Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3641-3643
CORRESPONDENCE
 |
To the Editor: |
Fucose supplementation in leukocyte adhesion deficiency type II
We read with great interest the report by Marquardt et
al1 regarding the beneficial effect of fucose
supplementation in a patient with leukocyte adhesion deficiency type II
(LAD II). Not only was improvement noted in neutrophil adhesion
function, but also the patient's developmental delay was
reduced. It should be noted that fucose treatment was started when the
child was already more than 1 year of age.
From the time of our initial report of LAD II,2 we have
discovered another 2 infants, a male and a female, affected by LAD II.
They were both of Arabic origin, the parents were close relatives, and
they live in the same area as the previous ones.
No evidence of consanguinity between the families is present. Their
birth length and weight was normal for gestational age. Both children
presented with febrile illnesses, and a complete blood count showed a
very high leukocyte count (above 40 000 cells/mm3).
CD15a was not expressed on leukocyte surfaces, and the children had the
Bombay blood group phenotype. CD18, CD11a, and CD11b were expressed
normally, and the diagnosis of LAD II was made. Because no anti-H
antibodies were detected, we decided to start fucose supplementation at
the age of 4 weeks, after obtaining informed consent from the parents.
As no data regarding fucose administration in humans is currently
available, we estimated that around 200 mg/kg per day may achieve the
desired normal blood fucose level.3 A loading dose of
5-gram fucose (Pfanctiehl, IL) was given orally to patient
1 (in 4 divided doses). A day later a marked decrease in the leukocyte
count was noted (Figure).
Hypoglycemia
developed, and the dose was decreased to 2.5 g, followed by an increase
in leukocyte count to 80 000/mm3. Although the baseline of
blood-free fucose was less than 50% of normal (Figure), repeated
fucose measurements showed very high levels while the child was on
therapy, which were comparable to those obtained by Marquardt et
al1 using a high dose of fucose. Fucose concentration in
samples was determined using both gas chromatographs coupled with mass
spectrometry and the enzymatic assay with fucose
dehydrogenase.3-4
Urine fucose levels in the patient while on therapy were 10 times
higher (1.2 µmol/L) than in control (25-110 µmol/L). Still, no
decrease in leukocyte count was observed, and CD15a expression was not
detected on leukocyte surfaces.
After a month, the dose of fucose was decreased to 1 g/d and was
continued for up to 12 months. Unfortunately no change in leukocytes
counts or CD15a expression was noted, and her psychomotor retardation
continue to deteriorate. Her weight, length, and head circumference
were all below the third percentile for age. The same
unfavorable results were seen in the male infant with LAD II, with whom
therapy also started at age 1 month.
How can one explain the different results of fucose supplementation in
our 2 patients and Marquardt et al's patient? The widespread lack of L-fucose on several different glycoconjugates seems to exclude any impairment of fucosyl-transferase activities and to favor a
general defect in L-fucose metabolism. Lymphocytes from 1 of our
patients display a significant reduction of GDP-D-mannose-4,6 dehydratase (GMD) activity, even though no qualitative or quantitative defects are observed for this enzyme, thus suggesting the presence of
inhibitory mechanisms.5 Recently, Lubke et al6
found that the activity of this enzyme was normal in their LAD II
patient, but a defect in the import of GDP fucose into Golgi-enriched
vesicles was found. A decreased import of GDP-L-fucose into the
Golgi-enriched vesicles was also observed in our patient
(Tonetti, unpublished results), and the consequent increase in the
cytosolic concentration of GDP-L-fucose, which is a very good
noncompetitive feedback inhibitor for GMD, can explain the defect in
the enzymatic activity observed. Our in vitro studies on cells from the
LAD II patient indicated that administration of L-fucose is able to
restore the expression of fucosylated antigens on the cell
membrane.7 But the concentrations used in vitro were many
times higher than those achievable in vivo. In Marquardt et al's
patient, fucose administration was able to correct not only the
expression of various glycoproteins on the cell surface but even
some of the neurological defects,1 while in our 2 patients
no effect was observed.
We believe, therefore, that although the phenotype of LAD II in our 4 Arabic patients is very similar to the Turkish patient reported by
Marquardt et al, the biochemical defect is somehow different. It is
clear that the increased fucose delivery to the cell through the
scavenger pathway is enough to overcome the Golgi-uptake defect in
Marquardt et al's patient. Still, in our patients the specific defect
in fucose import by the Golgi apparatus seems more profound and could
not be overcome by increasing fucose delivery to the cell, at least at
the concentrations that can be obtained in vivo. In order to clarify
this very interesting issue, complementation studies using cells from
the different patients should be performed in order to find the primary
molecular genetic defect.
Amos Etzioni
Division of Pediatrics
Rambam Medical Center
B Rappaport
School of Medicine
Technion
Haifa, Israel
Michela Tonetti
Department of Experimental Medicine
Section of
Biochemistry
University of Genova
Genova, Italy
| |
References |
1.
Marquardt T, John K, Srikrishna G, Greeze HH, Harms E, Vestevebr D.
Correction of leukocyte adhesion deficiency type II with oral fucose.
Blood.
1999;94:3976-3985[Abstract/Free Full Text].
2.
Etzioni A, Frydman M, Pollack S, et al.
Recurrent severe infections caused by a novel leukocyte adhesion deficiency.
New Engl J Med.
1992;327:1789-1792[Medline]
[Order article via Infotrieve].
3.
Sakai T, Yamamoto K, Yokota H, Hakosaki-Usui K, Hino F, Kato I.
Rapid simple enzymetia assay of free L-fucose in serum and urine, and its use as a marker for cancer, cirrhosis of gastric ulcers.
Clin Chem.
1990;36:474-476[Abstract/Free Full Text].
4.
Garcia-Raso A, Fernandez-Diaz M, Perez M, Sano J, Martinez-Castro J.
Gas chromatographic retention of carbohydrate trimethylsilyl ethers IV disaccarides.
J Chromatogr.
1989;471:205-210.
5.
Sturla L, Etzioni A, Bisso A, et al.
Defective intracellular activity of GDP-D- mannose-4,6-dehydratase in leukocyte adhesion deficiency type II syndrome.
FEBS Lett.
1998;429:274-278[Medline]
[Order article via Infotrieve].
6.
Lubke T, Marquardt T, von Figura K, Korner C.
A new type of carbohydrate-deficient glycoprotein syndrome due to decreased import of GDP-fucose into the Golgi.
J Biol Chem.
1999;274:25986-25989[Abstract/Free Full Text].
7.
Karsan A, Cornejo CJ, Winn RK, et al.
Leukocyte adhesion deficiency type II is a generalized effect of de novo GDP-fucose biosynthesis.
J Clin Invest.
1998;101:2438-2445[Medline]
[Order article via Infotrieve].
| |
Response: |
Fucose supplementation in leukocyte adhesion deficiency type II
Drs Etzioni and Tonetti report on 2 newly discovered leukocyte
adhesion deficiency type II (LAD II) patients and their attempts to
treat these 2 patients with oral fucose. The treatment did not lead to
detectable improvements and did not cause re-expression of the
carbohydrate epitope sLex (CD15s). These results need to be compared
with our recently published results on the successful treatment of LAD
II by oral fucose.1
Taking a closer look at the quantitative details reveals that the
fucose doses used in the described therapy were considerably lower than
those used in our therapy. Those doses would not have been sufficient
to rescue expression of sLex (CD15s) in our patient. Although the
presented description of the therapy is very brief, we will try to
compare the outcome with our recently published results.
Comparing the fucose doses used by Dr Etzioni with ours clearly shows
that they are not sufficient to cause significant expression of the
carbohydrate epitope sLex (CD15s). Expression of P-selectin ligands
(monitored with P-selectin-IgG) is much earlier observed during fucose
therapy and would have been the better epitope to monitor the outcome
of the therapy.
The doses reported in the letter are not given as an amount of fucose
administered per kilogram of body weight (b.w.) but as a total amount
given per day per patient. The body weight of the patient is not
reported. Since the starting dose was aimed at 4 daily doses of 200 mg/kg, and a total loading dose of 5 g was given for 1 day, the weight
of the baby can be calculated as 6.25 kg. Since we usually give 5 doses
per day, this would amount to a single dose of 160 mg/kg in our
therapy. This dose was only given for 1 day during therapy of the
Arabic patient. We observed significant expression of sLex (15% to
20% of normal expression) only above 5 single daily doses of 250 mg/kg, a dose level that was slowly reached after more than 60 days of
treatment during which doses were steadily increased. We
continuously increased our doses up to 492 mg/kg, a dose that we
reached at day 277 of therapy. We observed the first signs of the
expression of P-selectin ligands at a dose above 100 mg/kg.
The starting dose of 5 g/d was given to the Arabic patient only for a
single day, followed by 1 week without fucose (according to the graph).
The therapy was restarted with half the dose (comparable to single
daily doses of 80 mg/kg if 5 doses per day are given, as in our
therapy). After 1 month of treatment, the dose was reduced to 1 g/d (32 mg/kg of 5 single doses per day). If we take into account the rapid
gain of body weight of a baby over time, these doses per kilogram would
in fact be even lower. Clearly, these doses would not have been
sufficient to rescue expression of significant amounts of sLex in our
patient. No details were reported about the attempted therapy of the
second patient (doses, duration of treatment).
In contrast to our paper,1 the letter only reports on total
peripheral leukocyte counts, not on neutrophil counts. Our patient
still has a mild lymphocytosis, leading to mild elevation of total
leukocyte counts (12 000 leukocytes/µL). Soon after the onset of
therapy, we observed a dramatic reduction of peripheral neutrophil
counts.1 We agree with Drs Etzioni and Tonetti that treatment of their patient with 2.5 grams fucose per day (corresponding to 5 daily doses of 80 mg/kg) for 1 month might possibly have had a
chance to reduce peripheral leukocyte counts in our patient, although
treatment with this dose was not long in duration and the dose was at
the lower limit. If this were the case, it would argue for a different
sensitivity of the LAD II defect to the rescue by externally added
fucose. Although unknown, it is still conceivable that the same genes
would be mutated in the different patients, since different parts of
the molecules could be affected (see below).
The total serum fucose concentrations determined during
therapy are surprisingly high, given the low doses of fucose that were administered. Unfortunately, only 2 measurements are documented.
The letter reports that a loading dose of 5 grams of fucose was
followed by hypoglycemia. We controlled for this over the whole period
of our therapy and never found a sign of hypoglycemia, although for
most of our therapy we gave much higher doses of fucose. Considering
the biochemical pathways of glucose and fucose and the lack of
interaction between them, it is unlikely that application of fucose
could cause hypoglycemia.
The letter compares the unpublished therapy data of the 2 new patients
with published in vitro data obtained with cells from 1 of the 2 first
patients. It is important to keep in mind that these data were obtained
with different patients. Determination of reduced GDP-D-mannose-4,6
dehydratase activity in lysates of lymphoblasts were probably obtained
with the cells of 1 of the older LAD II patients.2 The
rescue of 
1,6-core fucosylation in fibroblasts and lymphoblastoid
cells was also described for cells of 1 of the 2 older
patients.3 It is unclear whether similar results were
obtained for cells of the 2 new patients. It is also unclear from which
patient the cells that allowed us to determine that GDP-L-fucose import
into Golgi vesicles was defective were isolated (referred to as
Tonetti, unpublished results).
We stated in our paper that the psychomotor development of the patient
improved during fucose treatment. Indeed, before therapy the patient
showed a severe psychomotor retardation, as evidenced by a total score
of 28.5 EQ (Griffiths test; 100 EQ is equivalent to the fiftieth
percentile).4 Reassessment after 3 months of fucose therapy
showed a significant increase in psychomotor functioning, with a total
score of 45 EQ. But naturally we do not know how the psychomotoric
abilities of the patient would have developed in the absence of fucose
treatment. Of course, the developmental defects of the patient have not
been normalized. We have clearly stated in our paper that "at two
years of age the boy is still severely retarded. He does not speak yet,
but is able to actively turn around when lying on his back, takes toys
that are presented in his hands, and starts to sit briefly without support."
It is interesting and important that Drs Tonetti and Etzioni have
detected a defect in GDP-fucose import into Golgi vesicles of 1 of the
4 Arabic patients (unpublished). Indeed, this would hint at a genetic
defect similar to that published in our case.5 In agreement
with Drs Tonetti and Etzioni, we can well imagine that different
mutated alleles of a protein that mediates GDP-fucose import could lead
to functional defects of this protein of various severity. Thus, an
increase in the cytosolic GDP-fucose level that might be sufficient to
overcome a low-efficiency GDP-fucose import in cells that express one
mutated allele might not be sufficient to rescue the import in cells
expressing another, more severely defective allele. It would be
interesting to know whether expression of fucosylated glycoconjugates
in cells of the 2 new patients could be rescued by culturing these
cells in the presence of externally added fucose and, if so, what
fucose concentrations would be necessary. Cloning of the gene(s) coding
for a GDP-fucose transporter will allow testing of whether the defect
in LAD II is indeed due to mutations in such gene(s). This will also
allow researchers to finally determine if and to what extent the
molecular defects in different LAD II patients vary.
Dietmar Vestweber
Institut für Zellbiologie, ZMBE
Universität
Münster
Münster, Germany
Thorsten Marquardt
Klinik und Poliklinik für
Kinderheilkunde
Universität Münster
Münster,
Germany
| |
References |
1.
Marquardt T, Lühn K, Srikrishna G, Freeze HH, Harms E, Vestweber D.
Correction of leukocyte adhesion deficiency type II with oral fucose.
Blood.
1999;94:3976-3985.
2.
Sturla L, Etzioni A, Bisso A, et al.
Defective intracellular activity of GDP-Dmannose-4,6-dehydratase in leukocyte adhesion deficiency type II syndrome.
FEBS Lett.
1998;429:274-278.
3.
Karsan A, Cornejo CJ, Winn RK, et al.
Leukocyte adhesion deficiency type II is a generalized defect of de novo GDP-fucose biosynthesis: endothelial cell fucosylation is not required for neutrophil rolling on human nonlymphoid endothelium.
J Clin Invest.
1998;101:2438-2445.
4. Griffiths R. Abilities of young
children. A Comprehensive System of Mental Measurement for the First
Eight Years of Life. London, UK: Young and Son, 1970.
5.
Lübke T, Marquardt T, von Figura K, Körner C.
A new type of carbohydrate-deficient glycoprotein syndrome due to a decreased import of GDP-fucose into the Golgi.
J Biol Chem.
1999;274:25986-25989.
