Effects of hematopoietic stem cell transplantation on acyl-CoA oxidase deficiency: a sibling
comparison study
Raymond Y Wang, M.D., Edwin S Monuki, M.D. Ph.D., James Powers, M.D., Phillip H Schwartz,
Ph.D., Paul A Watkins, M.D. Ph.D., Yang Shi, M.D., Ph.D., Ann Moser, B.S., David A Shrier, M.D.,
Hans R Waterham, Ph.D., Diane J Nugent, M.D., Jose E Abdenur, M.D.
Fibroblast -oxidation Assay:
Control fibroblast line GM 5400 was obtained from the NIGMS Human Genetic Repository maintained
by the Coriell Institute, Camden, NJ. Fibroblasts were harvested by gentle trypsinization and neural
stem cells harvested with mechanical shaking, washed twice with phosphate-buffered saline, and
resuspended in ice-cold isotonic buffer (0.25M sucrose, 10 mM Tris, pH 8.0, and 1 mM EDTA). Cell
suspensions (100-300 μg protein) were incubated with either [1-14C]palmitic acid (C16:0) to measure
mitochondrial β-oxidation or [1-14C]lignoceric acid (C24:0) to measure peroxisomal β-oxidation
Plasma Very Long Chain Fatty Acid Quantification:
VLCFA were quantified via capillary gas chromatography / mass spectroscopy of pentafluorobenzyl
bromide fatty acid esters as described in:
Lagersted SA et al. (2001) Quantitative Determination of C8-C26 Total Fatty Acids for the Biochemical
Diagnosis of Nutritional and Metabolic Disorders. Mol. Genet. Metab. 73:38 – 49.
Moser AB, et al. (1999) Plasma Very Long Chain Fatty Acids in 3,000 Peroxisome Disease Patients
and 29,000 Controls. Ann. Neurol. 45:100-110.
Patient 1 Neuropathology:
Gray matter throughout the CNS was marked by profound neuronal loss and gliosis. Cerebellar
neuronal losses in the dentate nucleus were greater than those in the olive, while in the vermis and
hemispheres Purkinje and granular cell losses seemed equal (Supplemental Figure 1 A). Residual
pontine, dentate, and Purkinje neurons revealed various degenerative lesions. Neuronal loss in the
cerebrum was diffuse, but more severe in layers V-VI, and appeared greater in motor cortex than other
neocortical sections. Sections of the motor cortex showed microcavitation in layers V-VI and failed to
reveal any recognizable giant motoneurons of Betz. In their place, atrophic to achromatic neurons
were seen. Additionally, some areas of layer V showed small clusters of punctate mineralization.
The decussation of the superior cerebellar peduncle showed an equal loss of myelin and axons with
some axonal swellings. In deep cerebellar white matter and brainstem, less inflammation was present
affecting primarily the ventral and ventrolateral basis pontis. Isomorphic gliosis of pontine crossing
fibers and peripheral cerebellar white matter was noted, while the deep cerebellar white matter gliosis
was more anisomorphic (Supplemental Figure 1 C). The cerebral peduncle also had a severe, diffuse,
and comparable loss of myelin and axons, with corticopontine fiber loss more severe than that of the
corticospinal tract (Supplemental Figure 1 E, asterisk). Choroid plexus from the right atrium had
severe epithelial cell loss (Supplemental Figure 1 G).
Patient 2 Neuropathology:
Neuronal loss was milder than patient 1 (Supplemental Figure 1 B). In the motor cortex sections, two
Betz cells were identified: one was morphologically normal, while the other displayed a pyknotic
nucleus. No foci of mineralization were seen. The pallor of deep cerebellar white matter (Supplemental
Figure 1 D) and cerebral peduncles (Supplemental Figure 1 F) were not as severe as those of patient
1. The hippocampus was poorly-rotated, while the choroid plexus appeared to have normal epithelial
cells (Supplemental Figure 1 H). Residual neurons in basis pontis were few and there was an atypical
astrocytosis. The latter was more prominent in the crossing fiber areas. Electron microscopy of
cerebral white matter revealed macrophages with membrane-bound spiculated inclusions
(Supplemental Figure 4 B, 4 D), as seen in patient 1 (Supplemental Figure 4 A, 4 C), although patient
2 had relatively few macrophages had inclusions, and those with inclusions had fewer per cell,
compared to patient 1.
Supplemental Figure Legends:
Supplemental Figure 1. Microscopic neuropathology of other brain regions (H&E stains). In both
patients, severe cerebellar cortical atrophy (A,B) as well as pallor of deep cerebellar white matter
(C,D) and cerebral peduncles (asterisks in E,F) are present. Choroid plexus from the right atrium
shows severe epithelial loss in patient 1 (G), whereas the choroid plexus from patient 2 (H) appears
normal. Arrows designate the substantia nigra. Scale bars: 1 mm (all panels).
Supplemental Figure 2. Axial T2-weighted brain MR imaging from patient 2, pre-HSCT at 2.75 years
of age (A-C) and 3.75 years post-HSCT at 6.5 years of age (D-F). (A) Demyelination (arrowheads) of
cerebellar deep white matter and corticospinal tracts was incipient but progressed (D) to involve the
cerebellar peduncles; cerebellar atrophy (asterisk) had also developed. (B,C) There were no visible
supra-tentorial abnormalities just prior to transplant, but (E,F) 3.75 years later, there was marked
cortical atrophy (asterisk) and non-enhancing demyelination (arrowheads) of the corpus callosum,
periventricular, and subcortical white matter.
Supplemental Figure 3. Plasma VLCFA (C26:0 and C26:1) and ratios (C26:C22, C24:22) in patient 2
over his lifetime, demonstrating that all markers remained persistently elevated throughout the posttransplant period despite full donor engraftment.
Supplemental Figure 4. Ultrastructural neuropathology. (A,B) The cytoplasm of macrophages
contained spiculated inclusions in discrete membrane-bound aggregates that are indistinguishable
from inclusions described in other peroxisomal disorders. In patient 2, inclusions were difficult to find,
and when found, were present in smaller and less numerous aggregates (arrows in B). (C,D) The
spicules consist of paired, parallel fibers. Scale bars: 1 um (B), 200 nm (C,D).
ACOX1 Sequencing Information:
ACOX1-specific PCR primers
Primers
Sequence (5’3’)1
[21M13]-AgA TTg TCA CTg CCA CCT C
-55ACOX1f-21M13
IN2+100ACOX1rM13r [M13rev]-TgC ATC TTC AAA CAC CAA gC
IN2-64ACOX1f
[-21M13]-AGAAGGGGTTGTAGGATGCAG
IN3+72ACOX1r
[M13rev]-GTCAAATGCCAGCAATCACT
IN3-63ACOX1f,
[-21M13]-TGCTGATTCTCTCACACC TGA
IN4+66ACOX1r
[M13rev]-GCACTCCACACATGGTAAGC
IN4-178ACOX1f,
[-21M13]-AGTGTGGGAAATTCAGTGTC
IN5+110ACOX1r
[M13rev]-AGGATTTCACCACCAAGTCTG
IN5-47ACOX1f,
[-21M13]-TGTATGAACGTTTGCCCACC
IN7+61ACOX1r
[M13rev]-GAGTAACAGCCACTCAACTCCAC
IN7-144ACOX1f,
[-21M13]-TCAGGGTGGGGACTAAGATG
IN8+137ACOX1r
[M13rev]-ATTACAGGCCCAGTTATCAC
IN8-66ACOX1f,
[-21M13]-CTGGCATATAGAAAGTTCTG
IN10+89ACOX1r
[M13rev]-GTGAAGAAAAACCATGGGAACAAG
IN10-79ACOX1f
[-21M13]-GCAACAAGAATGAAACTCCATC
1556AOX-M13r
[M13rev]-GTTAGGTTCCAAGCTACCTC
IN11-27ACOX1f
[21M13]-TCAGACCTCCAATCTATGTCT
IN13+133ACOX1r
[M13rev]-GACCTCAAGTGATCACCGTA
IN13-63ACOX1f,
[-21M13]-CCGCTTTATAGCTGTATATG
IN14+153ACOX1r
[M13rev]-TCCCTTCTGAACCTGTTGCTC
IN14-60ACOX1f,
[-21M13]-GTATACTATTTAACATGGCAAACTC
IN15+192ACOX1r
[M13rev]-ACTGGGATCAGCAGCATTAC
1All primers contain -21M13 (5'-TGTAAAACGACGGCCAGT-3') or M13-Rev (5'CAGGAAACAGCTATGACC-3') extension to allow sequencing with these primers
PCR reaction
Component
Total
10x PCR buffer.
MgCl2
dNTPs mix
10 µM fw primer
10 µM rev primer
H2O
Platinum Taq polymerase
.
~0.05-0,4 g gDNA
Volume
15 l
1,5 l
1,5 l
1,2 l
0,75 l
0,75 l
9,2 - x l
0,5 l
x l
PCR program
1
2
3
4
5
6
7
8
9
10
11
12
96oC
96oC
55oC
72oC
4 times to 2
94oC
55oC
72oC
24 times to 6
72oC
14oC
END
Min:sec
2:00
0:30
0:30
1:00
0:30
0:30
1:00
7:00
0:00
Concentration in assay
20 mM Tris/HCl (pH 8,4), 50 mM KCl
1,0 or 1,5 mM MgCl2
0,2 mM dNTPs
0,5 M
0,5 M