Contiguous Deletion of the X-Linked Adrenoleukodystrophy Gene (ABCD1) and DXS1357E: A Novel Neonatal Phenotype Similar to Peroxisomal Biogenesis Disorders

Article (PDF Available)inThe American Journal of Human Genetics 70(6):1520-31 · June 2002with28 Reads
DOI: 10.1086/340849 · Source: PubMed
X-linked adrenoleukodystrophy (X-ALD) results from mutations in ABCD1. ABCD1 resides on Xq28 and encodes an integral peroxisomal membrane protein (ALD protein [ALDP]) that is of unknown function and that belongs to the ATP-binding cassette-transporter superfamily. Individuals with ABCD1 mutations accumulate very-long-chain fatty acids (VLCFA) (carbon length >22). Childhood cerebral X-ALD is the most devastating form of the disease. These children have the earliest onset (age 7.2 +/- 1.7 years) among the clinical phenotypes for ABCD1 mutations, but onset does not occur at <3 years of age. Individuals with either peroxisomal biogenesis disorders (PBD) or single-enzyme deficiencies (SED) in the peroxisomal beta-oxidation pathway--disorders such as acyl CoA oxidase deficiency and bifunctional protein deficiency--also accumulate VLCFA, but they present during the neonatal period. Until now, it has been possible to distinguish unequivocally between individuals with these autosomal recessively inherited syndromes and individuals with ABCD1 mutations, on the basis of the clinical presentation and measurement of other biochemical markers. We have identified three newborn boys who had clinical symptoms and initial biochemical results consistent with PBD or SED. In further study, however, we showed that they lacked ALDP, and we identified deletions that extended into the promoter region of ABCD1 and the neighboring gene, DXS1357E. Mutations in DXS1357E and the ABCD1 promoter region have not been described previously. We propose that the term "contiguous ABCD1 DXS1357E deletion syndrome" (CADDS) be used to identify this new contiguous-gene syndrome. The three patients with CADDS who are described here have important implications for genetic counseling, because individuals with CADDS may previously have been misdiagnosed as having an autosomal recessive PBD or SED


Am. J. Hum. Genet. 70:1520–1531, 2002
Contiguous Deletion of the X-Linked Adrenoleukodystrophy Gene
(ABCD1) and DXS1357E: A Novel Neonatal Phenotype Similar
to Peroxisomal Biogenesis Disorders
Deyanira Corzo,
William Gibson,
Kisha Johnson,
Grant Mitchell,
Guy LePage,
Gerald F. Cox,
Robin Casey,
Carolyn Zeiss,
Heidi Tyson,
Garry R. Cutting,
Gerald V. Raymond,
Kirby D. Smith,
Paul A. Watkins,
Ann B. Moser,
Hugo W. Moser,
and Steven J. Steinberg
Division of Genetics, The Children’s Hospital, Boston;
Medical Genetics and
Gastroeneterology Services, Hoˆpital Ste-Justine, Montreal;
The Kennedy Krieger Institute, and
Institute of Genetic Medicine and Departments of
Neurology and
Pediatrics, Johns Hopkins University
School of Medicine, Baltimore;
Departments of Medical Genetics and Pediatrics, Alberta Children’s Hospital and University of Calgary,
Calgary; and
Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT
X-linked adrenoleukodystrophy (X-ALD) results from mutations in ABCD1. ABCD1 resides on Xq28 and encodes
an integral peroxisomal membrane protein (ALD protein [ALDP]) that is of unknown function and that belongs
to the ATP-binding cassette–transporter superfamily. Individuals with ABCD1 mutations accumulate very-long-
chain fatty acids (VLCFA) (carbon length
122). Childhood cerebral X-ALD is the most devastating form of the
disease. These children have the earliest onset (age 7.2 1.7 years) among the clinical phenotypes for ABCD1
mutations, but onset does not occur at
!3 years of age. Individuals with either peroxisomal biogenesis disorders
(PBD) or single-enzyme deficiencies (SED) in the peroxisomal b-oxidation pathway—disorders such as acyl CoA
oxidase deficiency and bifunctional protein deficiency—also accumulate VLCFA, but they present during the neo-
natal period. Until now, it has been possible to distinguish unequivocally between individuals with these autosomal
recessively inherited syndromes and individuals with ABCD1 mutations, on the basis of the clinical presentation
and measurement of other biochemical markers. We have identified three newborn boys who had clinical symptoms
and initial biochemical results consistent with PBD or SED. In further study, however, we showed that they lacked
ALDP, and we identified deletions that extended into the promoter region of ABCD1 and the neighboring gene,
DXS1357E. Mutations in DXS1357E and the ABCD1 promoter region have not been described previously. We
propose that the term “contiguous ABCD1 DXS1357E deletion syndrome” (CADDS) be used to identify this new
contiguous-gene syndrome. The three patients with CADDS who are described here have important implications
for genetic counseling, because individuals with CADDS may previously have been misdiagnosed as having an
autosomal recessive PBD or SED
X-linked adrenoleukodystrophy (X-ALD [MIM 300100])
is a recessive neurodegenerative disorder that has a hemi-
zygote frequency of 1:21,000 in the United States (Bezman
et al. 2001). Mutations in the ALD gene, ABCD1, are
associated with a broad spectrum of clinical phenotypes.
Childhood cerebral X-ALD has the earliest onset (mean
age 7.2 1.7 years) and most devastating effects but
Received March 1, 2002; accepted for publication March 19, 2002;
electronically published April 29, 2002.
Address for correspondence and reprints: Dr. Steven J. Steinberg,
The Peroxisomal Diseases Laboratory, The Kennedy Krieger Insti-
tute, 707 North Broadway, Baltimore, MD 21205. E-mail: SteinbergS
* The first two authors contributed equally to the clinical aspects
of this study.
2002 by The American Society of Human Genetics. All rights reserved.
does not manifest clinically until age 3 years (Moser et
al. 2001). ABCD1 encodes the peroxisomal integral-
membrane ALD protein (ALDP) and belongs to the ATP-
binding cassette (ABC) transporter superfamily (Mosser
et al. 1993). The four known peroxisomal ABC proteins
are considered to be half-transporters that probably re-
quire dimerization in order to function (Liu et al. 1999;
Smith et al. 1999). The ligands transported by these pro-
teins have not been determined, although the role of the
nucleotide-binding fold in ATP binding and hydrolysis has
been demonstrated in vitro (Roerig et al. 2001). The re-
lationship between mutations in ABCD1 and the reduced
capacity of X-ALD fibroblasts to catabolize very-long-
chain fatty acids (VLCFA) (carbon length
122) by b-ox-
idation remains unknown. Although it has been reported
that X-ALD peroxisomes have reduced very-long-chain
acyl-CoA synthetase activity (Lazo et al. 1988; Wanders
et al. 1988), ALDP does not function as a synthetase and
Corzo et al.: Contiguous ABCD1 and DXS1357E Deletion 1521
is not required for localization of the human enzyme to
the peroxisome (Steinberg et al. 1999a). Without excep-
tion, however, individuals hemizygous for ABCD1 mu-
tations accumulate VLCFA in plasma (Moser et al. 1999).
Although X-ALD is the most common peroxisomal
disorder, other peroxisomal diseases also can be identified
on the basis of elevated VLCFA levels in plasma. Indi-
viduals with peroxisomal biogenesis disorders (PBD)
such as Zellweger syndrome (ZS) or single-enzyme de-
ficiencies (SED) in the peroxisomal b-oxidation path-
way—such as acyl-CoA oxidase deficiency (OxD),
functional protein deficiency (BD), and b-ketothiolase
deficiency—all accumulate VLCFA in complex lipid frac-
tions in plasma and other tissues (Gould et al. 2001;
Wanders et al. 2001). In spite of the overlap in biochem-
ical phenotype, it has previously been possible to distin-
guish between these syndromes and those in individuals
with ABCD1 mutations, by measurements of other bi-
ochemical markers of peroxisomal function and by their
clinical presentation. For instance, individuals with PBD
fail to assemble mature peroxisomes and thus have de-
ficiencies in multiple peroxisomal pathways, including
plasmalogen synthesis,
-pipecolic acid metabolism,
branched-chain fatty-acid a-oxidation, and cholesterol
metabolism (Gould et al. 2001). Individuals with BD
have elevated plasma VLCFA and urinary bile-acid in-
termediates dihydroxycholestanoic acid (DHCA) and
trihydroxycholestanoic acid (THCA), and their cultured
fibroblasts have a reduced capacity to a-oxidize phytanic
acid (Wanders et al. 2001). In contrast, individuals with
X-ALD have both normal levels of other peroxisomal
analytes and normal peroxisome structure (Moser et al.
2001), although fibroblasts representing at least two-
thirds of nonrecurrent ABCD1 mutations are immuno-
negative for ALDP (Watkins et al. 1995a; Feigenbaum
et al. 1996; Kemp et al. 2001). Newborns with either
PBD or SED commonly have congenital abnormalities,
failure to thrive, hypotonia, and developmental delay,
and many die during the first months to years of life. In
contrast, newborns with ABCD1 mutations accumulate
VLCFA in the adrenal gland and plasma but appear
healthy at birth and thrive postnatally.
The vast majority of individuals with an increase in
plasma VLCFA levels who have been identified through
diagnostic screening are X-ALD hemi- or heterozygotes
(Moser et al. 1999). Nonetheless, the Peroxisomal Dis-
eases Laboratory at the Kennedy Krieger Institute inves-
tigates 40 individuals a year who have increased plasma
VLCFA and clinical symptoms suggestive of a PBD. Ap-
proximately 20% of these individuals have a biochemical
phenotype consistent with a b-oxidation SED other than
X-ALD (hereafter, “SED” will refer only to OxD and
BD) (Moser 1999). We have identified three patients
whose early clinical symptoms and initial biochemical
results appeared to be consistent with BD or OxD; how-
ever, a study of cultured fibroblasts demonstrated that
they had defects consistent with an ABCD1 mutation.
Elsewhere, we have reported, in abstract form, prelimi-
nary investigations of patients 1 and 2 (Corzo et al. 1999;
Steinberg et al. 2001). Here we describe the unique clin-
ical features in three patients, document the biochemical
evidence for a defect in fibroblast peroxisomal b-oxida-
tion, and describe molecular investigations establishing
that these patients have a contiguous deletion spanning
the 5
ends of ABCD1 and DXS1357E.
Material and Methods
Biochemical Analyses
Whole-blood samples were collected in EDTA tubes.
Plasma was isolated and used for analysis of VLCFA
-pipecolic acid, as described elsewhere (Kelley
1991; Moser and Moser 1991). Plasmalogens were mea-
sured in washed erythrocyte membranes by capillary gas
chromatography (Bjorkhem et al. 1986). Organic acids
were extracted from urine, were derivatized, and were
analyzed by gas chromatography/mass spectrometry
(Tanaka et al. 1980). Cultures were initiated and main-
tained in Eagle’s minimum essential medium supple-
mented with 10% fetal calf serum (Moser et al. 1995)
and were assayed for VLCFA content, C24:0 b-oxida-
tion, plasmalogen synthesis, phytanic acid oxidation,
and catalase solubility, as described elsewhere (Wanders
et al. 1984; Roscher et al. 1985; Moser and Moser 1991;
Zenger-Hain et al. 1992; Watkins et al. 1995b). Cho-
lesterol-ester fractions were extracted from whole tissue
and were analyzed by gas chromatography (Rasmussen
et al. 1994). Somatic-cell hybridization was conducted
by use of polyethylene glycol as a fusogen and ficoll for
gradient separation (Moser et al. 1995). All research
activities were approved by the Joint Committee of Clin-
ical Investigation, and parental consents were obtained.
Immunocytochemical Analyses of Cultured Fibroblasts
Cells were cultured on glass coverslips, were fixed with
formaldehyde, were permeabilized, and were incubated
with primary antibodies and fluorescein- or rhodamine-
conjugated secondary antibodies, as described elsewhere
(Watkins et al. 1995a).
ABCD1 DNA Analyses
Genomic DNA was isolated from cultured fibroblasts
by use of a Puregene kit (Gentra). The 10 exons of
ABCD1 were amplified, by PCR, as 10 amplicons and
were directly sequenced (Boehm et al. 1999). For South-
ern blot analysis, 5 mg of genomic DNA was digested
with BamHI, separated by agarose-gel electrophoresis,
transferred to nitrocellulose membrane, and hybridized
1522 Am. J. Hum. Genet. 70:1520–1531, 2002
by use of a full-length ABCD1 cDNA
Other DNA Analyses
STS oligonucleotide primers for PCR were synthesized
at the DNA Analysis Facility at Johns Hopkins Univer-
sity, on the basis of sequences reported in the STS da-
tabase at the National Center for Biotechnology Infor-
mation (dbSTS: database of “Sequence Tagged Sites”).
Reaction mixtures containing 0.1 mg of genomic DNA
were prepared as described elsewhere (Steinberg et al.
1999b), by use of the Expand High Fidelity system
(Roche). The following PCR program was used: 94C
for 4 min (1 cycle); 94C for 30 s, 66C for 1 min, and
72C for 1 min (35 cycles); and 72C for 10 min (1
cycle). Further analysis of DXS1357E exon 5 was per-
formed with PCR primers CDM1-F (5
) and CDM1-R (5
) and with sequencing primer CDM1-S (5
Clinical Presentation Similar to That of Individuals with
a PBD or a SED
Patients 1, 2, and 3 (hereafter denoted by “Pt1,”
“Pt2,” and “Pt3,” respectively) were born to healthy
women with no known family history of adrenoleuko-
dystrophy or other peroxisomal disease. None of the
parental relationships were consanguineous. All three
patients were male. Two of the patients were French
Canadian, and the third patient was Vietnamese. Table
1 summarizes the clinical features of these cases and
compares them to those of individuals with either ZS,
BD, OxD, or X-ALD. All boys had profound neonatal
hypotonia, subsequent failure to thrive, and cholestatic
liver disease. Liver biopsies in all patients showed intra-
canalicular and ductal cholestasis. Pt2 and Pt3 developed
seizures at age 2 mo. Pt1 did not have seizures, but did
experience frequent episodes of opisthotonos and brux-
ism. Provocative adrenal-function testing was not per-
formed on any of the patients. However, at autopsy
small, fibrotic adrenal glands (0.5 g, combined) were
found in Pt3. Limited gross autopsy findings were avail-
able on Pt3 only. The total body weight was low (2.19
kg), and the liver appeared small, fibrotic, and jaundiced.
The cut brain revealed white-matter abnormalities. Post-
mortem magnetic-resonance images, compared with im-
ages obtained several months earlier, indicated that my-
elination had progressed but that overall myelination
was significantly delayed. As part of the extensive eval-
uations of all three patients, VLCFA were measured.
Biochemical Analyses—Results Consistent with a SED
of Peroxisomal b-Oxidation
Plasma VLCFA analysis in Pt1, Pt2, and Pt3 showed
elevations consistent with a deficiency in peroxisomal b-
oxidation (table 2). Erythrocyte plasmalogens and plas-
-pipecolic acid were normal for all three patients
(table 2). Epoxydicarboxylic acids and 2-hydroxysebacic
acid were not detected in the urine organic-acid profile
of Pt3. Cultured fibroblasts from each patient demon-
strated VLCFA accumulation and a reduced capacity to
b-oxidize the VLCFA C24:0 (table 2). Each cell line had
a normal capacity for a-oxidation and plasmalogen syn-
thesis (table 2). Catalase solubility in fractionated cells
demonstrated normal peroxisomal localization (table 2)
and provided further support for the presence of a SED.
THCA and DHCA were not detected in the urine of Pt2
and Pt3. Total bile acids were increased in Pt2, and Pt 3
had a moderate increase in cholestanol. Results of liver
biopsies were consistent with cholestasis in all three pa-
tients. Peroxisomes were present in liver tissue, as dem-
onstrated by electron microscopy in Pt1 and Pt2.
Fibroblasts—Immunonegative for Peroxisomal ALD
Protein (ALDP)
When fibroblasts from these cases were evaluated by
immunocytochemistry with an antibody to ALDP, no
peroxisomal staining was seen (fig. 1BD). In contrast,
when cells were stained with antibody to the matrix
protein catalase, peroxisomes of normal size and number
were observed (see fig. 1FH). Furthermore, peroxi-
somes of normal size and number were visualized when
cells were treated with antibody to the peroxisomal ABC
protein PMP70 (fig. 1JL).
Failure of Complementation Studies with X-ALD
Fibroblasts to Correct the Defect
Somatic-cell hybridization studies were performed
with an ALDP immunonegative cell line derived from
an individual with X-ALD and the ABCD1 mutation
Nt1876GrA (A626T). Cells from Pt1 and Pt2 were
fused with the X-ALD cells and then were separated into
two fractions, one enriched with multinuclear cells and
the other containing mainly cocultivated mononuclear
cells. A cross between the X-ALD and a PBD cell line
was used as a positive control. The ability to b-oxidize
lignoceric acid (i.e., C24:0) was used as a marker for
correction. C24:0 b-oxidation was restored to normal
control levels in the cross with the PBD cell line, but
cells from Pt1 and Pt2 did not complement the known
X-ALD cell line (table 3).
Table 1
Clinical Features in Pt1, Pt2, and Pt3, Compared to Other Peroxisomal Disorders
Pt1 Pt2 Pt3
Ethnicity French Canadian French Canadian Vietnamese
Age at death 11 mo 4 mo 4 mo
!1 year 9 mo 4 years !1 year 9.4 years
Cause of death Liver failure and gastro-
intestinal bleeding
Respiratory failure and gas-
trointestinal bleeding
Liver and respiratory
Clinical symptoms:
Neonatal hypotonia ⫹⫹
Neonatal seizures
At age 2 mo At age 2 mo /⫺⫹
Craniofacial dysmorphism ⫺⫺
Intrauterine growth retardation ⫺⫺Severe /⫺⫺
Liver disease ⫹⫹/⫺⫺
Neonatal cholestasis
⫹⫺ /⫺⫺
Cataract ⫹⫺/⫺⫺
Sensorineural deafness ⫹⫹/⫺⫹/⫺⫹/⫺⫹/⫺⫺
Reference Watkins et
al. (1995b)
Watkins et
al. (1995b)
Gould et al.
Moser et al.
For childhood cerebral disease; average of 167 patients.
Aplussign() denotes presence; a minus sign () denotes absence; a plus/minus sign (/) denotes that the clinical symptom may or may not be present (and is used only when
multiple patients are described).
Frequent episodes of opisthonos and bruxism.
Neonatal cholestasis, documented by liver biopsy.
Increased plasma and urine bile-acid levels and large amounts of several unknown compounds.
Mild increase in cholestanol, which can be related to defects in bile-acid synthesis.
1524 Am. J. Hum. Genet. 70:1520–1531, 2002
Table 2
Peroxisomal Biochemical Profile in Pt1, Pt2, and Pt3
Assay Units Pt1 Pt2 Pt3 Control PBD X-ALD
Blood analytes:
VLCFA mg/ml, for C26:0 2.09 2.48 2.98 .22.08 3.311.63 1.18.53
mg/ml, for C26:1 2.02 1.86 2.34 .12.05 1.55.55 .19.05
C24/C22 1.56 1.65 1.85 .84.08 1.95.42 1.49.45
C26/C22 .16 .21 .18 .01.01 .52.24 .07.04
C16 DMA/C16 .054 .059 .050 .051-.090 .001-.025 NM
C18 DMA/C18 .14 .151 .118 .137-.255 .001-.050 NM
-Pipecolic acid Micromolar 1.2 .2 1.1 1.8.9
VLCFA mg/mg protein, for C26:0 .335 .535 .329 .07.04 .87.44 .42.15
mg/mg protein, for C26:1 .116 .216 .057 .09.07 1.06.72 .17.1
C26/C22 .731 .828 .728 .08.03 1.00.33 .69.19
C24:0 b-oxidation nmol/h/mg protein .229 .298 .109 1.16.166 .10.05 .31.06
Plasmalogen synthesis
C .56 .93 .7 .67.19 9.924.4 NM
Phytanic acid oxidation % of control value 106 99.3 123 100 2.2 NM
Catalase solubility % Soluble 18 22 26
!25 185 NM
.—For each test and each disease category, the results are for 10 cases—except in the case of b-oxidation, for which the
results are from 10 control cell lines, 7 PBD cell lines, and 7 X-ALD cell lines.
NM p not measured routinely.
DMA p dimethylacetal.
Patients were 1–6 mo of age.
DNA Sequencing and Southern Blot Analysis—
Identification of Large ABCD1 Deletions
To determine whether patients had a mutation in the
ABCD1 gene, the 10 exons were amplified and directly
sequenced. We were unable to amplify product for exons
1–10 of Pt1 and Pt3 or for exons 1–5 of Pt2. No se-
quence variation was detected in exons 6–10 of Pt2.
Since we were able to amplify other gene targets from
the purified DNA for Pt1 and Pt3, the failed ABCD1
reactions suggested the presence of a large gene deletion.
Southern blot analysis was performed as described
above (see the Material and Methods section), by use of
a full-length ABCD1 cDNA probe. This region of Xq28
has undergone interchromosomal duplication several
times (Eichler et al. 1997). Loci sharing high sequence
identity to the 3
end of ABCD1 are found on 2p11,
10p11, 16p11, and 22q11. To discriminate Xq28 ge-
nomic DNA from the autosomal partial homologs, val-
idation studies were performed by use of the cell line
AHA11a, a mouse/human somatic hybrid harboring the
human X chromosome (Dorman et al. 1978; Smith et
al. 1999). AHA11a DNA had three bands that corre-
sponded to the three expected fragment sizes (10, 8.3,
and 5.6 kb, respectively) for BamHI digestion of human
Xq28 and one band that appeared in the wild-type
mouse alone (fig. 2B). In addition to the three bands
corresponding to ABCD1, the lane for the human con-
trol subject contained two bands that represent cross-
reactivity of the cDNA probe to autosomal homolog
DNA fragments. Pt1 and Pt3 lacked the three ABCD1
bands corresponding to all 10 exons (fig. 2B). In con-
trast, Pt2 lacked bands corresponding to exons 1–6 but
retained the 8.3-kb band representing exons 6–10 (fig.
2B). The two bands that represent DNA fragments from
autosomal partial homologs were present in all three
affected patients and serve as an internal control for the
Carrier Status of the Mothers
The mother of Pt1 had a second pregnancy with a
male fetus. Prenatal diagnosis of cultured amniocytes
indicated that the fetus had a defect in peroxisomal fatty-
acid metabolism (table 4), and the pregnancy was ter-
minated (Moser and Moser 1999). VLCFA comprised
36.6% of the total fatty acids in the cholesterol-ester
fraction extracted from fetal adrenal, compared with the
normal control mean of 1.02% ( ) (Moser et al.n p 4
1982). Thus, the mother of Pt1 is the only obligate het-
erozygote among these three women. Plasma and fibro-
blast VLCFA were normal in all three mothers. The dis-
criminant function for females, on the basis of three
plasma measures (Moser et al. 1999), was 4.6–4.8 for
these mothers, compared with a median normal value
of 5 ( ) and obligate-heterozygote level ofn p 11,800
9 ( ). In fibroblasts immunohistochemicallyn p 281
stained for ALDP, a normal peroxisomal pattern was
visualized in 75%, 94%, and
199% of cells from the
mothers of Pt1, Pt2, and Pt3, respectively (not shown).
Southern blot analysis of the mothers of Pt1 and Pt2
confirmed that, on one allele, they have the same deletion
as is seen in their sons (results not shown). In contrast,
Southern blot analysis of the mother of Pt3 showed that
she is not a carrier of the ABCD1 deletion detected in
her son.
Corzo et al.: Contiguous ABCD1 and DXS1357E Deletion 1525
Table 3
Complementation Analysis
C24:0 b-Oxidation in Mulinuclear
Cells/Mononuclear Cells [Ratio]
(nmol/h/mg protein)
Cell line 1/cell line 2:
X-ALD/PBD 1.338/.214 [6.3]
X-ALD/Pt1 .329/.361 [0.9]
X-ALD/Pt2 .372/.418 [0.9]
Control (not fused) NA/1.267
Cell line 1 is immunonegative for ALDP and is derived
from an individual with X-ALD who had classic childhood
cerebral onset and ABCD1 mutation Nt1876GrA. PBD p
cell line from an individual with ZS who had deficiencies in
multiple peroxisomal pathways and lacked peroxisomes.
NA p not applicable.
Figure 1 Peroxisomal proteins in cultured fibroblasts evaluated by immunocytochemical analysis. Fibroblasts were double-labeled for
ALDP and catalase and were labeled separately for PMP70. A–D, ALDP, a protein encoded by ABCD1. Pt 1–Pt3 are immunonegative for this
protein. Two-thirds of ABCD1 mutations result in immunonegative status (Watkins et al. 1995a; Feigenbaum et al. 1996). E–H, Catalase, a
peroxisomal matrix protein. Cells from Pt1, Pt2, and Pt3 and from a control subject have normal peroxisome size and number. In the control-
subject panel, the catalase signal colocalizes with that seen for ALDP. I–L, PMP70, a peroxisomal membrane protein that belongs to the same
ABC transporter subtype as does ALDP. Compared with those from the control subject, cell lines from Pt1, Pt2, and Pt3 have normal PMP70
localization. All cells were visualized at 1,000# magnification.
Further Molecular StudiesIndications That Xq28
Deletions Extend Beyond ABCD1
To determine whether the deletion in these three pa-
tients extended beyond the 5
region of ABCD1, two STS
markers upstream from the ABCD1-gene promoter were
selected for PCR analysis (fig. 2C). These markers are
associated with two genes that are immediately upstream
of ABCD1. Marker stSG39985 is in intron 4 and very
near exon 4 of DXS1357E, a gene that is in a head-to-
head orientation with ABCD1 (Mosser et al. 1994). The
stSG4965 marker lies at the extreme 3
end of SLC6A8,
a gene that encodes a creatine transporter. Mutations in
this gene cause a creatine-deficiency syndrome that im-
pairs neurological function (Salomons et al. 2001). The
marker in the middle of DXS1357E failed to amplify in
all three patients (fig. 2D) and indicated that these dele-
tions extend beyond exon 1 and the promoter of ABCD1
and into the neighboring, DXS1357E gene.
To verify and further delineate the deletions in this re-
gion, DXS1357E exon 5 was selected for PCR amplifi-
cation (figs. 2D and 3). A 26.5-kb duplication from Xq28
that includes the coding regions of DXS1357E and
SLC6A8 is located on 16p11 (Eichler et al. 1996). The
DXS1357E homolog on 16p11 appears to be a pseu-
Corzo et al.: Contiguous ABCD1 and DXS1357E Deletion 1527
Table 4
Prenatal Diagnosis in the Mother of Pt1
Source of Amniocytes
(mg/mg protein) C26:0/C22:0
Male fetus and mother of Pt1 .43 .64
Unaffected male subjects ( )
n p 127 .10 .07 .17 .10
X-ALD hemizygotes ( )
n p 45 .47 .17 1.06 .48
Source.—Moser and Moser (1999).
Figure 2 Large ABCD1 deletions that extend into the coding region of DXS1357E, in Pt1, Pt2, and Pt3. A, ABCD1, which spans 24
kb and has 10 exons (Sarde et al. 1994). Genomic DNA BamHI digestion was predicted to yield three ABCD1 fragments from Xq28. B, Results
of Southern blot validation studies and analyses of patients. Validation studies were conducted to identify genomic fragments containing DNA
from autosomal homologs (Eichler et al. 1997; Smith et al. 1999) that might cross-react with the full-length ABCD1 cDNA probe. DNA from
wild-type mouse, from a mouse cell line harboring one human X chromosome (AHA11a [Dorman et al. 1978]), and from a human control
subject (Ctl) were analyzed simultaneously. Four bands appeared for the AHA11a DNA, one corresponding to the band from wild-type mouse
and three matching the predicted sizes (i.e., 10, 8.3, and 5.6 kb) of fragments for ABCD1 on Xq28 and aligning with bands in Ctl. The two
additional bands in Ctl correspond to autosomal homologs that cross-react with the cDNA probe, since they did not appear in the mouse
hybrid cell line harboring the human X chromosome. Pt1 and Pt3 lack the three ABCD1 bands corresponding to all 10 exons. In contrast, Pt2
lacks bands corresponding to exons 1–6 but retains the 8.3-kb band representing exons 6–10. Ex p exons. C, DNA map for Xq28, showing
the location of four STS markers (gels A, B, D, and G), in relation to ABCD1 and surrounding genes (se the Entrez Nucleotide Web site of
the National Center for Biotechnology Information [accession number U52111]). In addition, the location of ABCD1 exons 2 and 10 (gels E
and F, respectively), the boundary between ABCD1 intron 5 and exon 6 (denoted by the asterisk [*]), and DXS1357E exon 5 (gel C) are shown.
DXS1357E shares a CpG island and is in a head-to-head orientation with ABCD1 (Mosser et al. 1994). D, PCR primers for the markers shown
were used for analysis in Pt1, Pt2, and Pt3 and in a male control subject (Ctl). These results are summarized in figure 3. E, Amplification of
template for Xq28 and 16P11. Because of sequence homology between Xq28 and 16p11, the primers for reaction C (DXS1357E exon 5)
amplify both templates. Compared with DNA from the control subject and Pt2, that from Pt1 and Pt3 yielded a small amount of product.
When the amplicon from Pt1 and Pt3 was sequenced by use of primer CDM-1S (see the “Material and Methods” section), a 16p11-specific
sequence was revealed, whereas the products from Pt2 and the control subject had DNA sequence specific to Xq28. Sequence specific to 16p11
was detectable only in the absence of the corresponding Xq28 region.
dogene (Eichler et al. 1997), but the 16p11 paralog to
SLC6A8 encodes a testes-specific transcript, and the gene
is named SLC6A10 (Iyer et al. 1996). DXS1357E exon
5 on Xq28 has 93% sequence identity to the 16p11 para-
log. The degree of sequence identity between the PCR
primers for the DXS1357E exon 5 reaction and exon 5
of both Xq28 and 16p11 suggests that both regions would
be amplified if both were present, although the primers
were designed to favor the Xq28 sequence. The exon 5
reaction yielded an intense band of the expected size for
the control subject and for Pt2 and yielded a barely de-
tectable amplicon for Pt1 and Pt3. Results from sequenc-
ing initiated by primer CDM-1S (see the “Material and
Methods” section, above) are shown across a region that
clearly distinguishes between 16p11 and Xq28 (fig. 2E).
The exon 5 product in Pt1 and Pt3 had a 16p11-specific
sequence, whereas Pt2 and the control amplicons were
Xq28 specific. The PCR primers and reaction conditions
favored the amplification of the Xq28 DNA to such an
extent that the 16p11 sequence was detectable only when
the homologous Xq28 region was deleted.
DXS1357E exon 5 was present in Pt2 and indicated
that the 5
breakpoint for this deletion resides between
exons 4 and 5. Since the 3
breakpoint resides between
ABCD1 exons 5 and 6, these studies predict, overall,
that the deletion in Pt2 spans 22.036.2 kb (fig. 3). This
deletion is the smallest in the three patients investigated
and suggests that the critical region responsible for this
disorder is between DXS1357E exon 5 and ABCD1
exon 6.
The three male patients reported here were referred to
the Peroxisomal Diseases Laboratory for evaluation of
an inborn error of peroxisomal function. All three new-
borns were profoundly hypotonic and developmentally
delayed, failed to thrive, and had cholestatic liver dis-
ease. The atrophied adrenal glands in Pt3 and the high
level of VLCFA in adrenals from the fetus from the
mother of Pt1 suggest that these patients may also have
had adrenal insufficiency (Govaerts et al. 1984; Moser
et al. 2001). Testing these patients to rule out either a
PBD such as ZS or a related SED was appropriate
(Moser and Raymond 1998). Each patient had both an
accumulation of VLCFA in plasma and fibroblasts and
reduced peroxisomal b-oxidation rates in fibroblasts, but
other peroxisomal biochemical pathways were normal,
and matrix-protein import remained intact. The bio-
chemical profile in blood, urine, and cultured fibroblasts
from Pt1, Pt2, and Pt3 supported the diagnosis of an
isolated deficiency in peroxisomal fatty-acid metabolism.
The neonatal onset of disease in Pt1, Pt2, and Pt3
appeared, on a clinical basis, to exclude X-ALD or a
mutation in ABCD1, because 2.75 years is the earli-
est age at onset of neurological symptoms that has been
observed in
12,000 documented patients with X-ALD
(H.W.M., unpublished data). Furthermore, cholestatic
liver disease has never been described in X-ALD. In
contrast to these differences, the biochemical abnor-
malities in the newborns in the present study more
1528 Am. J. Hum. Genet. 70:1520–1531, 2002
Figure 3 CADDS critical region, which lies between DXS1357E
exon 5 and ABCD1 exon 6. The minimum and maximum extent of
Xq28 deletion, on the basis of PCR and Southern blot analyses, is
shown for each patient. The black segments denote DNA demonstrated
to be absent. The horizontal dotted lines extending from the ends of
each box denote regions that have yet to be excluded. An enlargement
of the critical region that is missing in all three patients is shown. The
smallest deletion occurs in Pt2 and spans 22.0 36.2 kb.
closely resembled those in X-ALD. The bile-acid inter-
mediates DHCA and THCA, which are increased in BD
(Natowicz et al. 1996), were not detected in the urine
of Pt2 and Pt3. The epoxydicarboxylic acids frequently
observed in PBD and SED urine (Yamaguchi et al. 2001)
were absent in Pt3. All three patients had normal phy-
tanic acid oxidation, which is diminished in BD and, to
a lesser extent, in OxD (A.B.M., unpublished data).
Unlike the usual pattern in X-ALD, plasma levels of
C26:1 were increased in the patients reported here, a
difference that may be due to liver disease.
The absence of immunocytochemical staining for
ALDP in fibroblasts from Pt1, Pt2, and Pt3 provided the
first clue that there was a primary defect in ABCD1.
PMP70 and catalase localized to the peroxisome in each
cell line, establishing normal peroxisome assembly. The
normal localization of PMP70 argues against a unique
generalized assembly defect that would affect the routing
of multiple peroxisomal ABC proteins to the membrane.
Mutation identification in the three patients was ham-
pered initially by a failure to amplify all or most of the
exons. Southern blot analysis demonstrated that these
patients had large deletions that encompassed exons
1–10 of Pt1 and Pt3 and exons 1–5 of Pt2. Two-hundred
forty-six nonrecurrent ABCD1 mutations have been re-
ported (Kemp et al. 2001), only 4.5% of which rep-
resent deletions encompassing at least one entire exon.
There has been a single report of a partial deletion in-
volving exon 1 (Koike et al. 1995). This deletion elim-
inated 0.5 kb of sequence located near the 3
end of the
11,286-nt exon. The individual presented at age 19
years, with cerebellar and brain-stem signs and thus is
phenotypically and genotypically distinct from our three
patients. Although mutations in exon 1 occur at the
same per-nucleotide rate as in other exons (X-linked
Adrenoleukodystrophy Database), deletions involving
exon 1 are rare, and, to our knowledge, a complete
deletion of exon 1 has not previously been reported.
PCR analysis with STS markers established that the
first four exons of DXS1357E were absent in Pt1, Pt2,
and Pt3 (fig. 3), indicating that the ABCD1 deletions
extended beyond exon 1 and the promoter region. The
extent of the deletions was further supported by PCR
analyses, which demonstrated absence of DXS1357E
exon 5 in Pt1 and Pt3. These three cases are the first
described with ABCD1 deletions that extend beyond
the initiation codon. Pt2 had normal sequence for
ABCD1 exons 6–10, indicating that the genomic DNA
downstream of ABCD1 exon 5 is unlikely to be in-
volved in the pathogenetic mechanism. The smaller de-
letion size in Pt2 suggests that the critical region for this
novel phenotype is restricted to a 36.2-kb span residing
between DXS1357E exon 5 and ABCD1 exon 6 (fig.
3). Mutation of DXS1357E has not been reported pre-
viously in association with human disease, but its partial
deletion may be implicated in the new phenotype. Its
1.5-kb transcript is reportedly ubiquitously expressed
(Mosser et al. 1994). The translated protein is a B-cell
antigen receptor–associated protein, BAP31, that het-
erodimerizes with BAP29 (Adachi et al. 1996). Exten-
sive studies of the expression and function of this pro-
tein have not been reported in the literature.
Deletions involving the 5
ends of both ABCD1 and
DXS1357E suggest that this neonatal phenotype is due
to a contiguous-gene defect. We propose using the term
“contiguous ABCD1 DXS1357E deletion syndrome”
(CADDS) to describe this disorder. We strongly advise
against use of the term “neonatal X-ALD,” to avoid fur-
ther confusion between the terms “X-ALD” and “neo-
natal adrenoleukodystrophy,” a PBD. In addition, the
primary cause of neurological impairment in CADDS
most likely is not a leukodystrophy. The brain of Pt3,
for example, appeared to have delayed myelination. Fur-
ther studies to delineate the breakpoints of these deletions
and to explore their impact on gene expression are in
progress and may shed some light on either the ABCD1
promoter or the function of ALDP. Nevertheless, the
elimination of the promoter regions of both DXS1357E
and ABCD1 provides a putative mechanism for the new
phenotype. The early presentation and phenotypic sim-
ilarity of these three patients, which are distinct from
those of all previously described individuals with ABCD1
mutations, strongly suggest that DXS1357E or another
Corzo et al.: Contiguous ABCD1 and DXS1357E Deletion 1529
nearby gene regulated by this region contributes to this
neonatal phenotype.
There is no direct correlation between ABCD1 mu-
tations and clinical phenotype. The childhood- and
adult-onset forms are frequently found in the same kin-
dred (Moser et al. 2001). Although there is evidence for
an autosomal modifier gene (Smith et al. 1991; Maestri
and Beaty 1992), so far this has not been identified.
Recently, O’Neill et al. (2001) reported a kindred in
which there is a point mutation in the ABCD1 initiator
methionine and in which all affected members have
The three patients hemizygous for this 5
ABCD1 de-
letion are relatively uniform in their clinical phenotype.
They all had neonatal cholestasis and profound hypo-
tonia and died at age
!1 year. Nevertheless, at this time
we cannot rule out the possibility of a genotypic and
phenotypic spectrum that might include children who
have less neurological or liver impairment (or both) in
the neonatal period. Likewise, there may be heterozy-
gous girls with an unfavorable X-inactivation pattern
who are less severely affected than are the hemizygotes.
The origin and the precise mechanism of the deletion
in these three unrelated patients likely vary, because the
preliminary data indicate that the breakpoints are dif-
ferent for all three.
These three cases highlight the importance of careful
investigation of any individual presenting, during the
neonatal period, with a SED affecting VLCFA b-oxi-
dation. Although clinical findings in the patients re-
ported here resemble PBD, they differ profoundly with
respect to genetic counseling, because their mode of in-
heritance is X-linked recessive and not autosomal re-
cessive as in PBD. Pedigree analysis may help in the
identification of families that demonstrate an X-linked
pattern of inheritance, but, in the three families that we
thus far have identified, the only symptomatic individ-
ual was the index case. Although two of the mothers
are carriers, they may have mutations that arose from
the paternal gamete. Since both BD and OxD are au-
tosomal recessive disorders, at-risk families do not be-
come aware of their risk until the first affected child is
born. Prenatal diagnosis is available for these couples,
but, because of the low carrier frequency, further coun-
seling for the extended family is generally not required.
In families with a history of an X-linked disorder, it is
crucial to determine the origin of the mutation, so that
appropriate counseling can be offered to those at risk.
If our three patients had been left with the label of
having a SED other than X-ALD, then appropriate
counseling could not be offered.
We would like to thank Dr. Gerardo Jimenez-Sanchez for
PMP70 antibody; Drs. Richard Kelley and Lisa Kratz for
plasma cholesterol quantitation; Drs. Kenneth Setchell and
Gerald Salen for bile-acid analysis; Dr. Antonio R. Perez for
electron microscopy of liver on Pt 2; Dr. George Thomas for
urine organic acid analysis; Dr. David Valle for thoughtful
discussion; and Sheila Foreman, Anita Liu, Surinder Khan-
goora, and Anisa Chaudhry for their technical expertise in the
laboratory. This work was supported in part by U.S. Public
Health Service grants HD 10981 and RR 00052.
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    • "In fibroblasts of female ABCD1 carriers, a mosaic pattern can be observed due to Xchromosome inactivation (Kemp et al 2012). In patients with CCADS (contiguous ABCD1 DXS1357E (BAP31) deletion syndrome, OMIM 300475) there is also no expression of ALDP (Corzo et al 2002). The only ABCD3-deficient patient was initially diagnosed by the serendipitous finding of absence of ABCD3 staining upon routine immunofluorescence microscopy analysis when screening for peroxisomal disorders in fibroblasts was performed (Ferdinandusse et al 2015). "
    [Show abstract] [Hide abstract] ABSTRACT: Peroxisomes are dynamic organelles that play an essential role in a variety of metabolic pathways. Peroxisomal dysfunction can lead to various biochemical abnormalities and result in abnormal metabolite levels, such as increased very long-chain fatty acid or reduced plasmalogen levels. The metabolite abnormalities in peroxisomal disorders are used in the diagnostics of these disorders. In this paper we discuss in detail the different diagnostic tests available for peroxisomal disorders and focus specifically on the important role of biochemical and functional studies in cultured skin fibroblasts in reaching the right diagnosis. Several examples are shown to underline the power of such studies.
    Full-text · Article · Mar 2016
    • "Patients with ABCD1 mutations, the most common peroxisomal βoxidation disorder (better known as X-linked adrenoleukodystrophy) [40], and a single patient with SCPx deficiency [41] do not develop liver pathologies. However, in CADDS [42,43] , a clinical lethal phenotype caused by a contiguous deletion of both ABCD1 and BCAP1 (originally called DXS1357E) liver dysfunction occurs with fibrosis and cholestasis [43,44]. The hepatic cholestasis seems due to a synergism, occurring when both ABCD1 and BCAP1 are deleted [45] . "
    [Show abstract] [Hide abstract] ABSTRACT: The peroxisomal compartment in hepatocytes hosts several essential metabolic conversions. These are defective in peroxisomal disorders that are either caused by failure to import the enzymes in the organelle or by mutations in the enzymes or in transporters needed to transfer the substrates across the peroxisomal membrane. Hepatic pathology is one of the cardinal features in disorders of peroxisome biogenesis and peroxisomal β-oxidation although it only rarely determines the clinical fate. In mouse models of these diseases liver pathologies also occur, although these are not always concordant with the human phenotype which might be due to differences in diet, expression of enzymes and backup mechanisms. Besides the morphological changes, we overview the impact of peroxisome malfunction on other cellular compartments including mitochondria and the ER. We further focus on the metabolic pathways that are affected such as bile acid formation, dicarboxylic acid and branched chain fatty acid degradation. It appears that the association between deregulated metabolites and pathological events remain unclear.
    Full-text · Article · Oct 2015
    • "Defects in ABCD1 have been shown to be associated with impaired peroxisomal β-oxidation and accumulation of saturated VLCFA in all tissues of the body, and are considered to be the underlying cause of ALD [1,3]. ALD is a rare X-linked demyelinating disorder affecting the nervous system, adrenal cortex and testis [1,2], and is characterized by variation in phenotypic expres- sion [6,8,9] . To date, several clinical forms have been reported in male patients (Table 5) [3,4,101112. "
    [Show abstract] [Hide abstract] ABSTRACT: Background We report on two brothers with a distinct syndromic phenotype and explore the potential pathogenic cause.Methods Cytogenetic tests and exome sequencing were performed on the two brothers and their parents. Variants detected by exome sequencing were validated by Sanger sequencing.ResultsThe main phenotype of the two brothers included congenital language disorder, growth retardation, intellectual disability, difficulty in standing and walking, and urinary and fecal incontinence. To the best of our knowledge, no similar phenotype has been reported previously. No abnormalities were detected by G-banding chromosome analysis or array comparative genomic hybridization. However, exome sequencing revealed novel mutations in the ATP-binding cassette, sub-family D member 1 (ABCD1) and Dachshund homolog 2 (DACH2) genes in both brothers. The ABCD1 mutation was a missense mutation c.1126G¿>¿C in exon 3 leading to a p.E376Q substitution. The DACH2 mutation was also a missense mutation c.1069A¿>¿T in exon 6, leading to a p.S357C substitution. The mother was an asymptomatic heterozygous carrier. Plasma levels of very-long-chain fatty acids were increased in both brothers, suggesting a diagnosis of adrenoleukodystrophy (ALD); however, their phenotype was not compatible with any reported forms of ALD. DACH2 plays an important role in the regulation of brain and limb development, suggesting that this mutation may be involved in the phenotype of the two brothers. Conclusion The distinct phenotype demonstrated by these two brothers might represent a new form of ALD or a new syndrome. The combination of mutations in ABCD1 and DACH2 provides a plausible mechanism for this phenotype.
    Full-text · Article · Sep 2014
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