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The State of the Molecular Autopsy for Sudden Death in the Young
Michael J. Ackerman, MD, PhD Windland Smith Rice Cardiovascular Genomics Research Professor Professor of Medicine, Pediatrics, and Pharmacology Director, Long QT Syndrome Clinic and the Mayo Clinic Windland Smith
Rice Sudden Death Genomics Laboratory President, Sudden Arrhythmia Death Syndromes (SADS) Foundation
WINDLAND Smith Rice Sudden Death
Genomics Laboratory
Conflicts of Interest to Disclose: • Consultant – Boston Scientific, Gilead Sciences, Medtronic, St. Jude Medical, and Transgenomic/FAMILION • Royalties – Transgenomic/FAMILION
Learning Objectives to Disclose: • To DETAIL the state of postmortem genetic testing (AKA, “the molecular autopsy”) for autopsy positive and autopsy negative Sudden Unexplained Death Syndrome (SUDS) • To EXAMINE the three Achilles’ heels that threaten to derail the molecular autopsy
Nature Medicine 2013
Sudden Death in the Young (SDY)
Nature Medicine 2013
Sudden Death in the Young (SDY)
Tester and Ackerman. Pediatric Cardiology 33:461-467, 2012
Sudden Cardiac Death in the Young
Autopsy Positive
Autopsy Negative
Sudden Cardiac Death in the Young
Clinical Markers for Positive Genetic Test
Marker Pts
Age Dx < 45 yrs 1
MLVWT ≥ 20 mm 1
FH of HCM 1
FH SCD 1
Reverse-curve HCM 1
Hx of Hypertension -1
Scoring range: -1 – 5 pts
p < 0.0001
-1 4/84
0 32/225
1 45/238
2 82/201
3 102/173
4 64/95
5 30/37
Yiel
d of
Gen
etic
Tes
ting
(%)
Total Score of Clinical Markers
5%
14% 19%
41%
59%
67%
81%
Predicting Positive Genetic Test
Bos…Ackerman. Mayo Clin Proc 89:727-737, 2014
<5% ~80% Yield of Genetic Testing
L185R- TPM1
G C T G A G C T C T C A G
G C T G A G C T C T C A
Normal
Van Driest … Ackerman. AJC 90:1123-1127, 2002
HCM: A Disease of the Myofilament
Modified from Spirito P et al. NEJM 336:775, 1997
Genetic Testing for HCM
Van Driest … Ackerman. AJC 90:1123-1127, 2002
Genetic Testing for HCM
Van Driest … Ackerman. AJC 90:1123-1127, 2002
Mutation-specific genetic testing is recommended for family members and other appropriate relatives subsequently following the identification of the disease-causative mutation in an index case.
Ackerman, Priori, et al. Heart Rhythm 8:1308-1339, 2011 (HRS/EHRA) Gersh, Maron, et al. Circulation 124:2761-2796, 2011 (ACCF/AHA)
A Mother’s Question
Why did my 17-year-old son die?
State of Postmortem Genetic Testing for Autopsy Negative SUD
• March 1999 – previously well 17-year-old white male found dead in bed
Evaluation of Sudden Unexplained Death
• Autopsy negative • Local newspaper editorial positive –
“Parents talk to your kids” • Clue in the family history – mom “fell”
from 3-meter diving board at age 9 • Negative standard evaluation – ECG, Echo
L191fs/90
735-739delGCGCT
Ackerman et al. Am J of Forensic Medicine and Pathology 22:105-111, 2001
Genetic Diagnosis of
LQTS
60 yr 400
56 yr 459
38 yr 360
36 yr 420
31 yr 390
13 yr 14 yr 12 yr
SUD 3/26/99
17 yr
Nature Medicine 2013
Autopsy Negative Sudden Unexplained Death Syndrome
In Autopsy Negative SUD, How Often Would a
Molecular Autopsy Be Positive? 1. Don’t know what a molecular autopsy is.
2. 5-10%
3. 15-20%
4. 25-30%
5. > 50%
• N = 173 cases of SUD (106 males)
• Average age = 18 + 13 years (1 - 69 yrs)
• Personal or FHx of Cardiac Events = 70 (40%) Autopsy Negative
Sudden Unexplained Death
Tester … Ackerman. Mayo Clin Proc 79:1380 – 1384, 2004 Tester … Ackerman. JACC 49:240-246, 2007
Tester … Ackerman. Mayo Clin Proc 87:524-539, 2012
~ 25 - 30%
State of Postmortem Genetic Testing
0
10
20
30
40
50
60
All Ages (n=173)
1-10ys (n=43)
11-20ys (n=75)
21-30ys (n=27)
31-40ys (n=16)
41-50ys (n=9)
> 50ys (n=3)
M F M F M F M F M F M F M F 106 67
26
39%
19 24 25 50 10 17 8 8 4 5 1 2
5
6 32%
21%
9
12 48%
18%
4
4
40%
24%
1
3 38%
13%
1 25%
0% 0% 0%
18% 19
p<0.005
Per
cent
Yie
ld (%
) Age- and Sex-Specific Effect on the Yield of a Cardiac Channel Molecular Autopsy
Tester … Ackerman. Mayo Clin Proc 87:524-539, 2012
LQTS Mutations - Pathogenic Basis for ~10% of SIDS or SUID
Arnestad … Schwartz. Circulation 115:361-67, 2007
Ackerman et al. JAMA 286:2264-69, 2001 Tester … Ackerman. Cardiovasc Res 67:388-96, 2005
SUDS/SIDS Molecular Autopsy
Ackerman, Priori, et al. Heart Rhythm 8:1308-1339, 2011 (HRS/EHRA)
1. In the setting of autopsy negative SUDS, comprehensive or targeted (RYR2, KCNQ1, KCNH2, and SCN5A) ion channel genetic testing may be considered in an attempt to establish probable cause and manner of death and to facilitate the identification of potentially at-risk relatives and is recommended if circumstantial evidence points towards a clinical diagnosis of LQTS or CPVT specifically (such as emotional stress, acoustic trigger, drowning as the trigger of death).
Whole exome sequencing (WES)
allows for simultaneous
mutational analysis of a patient’s entire library of genes!
Next Generation Whole Exome Sequencing
Gene specific targeted analysis
Next Generation Whole Exome Sequencing
Gene specific targeted analysis
29 consecutive sudden death cases (21 males, 26.7 + 5.9 years) collected at the Office of the Medical Examiner, Cook County, Illinois from January 2012 to December 2013 were referred to Mayo Clinic for molecular autopsy. Will … Ackerman. HRS 2015
10%
N1
C4967
Cytosol
SR lumen
11
RyR2
FKBP12.6 Binding Domain
N4782S
Y462S 28 yo white male found in bed, equivocal autopsy.
14 yo white male, found unresponsive on floor after playing/exertion, negative autopsy.
“Clinically Actionable” Variants
Colied-Coil region IQ domain
ATP binding
Actin binding domains
Four MYH7 (β-myosin heavy chain) Subunits
R783H
27 yo black female, sudden collapse, equivocal autopsy noting cardiomyopathy.
MYH7
Molecular Autopsy’s 3 Achilles’ Heels 1. Cost
3. Interpreting the Molecular Autopsy
Insurance companies do NOT like to pay for things when you have died!
2. Medical Examiner’s SOP Paraffin-embedded tissue is NOT DNA friendly!
“X” does NOT always mark the spot!
Tissue is the Issue!! Carturan, Tester …Thiene, Ackerman. Am J Clin Pathol 129:391-397, 2008
- Need blood spot card, blood in EDTA, or frozen tissue
Molecular Autopsy of SCD
Tissue is the Issue!! Carturan, Tester …Thiene, Ackerman. Am J Clin Pathol 129:391-397, 2008
- Need blood spot card, blood in EDTA, or frozen tissue
1. For all SUDS and SIDS cases, collection of
a tissue sample is recommended for subsequent DNA analysis/genetic testing.
Ackerman, Priori, et al. Heart Rhythm 8:1308-1339, 2011
Molecular Autopsy of SCD
Is the “X” that marks the spot truly THE disease-causing mutation?
Genetic Testing’s Achilles’ Heel
What’s the “Background Noise Rate”? What’s the Signal-to-Noise Ratio?
Sudden Cardiac Death in the Young
Autopsy Positive
Autopsy Negative
Tester and Ackerman. Pediatric Cardiology 33:461-467, 2012
Autopsy Negative SUD is NOT a Good Phenotype!
LQTS – 15% CPVT – 10% BrS – 3%
KCNQ1, KCNH2, SCN5A, RYR2
Background Noise Issue 2003
13.5% KCNQ1, KCNH2, SCN5A, RYR2 = ~5%
- “Maybe” Test Result ? “Possible Deleterious”
“Variant of Uncertain Significance (VUS)”
Postmortem Genetic Testing
“Genetic Purgatory is a Real Place and its
Scary!”
Case Presentation
39 34
+
Ackerman. Heart Rhythm 2015
Case Presentation
L537P
Ackerman. Heart Rhythm 2015
Case Presentation
39 34
+
+ = L537P-SCN5A = BrS1
+ + + -
Genetic Purgatory is Real
L537P VUS
Kapplinger … Ackerman. Circ CV Genet 2015 (in press)
< 10% POP
Case Presentation
L537P WILDTYPE
Case Presentation
39 34
+
+ = L537P-SCN5A = BrS1
+ + + -
-
+ = L537P-SCN5A = “One Hell of Situation”! (PJS)
The State of the Molecular Autopsy - 1. ~25% of autopsy negative SUD and 10% of SIDS -
Channelopathic! 2. Should postmortem genetic testing become a
standard part of the conventional autopsy for “autopsy negative” SUD?
3. Formalin-fixed paraffin embedded tissue is the enemy of a “comprehensive” molecular autopsy.
4. “X” does NOT always mark the spot!
Dr. Scholl Foundation, CJ Foundation for SIDS Hannah Wernke Memorial Foundation
Sheikh Zayed Saif Mohammed Al Nahyan Fund National Institutes of Health
WINDLAND Smith Rice Sudden Death
Genomics Laboratory
“To heal the sick and advance the science” Dr. Charles W. Mayo
WINDLAND Smith Rice Sudden Death
Genomics Laboratory
