Enhanced osteoblastic differentiation of parietal bone in a novel murine model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II, OMIM 309900) is an X-linked disorder caused by a deficiency of lysosomal enzyme iduronate-2-sulfatase (IDS). The clinical manifestations of MPS II involve cognitive decline, bone deformity, and visceral disorders. These manifestations are closely associated with IDS enzyme activity, which catalyzes the stepwise degradation of heparan sulfate and dermatan sulfate. In this study, we established a novel Ids-deficient mice and further assessed the enzyme's physiological role. Using DNA sequencing, we found a genomic modification of the Ids genome, which involved the deletion of a 138-bp fragment spanning from intron 2 to exon 3, along with the insertion of an adenine at the 5' end of exon 3 in the mutated allele. Consistent with previous data, our Ids-deficient mice showed an attenuated enzyme activity and an enhanced accumulation of glycosaminoglycans. Interestingly, we noticed a distinct enlargement of the calvarial bone in both neonatal and young adult mice. Our examination revealed that Ids deficiency led to an enhanced osteoblastogenesis in the parietal bone, a posterior part of the calvarial bone originating from the paraxial mesoderm and associated with an enhanced expression of osteoblastic makers, such as Col1a and Runx2. In sharp contrast, cell proliferation of the parietal bone in these mice appeared similar to that of wild-type controls. These results suggest that the deficiency of Ids could be involved in an augmented differentiation of calvarial bone, which is often noticed as an enlarged head circumference in MPS II-affected individuals.

Intracerebroventricular enzyme replacement therapy in patients with neuronopathic mucopolysaccharidosis type II: Final report of 5-year results from a Japanese open-label phase 1/2 study.

Intravenous idursulfase is standard treatment for mucopolysaccharidosis II (MPS II) in Japan. In the interim analysis of this open-label, phase 1/2 study (Center for Clinical Trials, Japan Medical Association: JMA-IIA00350), intracerebroventricular (ICV) idursulfase beta was well tolerated, suppressed cerebrospinal fluid (CSF) heparan sulfate (HS) levels, and stabilized developmental decline over 100 weeks in Japanese children with MPS II. Here, we report the final study results, representing 5 years of ICV idursulfase beta treatment. Six male patients with MPS II and developmental delay were enrolled starting in June 2016 and followed until March 2021. Patients received up to 30 mg ICV idursulfase beta every 4 weeks. Outcomes included CSF HS levels, developmental age (DA) (assessed by the Kyoto Scale of Psychological Development), and safety (adverse events). Monitoring by laboratory biochemistry tests, urinary uronic tests, immunogenicity tests, and head computed tomography or magnetic resonance imaging were also conducted regularly. Following ICV idursulfase beta administration, mean CSF HS concentrations decreased from 7.75 µg/mL at baseline to 2.15 µg/mL at final injection (72.3% reduction). Mean DA increased from 23.2 months at screening to 36.0 months at final observation. In five patients with null mutations, mean DA at the final observation was higher than or did not regress compared with that of historical controls receiving intravenous idursulfase only, and the change in DA was greater in patients who started administration aged ≤3 years than in those aged >3 years (+28.7 vs -6.5 months). The difference in DA change versus historical controls in individual patients was +39.5, +40.8, +17.8, +10.5, +7.6 and - 4.5 (mean + 18.6). Common ICV idursulfase beta-related adverse events were vomiting, pyrexia, gastroenteritis, and upper respiratory tract infection (most mild/moderate). These results suggest that long-term ICV idursulfase beta treatment improved neurological symptoms in Japanese children with neuronopathic MPS II.

A novel mucopolysaccharidosis type II mouse model with an iduronate-2-sulfatase-P88L mutation.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder characterized by an accumulation of glycosaminoglycans (GAGs), including heparan sulfate, in the body. Major manifestations involve the central nerve system (CNS), skeletal deformation, and visceral manifestations. About 30% of MPS II is linked with an attenuated type of disease subtype with visceral involvement. In contrast, 70% of MPS II is associated with a severe type of disease subtype with CNS manifestations that are caused by the human iduronate-2-sulfatase (IDS)-Pro86Leu (P86L) mutation, a common missense mutation in MPS II. In this study, we reported a novel Ids-P88L MPS II mouse model, an analogous mutation to human IDS-P86L. In this mouse model, a significant impairment of IDS enzyme activity in the blood with a short lifespan was observed. Consistently, the IDS enzyme activity of the body, as assessed in the liver, kidney, spleen, lung, and heart, was significantly impaired. Conversely, the level of GAG was elevated in the body. A putative biomarker with unestablished nature termed UA-HNAc(1S) (late retention time), one of two UA-HNAc(1S) species with late retention time on reversed-phase separation,is a recently reported MPS II-specific biomarker derived from heparan sulfate with uncharacterized mechanism. Thus, we asked whether this biomarker might be elevated in our mouse model. We found a significant accumulation of this biomarker in the liver, suggesting that hepatic formation could be predominant. Finally, to examine whether gene therapy could enhance IDS enzyme activity in this model, the efficacy of the nuclease-mediated genome correction system was tested. We found a marginal elevation of IDS enzyme activity in the treated group, raising the possibility that the effect of gene correction could be assessed in this mouse model. In conclusion, we established a novel Ids-P88L MPS II mouse model that consistently recapitulates the previously reported phenotype in several mouse models.

Automated urinary sediment detection for Fabry disease using deep-learning algorithms.

Fabry disease is a congenital lysosomal storage disease, and most of these cases develop organ damage in middle age. There are some promising therapeutic options for this disorder, which can stabilize the progression of the disease. However, a long delay in diagnosis prevents early intervention, resulting in treatment failure. Because Fabry disease is a rare disease, it is not well recognized and disease specific screening tests are rarely performed. Hence, a novel approach to for detecting patients with a widely practiced clinical test is crucial for the early detection of the disease. Recently, decision support systems based on artificial intelligence (AI) have been developed in many clinical fields. However, the construction of these models requires datasets from a large number of samples; this aspect is one of the main obstacles in AI-based approaches for rare diseases. In this study, with a novel image amplification method to construct the dataset for AI-model training, we built the deep neural-network model to detect Fabry cases from their urine samples. Sensitivity, specificity, and the AUC of the models on validation dataset were 0.902 (95% CI, 0.900-0.903), 0.977 (0.950-0.980), and 0.968 (0.964-0.972), respectively. This model could also extract disease-specific findings that are interpretable with human recognition. These results indicate that we can apply novel AI models for rare diseases based on this image amplification method we developed. We expect this approach could contribute to the diagnosis of Fabry disease. Synopsis: This is the first reported AI-based decision support system to detect undiagnosed Fabry cases, and our new image amplification method will contribute to the AI models for other rare disorders.

LC-MS/MS-based enzyme assay for lysosomal acid lipase using dried blood spots.

Lysosomal acid lipase deficiency (LAL-D) (OMIM: 278000) is a lysosomal storage disorder with two distinct disease phenotypes such as Wolman disease and cholesteryl ester storage disorder (CESD), characterized by an accumulation of endocytosed cholesterol in the body. Due to the presence of multiple lipases in DBS, previous studies measured LAL enzyme activity in the presence of Lalistat-2, an established LAL-specific inhibitor (Hamilton J et al Chim Clin Acta (2012) 413:1207-1210). Alternatively, a novel substrate specific for LAL has been reported very recently (Masi S. et al Clin Chem (2018) 64:690-696). In this study, we examined the LAL enzyme activity of a Japanese population with the LAL-specific substrate using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based enzyme assay whether an affected individual can be identified among this population. To achieve this, we first performed assay validation using LC-MS/MS. Under our experimental setting, typically we obtained LAL enzyme activity for QC High (100% enzyme activity) as 261.9 ± 3.2 µmol/h/L (n = 5) and for QC Low as (5% enzyme activity) as 14.7 ± 0.5 µmol/h/L (n = 5). The percentage of coefficient of variation for interday assay for QC High was 9.6% (n = 4) and for QC Low was 7.9% (n = 4), respectively. Based on these results, we further examined the LAL enzyme activity of control Japanese population and that of affected individuals with Wolman disease and CESD. The averaged enzyme activity for control newborns, Wolman, and CESD was 123.9 ± 53.9 µmol/h/L (n = 131), 6.6 ± 0.9 µmol/h/L (n = 3), and 4.8 ± 0.3 µmol/h/L (n = 3), respectively. These results suggest that an LAL-D-affected individual can be readily identified by enzyme activity using LC-MS/MS-based technique.

Maternal uniparental disomy of chromosome 7 underlying argininosuccinic aciduria and Silver-Russell syndrome.

We describe a patient presenting with argininosuccinic aciduria and Silver-Russell syndrome (SRS). SRS was caused by maternal uniparental disomy of chromosome 7 (UPD(7)mat). UPD(7)mat also unmasked a maternally inherited splicing variant in ASL on chromosome 7, leading to the onset of argininosuccinic aciduria. The phenotype of the present case was more severe than that of a previous case, demonstrating a phenotypic variation in the combination of argininosuccinic aciduria and SRS.

Development of a Highly Sensitive and Rapid Liquid Chromatography-Tandem Mass Spectrometric Method Using a Basic Mobile Phase Additive to Determine the Characteristics of the Urinary Metabolites for Niemann-Pick Disease Type C.

As Niemann-Pick disease type C (NPC) is difficult to diagnose owing to its various clinical symptoms; biomarker tests have been developed. Previously, we revealed urinary sulfated cholesterol metabolites as noninvasive biomarkers for NPC. However, LC/tandem mass spectrometry (LC/MS/MS) requires long separation time and large urine volumes. Recently, a basic mobile phase was reported to increase the MS intensity. Thus, we developed a highly sensitive and rapid LC/MS/MS method for analyzing urinary cholesterol metabolites using a basic mobile phase additive. 3ß-Sulfooxy-7ß-N-acetylglucosaminyl-5-cholenic acid, its glycine and taurine conjugates, 3ß-sulfooxy-7ß-hydroxy-5-cholenic acid, and 7-oxo form were measured, with selected reaction monitoring in negative ion mode. Oasis HLB and L-column 3 were used for column-switching LC/MS/MS and urine diluted 10-fold was employed as the sample. After trapping, gradient separation was performed using solutions containing 1% (v/v) ammonium solution. On average, a 16-fold increase in peak areas was observed compared to that obtained at pH 5.5 with the mobile phases. Although the previous method needed 60 min for separation from interference peaks, we succeeded to separate them in 7 min with optimized LC condition. Further, all compounds showed good linearity from 0.3-1000 ng/mL, with satisfactory intra- and inter-day reproducibility. The developed method was applied to the urinalysis of healthy participants and NPC patients. Overall, the concentrations of metabolites correlated with those obtained using the previous method. Therefore, we succeeded to increasing MS intensity and shorten LC running time; and the method is useful for the noninvasive diagnostic screening of patients with NPC.

Physiology and Pathophysiology of Heparan Sulfate in Animal Models: Its Biosynthesis and Degradation.

Heparan sulfate (HS) is a type of glycosaminoglycan that plays a key role in a variety of biological functions in neurology, skeletal development, immunology, and tumor metastasis. Biosynthesis of HS is initiated by a link of xylose to Ser residue of HS proteoglycans, followed by the formation of a linker tetrasaccharide. Then, an extension reaction of HS disaccharide occurs through polymerization of many repetitive units consisting of iduronic acid and N-acetylglucosamine. Subsequently, several modification reactions take place to complete the maturation of HS. The sulfation positions of N-, 2-O-, 6-O-, and 3-O- are all mediated by specific enzymes that may have multiple isozymes. C5-epimerization is facilitated by the epimerase enzyme that converts glucuronic acid to iduronic acid. Once these enzymatic reactions have been completed, the desulfation reaction further modifies HS. Apart from HS biosynthesis, the degradation of HS is largely mediated by the lysosome, an intracellular organelle with acidic pH. Mucopolysaccharidosis is a genetic disorder characterized by an accumulation of glycosaminoglycans in the body associated with neuronal, skeletal, and visceral disorders. Genetically modified animal models have significantly contributed to the understanding of the in vivo role of these enzymes. Their role and potential link to diseases are also discussed.

Enzyme Replacement Therapy with Pabinafusp Alfa for Neuronopathic Mucopolysaccharidosis II: An Integrated Analysis of Preclinical and Clinical Data.

Enzyme replacement therapy (ERT) improves somatic manifestations in mucopolysaccharidoses (MPS). However, because intravenously administered enzymes cannot cross the blood-brain barrier (BBB), ERT is ineffective against the progressive neurodegeneration and resultant severe central nervous system (CNS) symptoms observed in patients with neuronopathic MPS. Attempts to surmount this problem have been made with intrathecal and intracerebroventricular ERT in order to achieve CNS effects, but the burdens on patients are inimical to long-term administrations. However, since pabinafusp alfa, a human iduronate-2-sulfatase fused with a BBB-crossing anti-transferrin receptor antibody, showed both central and peripheral efficacy in a mouse model, subsequent clinical trials in a total of 62 patients with MPS-II (Hunter syndrome) in Japan and Brazil substantiated this dual efficacy and provided an acceptable safety profile. To date, pabinafusp alfa is the only approved intravenous ERT that is effective against both the somatic and CNS symptoms of patients with MPS-II. This article summarizes the previously obtained preclinical and clinical evidence related to the use of this drug, presents latest data, and discusses the preclinical, translational, and clinical challenges of evaluating, ameliorating, and preventing neurodegeneration in patients with MPS-II.

Production of therapeutic iduronate-2-sulfatase enzyme with a novel single-stranded RNA virus vector.

The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate-2-sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK-21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose-6-phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross-correction. These results suggest that our novel RNA vector may be applicable for further clinical settings.

Prevalence of patients with lysosomal storage disorders and peroxisomal disorders: A nationwide survey in Japan.

INTRODUCTION: Lysosomal storage disorders and peroxisomal disorders are rare diseases caused by the accumulation of substrates of the metabolic pathway within lysosomes and peroxisomes, respectively. Owing to the rarity of these diseases, the prevalence of lysosomal storage disorders and peroxisomal disorders in Japan is unknown. Therefore, we conducted a nationwide survey to estimate the number of patients with lysosomal storage disorders and peroxisomal disorders in Japan. METHODS: A nationwide survey was conducted following the "Manual of nationwide epidemiological survey for understanding patient number and clinical epidemiology of rare diseases (3rd version)". A questionnaire asking for detailed information, such as disease phenotypes and medical history, was created and sent to 504 institutions with doctors who have experience in treating patients with lysosomal storage disorders and peroxisomal disorders. Result A total of 303 completed questionnaires were collected from 504 institutions (response rate: 60.1%). The number of patients was estimated by calculating the rate/frequency of overlap. The estimated number of patients was 1658 (±264.8) for Fabry disease, 72 (±11.3) for mucopolysaccharidosis I, 275 (±49.9) for mucopolysaccharidosis II, 211 (±31.3) for Gaucher disease, 124 (±25.8) for Pompe disease, 83 (±44.3) for metachromatic leukodystrophy, 57 (±9.4) for Niemann-Pick type C, and 262 (±42.3) for adrenoleukodystrophy. In addition the birth prevalence was calculated using the estimated number of patients and birth year data for each disease, and was 1.25 for Fabry disease, 0.09 for mucopolysaccharidosis I, 0.38 for mucopolysaccharidosis II, 0.19 for Gaucher disease, 0.14 for Pompe disease, 0.16 for metachromatic leukodystrophy, 0.16 for Niemann-Pick type C, and 0.20 for adrenoleukodystrophy. DISCUSSION: Among the diseases analyzed, the disease with the highest prevalence was Fabry disease, followed by mucopolysaccharidosis II, adrenoleukodystrophy, Gaucher disease and metachromatic leukodystrophy. In particular, the high prevalence of mucopolysaccharidosis II and Gaucher disease type II was a feature characteristic of Japan. CONCLUSION: We estimated the number of patients with lysosomal storage disorders and peroxisomal disorders in Japan. The details of the age at diagnosis and treatment methods for each disease were clarified, and will be useful for the early diagnosis of these patients and to provide appropriate treatments. Furthermore, our results suggest that supportive care and the development of an environment that can provide optimal medical care is important in the future.

Impact of intracerebroventricular enzyme replacement therapy in patients with neuronopathic mucopolysaccharidosis type II.

This open-label, phase 1/2 study (JMACCT CTR JMA-IIA00350) evaluated the efficacy and safety of intracerebroventricular idursulfase beta in patients with mucopolysaccharidosis II (MPS II). Herein, we report the 100-week results. Six patients with severe MPS II aged 23-65 months were enrolled. Idursulfase beta (increasing from 1 to 30 mg between weeks 0 and 24, followed by a 30-mg final dose) was administered intracerebroventricularly once every 4 weeks using an implanted cerebrospinal fluid (CSF) reservoir; intravenous administration of idursulfase was also continued throughout the study. Efficacy endpoints included developmental age by the Kyoto Scale of Psychological Development 2001 and heparan sulfate (HS) concentration in CSF (primary outcome). In all six patients, HS concentrations decreased (40%-80%) from baseline to week 100. For overall developmental age, the difference in change from baseline to week 100 in each patient compared with patients treated by intravenous idursulfase administration (n = 13) was +8.0, +14.5, +4.5, +3.7, +8.2, and -8.3 months (mean, +5.1 months). Idursulfase beta was well tolerated. The most common adverse events were pyrexia, upper respiratory tract infection, and vomiting. The results suggest that intracerebroventricular idursulfase beta is well tolerated and can be effective at preventing and stabilizing developmental decline in patients with neuronopathic MPS II.

Guide for diagnosis and treatment of hyperphenylalaninemia.

IMPORTANCE: Sapropterin hydrochloride, a natural coenzyme (6R-tetrahydrobiopterin) of phenylalanine hydroxylase, was first approved as a treatment for tetrahydrobiopterin deficiency in 1992 in Japan, and was then approved as a treatment for a tetrahydrobiopterin-responsive hyperphenylalaninemia in 2007 and 2008, in the USA and Japan, respectively. Guidelines are required on the proper use of sapropterin hydrochloride for tetrahydrobiopterin-responsive hyperphenylalaninemia. OBSERVATIONS: It is recommended that tetrahydrobiopterin-responsive hyperphenylalaninemia should be diagnosed in all cases of hyperphenylalaninemia, including phenylketonuria, by tetrahydrobiopterin administration tests rather than by phenotype or blood phenylalanine levels. CONCLUSIONS AND RELEVANCE: If tetrahydrobiopterin-responsive hyperphenylalaninemia is diagnosed, all ages can be treated with sapropterin hydrochloride. Although there are reports that sapropterin hydrochloride is effective and safe for the prevention of maternal phenylketonuria, further investigation is required.

A Phase 2/3 Trial of Pabinafusp Alfa, IDS Fused with Anti-Human Transferrin Receptor Antibody, Targeting Neurodegeneration in MPS-II.

Pabinafusp alfa (JR-141) is a novel enzyme drug that crosses the blood-brain barrier by transcytosis via transferrin receptors. In order to establish its efficacy and safety, a multicenter, single-arm, open-label phase 2/3 clinical trial was conducted in 28 Japanese patients with mucopolysaccharidosis II (MPS-II, Hunter syndrome) by intravenous administrations of 2.0 mg/kg of pabinafusp alfa for 52 weeks. The primary efficacy endpoint was changes in heparan sulfate (HS) concentrations in the cerebrospinal fluid (CSF). Secondary endpoints included assessments of neurocognitive development for central efficacy, and changes in plasma HS and dermatan sulfate (DS) concentrations for peripheral efficacy. HS concentrations in the CSF significantly decreased from baseline to week 52 (p < 0.001), suggesting continuous inhibition of substrate accumulations in the CNS, i.e., hitherto unaddressed progressive neurodegeneration. Evaluations of neurocognitive developments showed positive changes in 21 of the 28 patients. Serum HS and DS concentrations, liver and spleen volumes, and other assessments suggested the peripheral efficacy of pabinafusp alfa was comparable to that of idursulfase. Drug-related adverse events were mild or moderate in severity, transient, and manageable. The results establish delivery across the BBB of pabinafusp alfa as an effective therapeutic for treating both the CNS and peripheral symptoms of patients with MPS-II.

A cDNA analysis disclosed the discordance of genotype-phenotype correlation in a patient with attenuated MPS II and a 76-base deletion in the gene for iduronate-2-sulfatase.

We previously showed that the genotype-phenotype correlation in MPS II is well-conserved in Japan (Kosuga et al., 2016). Almost all of our patients with attenuated MPS II have missense variants, which is expected to result in residual activity of iduronate-2-sulfatase. In contrast, our patients with severe MPS II have so-called null-type disease-associated variants, such as nonsense variants, frame-shifts, gene insertions, gene deletions and rearrangement with pseudogene (IDS2), none of which are expected to result in residual activity. However, we recently encountered a patient with attenuated MPS II who had a presumable null-type disease-associated variant and 76-base deletion located in exon 1 that extended into intron 1. To investigate this discordance, we extracted RNA from the leukocytes of the patient and performed reverse transcription polymerase chain reaction. One of the bands of the cDNA analysis was found to include a nucleotide sequence whose transcript was expected to generate an almost full-length IDS mature peptide lacking only part of its signal peptide as well as only one amino acid at the end of the N-terminus. This suggests that an alternative splicing donor site is generated in exon 1 upstream of the deleted region. Based on these observations, we concluded that the phenotype-genotype discordance in this patient with MPS II was due to the decreased amount of IDS protein induced by the low level of the alternatively spliced mRNA, lacking part of the region coding for the signal peptide but including the region coding almost the full mature IDS protein. The first 25 amino acids at the N-terminus of IDS protein are a signal peptide. The alternative splice transcript has only 13 (1 M-13 L) of those 25 amino acids; 14G-25G are missing, suggesting that the exclusively hydrophobic 1 M-13 L of the signal peptide of IDS might have a crucial role in the signal peptide.

Natural history of cognitive development in neuronopathic mucopolysaccharidosis type II (Hunter syndrome): Contribution of genotype to cognitive developmental course.

The natural history of cognitive growth in the neuronopathic form of Mucopolysaccharidosis type II (MPS II) is not well defined especially their patterns of development and decline. The ability to predict the developmental course of the neurologically impaired patient is necessary to assess treatment outcomes aimed at the brain. Thirteen intravenous enzyme replacement therapy-treated Japanese patients with neuronopathic MPSII who had mutation analysis were followed on one standard measure of cognitive development over time. Six children in Group MS had missense mutations and 7 children in Group NT had null type mutations such as deletions, recombination with the pseudogene, and nonsense mutations. The patients as a whole demonstrated cognitive growth until about 36-42 months of age, followed by a plateau in development. The mean age equivalent score at age 3 was similar to that at age 6. While the decline was slow for the entire group, the patients in Group NT showed a more rapid decline than those in Group MS. Two patients with deletions showed decline to a very low level by age 5. The long plateau in cognitive development in patents with MPS II was substantiated and was consistent with other studies. This is the first demonstration that different mutation types within the neuronopathic MPS II patients are associated with different rates of decline. We also were able to identify the chronological age before which a trial would need to start in order to maintain cognitive growth and a ceiling beyond which a relatively normal outcome would not be likely.

Development of a Diagnostic Screening Strategy for Niemann-Pick Diseases Based on Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Analyses of N-Palmitoyl-O-phosphocholine-serine and Sphingosylphosphorylcholine.

Early diagnosis of Niemann-Pick diseases (NPDs) is important for better prognosis of such diseases. N-Palmitoyl-O-phosphocholine-serine (PPCS) is a new NPD biomarker possessing high sensitivity, and with its combination with sphingosylphosphocholine (SPC) it may be possible to distinguish NPD-C from NPD-A/B. In this study, a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (method 1) and a validated LC-MS/MS analysis (method 2) of PPCS and SPC were developed, and we have proposed a diagnostic screening strategy for NPDs using a combination of serum PPCS and SPC concentrations. Nexera and API 5000 were used as LC-MS/MS systems. C18 columns with lengths of 10 and 50 mm were used for method 1 and 2, respectively. 2H3-Labeled PPCS and nor-SPC were used as internal standards. Selective reaction monitoring in positive-ion mode was used for MS/MS. Run times of 1.2 and 8 min were set for methods 1 and 2, respectively. In both methods 1 and 2, two analytes showed high linearity in the range of 1-4000 ng/mL. Method 2 provided high accuracy and precision in method validation. Serum concentrations of both analytes were significantly higher in NPD-C patients than those of healthy subjects in both methods. Serum PPCS correlated between methods 1 and 2; however, it was different in the case of SPC. The serum PPCS/SPC ratio was different in healthy subjects, NPD-C, and NPD-A/B. These results suggest that using a combination of the two LC-MS/MS analytical methods for PPCS and SPC is useful for diagnostic screening of NPDs.

Pharmacokinetics and pharmacodynamics of JR-051, a biosimilar of agalsidase beta, in healthy adults and patients with Fabry disease: Phase I and II/III clinical studies.

Fabry disease is a rare X-linked lysosomal disease, in which mutations in the gene encoding α-galactosidase A result in progressive cellular accumulation of globotriaosylceramide (GL-3) in various organs including the skin, kidney, and heart, often leading to life-threatening conditions. Enzyme replacement therapy is currently the standard therapy for the disease, to which two α-galactosidase A formulations have been approved: agalsidase α (Replagal®, Shire) and agalsidase ß (Fabrazyme®, Sanofi). We have recently developed a biosimilar of agalsidase ß, JR-051, and investigated its pharmacokinetics and pharmacodynamics to assess its bioequivalence to agalsidase ß. In a randomized phase I study, healthy adult male volunteers were treated with JR-051 or agalsidase ß and the pharmacokinetics of the drugs were compared. The ratio of geometric means (90% confidence interval [CI]) of the AUC0-24 and Cmax for JR-051 over agalsidase ß were 0.91 (0.8294, 1.0082) and 0.90 (0.7992, 1.0125), respectively. In a 52-week, single-arm, phase II/III study, patients with Fabry disease switched therapy from agalsidase ß to JR-051 to evaluate its pharmacodynamics. The mean (95% CI) plasma GL-3 concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.03 (0.91, 1.15) and 0.96 (0.86, 1.06), respectively, which were within the pre-determined bioequivalence acceptance range (0.70, 1.43). The mean (95% CI) plasma globotriaosylsphingosine (lyso-GL-3) concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.07 (0.92, 1.23) and 1.13 (1.03, 1.22), respectively. Estimated glomerular filtration rate and left ventricular mass index, as renal and cardiac function indicators, showed no notable changes from baseline throughout the study period, and no new safety concerns were identified. In conclusion, these studies demonstrated bioequivalence of JR-051 to agalsidase ß in terms of its pharmacokinetics and pharmacodynamics. JR-051 offers a potential new treatment option for patients with Fabry disease.

Biomarkers for Lysosomal Storage Disorders with an Emphasis on Mass Spectrometry.

Lysosomal storage disorders (LSDs) are characterized by an accumulation of various substances, such as sphingolipids, mucopolysaccharides, and oligosaccharides. The LSD enzymes responsible for the catabolism are active at acidic pH in the lysosomal compartment. In addition to the classically established lysosomal degradation biochemistry, recent data have suggested that lysosome plays a key role in the autophagy where the fusion of autophagosome and lysosome facilitates the degradation of amino acids. A failure in the lysosomal function leads to a variety of manifestations, including neurovisceral disorders. While affected individuals appear to be normal at birth, they gradually become symptomatic in childhood. Biomarkers for each condition have been well-documented and their proper selection helps to perform accurate clinical diagnoses. Based on the natural history of disorders, it is now evident that the existing treatment becomes most effective when initiated during presymptomatic period. Neonatal screening provides such a platform for inborn error of metabolism in general and is now expanding to LSDs as well. These are implemented in some areas and countries, including Taiwan and the U.S. In this short review, we will discuss several issues on some selected biomarkers for LSDs involving Fabry, Niemann-Pick disease type C, mucopolysaccharidosis, and oligosaccharidosis, with a focus on mass spectrometry application to biomarker discovery and detection.

Novel HADHB mutations in a patient with mitochondrial trifunctional protein deficiency.

We encountered a patient with mitochondrial trifunctional protein deficiency in whom the corresponding mutations were not identified by a DNA panel for newborn screening for targeted diseases. After diagnosis confirmation by an enzyme assay and immunoblotting using the autopsied liver, the re-evaluation of the panel data indicated a heterozygous deletion of exons 6-9 that was later confirmed at the genomic level. cDNA analysis also identified exonization of the 5' region of intron 9 caused by a deep intronic mutation, c.811 + 82A>G.