Biochemical and molecular predictors for prognosis in nonketotic hyperglycinemia

Abstract

Objective: Nonketotic hyperglycinemia is a neurometabolic disorder characterized by intellectual disability, seizures, and spasticity. Patients with attenuated nonketotic hyperglycinemia make variable developmental progress. Predictive factors have not been systematically assessed.

Methods: We reviewed 124 patients stratified by developmental outcome for biochemical and molecular predictive factors. Missense mutations were expressed to quantify residual activity using a new assay.

Results: Patients with severe nonketotic hyperglycinemia required multiple anticonvulsants, whereas patients with developmental quotient (DQ) > 30 did not require anticonvulsants. Brain malformations occurred mainly in patients with severe nonketotic hyperglycinemia (71%) but rarely in patients with attenuated nonketotic hyperglycinemia (7.5%). Neonatal presentation did not correlate with outcome, but age at onset ≥ 4 months was associated with attenuated nonketotic hyperglycinemia. Cerebrospinal fluid (CSF) glycine levels and CSF:plasma glycine ratio correlated inversely with DQ; CSF glycine > 230 μM indicated severe outcome and CSF:plasma glycine ratio ≤ 0.08 predicted attenuated outcome. The glycine index correlated strongly with outcome. Molecular analysis identified 99% of mutant alleles, including 96 novel mutations. Mutations near the active cleft of the P-protein maintained stable protein levels. Presence of 1 mutation with residual activity was necessary but not sufficient for attenuated outcome; 2 such mutations conferred best outcome. Divergent outcomes for the same genotype indicate a contribution of other genetic or nongenetic factors.

Interpretation: Accurate prediction of outcome is possible in most patients. A combination of 4 factors available neonatally predicted 78% of severe and 49% of attenuated patients, and a score based on mutation severity predicted outcome with 70% sensitivity and 97% specificity.

Figure 1

Differences in antiepileptic drugs, glycine levels, and glycine index by disease category. (A) The differences between severe nonketotic hyperglycinemia (NKH), poor attenuated NKH, intermediate attenuated NKH, and mild attenuated NKH are shown for the number of antiepileptic drugs (A), the cerebrospinal fluid (CSF) glycine levels (B), the CSF:plasma glycine ratios (C), and the glycine index (D). [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]

Figure 2

Relation between cerebrospinal fluid (CSF) glycine levels and developmental quotient (DQ). There is a direct linear correlation between CSF glycine levels and DQ. The cutoff at 230 μM is shown.

Figure 3

Mutations in GLDC. Missense mutations are shown above the diagram of the exonic structure of the GLDC gene. Missense mutations with residual activity are shown in green, missense mutations without residual activity are shown in red, and mutations not expressed are shown in yellow. Frameshift mutations (gray) and splice site mutations (orange) are shown below the exonic structure. The length of observed exonic deletions is shown in red and duplications in blue. The frequency of occurrence is provided for recurring mutations.

Figure 4

Mutations in AMT. Missense mutations (yellow) are shown above the diagram of the exonic structure of the AMT gene. Frameshift mutations (gray) and splice site mutations (orange) are shown below the exonic structure.

Figure 5

Number of residual activity–conferring mutations and disease severity. (A) The frequency of developmental outcome in categories of severe, attenuated (Att) poor, attenuated intermediate, attenuated mild, or attenuated not otherwise specified (NOS; without known developmental quotient [DQ]) is shown in relation to the number of alleles with a mutation conferring no residual activity (severe mutation) or alleles with a mutation conferring residual activity (mild mutation). (B) The frequency of patients requiring a number of antiepileptic medications in relation to the number of severe or mild alleles.

Figure 6

Mutation score by disease category. The mutation score is compared between neonatal deceased, severe nonketotic hyperglycinemia (NKH), poor attenuated NKH, intermediate attenuated NKH, mild attenuated NKH, and attenuated NKH not otherwise specified (NOS). The mutation score is significantly higher in attenuated NKH than in severe or neonatal decreased NKH. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]

Figure 7

Molecular modeling of mutated amino acids on the structure of the P‐protein. The homology structure of the human P‐protein dimer modeled from Synechocystis species is shown with the amino acids of the expressed missense mutations highlighted. Amino acids involved in missense mutations without residual activity on expression study are shown in red, with <1% residual activity in blue, with 1 to 10% residual activity in yellow, and with >10% residual activity in green. The active site lysine 754 is shown in pink. Amino acids that when mutated result in a stable protein (identified in the lower part) tend to cluster around the active site fold (red circle), whereas amino acids that when mutated result in unstable protein (identified in the upper part) tend to be located away from the active site fold.

Figure 8

Western blot analysis of expressed mutations in GLDC. The missense mutations present in 2 patients were expressed in COS cells, and the level of P‐protein identified by Western blot. The amount of β‐actin protein is shown as a loading control. WT = wild type. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]