Urine N-Acetylaspartate Distinguishes Phenotypes in Canavan Disease

Abstract

Canavan disease (CD) is an ultra-rare autosomal recessive leukodystrophy caused by loss-of-function mutations in ASPA, which encodes aspartoacylase (ASPA), leading to accumulation of N-acetylaspartate (NAA). Patients with CD typically present with profound psychomotor deficits within the first 6 months of life and meet few motor milestones. Within CD a subset of patients exhibits a milder phenotype with more milestone acquisition, possibly related to greater residual ASPA activity. An ongoing CD natural history study and a literature search were leveraged to compare urine NAA levels and associated genotypes in patients classified with mild or typical CD, with the hypothesis that urine NAA levels reflect ASPA activity and therefore can distinguish between the two phenotypes. Urine NAA levels were lower, on average (p < 0.0001), in individuals with mild (mean 525.3, range 25.2-1,335 mmol/mol creatinine [Cr]) compared with typical CD (mean 1,369, range 391.7-2,420 mmol/mol Cr). Mutations R71H and Y288C, variants that may harbor residual ASPA activity, were unique to the mild phenotype population (56%, 14/25) and not found in individuals with a typical phenotype (0%, 0/39). In aggregate, urine NAA levels can distinguish between mild and typical CD phenotypes, suggesting the ability to reflect ASPA activity.

Keywords: Canavan disease; N-acetylaspartic acid (NAA); aspartoacylase (ASPA); biomarker; genotype; phenotype.

Figure 1.

Urine NAA levels are different between mild and typical CD phenotypes. (A) Comparison of urine NAA levels utilizing all data (literature, retrospective, and prospective Natural History) demonstrates a significant difference (p < 0.0001) between mild (n = 20, mean 525.3 mmol/mol Cr, range 25.2–1,335 mmol/mol Cr) and typical phenotypes (n = 39, mean 1,369 mmol/mol Cr, range 391.7–2,420). (B) Restricting the comparison of urine NAA levels to results run only at the central laboratory still showed a significant difference (p = 0.0001) between phenotypes with mild (n = 6) having a mean of 580.4 mmol/mol Cr (range 249.5–958.4 mmol/mol Cr) and typical phenotypes (n = 24) having a mean of 1,412 mmol/mol Cr (range 426.2–2,209 mmol/mol Cr). (C) Restricting the comparison of urine NAA levels to only literature results showed a significant difference (p = 0.0074) between phenotypes with mild (n = 14) having a mean of 487.3 mmol/mol Cr (range 25.2–1,335 mmol/mol Cr) and typical phenotypes (n = 7) having a mean of 1,300 mmol/mol Cr (range 391.7–2420 mmol/mol Cr). CD, Canavan disease; Cr, creatinine; NAA, N-acetylaspartate.

Figure 2.

Scatter plot of urine NAA versus age. All urine NAA results were plotted vs age with phenotype denoted by filled black circles (typical) or filled gray circles (mild). Literature results are differentiated from Natural History results by having a colored outline to the circle, red for a literature result with a typical phenotype, and green for a literature result from a mild phenotype. Note that some individuals have multiple results represented on this plot, for longitudinal representation of each individual see Supplementary Figure S1. The red dashed line at 1,062 mmol/mol Cr represents the 10th percentile of all typical (Literature + Natural History) results from individuals between the ages of 0–8 years. Most urine NAA results from individuals classified with a mild phenotype fall below this 10th percentile line, while most individuals with a typical phenotype aged 0–8 years are above this line.

Figure 3.

Age effect on urine NAA levels. Urine NAA results only from natural history participants with a typical phenotype whose sample was run at the central laboratory were used in this analysis. There is no statistical difference between the means of the selected age groups until age 8 years or older. The average from the 8 years and up group were lower, (p = 0.009) than the 0–1-year age category.

Figure 4.

Age restricted to 0–8 years urine NAA comparison between mild and typical phenotypes. (A) Comparison of urine NAA levels utilizing all data and age restricted to 0–8 years demonstrates a significant difference (p < 0.0001) between mild (n = 17, mean 569 mmol/mol Cr, range 25.2–1,335 mmol/mol Cr) and typical phenotypes (n = 35, mean 1,434 mmol/mol Cr, range 391.7–2,420). (B) Restricting the comparison of urine NAA levels to results only run at the central laboratory still showed a significant difference (p = 0.0002) between phenotypes with mild (n = 4) having a mean of 699.4 mmol/mol Cr (range 572.1–958.4 mmol/mol Cr) and typical phenotypes (n = 20) having a mean of 1,536 mmol/mol Cr (range 1,076–2,209 mmol/mol Cr). (C) Restricting the comparison of urine NAA levels to only the literature results also show a significant difference (p = 0.0098) between phenotypes with mild (n = 12) having a mean of 507.4 mmol/mol Cr (range 25.2–1,335 mmol/mol Cr) and typical phenotypes (n = 7) having a mean of 1,300 mmol/mol Cr (range 391.7–2,420 mmol/mol Cr).

Figure 5.

Intraindividual urine NAA variability over time. (A) Scatter plot of natural history participants, classified with a typical phenotype, with more than 1 measurement to show the variability of changes per individual. (B) Bar graph showing the percent change ranged from −37.8% to 31.5% with a mean change of −0.18%.