Validation of Aspartylglucosaminidase Activity Assay for Human Serum Samples: Establishment of a Biomarker for Diagnostics and Clinical Studies.

Novel treatment strategies are emerging for rare, genetic diseases, resulting in clinical trials that require adequate biomarkers for the assessment of the treatment effect. For enzyme defects, biomarkers that can be assessed from patient serum, such as enzyme activity, are highly useful, but the activity assays need to be properly validated to ensure a precise, quantitative measurement. Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by the deficiency of the lysosomal hydrolase aspartylglucosaminidase (AGA). We have here established and validated a fluorometric AGA activity assay for human serum samples from healthy donors and AGU patients. We show that the validated AGA activity assay is suitable for the assessment of AGA activity in the serum of healthy donors and AGU patients, and it can be used for diagnostics of AGU and, potentially, for following a treatment effect.

Topological Structural Brain Connectivity Alterations in Aspartylglucosaminuria: A Case-Control Study.

BACKGROUND AND PURPOSE: We investigated global and local properties of the structural brain connectivity networks in aspartylglucosaminuria, an autosomal recessive and progressive neurodegenerative lysosomal storage disease. Brain connectivity in aspartylglucosaminuria has not been investigated before, but previous structural MR imaging studies have shown brain atrophy, delayed myelination, and decreased thalamic and increased periventricular WM T2 signal intensity. MATERIALS AND METHODS: We acquired diffusion MR imaging and T1-weighted data from 12 patients with aspartylglucosaminuria (mean age, 23 [SD, 8] years; 5 men), and 30 healthy controls (mean age, 25 [SD, 10] years; 13 men). We performed whole-brain constrained spherical deconvolution tractography, which enables the reconstruction of neural tracts through regions with complex fiber configurations, and used graph-theoretical analysis to investigate the structural brain connectivity networks. RESULTS: The integration of the networks was decreased, as demonstrated by a decreased normalized global efficiency and an increased normalized characteristic path length. In addition, the average strength of the networks was decreased. In the local analyses, we found decreased strength in 11 nodes, including, for example, the right thalamus, right putamen, and, bilaterally, several occipital and temporal regions. CONCLUSIONS: We found global and local structural connectivity alterations in aspartylglucosaminuria. Biomarkers related to the treatment efficacy are needed, and brain network properties may provide the means for long term follow-up.

[Analysis of genetic variant in a child with Aspartylglucosaminuria].

OBJECTIVE: To explore the genetic basis for a child with Aspartylglucosaminuria (AGU). METHODS: Clinical data of the patient was analyzed. The child was subjected to trio-whole exome sequencing (WES) and copy number variation sequencing (CNV-seq), and candidate variant was verified by Sanger sequencing. RESULTS: The child was found to harbor homozygous c.319C>T (p.Arg107*) nonsense variant of the AGA gene, for which both of his parents were heterozygous carriers. No abnormality was found by CNV-seq analysis. The c.319C>T (p.Arg107*) variant was not found in population database, HGMD and other databases. Based on guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic (PVS1+PM2+PP3). CONCLUSION: The c.319C>T variant of the AGA gene probably underlay the autosomal recessive AGU in this child. Above finding has enabled genetic counseling and prenatal diagnosis for his parents.

Towards Splicing Therapy for Lysosomal Storage Disorders: Methylxanthines and Luteolin Ameliorate Splicing Defects in Aspartylglucosaminuria and Classic Late Infantile Neuronal Ceroid Lipofuscinosis.

Splicing defects caused by mutations in the consensus sequences at the borders of introns and exons are common in human diseases. Such defects frequently result in a complete loss of function of the protein in question. Therapy approaches based on antisense oligonucleotides for specific gene mutations have been developed in the past, but they are very expensive and require invasive, life-long administration. Thus, modulation of splicing by means of small molecules is of great interest for the therapy of genetic diseases resulting from splice-site mutations. Using minigene approaches and patient cells, we here show that methylxanthine derivatives and the food-derived flavonoid luteolin are able to enhance the correct splicing of the AGA mRNA with a splice-site mutation c.128-2A>G in aspartylglucosaminuria, and result in increased AGA enzyme activity in patient cells. Furthermore, we also show that one of the most common disease causing TPP1 gene variants in classic late infantile neuronal ceroid lipofuscinosis may also be amenable to splicing modulation using similar substances. Therefore, our data suggest that splice-modulation with small molecules may be a valid therapy option for lysosomal storage disorders.

Aspartylglucosaminuria: Clinical Presentation and Potential Therapies.

Aspartylglucosaminuria (AGU) is a recessively inherited neurodegenerative lysosomal storage disease characterized by progressive intellectual disability, skeletal abnormalities, connective tissue overgrowth, gait disturbance, and seizures followed by premature death. AGU is caused by pathogenic variants in the aspartylglucosaminidase (AGA) gene, leading to glycoasparagine accumulation and cellular dysfunction. Although more prevalent in the Finnish population, more than 30 AGA variants have been identified worldwide. Owing to its rarity, AGU may be largely underdiagnosed. Recognition of the following early clinical features may aid in AGU diagnosis: developmental delays, hyperactivity, early growth spurt, inguinal and abdominal hernias, clumsiness, characteristic facial features, recurring upper respiratory and ear infections, tonsillectomy, multiple sets of tympanostomy tube placement, and sleep problems. Although no curative therapies currently exist, early diagnosis may provide benefit through the provision of anticipatory guidance, management of expectations, early interventions, and prophylaxis; it will also be crucial for increased clinical benefits of future AGU disease-modifying therapies.

Pre-clinical Gene Therapy with AAV9/AGA in Aspartylglucosaminuria Mice Provides Evidence for Clinical Translation.

Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal storage disease caused by loss of the enzyme aspartylglucosaminidase (AGA), resulting in AGA substrate accumulation. AGU patients have a slow but progressive neurodegenerative disease course, for which there is no approved disease-modifying treatment. In this study, AAV9/AGA was administered to Aga-/- mice intravenously (i.v.) or intrathecally (i.t.), at a range of doses, either before or after disease pathology begins. At either treatment age, AAV9/AGA administration led to (1) dose dependently increased and sustained AGA activity in body fluids and tissues; (2) rapid, sustained, and dose-dependent elimination of AGA substrate in body fluids; (3) significantly rescued locomotor activity; (4) dose-dependent preservation of Purkinje neurons in the cerebellum; and (5) significantly reduced gliosis in the brain. Treated mice had no abnormal neurological phenotype and maintained body weight throughout the whole experiment to 18 months old. In summary, these results demonstrate that treatment of Aga-/- mice with AAV9/AGA is effective and safe, providing strong evidence that AAV9/AGA gene therapy should be considered for human translation. Further, we provide a direct comparison of the efficacy of an i.v. versus i.t. approach using AAV9, which should greatly inform the development of similar treatments for other related lysosomal storage diseases.

Detailed profile of cognitive dysfunction in children with aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a rare, recessively inherited lysosomal disease with relatively high prevalence in Finnish population. This progressive disease has a vast impact on patient's cognition and physical health, leading to intellectual disability and shorter life expectancy. Cognitive functions of 21 7- to 14-year-old children with AGU were studied cross-sectionally using Wechsler's Intelligence Scale for Children IV and the results were compared with a standardized Finnish sample. In addition to overall cognitive performance, abilities in discrete cognitive domains, including verbal comprehension, perceptual reasoning, working memory, and processing speed, were examined. The results showed that despite the very low overall level of cognitive performance, there were notable differences between individuals. All those children whose performance was closer to their own age level were 7 to 10 years old. Processing speed appeared more compromised, as compared with verbal comprehension. Furthermore, examining the subtest raw scores, there were no significant positive correlations between age and subtest scores, suggesting that the developmental level of AGU children could be rather stable throughout ages 7 to 14. This study gives insight to the severe nature of AGU disease. Since younger children performed better compared to their age norms than older children, the results raise a question whether the highest peak in cognitive functions is reached at an earlier age than previously thought.

Susceptibility-Weighted Imaging Findings in Aspartylglucosaminuria.

BACKGROUND AND PURPOSE: Aspartylglucosaminuria is a rare lysosomal storage disorder that causes slowly progressive, childhood-onset intellectual disability and motor deterioration. Previous studies have shown, for example, hypointensity in the thalami in patients with aspartylglucosaminuria on T2WI, especially in the pulvinar nuclei. Susceptibility-weighted imaging is a neuroimaging technique that uses tissue magnetic susceptibility to generate contrast and is able to visualize iron and other mineral deposits in the brain. SWI findings in aspartylglucosaminuria have not been reported previously. MATERIALS AND METHODS: Twenty-one patients with aspartylglucosaminuria (10 girls; 7.4-15.0 years of age) underwent 3T MR imaging. The protocol included an SWI sequence, and the images were visually evaluated. Thirteen patients (6 girls, 7.4-15.0 years of age) had good-quality SWI. Eight patients had motion artifacts and were excluded from the visual analysis. Thirteen healthy children (8 girls, 7.3-14.1 years of age) were imaged as controls. RESULTS: We found a considerably uniform distribution of decreased signal intensity in SWI in the thalamic nuclei in 13 patients with aspartylglucosaminuria. The most evident hypointensity was found in the pulvinar nuclei. Patchy hypointensities were also found especially in the medial and anterior thalamic nuclei. Moreover, some hypointensity was noted in globi pallidi and substantia nigra in older patients. The filtered-phase images indicated accumulation of paramagnetic compounds in these areas. No abnormal findings were seen in the SWI of the healthy controls. CONCLUSIONS: SWI indicates accumulation of paramagnetic compounds in the thalamic nuclei in patients with aspartylglucosaminuria. The finding may raise the suspicion of this rare disease in clinical practice.

The T99K variant of glycosylasparaginase shows a new structural mechanism of the genetic disease aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is an inherited disease caused by mutations in a lysosomal amidase called aspartylglucosaminidase (AGA) or glycosylasparaginase (GA). This disorder results in an accumulation of glycoasparagines in the lysosomes of virtually all cell types, with severe clinical symptoms affecting the central nervous system, skeletal abnormalities, and connective tissue lesions. GA is synthesized as a single-chain precursor that requires an intramolecular autoprocessing to form a mature amidase. Previously, we showed that a Canadian AGU mutation disrupts this obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterization of a model enzyme corresponding to a new American AGU allele, the T99K variant. Unlike other variants with known 3D structures, this T99K model enzyme still has autoprocessing capacity to generate a mature form. However, its amidase activity to digest glycoasparagines remains low, consistent with its association with AGU. We have determined a 1.5-Å-resolution structure of this new AGU model enzyme and built an enzyme-substrate complex to provide a structural basis to analyze the negative effects of the T99K point mutation on KM and kcat of the amidase. It appears that a "molecular clamp" capable of fixing local disorders at the dimer interface might be able to rescue the deficiency of this new AGU variant.

Biochemical and structural insights into an allelic variant causing the lysosomal storage disorder - aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by defects of the hydrolase glycosylasparaginase (GA). Previously, we showed that a Canadian AGU mutation disrupts an obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterizations of a model enzyme corresponding to a Finnish AGU allele, the T234I variant. Unlike the Canadian counterpart, the Finnish variant is capable of a slow autoprocessing to generate detectible hydrolyzation activity of the natural substrate of GA. We have determined a 1.6 Å-resolution structure of the Finnish AGU model and built an enzyme-substrate complex to provide a structural basis for analyzing the negative effects of the point mutation on KM and kcat of the mature enzyme. ENZYME: Glycosylasparaginase or aspartylglucosaminidase, EC3.5.1.26.

Amlexanox provides a potential therapy for nonsense mutations in the lysosomal storage disorder Aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by mutations in the gene for aspartylglucosaminidase (AGA). This enzyme participates in glycoprotein degradation in lysosomes. AGU results in progressive mental retardation, and no curative therapy is currently available. We have here characterized the consequences of AGA gene mutations in a compound heterozygous patient who exhibits a missense mutation producing a Ser72Pro substitution in one allele, and a nonsense mutation Trp168X in the other. Ser72 is not a catalytic residue, but is required for the stabilization of the active site conformation. Thus, Ser72Pro exchange impairs the autocatalytic activation of the AGA precursor, and results in a considerable reduction of the enzyme activity and in altered AGA precursor processing. Betaine, which can partially rescue the AGA activity in AGU patients carrying certain missense mutations, turned out to be ineffective in the case of Ser72Pro substitution. The Trp168X nonsense allele results in complete lack of AGA polypeptide due to nonsense-mediated decay (NMD) of the mRNA. Amlexanox, which inhibits NMD and causes a translational read-through, facilitated the synthesis of a full-length, functional AGA protein from the nonsense allele. This could be demonstrated as presence of the AGA polypeptide and increased enzyme activity upon Amlexanox treatment. Furthermore, in the Ser72Pro/Trp168X expressing cells, Amlexanox induced a synergistic increase in AGA activity and polypeptide processing due to enhanced processing of the Ser72Pro polypeptide. Our data show for the first time that Amlexanox might provide a valid therapy for AGU.

Crystal structure of a mutant glycosylasparaginase shedding light on aspartylglycosaminuria-causing mechanism as well as on hydrolysis of non-chitobiose substrate.

Glycosylasparaginase (GA) is an amidase that cleaves Asn-linked glycoproteins in lysosomes. Deficiency of this enzyme causes accumulation of glycoasparagines in lysosomes of cells, resulting in a genetic condition called aspartylglycosaminuria (AGU). To better understand the mechanism of a disease-causing mutation with a single residue change from a glycine to an aspartic acid, we generated a model mutant enzyme at the corresponding position (named G172D mutant). Here we report a 1.8Å resolution crystal structure of mature G172D mutant and analyzed the reason behind its low hydrolase activity. Comparison of mature G172D and wildtype GA models reveals that the presence of Asp 172 near the catalytic site affects substrate catabolism in mature G172D, making it less efficient in substrate processing. Also recent studies suggest that GA is capable of processing substrates that lack a chitobiose (Glycan, N-acetylchiobios, NAcGlc) moiety, by its exo-hydrolase activity. The mechanism for this type of catalysis is not yet clear. l-Aspartic acid ß-hydroxamate (ß-AHA) is a non-chitobiose substrate that is known to interact with GA. To study the underlying mechanism of non-chitobiose substrate processing, we built a GA-ß-AHA complex structure by comparing to a previously published G172D mutant precursor in complex with a ß-AHA molecule. A hydrolysis mechanism of ß-AHA by GA is proposed based on this complex model.

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.

Aspartylglycosaminuria: a review.

Aspartylglucosaminuria (AGU), a recessively inherited lysosomal storage disease, is the most common disorder of glycoprotein degradation with a high prevalence in the Finnish population. It is a lifelong condition affecting on the patient's appearance, cognition, adaptive skills, physical growth, personality, body structure, and health. An infantile growth spurt and development of macrocephalia associated to hernias and respiratory infections are the key signs to an early identification of AGU. Progressive intellectual and physical disability is the main symptom leading to death usually before the age of 50 years.The disease is caused by the deficient activity of the lysosomal enzyme glycosylasparaginase (aspartylglucosaminidase, AGA), which leads to a disorder in the degradation of glycoasparagines - aspartylglucosamine or other glycoconjugates with an aspartylglucosamine moiety at their reducing end - and accumulation of these undegraded glycoasparagines in tissues and body fluids. A single nucleotide change in the AGA gene resulting in a cysteine to serine substitution (C163S) in the AGA enzyme protein causes the deficiency of the glycosylasparaginase activity in the Finnish population. Homozygosity for the single nucleotide change causing the C163S mutation is responsible for 98% of the AGU cases in Finland simplifying the carrier detection and prenatal diagnosis of the disorder in the Finnish population. A mouse strain, which completely lacks the Aga activity has been generated through targeted disruption of the Aga gene in embryonic stem cells. These Aga-deficient mice share most of the clinical, histopathologic and biochemical characteristics of human AGU disease. Treatment of AGU mice with recombinant AGA resulted in rapid correction of the pathophysiologic characteristics of AGU in non-neuronal tissues of the animals. The accumulation of aspartylglucosamine was reduced by up to 40% in the brain tissue of the animals depending on the age of the animals and the therapeutic protocol. Enzyme replacement trials on human AGU patients have not been reported so far. Allogenic stem cell transplantation has not proved effective in curing AGU.

Identification of Small Molecule Compounds for Pharmacological Chaperone Therapy of Aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder that is caused by genetic deficiency of the enzyme aspartylglucosaminidase (AGA) which is involved in glycoprotein degradation. AGU is a progressive disorder that results in severe mental retardation in early adulthood. No curative therapy is currently available for AGU. We have here characterized the consequences of a novel AGU mutation that results in Thr122Lys exchange in AGA, and compared this mutant form to one carrying the worldwide most common AGU mutation, AGU-Fin. We show that T122K mutated AGA is expressed in normal amounts and localized in lysosomes, but exhibits low AGA activity due to impaired processing of the precursor molecule into subunits. Coexpression of T122K with wildtype AGA results in processing of the precursor into subunits, implicating that the mutation causes a local misfolding that prevents the precursor from becoming processed. Similar data were obtained for the AGU-Fin mutant polypeptide. We have here also identified small chemical compounds that function as chemical or pharmacological chaperones for the mutant AGA. Treatment of patient fibroblasts with these compounds results in increased AGA activity and processing, implicating that these substances may be suitable for chaperone mediated therapy for AGU.

Brain MRI findings in two Turkish pediatric patients with aspartylglucosaminuria.

Aspartylglucosaminuria is a rare lysosomal storage disorder that occurs as a result of a deficiency of the aspartylglucosaminidase enzyme. Because the disease is commonly referred to as the Finnish disease heritage, it is underdiagnosed outside of Finland. To date, only three Turkish patients are described in the literature. Here we describe the clinical and brain magnetic resonance imaging findings in two Turkish cousins with aspartylglucosaminuria, which can raise the suspicion of this rare disease in clinical practice.

Brain MRI findings in aspartylglucosaminuria.

BACKGROUND AND PURPOSE: The aim of this study was to identify characteristic 3.0 T brain MRI findings in patients with aspartylglucosaminuria (AGU), a rare lysosomal storage disorder. Previous AGU patient material imaged at 1.0 and 1.5 T was also re-evaluated. MATERIALS AND METHODS: Twenty-five brain MRI examinations from 20 AGU patients were included in the study. Thirteen patients underwent a prospective 3.0 T MRI (5 male, 8 female, aged 9-45 years). Twelve examinations from nine patients (4 male, 5 female, aged 8-33 years) previously imaged at 1.0 or 1.5 T were re-evaluated. Two patients were included in both the prospective and the retrospective groups. Visual analysis of the T1- and T2-weighted images was performed by two radiologists. RESULTS: The previously reported signal intensity changes in T2-weighted images were visible at all field strengths, but they were more distinct at 3.0 T than at 1.0 or 1.5 T. These included signal intensity decrease in the thalami and especially in the pulvinar nuclei, periventricular signal intensity increase and juxtacortical high signal foci. Poor differentiation between gray and white matter was found in all patients. Some degree of cerebral and/or cerebellar atrophy and mild ventricular dilatation were found in nearly all patients. This study also disclosed various unspecific findings, including a higher than normal incidence of dilated perivascular spaces, arachnoid cysts, pineal cysts and mildly dilated cavum veli interpositi. CONCLUSION: This study revealed particular brain MRI findings in AGU, which can raise the suspicion of this rare disease in clinical practice.

Structural basis of a point mutation that causes the genetic disease aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a lysosomal storage disease caused by a metabolic disorder of lysosomes to digest Asn-linked glycoproteins. The specific enzyme linked to AGU is a lysosomal hydrolase called glycosylasparaginase. Crystallographic studies revealed that a surface loop blocks the catalytic center of the mature hydrolase. Autoproteolysis is therefore required to remove this P loop and open up the hydrolase center. Nonetheless, AGU mutations result in misprocessing of their precursors and are deficient in hydrolyzing glycoasparagines. To understand the catalytic and structural consequences of AGU mutations, we have characterized two AGU models, one corresponding to a Finnish allele and the other found in a Canadian family. We also report a 2.1 Å resolution structure of the latter AGU model. The current crystallographic study provides a high-resolution structure of an AGU mutant. It reveals substantial conformation changes at the defective autocleavage site of the AGU mutant, which is trapped as an inactive precursor.

[A family with two children diagnosed with aspartylglucosaminuria-case report and literature review].

OBJECTIVE: The authors sought to investigate the clinical features and characteristics of genetic mutation in patients with aspartylglucosaminuria. METHOD: Clinical data of two pediatric siblings in a family were analyzed retrospectively and relative literature was reviewed in order to study the clinical features, imaging and enzymatic characteristics and genetic mutations. RESULT: Case 1, the proband, male, he was hospitalized at 20 months of age because of fever and hepatosplenomegaly for nine days. This child was of moderate nutritional status and normal development. Blood tests showed hemoglobin 78.0 g/L, RBC3.18 × 10¹²/L, WBC 4.06 × 109/L, neutrophils 0.236, lymphocytes 0.631, platelets 34 × 109/L, C-reactive protein 17 mg/L. Blood biochemistry showed alanine aminotransferase 67.1 U/L, aspartate aminotransferase 74.1 U/L, serum albumin 32.8 g/L, direct bilirubin 10.5 µmol/L, lactate dehydrogenase 301.7 U/L. Bone marrow cytology showed reactive morphological changes in bone marrow cells. Atypical lymphocytes could be seen in both peripheral blood and bone marrow smears. Cranial MRI showed poor myelination. Aspartylglucosaminidase activity in peripheral leucocytes of the proband 5.7 nmol/(g × min) vs. normal control>26.6 nmol/(g × min). On his AGA gene and that of his parents, a heterozygous mutation site located in exon 3, c.392C>T (p.S131L), was identified as a novel mutation inherited from his father. The mutation from his mother has not been detected. The proband was not responsive to the anti-infectious medication, nutritional intervention and symptomatic treatment.He died one month after diagnosis.His elder brother, Case 2, showed fever, recurrent respiratory tract infection and progressive psychomotor regression with hepatosplenomegaly from the age of four years. Cranial MRI revealed extensive symmetrical leukodystrophy in bilateral cerebra, cerebellum and brainstem.He died at the age of six years.Related literature was summarized, and no Chinese AGU cases had been reported; 221 foreign cases were collected. The clinical and imaging characteristics were summarized. Delay in language development was one of the clinical symptoms that the majority of parents of AGU children first noted. CONCLUSION: Patients with aspartylglucosaminuria lack of specific symptoms.For children with unexplained delayed speech and progressive mental retardation, the possibility of AGU should be considered, and efforts be made for enzymatic and genetic diagnosis. c.392C> T (p.S131L) was identified as a novel mutation of AGA gene.