Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is an autosomal recessive lysosomal storage disorder resulting from a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Diagnosis can be challenging and requires agreement of clinical, radiographic, and laboratory findings. A group of biochemical genetics laboratory directors and clinicians involved in the diagnosis of MPS IVA, convened by BioMarin Pharmaceutical Inc., met to develop recommendations for diagnosis. The following conclusions were reached. Due to the wide variation and subtleties of radiographic findings, imaging of multiple body regions is recommended. Urinary glycosaminoglycan analysis is particularly problematic for MPS IVA and it is strongly recommended to proceed to enzyme activity testing even if urine appears normal when there is clinical suspicion of MPS IVA. Enzyme activity testing of GALNS is essential in diagnosing MPS IVA. Additional analyses to confirm sample integrity and rule out MPS IVB, multiple sulfatase deficiency, and mucolipidoses types II/III are critical as part of enzyme activity testing. Leukocytes or cultured dermal fibroblasts are strongly recommended for enzyme activity testing to confirm screening results. Molecular testing may also be used to confirm the diagnosis in many patients. However, two known or probable causative mutations may not be identified in all cases of MPS IVA. A diagnostic testing algorithm is presented which attempts to streamline this complex testing process.

Human first-trimester floating mesenchymal villi explanted onto gels of collagen I or Matrigel were observed to undergo de novo development of anchoring sites. These consisted of cytotrophoblast columns that formed by proliferation of stem villous cytotrophoblast cells, as revealed by whole-mount and thin-section microscopy and incorporation of bromodeoxyuridine into DNA. Column formation occurred exclusively at the distal tips of the villi. No column formation was observed in tissue explanted onto agarose. On Matrigel, the developing columns penetrated downwards into the matrix, whereas on collagen I, cytotrophoblast sheets spread across the surface of the gel and merged to form a shell. The developing columnar cytotrophoblast up-regulated integrins alpha1beta1 and alpha5beta1 and produced an extracellular matrix containing oncofetal fibronectin, as in vivo. Function-blocking antibodies were used to investigate the role of the integrin-fibronectin interaction in anchoring villus development on collagen I. Antibodies to fibronectin and the integrin subunits alpha5 and beta1, added at 24 h, all changed the pattern of cytotrophoblast outgrowth. Anti-fibronectin caused cell rounding within the cytotrophoblast sheet and increased the population of single cells at its periphery. Anti-integrin alpha5 caused rounding and redistribution of cells within the outgrowth. In the presence of anti-integrin beta1, cell-collagen interactions within the sheet were destabilized, often leading to the appearance of an annulus of aggregated cells at the periphery. These results show that 1) mesenchymal villi retain the potential to form anchoring sites until at least the end of the first trimester, 2) adhesion to a permissive extracellular matrix stimulates cytotrophoblast proliferation and differentiation along the extravillous lineage, 3) integrin alpha5beta1-fibronectin interactions contribute significantly to anchorage of the placenta to uterine extracellular matrix. We suggest that as the developing placenta ramifies, new sites of anchorage form whenever peripheral villi contact decidua. This process is predicted to contribute to the stability of the placental-decidual interface.

A rare lysosomal disease resembling a mucopolysaccharidosis with unusual systemic features, including renal disease and platelet dysfunction, caused by the defect in a conserved region of the VPS33A gene on human chromosome 12q24.31, occurs in Yakuts—a nomadic Turkic ethnic group of Southern Siberia. VPS33A is a core component of the class C core vacuole/endosome tethering (CORVET) and the homotypic fusion and protein sorting (HOPS) complexes, which have essential functions in the endocytic pathway. Here we show that cultured fibroblasts from patients with this disorder have morphological changes: vacuolation with disordered endosomal/lysosomal compartments and—common to sphingolipid diseases—abnormal endocytic trafficking of lactosylceramide. Urine glycosaminoglycan studies revealed a pathological excess of sialylated conjugates as well as dermatan and heparan sulphate. Lipidomic screening showed elevated β-D-galactosylsphingosine with unimpaired activity of cognate lysosomal hydrolases. The 3D crystal structure of human VPS33A predicts that replacement of arginine 498 by tryptophan will de-stabilize VPS33A folding. We observed that the missense mutation reduced the abundance of full-length VPS33A and other components of the HOPS and CORVET complexes. Treatment of HeLa cells stably expressing the mutant VPS33A with a proteasome inhibitor rescued the mutant protein from degradation. We propose that the disease is due to diminished intracellular abundance of intact VPS33A. Exposure of patient-derived fibroblasts to the clinically approved proteasome inhibitor, bortezomib, or inhibition of glucosylceramide synthesis with eliglustat, partially corrected the impaired lactosylceramide trafficking defect and immediately suggest therapeutic avenues to explore in this fatal orphan disease.

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BACKGROUND Mucopolysaccharidoses (MPS) are a group of inborn errors of metabolism that are progressive and usually result in irreversible in skeletal, visceral and/or brain damage, highlighting a need for early diagnosis. METHODS This pilot study analyzed 2,862 dried blood spots (DBS) from newborns and 14 DBS from newborn patients with MPS (MPS I, n = 7; MPS II, n = 2; MPS III, n = 5). Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Heparan sulfate (0S, NS), dermatan sulfate (DS) and mono- and di-sulfated KS were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Median absolute deviation (MAD) was used to determine cutoffs to distinguish patients from controls. Cutoffs were defined as median + 7× MAD from general newborns. RESULTS The cutoffs were as follows: HS-0S> 90ng/mL; HS-NS> 23 ng/mL, DS> 88 ng/mL; mono-sulfated KS> 445 ng/mL; di-sulfated KS> 89 ng/mL and ratio di-KS in total KS> 32%. All MPS I and II samples were above the cutoffs for HS-0S, HS-NS and DS, and all MPS III samples were above cutoffs for HS-0S and HS-NS. The rate of false positives for MPS I and II was 0.03% based on a combination of HS-0S, HS-NS, and DS, and for MPS III was 0.9% based upon a combination of HS-0S and HS-NS. CONCLUSIONS Combination of levels of two or more different GAGs improves separation of MPS patients from unaffected controls, indicating that GAG measurements are potentially valuable biomarkers for newborn screening for MPS.

Haematopoietic stem cell transplantation is the treatment of choice for the severe form of Mucopolysaccharidosis Type I, or Hurler syndrome. In many centres standard practice is to deliver enzyme replacement therapy alongside haematopoietic stem cell transplantation to improve the condition of the patient prior to transplant. We report the combined 10 year experience of this approach in two paediatric metabolic and transplant centres. Of 81 patients who underwent a first transplant procedure for Hurler, 88% (71/81) survived and 81% (66/81) were alive and engrafted at a median follow-up of 46 months (range 3–124 months). The incidence of grade II–IV acute and any chronic graft versus host disease was 17% and 11% respectively. Urinary glycosaminoglycans were significantly reduced after a period of enzyme replacement therapy, and further reductions were seen at 13–24 months and 25+ months after transplantation. In several individuals with decreased cardiac contractility, an improvement of their condition during enzyme replacement therapy enabled them to undergo transplantation, with one individual receiving full intensity conditioning.

BackgroundThe lysosomal storage disorder, mucopolysaccharidosis I (MPS I), commonly manifests with upper airway obstruction and sleep disordered breathing (SDB). The success of current therapies, including haematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) may be influenced by a number of factors and monitored using biomarkers of metabolic correction. We describe the pattern of SDB seen in the largest MPS I cohort described to date and determine therapies and biomarkers influencing the severity of long-term airway disease.MethodsTherapeutic, clinical and biomarker data, including longitudinal outcome parameters from 150 sleep oximetry studies were collected in 61 MPS I (44 Hurler, 17 attenuated) patients between 6 months pre to 16 years post-treatment (median follow-up 22 months). The presence and functional nature of an immune response to ERT was determined using ELISA and a cellular uptake inhibition assay. Multivariate analysis was performed to determine significant correlators of airway disease.ResultsThe incidence of SDB in our cohort is 68%, while 16% require therapeutic intervention for airway obstruction. A greater rate of progression (73%) and requirement for intervention is seen amongst ERT patients in contrast to HSCT treated individuals (24%). Multivariate analysis identifies poorer metabolic clearance, as measured by a rise in the biomarker urinary dermatan sulphate: chondroitin sulphate (DS:CS) ratio, as a significant correlator of increased presence and severity of SDB in MPS I patients (p = 0.0017, 0.008). Amongst transplanted Hurler patients, delivered enzyme (leukocyte iduronidase) at one year is significantly raised in those without SDB (p = 0.004). Cellular uptake inhibitory antibodies in ERT treated patients correlate with reduced substrate clearance and occurrence of severe SDB (p = 0.001).ConclusionWe have identified biochemical and therapeutic factors modifying airway disease across the phenotypic spectrum in MPS I. Interventions maximising substrate reduction correlate with improved long-term SDB, while inhibitory antibodies impact on biochemical and clinical outcomes. Monitoring and tolerisation strategies should be re-evaluated to improve detection and minimise the inhibitory antibody response to ERT in MPS I and other lysosomal storage diseases. Future studies should consider the use of sleep disordered breathing as an objective parameter of clinical and metabolic improvement.

BackgroundMucopolysaccharidosis type I is caused by deficiency of a-L-iduronidase. Currently available treatment options include an allogeneic hematopoietic stem cell transplant and enzyme replacement therapy. Exogenous enzyme therapy appears promising but the benefits may be attenuated, at least in some patients, by the development of an immune response to the delivered enzyme. The incidence and impact of allo-immune responses in these patients remain unknown. Design and MethodsWe developed an immunoglobulin G enzyme-linked immunosorbent assay as well as in vitro catalytic enzyme inhibition and cellular uptake inhibition assays and quantified enzyme inhibition by allo-antibodies. We determined the impact of these antibodies in eight patients who received enzyme therapy before and during hematopoietic stem cell transplantation. In addition, 20 patients who had previously received an allogeneic stem cell transplant were tested to evaluate this treatment as an immune tolerance induction mechanism. ResultsHigh titer immune responses were seen in 87.5% (7/8) patients following exposure to a-Liduronidase. These patients exhibited catalytic enzyme inhibition (5/8), uptake inhibition of catalytically active enzyme (6/8) or both (4/8). High antibody titers generally preceded elevation of previously described biomarkers of disease progression. The median time to development of immune tolerance was 101 days (range, 26-137) after transplantation. All 20 patients, including those with mixed chimerism (22%), tested 1 year after transplantation were tolerized despite normal enzyme levels. ConclusionsWe found a high incidence of neutralizing antibodies in patients with mucopolysaccharidosis type I treated with enzyme replacement therapy. We also found that allogeneic hematopoietic stem cell transplantation was an effective and rapid immune tolerance induction strategy. © F e r r a t a S t o r t i F o u n d a t i o n © F e r r a t a S t o r t i F o u n d a t i o n