence
Homozygous
GAA expansion
in FRDA
96%
Heterozygous
GAA expansion
in FRDA
Heterozygous
point mutation in
FRDA
Test
Availability
Clinical
4%
Case 6: Test Result
Sequence analysis of FRDA gene: G130V
missense mutation
Interpretation: Patient is a compound
heterozygote for two abnormal alleles:
–
–
an expanded GAA repeat
a missense mutation
Diagnosis: Friedreich ataxia
Focus
Test results. Presence of one mutant
allele does not confirm the diagnosis
of an autosomal recessive disorder.
Test methods. Additional test methods
might be available to clarify an
ambiguous test result.
Case Vignette 7
42 year old woman with family history
of Huntington disease.
– She has a normal exam and requests
presymptomatic testing for HD
– Father, uncle and younger sister had
progressive neurologic disease; all are
deceased.
– Sister’s brain at autopsy reported as
“compatible with HD.” Frozen brain
tissue was saved.
Case 7: Question
Would you test this woman for the
HD gene mutation?
A. Yes
B. No
Case 7
No! You should not test an asymptomatic
at-risk relative without a molecular
diagnosis in an affected family member.
HD gene testing on frozen tissue from the
deceased sister was normal.
Case 7
More family history was obtained:
– The deceased sister also had visual
loss resulting in blindness
– Records on other family members
showed a diagnosis of severe
cerebellar ataxia
Case Vignette 7
For which of the following genetic
disorders would you test the frozen
tissue?
A.
B.
C.
D.
E.
SCA 1
SCA 2
SCA 3
SCA 6
SCA 7
Case 7: Test Results
The tissue had 80 CAG repeats
in the SCA7 gene
Spinocerebellar Ataxia Type 7
Molecular genetic testing
Allele sizes
• Normal alleles: 19 or fewer CAG repeats.
• Mutable normal alleles: 30 to 35 repeats
• Reduced penetrance alleles: alleles with
34-36 repeats may be provisionally
defined as alleles with reduced penetrance
• Full penetrance alleles: 36 to 460 CAG
repeats.
Case 7: Test Results
The consultand tests normal for the
SCA 7 CAG repeat
Focus
Testing strategy in a family.
You must confirm the diagnosis in an affected
relative before offering presymptomatic testing to
at-risk family members.
Case Vignette 8
A 38 year old woman has a family history
of ALS.
– Her father, paternal aunt, and paternal
grandfather have all died from ALS.
– Her 40 year old sister has ALS.
– The affected sister has a negative DNA test
for SOD1 mutations.
Case 8: Questions
Does this family have familial ALS
(FALS)?
A. Yes
B. No
Case 8: Questions
What proportion of ALS is familial?
Amyotrophic Lateral Sclerosis Overview
Causes
About 90% of ALS occurs in individuals with
no family history of ALS; such individuals are
said to have sporadic ALS (SALS).
About 10% of individuals with ALS have at
least one other affected family member and
are said to have familial ALS (FALS).
Case 8: Questions
What proportion of familial ALS can be
attributed to SOD1 mutations?
Amyotrophic Lateral Sclerosis Overview
Table 1. Molecular Genetics of Autosomal Dominant ALS
% Individuals
with Familial ALS
Locus Name
Disease Name
Gene
20%
ALS1
FALS
SOD1
ALS with
frontotemporal
dementia (FTD)
Rare (<5%)
Rare
ALS3
Rare
ALS4
Rare
ALS6
Rare
ALS7
Case 8: Questions
What is the proband’s risk for ALS?
A.
B.
C.
D.
E.
50 %
25 %
10 %
Background (population) risk
Unknown but higher than
background risk
Case Vignette 9
• A male diagnosed prenatally to have
cardiac rhabdomyoma is stillborn
• His parents are healthy
• The father has a 30 yo paternal
female first cousin with a seizure
disorder and pulmonary disease
Case 9
Stillborn
Cardiac rhabdomyoma
Seizures; pulmonary disease
Case 9: Question
Which of the following disorders best
explains the findings in this family?
A.
B.
C.
D.
Neurofibromatosis type 1
Tuberous sclerosis complex
Von Hippel Lindau disease
Myotonic dystrophy type 1
Case 9: Question
Which of the following is the next
best step in the evaluation of this
family?
A. Examine and test the parents of the
stillborn
B. Examine the parents and test tissue
from the stillborn
C. Examine and test the cousin
Case 9: Results
Skin and eye examinations, brain MRIs and renal
US examinations are normal in both parents
Tissue from the stillborn shows a missense
mutation in the TSC1 gene
What should you do next?
A. Test the mother
B. Test the father
C. Test both parents
Case 9: Results
The father has the same TSC1 missense
mutation as the stillborn
The mother does not have a TSC1 mutation
The father’s cousin has pulmonary
lymphangiomyomatosis (LAM) and
the same TSC1 mutation.
How do you explain the findings
in this family?
Case 9
Variable expressivity: Variation in clinical
features (type and severity) of a genetic
disorder between affected individuals,
even within the same family
Reduced penetrance: The proportion of
individuals with a mutation causing a
disorder who exhibit clinical symptoms is
less than 100%
Focus
Commonly used and abused genetic
terms: penetrance and variable
expressivity.
Testing strategy for at-risk relatives. You
must identify the specific mutation in an
affected family member before testing
relatives who might be affected.
Case Vignette 10
12 year old girl with:
–
–
–
–
Recent onset stimulus sensitive myoclonus
Tonic-clonic seizures
EEG photosensitivity
Normal brain MRI
Brother died at age 19 years after
progressively worsening similar syndrome
treated with phenytoin
Normal parents
Case 10: Question
For which of the following reasons
should genetic testing be done?
A. Diagnosis
B. Prognosis
C. Management
D. Genetic counseling
Case 10: Question
For which of the following disorders would
you test first?
A. Tuberous sclerosis complex
B. Prion disease
C. Unverricht-Lundborg (EPM1)
D. MERRF
E. Lafora body (EPM2)
Case 10:Test Results
The EPM1 gene is homozygous for a 40
dodecamer (CCC-CGC-CCC-GCG)
repeat expansion
Unverricht-Lundborg Disease
Molecular Genetic Testing
Allele sizes
• Normal alleles: 2-3 dodecamer
repeats
• Full penetrance alleles: 30 or
more dodecamer repeats
Case 10: Relevance of the Genetic
Test Result
• Diagnosis: EPM1 (Unverricht-Lundborg
Disease)
• Prognosis: Progressive
• Management: Valproate best; Phenytoin
detrimental
• Genetic counseling: Autosomal recessive
Focus
Uses of genetic testing
• Diagnosis
• Management (including avoidance
of agents/circumstances that can
aggravate disease manifestations)
• Genetic Counseling
Clinical Implications of
Genetic Testing with a Focus on:
• Test results
• Common test methods
• Testing strategies
• Commonly used and abused
genetic terms
• Uses of genetic testing