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.

RNA-Based Therapeutic Technology.

RNA-based therapy has been an expanding area of clinical research since the COVID-19 outbreak. Often, its comparison has been made to DNA-based gene therapy, such as adeno-associated virus- and lentivirus-mediated therapy. These DNA-based therapies show persistent expression, with maximized therapeutic efficacy. However, accumulating data indicate that proper control of gene expression is occasionally required. For example, in cancer immunotherapy, cytokine response syndrome is detrimental for host animals, while excess activation of the immune system induces supraphysiological cytokines. RNA-based therapy seems to be a rather mild therapy, and it has room to fit unmet medical needs, whereas current DNA-based therapy has unclear issues. This review focused on RNA-based therapy for cancer immunotherapy, hematopoietic disorders, and inherited disorders, which have received attention for possible clinical applications.

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.

Lysosomal Acid Lipase Deficiency: Genetics, Screening, and Preclinical Study.

Lysosomal acid lipase (LAL) is a lysosomal enzyme essential for the degradation of cholesteryl esters through the endocytic pathway. Deficiency of the LAL enzyme encoded by the LIPA gene leads to LAL deficiency (LAL-D) (OMIM 278000), one of the lysosomal storage disorders involving 50-60 genes. Among the two disease subtypes, the severe disease subtype of LAL-D is known as Wolman disease, with typical manifestations involving hepatomegaly, splenomegaly, vomiting, diarrhea, and hematopoietic abnormalities, such as anemia. In contrast, the mild disease subtype of this disorder is known as cholesteryl ester storage disease, with hypercholesterolemia, hypertriglyceridemia, and high-density lipoprotein disappearance. The prevalence of LAL-D is rare, but several treatment options, including enzyme replacement therapy, are available. Accordingly, a number of screening methodologies have been developed for this disorder. This review summarizes the current discussion on LAL-D, covering genetics, screening, and the tertiary structure of human LAL enzyme and preclinical study for the future development of a novel therapy.

Lipid Nanoparticles: A Novel Gene Delivery Technique for Clinical Application.

Lipid nanoparticles (LNPs) are an emerging vehicle for gene delivery that accommodate both nucleic acid and protein. Based on the experience of therapeutic liposomes, current LNPs have been developed based on the chemistry of lipids and RNA and on the biology of human disease. LNPs have been used for the development of Onpattro, an siRNA drug for transthyretin-mediated amyloidosis, in 2018. The subsequent outbreak of COVID-19 required a vaccine for its suppression. LNP-based vaccine production received much attention for this and resulted in great success. In this review, the essential technology of LNP gene delivery has been described according to the chemistry for LNP production and biology for its clinical application.

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.

Mammalian Sulfatases: Biochemistry, Disease Manifestation, and Therapy.

Sulfatases are enzymes that catalyze the removal of sulfate from biological substances, an essential process for the homeostasis of the body. They are commonly activated by the unusual amino acid formylglycine, which is formed from cysteine at the catalytic center, mediated by a formylglycine-generating enzyme as a post-translational modification. Sulfatases are expressed in various cellular compartments such as the lysosome, the endoplasmic reticulum, and the Golgi apparatus. The substrates of mammalian sulfatases are sulfolipids, glycosaminoglycans, and steroid hormones. These enzymes maintain neuronal function in both the central and the peripheral nervous system, chondrogenesis and cartilage in the connective tissue, detoxification from xenobiotics and pharmacological compounds in the liver, steroid hormone inactivation in the placenta, and the proper regulation of skin humidification. Human sulfatases comprise 17 genes, 10 of which are involved in congenital disorders, including lysosomal storage disorders, while the function of the remaining seven is still unclear. As for the genes responsible for pathogenesis, therapeutic strategies have been developed. Enzyme replacement therapy with recombinant enzyme agents and gene therapy with therapeutic transgenes delivered by viral vectors are administered to patients. In this review, the biochemical substrates, disease manifestation, and therapy for sulfatases are summarized.

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.

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.

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.

Structural Determination of Lysosphingomyelin-509 and Discovery of Novel Class Lipids from Patients with Niemann-Pick Disease Type C.

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder caused by the mutation of cholesterol-transporting proteins. In addition, early treatment is important for good prognosis of this disease because of the progressive neurodegeneration. However, the diagnosis of this disease is difficult due to a variety of clinical spectrum. Lysosphingomyelin-509, which is one of the most useful biomarkers for NPC, was applied for the rapid and easy detection of NPC. The fact that its chemical structure was unknown until recently implicates the unrevealed pathophysiology and molecular mechanisms of NPC. In this study, we aimed to elucidate the structure of lysosphingomyelin-509 by various mass spectrometric techniques. As our identification strategy, we adopted analytical and organic chemistry approaches to the serum of patients with NPC. Chemical derivatization and hydrogen abstraction dissociation-tandem mass spectrometry were used for the determination of function groups and partial structure, respectively. As a result, we revealed the exact structure of lysosphingomyelin-509 as N-acylated and O-phosphocholine adducted serine. Additionally, we found that a group of metabolites with N-acyl groups were increased considerably in the serum/plasma of patients with NPC as compared to that of other groups using targeted lipidomics analysis. Our techniques were useful for the identification of lysosphingomyelin-509.

Biosynthesis of long chain base in sphingolipids in animals, plants and fungi.

Long chain base (LCB) is a unique building block found in sphingolipids. The initial step of LCB biosynthesis stems from serine:palmitoyl-CoA transferase enzyme, producing 3-ketodihydrosphingosine with multiple regulatory proteins including small subunit SPT a/b and orosomucoid-like protein1-3. 3-Ketodihydrosphingosine reductase and sphingolipid Δ4-desaturase, both of them poorly characterized mammalian enzymes, play key roles for neurological homeostasis based on their pathogenic mutation in humans. Ceramide synthase in mammals has six isoforms with distinct phenotype in each knockout mouse. In plants and fungi, sphingolipids also contain phytosphingosine due to sphingolipid C4-hydroxylase. In contrast to previous notion that dietary intake might be its major route in animals, emerging evidences suggested that phytosphingosine biosynthesis does occur in some tissues such as the skin by mammalian C4-hydroxylase activity of the DEGS2 gene. This short review summarizes LCB biosynthesis with their associating metabolic pathways in animals, plants and fungi.

Quantification of 11 enzyme activities of lysosomal storage disorders using liquid chromatography-tandem mass spectrometry.

Lysosomal storage disorders (LSDs) are characterized by the accumulation of lipids, glycolipids, oligosaccharides, mucopolysaccharides, and other biological substances because of the pathogenic deficiency of lysosomal enzymes. Such diseases are rare; thus, a multiplex assay for these disorders is effective for the identification of affected individuals during the presymptomatic period. Previous studies have demonstrated that such assays can be performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) detection. An assay procedure to quantify the activity of 11 enzymes associated with LSDs was provided. First, a validation study was performed using dried blood spot (DBS) samples with 100% and 5% enzyme activity for quality control (QC). Under the assay condition, the analytical range, defined as the ratio of the peak area of the enzyme reaction products from the DBS for QC with 100% enzyme activity to that from the filter paper blank sample, was between 14 for GALN and 4561 for GLA. Based on these results, the distribution of the enzyme activity for the 11 LSD enzymes was further examined. Consistent with the previous data, the enzyme activity exhibited a bell-shaped distribution with a single peak. The averaged enzyme activity for the healthy neonates was as follows: GLA, 3.80 ±â€¯1.6; GAA, 10.6 ±â€¯4.8; IDUA, 6.4 ±â€¯2.3; ABG, 8.6 ±â€¯3.1; ASM, 3.3 ±â€¯1.1; GALC, 2.8 ±â€¯1.3; ID2S, 16.7 ±â€¯6.1; GALN, 1.2 ±â€¯0.5; ARSB, 17.0 ±â€¯8.7; NAGLU, 4.6 ±â€¯1.5; and GUSB, 46.6 ±â€¯19.0 µmol/h/L (mean ±â€¯SD, n = 200). In contrast, the enzyme activity in disease-affected individuals was lower than the minimum enzyme activity in healthy neonates. The results demonstrate that the population of disease-affected individuals was distinguished from that of healthy individuals by the use of LC-MS/MS.

Elevation of plasma lysosphingomyelin-509 and urinary bile acid metabolite in Niemann-Pick disease type C-affected individuals.

Niemann-Pick disease type C (NPC) is a neurovisceral disorder associated with the accumulation of lipids such as cholesterol and sphingolipids. NPC is caused by either NPC1 or NPC2, which encode lysosomal proteins located at membraneous and soluble fractions, respectively. For the past decade, the oxidation products of cholesterol, such as cholestane-3ß,5α,6ß-triol and 7-ketocholesterol, have been considered selective biomarkers for NPC. However, recent evidence has indicated numerous novel biomarkers for NPC, which raises the possibility that the diagnosis of NPC might be associated with the elevation of multiple lipid biomarkers, rather than a single biomarker. Sphingosylphosphorylcholine (SPC) has been suggested to be one such biomarker for NPC, in which elevated sphingomyelin is a potential precursor. Thus, we first performed a validation study of plasma SPC using LC-MS/MS. The results showed the following plasma concentrations in the NPC-affected and control individuals, respectively: 8.2 ±â€¯2.8 nM (mean ±â€¯SD; median, 7.0 nM; max, 11.7 nM; min, 5.1 nM; n = 5) and 3.1 ±â€¯1.4 nM (median, 2.9 nM; max, 4.8 nM; min, 1.5 nM; n = 7). We further extended the study to plasma lysophingomyelin-509 for NPC, a newly reported biomarker with uncharacterized chemical nature. Based on these result with plasma SPC as a surrogate marker, the value of mean of median of plasma lysophingomyelin-509 in NPC-affected individuals elevated at 65.2 (max, 73.2; min, 26.7; n = 5). Furthermore, the efficacy of plasma SPC and lysosphingomyelin-509 as promising biomarkers for this disorder was supported by the finding that the urinary concentration of 3ß-sulfooxy-7ß-N-acetylglucosaminyl-5-cholen-24-oic acid, an established biomarker for NPC, was also elevated in the NPC-affected individuals. These results suggest that a novel combination of plasma biomarkers, such as SPC and/or lysophingomyelin-509, and urinary bile acid metabolite could offer a promising platform for the diagnosis of NPC.

Quantification of the enzyme activities of iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase and N-acetylgalactosamine-4-sulfatase using liquid chromatography-tandem mass spectrometry.

Mucopolysaccharidosis (MPS) is a genetic disorder characterized by the accumulation of glycosaminoglycans in the body. Of the multiple MPS disease subtypes, several are caused by defects in sulfatases. Specifically, a defect in iduronate-2-sulfatase (ID2S) leads to MPS II, whereas N-acetylgalactosamine-6-sulfatase (GALN) and N-acetylgalactosamine-4-sulfatase (ARSB) defects relate to MPS IVA and MPS VI, respectively. A previous study reported a combined assay for these three disorders in a 96-well plate using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technique (Kumar et al., Clin Chem 2015 61(11):1363-1371). In our study, we applied this methodology to a Japanese population to examine the assay precision and the separation of populations between disease-affected individuals and controls for these three disorders. Within our assay conditions, the coefficient of variation (CV, %) values for an interday assay of ID2S, GALN, and ARSB were 9%, 18%, and 9%, respectively (n = 7). The average enzyme activities of ID2S, GALN, and ARSB in random neonates were 19.6 ± 5.8, 1.7 ± 0.7, and 13.4 ± 5.2 µmol/h/L (mean ± SD, n = 240), respectively. In contrast, the average enzyme activities of ID2S, GALN, and ARSB in disease-affected individuals were 0.5 ± 0.2 (n = 6), 0.3 ± 0.1 (n = 3), and 0.3 (n = 1) µmol/h/L, respectively. The representative analytical range values corresponding to ID2S, GALN, and ARSB were 39, 17, and 168, respectively. These results raise the possibility that the population of disease-affected individuals could be separated from that of healthy individuals using the LC-MS/MS-based technique.

Lipid biomarkers for the peroxisomal and lysosomal disorders: their formation, metabolism and measurement.

Lipid biomarkers play important roles in the diagnosis of and monitoring of treatment in peroxisomal disorders and lysosomal storage disorders. Today, a variety of lipids, including very long chain fatty acids, glycolipids, bile acids and the oxidation products of cholesterol, have been considered as biomarkers for these disorders. In this brief review, the authors summarized the recent advances regarding these lipid biomarkers in terms of their formation, metabolism and measurement in these disorders. An understanding of these biomarkers will offer a key to the development of novel diagnoses and help create more effective therapies in the future.

A molecular analysis of the GAA gene and clinical spectrum in 38 patients with Pompe disease in Japan.

Pompe disease is an autosomal recessive disorder caused by acid α-glucosidase (GAA) deficiency, which results in the accumulation of glycogen in lysosomes in multiple tissues, including cardiac, skeletal, and smooth muscle cells. Thus far, 558 sequence variants of the GAA gene have been published in the Pompe Disease Mutation Database, and some mutations appear with considerable frequency in particular ethnic groups, such as Caucasians, Taiwanese, Chinese, and Koreans. However, the GAA mutation pattern in Japanese patients remains poorly understood. We analyzed the relationship between the genetic and clinical features of 38 mostly Japanese patients with Pompe disease from 35 unrelated families. We identified 28 different GAA gene mutations, including 7 novel mutations, by a GAA gene analysis. c.546G > T (22.9%) and c.1857C > G (14.3%) were the most common mutations and accounted for 37.1% of the total mutant alleles. In the six patients with infantile-onset Pompe disease (IOPD), c.1857C > G was also the most common mutation. In addition, there were 13 homozygotes (5 with the c.546G > T) among the 35 families, which is the highest frequency reported thus far. Regarding the initial symptoms, cardiomegaly was the most common (3/6 = 50%) in IOPD patients, while muscle weakness was observed the most frequently in patients with late-onset Pompe disease (LOPD) (15/30 = 50%). Notably, all IOPD patients who showed respiratory distress at the time of onset require respiratory assistance at present (4/4 = 100%). Regarding the presenting symptoms, cardiomegaly (6/6 = 100%) and hepatomegaly (4/6 = 66.7%) were more commonly seen in IOPD, and muscle weakness (24/29 = 82.7%) was observed more frequently in LOPD. Respiratory assistance is required at present in 33.3% of IOPD patients and 50% of LOPD patients, and 20% of IOPD patients and 29.6% of LOPD patients are wheelchair users. These individual clinical courses may be influenced by the timing of the diagnosis and treatment; for example, in 2007, an ERT orphan drug for treatment of Pompe disease, Alglucosidase alfa, was made available in Japan, and there were 5 (5/6 = 83.3%) wheelchair users diagnosed from 2008 to 2009 (cases 32-38) and 4 (4/27 = 14.8%) from 2010 to 2015 (cases 1-31). These findings underscore the importance of the early diagnosis and treatment.

Enzyme activities of α-glucosidase in Japanese neonates with pseudodeficiency alleles.

Lysosomal storage disorders (LSDs) are caused by defective enzyme activities in lysosomes, characterized by the accumulation of sphingolipids, glycolipids, oligosaccharides, mucopolysaccharides, the oxidation products of cholesterol, and other biological substances. A growing number of clinical studies have suggested the enhanced efficacy of existing therapies, including enzyme replacement therapy, which is effective when it is initiated during the presymptomatic period. Thus, the identification of disease-affected individuals by newborn screening has been considered an effective platform. Previous studies have suggested that the discrimination of infantile-onset Pompe disease (IOPD) requires multi-step examination of GAA enzyme activity using the fluorometric technique. In sharp contrast, the MS/MS-based technique can identify the population of IOPD and the pseudodeficiency alleles of the GAA enzyme [Liao HC et al. Clin Chem (2017) in press; doi: http://dx.doi.org/10.1373/clinchem.2016.269027]. To determine whether MS/MS-based assay can identify these two populations in Japanese neonates, we first performed a validation study of this assay using flow-injection analysis (FIA)-MS/MS and liquid chromatography (LC)-MS/MS followed by examination of GAA enzyme activity in our population. By minimizing the effect of substrate-derived in-source decomposition products, the activities of 6 LSD enzymes were quantified in FIA-MS/MS and LC-MS/MS. The mean value of GAA activity with IOPD, pseudodeficiency alleles, and healthy controls by FIA-MS/MS were 1.0 ± 0.3 µmol/h/L (max, 1.3; min, 0.7; median, 1.2; n = 3), 2.7 ± 0.7 µmol/h/L (max, 4.5; min, 1.5; median, 2.5; n = 19), and 12.9 ± 5.4 µmol/h/L (max, 29.6; min, 2.5; median, 11.0; n = 83), respectively. These results suggest that the population of GAA with pseudodeficiency alleles has approximately 20% of GAA enzyme activity compared to controls, providing the preliminary evidence to estimate the cut-off values in the Japanese population using this technique.

Aspartylglucosaminuria caused by a novel homozygous mutation in the AGA gene was identified by an exome-first approach in a patient from Japan.

BACKGROUND: Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme, aspartylglucosaminidase (AGA). This disorder is rare in the general population except in Finland. Since the most characteristic feature of this disorder is a progressive developmental regression, patients often show no specific symptoms in the initial stages, and thus early diagnosis is often challenging. CASE REPORT: We encountered a 16-year-old boy who began to show difficulties in his speech at the age of 6years. Due to a mild regression in his development, he gradually lost common daily abilities. His diagnosis was first obtained through exome sequencing that identified a novel homozygous mutation in the AGA gene. This result was reasonable because of parental consanguinity. Reduced enzymatic activity of AGA was then confirmed. His urine was retrospectively screened by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and a specific pattern of abnormal metabolites was identified. CONCLUSIONS: Because both exome sequencing and MALDI-TOF-MS screening are adaptable and comprehensive, future combinatory use of these methods would be useful for diagnosis of rare inborn errors of metabolism such as AGU.