Fateen, Ekram M.; Ibrahim, Mona M.; Gouda, Amr S.; Youssef, Zienab A.
Mucopolysaccharidoses (MPS) are a group of inherited lysosomal storage disorders with an autosomal recessive inheritance, except Hunter syndrome, which is inherited as an X-linked recessive syndrome. They result from the deficiency of certain enzymes that are required for the degradation of glycosaminoglycans (GAGs) (Muenzer, 2004). GAGs are long-chain complex carbohydrates that are usually linked to proteins to form proteoglycans. Proteoglycans are the major constituents of the ground substance of connective tissue. They are also present in mitochondrial, nuclear, and cellular membranes (Dietrich et al., 1976).
The major GAGs are chondroitin-4-sulfate, chondroitin-6-sulfate, heparan sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. Multiple lysosomal enzymes are involved in the degradation of GAGs. Deficiency of one of these enzymes leads to the accumulation of undegraded GAGs, which are either stored or excreted in urine. This results in a variety of heterogeneous progressive clinical manifestations that present usually in the second year of life, such as coarse facial features, intellectual disability, macrocephaly, short-trunked dwarfism, dysplasia (abnormal bone size and/or shape), thick skin, corneal clouding, hepatosplenomegaly, joint contractures, hearing and visual defects, and umbilical or inguinal hernias (Thomas et al., 2010). Many of the clinical features are common with the different MPS disorders, which vary in the severity depending on the type of MPS involved. Severe intellectual disability is usually associated with MPS type-IH (Hurler syndrome), MPS type-II (Hunter syndrome), and MPS type-III (Sanfilippo syndrome). MPS are rare and the incidence is ∼1 per 25 000 populations (Nash, 2007).
MPS type-IH is a progressive and multisystem disease. It results from the deficiency of the enzyme α-L-iduronidase (IDUA) and presents with severe symptoms and a lifespan of less than 10 years. This enzyme is involved in the degradation of dermatan and heparan sulfate in the lysosomes. Its absence leads to lysosomal accumulation of partially degraded mucopolysaccharides (Neufeld and Meunzer, 2001). MPS type-I patients exhibit a wide spectrum of clinical phenotypes. The most severe one with early onset is MPS type-IH (Hurler syndrome), the attenuated phenotype with late onset without intellectual disability is MPS type-IS (Scheie syndrome), and the intermediate one is MPS type-IH/S (Hurler/Scheie syndrome) (Amr et al., 2009). The severity of the disease is primarily because of the effect of various mutations on the IDUA gene, which lead to different amounts of its residual activity (Terlato and Cox, 2003).
MPS type-II (Hunter syndrome) is a rare X-linked disorder resulting from deficiency of the lysosomal enzyme iduronate-2-sulfatase, which leads to progressive accumulation of dermatan and heparan sulfate within various tissues and organs (Neufeld and Meunzer, 2001). This type exclusively affects male patients, although the phenotypic expression in female patients, which occurs because of X-chromosomal inactivation or chromosomal abnormalities, has been reported (Tusch et al., 2005). The phenotypic expression is quite variable with respect to the presence and severity of the signs and symptoms. There are two forms of MPS type-II, mild or attenuated and severe (Young and Haper, 1983). The most common presenting signs are coarseness of the facial features, short stature, abdominal distention, macroglossia, enlargement of the tonsils and adenoids, respiratory difficulties, sleep apnea, hepatosplenomegaly, dysostosis multiplex, progressive joint stiffness, and hearing defects. Seizures are common in MPS type-II and patients develop learning difficulties with progressive neurodegeneration. Most people with MPS type-II suffer from progressive episodes of diarrhea of unknown etiology (Ashworth et al., 2006; Kudo et al., 2006).
MPS type-III (Sanfilippo syndrome) is caused by the defects in four different enzymes that result in lysosomal storage of the GAGs heparan sulfate. The four types are MPS type-III A due to deficiency of heparan sulfaminidase, MPS type-III B due to deficiency of N-acetyl α-D-glucosaminidase, MPS type-III C due to deficiency of acetyl CoA-α-glucosaminidase N-acetyltransferase, and MPS type-III D due to deficiency of N-acetylglucosamine-6-sulfatase (Ashworth et al., 2006; Malinowska et al., 2009). Patients with MPS type-III are severely intellectually disabled with learning difficulties, have severe behavioral disturbances, hyperactivity, aggression that is very difficult to treat, sleep disturbances, sensorineural deafness, hernia, gastrointestinal symptoms, and joint contracture (Valstar et al., 2008; Ohmi et al., 2009).
MPS type-IV (Morquio syndrome) results from deficiency of the enzymes N-acetylgalactosamine-6-sulfatase (MPS type-IV A) or β-galactosidase (MPS type-IV B). MPS type-IV B is now considered to be a variant of GM1 gangliosidosis (Ashworth et al., 2006). Deficiency of the enzyme N-acetylgalactosamine-6-sulfatase (MPS type-IV A) leads to deposition and accumulation of keratan sulfate in various tissues. Patients are severely dwarfed with multiple severe deformities and normal intelligence (Van Hoof, 1974). The deformities include short neck and trunk, kyphoscoliosis, protrusion of the sternum, prognathism, and genu valgum. Deafness is frequent, corneal opacities are rare, and hepatomegaly is less pronounced than in Hunter syndrome. Restrictive respiratory disorders, cardiac valve lesions, and umbilical and inguinal hernia are also less prominent (Part et al., 2008; Bank et al., 2009).
In MPS type-VI (Maroteaux–Lamy syndrome), the deficiency is in arylsulfatase B enzyme (N-acetylgalactosamine-4-sulfatase), which leads to accumulation of dermatan sulfate in various tissues. This causes a progressive heterogeneous disorder that often results in death in the second decade of life (Harmatz et al., 2005). Patients present with short stature, large head, short neck, corneal opacity, open mouth associated with large tongue, and several oral and dental manifestations. The dental manifestations include unerupted dentition, dentigerous cyst-like follicles, and gingival hyperplasia (Harmatz et al., 2004; Alpöz et al., 2006).
MPS type-VII (Sly syndrome) results from deficiency of β-glucuronidase enzyme, resulting in accumulation of dermatan, heparan, and chondroitin sulfate and usually presents as nonimmune hydrops fetalis. Affected individuals rarely survive more than few months (Bergwerk et al., 2000).
Biochemical laboratory diagnosis of MPS is performed by estimating the amount of urinary GAGs. Although there are exceptions explained below, each type of MPS generally has a specific pattern of separation that must be confirmed by measuring the specific enzyme activity in blood.
Patients and methods
This study included 1249 suspected Egyptian patients (735 male patients and 514 female patients). They were referred to the Biochemical Genetics Department at the National Research Centre during the last 11 years for the diagnosis or exclusion of MPS. Their ages ranged between 1 month and 24 years. Positive history of parental consanguinity was present in 675 (54.1%) patients, whereas it was negative in 574 (45.9%) patients. From all patients, 4–5 ml urine samples were collected and centrifuged; 100 μl of the sample was used to measure the total levels of GAGs and 3 ml of it was used for the extraction of GAGs for electrophoretic separation. For assaying the activity of the deficient enzymes, 5 ml whole blood with EDTA was collected and the leukocytes were separated from the samples. However, in assaying the activity of N-acetylglucosaminidase (deficient in the MPS type B), we collected 2 ml of whole blood with EDTA and separated the plasma. Patients were subjected to the following:
The level of total GAGs in the urine samples was measured.
Two-dimensional electrophoretic separation of the urinary GAGs was carried out in:
samples with high levels of total GAGs,
clinically suspected patients with Morquio syndrome type B even with normal levels of total GAGs.
Blood samples were obtained and the specific enzyme activity was assayed:
for the type of MPS according to the pattern of separation to confirm the diagnosis,
in clinically suspected patients with Morquio syndrome type B even with normal pattern of separation.
Quantitative determination of total GAGs in all urine samples was carried out by the method of De Dong et al. (1989). This method is based on binding of the dye dimethylmethylene blue to the urinary GAGs and using dermatan sulfate as a standard (Amr et al., 2009). GAGs concentration (mg/dl) was subsequently normalized to urinary creatinine concentrations, which were determined by the method of Hortin and Goolsby (1997), to yield final reported values of GAGs concentrations in units of mg/mmol creatinine (Whiteman, 1973). Total amounts of GAGs in the urine samples were compared with the age-dependent reference values (De Dong et al., 1992).
Patients with high concentrations and those suspected clinically to have MPS type-IV A were subjected to extraction of GAGs from the urine samples according to the method of Whiteman (1973). In this procedure, 1–2 ml of urine was mixed with 10 volumes of Alcian blue reagent containing 50 mmol/l MgCl2. Blue complex was formed and after centrifugation it was separated and precipitated. The complex was dissociated by shaking with 4 mol/l NaCl and methanol. Free Alcian blue was precipitated by the addition of 0.1 mol/l Na2CO3 and water. The Alcian blue was removed by centrifugation. GAGs were precipitated from the clear supernatant by the addition of ethanol and the precipitate was taken up in 20–40 μl water (Whiteman, 1973).
Two-dimensional electrophoresis was carried out on cellulose acetate sheets, and 1–2 μl of urinary GAGs or reference standard GAGs solutions (1 mg/ml) were applied. The separation proceeded for 1 h in pyridine, glacial acetic acid, and water and then for 3 h in barium acetate buffer. The material was then stained for 1 h with 0.05% Alcian blue solution containing 50 mmol/l MgCl2 in 50 mmol/l sodium acetate buffer (pH 5.8) and washed with 5% acetic acid solution. Clear blue spots on a white background were obtained (Whiteman, 1973; Mossman and Patrick, 2005). According to the pattern of separation, each type of MPS was confirmed by assaying the activity of its specific deficient enzyme except for three forms of Sanfilippo syndrome, which are described below (Burlingame et al., 1981).
Activity of the enzyme α-iduronidase responsible for MPS type-I (Hurler syndrome) was assayed by the method of Hopwood et al. (1979) using 4-methylumbelliferyl α-L-iduronide as a substrate giving 4-methylumbelliferone, which was easily measured fluorometrically.
Assaying the activity of the enzyme iduronate-2-sulfate sulfatase, which is deficient in MPS type-II (Hunter syndrome), was performed fluorometrically using 4-methylumbelliferyl-α-iduronate 2-sulfate as a substrate (Shawky et al., 2008; Gabrielli et al., 2010).
The diagnosis of patients with Sanfilippo syndrome type B was confirmed by assaying the activity of the enzyme N-acetyl-α-D-glucosaminidase (Marsh and Fenson, 1985) and patients with Morquio syndrome type A by assaying the activity of the enzyme N-acetylgalactosamine-6-sulfatase in blood (Van Diggelen et al., 1990). The activity of the enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B) in patients with Maroteaux–Lamy syndrome was assayed according to Baum et al. (1959) to confirm the diagnosis.
A written consent was signed by the studied subjects or their parents after full explanation of the study. The approval of the Medical Ethics Committee of the National Research Centre was obtained.
The present study included 1249 patients suspected clinically to have MPS. They were referred to the Biochemical Genetics Department from all over Egypt. The distribution of sex, age, and the consanguinity rate are presented in Tables 1, 2, and 3, respectively.
The studied patients were divided according to their ages into four groups as shown in Table 2.
More than half of the studied patients were born to consanguineous parents. Percentage of parental consanguinity among the studied patients is shown in Table 3.
Quantitative determination of total GAGs in urine was carried out in all patients. In 548 patients (43.9% of the 1249), the levels of urinary GAGs were elevated with respect to their ages according to the age-dependent reference values as shown in Table 4 (De Dong et al., 1992). Mean levels of total urinary GAGs in each age group for all the studied patients are shown in Table 2.
Two-dimensional electrophoretic separation of urinary GAGs was performed in 548 patients with elevated levels and the results of separation are illustrated in Fig. 1. In 239 patients, the separation revealed abnormal patterns specific for the different types of MPS, whereas it was normal in 309 patients.
Examples of the abnormal electrophoretic patterns for MPS type-I, III, and VI compared with the normal pattern are illustrated in Fig. 2.
Each type was confirmed by assaying its specific enzyme activity in the leukocytes except for N-acetylglucosaminidase (deficient in the MPS type-III B), which was assayed in plasma. The activity of the enzyme N-acetylglucosaminidase was measured in blood of 43 patients, showing the electrophoretic pattern specific for MPS type-III with characteristic relatively big spots of heparan sulfate. The results of the enzyme assay proved that 18 patients (of the 43) had MPS type-III B and hence 25 patients (of the 43) had MPS type-III but types other than B (A, C, or D). Enzyme assays for those three types were not available in our laboratory.
In 104 patients (of the 1249) suspected clinically to have Morquio syndrome type A, the levels of total GAGs were within normal limit for their age except in eight patients (of the 548 with elevated levels) who had high levels. Their average level was 17 mg/mmol creatinine and the electrophoretic patterns of separation were normal for all 104 patients [56 male patients (53.8% of the 104) and 48 female patients (46.2% of the 104)]. Activity of the enzyme N-acetylgalactosamine-6-sulfatase was measured in all 104 patients and 39 of them proved to have Morquio syndrome type A.
Measurement of the enzyme activity in blood was performed in both patients who had a specific pathologic electrophoretic pattern (n=239) and those who were suspected clinically to have Morquio syndrome type A (n=104) even with normal levels of total urinary GAGs and normal pattern of electrophoretic separation. The total number of patients in this study proved to have MPS was 278 (22.3% of 1249). Their average enzyme activities and the percentage of the different types among the studied patients are shown in Table 5.
The MPS are a group of disorders caused by inherited defects in the lysosomal enzymes resulting in widespread intracellular and extracellular accumulation of GAGs. They have been subdivided according to the enzyme defect and systemic manifestations. MPS are caused by reduction in the activity of specific lysosomal enzymes involved in the breakdown of GAGs, which results in a wide spectrum of clinical phenotypes ranging from those disorders that are fatal in the first months of life to those compatible with a normal lifespan.
There are geographical variations in the incidence of the different MPS types. MPS type-II is more common in Israel and MPS type-IV is more common in Northern Ireland (Neufeld and Meunzer, 2001). MPS type-III is the most common type in the UK, whereas MPS type-VI and MPS type-VII are very rare (Wraith, 2001; Wraith, 2008). In this study, we are reporting the Egyptian experience in the diagnosis of MPS and the commonest type among the studied patients and its unique findings.
Consanguineous marriages are frequent among the Egyptians; the frequency ranged from 29.5 to 75% among the diagnosed patients in the study by Afifi et al. (2010). In many of our previous studies on the lysosomal disorders, parental consanguinity exceeded 80% among the diagnosed patients (Shawky et al., 2008). In this study, parental consanguinity was 54.1% among 1249 referred patients and reached 80% in the diagnosed patients except for Hunter syndrome, where it decreased to 24% because of the X-linked mode of inheritance of this disorder. This coincides with our previous publications regarding parental consanguinity among MPS patients (Aboul Nasr and Fateen, 2008).
The present study included 735 male patients (58.8%) and 514 female patients (41.2%). The male predominance in many of our previous studies reflected the cultural background in oriental countries with the preference of male siblings and giving them better medical care.
More than 50% of referred patients were in age group II (1–5 years). This is because most of the clinical features of the different MPS types appear after the age of 1 year. Some east European countries such as Poland, Russia, Lithuania, and Belarus reported that over 57% of MPS type-VI showed the first signs of the disease after the age of 5 years and even as late as 21 years (Jurecka et al., 2012), whereas, in this study, age group III (5–10 years) comprised only 24.1% of the total study and group IV (>10 years) only 12.7%. Accordingly, age group I and II constituted 63.2% of the referred patients in this study, which is a good indicator of referring the patients early. In addition, it indicates the early appearance of the clinical signs and symptoms, which are usually severe.
Our results of the mean amount of total GAGs with respect to the four age groups show clearly that the excretion of GAGs decreases by age. Quantitative determination of GAGs in urine must be in reference to the patient’s age. These tests rely on carefully established age-dependent reference value. It is entirely age dependent, as children excrete more GAGs during their first few months after birth and excretion declines with age.
Qualitative analysis of GAGs by electrophoresis in this study showed that 56.4% of patients with high GAGs levels had a normal pattern. Hence, quantitative determination of GAGs and high GAGs levels are not diagnostic of MPS, as it could be increased in other conditions. As GAGs are present in the bones and cartilages, their levels can be elevated in diseases affecting these tissues, such as mucolipidoses (Tomatsu et al., 2005), fucosidosis (OMIM: #230000), and GM1 gangliosidosis (OMIM: #230500).
Although each abnormal pattern is diagnostic of one type of MPS, still this must be confirmed by measuring the enzyme activity in each type. In many patients, Hunter and Hurler syndrome give similar electrophoretic patterns. Substantial experience is required in interpreting the pattern, and sometimes measuring both the α-iduronidase and iduronate-2-sulfate sulfatase enzyme activities is required to reach the proper diagnosis. This agrees with the study by Burlingame et al. (1981) who gave the same comment in their study on the direct quantitation of GAGs from 2 ml of MPS patient’s urine. They also stated that, in Maroteaux–Lamy syndrome, the urinary GAGs were similar in nature to those in the Hurler and Hunter patients, but no heparan sulfate or heparin were detected. Our results coincide with their observations.
Still patients with naturally trace amounts of heparan sulfate in addition to excess dermatan sulfate excretion due to MPS type-VI might be incorrectly identified as an MPS type-I or II patient. Ultimately, it is not feasible to conclusively differentiate MPS I, II, and VI using qualitative GAG analysis. Electrophoretic pattern in MPS type-I patients shows big spot of dermatan sulfate and regarding MPS type-II patients, the dermatan spots are relatively smaller. This coincides with what have been reported by Burlingame et al. (1981) that MPS type-I excreted more GAGs than any of the MPS type-II patients. In our patients, we found an overlap between the patterns of MPS type-I and II because the dermatan sulfate excretion was not in excess; we had to assay the deficient enzyme for both types.
Qualitative and quantitative methods currently used in the laboratory are only screening methods and require enzymatic and or molecular analysis to confirm the diagnosis of a specific disease; under no circumstances, they should be considered diagnostic (Wood et al., 2013). This statement by Wood et al. (2013) summarizes our experience. The enzyme activity was measured in all suspected patients to confirm the diagnosis.
Heparin and heparan sulfate were secreted in 7.8% of patients. This is specific for MPS type-III and coincides with the report by Sato and Gyorkey (1977) but is in contrast with the report by Burlingame et al. (1981). The latter group stated that in this disorder there is characteristic excretion of unusually high amounts of heparan sulfate only. They also stated that in Morquio syndrome there is an excretion of excess amount of chondroitin and keratan sulfate, whereas our Morquio patients had normal electrophoretic pattern and almost normal GAGs excretion in urine. This is because keratan sulfate is probably bound to or associated with certain amount of chondroitin sulfate as judged by its electrophoretic mobility before and after the use of enzymatic digestion by chondroitinase ABC. This suggests that keratan sulfate in urine of Morquio patients may be linked in some way to chondroitin sulfate (Burlingame et al., 1981). In the method we applied in this study, we did not use this enzyme digestion, which needs a modified and complicated method to obtain keratan sulfate as a separate spot. In the study by Wood et al. (2013), the authors stated that keratan sulfate can be present without elevating the total amount of GAGs in urine above the upper limits in unaffected individuals.
The diagnosis of Morquio syndrome can be challenging and requires agreements of clinical, radiographic, and laboratory findings. A group of biochemical genetics laboratory directors and clinicians involved in the diagnosis of MPS type-IV A, convened by BioMarin Pharmaceutical Inc., (Novato, California, USA) concluded that urinary GAGs analysis is particularly problematic in MPS type-IV A patients and it is strongly recommended to proceed to enzyme activity testing even if urine appears normal (Wood et al., 2013).
Although this recommendation appeared in the Journal of Inherited Metabolic Disease in March 2013, it agrees with our experience and guideline for the last 10 years. Morquio syndrome has distinct clinical and radiologic features that are characteristic and do not overlap with any other MPS types. In suspected patients with Morquio syndrome, we measure the N-acetylgalactosamine-6-sulfatase enzyme activity without measuring the GAGs levels, either quantitative or qualitative.
From our results, it is clear that the mean level of total urinary GAGs in MPS type-I and VI patients shows the highest concentration among the diagnosed patients followed by MPS type-II and then type-III.
Measuring different enzyme activities to confirm the diagnosis of these progressive disorders resulting from deficiency of lysosomal enzymes involved in the degradation of GAGs is the only diagnostic test for different types of MPS as stated by Wood et al. (2013) in his recommendations for the laboratory diagnosis of MPS type-VI. Enzyme activity analysis is universally acknowledged as a key component of diagnosis. Our experience over many years agrees totally with this recommendation, which summarizes our laboratory guidelines.
The main aim of this study was to analyze our data to demonstrate the presence of each type of MPS among our referred cohort of patients and compare it with the published data of other studies. MPS type-I comprised 28.5% of the diagnosed patients. Second in order came MPS type-VI with 25.5%, whereas MPS type-II came third in order with 16.5% incidence followed by type-III with 15.5% incidence (MPS type-III B with 6.5% and MPS type-III A, C, and D with 9% incidence) and then type-IV with 13.9% incidence. The study by Nieves Cobos et al. (2012) from Germany, which analyzed 211 samples including samples from Middle East, Mexico, and Europe, concluded that 8% were MPS type-I, 5% were type-II, and 5% type-VI. These results are totally different from ours, but they do not reflect one population.
The study conducted by Di Natale et al. (1993) summarized 15 years of experience in the diagnosis of MPS in Italy. They diagnosed 144 MPS patients, whereas we diagnosed 278 patients in 11 years, almost double the number of patients. The highest number of diagnosis in the Italian study was that of MPS type-II – 74 patients (51%), whereas MPS type-II in our study included 46 patients (16.5%); MPS type-I included 20 patients (13.9%) as against 79 patients (28.5%) in our study. Only three patients were diagnosed with MPS type-VI (2.1%), whereas we diagnosed 71 patients with MPS type-VI, which came in the second place (25.5%) after MPS type-I (28.5%).
Although there are geographical variations in the incidence of MPS, these disorders are common in our population because of the high consanguinity rate in our society compared with the Italian experience. However, the number of births per year over this interval is more than three times as many in Egypt as in Italy. The unique finding of our study is that MPS type-VI (Maroteaux–Lamy syndrome), which is considered a rare disease, comprised 25.5% of our diagnosed patients in our population.
Conflicts of interest
There are no conflicts of interest.
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