Newborn Screening for X-Linked Adrenoleukodystrophy (X-ALD)
and Other Peroxisomal Disorders: Measurement of 26:0 Lyso-PC by
LC-MSMS
Ann B. Moser 1, Walter C. Hubbard 2, and Gerald V. Raymond 3
1 Hugo W. Moser Research Institute, Kennedy Krieger Institute, Baltimore, MD
2 Dept of Clinical Pharmacology Johns Hopkins University School of Medicine,
Baltimore , MD 3 Univ. MN, Dept. of Pediatric Neurology
GFPD 7/27/13
LC-MSMS C26:0 lyso-PC Newborn Screening Test
Identifies The Following Peroxisomal Disorders:
X-linked Adrenoleukodystrophy (ALD) – genetically inherited; affects 1 in 21,000
births, more severe in males; 3 phenotypes:
1) Childhood Cerebral phenotype: 35-40% of subjects appear normal at birth
and at age 4 to 8 years symptoms occur, rapidly progressive to death in
childhood
2) Adrenomyeloneuropathy (AMN) phenotype starting in 20s slowly progressive
spinal cord disease
3) severe Addison’s disease live to 4th - 5th decades with treatment
Test also identifies: 80% of X-ALD Heterozygotes, Peroxisomal Biogenesis
Disorders (PBDs) (Zellweger spectrum disorders) and Single Enzyme Defects of
peroxisomal FA oxidation
Primary Purpose of Newborn Screening
Presymptomatic Diagnosis of Males with X-ALD in Order to:
1. Prevent overt adrenal insufficiency
2. Reduce the risk for childhood phenotype with LO diet
3. Improve prognosis of cerebral ALD by facilitating early
hematopoietic stem cell and gene transplants
4. Improve success of future therapies
Secondary Purpose of Newborn Screening
Identification of 80% of X-ALD Heterozygotes in order to:
1. Provide family screening for male X-ALD relatives.
Diagnosis of PBD/SED Patients in Order to:
1. Prevent diagnostic odyssey, provide genetic counseling
supportive therapy.
and early
Authentic Standards
H2-C-O-C-(CH2)24 -CH3
HO-C-H
O
+
H2-C-O-P-O-CH2-CH2-N(CH3)3
O
-
26:0-Lys o-PC, M W 635.5
Elevated in blood spots X-ALD
* = 2H
****
H2-C-O-C-(CH2)24 -CH3
HO-C-H
O
+
H2-C-O-P-O-CH2-CH2-N(CH3)3
O
2H
-
4-26:0-Lys o-PC, M W
Internal Standard
639.5
MS/MS Fragmentation of 26:0 Lyso-PC
26:0-Lys o-PC, M W 635.5
H2-C-O-C-(CH2)24 -CH3
HO-C-H
103
O
+
H2-C-O-P-O-CH2-CH2-N(CH3)3
OH
2H
4-26:0-Lys o-PC, M W
= 2H
*
****
H2-C-O-C-(CH2)24 -CH3
HO-C-H
103
O
+
H2-C-O-P-O-CH2-CH2-N(CH3)3
OH
183
M S/M S:
m /z 636 > 184
m /z 636 > 104
639.5
183
M S/M S:
m /z 640 > 184
m /z 640 > 104
Heel Stick from newborn
Methods 1
• Neonatal heel stick blood or Postnatal venous blood
on filter paper cards
• Stored @ -20o C. until sampling with a 1/8” punch
into test tube or 96 well plates
• Add a 10ul of purified water
• Add 150ul methanol containing 10.56pmoles
deuterium labeled internal standard, D-4 -26:0 LysoPC. Mix gently for 1 hour at room temp.
• Centrifuge and transfer supernatant to glass
injection vials with 100ul inserts for LC-MSMS, Inject
5ul API4000 LC-MSMS, 10ul API3200 LC-MSMS
Methods 2
Liquid Chromatography Tandem Mass Spectroscopy
Electrospray ionization (ESI) LC-MS/MS MRM Analysis:
Instruments: Applied Biosystems API 4000 or 3200
– MRM transitions: 1-acyl-lyso-PCs =[ M+H]+ >m/z 104, 1-alkyllyso-PCs =[ M+H]+ >m/z 104
– Reversed phase column: C8-MS, Waters XTerra, 3.5u particle,
4.6x50 mm (7min) or 1.0x50mm (2 min isocratic) analyses.
– HPLC Solvents: MPA H20:CH3CN:HCOOH 54.5:45:0.5
MPB HCOOH:CH3CN:CHCl3 0.5:90:10 (2mMHCOONH4)
– Flow: 0.5ml/minute for 4.6x50mm column 75% MPA to 0% at
6 min;
– .18ml/minute for 1.0x50mm column 50% MPA and MPB
isocratic for 2 min, for high throughput analysis.
– Optimized: declustering potential (DP), collision energy (CE,
collision exit potential (CXP)
API 4000 LC-MSMS
High throughput analysis
For accurate quantitation, need
to calculate C26:0-lyso-PC
based on retention time as well
as MW/MRM.
This newborn DBS C26:0-lysoPC
is within normal range.
Table 1: Summary of LC-MS/MS Data Obtained From Newborn
Blood Spots:
26:0-Lyso-PC Levels Expressed as Picomoles per 1/8” Blood
Spot
Normal newborns
X-ALD/PBD newborns
(retrieved from CA and MI)
Mean
Standard Deviation
Range
Number of Subjects
0.329
6.53
0.123
1.62
0.13 – 0.75
4000
4.69 – 9.71
16
Newborn Screening for ALD and PBD:
Together with the Maryland State Screening
Lab, we performed a prospective study to
offer optional testing for ALD / PBD to
parents of 5000 newborns born in 3
hospitals in the Baltimore area. We have
finished analyzing the consented newborn
samples with no positives. We hope to start
ALD newborn screening in Maryland in
November 2014.
Newborn Screening for ALD and 6
lysosomal disorders, a combined highthroughput assay:
Drs. Silvia Tortorelli and Dietrich Matern of the Mayo
Biochemical Genetics Laboratory and Dr. Fred Lorey of the
State of CA Newborn Screening Laboratory are screening
100,000 newborn blood spots from CA using a combined assay.
To date they have completed the anonymous screening on
60,000 newborns and have identified positives for the ALD
screening that will be confirmed by analysis of mutations in the
ALD gene and other genetic and biochemical tests if negative
for ALD.
By mid-September 2013 the screening of 100,000 newborn
blood spot samples is expected to be completed.
Prior to the September 13, 2012 SACHDNC ( the Secretary’s
Advisory Committee on Heritable Disorders in Newborns &
Children, section 1111 of Public Health, Newborn Screening
Saves Lives Act 0f 2008) meeting in Washington, DC, Drs.
Charlie Peters and Amber Salzman organized and sent the
documents supporting the petition to add ALD to the
Recommended Uniform Screening Panel (RUSP) for all state
newborn screening labs.
The lobbyists who were allowed to speak at the meeting in
support of ALD newborn screening were: Dr. Gerald
Raymond, and Ann Moser from KKI; Spencer Barsh ,son of
Amber Salzman, The Stop ALD Foundation; and Taylor Kane,
daughter of Diane Kane, Run 4 ALD. Despite the eloquent
speeches of the lobbyists and the compelling data showing
that the newborn screening test method is valid and that
ALD newborn screening would save lives of ALD boys if
identified before symptoms start, the advisory committee
decided to wait for the completion of Mayo Biochemical
Genetics Lab screening of 100,000 newborns from CA.
Importance of Lobbying for
ALD newborn screening by
ALD family support groups!!
Lobbying in NY, NJ, CT, MD
have resulted in legislative
approval to add ALD newborn
screening. Lobbying continues
in TX , IL and MA.
We Did It!
Bill Requiring ALD
Newborn Screening
Passed in CT 7/3/13
Brian's Hope
<jeankelley=brianshope.org@createsend4.com>
We Did It! Our bill
(Brian’s hope)
requiring ALD
newborn screening
passed in CT.
Governor Dannel
Malloy signed it
into law.
Brian had BMT 18 years
ago shortly after ALD
was diagnosed due to
brain disease.
Brian Kelly’s 25th Birthday, July 3. 2013
Update 7/20/13 by Ann B Moser
• *Mayo Medical Laboratory continues screening of 100,000
NBS from CA with 60,000 screened to date. Report on
validation of 2 ALD positives will be given to SACHDNC for the
September 2013 meeting.
• *NY state newborn screening lab will add ALD to their
newborn screening panel in January 2014. (200,000 births/yr)
• *The states of CT and NJ legislatures have voted to add ALD to
their newborn screening panels.
• *MD will add ALD newborn screening after the state lab
moves into their new building in the spring of 2014, provided
funds are available. We are applying for a grant to support
ALD newborn screening in MD.
Acknowledgements
Steven Steinberg, Anita Liu, Richard O. Jones – Kennedy Krieger Institute
Fred Lorey – Genetic Disease Screening Program, California
Susan R. Panny, Fizza Majid –Newborn Screening Program Maryland Dept. Mental
Health/Hygiene
Robert F. Vogt, Jr., Christopher Haines – CDC, Newborn Screening Branch
Silvia Tortorelli, Dietrich Matern – Mayo Biomedical Genetics Laboratory
Baltimore Hospitals: Johns Hopkins, Greater Baltimore Medical Center, Frederick Memorial
Funding: United Leukodystrophy Foundation,
European Leukodystrophy Association,
Myelin Project, Run 4 ALD, Brian’s Hope, Stop ALD Foundation
NIH: Newborn Screening Grant : HD057136
NIH: Instrumentation API4000 1-S10 RR16798 (WCH)