Aspartylglucosaminuria Research Articles

A new horizon in the phosphorylated sites of AGA: the structural impact of C163S mutation in aspartylglucosaminuria through molecular dynamics simulation

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by insufficient aspartylglucosaminidase (AGA) activity leading to chronic neurodegeneration. We utilized the PhosphoSitePlus tool to identify the AGA protein’s phosphorylation sites. The phosphorylation was induced on the specific residue of the three-dimensional AGA protein, and the structural changes upon phosphorylation were studied via molecular dynamics simulation. Furthermore, the structural behaviour of C163S mutation and C163S mutation with adjacent phosphorylation was investigated. We have examined the structural impact of phosphorylated forms and C163S mutation in AGA. Molecular dynamics simulations (200 ns) exposed patterns of deviation, fluctuation, and change in compactness of Y178 phosphorylated AGA protein (Y178-p), T215 phosphorylated AGA protein (T215-p), T324 phosphorylated AGA protein (T324-p), C163S mutant AGA protein (C163S), and C163S mutation with Y178 phosphorylated AGA protein (C163S-Y178-p). Y178-p, T215-p, and C163S mutation demonstrated an increase in intramolecular hydrogen bonds, leading to greater compactness of the AGA forms. Principle component analysis (PCA) and Gibbs free energy of the phosphorylated/C163S mutation structures exhibit transition in motion/orientation than Wild type (WT). T215-p may be more dominant among these than the other studied phosphorylated forms. It might contribute to hydrolyzing L-asparagine functioning as an asparaginase, thereby regulating neurotransmitter activity. This study revealed structural insights into the phosphorylation of Y178, T215, and T324 in AGA protein. Additionally, it exposed the structural changes of the C163S mutation and C163S-Y178-p of AGA protein. This research will shed light on a better understanding of AGA’s phosphorylated mechanism. Communicated by Ramaswamy H. Sarma

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.

Analysis of genetic variant in a child with Aspartylglucosaminuria

To explore the genetic basis for a child with Aspartylglucosaminuria (AGU). 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. 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). 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.

Detection of Aspartylglucosaminuria Patients from Magnetic Resonance Images by a Machine-Learning-Based Approach.

Magnetic resonance (MR) imaging data can be used to develop computer-assisted diagnostic tools for neurodegenerative diseases such as aspartylglucosaminuria (AGU) and other lysosomal storage disorders. MR images contain features that are suitable for the classification and differentiation of affected individuals from healthy persons. Here, comparisons were made between MRI features extracted from different types of magnetic resonance images. Random forest classifiers were trained to classify AGU patients (n = 22) and healthy controls (n = 24) using volumetric features extracted from T1-weighted MR images, the zone variance of gray level size zone matrix (GLSZM) calculated from magnitude susceptibility-weighted MR images, and the caudate-thalamus intensity ratio computed from T2-weighted MR images. The leave-one-out cross-validation and area under the receiver operating characteristic curve were used to compare different models. The left-right-averaged, normalized volumes of the 25 nuclei of the thalamus and the zone variance of the thalamus demonstrated equal and excellent performance as classifier features for binary organization between AGU patients and healthy controls. Our findings show that texture-based features of susceptibility-weighted images and thalamic volumes can differentiate AGU patients from healthy controls with a very low error rate.

A cross-sectional natural history study of aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a rare lysosomal storage disorder that causes stagnation of development in adolescence and neurodegeneration in early adulthood. Precision therapies, including gene transfer therapy, are in development with a goal of taking advantage of the slow clinical course. Understanding of disease natural history and identification of disease‐relevant biomarkers are important steps in clinical trial readiness. We describe the clinical features of a diverse population of patients with AGU, including potential imaging and electrophysiological biomarkers. This is a single‐center, cross‐sectional study of the clinical, neuropsychological, electrophysiological, and imaging characteristics of AGU. A comprehensive assessment of eight participants (5 Non‐Finnish) revealed a mean non‐verbal IQ (NVIQ) of 70.25 ± 10.33 which decreased with age (rs = −0.85, p = 0.008). All participants demonstrated deficits in communication and gross/fine motor dysfunction. Auditory and visual evoked potentials demonstrated abnormalities in one or both modalities in 7 of 8 subjects, suggesting sensory pathway dysfunction. Brain imaging demonstrated T2 FLAIR hypointensity in the pulvinar nuclei and cerebral atrophy, as previously shown in the Finnish AGU population. Magnetic resonance spectroscopy (MRS) showed a 5.1 ppm peak corresponding to the toxic substrate (GlcNAc‐Asn), which accumulates in AGU. Our results showed there was no significant difference between Finnish and Non‐Finnish patients, and performance on standardized cognitive and motor testing was similar to prior studies. Age‐related changes on functional assessments and disease‐relevant abnormalities on surrogate biomarkers, such as MRS, could be used as outcome measures in a clinical trial.

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.

Statistical Permutation Test Reveals Progressive and Region-Specific Iron Accumulation in the Thalami of Children with Aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a rare lysosomal storage disorder causing developmental delay, intellectual disability, and eventual death. A distinct feature in AGU is iron accumulation within the thalamus. Our aim is to demonstrate that susceptibility-weighted images (SWI) could be used as an MRI biomarker to evaluate the response within the AGU population to newly evolving treatments. SWI from 16 patients with AGU and 16 age-matched controls were used in the analysis. Thalamic volume with an iron accumulation was identified using a permutation test. Group differences were investigated for both the complete thalamus and the iron accumulation regions. Group-wise age correlation within these volumes were assessed with analysis of variance and multivariate regression. We found a statistically significant and large difference (p-value = 0.01, Cohen’s D = 0.97) for the whole thalamus comparison and an even greater difference in the iron accumulation regions (p-value < 0.01, Cohen’s D = 3.52). Furthermore, we found strong evidence for iron accumulation as a linear function of age with R2 = 0.65 only for AGU. The statistical analysis of SWI provides tools for assessing the degree of iron accumulation. This method could be used to study the response to treatments, in that a successful treatment would be expected to result in a decline in iron accumulation.

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.

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.

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.

Functional Analysis of the Ser149/Thr149 Variants of Human Aspartylglucosaminidase and Optimization of the Coding Sequence for Protein Production.

Aspartylglucosaminidase (AGA) is a lysosomal hydrolase that participates in the breakdown of glycoproteins. Defects in the AGA gene result in a lysosomal storage disorder, aspartylglucosaminuria (AGU), that manifests mainly as progressive mental retardation. A number of AGU missense mutations have been identified that result in reduced AGA activity. Human variants that contain either Ser or Thr in position 149 have been described, but it is unknown if this affects AGA processing or activity. Here, we have directly compared the Ser149/Thr149 variants of AGA and show that they do not differ in terms of relative specific activity or processing. Therefore, Thr149 AGA, which is the rare variant, can be considered as a neutral or benign variant. Furthermore, we have here produced codon-optimized versions of these two variants and show that they are expressed at significantly higher levels than AGA with the natural codon-usage. Since optimal AGA expression is of vital importance for both gene therapy and enzyme replacement, our data suggest that use of codon-optimized AGA may be beneficial for these therapy options.

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.

Brain MRI findings in aspartylglucosaminuria

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. 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. 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. This study revealed particular brain MRI findings in AGU, which can raise the suspicion of this rare disease in clinical practice.

Successful palliative valvuloplasty for critical aortic valve stenosis in aspartylglucosaminuria (AGU) syndrome

Successful palliative valvuloplasty for critical aortic valve stenosis in aspartylglucosaminuria (AGU) syndrome

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.

Oligosaccharide Analysis in Urine by MALDI-TOF Mass Spectrometry for the Diagnosis of Lysosomal Storage Diseases

There are 45 known genetic diseases that impair the lysosomal degradation of macromolecules. The loss of a single lysosomal hydrolase leads to the accumulation of its undegraded substrates in tissues and increases of related glycoconjugates in urine, some of which can be detected by screening of free oligosaccharides (FOS) in urine. Traditional 1-dimensional TLC for urine oligosaccharide analysis has limited analytical specificity and sensitivity. We developed fast and robust urinary FOS and glycoaminoacid analyses by MALDI-time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry for the diagnosis of oligosaccharidoses and other lysosomal storage diseases. The FOS in urine equivalent to 0.09 mg creatinine were purified through sequential passage over a Sep-Pak C18 column and a carbograph column and were then permethylated. MALDI-TOF/TOF was used to analyze the permethylated FOS. We studied urine samples from individuals in 7 different age groups ranging from 0-1 months to ≥ 17 years as well as urine from known patients with different lysosomal storage diseases. We identified diagnostic urinary FOS patterns for α-mannosidosis, galactosialidosis, mucolipidosis type II/III, sialidosis, α-fucosidosis, aspartylglucosaminuria (AGU), Pompe disease, Gaucher disease, and GM1 and GM2 gangliosidosis. Interestingly, the increase in urinary FOS characteristic of lysosomal storage diseases relative to normal FOS appeared to correlate with the disease severity. The analysis of urinary FOS by MALDI-TOF/TOF is a powerful tool for first-tier screening of oligosaccharidoses and lysosomal storage diseases.

In Memoriam

Leena Peltonen-Palotie 1952–2010 Professor Leena Peltonen-Palotie, a leading human geneticist and former editor of HEREDITAS, died 11 March 2010 in Helsinki of osteosarcoma. She used her maiden name Peltonen in international contexts while she was known as Leena Palotie in Finland. Leena Peltonen studied medicine and genetics at the University of Oulu. After having obtained her PhD in Oulu in 1978 she worked as a postdoctoral fellow at the Dept of Biochemistry of Rutgers University in New Jersey. From 1981 to 1984 she held research positions at the Dept of Cell Biology in Oulu. After that she was a professor both at the National Public Health Institute of Finland and the University of Helsinki. In addition, from 1995 through 1998 she was Chair and Professor at the Dept of Human Genetics at UCLA, Los Angeles, and later Visiting professor at the Broad Institute, MIT, Boston as well as Head of the Wellcome Trust Sanger Institute near Cambridge. Dr. Peltonen and her colleagues specialized in locating and sequencing genes responsible or associated with human diseases. In grand total they isolated and characterized 15 loci for arterial hypertension, multiple sclerosis, arthrosis, lactose intolerance, schizophrenia, Marfan syndrome and several diseases such as aspartylglucosaminuria (AGU) of the Finnish Disease Heritage. Finland, northern Sweden and parts of Norway each have a characteristic set of inherited diseases that are rare in other parts of the world while otherwise common diseases may be extremely rare. Such an uneven distribution is attributable to founder effects, Wahlund effect, and in the case of Finland, at least one prehistoric bottleneck. The church records of the Kingdom of Sweden are a unique resource that helps locating the ancestry of a proband in Finland and Sweden back in time to the years 1600 and 1640. Leena Peltonen published about 500 research papers, some 80 review articles and supervised 80 doctoral theses. She was awarded both national and international recognition. Her merits include the membership of the Finnish Academy of Sciences, she was an Academician of the Academy of Finland, President of the European Society of Human Genetics, President of HUGO, Honorary Doctor of the University of Uppsala and recipient of Stora Fernströmpriset of the Eric K. Fernström Foundation. The Finnish Postal Service issued a stamp in her honor. It came out three days before her death. HEREDITAS remembers Leena Peltonen as an editor who was always helpful and responded to any inquiries immediately.

Sleep‐related hypermotor seizures in aspartylglucosaminuria: A case report

From the age of 24 years a young man with a definitive diagnosis of aspartylglucosaminuria (AGU) presented short-lasting sleep-related paroxysmal events characterized by sudden awakening with a frightened look, hyperventilation, and complex bilateral motor activity. Nocturnal video-polysomnography recorded several events consistent with a diagnosis of hypermotor epileptic seizures. This pattern of sleep-related epileptic seizures has been reported in rare familial cases, more often in cryptogenic and symptomatic patients in the spectrum of nocturnal frontal lobe epilepsy. Epilepsy and sleep-related nonepileptic problems are common in patients with AGU, but no case of hyperkinetic nocturnal frontal lobe seizures has been reported to date. Differential diagnosis of abnormal paroxysmal motor events in sleep is frequently a challenge for the clinician: Video-polysomnographic recordings might serve to identify the possible epileptic origin of some of the excessive motor activities during sleep referred in patients with AGU.