The GALNS p.P77R variant is a probable Gujarati-Indian founder mutation causing Mucopolysaccharidosis IVA syndrome.

BACKGROUND: Mucopolysaccharidosis IVA (Morquio syndrome A, MPS IVA) is an autosomal recessive lysosomal storage disorder caused due to biallelic variants in the N-acetylgalactoseamine-6-sulfate sulfatase (GALNS) gene. The mutation spectrum in this condition is determined amongst sub-populations belonging to the north, south and east India geography, however, sub-populations of west Indian origin, especially Gujarati-Indians, are yet to be studied. We aimed to analyse the variants present in the GLANS gene amongst the population of Gujarat by sequencing all exons and exon-intron boundaries of the GALNS gene in patients from 23 unrelated families. RESULTS: We report 11 variants that include eight missense variants: (p.L36R, p.D39G, p.P77R, p.C79R, pP125L, p.P151L, p.G255A and p.L350P), one splice site variant: (c.121-7C > G), one small insertion: (c.1241_1242insA, p.I416HfsTer2) and one small deletion: (c.839_841delACA). Of these, three missense variants (p.D39G, p.G255A and p.L350P), one splice site and the two indels mentioned above are novel. Interestingly, we observed a higher than anticipated prevalence of p.P77R variant in our cohort (n = 14/25, 56%). Haplotype analysis in cases with p.P77R variant and 63 ethnicity matched healthy population controls suggested a 4 SNP haplotype block present in cases compared to controls (likelihood ratio test p-value = 1.16 × 10-13), thereby suggesting p.P77R variant as a founder variant in the Gujarati-Indian population. Furthermore, age of mutation analysis suggested the variant to have arisen approximately 450 years ago in the population. CONCLUSION: p.P77R variant in the GLANS gene is likely to be a founder variant in MPS IVA patients of Gujarati-Indian ancestry and appeared approximately 450 years ago in the population. To our knowledge, this is the first variant to be posited as a founder variant in the GLANS gene in patients with MPS IVA syndrome.

Treatment of skeletal and non-skeletal alterations of Mucopolysaccharidosis type IVA by AAV-mediated gene therapy.

Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9-Galns) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9-Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients.

A Case Report of a Japanese Boy with Morquio A Syndrome: Effects of Enzyme Replacement Therapy Initiated at the Age of 24 Months.

BACKGROUND: Morquio A syndrome, mucopolysaccharidosis type IVA (MPS IVA), is a lysosomal storage disorder caused by the deficient activity of N-acetylgalactosamine-6-sulfatase (GalNac6S), due to alterations in the GALNS gene. This disorder results in marked abnormalities in bones and connective tissues, and affects multiple organs. Here, we describe the clinical course of a Japanese boy with MPS IVA who began enzyme replacement therapy (ERT) at the age of 24 months. PATIENT: the patient presented for kyphosis treatment at 22 months of age. An X-ray examination revealed dysostosis multiplex. Uronic acids were elevated in the urine and the keratan sulfate (KS) fraction was predominant. The leukocyte GalNac6S enzyme activity was extremely low. The patient exhibited the c.463G > A (p.Gly155Arg) mutation in GALNS. Based on these findings, his disease was diagnosed as classical (severe) Morquio A syndrome. An elosulfase alfa infusion was initiated at the age of 24 months. The patient's body height improved from -2.5 standard deviation (SD) to -2 SD and his physical activity increased during the first 9 months on ERT. However, he gradually developed paralysis in the lower legs with declining growth velocity, which required cervical decompression surgery in the second year of the ERT. The mild mitral regurgitation, serous otitis media, and mild hearing loss did not progress during treatment. CONCLUSION: early initiation of the elosulfase alfa to our patient showed good effects on the visceral system and muscle strength, while its effect on bones appeared limited. Careful observation is necessary to ensure timely surgical intervention for skeletal disorders associated with neurological symptoms. Centralized and multidisciplinary management is essential to improve the prognosis of pediatric patients with MPS IVA.

Enzyme replacement therapy in mice lacking arylsulfatase B targets bone-remodeling cells, but not chondrocytes.

Mucopolysaccharidosis type VI (MPS-VI), caused by mutational inactivation of the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), is a lysosomal storage disorder primarily affecting the skeleton. We have previously reported that Arsb-deficient mice display high trabecular bone mass and impaired skeletal growth. In the present study, we treated them by weekly injection of recombinant human ARSB (rhARSB) to analyze the impact of enzyme replacement therapy (ERT) on skeletal growth and bone remodeling. We found that all bone-remodeling abnormalities of Arsb-deficient mice were prevented by ERT, whereas chondrocyte defects were not. Likewise, histologic analysis of the surgically removed femoral head from an ERT-treated MPS-VI patient revealed that only chondrocytes were pathologically affected. Remarkably, a side-by-side comparison with other cell types demonstrated that chondrocytes have substantially reduced capacity to endocytose rhARSB, together with low expression of the mannose receptor. We finally took advantage of Arsb-deficient mice to establish quantification of chondroitin sulfation for treatment monitoring. Our data demonstrate that bone-remodeling cell types are accessible to systemically delivered rhARSB, whereas the uptake into chondrocytes is inefficient.

Development of Substrate Degradation Enzyme Therapy for Mucopolysaccharidosis IVA Murine Model.

Mucopolysaccharidosis IVA (MPS IVA) is caused by a deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Conventional enzyme replacement therapy (ERT) is approved for MPS IVA. However, the fact that the infused enzyme cannot penetrate avascular lesions in cartilage leads to minimal impact on the bone lesion. Moreover, short half-life, high cost, instability, and narrow optimal pH range remain unmet challenges in ERT. Thermostable keratanase, endo-ß-N-acetylglucosaminidase, has a unique character of a wide optimal pH range of pH 5.0-7.0. We hypothesized that this endoglycosidase degrades keratan sulfate (KS) polymer in circulating blood and, therefore, ameliorates the accumulation of KS in multiple tissues. We propose a novel approach, Substrate Degradation Enzyme Therapy (SDET), to treat bone lesion of MPS IVA. We assessed the effect of thermostable keratanase on blood KS level and bone pathology using Galns knock-out MPS IVA mice. After a single administration of 2 U/kg (= 0.2 mg/kg) of the enzyme at 8 weeks of age via intravenous injection, the level of serum KS was significantly decreased to normal range level, and this suppression was maintained for at least 4 weeks. We administered 2 U/kg of the enzyme to MPS IVA mice every fourth week for 12 weeks (total of 3 times) at newborns or 8 weeks of age. After a third injection, serum mono-sulfated KS levels were kept low for 4 weeks, similar to that in control mice, and at 12 weeks, bone pathology was markedly improved when SDET started at newborns, compared with untreated MPS IVA mice. Overall, thermostable keratanase reduces the level of KS in blood and provides a positive impact on cartilage lesions, demonstrating that SDET is a novel therapeutic approach to MPS IVA.

Advances in the Development of Pharmacological Chaperones for the Mucopolysaccharidoses.

The mucopolysaccharidoses (MPS) are a group of 11 lysosomal storage diseases (LSDs) produced by mutations in the enzymes involved in the lysosomal catabolism of glycosaminoglycans. Most of the mutations affecting these enzymes may lead to changes in processing, folding, glycosylation, pH stability, protein aggregation, and defective transport to the lysosomes. It this sense, it has been proposed that the use of small molecules, called pharmacological chaperones (PCs), can restore the folding, trafficking, and biological activity of mutated enzymes. PCs have the advantages of wide tissue distribution, potential oral administration, lower production cost, and fewer issues of immunogenicity than enzyme replacement therapy. In this paper, we will review the advances in the identification and characterization of PCs for the MPS. These molecules have been described for MPS II, IVA, and IVB, showing a mutation-dependent enhancement of the mutated enzymes. Although the results show the potential of this strategy, further studies should focus in the development of disease-specific cellular models that allow a proper screening and evaluation of PCs. In addition, in vivo evaluation, both pre-clinical and clinical, should be performed, before they can become a real therapeutic strategy for the treatment of MPS patients.

4-epi-Isofagomine derivatives as pharmacological chaperones for the treatment of lysosomal diseases linked to ß-galactosidase mutations: Improved synthesis and biological investigations.

(5aR)-5a-C-pentyl-4-epi-isofagomine 1 is a powerful inhibitor of lysosomal ß-galactosidase and a remarkable chaperone for mutations associated with GM1-gangliosidosis and Morquio disease type B. We report herein an improved synthesis of this compound and analogs (5a-C-methyl, pentyl, nonyl and phenylethyl derivatives), and a crystal structure of a synthetic intermediate that confirms its configuration resulting from the addition of a Grignard reagent. These compounds were evaluated as glycosidase inhibitors and their potential as chaperones for mutant lysosomal galactosidases determined. Based on these results and on docking studies, the 5-C-pentyl derivative 1 was selected as the optimal structure for further investigations: this compound induces the maturation of mutated ß-galactosidase in fibroblasts of a GM1-gangliosidosis patient and promote the decrease of keratan sulfate and oligosaccharide load in patient cells. Compound 1 is clearly capable of restoring ß-galactosidase activity and of promoting maturation of the protein, which should result in significant clinical benefit. These properties strongly support the development of compound 1 for the treatment of GM1-gangliosidosis and Morquio disease type B patients harboring ß-galactosidase mutations sensitive to pharmacological chaperoning.

Safety, immunogenicity, and clinical outcomes in patients with Morquio A syndrome participating in 2 sequential open-label studies of elosulfase alfa enzyme replacement therapy (MOR-002/MOR-100), representing 5 years of treatment.

Elosulfase alfa is an enzyme replacement therapy for Morquio A syndrome (mucopolysaccharidosis IVA), a multisystemic progressive lysosomal storage disorder. This report includes the primary treatment outcomes and immunogenicity profile of elosulfase alfa in patients with Morquio A syndrome from 2 sequential studies, MOR-002 (ClinicalTrials.govNCT00884949) and MOR-100 (NCT01242111), representing >5 years of clinical study data. MOR-002 was an open-label, single-arm phase 1/2 study that evaluated the pharmacokinetics, safety, immunogenicity, and preliminary efficacy of 3 sequential doses of elosulfase alfa (0.1, 1.0, and 2.0 mg/kg/week) in patients with Morquio A syndrome (n = 20) over 36 weeks, followed by an optional 36- to 48-week treatment period using elosulfase alfa 1.0 mg/kg once weekly (qw). During the 0.1 mg/kg dosing phase, 1 patient discontinued due to a type I hypersensitivity adverse event (AE), and that patient's sibling voluntarily discontinued in the absence of AEs. An additional patient discontinued due to recurrent infusion reactions during the 1.0 mg/kg continuation phase. The remaining 17 patients completed MOR-002 and enrolled in MOR-100, an open-label, long-term extension study that further evaluated safety and clinical outcomes with elosulfase alfa administered at 2.0 mg/kg qw. During the course of MOR-100, patients were given the option of receiving elosulfase alfa infusions at home with nursing assistance. Over the course of both studies, all patients experienced ≥1 AE and most patients experienced a drug-related AE, generally of mild or moderate severity. Hypersensitivity reactions reported as related to study drug occurred in 25% of patients. Thirteen patients who chose to receive infusions at home had the same tolerability and safety profile, as well as comparable compliance rates, as patients who chose to receive on-site infusions. All patients developed antibodies to elosulfase alfa. Positivity for neutralizing antibodies was associated with increased drug half-life and decreased drug clearance. Despite formation of antidrug-binding (total antidrug antibodies, TAb) and in vitro neutralizing antibodies (NAb) in all patients, these types of immunogenicity to elosulfase alfa were not correlated with safety or clinical outcomes. In contrast with the reported natural history of Morquio A, no trends toward decreasing endurance, respiratory function, or ability to perform activities of daily living were observed in this cohort over the 5-year period.

Impact of long-term elosulfase alfa on activities of daily living in patients with Morquio A syndrome in an open-label, multi-center, phase 3 extension study.

BACKGROUND: Long-term safety and efficacy of elosulfase alfa enzyme replacement therapy (ERT) were assessed in 173 patients with Morquio A syndrome (mucopolysaccharidosis IVA) in a 96-week, open-label, multi-center, phase 3 extension study (MOR-005) of the pivotal 24-week, placebo-controlled study (MOR-004). Changes in efficacy endpoints were evaluated over 120weeks, from MOR-004 baseline to MOR-005 week 96. We report the impact of ERT on activities of daily living (ADL) across three domains (mobility, self-care, and caregiver-assistance), as assessed by the Mucopolysaccharidosis Health Assessment Questionnaire (MPS-HAQ) after 72 and 120weeks or approximately 1 and 2years. RESULTS: Mean baseline MPS-HAQ domain scores showed impairments in mobility, self-care, and independence. The MOR-005 intent-to-treat population (ITT; N=169, including 158 with 2years follow-up) showed sustained significant reductions (representing improvements) in mobility and self-care domain least square (LS) mean scores vs. baseline at 1 and 2years and a non-significant decrease in the caregiver-assistance domain at 2years. At week 120, LS mean (SE) changes from baseline were -0.5 (0.1) for mobility (P=0.002), -0.4 (0.1) for self-care (P=0.001), and -1.0 (0.5) for caregiver-assistance (P=0.06) (ITT population). Improvements in MPS-HAQ domain scores vs. baseline at 1 and 2years were greater in patients continuously treated with the weekly dosing regimen than in the total MOR-005 population and statistically significant across domains. A comparable untreated cohort of patients from the Morquio A Clinical Assessment Program (MorCAP) natural history study (ITT population, N=94, including 37 with 2years follow-up) showed no improvement over 2years, with two of the three domains worsening (LS mean (SE) changes from baseline: 0.3 (0.3) for mobility, 0.4 (0.2) for self-care, -0.5 (0.8) for caregiver-assistance). Changes in LS mean scores vs. baseline were statistically significantly different between MOR-005 and MorCAP for the mobility domain (-0.7 (SE 0.4), P=0.0490) and the self-care domain (-0.7 (SE 0.3), P=0.0146) at 2years. CONCLUSIONS: Together, these findings suggest that long-term elosulfase alfa ERT is associated with partial recovery of functional abilities, improving Morquio A patients' abilities to perform ADL. TRIAL REGISTRATION: ClinicalTrials.govNCT01415427. Registered 8 August 2011, retrospectively registered.

[Analysis of clinical features and GALNS gene mutation in a patient with mucopolysaccharidosis type IV A].

OBJECTIVE: To detect potential mutation of galactosamine-6-sulfate (GALNS) gene in a Chinese girl affected with mucopolysaccharidosis type IV A (Morquio A syndrome). METHODS: The patient was diagnosed by assaying the activities of mucopolysaccharidosis-related enzymes in leukocytes. Potential mutation in the GALNS gene was detected with PCR and Sanger sequencing. RESULTS: The patient was characterized by short stature, skeletal deformities, normal intelligence, and auditory dysfunction. The activities of GALNS enzymes were low. A compound heterozygous missense mutation, c.1094G>T (p.Gly365Val)/c.938C>T (p.Thr313Met), was detected in the GALNS gene. The mutations were respectively inherited from her father and mother. Among them, the c.1094G>T (p.Gly365Val) mutation was not reported previously. CONCLUSION: The mutations c.1094G>T (p.Gly365Val)/c.938C>T (p.Thr313Met) probably underlie the pathogenesis of the disease in our patient.

Four novel mutations in the N-acetylgalactosamine-6-sulfate sulfatase gene among Egyptian patients with Morquio A disease.

Morquio A disease (Mucopolysaccharidosis IVA, MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficient activity of the enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) encoded by the GALNS gene. This deficiency leads to a decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing their accumulation within the lysosomes and consequently prominent skeletal and visceral abnormalities. Clinical evaluation and biochemical GALNS enzyme activity determination were carried out for the patients from four unrelated Egyptian families. Mutational analysis was performed to PCR products by sequencing of the 14 exons and exon-intron boundaries of GALNS gene for the 4 patients. Sequence analysis revealed four novel mutations; three nonsense mutations (p.Q12X, p.Q220X, p.Y254X) and one missense mutation, p.D40G. All four patients were offspring of consanguineous marriages and were homozygous for the corresponding mutation. The activity of the GALNS enzyme was below normal reference range in all of them. The p.Q12X and p.Y254X were associated with severe MPS IVA phenotype. Molecular analysis of GALNS gene revealed four novel mutations in four different Morquio A Egyptian patients.

(5aR)-5a-C-Pentyl-4-epi-isofagomine: A powerful inhibitor of lysosomal ß-galactosidase and a remarkable chaperone for mutations associated with GM1-gangliosidosis and Morquio disease type B.

This report is about the identification, synthesis and initial biological characterization of derivatives of 4-epi-isofagomine as pharmacological chaperones (PC) for human lysosomal ß-galactosidase. The two epimers of 4-epi-isofagomine carrying a pentyl group at C-5a, namely (5aR)- and (5aS)-5a-C-pentyl-4-epi-isofagomine, were prepared by an innovative procedure involving in the key step the addition of nitrohexane to a keto-pentopyranoside. Both epimers were evaluated as inhibitors of the human ß-galactosidase: the (5aR)-stereoisomer (compound 1) was found to be a very potent inhibitor of the enzyme (IC50 = 8 nM, 30× more potent than 4-epi-isofagomine at pH 7.3) with a high selectivity for this glycosidase whereas the (5aS) epimer was a much weaker inhibitor. In addition, compound 1 showed a remarkable activity as a PC. It significantly enhanced the residual activity of mutant ß-galactosidase in 15 patient cell lines out of 23, with enhancement factors greater than 3.5 in 10 cell lines and activity restoration up to 91% of normal. Altogether, these results indicated that (5aR)-5a-C-pentyl-4-epi-isofagomine constitutes a promising PC-based drug candidate for the treatment of GM1-gangliosidosis and Morquio disease type B.

Antibodies that neutralize cellular uptake of elosulfase alfa are not associated with reduced efficacy or pharmacodynamic effect in individuals with Morquio A syndrome.

Many enzyme replacement therapies (ERTs) for lysosomal storage disorders use the cell-surface cation-independent mannose-6 phosphate receptor (CI-M6PR) to deliver ERTs to the lysosome. However, neutralizing antibodies (NAb) may interfere with this process. We previously reported that most individuals with Morquio A who received elosulfase alfa in the phase 3 MOR-004 trial tested positive for NAbs capable of interfering with binding to CI-M6PR ectodomain in an ELISA-based assay. However, no correlation was detected between NAb occurrence and clinical efficacy or pharmacodynamics. To quantify and better characterize the impact of NAbs, we developed a functional cell-based flow cytometry assay with a titer step that detects antibodies capable of interfering with elosulfase alfa uptake. Serum samples collected during the MOR-004 trial were tested and titers were determined. Consistent with earlier findings on NAb positivity, no correlations were observed between NAb titers and the clinical outcomes of elosulfase alfa-treated individuals with Morquio A.

Safety and clinical activity of elosulfase alfa in pediatric patients with Morquio A syndrome (mucopolysaccharidosis IVA) less than 5 y.

BACKGROUND: Previous studies have shown that elosulfase alfa has a favorable efficacy/safety profile in Morquio A patients aged ≥5 y. This study evaluated safety and impact on urine keratan sulfate (uKS) levels and growth velocity in younger patients. METHODS: Fifteen Morquio A patients aged <5 y received elosulfase alfa 2.0 mg/kg/week for 52 wk during the primary treatment phase of a phase II, open-label, multinational study. Primary endpoint was safety and tolerability; secondary endpoints were change in uKS and growth velocity over 52 wk. RESULTS: All 15 patients completed the primary treatment phase. Six of 743 infusions (0.8%) administered led to adverse events (AEs) requiring infusion interruption and medical intervention. Eleven patients (73.3%) had ≥1 study drug-related AE, mostly infusion-associated reactions. Mean z-score growth rate per year numerically improved from -0.6 at baseline to -0.4 at week 52. Comparison to untreated subjects of similar age in the Morquio A Clinical Assessment Program study showed a smaller decrease in height z-scores for treated than for untreated patients. Mean percent change from baseline in uKS was -30.2% at 2 wk and -43.5% at 52 wk. CONCLUSION: Early intervention with elosulfase alfa is well-tolerated and produces a decrease in uKS and a trend toward improvement in growth.

Novel missense mutation in the GALNS gene in an affected patient with severe form of mucopolysaccharidosis type IVA.

Mucopolysaccharidosis type IVA (MPS IVA), also known as Morquio A, is an autosomal recessive disorder characterized by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which causes major skeletal and connective tissue abnormalities and affects multiple organ systems. In this study, one MPS IVA patient with a severe form from consanguine large Iranian family has been investigated. To find a mutation, all of the 14 exons and intron-exon junctions of GALNS gene were sequenced. Sequencing results were analyzed using bioinformatic analysis in order to predict probable pathogenic effect of the variant. One novel homozygous missense mutation in exon 5, c.542A>G (p.Y181C), was found in the proband. That was predicted as being probably pathogenic by bioinformatics analysis. Segregation and familial study confirmed this pathogenic mutation. In conclusion, we have identified the novel mutation responsible for MPS IVA in an Iranian patient to assist in the diagnosis, genetic counseling and prenatal diagnosis of the affected families.

Safety and physiological effects of two different doses of elosulfase alfa in patients with morquio a syndrome: A randomized, double-blind, pilot study.

The primary treatment outcomes of a phase 2, randomized, double-blind, pilot study evaluating safety, physiological, and pharmacological effects of elosulfase alfa in patients with Morquio A syndrome are herewith presented. Patients aged ≥7 years and able to walk ≥200 m in the 6-min walk test (6MWT) were randomized to elosulfase alfa 2.0 or 4.0 mg/kg/week for 27 weeks. The primary objective was to evaluate the safety of both doses. Secondary objectives were to evaluate effects on endurance (6MWT and 3-min stair climb test [3MSCT]), exercise capacity (cardio-pulmonary exercise test [CPET]), respiratory function, muscle strength, cardiac function, pain, and urine keratan sulfate (uKS) levels, and to determine pharmacokinetic parameters. Twenty-five patients were enrolled (15 randomized to 2.0 mg/kg/week and 10 to 4.0 mg/kg/week). No new or unexpected safety signals were observed. After 24 weeks, there were no improvements versus baseline in the 6MWT, yet numerical improvements were seen in the 3MSCT with 4.0 mg/kg/week. uKS and pharmacokinetic data suggested no linear relationship over the 2.0-4.0 mg/kg dose range. Overall, an abnormal exercise capacity (evaluated in 10 and 5 patients in the 2.0 and 4.0 mg/kg/week groups, respectively), impaired muscle strength, and considerable pain were observed at baseline, and there were trends towards improvements in all domains after treatment. In conclusion, preliminary data of this small study in a Morquio A population with relatively good endurance confirmed the acceptable safety profile of elosulfase alfa and showed a trend of increased exercise capacity and muscle strength and decreased pain.

Computational analysis of human N-acetylgalactosamine-6-sulfate sulfatase enzyme: an update in genotype-phenotype correlation for Morquio A.

Mucopolysaccharidosis IV A (MPS IV A) is a lysosomal storage disease produced by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme. Although genotype-phenotype correlations have been reported, these approaches have not enabled to establish a complete genotype-phenotype correlation, and they have not considered a ligand-enzyme interaction. In this study, we expanded the in silico evaluation of GALNS mutations by using several bioinformatics tools. Tertiary GALNS structure was modeled and used for molecular docking against galactose-6-sulfate, N-acetylgalactosamine-6-sulfate, keratan sulfate, chondroitin-6-sulfate, and the artificial substrate 4-methylumbelliferyl-ß-D-galactopyranoside-6-sulfate. Furthermore, we considered the evolutionary residue conservation, change conservativeness, position within GALNS structure, and the impact of amino acid substitution on the structure and function of GALNS. Molecular docking showed that amino acids involved in ligand interaction correlated with those observed in other human sulfatases, and mutations within the active cavity reduced affinity of all evaluated ligands. Combination of several bioinformatics approaches allowed to explaine 90% of the missense mutations affecting GALNS, and the prediction of the phenotype for another 21 missense mutations. In summary, we have shown for the first time a docking evaluation of natural and artificial ligands for human GALNS, and proposed an update in genotype-phenotype correlation for Morquio A, based on the use of multiple parameters to predict the disease severity.

Elosulfase alfa.

Mucopolysaccharidosis type IVA (MPS IVA), also known as Morquio A syndrome, is an inherited, lysosomal storage disorder caused by genetic mutations in N-acetylgalactosamine-6-sulfatase (GALNS) enzyme gene. GALNS is essential for breakdown of glycosaminoglycans. The disease is characterized by the early onset of severe skeletal dysplasia resulting in significant disability by the second decade of life. Until recently there have been no available treatments other than surgery and palliative care. BioMarin Pharmaceutical developed elosulfase alfa, a recombinant human GALNS coproduced with sulfatase-modifying factor 1, as an enzyme replacement therapy for patients with MPS IVA. In clinical studies, enzyme replacement therapy with elosulfase alfa significantly improved physical endurance, respiratory function, growth and quality of life in patients with MPS IVA. Treatment increased clearance of glycosaminoglycans and induced gene expression consistent with improved chondrocyte function. Elosulfase alfa is approved for the treatment of MPS IVA in the U.S. and Europe.

Structural basis of pharmacological chaperoning for human ß-galactosidase.

GM1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal ß-galactosidase (ß-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human ß-Gal(WT) and two representative mutations in GM1 gangliosidosis Japanese patients, ß-Gal(R201C) and ß-Gal(I51T). We have also evaluated the PC effect of two competitive inhibitors of ß-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of ß-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of ß-Gal selective chaperoning by newly developed PC compounds.

[Analysis of GALNS gene mutation in thirty-eight Chinese patients with mucopolysaccharidosis type IVA].

OBJECTIVE: Mucopolysaccharidosis (MPS) type IVA (MPS IVA) is an autosomal recessive lysosomal storage disease caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) needed to degrade glycosaminoglycanes (GAGs), accumulation of GAGs in the tissue resulting in disorder of function. So far, the small number of articles about clinical study of Chinese MPS IVA were published and only one paper about gene mutation analysis was published. This study aimed to investigate the mutation spectrum and characteristic of GALNS gene in Chinese patients with MPS IVA who were diagnosed in our hospital. METHOD: Thirty-eight patients from 36 families (male 17, female 21) were diagnosed as MPS IVA by GALNS activity determination [(0.85 ± 1.33) nmol/(17 h·mg)] and clinical symptoms during 2006-2012. The average age of diagnosis was (5.7 ± 3.6) years. Mutation analysis of GALNS gene performed performed by PCR-direct DNA sequencing for 38 patients. PCR-restriction fragment length polymorphism analysis was used for validating novel mutation, and also to assess amino acid conservation for novel missense variants in five different species. PolyPhen-2 tool was used to predict the possible impact of missense mutations on the structure and function of the human GALNS protein, etc. Analysis of GALNS activity and gene mutation in amniotic fluid were performed to provide the prenatal diagnosis for some families with MPS type IVA. RESULT: (1) Thirty-eight kinds of mutation in GALNS gene were identified in 38 patients of them, 71% were missense mutations. p. M318R was a hot-spot mutation (21%) tested. Five kinds of mutation i.e., p. P163H, p.G168L, p. A324E, p. L366P and p. F452L were only found in Chinese patients with MPS IVA. Eighteen kinds of novel mutation were detected including p. E315K, p.G304D, p.R251Q, p.Y240C, p.G161E, p.N32D, p.L390P, p. D60E, p. P420S, W403C/T404S, p.L454P, for p.W405X, p. M1I, c.409_ c.420del12, c.1176_1178del3, c.1046delG, c.1188delG and IVS9-2A>C. (2) The polymorphism of novel missense variants were ruled out by the PCR-restriction fragment length polymorphism analysis and no related mutations were found in 50 normal controls. A splice site mutation IVS9-2A>C had been validated by reverse transcription PCR direct sequencing. The amino acid of mutant position of 10 kinds of missense variants are highly conserved and only p. L454 is moderately conserved position. These missense variants were predicted to cause damage to the structure and function of human GALNS protein possibly according to the PolyPhen-2 tool, so these novel missense variants may be disease-causing mutations. (3) Prenatal diagnosis was provided for 7 families and three fetuses were diagnosed as MPS IVA. CONCLUSION: The GALNS gene mutation spectrum in Chinese patients with MPS IVA is really different from that in other countries, five kinds of mutation were only found in Chinese patients with MPS IVA. The reports of hot-spot mutation in Chinese patients were also different, and should be analyzed by more data of gene mutation analysis and epidemiological study.