Mucolipidosis Type III α/β: The First Characterization of This Rare Disease by Autopsy

Mucolipidosis type III α/β (ML III, also known as pseudo-Hurler polydystrophy) is a rare autosomal recessive disorder of lysosomal hydrolase trafficking that displays prominent skeletal involvement. The enzymatic defect occurs in catalyzing the first step of mannose-6-phosphate synthesis, a step that is necessary to target a wide range of hydrolytic enzymes to the lysosome. Thus multiple enzymes and pathways are affected in this disorder in contrast to mucopolysaccharide diseases (MPSs), wherein individual lysosomal enzymes are defective because of mutated genes.1,2 Spranger and Wiedmann first described in 1970 that both mucopolysaccharides and lipids accumulate within lysosomes of patients with ML III.3 They observed that these patients clinically exhibited features of both MPSs and the sphingolipidoses and proposed the term mucolipidosis.3–6 Mucolipidosis type III is milder than other forms of mucolipidosis, and its clinical features most significantly involve abnormalities in cartilage and bone with a mild coarsening of facial features.1,4 Valvular heart disease and mild intellectual disability may also be seen.6–8 Features of this disease are evident in early childhood and slowly progress throughout life, generally becoming fatal in early adulthood.4 Diagnosis and typing of the mucolipidoses are performed by identifying specific enzyme level disparities between serum and cell extracts from cultured fibroblasts as well as through mutation analysis of the GNPTAB and GNPTG genes.4,6 

The patient was a 45-year-old woman with ML III whose disease manifested most prominently with tracheal-bronchial malacia and orthopedic problems. Early development was normal. At the age of 3 years she was noted to have stiff fingers and an inability to make a fist. The diagnosis of juvenile rheumatoid arthritis was assigned. Her growth slowed. Joint pain and limited range of motion progressed, particularly in her hands and hips. Radiographs were taken and were noted to be atypical for juvenile rheumatoid arthritis. Mucopolysaccharidosis VI and pseudo-Hurler polydystrophy (ML III) were considered in the differential diagnosis. Enzyme analyses distinguished the patient's disorder from the MPS conditions. The definitive diagnosis of ML III was confirmed with skin biopsy and fibroblast analyses at age 10. At age 42 years she underwent mutation analysis of the N-acetylglucosamine-1-phosphotransferase α/β gene (GNPTAB) for genotype-phenotype correlation studies in ML III. This analysis revealed an abnormal α/β subunit of the relevant gene with 2 point mutations that each led to a change in amino acid sequence (S15Y and R334Q).

Although neuropsychologic testing at age 6 years revealed a 1-year intellectual delay compared with peers, repeat cognitive testing when the patient was 43 years old by the Kaufman Brief Intelligence Test revealed a verbal score of 100, a nonverbal score of 90, and a normal composite IQ of 94. Her medical history contained many hospitalizations for bronchitis. Airway complications arose at 16 years during surgery to extract impacted wisdom teeth. Subsequent bronchoscopies confirmed tracheal deformity and tracheal malacia. At age 26 years she underwent reconstructive tracheal surgery that failed, which led to the placement of a tracheal stent that acted as a functional airway. This stent was replaced yearly for most of her life. A tracheobronchial fistula was discovered 2 years before her death. An umbilical hernia was repaired. Surgical history was also significant for numerous orthopedic procedures, including bilateral carpal tunnel releases, hallux valgus repair and revision, multiple arthroscopies, and joint replacements (bilateral knees and hips with revisions). In her 40s she received 2 years of high-dose bisphosphonate therapy. She ambulated independently for the majority of her life. For her terminal hospital course, she presented with progressive dysphagia, was treated for pneumonia, and died after 3 weeks.

The external examination revealed a patient of short stature (138 cm) weighing 43 kg with slightly coarsened facial features. She appeared younger than her stated age with pale white to waxy skin. Prominent kyphosis and scoliosis of the spine were noted, and her neck was short. On internal examination, osteoporosis was evident grossly throughout her ribs and vertebral column. Her tracheobronchial tree was thin-walled and small in caliber with focal areas of bone formation. There was a 2 × 1.5-cm chronic tracheoesophageal fistula without evidence of acute inflammation or injury. A recent tracheostomy site (0.3 × 0.2 cm) just inferior to the thyroid cartilage was noted, and the remaining portion of her tracheal stent spanned from just inferior to this site to the carina. The stent was well epithelialized with nodular granulation tissue distally. Evidence of a prior attempt at surgical reinforcement of the trachea using pericardium was seen with a 4.7 × 3.2-cm portion of intact tissue patch superficial to the right atrium. The lung parenchyma was variegated, purple-black to red-gray and friable. Diffuse adhesions were present along all visceral and parietal pleural surfaces, particularly between lung lobes bilaterally. The mediastinal and hilar lymph nodes were prominent and anthracotic. The mitral and tricuspid valves exhibited a firm but not calcific nodularity. There was slight hypertrophy of the myocardial ventricular walls (left ventricle thickness 1.3 cm [reference range, 0.8–1.2 cm], right ventricle 0.4 cm [reference range, 0.2–0.3 cm]) with minimal to no atherosclerosis throughout the vasculature. The liver appeared congested, and cholelithiasis was present. Her brain appeared grossly normal, weighing 1270 g in the fresh state, and the pituitary gland was small.

The mitral valve showed an increased cellularity with balloon-type cells (Figure 1, A), some of which contained periodic acid–Schiff (PAS)-positive granules. Alcian blue and trichrome stains (Figure 1, B) also highlighted the cytoplasmic lysosomal granules in these cells. Myocytes throughout the myocardium contained finely granular to chunky amorphous PAS-positive granules (Figure 1, C). Abundant lipofuscin granules were also seen surrounding nuclei. Trichrome staining highlighted increased connective tissue surrounding the large vessels. The right ventricle contained numerous hypertrophic myocytes with enlarged nuclei.

The lungs revealed patchy fibrosis and prominent chronic inflammation with increased intra-alveolar macrophages and lymphocytes as well as evidence of resolving aspiration pneumonia. The trachea showed a loss of cartilage with metaplastic bone formation. The cytoplasm of chondrocytes appeared finely vacuolated throughout the tracheobronchial tree (Figure 2, B and C). A portion of stratified squamous mucosa in a background of dense fibrous tissue with chronic inflammation was seen, consistent with tissue adjacent to a chronic tracheoesophageal fistula. The hyaline cartilage and perichondrium of the bronchi were structurally disorganized and contained areas of calcification.

Sections of ribs and vertebrae showed left-shifted trilineage hematopoiesis. Bone malformation was prominent with irregular defects in the subchondral bone of the vertebral body (Figure 2, A). Portions of the disk cartilage were seen herniating into the vertebral body, surrounded by granulation tissue and areas of partial mineralization. Osteoblastic and chondroblastic resorption were notable. Bone spicules showed scalloped and ragged edges with clusters of osteoclastic giant cells. Overall there was a slightly decreased bone mass with some thin trabeculae present. Fibrosis and repair were prominent and occurred in a slightly disorganized fashion, focused particularly around the subchondral bone plate. Polarization highlighted a slight reversal of the cement lines in some areas, resembling localized Paget disease. The overall picture appeared consistent with a metabolic bone disease after a history of bisphosphonate treatment. Cartilage of the trachea was notable for disordered collagen deposition and increased cellularity. Some of the chondrocytes were enlarged with granular, vacuolated cytoplasm. The spleen was congested with underlying histologically normal red and white pulp. Periodic acid–Schiff staining highlighted granular material in the cytoplasm of some of the cells, but whether this represented normal myeloid lineage granules or disease-related lysosomal granules is unclear. Lymph nodes showed lymphoid hyperplasia with reactive nodal architecture and scattered anthracotic pigment.

The liver appeared passively congested with a normal architecture. Some hepatocytes contained small, subtle PAS-positive lysosomal granules. This material was also slightly more prominently found in scattered fibroblasts within the portal triads as a focal process. Iron staining did not reveal increased iron stores.

Sections of the right frontal, hippocampal, and occipital cortices showed overall preservation of cytoarchitecture with a normal myelination pattern of the white matter. The leptomeninges contained occasional vacuolated macrophages and fibroblasts. Overall there appeared to be an increase in neuronal lipofuscin, and cortical neurons appeared slightly increased in size. Occasional reactive microglia and metabolic glia were seen throughout the brain. In the Sommer sector of the hippocampus there were areas of occasional neuronal dropout suggestive of subacute to remote hypoxic-ischemic injury. Periodic acid–Schiff and toluidine blue staining highlighted small, vesicular cytoplasmic lysosomal granules in neurons, oligodendrocytes, and astrocytes that appeared distinct from the neuronal lipofuscin (Figure 3, A and B). Alcian blue staining revealed a suggestion of finely granular cytoplasmic granules in neurons and glia. In the left occipital cortex, Luxol fast blue staining showed many neurons with Luxol fast blue positivity in cytoplasmic granular material. In the cerebellum, PAS highlighted cytoplasmic granules in the granule neurons, in the dentate nucleus, and particularly in the Bergmann glia. Within the spinal cord, PAS staining highlighted lysosomal granules in the anterior horn cells distinct from lipofuscin accumulation. Colloidal iron staining throughout the central nervous system sections was negative. The pituitary was histologically unremarkable.

Many organs showed cells with ultrastructural changes of increased cytoplasmic lysosomal granules. Electron microscopy of cerebral cortex sections showed scattered, diffuse vesicular cytoplasmic lysosomal granules in glia and neurons (Figure 3, C and D). Lysosomes contained a fine electron-lucent granular material. The increased lysosomes were not concentrated in particular areas of the cells but were found throughout the cytoplasm. Some lamellated phagolysosomes or autolysosomes were also seen with variably electron-dense material. The myocardium showed myocytes with prominent lipofuscin and a fibroblast filled with many finely granular lysosomes. The mitral valve showed a similar increase in lysosomal structures in some cells (Figure 1, D). Thyroid cartilage also showed increased finely granular lysosomes as well as electron-dense phagolysosomes or autolysosomes (Figure 2, D). In the skin, fibroblasts with similar fine and dense lysosomes were present though fewer in number. Hepatocytes displayed large, glycogenated vesicles along with lipid droplets and prominent terminal ischemic changes, but these cells did not have the characteristic lysosomes seen in other organs (with the exception of fibroblasts within periportal fibrous tissue). Mononuclear cells contained lysosomes similar to those seen in fibroblasts. Overall, the electron microscopic examination was significant for an increased number of intracellular lysosomal granules, particularly in the brain but also in cells of the heart, mitral valve, cartilage, and fibroblasts within the skin and liver.

Mucolipidosis type III α/β is a rare autosomal recessive lysosomal storage disease with prominent skeletal involvement. It resembles Hurler syndrome (and is also called pseudo-Hurler polydystrophy), though it is not characterized by organomegaly or mucopolysacchariduria. Accumulation of mucopolysaccharides along with lipids within lysosomes of patients with mucolipidosis is the characteristic abnormality. Clinically, these patients exhibit features of both the mucopolysaccharidoses and the sphingolipidoses.2,6 Clinical manifestations of ML III include moderate to severe dysostosis multiplex, stiffness of fingers and shoulders, carpal and tarsal tunnel syndromes, claw-hand deformity, short stature, and scoliosis. Other clinical characteristics include mild coarsening of facial features, mild corneal clouding, and valvular heart disease, and about half of patients experience mild intellectual disability.6–8 Diagnosis and typing of the mucolipidoses is performed by cultured fibroblast studies (showing that the enzymatic activity of different lysosomal hydrolases is decreased in fibroblasts and increased in serum and cell culture media) as well as through genetic techniques.6–8 There is significant variability in the expression and severity of this disease.6,9 

Classically, 4 distinct mucolipidoses have been recognized, and classified as type I through type IV (ML I–ML IV).2,4,6 However, now it is evident that only ML II and ML III are related allelic disorders of lysosomal trafficking. A deficiency in neuraminidase causes ML I (sialidosis), whereas ML IV is a neurodevelopmental disorder with retinal degeneration and normal lysosomal hydrolase activity.4 Patients with ML II and ML III are both characterized by a deficiency of UDP-G1cNAc 1-phosphotransferase enzyme activity. This enzyme is necessary for appropriate mannose 6-phosphate tagging, the phosphorylation of mannoses in oligosaccharide side chains that marks lysosomal hydrolases for uptake into the lysosome. Without these important recognition markers, lysosomal enzymes are not transported to the lysosomes; instead, they leak out of cells, and the serum levels of these enzymes are higher than normal. Macromolecules are thus incompletely degraded and subsequently accumulate in tissues.2,7 Whereas patients with ML II have complete loss of UDP-G1cNAc 1-phosphotransferase activity, patients with ML III have residual enzyme activity and thus a later onset and slower progression of signs and symptoms.7,10 Patients with ML II have severe progressive skeletal disease that is evident at birth and leads to death usually within the first decade of life. In contrast, patients with ML III have a more attenuated course and survive into adulthood, with minimal delays in childhood milestones but with a slowing of growth beginning at 4 years of age.1,4 

ML III was historically further subdivided into groups A, B, and C by complementation studies, although type B was represented by a single patient from whom there is no available cell line.11 The contemporary nomenclature for classification of ML II and III was established at the Second International Conference on Glycoprotein and Related Storage Diseases and incorporates current molecular and biochemical knowledge, designating which subunit of the gene is mutated (Table).7,10 The UDP-G1cNAc 1-phosphotransferase is a heterohexamer consisting of 3 subunits: 2 α, 2 β, and 2 γ subunits. It is a product of 2 separate, unlinked genes: GNPTAB, on chromosome 12q23.3, and GNPTG, on chromosome 16p13.3.1,4,11,12 Mucolipidosis type II α/β and ML III α/β, the principal focus of the current report, are the result of mutations in the gene coding for the α/β subunit where the catalytic domain resides. Mucolipidosis type III γ, a disorder clinically indistinguishable from ML III, results from mutations in the gene coding for the γ subunit for substrate recognition and binding.1,11 Genetic analysis in our case revealed an abnormal α/β subunit of GNPTAB. A recent study by Cathey et al4 (which included our case patient) analyzed the clinical and genetic features of 61 probands with ML II and ML III. It expanded upon previous, more general phenotype-genotype correlations, cataloged 42 novel mutations, and showed that the phenotypic spectrum of disease is more dichotomous than continuously variable.

The current case helps illustrate that although ML III has some clinical and pathologic overlap with MPS disorders, it is distinct. A genetically heterogeneous group, MPSs are inborn errors of lysosomal glycosaminoglycan metabolism and lead to the excessive accumulation of partially degraded glycosaminoglycan in essentially all tissues. The storage material depends on the deficient enzyme, and the spectrum of clinical severity is wide. However, the cellular pathology of affected tissues is similar in all MPS cases and is characterized by the presence of cells distended with large, clear vacuoles (clear cells) associated with extensive fibrosis in visceral organs. In the brain the clear cells represent the accumulation of gangliosides and are often associated with significant neuronal loss and gliosis.2 Hanai et al13 noted in 1971 that inclusions from cultured fibroblasts of patients with ML II were different from those of patients with MPSs, the former less metachromatic by light microscopy and containing osmophilic membranous structures by electron microscopy. In general, ML II and ML III more selectively affect the connective tissue as compared with MPSs and radiographic changes are more pronounced.1,6,14 

In MPS disorders, the pathology of the brain and liver has been studied most extensively, given that many patients manifest with hepatomegaly and mental retardation.15 Resnick et al15 studied liver biopsies from 27 patients with MPS and found characteristic membrane-bound inclusions within hepatocytes and Kupffer cells in all cases by electron microscopy. This contrasts with our current case of ML III, in which the liver findings were largely unremarkable with the exception of lysosomal granules contained within periportal fibroblasts. Jones et al16 characterized the pathology for 2 patients with MPS IIID (one of the rarest of the Sanfilippo syndromes) and found that the central nervous system was the most severely affected tissue because of secondary accumulation of gangliosides in addition to the primary lysosomal accumulation of heparin sulfate. In contrast to our case patient, who had minimal if any central nervous system symptoms in life, these 2 MPS IIID patients had severe intellectual disabilities, suffered loss of developmental milestones including speech, and died at ages 14 and 17 years. Grossly their brains showed gyral atrophy and mild dilatation of the lateral ventricles. By microscopic examination, neuronal cell bodies were distended, and the cytoplasm had a fine, foamy appearance due to fine and coarse PAS- and Luxol fast blue–positive granules. Storage patterns varied within and among different neurons. Heparin sulfate was found to preferentially accumulate in dendrites of the cerebral cortex, whereas gangliosides accumulated in the cell bodies. Oligodendrocytes, astrocytes, endothelial cells, and perithelial cells showed minimal evidence of lysosomal storage. Fibrosis, gliosis, and loss of neurons and neuropil were noted. Ultrastructural examination of neurons revealed complex arrangements of whorled or stacked membranes admixed with finely granular material and homogenous lipid droplets, with costorage of diverse materials within the same lysosome.16 In contrast, the brain of our patient with ML III was normal upon gross inspection. Histologic and ultrastructural examination revealed cytoplasmic lysosomal granules in neurons, oligodendrocytes, and astrocytes that were as diverse as those seen in MPSs but less dense in concentration and more evenly distributed throughout the cytoplasm of affected cells.

There is a paucity of literature regarding the pathology of ML III, and, to the best of the authors' knowledge, this is the first report published documenting full autopsy findings. Accordingly, the pathology of ML III is not as well understood as that of ML II. Given that the 2 diseases are now known to be allelic variants of dysfunction of a single enzyme, one can infer that the pathology of ML III might be similar to but less severe than that of ML II. Cultured fibroblasts from patients with ML III contain identical (though perhaps fewer) inclusion bodies compared with ML II.6 Notable gross pathologic findings in patients with ML II include Hurler-like facial features, gingival and tongue hypertrophy, dermal nodules, a short neck and thoracic cage, knoblike costochondral junctions, a thick pericardium, cardiomegaly, rigid and retracted valves, and lipid plaques in the aorta and major arteries.2,6 On a histologic level, lysosomal granules or inclusions are the most striking aspect of ML II, a disease also known as I-cell (for inclusion-cell) disease because of this feature.5,6 The histopathology of ML II is characterized by intracytoplasmic membrane-bound vacuoles in lymphocytes and fibroblasts in various tissues such as skin, conjunctiva, lymph nodes, spleen, gingiva, heart valves, and bones in areas of endochondral and membranous bone formation. Vacuoles have also been reported in Schwann cells, endothelial cells, pericytes, hepatocytes, myocardial fibers, and epithelial cells of renal glomeruli and tubules. Histochemically, these lysosomal granules stain for colloidal iron, PAS, Alcian blue at pH 2.5, toluidine blue at pH 2.0, and Sudan III and IV.2,6 A review17 of 56 cases, including predominantly patients with MPS but also a patient with ML II and recurrent pneumonia who died at age 8, examined the prevalence of narrowed airway due to storage material accumulation as a risk for anesthetic complications. The autopsy exemplified some of the aforementioned features: it revealed a thickened tongue base, epiglottis, larynx, and trachea grossly. Histology showed the presence of prominent balloon cells with acid mucopolysaccharide and lipids in connective tissue.

On an ultrastructural level, the intracellular lysosomal granules characteristic of the mucolipidoses have multiple appearances. The stored mucopolysaccharide substance is finely granular to flocculent and moderately electron-dense, whereas the lipid material appears as osmiophilic multilamellar bodies or fragments of membranelike material.6 Zebra bodies and membranous cytoplasmic bodies have been described in neurons. Electron-lucent vacuoles have also been found in astrocytes, oligodendrocytes, mesenchymal cells, and endothelial cells.2 

Skeletal and connective tissue problems tend to be prominent in patients with ML III, and the corresponding pathologic features have previously been characterized.9,14,18 A case series by Brik et al14 highlighted the rheumatologic presentation of ML III via 3 previously misdiagnosed patients with ML III and called for awareness of this disorder by pediatricians, rheumatologists, and orthopedic surgeons. Bone histopathology has been characterized by vigorous osteoclastic subperiosteal bone resorption, endosteal modeling, slight marrow fibrosis, and an increase in the amount of osteoid present. Normal cortical widths and low trabecular bone volume are typical.9 In our autopsy case, the underlying metabolic bone disease is seen after the remodeling influence of 2 years of high-dose bisphosphonate therapy ending a year and a half before the patient's death, and changes consistent with both disease and therapy are seen.

Valvular cardiac disease has also been well established in ML III.4,18 Fourteen of 15 patients with ML III in the previously mentioned series by Cathey et al4 exhibited clinical evidence of at least mild cardiac valve thickening, involving most commonly the mitral followed by the aortic valves. The one remaining patient is the subject of this case report; although there were no apparent valvular abnormalities clinically, upon autopsy she had nodular valve cusps with increased cellularity and balloon-type cells microscopically. Similarly, a small series reported by Satoh et al18 focused on the cardiac manifestations of mucolipidosis and promoted using echocardiography to follow patients throughout life. Of the 4 cases of mucolipidosis, 1 was a 16-year-old boy with ML III found to have asymmetric and thickened aortic cusps, a fixed mitral valve, and severe mitral prolapse with thickened leaflets on echocardiography.

For this case, we described what is thought to be the first autopsy report of a patient with ML III. In doing so, we reviewed the available literature, compared and contrasted findings in ML II and related diseases, corroborated the few areas of previously described histopathologic findings in ML III, and expanded the characterization of pathologic alterations possible in ML III. Our principal findings showed similar though less pronounced pathology compared with ML II and help further distinguish ML III and ML II from MPSs. Significant findings included gross abnormalities in bone and cartilage as well as cellular and ultrastructural abnormalities in bone, cartilage, the brain, myocardium, heart valves, and fibroblasts of the skin and liver.

We would like to thank Roger E. Stevenson, MD, of Greenwood Genetic Center, Greenwood, SC, for his expert review of our manuscript, and Arief A. Suriawinata, MD, of Dartmouth Hitchcock Medical Center, Lebanon, NH, for his excellent assistance in interpreting the liver pathology in this case. This study was supported in part by award number U54NS065768 from the National Institute of Neurological Disorders and Stroke, Bethesda, Maryland (Dr Cathey).