Assessment of urinary 6-oxo-pipecolic acid as a biomarker for ALDH7A1 deficiency

Abstract

ALDH7A1 deficiency is an epileptic encephalopathy whose seizures respond to treatment with supraphysiological doses of pyridoxine. It arises as a result of damaging variants in ALDH7A1, a gene in the lysine catabolism pathway. α-Aminoadipic semialdehyde (α-AASA) and Δ¹-piperideine-6-carboxylate (P6C), which accumulate because of the block in the lysine pathway, are diagnostic biomarkers for this disorder. Recently, it has been reported that 6-oxo-pipecolic acid (6-oxo-PIP) also accumulates in the urine, CSF and plasma of ALDH7A1-deficient individuals and that, given its improved stability, it may be a more suitable biomarker for this disorder. This study measured 6-oxo-PIP in urine from a cohort of 30 patients where α-AASA was elevated and showed that it was above the normal range in all those above 6 months of age. However, 6-oxo-PIP levels were within the normal range in 33% of the patients below 6 months of age. Levels increased with age and correlated with a decrease in α-AASA levels. Longitudinal analysis of urine samples from ALDH7A1-deficient patients who were on a lysine restricted diet whilst receiving supraphysiological doses of pyridoxine showed that levels of 6-oxo-PIP remained elevated whilst α-AASA decreased. Similar to α-AASA, we found that elevated urinary excretion of 6-oxo-PIP can also occur in individuals with molybdenum cofactor deficiency. This study demonstrates that urinary 6-oxo-PIP may not be a suitable biomarker for ALDH7A1 deficiency in neonates. However, further studies are needed to understand the biochemistry leading to its accumulation and its potential long-term side effects.

Keywords: 6‐oxo‐pipecolic acid; ALDH7A1 deficiency; aminoadipic semialdehyde; piperideine‐6‐carboxylate; pyridoxine‐dependent epilepsy.

FIGURE 1

The lysine oxidation pathway. Biomarkers of ALDH7A1 deficiency are highlighted with a red arrow. 6‐oxo‐PIP and its enol form are formed by the oxidation of 6‐OH‐PIP, an α‐AASA and Δ¹‐P6C intermediate. PIP, pipecolate; Δ¹‐P6C, Δ¹‐piperideine‐6‐carboxylate; 6‐OH‐PIP, 6‐hydroxy‐pipecolate; 2‐OPP, 2S,6S‐/2S,6R‐oxopropylpiperidine‐2‐carboxylic acid; 6‐oxo‐PIP, 6‐oxo‐pipecolate; Δ¹‐P2C, Δ¹‐piperideine‐2‐carboxylate; P2H2C, piperidine‐2‐hydroxy‐2‐carboxylate; HACA, 6‐hydroxy‐2‐aminocaproic acid; α‐AASA, aminoadipic semialdehyde; AAA, aminoadipic acid; AASS, aminoadipic semialdehyde synthase; AADAT, aminoadipate aminotransferase; CRYM/KR, μ‐crystallin/ketimine reductase; DHTKD1, 2‐oxoadipate dehydrogenase complex component E1; P5CR, pyrroline‐5‐carboxylate reductase 1; PIPOX, pipecolic acid and sarcosine oxidase. Asterisk indicates a speculative step. Dashed arrow indicates a multi‐enzyme step.

FIGURE 2

Urine α‐AASA and 6‐oxo‐PIP control ranges. Urine was from a cohort of 80 individuals with seizures of unknown origin but with α‐AASA concentrations within the normal range. Samples were grouped based on established age‐related urine α‐AASA control ranges (mmol/mol creatinine): <4, under 6 months; <2.5, 6–12 months; <2, above 12 months of age. Urine α‐AASA levels were significantly higher in individuals under 6 months of age. Urine 6‐oxo‐PIP control levels were not significantly different based on age. Thus, a control range was established based on the 97.5th centile (0–3.2 mmol/mol creatinine). Seven control samples with 6‐oxo‐PIP levels above the control range were determined as outliers (open symbols). P‐values: **<0.01; ****<0.0001; ns, not significant.

FIGURE 3

6‐oxo‐PIP levels in urine samples shown to have elevated α‐AASA levels. Dotted line, 6‐oxo‐PIP upper control limit; dashed line, α‐AASA upper control limit below 6 months of age; solid line, α‐AASA upper control limit above 12 months of age; black bar, median. α‐AASA control range for 6–12 months of age = 2.5 mmol/mol creatinine. P‐values: **<0.01; ****<0.0001.

FIGURE 4

Age‐related changes of 6‐oxo‐PIP concentrations in samples from patients with elevated α‐AASA concentrations. Open symbols, patients confirmed as having biallelic ALDH7A1 variants. Dashed line, α‐AASA control range <6 months old; dotted line, α‐AASA control range >12 months old; solid line, 6‐oxo‐PIP upper limit of control range for all age groups. The genetically confirmed patients will have been on pyridoxine supplementation and in some instances lysine restriction while for the others the treatment regime is unknown.

FIGURE 5

Longitudinal analysis of urinary α‐AASA and 6‐oxo‐PIP for ALDH7A1‐deficient patients. The diagnosis of all of these patients was confirmed genetically. All patients had been on pyridoxine supplementation and a lysine restricted diet as described in Table 2.

FIGURE 6

Urine α‐AASA and 6‐oxo‐PIP levels in MoCD‐A/B and SOXD patients. Dotted lines, α‐AASA control upper limits for <6 and >12 months old (4 and 2 mmol/mol creatinine, respectively); Dashed line, 6‐oxo‐PIP control upper limit.