toward newborn screening for cerebrotendinous ......j lipid res 42 (2001) 291-300 a honda, et al...
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![Page 1: Toward Newborn Screening for Cerebrotendinous ......J Lipid Res 42 (2001) 291-300 A Honda, et al Biochemical Phenotype of CTX: J Lipid Res 22 (1981) 191-200 I Bjorkhem, et al J Lipid](https://reader036.vdocuments.site/reader036/viewer/2022071611/614a9e0312c9616cbc698832/html5/thumbnails/1.jpg)
Andrea DeBarber, PhDResearch Assistant ProfessorPhysiology & Pharmacology DepartmentAssociate Director, Bioanalytical Shared Resource Facility Oregon Health & Science [email protected]
Toward Newborn Screening for Cerebrotendinous Xanthomatosis (CTX); Identifying Informative
Markers for CTX in Newborns
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Disclosures & Acknowledgements:
OHSU - Robert Steiner, Jenny Luo, Louise Merkens, Anu Pappu U Rio Grande do Sul - Roberto Giugliani, Carolina Souza NWR NBS Program - Cheryl Hermerath, Dave Sesser, Mike Skeels Ontario NBS Program - Michael Geraghty, Pranesh Chakraborty CA NBS Program - Fred Lorey OCTRI - grant 5KL2 RR024141-04 NCRR/NCATS STAIR - grant U54HD061939 NICHD/NCATS AB SCIEX - Michal Star-Weinstock, Babu Purkayastha
(patent application filed using QAO methodology to test for CTX)
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Clinical Phenotype of CTX:
0 10 20 30 40 50AGE
J Inherit Metab Dis 25 (2002) 501-513 PT Clayton et alArch Neurol 57 (2000) 520-524 A Verrips et al
![Page 4: Toward Newborn Screening for Cerebrotendinous ......J Lipid Res 42 (2001) 291-300 A Honda, et al Biochemical Phenotype of CTX: J Lipid Res 22 (1981) 191-200 I Bjorkhem, et al J Lipid](https://reader036.vdocuments.site/reader036/viewer/2022071611/614a9e0312c9616cbc698832/html5/thumbnails/4.jpg)
Neonatal cholestatic jaundice
Clinical Phenotype of CTX:
0 10 20 30 40 50AGE
J Inherit Metab Dis 25 (2002) 501-513 PT Clayton et alArch Neurol 57 (2000) 520-524 A Verrips et al
![Page 5: Toward Newborn Screening for Cerebrotendinous ......J Lipid Res 42 (2001) 291-300 A Honda, et al Biochemical Phenotype of CTX: J Lipid Res 22 (1981) 191-200 I Bjorkhem, et al J Lipid](https://reader036.vdocuments.site/reader036/viewer/2022071611/614a9e0312c9616cbc698832/html5/thumbnails/5.jpg)
Neonatal cholestatic jaundice
Clinical Phenotype of CTX:
0 10 20 30 40 50AGE
J Inherit Metab Dis 25 (2002) 501-513 PT Clayton et alArch Neurol 57 (2000) 520-524 A Verrips et al
![Page 6: Toward Newborn Screening for Cerebrotendinous ......J Lipid Res 42 (2001) 291-300 A Honda, et al Biochemical Phenotype of CTX: J Lipid Res 22 (1981) 191-200 I Bjorkhem, et al J Lipid](https://reader036.vdocuments.site/reader036/viewer/2022071611/614a9e0312c9616cbc698832/html5/thumbnails/6.jpg)
Neonatal cholestatic jaundice
Clinical Phenotype of CTX:
0 10 20 30 40 50AGE
Mean age symptom
onset 14-19 years
J Inherit Metab Dis 25 (2002) 501-513 PT Clayton et alArch Neurol 57 (2000) 520-524 A Verrips et al
![Page 7: Toward Newborn Screening for Cerebrotendinous ......J Lipid Res 42 (2001) 291-300 A Honda, et al Biochemical Phenotype of CTX: J Lipid Res 22 (1981) 191-200 I Bjorkhem, et al J Lipid](https://reader036.vdocuments.site/reader036/viewer/2022071611/614a9e0312c9616cbc698832/html5/thumbnails/7.jpg)
Neonatal cholestatic jaundice
Clinical Phenotype of CTX:
0 10 20 30 40 50AGE
Mean age symptom
onset 14-19 years
J Inherit Metab Dis 25 (2002) 501-513 PT Clayton et alArch Neurol 57 (2000) 520-524 A Verrips et al
![Page 8: Toward Newborn Screening for Cerebrotendinous ......J Lipid Res 42 (2001) 291-300 A Honda, et al Biochemical Phenotype of CTX: J Lipid Res 22 (1981) 191-200 I Bjorkhem, et al J Lipid](https://reader036.vdocuments.site/reader036/viewer/2022071611/614a9e0312c9616cbc698832/html5/thumbnails/8.jpg)
Neonatal cholestatic jaundice
Clinical Phenotype of CTX:
0 10 20 30 40 50AGE
Mean age symptom
onset 14-19 years
J Inherit Metab Dis 25 (2002) 501-513 PT Clayton et alArch Neurol 57 (2000) 520-524 A Verrips et al
![Page 9: Toward Newborn Screening for Cerebrotendinous ......J Lipid Res 42 (2001) 291-300 A Honda, et al Biochemical Phenotype of CTX: J Lipid Res 22 (1981) 191-200 I Bjorkhem, et al J Lipid](https://reader036.vdocuments.site/reader036/viewer/2022071611/614a9e0312c9616cbc698832/html5/thumbnails/9.jpg)
Neonatal cholestatic jaundice
Clinical Phenotype of CTX:
0 10 20 30 40 50AGE
Mean age diagnosis
35-37 years
Mean age symptom
onset 14-19 years
J Inherit Metab Dis 25 (2002) 501-513 PT Clayton et alArch Neurol 57 (2000) 520-524 A Verrips et al
![Page 10: Toward Newborn Screening for Cerebrotendinous ......J Lipid Res 42 (2001) 291-300 A Honda, et al Biochemical Phenotype of CTX: J Lipid Res 22 (1981) 191-200 I Bjorkhem, et al J Lipid](https://reader036.vdocuments.site/reader036/viewer/2022071611/614a9e0312c9616cbc698832/html5/thumbnails/10.jpg)
Neonatal cholestatic jaundice
Clinical Phenotype of CTX:
0 10 20 30 40 50AGE
neurological symptoms (95-97%):cognitive impairment,cerebellar signs (ataxia) & pyramidal signs (spasticity) Mean age diagnosis
35-37 years
Mean age symptom
onset 14-19 years
J Inherit Metab Dis 25 (2002) 501-513 PT Clayton et alArch Neurol 57 (2000) 520-524 A Verrips et al
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1. N Engl J Med (1984) 311: 1649-1652 VM Berginer, et al2. Neurol Sci (2001) 190: 29-33 M Mondelli, et al3. J Inherit Metab Dis (2008) Suppl:S 241-5 G Pierre, et al4. Pediatrics (2009) 123: 143-147 VM Berginer et al
Effective Oral Treatment for CTX:
Supplementation deficient bile acids1,2
Treatment pre-symptomatic children prevents disease complications3,4
Unfortunately treatment after many years of disease progression does not reverse neurological deterioration2
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Nomination requirements:
Clinical characteristics
High-throughput lab test
Optimally dried bloodspot (DBS) ESI-MS/MS biochemical test
Is CTX a Good Candidate Disorder to Screenfor in Newborns?
http://www.hrsa.gov/advisorycommittees/mchbadvisory/heritabledisorders/nominatecondition/index.html
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Biochemical Phenotype of CTX:
J Lipid Res 22 (1981) 191-200 I Bjorkhem, et alJ Lipid Res 42 (2001) 291-300 A Honda, et al
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Biochemical Phenotype of CTX:
J Lipid Res 22 (1981) 191-200 I Bjorkhem, et alJ Lipid Res 42 (2001) 291-300 A Honda, et al
![Page 15: Toward Newborn Screening for Cerebrotendinous ......J Lipid Res 42 (2001) 291-300 A Honda, et al Biochemical Phenotype of CTX: J Lipid Res 22 (1981) 191-200 I Bjorkhem, et al J Lipid](https://reader036.vdocuments.site/reader036/viewer/2022071611/614a9e0312c9616cbc698832/html5/thumbnails/15.jpg)
Biochemical Phenotype of CTX:
J Lipid Res 22 (1981) 191-200 I Bjorkhem, et alJ Lipid Res 42 (2001) 291-300 A Honda, et al
![Page 16: Toward Newborn Screening for Cerebrotendinous ......J Lipid Res 42 (2001) 291-300 A Honda, et al Biochemical Phenotype of CTX: J Lipid Res 22 (1981) 191-200 I Bjorkhem, et al J Lipid](https://reader036.vdocuments.site/reader036/viewer/2022071611/614a9e0312c9616cbc698832/html5/thumbnails/16.jpg)
Derivatization with quaternary amonoxy (QAO) O-(3-trimethylammonium-propyl) hydroxylamine reagent
Improve LLOQ & synthesize IS
Derivatization to Characterize Diagnostic Utility of Ketosterols:
Analytical Chemistry 84 (2012) 9310-9317 M Star-Weinstock, et al
+
7C4
+ +
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m/z (Da)
Rel
ativ
e in
tens
ity (%
)
152.3
438.6[M-59-18]+
556.5[M-59]+
515.5[M]+
NO
QAO Derivatization Signal Enhancement:
+
Time (min)
Inte
nsity
(cps
)
underivatized 7C4 (10ng/ml)m/z 401.6>97S/N = 4:1 QAO 7C4
(5ng/ml)m/z 515.5>152
S/N = 40:1
QAO 7C4 MS/MS spectrum
Analytical Chemistry 84 (2012) 9310-9317 M Star-Weinstock, et al
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4l plasma or 3.2mm DBS
80µl (210g) QAO reagent
in MeOH -5% acetic acid
LC-ESI-MS/MSanalysis
5500 QTRAP
10µl ISin MeOHOption 1
QAO Derivatization Workflow:
20µl ISin MeOH:H20
Option 2
Vortex mixRT
1-2 hours
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QAO Derivatization 7C4 in 4l plasma LC-ESI-MS/MS analysis 4-6 min LOD 7C4 spiked DCS plasma 5ng/ml Satisfactory linearity, precision & accuracy
across range 20-250ng/ml QAO 7C4-d7 IS (Option 1)
Quantification 7C4 in Plasma:
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QAO Derivatized 7C4 in Plasma:
QAO 7C4m/z 515.5 > 152.3QAO 7C4-d7m/z 522.5 > 152.3
Abs
olut
e In
tens
ity (c
ps)
Time (min)
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Synthesis IS using stable-isotope enriched QAO reagent:
OHNON
CD3
CD3
CD3
OHNON
D
Internal Standard (IS) options:
IS Option 1: Analyte-d7 + QAO-d0 reagent
IS Option 2: Analyte-d0 + QAO-d3 reagent
Exact Mass: 522.5
Exact Mass: 518.5
+
+
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0.00
50.00
100.00
150.00
200.00
250.00
300.00
350.00
0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00
y = 0.994x + 1.016
R2 = 0.984
Calculated conc (ng/ml)using IS Option 1
Cal
cula
ted
conc
(ng/
ml)
usin
g IS
Opt
ion
2
n = 200 samples & calibrants
Synthesis IS using stable-isotope enriched QAO reagent:
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QAO Derivatized 712C4 in Plasma:
QAO 712C4m/z 531.5 > 152.3QAO-d3 712C4m/z 534.5 > 152.3
Time (min)
Abs
olut
e In
tens
ity (c
ps)
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7C4 & 712C4 as Improved Markers for CTX:
Plasma concentrations:
7C4 712C4 5-cholestanol ng/ml ng/ml g/ml
CTX 1174 ± 711 1545 ± 1141 31 ± 19 patients (n=10) [204-1828] [57-2420] [8.4-66]
Unaffected 6.4 ± 5.3 0.4 ± 0.27 1.3individuals (n=20) [1.6-22] [0.1-0.8] [0.8-1.8]
Mean ± SD and [range of results] given
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Plasma concentrations:
>183-fold
CTX 1174 ± 711 1545 ± 1141 31 ± 19 patients (n=10) [204-1828] [57-2420] [8.4-66]
Unaffected 6.4 ± 5.3 0.4 ± 0.27 1.3individuals (n=20) [1.6-22] [0.1-0.8] [0.8-1.8]
Mean ± SD and [range of results] given
7C4 712C4 5-cholestanol ng/ml ng/ml g/ml
7C4 & 712C4 as Improved Markers for CTX:
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7C4 712C4 5-cholestanol ng/ml ng/ml g/ml
CTX 1174 ± 711 1545 ± 1141 31 ± 19 patients (n=10) [204-1828] [57-2420] [8.4-66]
Unaffected 6.4 ± 5.3 0.4 ± 0.27 1.3individuals (n=20) [1.6-22] [0.1-0.8] [0.8-1.8]
Mean ± SD and [range of results] given
Plasma concentrations:
>3862-fold
7C4 & 712C4 as Improved Markers for CTX:
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CTX 1174 ± 711 1545 ± 1141 31 ± 19 patients (n=10) [204-1828] [57-2420] [8.4-66]
Unaffected 6.4 ± 5.3 0.4 ± 0.27 1.3individuals (n=20) [1.6-22] [0.1-0.8] [0.8-1.8]
Mean ± SD and [range of results] given
7C4 712C4 5-cholestanol ng/ml ng/ml g/ml
Plasma concentrations:
>24-fold
7C4 & 712C4 as Improved Markers for CTX:
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Clinical phenotype:Older female Cataracts/xanthoma
Biochemical phenotype:Cholestanol 8.4 mg/ml [unaffected <6-8 mg/ml]1,2
7C4 1548 ng/ml [unaffected <22 ng/ml]712C4 795 ng/ml [unaffected <0.8 ng/ml]
Genotype:Homozygous c.1421G>A mutation CYP27A1
Case Study; A Better Blood Test for CTX?
1. Ann Intern Med 75 (1971) 843-851 G Salen, et al2. J Biochem 80 (1976) 223-228 Y Seyama, et al
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QAO Derivatization 712C4 in 3.2 mmDBS punches
LC-ESI-MS/MS analysis 4-6 min LOD 712C4 spiked blood 10ng/ml Satisfactory linearity, precision & accuracy
across range 50-250ng/ml QAO-d3 712C4 IS
Quantification 7 12C4 in DBS:
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QAO Derivatized 712C4 in Adult DBS:
QAO 712C4m/z 531.7 > 152.3QAO-d3 712C4m/z 534.7 > 152.3
Time (min)
Abs
olut
e In
tens
ity (c
ps)
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QAO Derivatized 712C4 in Newborn DBS:
QAO 712C4m/z 531.7 > 152.3QAO-d3 712C4m/z 534.7 > 152.3
Time (min)
Abs
olut
e In
tens
ity (c
ps)
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DBS concentrations:
712C4 cholestanol ng/ml g/ml
CTX n=4 1385 ± 467 n=3 17.4 ± 6.5 adults [852-1990] [6.8-24.4]
Unaffected n=3 1.0 ±1.3 n=6 2.3 ± 0.9adults [0-2.5] [1.4-3.8]CTX n=2 120-214 n=2 8.4-8.7
newbornsUnaffected n=6 16.4 ± 6.0 n=4 4.7 ± 1.8newborns [12.3-26.3] [2.5-7.0]
Mean concentration ±SD and [range of results] given
712C4 as a DBS Marker for CTX:
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DBS concentrations:
712C4 cholestanol ng/ml g/ml
CTX n=4 1385 ± 467 n=3 17.4 ± 6.5 adults [852-1990] [6.8-24.4]
Unaffected n=3 1.0 ±1.3 n=6 2.3 ± 0.9adults [0-2.5] [1.4-3.8]CTX n=2 120-214 n=2 8.4-8.7
newbornsUnaffected n=6 16.4 ± 6.0 n=4 4.7 ± 1.8newborns [12.3-26.3] [2.5-7.0]
Mean concentration ±SD and [range of results] given
10-fold
712C4 as a DBS Marker for CTX:
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DBS concentrations:
712C4 cholestanol ng/ml g/ml
CTX n=4 1385 ± 467 n=3 17.4 ± 6.5 adults [852-1990] [6.8-24.4]
Unaffected n=3 1.0 ±1.3 n=6 2.3 ± 0.9adults [0-2.5] [1.4-3.8]CTX n=2 120-214 n=2 8.4-8.7
newbornsUnaffected n=6 16.4 ± 6.0 n=4 4.7 ± 1.8newborns [12.3-26.3] [2.5-7.0]
Mean concentration ±SD and [range of results] given
10-fold
712C4 as a DBS Marker for CTX:
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DBS concentrations:
712C4 cholestanol ng/ml g/ml
CTX n=4 1385 ± 467 n=3 17.4 ± 6.5 adults [852-1990] [6.8-24.4]
Unaffected n=3 1.0 ±1.3 n=6 2.3 ± 0.9adults [0-2.5] [1.4-3.8]CTX n=2 120-214 n=2 8.4-8.7
newbornsUnaffected n=6 16.4 ± 6.0 n=4 4.7 ± 1.8newborns [12.3-26.3] [2.5-7.0]
Mean concentration ±SD and [range of results] given
2-fold
712C4 as a DBS Marker for CTX:
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cortisolm/z 363>12121-deoxycortisolm/z 347>31111-deoxycortisolm/z 347>9711-deoxycortico-steronem/z 331>9717OH-progesteronem/z 331>977C4m/z 401.6>97712C4m/z 417.6>97
1st Step Toward ESI-MS/MS Based Newborn Screening for CTX:
cortisol21-deoxycortisol
11-deoxycortisol
11-deoxy-corticosterone
17OH-progesterone
712C47C4
Abs
olut
e In
tens
ity (c
ount
s)
Time (min)
Clin Chem 50 (2004) 621-625 JM Lacey, et alSteroids 76 (2011) 1437-1442 N Janzen, et al
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