Approach Considerations
Clinical recognition of unique skeletal abnormalities, combined with radiographic and biochemical analyses, is important in the diagnosis of Morquio syndrome (mucopolysaccharidosis type IV [MPS IV]). Although radiographic findings provide substantial insight, they need to be combined with biochemical analysis (enzyme activity), substrate analysis (KS and C6S), and molecular analysis to diagnose Morquio syndrome.
Laboratory Studies
Biochemical Analysis
Morquio A syndrome
GALNS enzyme activity in fibroblasts and leukocytes is assessed when Morquio syndrome is suspected. However, GALNS activity in fibroblasts and leukocytes is sensitive to temperature changes, so strict adherence to guidelines for shipping specimens and interpretation of results is required. Dried blood spots (DBS) have been used as an alternative sample source when there are difficulties in shipping of viable cells. [44, 45] However, enzyme activity in DBS samples is also affected by temperature changes. Therefore, the delay between collection and assay can provide false-positive results. [45]
Currently, many laboratories use an assay based on the fluorogenic substrate. [46] The fluorometric assay needs 4-methylumbelliferyl-ß-D-galactopyranoside-6-sulfate (4MU-Gal6S) as a substrate. GALNS and exogenous ß-galactosidase in samples remove 6-sulfate and galactoside, respectively. The released methylumbelliferone fluoresces at a high pH. [47]
A quantitative GALNS assay method has been established by measuring processing of a novel substrate via tandem mass spectrometry. [44, 48, 49, 50] A careful interpretation of enzyme activity results is required since the reference range of GALNS activity is affected by the sample type, laboratory techniques, and specific methodologies. [51]
Morquio B syndrome
β-galactosidase activity is tested in fibroblasts or leukocytes. The fluorometric assay often uses p-nitrophenyl β-galactopyranoside or 4-methylumbelliferyl-ß-galactopyranoside as a substrate.
Substrate Analysis
Morquio A syndrome
Serum and urinary KS levels show a positive correlation with clinical severity in Morquio A syndrome (mucopolysaccharidosis type IVA [MPS IVA]). [52, 53, 54, 55, 56] Results of these studies indicate that serum and urinary KS levels may be used as a potential biomarker for evaluating the clinical severity of Morquio A syndrome at an early stage and may be valuable for monitoring therapeutic effects.
A range of assay methods have been developed to measure serum and urinary KS, both experimentally and clinically, including an inhibition enzyme-linked immunoassay (ELISA), [57] a sandwich ELISA, [57, 58] and liquid chromatography–tandem mass spectrometry (LC-MS/MS). [55, 59, 60, 61, 62] Oguma et al first reported a detection method to specifically measure abnormal GAG levels including KS by using LC-MS/MS. [59, 60] These methods have been revised and refined by other groups. [54, 55, 61, 62, 63, 64, 65, 66, 67] Therefore, LC-MS/MS has become a highly specific, sensitive, and cost-effective quantitative method for measuring KS and C6S levels in the blood, urine, and DBS specimens. [66, 67]
Mono-sulfated and di-sulfated KS levels in blood and urine were significantly higher in young patients with Morquio A syndrome compared with age-matched controls. The elevation of di-sulfated KS levels was more significant compared with that of mono-sulfated KS. The proportion of di-sulfated KS in total KS level increases with age in control patients but is age-independent among patients with Morquio A syndrome, suggesting that the proportion of di-sulfated KS in total KS is more discriminating for younger patients with Morquio A syndrome than for older patients.
A quantitative method to measure C6S was also established by using LC-MS/MS. [67] This assay method separates C6S disaccharides from other CS disaccharides in blood and urine. Levels in both tissues were significantly higher in patients with Morquio A syndrome than in age-matched controls. Combining KS and C6S data is better than either C6S or KS alone at differentiating patients with Morquio A syndrome from age-matched controls.
Overall, KS and C6S levels are potential biomarkers not only for screening and diagnosing Morquio A syndrome but also for assessing the clinical severity and therapeutic efficacy.
Morquio B syndrome
Patients with Morquio B syndrome have increased excretion and defective degradation of KS. [68] Urinary specimens in patients with Morquio B syndrome also contain much smaller chondroitin sulfate amounts than in patients with Morquio A syndrome.
Molecular Analysis
Morquio A syndrome
The human GALNS gene is localized at 16q24.3, [69, 70] and the entire gene is approximately 50 kb long, containing 13 introns and 14 exons. [71] The spliced mRNA is 1566-bp, encoding a protein of 522 amino acid residues. After a 21–amino acid signal peptide and N-glycosylation are removed by cleavage, the protein is processed, yielding the mature active GALNS enzyme, which consists of 40- and 15-kDa subunits. [71, 72, 73] GALNS is one of 13 evolutionary related sulfatases in the human genome. All sulfatases show 20%-35% similarity at the amino acid level, and the C79 residue in exon 2 of human GALNS is conserved in all sulfatase proteins from many species. This cysteine is post-translationally modified to a formylglycine residue by sulfatase modifying factor 1, [74] and structural and homology analysis shows that it is a vital part of the active GALNS site.
More than 400 mutant GALNS alleles have been identified from 250 patients with Morquio A syndrome. Of these mutant alleles, 328 different mutations have been shown to cause the disease phenotype as of October 2016; [52, 56, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85] missense/nonsense mutations account for 243 mutations (74.1%), while the rest consist of deletions (32 [9.8%]), splicing site mutations (32 [9.8%]), and insertions (5 [1.5%]). [6] The most common mutations are reported to be missense mutations: C1156>T (p.R386C), G901>T (p.G301C), and A337>T (p.l113F). These mutations have been detected in various ethnic groups. [80, 86, 87, 88, 89] The 10 most prevalent GALNS mutations account for 35% of all known Morquio A syndrome cases. Molecular analysis is commonly performed using a blood or DBS sample.
Morquio B syndrome
The human β-galactosidase gene is localized on chromosome 3 at 3p21.33, and the entire gene is approximately 60 kb long, containing 16 exons. The cDNA encodes 677 amino acid residues. Paschke et al reported that 14 of 15 European patients with Morquio B syndrome had a missense mutation, p.W273L. [90]
Imaging Studies
Morquio A syndrome
Imaging techniques such as radiography, computed tomography (CT), and magnetic resonance imaging (MRI) are often used to help management of Morquio A syndrome. Radiography, CT, and MRI are used to evaluate compression and instability in the upper cervical spine and spinal stenosis. These imaging studies commonly reveal anterior beaking, kyphoscoliosis, platyspondyly, and vertebrae irregularity. Imaging before age 2 years is important. Patients should be monitored annually to determine whether orthopedic surgery is required.
Regular follow-up of tracheal obstruction is also necessary, as tracheal obstruction in individuals with Morquio A syndrome is life-threatening and places them at high risk of mortality and extubation failure in the anesthetic procedure owing to sleep apnea and airway complications. [34, 91] CT angiography is required to observe anatomy of the trachea, artery, cervicothoracic spine, manubrium, and thoracic inlet. CT angiography has identified multiple factors that cause tracheal obstruction in Morquio A syndrome, including (1) disproportionate development of the trachea, brachiocephalic artery, cervicothoracic spine, and chest cavity and (2) a severe pectus carinatum and crowding of thoracic inlet (see image below). [91]
Tracheal obstruction. CT angiography in a 29-year-old patient shows severe tracheal obstruction. Tracheal narrowing (T; trachea), often due to compression from the crossing brachiocephalic (innominate) artery, increases with age. Note the position of the brachiocephalic artery (A) anterior to the trachea. Cervicothoracic spine moves forward while a severe pectus carinatum (M: manubrium) compresses backward. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
Morquio B syndrome
Gucev et al reported on a 24-year-old female with Morquio B syndrome in whom bone radiography showed platyspondyly with ovoid vertebrae and anterior projection. Her hip joint had coxa valga. The femoral head was flattened, and acetabulum was dysplastic and wavelike. Her hand had a deformity of radiocarpal articulation, and her metacarpals had conical bases. [92]
Histologic Findings
Morquio A syndrome
An autopsy case study in a patient with Morquio A syndrome showed systemic storage materials in multiple tissues (trachea, lung, thyroid, humerus, aorta, heart, liver, spleen, kidney, testes, bone marrow, and lumbar vertebrae) beyond cartilage. [93] Severely vacuolated and ballooned chondrocytes are found in the trachea, humerus, vertebrae, and thyroid cartilage with disorganized extracellular matrix and poor ossification. The appearance of foam cells and macrophages is shown in the lung, aorta, heart valves, heart muscle, trachea, visceral organs, and bone marrow.
Morquio B syndrome
Bulbar conjunctival biopsies showed intracytoplasmic vacuoles. [11]
Animal Models
To fully understand and treat rare genetic diseases in humans, animal models are typically developed. [94, 95] Molecular biology techniques and homologous recombination have been used to engineer animal models for Morquio A syndrome. [96]
In 2000, Montaño et al reported on the isolation and expression of mouse Galns cDNA, its chromosomal localization, genomic organization, and promoter analysis of the mouse Galns gene. [97] The full-length mouse Galns cDNA encodes 520 amino acids, two residues shorter than the human protein. The mouse and human nucleotide and amino acid sequences are 83% and 84% identical, respectively. The mouse Galns gene was mapped to the distal region of mouse chromosome 8, which is in a syntenic group on 16q24.3 that contains human GALNS. Both the mouse Galns gene and human GALNS gene have 14 exons and 13 introns, and both are approximately 50 kb in length, indicating high conservation of gene structure between these two species. [97]
After the elucidation of the mouse Galns gene structure, the first mouse model of Morquio A syndrome was produced via homologous recombination in 2003. This mouse model is a classic knock-out type mouse (Galns-/-), which produces no Galns mRNA because of a gene deletion in part of intron 1 and exon 2. [98] Lysosomal storage was present primarily within reticuloendothelial cells such as Kupffer cells and cells of the sinusoidal lining of the spleen at age 2 months. In addition, by age 12 months, vacuolar changes were observed in the visceral epithelial cells of glomeruli and cells at the base of heart valves, but not in parenchymal cells such as hepatocytes and renal tubular epithelial cells. In the brain, hippocampal and neocortical neurons and meningeal cells had lysosomal storage. Immunohistochemistry showed that KS and C6S were more abundant in the cytoplasm of corneal epithelial cells of Galns-/- mice compared with wild-type mice. Radiographs revealed no change in the bones of mice up to age 12 months. [98] After 50 mouse generations, the phenotype of the Galns-/- mouse model became more severe, with GAG accumulation in chondrocytes and other tissues. The genetic characteristics of this mouse model correspond to patients who have a large deletion in the GALNS gene and either no functional protein or no protein at all (unpublished).
In 2005, a mouse model tolerant to human GALNS (Galns tm (hC79S.mC76S) slu) was engineered. [99] This mouse model contains a point mutation in the active site (C76S) and a transgene (cDNA) expressing inactive human GALNS in intron 1 in which the active site cysteine is substituted with serine (C79S). This tolerant mouse model contains the inactive human cDNA and the C76S mouse mutation by targeted mutagenesis. This model showed an irregular growth plate region, with ballooned vacuolated chondrocytes in histopathological studies. The cartilage layer, especially the proliferative layer, was narrower than that in wild-type mice. The hypertrophic zone was thicker, and cells were disarrayed. [99] In addition, this tolerant mouse model had a ubiquitous expression of the inactive human GALNS, which resulted in tolerance to the immune response to human GALNS enzyme. This model has been very useful in the evaluation of enzyme replacement therapy or gene therapy in adult mice [100, 101] without the adverse effects of an immune response.
In 2007, a knock-in mouse model was developed (Galnstm (C76S) slu) in which the active site Cys was replaced with Ser (C76S) in the endogenous murine Galns by targeted mutagenesis. [102] The Galnstm(C76S)slu mouse model had lysosomal storage in Kupffer and spleen sinus-lining cells, heart valve stoma cells, glomerular visceral epithelial kidney cells, chondrocytes, and neocortical and hippocampal neurons. [102] Overall, the lysosomal accumulation in this mouse model was milder than that observed in the tolerant mouse model. The genetic characteristics of this mouse model correspond to patients with a point mutation in the GALNS gene who cannot produce a functional protein.
The bone phenotype of these mouse models is milder than that in patients with Morquio A syndrome. This can be explained by the lack of di-sulfated KS in mice and rats. [103] However, these mouse models still have mono-sulfated KS, which is present in proteoglycans that are found in cartilage, tendons, ligaments, and bone. [104] The phenotype of Morquio A syndrome mouse models has been questioned for several years owing to the milder appearance compared with patients who have Morquio A syndrome. The authors of this article have found that (1) the phenotype appears to be more severe after more than 50 generations of mice have been produced, (2) disease phenotype appears to progress with age, and (3) histological analysis of Morquio A mouse skeletal system has shown GAG accumulation and disarray in growth plate and chondrocytes.
Overall, the mouse models for Morquio A syndrome disease have helped significantly in the evaluation of therapies, including enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), and gene therapy. They remain essential tools in the development and evaluation of novel treatments for this devastating disease.
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Lateral view of spine in a child aged 8 years and 7 months. This radiograph shows advanced platyspondyly, irregularity, and anterior beaking of vertebral bodies characteristic of dysostosis multiplex. Note also the gibbus deformity and lordosis, which are characteristic of Morquio syndrome.
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Cervical spine, flexion and extension views, in a child aged 5 years and 11 months. These flexion and extension images depict anterior and posterior subluxation, respectively, of the atlas secondary to odontoid hypoplasia.
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Bilateral lower extremity views in a patient aged 22 years and 6 months. Metaphyseal irregularities and the characteristic genu valgus deformity are easily observed in this image.
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Bilateral hand radiographs in a patient aged 22 years and 6 months. Note the tapering of the proximal portion of metacarpals 2 through 5 and small irregular carpal bones. The epiphyseal involvement characteristic of Morquio syndrome is exemplified by the tapered irregular distal radius and ulna. Overall, the bones are osteopenic with cortical thinning.
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Upper extremities in a child aged 6 years and 11 months. Note the irregular epiphyses and widened metaphyses. Cortical thinning and mild widening of the diaphysis of the humerus are visible.
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Multiple abnormalities are present in the pelvis, including dysplastic femoral heads and oblique acetabular roof with coxa valgus deformity. Flared iliac wings usually observed in Morquio syndrome are not well represented in this radiograph.
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Anteroposterior view of the chest in a child aged 8 years and 4 months with Morquio syndrome. To reference the relatively small size of this chest, this patient's vital capacity was 500 cc, but the expected value based on height and weight was 1400 cc. Widened metaphyses and irregular epiphyses of the humeri and generalized platyspondyly are present. Oar-shaped ribs (widening ribs anteriorly and narrowing at the vertebrae) are easily observed and are another key characteristic of dysostosis multiplex.
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Defects in keratan sulfate (KS) degradation resulting in Morquio syndrome.
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The individual on the front of the scooter is 19 years old and has Morquio syndrome. Her friend on the back is an average-stature 10 year old without Morquio syndrome. On the driver, note the enlargement at the knees and the wrist deformity. Also, note the successful adaptation of the scooter to ambulate.
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Note the short trunk and protuberant rib structure in this child with Morquio syndrome. More importantly, notice that Morquio syndrome is not preventing this child from being active and fishing.
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Clinical pictures of patients with Morquio A syndrome (mucopolysaccharidosis type IVA [MPS IVA]). Left panel: a 31-year-old female patient with a severe form. Middle panel: an 18-year-old male patient with an intermediate form. Right panel: a 25-year-old male patient with a mild form. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
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Clinical features of a patient with Morquio A syndrome (mucopolysaccharidosis type IVA [MPS IVA]). This patient had a severe form of MPS IVA at age 3 years and had bone abnormalities of pectus carinatum, kyphoscoliosis, genu valgum, short stature, prominent forehead, and abnormal gait (height, 90 cm; 50th percentile of male MPS IVA growth chart; body weight, 14 kg). Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
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MRI of the cervical spine in a patient aged 4 years. A baseline study of the upper cervical anatomy is recommended no later than age 2 years or at diagnosis using flexion/extension radiography. If severe pain or pain associated with weakness or strength or tremors (or clonus) in the arms or legs occurs, the patient should undergo studies of the neck to evaluate for the slippage (subluxation) of the neck vertebrae and compression of the spinal cord. Radiography and MRI are performed with the head bent forward (flexion) and with the neck back (extension) and is performed annually for monitoring. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
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Clinical picture of hip deformity in an 8-year-old patient with Morquio A syndrome (mucopolysaccharidosis type IVA [MPS IVA]). Multiple abnormalities are shown in the hip, including spondyloepiphyseal dysplastic femoral heads, oblique acetabular roof with coxa valgus deformity, and flared iliac wings. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
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Skeletal/joint disease - hands. Bilateral hand radiographs in a patient aged 6 years. Note the tapering of the proximal portion of metacarpals 2 through 5 and small irregular carpal bones. The joints may become hyperlaxity by age 2 years. Eventually, the hands take on a characteristic tilting of the radial epiphysis toward the ulna due to a combination of metaphyseal deformities, hypoplasia of the bones, and degradation of connective tissues near the joint secondary to GAG accumulation. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
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Skeletal/joint disease - hands. The epiphyseal involvement characteristic of Morquio syndrome is exemplified by the tapered irregular distal radius and ulna. Overall, the bones are osteopenic with cortical thinning. Upper extremities in a child aged 2 years, 3 months (left panel). Note the irregular epiphyses and widened metaphyses. Cortical thinning and mild widening of the diaphysis of the humerus are visible. With aging, the bone deformity progresses, eg, with the tilting of the radial epiphysis toward the ulna. The humerus usually appears shortened later. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
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Skeletal/joint disease - hands. Unlike other mucopolysaccharidoses, Morquio syndrome is associated with ligamentous laxity. The cause of abnormal joint function remains unknown, presumably derived from a combination of metaphyseal deformities, hypoplasia of the bones, and degradation of connective tissues near the joint secondary to GAG accumulation. The wrist and fingers (small joints) are usually extremely weak, resulting in a very weak grip. Difficulties with dressing, personal hygiene, and writing can result from this hypermobile ligament. Range-of-motion exercises, swimming, and computer typing appear to offer some benefits in preserving joint function and fine motor skills and should be started early in the clinical course. Wrist splints and plastic braces may benefit fine motor functioning. The indication of physical therapy and its benefits in Morquio syndrome should be studied further. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
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Skeletal/joint problem – hands and legs. This 10-year-old patient with Morquio A syndrome (mucopolysaccharidosis type IVA [MPS IVA]) showed skeletal habitus with a disproportionate short stature of short trunk type with relatively long extremities. The upper extremities show lateral bowing and prominence of the radius. In the lower extremities, there is genu valgum (knocked knee). A skew foot posture is seen in both feet, as well as increased sandal gap between toes 1 and 2. Extreme ligamentous laxity permits very hypermobile knees, fingers, and wrists. The feet show pronated pes planus. Muscle bulk is reduced, particularly in the extremities. Generalized mild to moderate hypotonia is present. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
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Spinal cord pathophysiology. Odontoid hypoplasia is the most critical skeletal feature to be recognized in any patient with Morquio syndrome (mucopolysaccharidosis type IV [MPS IV]). This, in combination with ligamentous laxity and extradural mucopolysaccharide deposition, results in atlantoaxial subluxation, with consequential quadriparesis or even death. Another potential complication is cervical myelopathy. A history of exercise intolerance in patients with MPS IV often predicts occult cervical myelopathy, which can also cause bowel and bladder dysfunction and compression of the spinal cord, leading to weakness or paralysis. Mortality and morbidity are primarily related to the atlantoaxial instability and subsequent cervical myelopathy, and patients with a severe form, primarily related to cervical instability, often do not survive beyond the second or third decade of life. A minor fall or extension of the neck can result in spinal cord injury and subsequent quadriparesis or sudden death. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
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Pathophysiology of difficult airway in Morquio A syndrome. Both restrictive and obstructive respiratory pathology are common in patients with Morquio A syndrome. The restrictive defect results from thoracic cage deformity, and the obstructive defect is caused by tracheobronchial abnormalities, large tongue and mandible, adenoidal, tonsillar, and vocal cord hypertrophy by the accumulation of storage materials. An imbalance of growth between trachea, cervical spine, and brachiocephalic artery causes tracheal obstruction. Moreover, individuals with Morquio A syndrome have small nasal passages caused by thickened mucous membranes and thick and copious secretions. Chronic upper respiratory tract infection further decreases the already diminished airway lumen. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
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Tracheal obstruction. CT angiography in a 29-year-old patient shows severe tracheal obstruction. Tracheal narrowing (T; trachea), often due to compression from the crossing brachiocephalic (innominate) artery, increases with age. Note the position of the brachiocephalic artery (A) anterior to the trachea. Cervicothoracic spine moves forward while a severe pectus carinatum (M: manubrium) compresses backward. Courtesy of the Carol Ann Foundation; image adapted from Educational CD for International Morquio Organization.
