Pyridoxine-dependent epilepsy seizure (PDE; OMIM 266100) is a rare autosomal recessive disorder characterized by recurrent seizures in the prenatal, neonatal, and/or postnatal periods. Untreated patients may die, although the problem can be controlled with pyridoxine monotherapy. Most patients show mild-to-moderate postnatal developmental delay (Baxter, 2001). Prompt diagnosis is important if treatment is to prevent irreversible neurologic and cognitive damage.
The underlying enzyme defect associated with the disease was shown to be located at the level of α-aminoadipic semialdehyde (α-AASA) dehydrogenase (antiquitin protein), which is encoded by the ALDH7A1 gene. This has been mapped to 5q31. The enzyme takes part in the pipecolic acid (PA) pathway of lysine catabolism. Patients show high plasma and cerebrospinal fluid (CSF) levels of PA (which is also elevated in peroxisome biogenesis disorder and chronic liver dysfunction) as well as pathognomic urine levels of α-AASA (Mills et al., 2006).
More than 78 documented mutations have been associated with PDE. Missense mutations account for nearly 60% of the alleles, the remainder being truncation mutations, namely nonsense mutations, splicing mutations, small insertions and deletions, and gross rearrangements (Human Gene Mutation Database at the Institute of Medical Genetics, Cardiff, United Kingdom [ HGMD] professional release).
This work reports the clinical, biochemical, and mutational spectrum of 12 patients with clinically proven PDE. The mutation spectrum included 12 variant changes, 7 of which are previously undescribed. The successful antisense therapy rescue of a splicing defect produced by an exonic change is also described.
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- Patients and Methods
The onset of seizures in the present patients varied from soon after birth to the first months of life, as described by other authors (Bankier et al., 1983; Goutieres & Aicardi, 1985; Coker, 1992; Chou et al., 1995). Most patients initially showed focal or multifocal, clonic or myoclonic seizures, and generalized tonic seizures. Only one patient also had oculogyric seizures. The presence of perinatal adverse events such as hypoxic–ischemic encephalopathy may coexist with convulsions; it should not, therefore, rule out the possibility of PDE (Wolf et al., 2005) (unfortunately this was what happened with patient P4). It is remarkable that 8 of the 12 patients showed atypical transient control of their seizures for more than 15 days with conventional AEDs. This response led to a delay in the correct diagnosis, especially in those for whom α-AASA or plasma PA screening was not possible when the disease was first suspected, and in those who provided no samples for testing. The effective dose of pyridoxine to suppress seizures was variable, ranging from 10 to 30 mg/kg/day, although in most patients a dose of 15 mg/kg/day was enough.
The clinical spectrum of our PDE patients was not limited to seizures; many patients showed associated neurologic dysfunctions such as muscle tone alterations, irritability, and psychomotor retardation. These symptoms may be attributed to a delay in the onset of treatment with pyridoxine. However, in some patients, neurologic signs were already present at the onset of seizures, and persisted even with early and adequate treatment with pyridoxine. Altered neuroimaging findings, mainly of the dysgenetic type, were recorded for the majority of patients. In our series, MRI was not available in two cases, and was normal in another two, whereas eight cases showed some type of abnormality, most often in combination. The abnormalities included mega cisterna magna (five cases), corpus callosum dysgenesia (two cases), periventricular white matter abnormalities (two cases), ventriculomegaly (two cases), posterior fossa arachnoid cyst (one case), and Dandy-Walker variant (one case). In one case (P4) MRI showed findings suggestive of hypoxic–ischemic encephalopathy. In a recent report of a cohort of 14 patients with PDE (Bok et al., 2012), MRI abnormalities were present in 10 cases, most of them with corpus callosum dysplasia/hypoplasia and/or white matter abnormalities on T2 or on diffusion MRI (in the neonatal period). Taken together, the associated neurologic dysfunctions and anatomic alterations suggest that the deleterious effects of ALDH7A1 gene mutations on the central nervous system in some patients precede and go beyond the mere onset of seizures.
About 50% of the present patients had status epilepticus at some time. A significant number showed recurrent brief seizures despite pyridoxine therapy, as described by (Gospe, 2002) and (Mills et al., 2010). Although good cognitive outcome has been reported previously following prolonged status epilepticus (Kluger et al., 2008), three of the present patients who had prolonged status epilepticus developed moderate/severe mental retardation. Despite seizure control with pyridoxine, some degree of developmental delay was evident in most patients. In two, P6 and P9, chronic treatment with doses of pyridoxine higher than those necessary to suppress the seizures might have improved their intellectual development, as suggested by (Baxter, 2001). Long-term prognosis of patients with ALDH7A1 mutations in our series is limited because the follow-up period in many of them is short. However cases P6, P7, P8, P9, P10, and P12 have been followed for more than 5 years, and all of them show some degree of cognitive dysfunction and in most of them IQs are in the range of borderline to mild mental retardation. In case P10, mental retardation is severe. In a recent report of two sibs, which include long-term observation, both patients showed moderate to severe mental retardation despite an adequate and early pyridoxine administration and seizure control (Yeghiazaryan et al., 2011). Another recent publication has described the long-term neurodevelopment outcome in a cohort of 14 patients with PDE (Bok et al., 2012). In this report mental development was delayed in most cases, with a median IQ or developmental index of 72. The authors also found that a delayed initiation of treatment with pyridoxine, and corpus callosum abnormalities were associated with a most unfavorable neurodevelopmental outcome. The evolution described in these two recent reports, as well as that of our cases with a longer follow-up, suggests that treatment with vitamin B6 is effective for suppressing the seizures and normalizing the EEG, but that it is not so effective to ensure normal neurodevelopment.
Despite the limited number of patients in the present series, urine levels of α-AASA and plasma/CSF levels of PA appear to serve as biomarkers of PDE, even in older patients who have undergone several years of pyridoxine treatment and who have normal or high levels of plasma PLP. It is worth noting that most of the present patients analyzed before pyridoxine treatment had low levels of plasma PLP, unlike those described previously (Shin et al., 1984), showing this metabolite to be an adjuvant marker for treatment monitoring. In addition, irrespective of treatment status, monoamine neurotransmitter analysis by HPLC detected peak X in the CSF. The identity of this pathognomonic compound is yet to be determined, although it seems that it is neither α-AASA nor the Δ1-piperidine-6-carboxylate (P6C)-PLP complexation product (Stockler et al., 2011). The permanent presence of small accumulations of PA and compounds of yet unknown identity (peak X) in the CSF, as well as of α-AASA (a highly reactive and probably toxic compound) in blood and urine, may be related to brain dysfunction; despite their seizures being controlled by pyridoxine treatment, most of the present patients had intellectual disability. The restriction of lysine in the diet may contribute to the normalization of α-AASA levels, and thereby improve the general outcome of patients.
The mutational spectrum of the 11 Spanish patients was different from those reported for other populations. Indeed, the common mutation p.Glu399Gln (Plecko et al., 2007; Salomons et al., 2007; Bennett et al., 2009; Mills et al., 2010) was absent. Private mutations affected only one family, and only one change (p.Gly477Arg) was identified in four different alleles, highlighting the genetic heterogeneity of the disease in this population. The list of mutations reported expands the mutational database of the disease and shows the importance of genetically analyzing specific populations for use as an interpretative aid in diagnostic laboratories.
The novel nucleotide changes identified in this study are probably pathogenic mutations. Variations affecting the splicing process (c.75C>T and c.787 + 1G>T), the two nonsense mutations (c. 319G>T and c.757C>T), the small deletion (c. 554_555delAA), and the large deletion (c.1093-? _1620+?) predictably result in early truncation of the protein; indeed, they are loss-of-function mutations. The pathogenicity of the new missense change p.Ser492Pro is based on circumstantial evidence provided by the evolutionary conservation of the amino acid involved, and by its prediction as “damaging” by the Polyphen computational prediction algorithm (genetics.bwh.harvard.edu/pph2).
The specific transcriptional profile analysis performed in cells of patients allowed three different nucleotide changes affecting the splicing process to be identified, one of them in the coding sequence of the gene. The effects of c.787 + 1G>T and c.1482-1G>T are severe. mRNA analysis combined with ex vivo minigene expression analysis showed the exonic change c.75C>T to create a new splice site. This shows that exonic changes may also affect the splicing process, as described for another disease-causing mutation, c.750G>A, in this gene (Salomons et al., 2007). These two changes, c.75C>T and c.750G>A, could easily be wrongly classified as silent changes, highlighting the importance of mutation-detection techniques based on the combination of RNA and DNA analysis. In addition, to demonstrate that c.75C>T is a pathogenic mutation, an antisense therapy assay was performed. Normal splicing processes and functional proteins were successfully rescued in a sequence- and dose-specific manner. These results lend further support to the notion that this therapeutic approach could be used to rescue exonic changes (Perez et al., 2010). Given in combination with pyridoxine treatment, it might improve outcomes by avoiding the production of toxic metabolites.
It was impossible to establish clear-cut phenotype–genotype correlations, since the number of patients in this study was small, and most had a unique genotype. Moreover, the final neurologic outcome may be more greatly affected by phenotypic modifying factors than by the actual genotype (Scharer et al., 2010). In this regard, the dual subcellular localization of antiquitin protein (Wong et al., 2010) may also contribute to the neuropathology of the disease by increasing oxidative stress and subsequent mitochondrial dysfunction, as described for other mitochondrial diseases (Richard et al., 2007).
In conclusion, the present work highlights the need to improve the prevention of persistent neurologic damage in PDE despite vitamin therapy. The results suggest that antisense therapy may hold promise as a means of rescuing splicing changes in ALDH7A1 (Milh et al., 2007), thereby avoiding the production of toxic metabolites. Finally, PDE should always be considered in any infant with intractable or poorly controlled seizures until biochemical or genetic test results rule out the disease.