• Pyridoxine-dependent;
  • ALDH7A1;
  • Vitamin B6;
  • Neonatal seizures


  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Purpose: Pyridoxine-dependent seizure (PDS) is a rare disorder characterized by seizures that are resistant to common anticonvulsants, and that are ultimately controlled by daily pharmacologic doses of pyridoxine (vitamin B6). Mutations of the antiquitin gene (ALDH7A1) are now recognized as the molecular basis of cases of neonatal-onset PDS.

Methods: Bidirectional DNA sequence analysis of ALDH7A1 was undertaken along with plasma pipecolic acid (PA) measurements to determine the prevalence of ALDH7A1 mutations in a cohort of 18 North American patients with PDS.

Results: In patients with neonatal-onset PDS, compound heterozygous or homozygous ALDH7A1 mutations were detected in 10 of 12 cases, and a single mutation was found in the remaining 2. In later-onset cases, mutations in ALDH7A1 were detected in three of six cases. In two patients with infantile spasms responsive to pyridoxine treatment and with good clinical outcomes, no mutations were found and PA levels were normal. In total, 13 novel mutations were identified.

Discussion: Our study advances previous findings that defects of ALDH7A1 are almost always the cause of neonatal-onset PDS and that defects in this gene are also responsible for some but not all later-onset cases. Later-onset cases of infantile spasms with good outcomes lacked evidence for antiquitin dysfunction, suggesting that this phenotype is less compelling for PDS.

Pyridoxine-dependent seizures (PDS: OMIM, 266100), also known as pyridoxine-dependent epilepsy, is a rare, autosomal recessive disease with an estimated prevalence of between 1 per 276,000 and 1 per 700,000 births (Baxter, 2001; Bennett et al., 2005; Gospe, 2006; Bok et al., 2007). Patients classically present with neonatal seizures that are unresponsive to conventional anticonvulsant therapy, but which can be controlled with pyridoxine monotherapy; less commonly, later-onset cases present through the second to third years of life. A variety of clinical seizure types are seen including myoclonic seizures, atonic seizures, partial and generalized events, and infantile spasms. Although most patients with PDS show a rapid response to pyridoxine treatment, some show a transient response to common anticonvulsants or a poor initial response to pyridoxine (Gospe, 2002). Patients with PDS display varying degrees of developmental delay, which may, in part, be independent of the timing of pyridoxine therapy (Baynes et al., 2003; Rankin et al., 2007). A related condition designated as pyridoxine-responsive seizure (PRS) includes infants and young children with seizures that respond initially to pyridoxine but lack seizure recurrence with pyridoxine withdrawal; familial cases of PRS have not been described (Baxter, 1999, 2001).

Until recently, the unequivocal diagnosis of PDS was made by withdrawing pyridoxine treatment from responsive patients, to provoke seizures, followed by the reintroduction of pyridoxine and the return of seizure control (Gospe, 1998, 2002; Baxter, 2001). Since the discovery of biomarkers for PDS—α-aminoadipic semialdehyde (AASA) and pipecolic acid (PA)—and available clinical DNA tests for the antiquitin gene, it is no longer necessary to withdraw patients from pyridoxine treatment to confirm the diagnosis (Plecko et al., 2000, 2005; Willemsen et al., 2005; Gospe, 2006).

The underlying enzymatic defect of PDS has been located at the level of AASA dehydrogenase within the cerebral lysine catabolism pathway. The significance of elevated PA in PDS was disclosed from understanding the biochemical mechanism leading to seizures in hyperprolinemia type II (Farrant et al., 2001). A defect in L-Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase causes accumulation of P5C, which in turn reacts with pyridoxal-5-phosphate (PLP) by Knoevenagel condensation, resulting in PLP depletion. A similar mechanism was shown to cause PLP depletion in PDS patients with mutations in the antiquitin gene (ALDH7A1). When antiquitin function is defective, piperideine-6-carboxylic acid (P6C) accumulates and reacts with PLP, thereby causing its concomitant depletion (Mills et al., 2006). The precise mechanism by which cerebral PLP depletion leads to seizures is not fully understood but undoubtedly involves compromised neurotransmitter synthesis where PLP is required as a coenzyme. In addition, given that PLP depletion is common to both hyperprolinemia type II and PDS, it may be suggested that metabolite or adduct build-up could directly contribute to the disease phenotypes.

We undertook clinical evaluation of 18 patients with PDS and performed DNA sequence analysis of the ALDH7A1 gene to evaluate the hypothesis that the prevalence of ALDH7A1 mutations is discordant between early (neonatal) and later-onset cases of PDS.

Materials and Methods

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was approved by the University of Washington Institutional Review Board and informed consent was obtained from all subjects studied.


All affected pedigree members described in this manuscript met clinical criteria for PDS diagnosis (OMIM, 266100) (Gospe, 2002; Baxter, 2003) and were designated as definite, probable, or possible cases according to Baxter’s classification system, with slight modifications. Definite cases were proved by either a formal trial of pyridoxine withdrawal (or an interval of pyridoxine noncompliance) followed by seizure recurrence that again responded completely to pyridoxine monotherapy, or breakthrough seizures that were controlled subsequently with higher daily doses of pyridoxine. Probable cases either have seizures that are controlled by pyridoxine and a similarly affected sibling who presented at the same age, or have a history of neonatal seizures that responded to a single dose of pyridoxine, followed by a later recurrence of seizures that again were controlled with pyridoxine, which was then continued. Possible cases were those for which no trial of pyridoxine withdrawal has been performed, and, therefore, may include some cases of PRS (Baxter, 1999). All patients are either U.S. or Canadian nationals and, unless otherwise stated, are Caucasian.

Twelve kindreds with neonatal-onset PDS and six kindreds with later-onset PDS are included. One kindred with two affected children (K3008) has one child with seizure onset at 3 weeks of age and the sibling with seizure onset at 9 months of age. We elected to assign this kindred to the neonatal-onset group. Clinical details of all patients are listed for neonatal-onset PDS (Table 1a) and for later-onset PDS (Table 1b). We have described the following pedigrees previously: K3001; K3002; K3005; K3006; K3008; K3009 (Bennett et al., 2005); K3003 (Battaglioli et al., 2000); K3004 (Gospe & Bell, 2005); and K3010 (Baynes et al., 2003). Clinical details of nine additional kindreds are as follows.

Table 1.   Clinical description of affected family members with (a) neonatal onset PDS; (b) later onset PDS
No.KindredCaseGenderAge at presentationInitial seizure type and clinical featuresAge at diagnosisCurrent ageBaxter Classification
 1K3001abII.1MaleNeonatePartial motor, encephalopathy8 months18 yearsDefinite
II.2FemaleNeonatePartial motor, encephalopathyNeonate11 yearsDefinite
 2K3002abdII.1FemaleNeonate (fetal)Myoclonus and partial motor3 months14 yearsDefinite
II.2MaleNeonateGeneralized tonicNeonate10 yearsDefinite
 3K3004dII.1FemaleNeonatePartial motor, encephalopathy9 weeks6 yearsPossible
 4K3006aII.1MaleNeonatePartial motor apnea, encephalopathyNeonate11 yearsDefinite
II.2MaleNeonatePartial motor, encephalopathyNeonate6 yearsDefinite
 5K3007II.1MaleNeonatePartial motor, encephalopathy10 months9 yearsDefinite
II.2FemaleNeonate (fetal)Partial motor, encephalopathyNeonate2 yearsDefinite
 6K3008aII.1Male3 weeksPartial motor8 months16 yearsDefinite (see text)
II.2Male9 monthsPartial motor9 months14 yearsDefinite (see text)
 7K3010cII.1MaleNeonate (fetal)Partial motorNeonate40 yearsDefinite
 8K3011II.1FemaleNeonatePartial motor and tonic14 months9 yearsDefinite
 9K3014II.1MaleNeonatePartial motor, encephalopathy1 month2 yearsPossible
10K3015II.1FemaleNeonateApnea, tonic, encephalopathyNeonate9 yearsPossible
11K3016II.1MaleNeonateEpileptic (myoclonic) spasmsNeonate3 yearsDefinite
12K3020II.1MaleNeonatePartial seizures1–2 months17 yearsPossible (see text)
13K3003bII.1Male6 monthsInfantile spasms8 years18 yearsProbable
II.2Female6 monthsInfantile spasms6 months10 yearsProbable
14K3005aII.1Female2 monthsMixed4 months7 yearsPossible
15K3009aII.1Female3 monthsInfantile spasms9 months9 yearsPossible
16K3017II.1Female6 monthsGeneralized convulsions8 months3 yearsDefinite
17K3018II.1Male5 monthsInfantile spasms6 months4 yearsPossible
18K3019II.1Male2 monthsNot specified5 months29 yearsDefinite
II.2Female2 monthsNot specified2 monthsDeceasedDefinite

The proband, a now 9-year-old boy (II.1) presented at one week of age with partial motor seizures that were initially controlled with phenobarbital, successfully discontinued at 2 months of age. Seizures recurred at 5 months of age and became refractory to multiple medications, with pyridoxine-dependency diagnosed at 10 months of age when a prolonged seizure was terminated after the administration of intravenous pyridoxine under video-EEG monitoring. Since then, the boy has been maintained on pyridoxine monotherapy, and a trial of pyridoxine withdrawal was never attempted. He has significant cognitive developmental delay with no expressive language. Six years later a sister was born (II.2) who developed clinical seizures at several hours of age. Fetal seizures may have been present. Since presentation she has been continuously treated with pyridoxine, and at 2 years of age she has a slight degree of expressive language delay but is otherwise developing appropriately.


This 9-year-old Hispanic girl developed neonatal partial motor seizures that were treated with phenobarbital and phenytoin. Complete seizure control was never achieved, and at 14 months of age a prolonged partial motor seizure ceased after the administration of intravenous pyridoxine during video-EEG monitoring. Although a formal trial of pyridoxine withdrawal was not attempted, the child had a breakthrough seizure at approximately 20 months of age after her pyridoxine preparation had been substituted with a new product by a pharmacy. The child is microcephalic with cognitive and language developmental deficits.


This 2-year-old male of mixed Hispanic/European heritage developed refractory neonatal seizures that were controlled at one month of age with intravenous pyridoxine administered during continuous EEG monitoring. A trial of pyridoxine withdrawal was not attempted. At 2 years of age he has motor and language developmental delay.


A now 9-year-old girl presented with neonatal tonic seizures, apnea, and encephalopathy. These anticonvulsant-resistant seizures were controlled with pyridoxine. She remains seizure free on pyridoxine monotherapy and has never had a trial of pyridoxine withdrawal. She has significant cognitive and language impairment with autistic features.


This 3-year-old boy of mixed Hispanic/European heritage had refractory neonatal epileptic (myoclonic) spasms that responded clinically and electrographically to intravenous pyridoxine. Anticonvulsants and pyridoxine were withdrawn, and clinical myoclonic events recurred that were again controlled with pyridoxine. Subsequently, the boy has experienced several breakthrough seizures, resulting from either intercurrent illness or missed doses of pyridoxine. He has expressive language deficits.


A now 3-year-old girl, developed generalized convulsions at the age of 6 months that did not respond to anticonvulsants. At 8 months of age, seizures were controlled both clinically and electrographically after the intravenous administration of pyridoxine. While she has been maintained on pyridoxine monotherapy without a formal trial of pyridoxine withdrawal, she did experience a flurry of breakthrough seizures that were controlled with a higher daily dose of the vitamin. There have been no further recurrences.


This 5-year-old boy developed infantile spasms and hypsarrhythmia at 5 months of age that were unresponsive to multiple anticonvulsants. Within 2 days of starting oral pyridoxine, the spasms stopped. He was eventually taken off all anticonvulsants but has remained on daily pyridoxine. He has some mild behavioral concerns, but otherwise he is developmentally normal.


A 29-year-old man (II.1) had a first seizure at 2 months of age and was treated with phenobarbital. Intractable seizures developed at 5 months of age, which then responded to intravenous pyridoxine. The vitamin was not continued and seizures recurred several days later. With the exception of an episode of status epilepticus at 27 years of age, he has remained seizure free on pyridoxine monotherapy. He is a high school graduate, lives independently, and is employed as a machine operator. A younger sister (II.2) also had pyridoxine-dependent seizures and died in young adulthood secondary to status epilepticus, possibly complicated by poor compliance.


This 17-year-old male with intractable epilepsy presented with neonatal seizures and pyridoxine dependency and was diagnosed within the first month of life after clinical seizures and electrographic features improved with pyridoxine therapy. The boy had frequent breakthrough seizures and continued on pyridoxine and high doses of several anticonvulsants. During a subsequent hospitalization, pyridoxine inadvertently was not ordered and his seizure control deteriorated further until the vitamin was restarted. Because of continued breakthrough seizures, at 5 years of age he was started on the ketogenic diet, which along with pyridoxine, was continued for 5 years. Subsequently, he remained on pyridoxine monotherapy but experienced brief nocturnal tonic seizures several times per week. For the last year he has also experienced daytime partial motor seizures with secondary generalization, and he is now treated with high-dose pyridoxine, pyridoxal-5-phosphate, and oxcarbazepine. Brain imaging demonstrated hypoplasia of the cerebellar vermis, cavum septum pellucidum, a large posterior fossa cyst, and hydrocephalus. The cyst and ventricular system were shunted at 16 months of age, and he has required several shunt-revision procedures. He has a history of delayed ambulation, along with expressive language and other cognitive deficits.

DNA extraction and ALDH7A1 sequence analysis

DNA samples were extracted from peripheral blood samples from members of all 18 pedigrees, each consisting of the proband, parents, and affected and unaffected siblings, using standard methods (Puregene DNA isolation Kits, Gentra, Minneapolis, MN, U.S.A.). To sequence all 18 coding exons of ALDH7A1, intronic primers were designed to amplify each exon and at least 50bp of the intron/exon boundary (primer sequences available on request). Cycle sequencing was performed using an incorporated customer primer sequence, which is attached to the gene specific primer, and separation was performed by high-throughput capillary sequencers. Electropherograms were examined by an experienced analyst and at least one of the authors to ensure accuracy.

ALDH7A1 ex17 genomic deletion analysis by long-range polymerase chain reaction

To detect the previously reported exon 17 genomic deletion within the ALDH7A1 locus (Kanno et al., 2007), primers were designed to generate a 5.21-kb amplicon encompassing exon 16 through to 96 bp beyond the stop codon of exon 18. Forward and reverse primers used were: forward, 5′-ttg ctc tca cca tgc ctc cct ttt tac aca-3′ and reverse, 5′-ggg cttt ggg gtc ata ggg gga tta gtc ac-3′. The Expand Long Template PCR System (Roche, Indianapolis, IN, U.S.A.) was used according to standard protocols with annealing temperature set at 62°C for 30 s and a elongation step at 70°C for 6 min. Amplified polymerase chain reaction (PCR) products were resolved by 0.8% agarose gel electrophoresis and visualized with ethidium bromide staining.

Analysis of pipecolic acid (PA) levels in plasma and urine

PA was determined by isotope-dilution gas chromatography/mass spectrometry in plasma as a diagnostic service at either the Biochemical Genetics Laboratory of the Mayo Medical Laboratories (Rochester, MN, U.S.A.) or by the diagnostic services of the Kennedy Krieger Institute Peroxisomal Diseases Laboratory (Baltimore, MD, U.S.A.). Normal values are age-dependent, as follows: up to 1 month, <5.3 μmol/L; 1–6 months, <3.9 μmol/L; 7 months to 5 years, <4.2 μmol/L; and 5 years and older, <4.0 μmol/L (Anne Moser, personal communication).


  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Our testing revealed ALDH7A1 mutations in 15 of 18 PDS patients (Table 2). Of those found with mutations, one patient (K3008-II.I) harbored only a single mutation, but plasma PA level was significantly elevated (Table 2) strongly suggesting antiquitin deficiency. In patient K3007-II.1, two in cis intronic alterations, c.109 -9t>g and c.109-15t>c, which were absent from all other samples tested, suggest a potential splicing aberration. Splice site prediction software (e.g.,, suggest the normal splice site with a probability score of 0.81 (0.1–1) would be reduced to 0.43 by both alterations. Therefore, it is a strong possibility that alternate cryptic splice sites would then be used in preference, leading to an aberrant message. In two of the later-onset PDS cases (K3003-II.1 and K3018-II.1), no ALDH7A1 mutations were detected, and normal PA levels were apparent in both cases.

Table 2.   Clinical classification and genetic testing results
 Kindred-caseGene allelesMutation outcomePA in plasma (μmol/L) CA repeats/intron 2
  1. NT, not tested; fs, frameshift; ins, insertion mutation; ?, indicates no mutation detected in the “Gene alleles” column and no protein change anticipated in the “Mutation outcome” column.

  2. aElevated pipecolic acid level for age.

  3. bNormal pipecolic acid level for age.

 1K3001-II.1c.[1195g>c] + [1004g>a]p.(E399Q) + (W335X)NT17/22
 2K3002-II.1c.[1260t>a] + [1195g>c]p.(N420K) + (E399Q)NT21/22
 3K3004-II.1c.[1121 ins-A] + [750g>a]p.(fsI373>409-stop) + (mutant splice site)6.2a17/21
 4K3006-II.1c.[890c>a] + [1396g>c]p.(T297K) + (G466R)4.4a17/19
 5K3007-II.1c.[750g>a] + [c.109 -9t>g, c.109-15t>c]p.(mutant splice site) + (possible splice defect)4.7a16/18
 6K3008-II.1c.[1197g>t] + [?]p.(E399D) + (?)19.7a17/22
 7K3010-II.1c.[1195g>c] + [1195g>c]p.(E399Q) + (E399Q)NT17/18
 8K3011-II.1c.[764g>a] + [1429g>c]p.(G255D) + (G477R)NT17/18
 9K3014-II.1c.[del -1-3, IVS6)] + [1195g>c]p.(exon 7, spliced out) + (E399Q)8.7a17/19
10K3015-II.1c.[712c>t] + [500a>g]p.(R238X) + (N167S)2.6b17/22
11K3016-II.1c.[1195g>c] + [1429g>c]p.(E399Q) + (G477R)7.8a17/19
12K3020-II.1c.[1195g>c] + [1195g>c]p.(E399Q) + (E399Q)5.6a17/18
13K3003-II.1c.[?] + [?]p.(?) + (?)1.3b20/22
14K3005-II.1c.[1196a>g] + [818a>t]p.(E399G) + (N273I)8.3a17/18
15K3009-II.1c.[?] + [?]p.(?) + (?)7.3a17/18
16K3017-II.1c.[1195g>c] + [1195g>c]p.(E399Q) + (E399Q)9.4a17/19
17K3018-II.1c.[?] + [?]p.(?) + (?)1.7b17/21
18K3019-II.1c.[890c>g] + [1195g>c]p.(T297R) + (E399Q)4.8a17/18

ALDH7A1 mutation detection

In total, 13 novel ALDH7A1 mutations were identified in 18 patients (Table 2). Two previously reported mutations, N273I (Plecko et al., 2007) and the common E399Q mutation (Mills et al., 2006; Plecko et al., 2007) were also observed. Two additional substitutions of the E399 residue were detected; E399D and E399G. With the exception of pedigrees, K3010, K3011, K3017, K3019, and K3020 (unavailable parents), the segregation of mutant alleles were confirmed by parental DNA sequence analysis in all cases.

In all cases, novel putative antiquitin missense substitutions (Table 2) were not detected in 96 control chromosomes. To demonstrate conservation of mutated resides, the location of new ALDH7A1 missense mutations are shown within short segments of antiquitin orthologues representing seven diverse species (Fig. 1).


Figure 1.  Amino acid sequence alignments for novel missense mutations. Substituted residues are indicated by arrows, and arrowheads indicate mutations that have been previously reported.

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A c.750g>a transition within exon 9 was detected in two patient’s, K3004-II.1 and K3007-II.1. The wild type sequence (c.750g) is part of a cryptic splice site. In a previous study, a patient with PDS who was homozygous for the c.750g>a mutation was found to use the frame-shifting, mutant splice donor almost exclusively in preference to the native splice site (Salomons et al., 2007), confirming the functional significance of this mutation.

In proband K3011-II.1, both mutations are novel missense mutations. One mutant allele is a c.764g>a change in exon 9, resulting in a G255D substitution. This specific residue is a G (glycine) or A (alanine) in vertebrate orthologues of antiquitin. Unlike other substitutions associated with PDS, the G255 targeted residue is not as conserved (Fig. 1). However, the SIFT program (Ramensky et al., 2002) predicts this mutation would not be tolerated.

One mutant allele in proband K3014-II.1 harbors a three-nucleotide deletion at the splice-acceptor site of intron 6, at positions minus 1, 2, and 3 (Table 2). This likely causes skipping of exon 7, 45 bp in length. Although this expected defect of splicing may result in an in-frame message, the highly conserved nature of the protein suggests that a protein encoded by such a transcript, missing 15 amino acids would be nonfunctional.

ALDH7A1 ex17 genomic deletions

In one neonatal-onset case the second ALDH7A1 mutation could not be detected (K3008-II.1), yet elevated plasma PA was evident (Table 2). In a second proband, K3007-II.1, although a second splice mutation was suspected (Table 2), its causality has not been functionally proven. Within the later-onset cases, no mutation was found in proband K3009-II.1, and plasma PA levels were elevated. Proband K3003-II.1 did not harbor ALDH7A1 mutations and PA levels had not been tested at the time this molecular analysis was undertaken. In each of these four cases we sought to determine if a common ALDH7A1 genomic deletion of 1937 bp (Kanno et al., 2007) was present. We found no evidence of deletion, as only the 5.21kb band was observed in each lane, representing nondeleted ALDH7A1 alleles (data not shown).


  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In this study, we have identified ALDH7A1 mutations in a high percentage of patients with PDS (81%), including: missense, nonsense, frame-shift, and splice-site mutations encompassing 13 mutations in this gene. Each of the nine new missense mutations (Table 2) was predicted to be deleterious from analysis by the Sort Intolerant from Tolerant (SIFT) program (Ramensky et al., 2002) and the Polymorphism Phenotype (PolyPhen) program (Ng & Henikoff, 2001). This is perhaps not a surprising finding given that disease-associated amino acid substitutions of this metabolic enzyme were located largely in highly conserved regions of the protein (Fig. 1). The single exception to this is the ALDH7A1 G255D substitution, which is significantly less conserved than all other mutated residues associated with PDS.

We hypothesized that the prevalence of ALDH7A1 mutations, with or without indirect biochemical evidence of antiquitin dysfunction, would be higher for neonatal-onset subjects, versus later-onset PDS (Tables 1 and 2). To this end we undertook genomic DNA sequencing of the ALDH7A1 gene, allelic-specific detection of an intragenic microsatellite marker to exclude an intragenic deletion (Kanno et al., 2007), and whenever possible, biochemical analyses of plasma PA levels. Although elevation of either plasma or urine AASA represents a more specific biomarker of PDS than PA (Mills et al., 2006), methods to detect and measure this particular substance are not yet readily available. Detection of elevated PA levels was able to support the causal status of the novel mutations we identified in most cases, yet this is not definitive. When patients are receiving large doses of pyridoxine, the fold-elevation observed is significantly reduced over time. For example, in the study by Plecko et al. (2005), patient 1 had PA levels of 17.6 μmol/L, which were later reduced to 5.3 μmol/L with pyridoxine treatment. We observed PA levels in the normal range of 2.6 μmol/L in one patient harboring two ALDH7A1 mutations, R238X and N167S (K3015-II.1). Ideally, incorporation of AASA testing would be more sensitive and specific, but this assay was not available for this study.

Within neonatal-onset PDS, the combination of ALDH7A1 sequencing and the measurement of plasma PA levels suggested that recessive defects of antiquitin function were causal in all 12 cases, confirming our hypothesis for this group of subjects. In later-onset PDS, ALDH7A1 mutations were identified in three of six cases. In one additional case, K3009-II.1, no mutations were detected, yet plasma PA levels were significantly elevated, suggesting antiquitin deficiency to be causal.

These three cases represent the first patients with late-onset PDS in whom ALDH7A1 mutations have been identified. In addition, within this group, we effectively excluded a causal role for loss of antiquitin function in two of the kindreds. In K3003-II.1 and K3018-II.1, no ALDH7A1 mutations were detected and plasma PA levels were in the mid-normal range. These patients (K3003-II.1 and K3018-II.1) also manifested interesting seizure types, as both siblings of pedigree K3003 (II.1 and II.2) and patient K3018-II.1 had infantile spasms. Although infantile spasms are not a typical PDS seizure type, they are occasionally observed (Baxter, 1999). These three subjects have had an unusually good outcome following the resolution of infantile spasms, with normal intellectual development resulting. Given the lack of ALDH7A1 mutations and these particular clinical features, these patients may, in fact, represent examples of PRS. If this should be the case, the two siblings in kindred K3003 would represent the first example of PRS recurring within a family. However, none of these patients have had trials of pyridoxine withdrawal, and, therefore, neither PDS nor PRS has been clinically confirmed. If a trial of pyridoxine withdrawal is eventually conducted in these patients and definite PDS is assigned, the absence of ALDH7A1 mutations along with this unique phenotype of infantile spasms with a good outcome would support genetic heterogeneity for PDS (Bennett et al., 2005).

A fourth late-onset patient, K3009-II.1 also presented with infantile spasms, and although DNA sequencing did not detect ALDH7A1 mutations, plasma PA was significantly elevated, indicative of antiquitin dysfunction and suggesting either incomplete sensitivity of DNA analysis of ALDH7A1 or another cause for the PDS. In this particular case, the poor long-term outcome is in line with cognitive outcomes for many patients with PDS (Gospe, 2002; Baxter, 2003; Rankin et al., 2007), and, indeed, as is commonly the outcome for infantile spasms. Therefore, for clinicians facing patients with infantile spasms, responsive to pharmacologic doses of pyridoxine (and, therefore, meeting at least one PDS diagnostic criterion), antiquitin deficiency cannot be presumed. The response to pyridoxine along with an elevation in plasma PA, in such cases, may suggest that another downstream protein in the cerebral lysine catabolic pathway, or an overlapping pathway, maybe involved.

Our prior segregation analysis of the chromosome 5q31 PDS locus (ALDH7A1) in six small multi-sib North American families was consistent with linkage in five of the six families. In pedigree K3001, linkage to 5q31 appeared to be excluded, as haplotype sharing was apparent between siblings discordant for PDS (Bennett et al., 2005). This appeared to confirm earlier suggestions of genetic heterogeneity for PDS (Baxter, 2003), but in our present study, K3001 was found to harbor an E399Q mutation and a W335X nonsense mutation, confirming antiquitin dysfunction as the basis of disease in K3001.

Interestingly, interfamilial variation of PDS onset is observed for patients K3010-II.1 and K3020-II.1 (neonatal/fetal) versus patient K3017-II.1 (6 months of age), all harboring two copies of the E399Q common mutation. The same genotype was seen four times in a recent report (Plecko et al., 2007), with one possible, one probable, and two definite patient classifications. Intrafamilial variation was also present in pedigree K3008 as the onset of ALDH7A1-related PDS varied from neonatal onset (II.1) to onset at 9 months (II.2) suggesting that other genetic and environmental modifiers are influencing phenotype.

In case K3020-II.1, despite early clinical and electrographic support of a diagnosis of pyridoxine dependency, this patient has only experienced a short period of complete seizure freedom during his 17 years of life. In reviewing his complex history, many clinicians would conclude that he either does not have PDS, or alternatively has a secondary cause for his intractable epilepsy. However, K3020-II.1 is homozygous for the common E399Q mutation. As he has been treated appropriately for PDS, his intractable epilepsy must be secondary, potentially due to inherent PDS-associated cerebral dysgenesis, a finding that has been reported in many individuals with PDS (Gospe & Hecht, 1998; Baxter, 2003). This case emphasizes the utility of measuring PA or AASA levels and screening ALDH7A1 for mutations in patients with preliminary diagnoses of PDS.

Taken together, our data confirm that PDS is commonly the result of recessive mutations of the ALDH7A1 gene (Mills et al., 2006; Bok et al., 2007; Plecko et al., 2007), but that genetic heterogeneity is also present. One might posit that genetic causes other than ALDH7A1 mutations are more likely to be seen in later-onset cases. However, in the recent Japanese study, genetic heterogeneity was evident in a patient with neonatal-onset PDS (onset of symptoms, 18 days). In this patient, no ALDH7A1 mutations were found and PA levels were in the normal range. It is of interest to note that as with our three cases with infantile spasms and putative PRS in the later-onset group, mental retardation was not evident (Kanno et al., 2007).

In the initial PDS gene identification report (Mills et al., 2006), and in a subsequent study of 18 patients with PDS (Plecko et al., 2007), the E399Q mutation emerged at a high frequency of 33%. We found the E399 residue was mutated at the same high frequency of 33% (12 of 36 alleles) in our total cohort of predominantly Caucasian patients. In the limited patient subset of Hispanic or mixed Hispanic/European ancestry, the E399Q substitution represented a similar 33% of mutant alleles (2 of 6 alleles). In a recent study of ALDH7A1 mutations in Dutch patients, E399Q was again found at high frequency (Salomons et al., 2007). This common mutation may represent a possible founder effect in this latter population, yet no haplotype analysis was undertaken. Also of interest, in the recent Japanese study, no mutation of the E399 residue was present suggesting ethnic differences (Kanno et al., 2007). We found no evidence of a founder effect in our patient cohort, as no specific microsatellite genotype segregated with the E399Q mutation (Table 2, CA repeats). Irrespective, this particular residue has been clearly established as a hot-spot and critical for several reasons (Mills et al., 2006). First, it is highly conserved throughout evolution. Second, this residue in other human aldehyde dehydrogenase superfamily members was shown to interact with the ribose moiety of NAD, and that substitution of glutamine converts the rate-limiting step from deacylation to hydride transfer (Perozich et al., 1999). Third, transfection of CHO cells with the E399Q mutant produced no detectable AASA dehydrogenase activity (Mills et al., 2006).

In our patient cohort, we also identified a coding c.750g>a transversion that at first appeared to be a harmless synonymous codon substitution (V250V). However, because it was present in two patients, K3007-II.1 and K3004-II.1, both with raised PA levels, we speculated that it may represent a cryptic splice-site mutation. Recently, this mutation was confirmed to generate a mutant splice donor site that is used almost exclusively, generating a frame-shift and nonsense mediated decay of the message (Salomons et al., 2007).

In conclusion, we confirmed in this investigation that ALDH7A1 mutations underlie all cases of classic neonatal-onset PDS and a lesser proportion of later-onset cases. Within late-onset cases, three cases manifested infantile spasms, with a surprisingly good long-term outcome but lacked evidence for antiquitin dysfunction suggesting this phenotype to be less compelling for PDS. We confirm that E399Q is a common mutation within ALDH7A1 and may provide a means of triage to diagnostic molecular testing for this gene. The utility of biochemical markers combined with DNA analysis of ALDH7A1 obviate traditional means of PDS diagnosis, and enable the provision of prenatal diagnosis using this approach.


  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported, in part, by the research endowments of the Division of Neurology, Children’s Hospital and Regional Medical Center, Seattle, WA. We thank all the participating families and thank the University of Washington High-Throughput Genomics Unit (HTGU), Department of Genome Sciences for the design of ALDH7A1 primers and all DNA sequencing. We thank Dr. Valeria Vasta who helped with analyzing the sequencing data from patients.

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Disclosure of conflicts of interest: All authors confirm that no conflict of interest exists in the publication of this research.


  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References