American Journal of Medical Genetics 132A:369–375 (2005)
A Splicing Mutation in the a/b GlcNAc-1-
Phosphotransferase Gene Results in an Adult
Onset Form of Mucolipidosis III Associated
With Sensory Neuropathy and Cardiomyopathy
Richard A. Steet,1Roger Hullin,2Mariko Kudo,3Michele Martinelli,2Nils U. Bosshard,5
Thomas Schaffner,4Stuart Kornfeld,1* and Beat Steinmann5
1Department of Internal Medicine, Washington University School of Medicine, Saint Louis, Missouri
2Department of Cardiology, University Hospital, Bern, Switzerland
3Genzyme Corporation, Oklahoma City, Oklahoma
4Institute of Pathology, Medical School, University Bern, Bern, Switzerland
5Division of Metabolism and Molecular Pediatrics, University Children’s Hospital, Zurich, Switzerland
A47-year-old female who presented with adilated
cardiomyopathy and mild neuropathy was found
to have pseudoHurler polydystrophy (mucolipi-
dosis III). The serum lysosomal enzymes were
strikingly elevated and GlcNAc-1-phosphotrans-
ferase activity in the patient’s fibroblasts was 3%
of normal. Sequence analysis of the patient’s
genomic DNA revealed a homozygous mutation
of the last nucleotide of the 135-bp exon 7 of the
phosphotransferase gene encoding the a/b sub-
units, resulting in aberrant splicing and skipping
of this exon. Remarkably, none of the skeletal and
connective tissue anomalies characteristic of the
and further broadens the clinical spectrum of the
? 2005 Wiley-Liss, Inc.
KEY WORDS:mucolipidosis III; lysosomal sto-
rage disorder; GlcNAc-1-phospho-
I-cell disease (mucolipidosis II, ML II; MIM 252500) and
pseudoHurler polydystrophy (mucolipidosis III, ML III; MIM
252600) are autosomal recessive lysosomal storage disorders
defined by decreased activity of the N-acetylglucosamine
enzyme in the synthesis of the mannose 6-phosphate target-
ing signal for lysosomal enzymes [Kornfeld and Sly, 2001].
Unlike lysosomal storage disorders affecting single enzymes
in a catabolic pathway, these disorders are characterized by
impaired sorting of multiple enzymes to lysosomes and their
subsequent secretion from cells. ML II and ML III share
similar clinical features including skeletal abnormalities.
Mucolipidosis II is clinically more severe, often noted shortly
after birth, whereas mucolipidosis III has a later onset,
normally in early childhood, and a slower course.
The targeting of acid hydrolases to lysosomes requires the
synthesis of mannose 6-phosphate residues on the oligosac-
charides of these enzymes [Kornfeld, 1986]. This post-transla-
tional modification of high-mannose type oligosaccharides of
enzymes, GlcNAc-1-phosphotransferase and phosphodiester
glycosidase (uncovering enzyme). The first enzyme catalyzes
the transfer of GlcNAc-1-phosphate to mannose residues on
the oligosaccharides; the second enzyme removes the outer
GlcNAc residues to produce the phosphomonoester mannose-
6-phosphate [Kornfeld et al., 1982]. GlcNAc-1-phosphotrans-
ferase exists as a complex containing three distinct subunits
(a2, b2, g2) [Bao et al., 1996]. The a/b subunits, encoded by one
gene, contain the catalytic activity of the enzyme while the
regulatory gsubunit, encoded byaseparate gene, is thought to
be responsible for binding specifically to lysosomal enzymes.
with dilated cardiomyopathy and mild neuropathy but none of
asplicejunction ofthe a/bsubunit GlcNAc-1-phosphotransfer-
ase gene was identified in the patient that causes skipping of
exon 7 and results in deficient enzyme activity in patient
fibroblasts. Our findings suggest that genetic variants of ML
MATERIALS AND METHODS
Primary cell lines were established from patient and control
skinfibroblasts.Thefibroblasts weremaintainedat 378Cinan
atmosphere of 5% CO2in aMEM supplemented with 10% fetal
All reagents were obtained from Sigma, Saint Louis,
Missouri, USA unless otherwise noted. [32P]-UDP-GlcNAc
was kindly provided as a gift by Dewan Haque at Genzyme
(Oklahoma City, OK). Cathepsin D anti-serum was provided
by Walter Gregory (Kornfeld Laboratory, Washington Uni-
versity School of Medicine, Saint Louis, Missouri).
The activity of the following enzymes in serum, leukocytes,
and fibroblasts was determined fluorimetrically according
Richard A. Steet and Roger Hullin contributed equally to this
*Correspondence to: Stuart Kornfeld, Department of Internal
Medicine, Washington University School of Medicine, 660 S.
Euclid Avenue, Saint Louis, MO 63110.
Received 25 May 2004; Accepted 15 October 2004
? 2005 Wiley-Liss, Inc.
to Galjaard : a-iduronidase (EC 126.96.36.199), a-fucosidase
(EC 188.8.131.52), a-galactosidase (EC 184.108.40.206), b-galactosidase
220.127.116.11), and b-hexosaminidase (EC 18.104.22.168). Assay of acid
phosphatase (EC 22.214.171.124) was done according to Beutler et al.
GlcNAc-1-phosphotransferase activity was measured in
microsomal preparations from fibroblasts as described earlier
[Reitman and Kornfeld, 1981]. Briefly, cells were lysed by
sonication in Tris buffer pH 7.5 containing 0.25M sucrose and
microsomes prepared by ultracentrifugation. Reactions using
a-methylmannoside as an acceptor and [32P]-UDP-GlcNAc
as substrate were conducted in the following reaction buffer:
50 mM Tris pH 7.5, 0.3% Lubrol, 50 mM N-acetylglucosamine,
250 mM DTT, 20 mg/ml bovine serum albumin, 10 mM sodium
molybdate, 1 mM MgCl2, and 1 mM MnCl2. Activity units are
calculated as pmoles of GlcNAc-[32P] transferred/hr. Protein
concentration was measured using the micro BCA protein
assay kit (Pierce, Rockford, IL, USA).
Analysis of Phosphorylated
Fibroblasts cultures (80%–90% confluent) were labeled in
(ICN Radiochemicals, Irvine, CA) for 4 hr followed by a 1 hr
chase in complete media. Cell pellets were harvested and
extracted sequentially with 2?2 ml chloroform:methanol
(2:1), 50% ethanol, and chloroform:methanol:water (10:10:3).
The remaining protein pellet was solubilized in 50 mM Tris
pH 6.8, 0.5% SDS by boiling and incubated overnight at 378C
with 2 U of endoglycosidase H (New England Biolabs, Beverly,
MA, USA) following addition of citrate buffer, pH 5.5. Endo
H-sensitive oligosaccharides were separated from resistant
material using a Centricon10 filtration unit. The Endo H-
sensitive oligosaccharides were analyzed by QAE-sephadex
chromatography following mild acid hydrolysis (pH 2.0, 1008C
for 30 min) which removes any covering GlcNAc residues
[Cantor et al., 1992]. In some cases, samples were digested
overnight with 10 U of alkaline phosphatase prior to analysis.
Cathepsin D Sorting
Metabolic labeling and sorting of cathepsin D in control and
patient fibroblasts was done as previously described [Braulke
etal., 1987]. Briefly, cell monolayers at ?70%–80% confluency
were washed twice with cysteine/methionine-free DMEM
and labeled for 1 hr with 1 ml of 1 mCi/ml Tran35S-label
(ICN Radiochemicals) in the presence of 20 mM Hepes and
5 mM mannose 6-phosphate. Excess unlabeled methionine
(10 mM final concentration) was added to initiate a 4 hr chase.
The amount of cathepsin D sorted was determined by over-
night immunoprecipitation of equivalent aliquots of the lysed
cell and media high-speed supernatants at 48C with rabbit
anti-human cathepsin D antiserum and protein A-Sepharose
beads (Repligen, Cambridge, MA). After five washes with
buffer containing 1M KCl and 1% Triton X-100, immuno-
precipitates were subjected to 10% SDS–PAGE under non-
reducing conditions. Gels were treated with EN3HANCE
(PerkinElmer Life Sciences, Boston, MA, USA), dried, and
exposed to a Phosphoimager screen (Molecular Dynamics,
Piscataway, NJ, USA). Quantitation of the various bands was
performed using Phosphoimager software. The percentage of
cathepsin D sorted was calculated as the ratio of radioactivity
in the processed form to the sum of the processed and secreted
The sequence of GlcNAc-1-phosphotransferase g subunit
[Raas-Rothschild et al., 2000] with the exception of the
first primer. 50-CTGGCGCGGCTCCTGTTGCTC-30was used
instead. In all cases, total RNA was isolated using the Trizol
method and single-stranded cDNA generated using Super-
script RTII system (Invitrogen, Carlsbad, CA, USA). PCR
amplification and sequencing of the gene encoding GlcNAc-1-
phosphotransferase subunits a and b was done using primer
sets to be reported elsewhere (Kudo et al., submitted). The
identified by RT-PCR using primer sets specific for regions
exon 7 (EX7R: 50-GCATTAGCACTAATCCAGGG-30) as well as
8 (EX8R: 50-CTGCTTAGACTGGCTGATGG-30). Skipping of
exon 7 in the patient0s mRNA was confirmed by direct
sequencing of amplified PCR products following synthesis of
single-stranded cDNA (primers: 50-GTTGAAGATGCCCA-
GAAGC-30). The presence of the homozygous mutation in the
patient and identification of carriers for this mutation were
verified by Spe1 digestion of a PCR product spanning the
mutated base generated using the following primers: 50-
was prepared from blood cells using a Qiagen DNA extrac-
The sequence environment of all splice mutations was
analyzed using the SSPNN program, and a splice-site score
(SSS) was obtained (URL address: http://www.fruitfly.org/
Patient HE, a 47-year-old woman, was the second of three
children born to parents originating from the same Swiss
valley (Emmental) (Fig. 1); consanguinity of the parents could
not be ascertained positively. The patient’s childhood and
adolescence were normal. She finished secondary school,
married, and delivered two healthy boys. In 1997, the patient
was admitted for a non-familial subacute tetraspastic
syndrome in combination with hyposensibility and paresthe-
sias of the distal upper and lower extremities. Neurological
members; squares, male family members; symbols with a slash, deceased
problems. Carriers detected by genotyping are indicated with a þ/? symbol.
Pedigree of ML III patient. Circles denote female family
370Steet et al.
workup initially revealed a sensory neuropathy and months
later, a discrete lower extremity motor neuropathy was also
Sensory neuropathy was documented by orthodromic neu-
rography comparing left and right sural nerve. The affected
side was demonstrated by slower impulse propagation (39 vs.
45m/sec), reduced amplitude ofimpulse (1.18 vs.7.25mV), and
increased number of phases (14 vs. 4). Evoked motor nerve
potentials in upper and lower extremities were normal but
demonstrated a discrete asymmetry with respect to the
from interventricular septum; Sudan black (a), transmission electron
microscopy (TEM; b–e). Dark, coarse sudanophilic inclusions (arrow in a)
are rich in phospholipids resistant to solvent extraction by the embedding
procedure. The hemalum-eosin stain (not shown) revealed the same, often
ellipsoid paranuclear structures as yellow to tan inclusions, similar in color
to lipofuscin. This unusual finding prompted the study by TEM. The larger
inclusions consisted of aggregated whorls of osmiophilic membranes
Clinical features of the patient. a–e: Endomyocardial biopsy
reminiscent of myelin figures (arrow in b). Smaller structures comprised
degenerate lysosomal organelles (arrow in c) and aggregated or isolated
structures bound by a single membrane containing stacked electron-dense
membranes (arrows in d and e) similar in size to mitochondria (left in
panel e). f–h: Cardiac MRI. Arrows mark dilated right ventricle (f), dilated
right ventricular outflow tract (g), and thinned left ventricular apex (h).
(i) Unremarkable X-rays film of the right hand.
Adult Onset Form of Mucolipidosis III 371
anterior tibial muscle. Spinal cord and cerebral magnetic
resonance imaging was normal except for an old thromboem-
bolic cerebral infarction in the left frontal lobe that was not
Further workup revealed a biventricular cardiomyopathy
(Fig. 2f–h). The left ventricular ejection fraction was reduced
to 25%, but hemodynamic parameters were normal in the
pulmonary and systemic circulation. Over the next few years
theneurologic featuresremainedunchanged, butsymptoms of
heart failure progressed despite medical treatment. In 2001,
an endomyocardial biopsy demonstrated hypertrophied cardi-
omyocytes with birefringent inclusion bodies (Fig. 2a). Elec-
tron microscopy revealed prominent lysosomes containing
rolledlipid bilayers(Fig. 2b–e).Thisledtoaninvestigation for
Fabry disease. As shown in Table I, the activity of multiple
lysosomal enzymes was distinctly elevated in the patient’s
serum suggestive of mucolipidosis III.
Due to worsening heart failure, the patient underwent
ogical evidence of lysosomal storage disease in the allograft
at 1 year post-transplant. At the time of transplantation, no
obvious pathology of the cardiac valves was described (not
shown). The patient died in 2004 from intractable chronic
infections with pneumocystic carini and pseudomonas despite
relaxation of immunosuppressive therapy. At autopsy, neural
tissue was examined and shown to contain inclusions similar
to those observed in myocardial tissue (data not shown),
however, the number of inclusion bodies per cell was smaller.
No other manifestations of mucolipidosis apart from cardiac
and neural tissue were observed. Together, the similar histol-
ogical changes observed in the two organs of the patient
suggest a common underlying disease process.
The family history is remarkable in that the father died at
age 72 due to cardiac failure and that cardiac disease was also
noted in other members of the patient’s family (Fig. 1). Family
tion or presented with coronary heart disease.
Consistent with the diagnosis of mucolipidosis III, cultured
skin fibroblasts from the patient were found to be deficient in
a subset of lysosomal enzymes (Table II). Notably, the levels of
b-glucuronidase, b-galactosidase, and a-fucosidase were not
decreased to the same extent as usually observed in ML II/ML
III patients. Joint mobility and skin were normal. Radiological
examination revealed minor degenerative changes with osteo-
chondrosis in the cervical, thoracic, and lumbar spine, slight
osteoporosis of the pelvis, and unremarkable right hand films
(Fig. 2i). Analysis of bone tissue at autopsy, however, suggests
that the degenerative changes are not likely due to the diag-
nosed disease. Taken together, these findings indicate the
presence of a mild form of ML III with an atypical clinical
The activity of the GlcNAc-1-phosphotransferase enzyme
was measured in control and patient fibroblast microsomes
using a-methylmannoside as an acceptor. The activity of
the GlcNAc-1-phosphotransferase enzyme from the patient’s
TABLE I. Enzyme Activities in Serum of Patient, Controls, and Other ML II/III Patients
Activity (mU/ml serum)
Patient range (n)a
Average Range (n)
aNumber of determinations during 1.5 years, before, and after heart transplantation; there were no tendencies
towardsincreased ordecreasedenzyme activities during thisperiod.Threesubjects takingsimilar medications as
the patient had enzyme activities in the normal range.
bDifferent batches of substrate.
TABLE II. Enzyme Activities in Fibroblasts of Patient, Controls, and Other ML II/III Patients
Activity (mU/mg protein)
ML II/III (n¼5)
data not shown); note that acid phosphatase and b-glucosidase activity is normal in ML II/III patients.
372Steet et al.
fibroblast extracts was 3.2?2.7% that of control samples
(2.3?0.4 vs. 51.4?6.2 U/mg protein, respectively). The
deficient enzyme activity in this patient is consistent with
previously reported ML III cases (range: 2%–20% activity
relative to control) and warranted further genetic investiga-
tion of the two genes encoding GlcNAc-1-phosphotransferase.
Mutation Analysis of Phosphotransferase Gene
The sequence of the cDNA encoding the g subunit of phos-
photransferase was analyzed by RT-PCR. Sequencing of the
PCR products revealed no mutations in the g subunit of
phosphotransferase, consistent with the previous observation
that mutations in this subunit result in normal phosphotrans-
[Varki et al., 1981].
The sequence of the gene encoding the a/b subunit was
analyzed using genomic DNA isolated from patient’s fibro-
blasts. The complete sequence of this gene will be published
elsewhere (Kudo et al., submitted). Sequencing analysis of the
patient’s amplified PCR products revealed a homozygous
G!A substitution in the last nucleotide of exon 7 (Fig. 3A).
Since such mutations have been previously reported to cause
pathological splicing events such as exon skipping (see
‘‘Discussion’’), we examined this possibility using RT-PCR.
PCR products were amplified corresponding to regions that
either span exon 7 (Fig. 3B; EX6F/EX8R) or originate within
exon 7 (Fig. 3B; EX5F/EX7R) using cDNA prepared from
amplification of products corresponding to exons 5–7 and
exons 6–8 generated the expected fragment sizes of ?220 and
?370 bases, respectively (Fig. 3C, lanes 2 and 3). No fragment
was amplified, however, in the patient sample when a forward
primer that hydridizes to exon 7 was used (Fig. 3C, lane 4).
Furthermore, the amplified exon 6–8 fragment in the patient
sample migrated at a smaller size (?235 bases, lane 5) com-
135 bp likely corresponds to the 135-nt of exon 7 and strongly
indicated that this exon is skipped in the patient’s GlcNAc-
1-phosphotransferase mRNA. The skipping of exon 7 was
directly confirmed by sequencing of an amplified fragment of
1-phosphotransferase gene. A faint band of 150 bp in the
patient sample (lane 5) was sequenced but found not to cor-
respond to any sequence within the phosphotransferase gene.
Genomic DNA from the patient’s family was analyzed by
restriction enzyme digestion of amplified PCR products. The
the presence of a homozygous mutation in the patient was
confirmed following Spe1 digestion. Several family members
including the mother of the patient were found to be hetero-
zygous for this mutation (Figs. 1 and 4). This heterozygosity
correlated well with the elevation of serum lysosomal enzyme
activity above the normal range (Fig. 5) in the affected family
members, confirming and extending earlier findings [Varki
et al., 1982].
Effect of GlcNAc-1-Phosphotransferase Deficiency on
Lysosomal Enzyme Phosphorylation and Sorting
We next measured the total phosphorylated high mannose
whether this decrease in phosphotransferase activity cor-
related with a reduction in the synthesis of the lysosomal
enzyme targeting signals. To do so, confluent fibroblast cul-
tures were pulse-labeled with mannose, high mannose oligo-
saccharides released by Endo H treatment and analyzed by
skipping. A: G!A in the last base of exon 7 in the patient. B: Schematic
representation of cDNA regions around exons 5–8. Primers used are shown
with arrows. C: Agarose gel electrophoresis of RT/PCR fragments of normal
(lanes 2 and 3) and patient (lanes 4 and 5) fibroblast RNA. Lanes 2 and 4,
primers EX5F/EX7R; lanes 3 and 5, primers EX6F/EX8R; lanes 1 and 6,
DNA. The G!A mutation was confirmed by RFLP analysis using Spe1.
non-carrier (7/I in pedigree).
Restriction enzyme digestion of patient and relative genomic
Adult Onset Form of Mucolipidosis III 373
to uncover mannose 6-phosphate residues. Oligosaccharides
containing one Man-6-P residue eluted from the column with
70 mM NaCl and those with two Man-6-P residues eluted with
140 mM NaCl. In control cells, nearly 2% of the total radio-
activity bound to the QAE-sephadex column indicating the
presence of charged oligosaccharides. Alkaline phosphatase
abolished the ability of the released oligosaccharides to bind to
residues. In contrast, only 0.5% of the total radioactivity in
the patient’s samples bound to the column demonstrating a
substantial decrease in the synthesis of oligosaccharides
bearing mannose 6-phosphate residues. Furthermore, in the
patient the oligosaccharides containing two residues were
more reduced (20% of control) as compared with those con-
taining just one residue (30% of control). This decrease in the
amount of total phosphorylated high mannose oligosacchar-
ides is similar to those observed for a known ML III patient
(data not shown). These findings indicate that although the
patient’s fibroblasts contain only 3% normal phosphotransfer-
ase activity, they are still able to phosphorylate the high
mannose oligosaccharides of lysosomal enzymes to roughly
25% the level found in control fibroblasts.
Next, we looked at whether the decrease in phosphotrans-
ferase activity led to the missorting of a specific lysosomal
enzyme, cathepsin D. The processing of newly-synthesized
cathepsin D molecules was examined in patient and normal
fibroblasts by pulse-chase experiments (Fig. 6). Fibroblasts
from the patient missorted cathepsin D, indicating that the
targeting of this enzyme was strongly affected by the phospho-
transferase deficiency. Thismissorting wasnot due tochanges
in the levels of the cation-independent mannose 6-phosphate
receptor since levels of this protein were identical in patient
and control cell lysates as determined by Western blotting
(data not shown).
The ML III patient described in this report is unique in two
respects: age of onset and clinical presentation. At 47 years of
age, this is the oldest presentation for an ML III patient
described to date. The typical appearance of symptoms in ML
a few instances, the onset of symptoms is delayed until the
teenage years [Tylki-Szymanska et al., 2002].
Cardiac manifestations have been reported in both ML II
and ML III patients. Notably, cardiomyopathy has been
observed in ML II patients [Okada et al., 1985; Schulz et al.,
1987; Muller et al., 2000] and valvular heart disease detected
in ML III patients [Raas-Rothschild et al., 2004]. However,
sensory neuropathy and cardiomyopathy as the presenting
symptomsinanadult MLIIIpatient isanovelfinding. Inlight
ofourobservations, analysisofserum lysosomal enzyme activ-
ity in patients with cardiomyopathy or sensory neuropathy of
unknown etiology may prove fruitful.
are typical manifestations of ML III, although in rare cases
the involvement may be limited to specific joints, such as the
hip joint [Freisinger et al., 1992].
We have demonstrated that the homozygous mutation
(G!A) in the last nucleotide of exon 7 in the patient results
in the skipping of this exon and the production of a minimal
amount of functional enzyme. Although the splice-site muta-
tion does not result in a large change in a quantitative splice
score (1.00 vs. 0.91 SSPNN; see ‘‘Materials and Methods’’),
no normally spliced product was detected (Fig. 4). One
possibility to account for this observation is the existence of a
very long intron (?10,000 bases) following exon 7. Previous
reports have shown that intron length can substantially affect
splicing efficiency [Bell et al., 1998]. Therefore, even small
decreases in the theoretical splicing efficiency can result in
members. The mean enzyme activities of the non-carriers are expressed as
100%. The mean values for carriers are increased to 223% (P<0.0045;
Wilcoxon test) for b-glucuronidase (closed circles) and to 182% (P<0.0014)
for b-hexosaminidase AþB (closed triangles). The patient’s enzyme
Enzyme activities in the serum of the patient and family
cathepsin D molecules were immunoprecipitated from fibroblast cell
extracts (C) and culture media (M) and quantified. The sorting efficiency is
calculated with the intermediate and mature forms of cathepsin D from the
cell extracts (with int.) and the mature form only (without int.). In either
case, thepatient’scells hypersecrete cathepsinD.Theresultsshownare the
average of two independent experiments.
Cathepsin D sorting in fibroblasts. Newly synthesized labeled
374Steet et al.
literature where a G!A transition in the last base of an exon
results in exon skipping [Grandchamp et al., 1989; Weil et al.,
1989; Akli et al., 1990; Krawczak et al., 1992; Klima et al.,
We demonstrated that only 3% normal GlcNAc-1-phospho-
transferase activity in patient’s fibroblasts is sufficient to
phosphorylate 25% of the total lysosomal enzyme oligosac-
charides. It is likely, therefore, that increases in total enzyme
activity to as little as 6%–10% may be sufficient to phos-
phorylate lysosomal enzymes to a level that would avoid a
pathological outcome. In other lysosomal storage disorders
[Leinekugel et al., 1992] as well as other genetic disorders like
cystic fibrosis [Rowntree and Harris, 2003], it has been
demonstrated that minimal activity can avoid disease. There-
fore, stimulation of GlcNAc-1-phosphotransferase activity via
pharmacological intervention may represent a promising
The residual phosphotransferase activity in the fibroblasts
could arise by two mechanisms. The deletion of the exon could
result in a splicing isoform with a low level of residual activity.
Alternatively, the splicing isoform may be completely
inactive, and the 3% of activity observed is the result of a
small amount of proper splicing. Since some misfolding
such as glycerol or DMSO [Ohashi et al., 2003], we tested the
effect of these agents on the activity of phosphotransferase in
(data not shown). In our patient, organ damage was most
pronounced in the heart. It is known from the literature that
splicing efficiency within different tissues depends on numer-
ous factors including the relative level of various splicing
factors [Rio, 1993; Gallego et al., 1996; Black, 2003]. It is
plausible, therefore, that most tissues of the patient can
sufficiently splice phosphotransferase mRNA and produce
enough normal mRNA and enzyme to avoid disease in those
tissues. Likewise, the level of enzyme needed in different
tissues likely varies as well.
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