Adriana M. Montaño

International Morquio A Registry: Clinical manifestation and natural course of Morquio A disease

SummaryMucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase. The natural history of this disease is incompletely understood. To study which variables influence the clinical outcome, we conducted a study in which MPS IVA patients were asked to fill out a questionnaire with inquiries regarding family history, diagnosis, signs and symptoms, height, weight, surgical history, physical activity, and general complaints. A total of 326 patients (172 male, 154 female) from 42 countries enrolled in the Morquio A Registry programme. The mean age of patients enrolled was 14.9 years for males and 19.1 years for females, with a wide range of 1–73 years. Sixty-four per cent of the patients were under 18 years. Initial symptoms were recognized between 1 and 3 years of age (mean age 2.1 years) and mean age at diagnosis for the patients was 4.7 years. A progressive skeletal dysplasia was commonly observed among the MPS IVA patients. Fifty per cent of patients underwent surgical operations to improve their quality of life. The most frequent surgical sites include neck (51%), ear (33%), leg (26%) and hip (25%). The birth length for affected males and females was 52.2 ± 4.7 cm and 52.2 ± 4.5 cm, respectively. The final adult height for affected males and females was 122.5 ± 22.5 cm and 116.5 ± 20.5 cm, respectively. The results of this study provide a reference for assessment of efficacy for studies of novel therapies.

Mucopolysaccharidosis Type IVA (Morquio A Disease): Clinical Review and Current Treatment: A Special Review

Mucopolysaccharidosis IVA (MPS IVA), also known as Morquio A, is a rare, autosomal recessive disorder caused by a deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate-sulfatase (GALNS), which catalyzes a step in the catabolism of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate (C6S). It leads to accumulation of the KS and C6S, mainly in bone and cornea, causing a systemic skeletal chondrodysplasia. MPS IVA has a variable age of onset and variable rate of progression. Common presenting features include elevation of urinary and blood KS, marked short stature, hypoplasia of the odontoid process, pectus carinatum, kyphoscoliosis, genu valgum, laxity of joints and corneal clouding; however there is no central nervous system impairment. Generally, MPS IVA patients with a severe form do not survive beyond the third decade of life whereas those patients with an attenuated form may survive over 70 years. There has been no effective therapy for MPS IVA, and care has been palliative. Enzyme replacement therapy (ERT) and hematopoietic stem cell therapy (HSCT) have emerged as a treatment for mucopolysaccharidoses disorders, including Morquio A disease. This review provides an overview of the clinical manifestations, diagnosis and symptomatic management of patients with MPS IVA and describes potential perspectives of ERT and HSCT. The issue of treating very young patients is also discussed.

Growth Charts for Patients Affected With Morquio A Disease

Children with Morquio A disease grow poorly and become physically handicapped because of systemic bone disease. The purpose of this study was to describe observed growth patterns and their relationship with the physical condition of patients with Morquio A. In a one‐center study, questionnaire‐based longitudinal and cross sectional data were used to develop growth curves, to assess physical activity and to determine the incidence of surgical procedures in 354 patients with Morquio A. Mean birth lengths of boys and girls were 52.6 and 52.1 cm, respectively. The mean final heights for males and females at 18 years and older were 122.4 ± 21.5 and 113.1 ± 22.6 cm, respectively. These results corresponded to −7.4 SD for males and −7.7 SD for females compared to the normal healthy controls. Mean birth weights for boys and girls were 3.59 ± 0.58 and 3.5 ± 0.7 kg, respectively. The mean body mass index for males and females at over 18 years of age was 24.7 ± 6.1 and 25.6 ± 5.4 kg/m2, respectively. The growth pattern in Morquio A patients was characterized by impaired growth velocity after 1 year of age. This is the first report providing growth charts for patients with Morquio A, which can help with monitoring the disease and assessing the clinical efficacy of treatments.

Newborn screening and diagnosis of mucopolysaccharidoses

Mucopolysaccharidoses (MPS) are caused by deficiency of lysosomal enzyme activities needed to degrade glycosaminoglycans (GAGs), which are long unbranched polysaccharides consisting of repeating disaccharides. GAGs include: chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and hyaluronan. Their catabolism may be blocked singly or in combination depending on the specific enzyme deficiency. There are 11 known enzyme deficiencies, resulting in seven distinct forms of MPS with a collective incidence of higher than 1 in 25,000 live births. Accumulation of undegraded metabolites in lysosomes gives rise to distinct clinical syndromes. Generally, the clinical conditions progress if untreated, leading to developmental delay, systemic skeletal deformities, and early death. MPS disorders are potentially treatable with enzyme replacement therapy or hematopoietic stem cell transplantation. For maximum benefit of available therapies, early detection and intervention are critical. We recently developed a novel high-throughput multiplex method to assay DS, HS, and KS simultaneously in blood samples by using high performance liquid chromatography/tandem mass spectrometry for MPS. The overall performance metrics of HS and DS values on MPS I, II, and VII patients vs. healthy controls at newborns were as follows using a given set of cut-off values: sensitivity, 100%; specificity, 98.5-99.4%; positive predictive value, 54.5-75%; false positive rate, 0.62-1.54%; and false negative rate, 0%. These findings show that the combined measurements of these three GAGs are sensitive and specific for detecting all types of MPS with acceptable false negative/positive rates. In addition, this method will also be used for monitoring therapeutic efficacy. We review the history of GAG assay and application to diagnosis for MPS.

Enhancement of Drug Delivery: Enzyme-replacement Therapy for Murine Morquio A Syndrome

Mucopolysaccharidosis IVA (MPS IVA, Morquio A disease) is an inherited lysosomal storage disorder that features skeletal chondrodysplasia caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Human GALNS was bioengineered with the N-terminus extended by the hexaglutamate sequence (E6) to improve targeting to bone (E6-GALNS). We initially assessed blood clearance and tissue distribution. Next, to assess the effectiveness of storage clearance and reversal of pathological phenotype, a dose of 250 U/g of enzyme was given weekly to Morquio A mice (adults: 12 or 24 weeks, newborn: 8 weeks). Sulfatase modifier factor 1 (SUMF1) was co-transfected to activate the enzyme fully. The E6-GALNS tagged enzyme had markedly prolonged clearance from circulation, giving over 20 times exposure time in blood, compared to untagged enzyme. The tagged enzyme was retained longer in bone, with residual enzyme activity demonstrable at 48 hours after infusion. The pathological findings in adult mice treated with tagged enzyme showed substantial clearance of the storage materials in bone, bone marrow, and heart valves, especially after 24 weekly infusions. Mice treated from the newborn period showed marked reduction of storage materials in tissues investigated. These findings indicate the feasibility of using tagged enzyme to enhance delivery and pathological effectiveness in Morquio A mice.

Mutations and Polymorphisms in GUSB Gene in Mucopolysaccharidosis VII (Sly Syndrome)

Mucopolysaccharidosis VII (MPS VII; Sly syndrome) is an autosomal recessive disorder caused by a deficiency of β‐glucuronidase (GUS, EC 3.2.1.31; GUSB). GUS is required to degrade glycosaminoglycans (GAGs), including heparan sulfate (HS), dermatan sulfate (DS), and chondroitin‐4,6‐sulfate (CS). Accumulation of undegraded GAGs in lysosomes of affected tissues leads to mental retardation, short stature, hepatosplenomegaly, bone dysplasia, and hydrops fetalis. We summarize information on the 49 unique, disease‐causing mutations determined so far in the GUS gene, including nine novel mutations (eight missense and one splice‐site). This heterogeneity in GUS gene mutations contributes to the extensive clinical variability among patients with MPS VII. One pseudodeficiency allele, one polymorphism causing an amino acid change, and one silent variant in the coding region are also described. Among the 103 analyzed mutant alleles, missense mutations accounted for 78.6%; nonsense mutations, 12.6%; deletions, 5.8%; and splice‐site mutations, 2.9%. Transitional mutations at CpG dinucleotides made up 40.8% of all the described mutations. The five most frequent mutations (accounting for 44/103 alleles) were exonic point mutations, p.L176F, p.R357X, p.P408S, p.P415L, and p.A619 V. Genotype/phenotype correlation was attempted by correlating the effects of certain missense mutations or enzyme activity and stability within phenotypes. These were in turn correlated with the location of the mutation in the tertiary structure of GUS. A total of seven murine, one feline, and one canine model of MPS VII have been characterized for phenotype and genotype. Hum Mutat 0,1–10, 2009,

Dermatan sulfate and heparan sulfate as a biomarker for mucopolysaccharidosis I

Mucopolysaccharidosis I (MPS I) is an autosomal recessive disorder caused by deficiency of α-L-iduronidase leading to accumulation of its catabolic substrates, dermatan sulfate (DS) and heparan sulfate (HS), in lysosomes. This results in progressive multiorgan dysfunction and death in early childhood. The recent success of enzyme replacement therapy (ERT) for MPS I highlights the need for biomarkers that reflect response to such therapy. To determine which biochemical markers are better, we determined serum and urine DS and HS levels by liquid chromatography tandem mass spectrometry in ERT-treated MPS I patients. The group included one Hurler, 11 Hurler/Scheie, and two Scheie patients. Seven patients were treated from week 1, whereas the other seven were treated from week 26. Serum and urine DS (ΔDi-4S/6S) and HS (ΔDiHS-0S, ΔDiHS-NS) were measured at baseline, week 26, and week 72. Serum ΔDi-4S/6S, ΔDiHS-0S, and ΔDiHS-NS levels decreased by 72%, 56%, and 56%, respectively, from baseline at week 72. Urinary glycosaminoglycan level decreased by 61.2%, whereas urine ΔDi-4S/6S, ΔDiHS-0S, and ΔDiHS-NS decreased by 66.8%, 71.8%, and 71%, respectively. Regardless of age and clinical severity, all patients showed marked decrease of DS and HS in blood and urine samples. We also evaluated serum DS and HS from dried blood-spot samples of three MPS I newborn patients, showing marked elevation of DS and HS levels compared with those in control newborns. In conclusion, blood and urine levels of DS and HS provide an intrinsic monitoring and screening tool for MPS I patients.

Validation of disaccharide compositions derived from dermatan sulfate and heparan sulfate in mucopolysaccharidoses and mucolipidoses II and III by tandem mass spectrometry

Glycosaminoglycans (GAGs) are accumulated in various organs in both mucopolysaccharidoses (MPS) and mucolipidoses II and III (ML II and III). MPS and ML II and III patients can not properly degrade dermatan sulfate (DS) and/or heparan sulfate (HS). HS storage occurs in the brain leading to neurological signs while DS storage involves mainly visceral and skeletal manifestations. Excessive DS and HS released into circulation and thus blood levels of both are elevated, therefore, DS and HS in blood could be critical biomarkers for MPS and ML. Such measurement can provide a potential early screening, assessment of the clinical course and efficacy of therapies. We here assay DS and HS levels in MPS and ML patients using liquid chromatography tandem mass spectrometry (LC/MS/MS). Plasma samples were digested by heparitinase and chondroitinase B to obtain disaccharides of DS and HS, followed by LC/MS/MS analysis. One hundred-twenty samples from patients and 112 control samples were analyzed. We found that all MPS I, II, III and VI patients had a significant elevation of all DS+HS compositions analyzed in plasma, compared with the controls (P<0.0001). Specificity and sensitivity was 100% if the cut off value is 800 ng/ml between control and these types of MPS group. All MPS I, II and III patients also had a significant elevation of plasma HS, compared with the controls (P<0.0001). All MPS VI patients had a significant elevation of plasma DS, compared with the controls (P<0.0001). These findings suggest measurement of DS and/or HS levels by LC/MS/MS is applicable to the screening for MPS I, II, III and VI patients.

Assessment of bone dysplasia by micro-CT and glycosaminoglycan levels in mouse models for mucopolysaccharidosis type I, IIIA, IVA, and VII

Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by mutations in lysosomal enzymes involved in degradation of glycosaminoglycans (GAGs). Patients with MPS grow poorly and become physically disabled due to systemic bone disease. While many of the major skeletal effects in mouse models for MPS have been described, no detailed analysis that compares GAGs levels and characteristics of bone by micro-CT has been done. The aims of this study were to assess severity of bone dysplasia among four MPS mouse models (MPS I, IIIA, IVA and VII), to determine the relationship between severity of bone dysplasia and serum keratan sulfate (KS) and heparan sulfate (HS) levels in those models, and to explore the mechanism of KS elevation in MPS I, IIIA, and VII mouse models. Clinically, MPS VII mice had the most severe bone pathology; however, MPS I and IVA mice also showed skeletal pathology. MPS I and VII mice showed severe bone dysplasia, higher bone mineral density, narrowed spinal canal, and shorter sclerotic bones by micro-CT and radiographs. Serum KS and HS levels were elevated in MPS I, IIIA, and VII mice. Severity of skeletal disease displayed by micro-CT, radiographs and histopathology correlated with the level of KS elevation. We showed that elevated HS levels in MPS mouse models could inhibit N-acetylgalactosamine-6-sulfate sulfatase enzyme. These studies suggest that KS could be released from chondrocytes affected by accumulation of other GAGs and that KS could be useful as a biomarker for severity of bone dysplasia in MPS disorders.

Mucopolysaccharidosis IVA: Correlation between genotype, phenotype and keratan sulfate levels

Mucopolysaccharidosis IVA (MPS IVA) is caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to systemic skeletal dysplasia because of excessive storage of keratan sulfate (KS) in chondrocytes. In an effort to determine a precise prognosis and personalized treatment, we aim to characterize clinical, biochemical, and molecular findings in MPS IVA patients, and to seek correlations between genotype, phenotype, and blood and urine KS levels. Mutation screening of GALNS gene was performed in 55 MPS IVA patients (severe: 36, attenuated: 13, undefined: 6) by genomic PCR followed by direct sequence analysis. Plasma and urine KS levels were measured by ELISA method. Genotype/phenotype/KS correlations were assessed when data were available. Fifty-three different mutations including 19 novel ones (41 missense, 2 nonsense, 4 small deletions, 1 insertion, and 5 splice-site) were identified in 55 patients and accounted for 93.6% of the analyzed mutant alleles. Thirty-nine mutations were associated with a severe phenotype and ten mutations with an attenuated one. Blood and urine KS concentrations in MPS IVA patients were age-dependent and markedly higher than those in age-matched normal controls. Plasma and urine KS levels in MPS IVA patients with the severe phenotype were higher than in those with an attenuated form. This study provides evidence for extensive allelic heterogeneity of MPS IVA. Accumulation of mutations as well as clinical descriptions and KS levels allows us to predict clinical severity more precisely and should be used for evaluation of responses to potential treatment options.