Maple syrup urine disease
Jan. 08, 2023
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Canavan disease is a leukodystrophy that causes progressive degeneration, resulting in a spongy deterioration of the brain. The disease is due to mutations in the ASPA gene (OMIM *608034), which encodes aspartoacylase, an enzyme that catalyzes the conversion of N-acetylaspartic acid to aspartate and acetate. In this article, the authors present the history of Canavan disease, current understanding of the pathophysiology, diagnostic work-up, and new therapies that are being developed to treat this rare and devastating condition.
• Aspartoacylase deficiency in Canavan disease leads to spongy degeneration of the white matter.
• Aspartoacylase deficiency leads to increased N-acetylaspartic acid in blood, CSF, brain tissue, and urine.
• Diagnosis of Canavan disease can be achieved through imaging, biochemistry, and molecular assay.
• Brain magnetic resonance imaging (MRI) findings in patients with Canavan disease show early involvement of the subcortical U-fibers. Magnetic resonance spectroscopy (MRS) shows an elevated N-acetylaspartate peak.
• Carrier screening is recommended for people of Ashkenazi Jewish descent.
• New therapies, including new gene therapies, are currently being investigated for Canavan disease.
Canavan disease, or Canavan-Van Bogaert-Bertrand disease, was first described in 1931 by Myrtelle Canavan (13). The description by Canavan subsequently dominated the American medical literature when spongy degeneration of the brain came to be recognized as a specific entity. A detailed report of three Jewish children with spongy degeneration of the brain was published in 1949 and is widely quoted in the European literature (59). Since then, numerous cases of Canavan disease have been reported, and Canavan disease has been shown to have a higher prevalence in the Ashkenazi Jewish population (06).
The enzyme responsible for Canavan disease, aspartoacylase, was identified by Matalon and colleagues in 1988 (41). The gene was cloned in 1993 (29). As a result of these advancements, mutations can now be identified in patients, family members, and at-risk populations (29; 39). Additionally, the understanding that deficiency of aspartoacylase leads to increased concentration of N-acetylaspartic acid in CSF, serum, and urine has enabled the diagnosis of Canavan disease through biochemical methods.
• Two subtypes of Canavan disease have been described based on age of onset: infantile and juvenile.
Infantile form. The infantile form of Canavan disease is the more common form and is characterized by the triad of developmental delay, hypotonia, and acquired macrocephaly. Babies born with Canavan disease do not have distinctive clinical features in their first few months of life. However, delayed development is often first noted at about 3 months of age. Likewise, in early infancy, the head circumference and brain may not be remarkably increased and may be at the upper limit of normal in some cases. However, in the majority of cases, head circumference and brain size will dramatically increase after the age of 6 months and is usually above the 90th percentile by 1 year. In these infants, MRI often shows diffuse cerebral white matter degeneration with early involvement of the subcortical U-fibers. T2 hyperintensities in the cerebellum, periventricular white matter, basal ganglia, thalami, and brainstem can also be seen (62; 53). As the infant grows older, developmental milestones are not achieved. This includes developmental delays in gross motor, fine motor, and language development. As the disease progresses, developmental regression occurs. Patients with Canavan disease also develop nystagmus, poor visual tracking, pronounced startle reflex, feeding difficulties, sleep disturbances, and epilepsy (11). Although possible in the first year of life, seizures and optic atrophy usually develop in the second year of life (39; 11).
In order to better characterize the phenotype of Canavan disease, a new symptom scoring system, known as the Canavan disease severity score, has been proposed (11). The new scoring system is 22 points and is based on 11 clinical features, including epileptic seizures, truncal hypotonia, appendicular hypertonia/spasticity, macrocephaly, difficulties with feeding, difficulties with language, the lack of a responsive social smile, difficulties with visual track, difficulties with head control, difficulty with reaching for objects, and difficulties in sitting without support. In the new scoring system, a 0 is assigned if no deficit is present, a 1 is assigned if there is a mild deficit, and a 2 is assigned if there is a constant or pronounced deficit. The new scoring system can help keep track of disease progression and may be useful to determine the efficacy of new therapies that are developed.
Juvenile form. In contrast to the infantile form of Canavan disease, the juvenile form tends to have a much milder phenotype (43). In the juvenile form of Canavan disease, macrocephaly and spongiform changes on MRI may not develop. However, juvenile-onset cases with macrocephaly, seizures, and retinitis pigmentosa have been reported (56; 57; 08). Patients with the juvenile form of Canavan disease tend to present in early childhood as opposed to infancy and tend to have mild, if any, developmental delays without regression (38). MRI abnormalities that have been seen in the juvenile cases have included mild T2-weighted and FLAIR hyperintensity changes in the arcuate fibers, striatum, dentate nuclei, and the pons (50; 12).
The prognosis for infantile Canavan disease is poor but improving. Previously, most children with the infantile form of Canavan disease succumbed to the disease by the age of 3 years. A new natural history study revealed that 73% of patients are now surviving at 10-year follow-up (11). With age, contractures and spasticity develop and resemble symptoms of severe cerebral palsy (40). Due to problems with feeding, including dysphagia and gastroesophageal reflux, as well as poor weight gain, some children with Canavan disease require nasogastric feeding or a gastrostomy tube. Children with the juvenile form of Canavan disease tend to have mild developmental delay and may have eye findings of optic atrophy or retinitis pigmentosa.
A white, male infant was born at term with an unremarkable pregnancy and delivery. Birth length, weight, and head circumference were all at the 50th percentile. The neonatal exam was normal. At 3 months of age, he was noted to be irritable with poor sleep, and he gagged during feedings. A diagnosis of gastroesophageal reflux was suspected, and he was treated with cisapride. At 6 months of age, he continued to be irritable, and delayed milestones were noted. He could raise his head when prone but could not maintain head control when in an upright position. There was marked hypotonia noted on examination, and he was referred for a neurologic consultation. Because of developmental delays, hypotonia, and an increase of head circumference to the 90th percentile, MRI of the brain was performed. The MRI showed delayed maturation of the white matter. Perinatal injury was suggested as a possible etiology. At 14 months of age, he still could not sit, stand, or support his head. At that time, a second brain MRI showed widespread white matter T2 hyperintensity that involved U-fibers. Urine studies revealed an elevated N-acetylaspartic acid concentration of 1650 µmol/mmol creatinine (normal less than 40 µmol/mmol creatinine), and the diagnosis of Canavan disease was made.
• Aspartoacylase deficiency in Canavan disease leads to accumulation of N-acetylaspartic acid and spongy degeneration of the white matter, although the specific mechanism for this is not fully understood.
Canavan disease is an autosomal recessive disorder due to aspartoacylase deficiency that results from pathogenic variants affecting the aspartoacylase gene (ASPA) located on chromosome 17 (41). Histologically, it is characterized by spongiform vacuolization, which is seen throughout the subcortical white matter in both the cerebrum and cerebellum. The spongiform vacuolization seen in histological sections is due to vacuole formation within individual astrocytes and between myelin lamellae (02; 01).
Pathogenic variants in ASPA in patients with Canavan disease cause decreased aspartoacylase activity. Disease severity is inversely proportional to the amount of residual enzyme activity. The most severe forms of Canavan disease have less than 1% of normal enzyme activity (65; 43).
Although the enzyme defect is known, much about the pathogenesis is still unclear. Mouse models of the disease have provided evidence that the pathogenesis of Canavan disease involves steps in synthesis, transport, and breakdown of N-acetylaspartic acid. N-acetylaspartic acid is the second most common amino acid in the brain (https://pubs.acs.org/doi/pdf/10.1021/acschemneuro.1c00455). It is made by the enzyme aspartate N-acetyltransferase (ANAT) in humans, which is encoded by the N-acetyltransferase-8 like (NAT8l) gene. After its synthesis, primarily in the cortex, N-acetylaspartic acid is taken up by astrocytes through transporters, including SLC13A3, for subsequent transport to oligodendrocytes via astrocyte-oligodendrocyte contacts (47). In oligodendrocytes, N-acetylaspartic acid is broken down into L-aspartate and acetate by aspartoacylase. There are three leading hypotheses regarding the pathophysiology of Canavan disease: the acyl-myelin theory, the N-acetylaspartic acid toxicity theory, and the molecular water pump theory.
The acyl-myelin theory is centered on the idea that oligodendrocytes deficient in aspartoacylase are unable to form myelin. This deficiency prevents oligodendrocytes from forming acetate. The inability to derive acetate from N-acetylaspartic acid interferes with myelin production through two mechanisms. First, acetate serves as a precursor molecule for myelin lipid production, and second, acetyl-CoA derived from acetate is a substrate for ATP formation through the citric acid cycle (47; 36). It has also been found that the acetate generated from the hydrolysis of N-acetylaspartic acid is used in other ways, such as epigenetic regulation of gene expression, including acetylation of histones (49). Acetate deficiency likely causes myelin pathology through a combination of these mechanisms.
The N-acetylaspartic acid toxicity theory is based on the idea that elevated levels of N-acetylaspartic acid are toxic to astrocytes and oligodendrocytes. According to this theory of pathogenesis, astrocytes take up N-acetylaspartic acid through SLC13A3 before transporting it to oligodendrocytes. Elevated N-acetylaspartic acid in both astrocytes and oligodendrocytes can lead to oxidative stress and vacuolization (45). N-acetylaspartic acid also binds to and activates NMDA receptors (32). It is speculated that the activation of NMDA receptors by N-acetylaspartic acid could lead to the seizures that are seen in Canavan disease (36). Previous studies have also shown that aspartoacylase-deficient mice can be rescued from developing spongiform degeneration by homozygous knockout of Nat8l (the enzyme that forms N-acetylaspartic acid). Lastly, in cultured stomach cells, N-acetylaspartic acid can activate NF kappa B activity and MAP kinases, which leads to the expression of inducible nitric oxide synthase and cell death (54; 55).
The molecular water pump theory is based on the idea that N-acetylaspartic acid serves as an osmolyte in the brain. In a study in which the dorsal lateral striatum of rats was exposed to increasing hypoosmotic media, the concentration of N-acetylaspartic acid increased in the extracellular matrix, implying that N-acetylaspartic acid may be protective against hyposmolar damage at physiologic levels (58). The osmotic activity of N-acetylaspartic acid may require a delicate balance, such that excess intracellular N-acetylaspartic acid could lead to vacuole formation within cells such as astrocytes, and excess extracellular N-acetylaspartic acid between oligodendrocyte lamellae could lead to splitting of the myelin lamellae and extracellular vacuolization.
None of these theories are mutually exclusive, and they may all contribute to the pathology seen in Canavan disease.
• Canavan disease occurs in people of all ethnicities; however, it is most prevalent among those of Ashkenazi Jewish descent.
Canavan disease is panethnic. It is most prevalent among Ashkenazi Jews of Eastern European descent; however, there is increasing recognition that it may be more prevalent in other ethnic groups than previously thought (11). In the Ashkenazi Jewish population, two specific mutations predominate. A missense mutation on codon 285 with substitution of glutamic acid to alanine (p.Glu285Ala) accounts for 84% of pathogenic variants identified in a large number of Jewish patients, and a nonsense mutation, tyrosine to termination, on codon 231 (p.Tyr231X) for 13.4% (37). Together these two mutations account for over 97% of the alleles in Jewish Canavan patients.
Study of blood specimens from over 5000 healthy Ashkenazi Jewish people indicated that one out of 37.7 were carriers for the two common mutations (42). Carrier frequency for these two mutations in a Jewish population was 1:82 in Israel and 1:57 in Canada (18; 17). These frequencies depend on the population tested and their countries of origin. Ashkenazi Jewish people from Ukraine or Lithuania are probably the main founders of the Jewish mutations.
In non-Jewish patients, the mutations are more varied. The most common Canavan mutation in non-Jewish patients is in codon 305, a missense mutation that substitutes alanine with glutamic acid (p.Ala305Glu) (30; 40). Molecular characterization of Canavan disease in the Indian subcontinent has revealed two novel mutations (p.Ile243Ser and p.Leu301Pro) and two known mutations that were subsequently used for prenatal diagnosis (09).
Patients with juvenile or mild forms of Canavan disease are often compound heterozygotes, with a mild mutation on one allele and a severe mutation on the other allele. Mild mutations include p.Tyr288Cys, p.Phe295Ser, p.Lys213Glu, p.Arg305Glu, and p.Arg71His (56; 57; 62; 27).
• Prevention can be achieved through carrier testing and preimplantation genetic diagnosis.
The risk of an affected baby is 1:4 if both parents are carriers of the disease. Carrier determination and preventive counseling can now be attained using DNA analysis. The high carrier rate observed in Ashkenazi Jews warrants screening of this population similar to screening programs for Tay-Sachs disease. The Committee on Genetics of the American College of Obstetrician Gynecologists and the American College of Medical Genetics and Genomics recommends genetic screening of Canavan disease in Jewish couples (04; 22). Prenatal diagnosis can be made through DNA analysis, determination of N-acetylaspartic acid levels in the amniotic fluid, or preimplantation genetics (16). Preimplantation genetics allows identification and implantation of an embryo that does not have an allele known to cause Canavan disease (63).
Macrocephaly and developmental delay, which are characteristics of Canavan disease, can also be found in many inborn errors of metabolism and leukodystrophies, including Alexander disease, Tay-Sachs disease, and megalencephalic leukoencephalopathy with subcortical cysts. Autosomal dominant megalencephaly should be considered for patients with normal development, normal brain imaging, and large head size, particularly if there is a family history of macrocephaly. The involvement of subcortical U-fibers in Canavan disease aids in the diagnosis, although this can be found in Alexander disease, megalencephalic leukoencephalopathy, and Pelizaeus-Merzbacher disease (which generally involves microcephaly). MRS can also be used to help differentiate between these disorders due to its ability to detect N-acetylaspartic acid elevations. In addition to Canavan disease, spongy degeneration of the brain may be found in viral infections, mitochondrial diseases, and other metabolic diseases, such as homocystinuria. Elevation in the excretion of N-acetylaspartic acid and deficiency of aspartoacylase enzyme activity are the distinguishing features that are specific to Canavan disease.
Optic atrophy has been found in infantile Canavan disease. Retinitis pigmentosa has been found in patients with the juvenile form of Canavan disease (39; 56; 57; 08; 11).
• Imaging of the brain is the first step in the diagnostic workup.
• Elevation of N-acetylaspartic acid in blood, urine, CSF, or on MRS is a specific indicator of Canavan disease.
Imaging of the brain, via computed tomography (CT) or MRI of the brain, is useful in the diagnostic work-up of Canavan disease. CT will often reveal diffuse white matter degeneration (51). MRI is the preferred modality for visualizing white matter abnormalities and can reveal diffuse white matter degeneration with early involvement of the subcortical U-fibers and variable T2 hyperintensity in the cerebellum, basal ganglia, thalami, and brainstem (62; 53). It is important to note that the initial MRI or CT may be interpreted as normal early in life. As a result, follow-up imaging is indicated if there remains a suspicion for metabolic brain disease (10). MRS shows an increased N-acetylaspartic acid peak (23; 61), even if serum and urine N-acetylaspartic acid are normal (28). Patients with mild variants of Canavan disease have mild T2-weighted and FLAIR hyperintensity changes in the arcuate fibers, striatum, dentate nuclei, and pons (50; 12).
Although imaging is helpful, biochemical and molecular tests are more specific than MRI findings and are used to diagnose Canavan disease. Patients with severe Canavan disease typically have highly elevated levels of urine N-acetylaspartic acid (1440 + 873 mmol/mmol creatinine vs. normal 23 +16 mmol/mmol creatinine), although normal or slightly elevated levels can be found in some patients. In those cases, diagnosis is made through other methods, such as molecular testing (24). Molecular testing to obtain ASPA genotype can be accomplished through single gene testing, genetic panels, or whole exome or genome sequencing. Enzyme activity can be measured in cultured skin fibroblasts but is not necessary if N-acetylaspartic acid is elevated in blood, urine, or CSF or if a pathogenic APSA mutation is identified.
Once a biochemical diagnosis of Canavan disease is reached, DNA mutation analysis of the ASPA gene should be performed on the proband as well as on family members for counseling and preventive measures (39). Prenatal and preimplantation testing is available (63).
• Management of Canavan disease is symptomatic.
• Advances in the understanding of the pathophysiologic mechanisms behind Canavan disease will aid in the discovery of new therapies.
• Clinical trials of promising therapies are underway.
There are currently no cures for Canavan disease, and therapy focuses on mitigating complications. For instance, physical therapy may help prevent contractures, and seizures are managed with antiseizure medication. In addition, special care is needed to avoid aspiration with feedings. Nasogastric or gastrostomy feeding is often necessary. In addition to the aforementioned recommendations, reviews have been published to help guide clinicians in the treatment of patients with leukodystrophies (03; 31). Once a diagnosis of Canavan disease is made, patients should receive recurrent neurologic evaluations along with developmental assessments. Due to optic atrophy and associated retinitis pigmentosa, recurrent ophthalmologic exams should also be performed. In addition, carriers should have access to genetic counseling prior to planning a pregnancy.
Therapies under investigation. Significant efforts have been made to find disease-modifying therapies as our understanding of the pathophysiology of Canavan disease has advanced. This includes the study of nutritional supplements, lithium, small molecules, enzyme replacement therapy, and cell-based therapies as well as the development of gene therapy products. Multiple knockout mouse models for Canavan disease have been created, which are useful in the preclinical evaluation of potential therapies (36).
Nutritional supplements and lithium. In the past, a clinical trial of glycerol triacetate (inspired by the acyl-myelin hypothesis) in Canavan disease did not demonstrate clinical efficacy (52). Resveratrol is thought to have therapeutic potential for Canavan disease due to its ability to upregulate the ASPA gene, which is mutated in Canavan disease (14). In addition, resveratrol, which generally plays a prominent role in metabolism and in mitochondrial function, restores fatty acid beta-oxidation capacities in the Canavan patient–derived cell line, GM04268.
In addition to resveratrol, Francis and colleagues found that in a mouse model of Canavan disease, dietary triheptanoin promoted oligodendrocyte survival, increased myelination, decreased vacuolization, and improved motor activity in the mice that were given triheptanoin compared to control mice (20). Lipoic acid may act as an antioxidant and prevent damage from N-acetylaspartic acid (45; 46). Lastly, lithium citrate given to six patients for 60 days was associated with decreased N-acetylaspartic acid in the basal ganglia, mild improvement in myelination in frontal white matter, increased myelin density in the corpus callosum, and increased levels of alertness (05).
Small molecules. Small molecules that inhibit aspartate N-acetyltransferase (ANAT)–mediated production of N-acetylaspartic acid are being investigated. Large-scale screening of 10,000 compounds identified two new potential inhibitors (44). In addition to ANAT inhibitors, inhibiting N-acetylaspartic acid transport through SLC13A3 is also being proposed as a therapeutic mechanism. Wang and colleagues showed that mice that had both the aspartylacylase and the SLC13A3 genes knocked down had normalized N-acetylaspartic acid levels in the CSF, developed less vacuolization in the brain, and had improved motor performance (60). However, it should be noted that at least two patients with SLC13A3 variants developed leukoencephalopathy after febrile illnesses (15).
Enzyme replacement. Enzyme replacement therapy is a therapeutic option that has been used in other disorders, such as Gaucher disease, and strategies to create an enzyme replacement therapy for Canavan disease are underway (35). Aspartoacylase injected into knockout mice with Canavan disease leads to increased aspartoacylase activity in the mouse brain (64; 48). Despite the success in mice, efficacy of such treatment in Canavan disease has yet to be demonstrated in clinical trials.
Cell-based therapies. Feng and colleagues transduced patient-derived pluripotent stem cells with wildtype ASPA and transformed them into induced oligodendrocyte precursor cells (iOPCs) or induced neuron precursor cells (iNPCs) that express aspartoacylase (19). When transplanted into a mouse model of Canavan disease, these transformed cells resulted in significant improvement in motor activity and decreased vacuolization and N-acetylaspartic acid levels. Such patient-specific cell-based therapies do not trigger rejection as allogenically derived stem cells can; however, they are costly and carry a risk of malignant transformation.
Gene therapy. Gene therapy for the treatment of Canavan disease is under investigation in both mouse models and humans. In mice, spongiform degeneration can be suppressed by the addition of acetyl-aspartase or the deletion of the enzyme that creates N-acetylaspartic acid (Nat8l) (33; 34; 26; 07; 25; 21).
At least four different gene therapies for the treatment of Canavan disease in humans have been developed. In the first study, the aspartoacylase gene was transferred to two children with Canavan disease using liposomes (33). Subsequently, gene therapy of Canavan disease was achieved using adeno-associated virus 2 (AAV2) as a vector for aspartoacylase in a phase 1 study, which did not identify substantial toxicity (26). The 5-year follow-up showed long-term safety, with some decrease in the elevation of N-acetylaspartic acid in brain, improvement in seizure frequency, and stabilization of overall clinical status (34). In addition to these previous studies, two open-label phase 1/phase 2 clinical trials are currently being conducted. In one trial (NCT04833907), the ASPA gene is being delivered intracerebroventricularly with AAV/Olig001, with the goal of aspartoacylase expression in oligodendrocytes. In the second trial (NCT04998396), the gene for aspartoacylase is being delivered intravenously with an AAV9 vector to facilitate aspartoacylase expression in both neuronal and non-neuronal cell types.
For Canavan disease, prenatal diagnosis was historically based on enzyme determinations in amniocytes (09). When N-acetylaspartic acid is measured in amniotic fluid, it may be increased in an affected pregnancy (16). Prenatal and preimplantation testing is available (63).
Anesthesia should be administered with caution because of the swallowing difficulties and risk of aspiration. No specific sensitivity to anesthesia in people with Canavan disease is known.
All contributors' financial relationships have been reviewed and mitigated to ensure that this and every other article is free from commercial bias.
Erika Augustine MD MS
Dr. Augustine of Kennedy Krieger Institute, Johns Hopkins University, and University of Rochester Medical Center received consulting fees from Amicus Therapeutics, Exicure, and Taysha Gene Therapies, a clinical trial agreement as Central Rater from Neurogene Inc, and an honorarium as a member of the Data Safety and Monitory Board for PTC Therapeutics.See Profile
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