A useful routine for biochemical detection and diagnosis of mucopolysaccharidoses

Mucopolysaccharidoses (MPS) constitute, owing to their biochemical, genetical and clinical characteristics, a large and heterogeneous subgroup among the lysosomal storage diseases (LSD). They are caused by deficiency of specific enzymes, which are responsible for glycosaminoglycan (GAG) breakdown during different steps of its degradation pathway. MPS are responsible for about 32% of inborn errors of metabolism (IEM) and 54% of LSD identified in our laboratory (Regional Laboratory of Inborn Errors of Metabolism (RLIEM), Medical Genetics Unit, Hospital de Clínicas in Porto Alegre), which is a reference center for LSD diagnosis in Brazil. Therefore, we decided to set up a specific laboratory routine for detection and differential diagnosis of MPS in patients with clinical features suggestive of this group of disorders.


INTRODUCTION
Mucopolysaccharidoses (MPS) are a large and heterogeneous subgroup among the lysosomal storage diseases (LSD).It results from a deficiency of one of 10 different lysosomal enzymes (Neufeld and Muenzer, 1995).Differential diagnosis is important for a correct prognosis, definition of management strategies, genetic counseling, prenatal diagnosis, and prediction of future cases in the family.
Since MPS diseases account for about 32% of inborn errors of metabolism (IEM) and 54% of LSD identified in our laboratory, we decided to set up a specific laboratory routine for detection and differential diagnosis of MPS.

Sample
The sample consisted of 177 patients referred to our laboratory from pediatric and genetic centers from all over Brazil due to clinical, radiological and/or laboratorial findings suggestive of MPS.
To obtain reference values for quantitative analysis of urinary glycosaminoglycans (GAGs), random urine samples were collected from 40 normal children aged 1 to 10 years.Ranges were established according to age.Reference values for enzyme activities were established from samples collected from normal subjects.

Collection and storage of blood and urine samples
A random urine specimen without preservative (at least 30 ml) and a sample of heparinized blood (10 ml) were obtained from all patients.Plasma and leukocytes (prepared according to Skoog and Beck, 1956) were isolated from blood samples.Samples collected in other centers were sent to our laboratory in Styrofoam containers with ice, arriving generally up to 48 h after collection.When possible leukocytes were separated immediately after arrival.Otherwise, samples were separated the following day.In these cases, the blood samples were kept at 4ºC.Urine and plasma were kept frozen at -20ºC and leukocytes at -40ºC until analysis.

Routine employed
All patients referred to our laboratory were submitted to cetyltrimetylammonium bromide (CTAB) screening and toluidine blue spot tests for MPS.
When results were positive or doubtful, the investigation was continued through quantitative analysis of urinary GAGs by the alcian blue method proposed by Whiteman (1973).This method was chosen due to its simplicity and value in detection of increased urinary GAGs even in the Morquio syndrome.Results were expressed in mg/g creatinine.
When increased levels of urinary GAGs were found, qualitative analysis of urinary GAGs (after fractionation by the alcian blue method) was performed by thin-layer chromatography (TLC) (Humbel and Chamoles, 1972).Three abnormal patterns were identified by TLC, allowing preliminary classification of patients among subgroups.This step reduced the number of enzyme assays necessary for final diagnosis.
Final diagnosis was performed by detection of specific enzyme deficiency in plasma ( -iduronidase, iduronate-2-sulfatase,  -N-acetylglycosaminidase,  -glucuronidase) or leukocytes (N-acetylgalactosamine 4-sulfatase, sulfamidase, galactose-6sulfatase,  -galactosidase).In the present work, some of these assays were carried out at the Paediatric Research Unit, Guys Hospital, London, UK. Figure 1 summarizes the routine used for detection and diagnosis of MPS.

RESULTS
Abnormal results in the screening tests were obtained in 69 of the 177 patients referred to our laboratory with clinical features suggestive of MPS.In 29 of these cases investigation was not completed.The remaining 40 patients were investigated until the enzyme defect was confirmed (Table I).In the qualitative analysis of urinary GAGs, we detected the presence of both dermatan and heparan sulfates in 22 cases, which may occur in MPS I, MPS II or MPS VII.This pattern was also observed in MPS VI, because a second band of dermatan sulfate overlaps the heparan sulfate band.In these 22 patients we found 8 patients with -iduronidase deficiency (MPS I), 9 with iduronate sulfatase deficiency (MPS II) and 5 with Nacetylgalactosamine 4-sulfatase deficiency (MPS VI).
In 9 patients the chromatographic pattern showed a marked excretion of heparan sulfate, which suggests Sanfilippo syndrome.In 3 patients we detected sulfamidase deficiency (MPS IIIA).The remaining 6 had N-acetyl--D-glucosaminidase deficiency (MPS IIIB).
In the remaining 9 patients the qualitative analysis detected increased amounts of keratan sulfate by TLC, suggesting Morquio syndrome.We found galactose-6-sulfatase deficiency in 7 patients (MPS IVA) and ß-galactosidase deficiency (MPS IVB) in 2 patients.

Efficiency of screening tests for MPS detection
According to Renuart (1966), the CTAB test shows positive results only in urine samples with high levels of GAGs.This fact was not observed in our patients, since CTAB results were not related to the results observed in the quantitative analysis.In the CTAB positive samples, urinary excretion of GAGs was approximately 7 times higher than age-related values.The same occurred in samples with negative or doubtful CTAB results.The CTAB test not only is difficult to interpret (Renuart, 1966), but also can be affected by ionic strength, pH and urinary concentration.For these reasons we expected false negative results, and indeed 14 false negative cases (35.9%) were observed.These patients would not have been detected if the CTAB test was the only one for MPS screening.If we consider the doubtful results as negative, we increase our percentage of false negative to 48.7%.
The toluidine blue spot test seemed to be more sensitive, with 20% false negatives (including negatives and doubtful results).This proportion decreased when we used a combination of the two tests (CTAB and toluidine blue).In this case we had 15% false negatives (negative and doubtful) in screening tests.These results show the importance of using the two tests together as recommended by Caportori and Ventura (1966).If this is not possible, the toluidine blue test is preferred.

Efficiency of quantitative and qualitative analysis of GAGs
Quantitative analysis of urinary GAGs showed, in all 40 patients with confirmed MPS, a level at least two times higher than normal age-related individuals.We did not register any false negative with this method.The lowest values were seen in MPS III and MPS IV patients.A possible explanation is the presence of endoheparinases in many tissues which cleave the heparan sulfate into smaller fragments.The low molecular weight fragments produced by this "endocleavage" can be detected in urine, but sometimes can be lost in the current analysis directed at the largest polymers.In MPS IV, the residual compounds found in keratan sulfate are galactose instead of uronic acid, and the majority of the techniques used are directed at the precipitation of uronic acid.Moreover, GAGs excretion in MPS IV decreases significantly with age (Neufeld and Muenzer, 1995).
Because GAGs excretion decreases with age, care should be taken with neonates as they can display apparent high levels of GAGs (Pennok, 1971).The opposite is also true, and care should be taken when interpreting as normal the apparent low levels observed in adolescents or adults.
We were able to classify the patients through TLC into three major groups (see Results) focusing the enzyme assays to be performed and consequently saving time and costs.
Although the literature reports that patients with MPS I excrete more dermatan than heparan sulfate, patients with MPS II excrete more heparan than dermatan sulfate, and MPS VI patients excrete exclusively dermatan sulfate (Neufeld and Muenzer, 1995), we couldnt observe specific patterns for these diseases with the TLC method employed here.This does not seem to be a problem, because the ultimate diagnosis is based solely on the specific enzyme assay.

Specific enzyme diagnosis
The final MPS diagnosis was made only after specific enzyme assays, which were determined by clinical features and urinary analysis.The task of establishing almost all the assays used in the present work in the RLIEM showed that it is feasible, even in a third world country like Brazil, to set up a reference laboratory for MPS diagnosis.
Even if there is no efficient treatment so far, for most cases, diagnostic confirmation of MPS by specific enzyme deficiency detection is valuable for the prevention of such diseases, through genetic counseling, prenatal diagnosis and carrier detection.Nowadays, with the advent of DNA technology, the characterization of the disease causing mutation is of immeasurable importance for carrier detection, specially for X-linked diseases like Hunter syndrome, where enzyme assays are not reliable due to nonrandom X-inactivation.Furthermore, gene therapy relies on the identification of each individual mutation, and it may be a suitable approach in the near future for treatment of this group of disorders, where bone marrow transplantation has shown little, if any, improvement.

ACKNOWLEDGMENTS
We are very grateful to Maria Luiza Pereira who initially set up some of the enzyme assays for MPS, to Dr. A.H. Fensom from the Paediatric Research Unit, Guys Hospital, London, who performed some of the assays in selected patients and to Ursula Matte for reviewing the manuscript.

Table I -
Results obtained in the 40 patients who went through the whole MPS routine.