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Technical Briefs |
-L-Iduronidase Deficiency in Dried Blood Spots on Filter Paper: The Possibility of Newborn Diagnosis
1
Laboratory of Neurochemistry, Uriarte 2383, 1425 Buenos Aires, Argentina
a author for correspondence: fax 5411-4774-5920, nachamoles{at}fibertel.com.ar
Mucopolisaccharidosis type I (MPS I), produced by a deficiency
of 
-L-iduronidase (EC 3.2.1.76) activity, can manifest
three major clinical phenotypes: Hurler, Scheie, and Hurler-Scheie
syndromes. The clinical phenotypes cannot be differentiated by routine
biochemical diagnostic procedures. Mutation analysis allows the
classification of some patients, but in most cases assignment to an MPS
I type can be made only on the basis of clinical criteria
(1).
In the last few years treatment of MPS I became possible by bone marrow transplantation (2), enzyme replacement therapy (3), and gene transfer or gene modification (4). The effectiveness of these therapies, particularly for MPS involving the central nervous system, may rely heavily on early diagnosis of the disorder. An additional consideration critical to bone marrow transplantation is that early diagnosis of the patient will allow clinicians to take advantage of the period of natural suppression of the immune system of the neonate to maximize the chances of a successful engraftment. Except for those cases with a family history of the disease, presymptomatic detection of MPS I can be achieved only by newborn screening (5). In any case, a simple technique suitable for dried blood spots on filter paper (DBFP) is needed.
The most widely used specimens for the diagnosis of iduronidase deficiency have been homogenates of cultured fibroblasts or leukocytes. Analysis of iduronidase in leukocytes is preferred because it avoids time-consuming and costly tissue culture. However, isolation of leukocytes from venous blood is also time-consuming. Additionally, transport of these samples from one city or country to another is difficult.
Several microtests for the assay of iduronidase in plasma and/or
leukocytes have been published (6)(7), but these methods
have not been applied to DBFP. We describe here an adaptation of the
fluorescence method (8) used for leukocyte assay of
-L-iduronidase activity to DBFP.
4-Methylumbelliferyl--L-iduronide,
4-methylumbelliferone, and D-saccharic acid-1,4-lactone
were purchased from Sigma. All other chemicals were of high-purity
grade.
Blood samples obtained from 40 healthy individuals 18–37 years of age were spotted on filter paper (Schleicher and Schuell no. 903) and allowed to dry 4 h at room temperature. Filter papers were stored at 4 °C in plastic bags until analysis. The assays were performed not more than 72 h after blood sampling. Twenty-five DBFP samples from our newborn screening program collected on the 3rd to 7th day postpartum were also analyzed. DBFP samples from five cases of MPS I type Scheie (age range, 10–34 years), four cases of MPS I type Hurler (age range, 2–4 years), and four obligate carriers were obtained after informed consent of the patients or families and processed in the same way. Diagnosis of MPS in these patients was established by clinical and standard biochemical procedures.
To duplicate disposable 1-mL test tubes containing a 3-mm diameter
circle paper (5.5 µL of blood) obtained with a standard paper punch
we added 40 µL of 50 mmol/L formate buffer (pH 2.8) containing 0.3
µg of D-saccharic acid-1,4-lactone as elution liquid and
20 µL of 2 mmol/L 4-methyhylumbelliferyl--L-iduronide
in distilled water as substrate. After vortex-mixing for 1 min,
the tubes were incubated for 20 h at 37 °C in a shaking
water bath. The tubes were then placed on ice, and 300 µL of
glycine-carbonate buffer (0.085 mol/L, pH 10.5) was added to stop the
reaction. The tubes were vortex-mixed and allowed to stand for 30 min
at room temperature.
The filter paper need not be removed during the analysis. Assays were
performed in duplicate. One blank tube was run for each sample of the
assay. Blanks were prepared by adding 300 µL of glycine-carbonate
buffer (0.085 mol/L, pH 10.5) to a mixture of the paper punch in the
elution liquid and the substrate, which had been incubated separately.
Fluorescence (excitation, 365 nm; emission, 450 nm) of the enzyme
product 4-methylumbelliferone was measured on a Farrand fluorometer
Model RF-2 (Farrand Optical Inc.). The fluorescence readings were
corrected for blanks, and the results were compared with the
fluorescence from a 4-methylumbelliferone calibrator. Enzymatic
activities were expressed as micromoles of substrate hydrolyzed per
liter of blood per 20 h. Different assays were performed to
establish the appropriate elution liquid, buffer, pH, and substrate
concentrations (data not shown). Assay imprecision was calculated by
replicate analysis of the DBFP from a health control (-iduronidase
activity, 61.1 µmol · L-1 blood · 20
h-1) and from an obligate carrier (-iduronidase
activity, 33.5 µmol · L-1 blood · 20
h-1). The within-assay CVs were 7.7% and 8.4% (n =
10), respectively. The interassay CVs for both DBFP samples studied on
five different occasions within 1 month were 7.1% and 9.1%,
respectively.
Blood from a healthy adult (-iduronidase activity, 61.1 µmol
· L-1 blood · 20 h-1) was mixed in
equal proportions with blood from a Hurler patient
(-L-iduronidase activity, 0 µmol ·
L-1 blood · 20 h-1); the resulting
activity was 32.9 µmol · L-1 blood
· 20 h-1. The effect of variable incubation times on the
activities of iduronidase is shown in Fig. 1
.
|
There were no significant changes in enzyme activity of DBFP samples after storage for 21 days at 4 °C or -20 °C (n = 5). When samples from a control, an obligate carrier, and a Hurler patient were stored at room temperature (26 °C) for the same period, the iduronidase activity decreased from of 128, 41, and 2.2 µmol · L-1 blood · 20 h-1 to 85.7, 29.1, and 1.7 µmol · L-1 blood · 20 h-1, respectively.
The iduronidase activities of healthy individuals, newborn controls,
obligate carriers, and MPS I patients are shown in Table 1
. There was no overlap among the results of MPS I patients and
carriers or controls. DBFP samples from MPS I patients showed
iduronidase activities 
8% and 6% of the mean values of adult and
newborn controls, respectively. No differences were found between the
Hurler and Scheie patients. In leukocytes, the absolute iduronidase
activity varies with assay conditions, but a finding of <10% of the
mean activity in healthy subjects is accepted as diagnostic of MPS I
(8). In our DBFP assay, the obligate carriers overlapped
with the healthy adult range.
|
The present methodology is easier, faster, and less expensive than the leukocyte assay. A drop of blood obtained through heel prick is sufficient to perform the assay in duplicate plus one blank. Sample transportation is safe. Minimal activity loss occurs during storage at room temperature up to 20 days. This fact does not modify the recognition of MPS I patients and controls, and it usually is enough time to mail the DBFP sample to a specialized laboratory for analysis. Our results suggest that this method allows the diagnosis of MPS I in DBFP samples. However, it will be necessary to study a larger number of patients and carriers to validate this methodology.
The possibility of an early diagnosis and the treatment of presymptomatic MPS I patients is desirable; the use of this methodology for a pilot newborn-screening program thus appears as a reasonable approach. Such a pilot study will eventually define whether massive newborn screening is indicated. In that case, microplate adaptation and automation of the method could be easy.
The iduronidase activity test in DBFP samples appears to be a reasonable approach for the initial diagnosis of MPS I. Because clinical differentiation among different types of MPS is difficult, especially with Maroteaux-Lamy syndrome and ß-glucuronidase deficiency, we are working on the development of similar methods using DBFP samples for the diagnosis of other MPS disorders. In abnormal cases, a blood sample for leukocyte isolation or a fibroblast skin culture can be requested for further characterization of the biochemical and molecular phenotype of the disorder.
Acknowledgments
We gratefully acknowledge the cooperation of Drs. A. Lemes (Department of Genetics, Hospital Italiano, Montevideo, Uruguay), R. Giugliani (Unidade de Genetica Medica, Hospital de Clinicas, Porto Alegre, Brazil), and J. Van Hove (Department of Pediatrics, University Hospital Gasthuisberg, Leuven, Belgium) for providing DBFP samples from affected patients. We thank Dr. J.E. Abdenur for useful discussions and suggestions.
References
-L-iduronidase deficiency (Hurler syndrome) in human bone marrow. Proc Natl Acad Sci U S A 1996;93:2025-2030.-L-iduronidase in serum for detection of affected and carrier animals in a canine model of mucopolysaccharidosis I. Clin Chem 1985;31:826-827.-L-iduronidase in plasma and leucocytes and its potential for diagnosing -L-iduronidase deficiency [Letter]. Lancet 1984;i:794.
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, adult control; 
, MPS I carrier;
, MPS I patient. Equations for lines: line 1 (control),
y = 7.4227x - 3.9656
(R2 = 0.997); line 2 (carrier),
y = 2.4879x + 0.6741
(R2 = 0.9957).