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Keywords:

  • Antiquitin protein;
  • Splicing mutation;
  • Antisense therapy;
  • Genomic rearrangement

Summary

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

Purpose

Pyridoxine-dependent epilepsy seizure (PDE; OMIM 266100) is a disorder associated with severe seizures that can be controlled pharmacologically with pyridoxine. In the majority of patients with PDE, the disorder is caused by the deficient activity of the enzyme α-aminoadipic semialdehyde dehydrogenase (antiquitin protein), which is encoded by the ALDH7A1 gene. The aim of this work was the clinical, biochemical, and genetic analysis of 12 unrelated patients, mostly from Spain, in an attempt to provide further valuable data regarding the wide clinical, biochemical, and genetic spectrum of the disease.

Methods

The disease was confirmed based on the presence of α-aminoadipic semialdehyde (α-AASA) in urine measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and pipecolic acid (PA) in plasma and/or cerebrospinal fluid (CSF) measured by high performance liquid chromatography (HPLC)/MS/MS and by sequencing analysis of messenger RNA (mRNA) and genomic DNA of ALDH7A1.

Key Findings

Most of the patients had seizures in the neonatal period, but they responded to vitamin B6 administration. Three patients developed late-onset seizures, and most patients showed mild-to-moderate postnatal developmental delay. All patients had elevated PA and α-AASA levels, even those who had undergone pyridoxine treatment for several years. The clinical spectrum of our patients is not limited to seizures but many of them show associated neurologic dysfunctions such as muscle tone alterations, irritability, and psychomotor retardation. The mutational spectrum of the present patients included 12 mutations, five already reported (c.500A>G, c.919C>T, c.1429G>C c.1217_1218delAT, and c.1482-1G>T) and seven novel sequence changes (c.75C>T, c.319G>T, c.554_555delAA, c.757C>T, c.787 + 1G>T, c.1474T>C, c.1093-?_1620+?). Only one mutation, p.G477R (c.1429G>C), was recurrent; this was detected in four different alleles. Transcriptional profile analysis of one patient's lymphoblasts and ex vivo splicing analysis showed the silent nucleotide change c.75C>T to be a novel splicing mutation creating a new donor splice site inside exon 1. Antisense therapy of the aberrant mRNA splicing in a lymphoblast cell line harboring mutation c.75C>T was successful.

Significance

The present results broaden our knowledge of PDE, provide information regarding the genetic background of PDE in Spain, afford data of use when making molecular-based prenatal diagnosis, and provide a cellular proof-of concept for antisense therapy application.

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.

Patients and Methods

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

Patients

Eleven patients with suspected PDE (Table 1 shows their characteristics) were recruited from different Spanish hospitals, and one patient was recruited from India (P1). The study included patients with early onset seizures refractory to antiepileptic drugs (AEDs) who showed a definite response to administration of pyridoxine, with effective cessation of seizures after a short period of time, as well as one patient with AED refractory seizures, who died in the neonatal period, in whom pyridoxine was not tried (P4). This patient was included as there was a strong clinical suspicion of PDE. Some of the patients showed a transient response to AEDs, but seizures recurred and were later effectively suppressed by pyridoxine. Patients with a “doubtful” response to pyridoxine as manifested by a diminished number of seizures after addition of pyridoxine to previous AED were excluded from the study.

Table 1. Personal, clinical, and neuroimaging data of the present patients
Pt/GenderOnsetEvolution
AgeSeizure typeEEGAssociated symptomsAED response (duration)Pyridoxine withdrawalCurrent agePsychomotor developmentSeizuresMRIPyridoxine doseOther AEDs
  1. AC, arachnoid cyst; AED, antiepileptic drug; ASD, autism spectrum disorder; BI, borderline intelligence; BS, burst suppression; CBZ, carbamazepine; CCH, corpus callosum hypoplasia; CNZ, clonazepam; CS, clonic seizures; DD, developmental delay; DWV, Dandy-Walker variant; F, female; FA, focal activity; FolA, Folinic acid; FS, focal seizures; GSWA, generalized slow-wave activity; GTCS, generalized tonic–clonic seizures; HIE, hypoxic-ischemic encephalopathy; IQ, Intelligence Quotient, (WISC-R); LEV, levetiracetam; M, male; MCM, mega cisterna magna; MCS, myoclonic seizures; MDZ, midazolam; MFS, multifocal seizures; MFA, multifocal activity; MR, mental retardation; NT, not tried; OS, oculogyric seizures; PB, phenobarbital; PCA, partial callosal agenesis; PHT, phenytoin; PF, posterior fossa; PVLM, periventricular leukomalacia; TS, tonic seizures; VM, ventriculomegaly; VPA, valproic acid.

P1/M1 dayCSBSNoNTNT2 yearsMild DDNoNot done30 mg/kg/dayNo
P2/M1 dayCSFARespiratory distress, irritability, acidosisPB (2 days)NT3 yearsNormalNoMCM15 mg/kg/dayNo
P3/M1 dayMCSBSHypotonia, irritabilityNoNT18 monthsModerate DDGTCS with feverNormal11 mg/kg/dayVPA+ FolA
P4/M1 dayTSBSHypertonia, acidosisNoNTDeath at 10 day  HIE-like  
P5/F1 dayCS, OSBSHypotoniaPB + PHT (26 days)NT8 monthsModerate DDGTCS with feverAC in PF15 mg/kg/dayNo
P6/F1 dayMCSGSWA, MFAHypotonia, deep resuscitationVPA (2 months)Yes (2 day)9 years

BI

IQ 75

NoVM, PVLM, MCM15–20 mg/kg/dayNo
P7/F1 dayMFS-CSMFAIrritabilityPB+ PHT (2 months)Yes (2 day)18 years

Mild DD

IQ 67

NoPCA, MCM300 mg/dayNo
P8/F4 daysTSBSHypotoniaPB+ CBZYes15 yearsMild MRNoDWV, CCH300 mg/dayCBZ
P9/M6 daysFSNormalHypotonia, DDPB (2 months)Yes (4 day)7 years

BI

IQ 78

NoMCM15–18 mg/kg/dayNo
P10/M1 monthFSMFANoPB (15 days)Yes (4 day)18 yearsSevere MR, ASDGTCS with feverNot done600 mg/dayVPA
P11/M3 monthsCSGSWA, MFAHypotonia, macrocephalyPHT, VPA, LEV, MDZ (4 days)NT23 monthsModerate DDNoNormal15 mg/kg/dayNo
P12/M5 monthsFSGSWANoPB (1 month)/PB+ PHT+ CNZ (1 month)Yes (5 day)8 years

Moderate MR

IQ 55

NoVM, PVLM, MCM15 mg/kg/dayNo

The study was approved by the ethics committees of the Universidad Autónoma de Madrid and other participating centers. For the genetic studies, written permission was obtained from all patients' parents or guardians.

Biochemical methods

α-AASA was measured in urine as previously described (Mills et al., 2006). Thirty microliters of 50-μm d3 α-aminoadipic acid, serving as an internal standard, was added to 10 μl of urine. After derivatization of the α-AASA molecule with fluorenylmethoxycarbonyl chloride, the sample was directly analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Pipecolic acid was analyzed underivatized in plasma/serum and CSF by high performance liquid chromatography (HPLC/MS/MS) with stable isotope dilution. Biogenic amine neurotransmitter metabolites in CSF were analyzed by HPLC with electrochemical detection (Coulochem III, ESA, Thermo Scientific, Wilmington, DE, U.S.A.) (Ormazabal et al., 2005). The plasma pyridoxal-phosphate (PLP) concentration was quantified by a commercially available kit (Chromsystems, Munich, Germany) using HPLC with fluorescence detection.

Genetic analysis

Total messenger RNA (mRNA) and genomic DNA were isolated from venous whole blood (four patients) cultured skin fibroblasts (seven patients), or lymphoblasts (one patient) using the MagnaPure system (Roche Applied Science, Indianapolis IN, U.S.A.) following the manufacturer's instructions. Mutational analysis was performed by complementary DNA (cDNA) sequencing and the changes identified confirmed by sequencing the corresponding genomic DNA region. This was also performed in samples taken from the patients' parents to ensure that the variant changes detected in the patients were on different alleles, and to rule out the presence of a large genomic rearrangement. The primers used for cDNA and genomic DNA amplifications were designed using the Genome Browser ENSEMBL (ENSG00000140650) and GenBank accession number NM_001182.2. The novel change c.1474T>C was analyzed by AccI restriction enzyme analysis in 96 ethnically matched controls.

Genomic rearrangement analysis was performed by a genome–wide scan of 610,000 single-nucleotide polymorphisms (SNPs) using the Illumina610-Quad Beadchip (Illumina, San Diego, CA, U.S.A.) according to the manufacturer's instructions, as well as a customized oligonucleotide-based comparative genomic hybridization array (Metabolarray; Agilent, Santa Clara, CA, U.S.A.) consisting of 62,979 oligo probes covering the whole genome (mainly the exonic regions) and 40,555 oligos covering 205 genes related to inherited metabolic diseases. The average probe spacing was about 300 bp.

In silico analysis of the effect on the splicing process was performed using different software: Berkeley Drosophila Genome Project BDGP (www.fruitfly.org/seq_tools/plice.html), MaxEnt (http://genes.mit.edu/12 burgelab/maxent/Xmaxentscan_scoreseqhtml), Splice Site Analyzer (http://ast.bioinfo.tau.ac.il/SpliceSiteFrame.htm), and Human Splicing Finder (http://www.umd.be/HSF/). For minigene assays, COS-7 cells were transiently transfected. The minigene system was used to analyze the functional effect of the nucleotide change c.75C>T. At 24 h posttransfection, the cells were harvested to isolate RNA (Rincon et al., 2007). Because the above nucleotide change is located in the first exon with no upstream 3′ splice site (3′ss), a reporter hybrid plasmid (constructed in our laboratory) was used to provide one (Arrabal et al., 2011). Briefly the region encompassing ALDH7A1 exon 1 and a fragment of intron 1 was fused to exon 2 of the sepiapterin reductase gene (SPR), which contains a 3′ss.

For antisense morpholino oligonucleotide (AMO) analysis, a 25-mer AMO targeted to the new exonic donor splice site in the pre-mRNA was designed, synthesized, and purified by Gene Tools (Philomath, OR, U.S.A.). A morpholino standard 25-mer control oligo provided by Gene Tools with the sequence 5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′ was used as a scrambled oligo. The AMO was used to transfect lymphoblasts from patient P8 as previously described (Rincon et al., 2007). Polymerase chain reaction (PCR) amplification of transcripts was performed using the primers 5′-GCAAAGACCAGCAAGCTCTC-3′ and 5′-GGCCAGATCTTTCAGAAGGC-3′ (forward and reverse, respectively). For antiquitin protein detection, a monoclonal antibody (Epitomics, Inc. Burlingame, CA, U.S.A.) was used in Western blot assays.

Results

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

Clinical features

Table 1 summarizes the main clinical features and neuroimaging findings of the patients. Although PDE is an autosomal recessive condition, a male preponderance was seen (male-to-female ratio 2:1). Seizure onset occurred in the neonatal period in the majority of patients (seven in the first day and two in the first week of life), whereas in three infants it started later (first, third, and fifth months of life, respectively). The mothers of two patients (P3 and P5) who showed onset on the first day after birth recalled intrauterine convulsions. Two neonates (P4 and P6) had clinical and/or analytical features compatible with perinatal hypoxic–ischemic encephalopathy. A family background of lethal epileptic encephalopathy in previous sibling was present in three cases (P1, P8, and P12); two patients (P1 and P3) had consanguineous parents. The seizures presented as status epilepticus in three patients (in P3 and P4 on the first day of life, and P5 at 1 month). Another three patients (P10, P11, and P12) had status epilepticus during postnatal development. In four patients this status continued for more than 24 h until resolution, except in one neonate (P4) in which it continued unabated for 10 days. The initial type of seizures were focal/multifocal, clonic, or myoclonic in 10 patients. Patient P7 had infantile spasms that subsided with pyridoxine. Abnormal neurologic signs such as hypotonia and irritability were recorded in nine patients. Initial electroencephalography (EEG) recordings showed different abnormalities: focal or multifocal discharges in five patients (P2, P6, P7, P10, and P11), burst-suppression pattern in another five (P1, P3, P4, P5 and P8), and a generalized slowing in three (P6, P11, and P12). Only one patient had a normal EEG (P9). This great variability in EEG recordings has been referred to in previous reports. It is also remarkable that a normal EEG does not rule out PDE. In eight patients (66%) a transient response was seen to treatment with conventional antiepileptic drugs (AEDs). Brain magnetic resonance imaging (MRI) was performed in 10 patients. The most common findings were mild posterior fossa abnormalities such as mega cisterna magna or Dandy-Walker variants (P2, P5, P6, P7, P8, P9, and P12). Periventricular leukomalacia-like abnormalities, ventriculomegaly, and corpus callosum dysgenesis were found in three patients (P6, P8, and P12).

The ages of the 11 surviving patients in the present series (patient P4 died at 10 days of age) ranged from 8 months to 18 years. Only one patient (P2) showed normal psychomotor development. EEG recordings became normal in all patients under pyridoxine treatment.

Biochemical data

Table 2 shows the diagnostic markers in the patients' biologic fluids. All patients, even those who had received pyridoxine treatment for several years, showed elevated PA levels in plasma and/or CSF, and elevated α-AASA in the urine. Plasma and CSF PA concentrations measured before treatment in patients P1, P2, P3, P4, and P11 were high (4.5–18 fold and 24–65-fold the upper normal limit, respectively). Plasma and CSF PA commonly remained high even with pyridoxine treatment (measured in P5, P6, P7, P8, P9, P10, and P12), although to a lesser extent (1.3–5.9-fold the upper normal limit) compared to infants studied before treatment. α-AASA excretion was very high (58–120-fold that of age-matched controls) before treatment in the four patients for whom such information was available (P2, P3, P4, and P11). To know the PLP status of the patients, plasma PLP levels were measured in three patients before pyridoxine treatment (P3, P4, and P11) and in four patients under treatment (P5, P9, P10, and P12). There would seem to be a negative correlation between urinary levels of α-AASA and plasma PLP, although patient P11, who experienced late-onset seizures, showed the greatest excretion of α-AASA but normal levels of plasma PLP. The excretion of α-AASA by patient P6, who had received pyridoxine treatment for several years, remained high (9.05 mmol/mol creatinine; normal value up to 1.36).

Table 2. Genetic and biochemical data for the present ALDH7A1-deficient patients
PatientAge of onset (age at time of biochemical study)Allele 1Allele 2Plasma P A (μm)CSF PA (μm)Urine α-AASA mmol/mol creatPlasma PLP (μm)CSF Neurotransmitters (μm)
  1. PA, pipecolic acid; CSF, cerebrospinal fluid; α-AASA, α-aminoadipic semialdehyde; PLP, pyridoxal-phosphate; HVA, homovanillic acid; OMD, O-methyldopa.

  2. Normal value of urinary α-AASA (0–6 month): 1.65 ± 1.2 (0.85–2.04) mmol/mol creat; (6 months–1 year): 1.3 ± 1.2 (0.5–1.45) mmol/mol creat; (>1 year): 0.6 ± 0.5 (0.15–1.36) mmol/mol creat.

  3. Normal values of plasma PA: 1.22 ± 0.59 (0.12–3.01) μm. Normal value of CSF PA: 0.05 ± 0.03 (0.01–0.14) μm.

  4. Normal values of plasma PLP (<1 month): 67 ± 30 (24–123) μm; (1–12 month):72 ± 26 (31–128) μm; (1–3 year) 50 ± 19 (20–84) μm; (3–20 year): 42 ± 19 (20–91) μm.

  5. Normal values of CSF homovanillic acid (<1 month): 428–1,268 nm; (1–6 month): 390–1,342 nm; (6 month–2 year): 339–926 nm; (2–5 year): 274–863 nm; (5–10 year): 190–786 nm.

  6. Normal values of CSF OMD (<1 month): <170 nm; (1–6 month): <150 nm; (6 month–2 year): <100 nm; (2–5 year): <40 nm; (5–10 year): <40 nm.

  7. The segregation of mutated alleles was confirmed in all families except those of patients P7 and P9 (no samples were available).

  8. a

    Patients analyzed before B6 treatment.

  9. b

    Novel mutations.

P1a1 day (1 day)p.Glu107Termb (c.319G>T)p.Glu107Term (c.319G>T) 5.2   
P2a1 day (2 day)p.Arg307Term (c.919C>T)p.Asn273 fsb (c.554_555delAA)54.99.1127.8 

HVA 425

OMD[UPWARDS ARROW][UPWARDS ARROW]

Peak X

P3a1 day (3 day)p.Ser492Prob (c.1474T>C)p.Ser492Pro (c.1474T>C)46.63.78117.74.6

HVA 390

OMD 225

Peak X

P4a1 day (6 day)

p.Gln253Termb

c.757C>T

p.Gly477Arg (c.1429G>C)26.83.4148.511

HVA 184

OMD 308

Peak X

P51 day (1 month)p.Thr495_Ser499del (c.1482-1G>T)p.Thr495_Ser499del (c.1482-1G>T)182.584.1139

HVA 466

OMD 98

Peak X

P61 day (4 year)p.Gly477Arg (c.1429G>C)p.Tyr406 fs (c.1217_1218delAT)5.04 9.05  
P71 day (16 year)p.Gly477Arg (c.1429G>C)?4.1    
P84 day (14 year)p.Val26 fsb (c.75C>T)p.Val26 fs (c.75C>T)4.2    
P96 day/(2.5 year)c. 1093- ?_1620+?bc.1093-?_1620+? 0.7934.4116

HVA 329

OMD 23

Peak X

P101 month (14 year)p.Gly230 fsb (c.787 + 1G>T)p.Gly230 fs(c.787 + 1G>T)3.88 26.1372
P11a3 month (3 month)p.Gly477Arg (c.1429G>C)p.Gly477Arg (c.1429G>C)145.2246.435

HVA 643

OMD 62

Peak X

P125 month (6.5 year)p.Asn167Ser (c.500A>G)p.Asn167Ser (c.500A>G)5.40.36 492

HVA 312

OMD:N

Peak X

Monoamine neurotransmitter analysis was performed on the CSF of seven patients (P1, P2, P3, P4, P9, P11, and P12) to investigate neurotransmitter alterations as consequence of cerebral PLP depletion. Peak X, an unidentified compound originally described in patients with folinic acid–responsive seizures, was present in all of them (Hyland & Arnold, 2002). In patients P2, P3, and P4 analyzed before treatment, O-methyldopa (O-MD) was found to be grossly elevated; in contrast, in the single patient showing atypical late onset disease (P11) it was normal. Patient P4, who developed severe disease and died at 10 days of age, had a very high O-MD concentration (308 nm, normal values <170), a low concentration of homovanillic acid (184 nm, normal values >428), undetectable levels of PLP in the CSF, and a high urine concentration of vanillacetic acid (27 mmol/mol creatinine, normal values: traces) mimicking a pyridoxamine 5-prime-phosphate oxidase (PNPO) deficiency.

Mutation analysis and functional studies of splicing mutations

Variant changes in ALDH7A1 were detected in all patients. One allele in patient P7 could not be characterized (Table 2) with the conventional methods used. This patient was therefore also studied by SNP array analysis; no large rearrangement was identified.

The mutational spectrum of the present patients included 12 mutations: 5 already reported (c.500A>G, c.919C>T, c.1429G>C, c.1217_1218delAT and c.1482-1G>T), and seven novel sequence changes (c.75C>T, c.319G>T, c.554_555delAA, c.757C>T, c.787 + 1G>T, c.1474T>C and c.1093-?_1620+?). Eight patients were homozygous for one mutation despite consanguinity being confirmed in only two families, and only four were compound heterozygous for two different changes. The segregation of mutated alleles was confirmed in all families except those of patients P7 and P9 (no samples were available).

SNP array analysis of DNA from the blood sample of patient P9 revealed homozygous gross genomic rearrangement of a 23-kb region containing ALDH7A1 encompassing exons 12–18 of ALDH7A1 (from SNP rs4836271 located in base pair 125872017 of chromosome 5q31 to SNP rs4835913 in base pair 125895509) (Fig. 1A). This result was validated using a customized high-resolution comparative genomic hybridization array (Metabolarray) (Fig. 1B).

image

Figure 1. Genomic rearrangement analysis of the DNA of patient P9. (A) Genome-wide SNP array (Illumina Human Hap610 BeadChip) analysis revealed a deletion of at least 23 kb encompassing exons 12–18 of ALDH7A1 (from SNP rs4836271 located in base pair 125872017 of chromosome 5q31 to SNP rs4835913 in base pair 125895509). (B) Comparative genome hybridization array study. The figure shows the whole-chromosome view of chromosome 5 and the deleted region. Green dots represent copy number loss.

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ALDH7A1 mRNA was analyzed to investigate the effect of the nucleotide changes in the splicing process. Transcriptional profile analysis of the blood of patient P10 showed the novel change c.787 + 1G>T to cause the out-of-frame skipping of exon 9, likely generating a substrate transcript for the nonsense-mediated mRNA decay system or a truncated protein (p.Gly230 fs). The nucleotide change c.1482-1G>T found in patient P5 activated a cryptic splice site inside exon 18; the transcriptional profile for the blood sample revealed a small in-frame deletion (p.Thr495_Ser499del). The reverse transcription-PCR (RT-PCR) pattern for the whole blood and lymphoblasts of patient P8 revealed the “silent” nucleotide change c.75C>T to activate a new splice site inside exon 1, potentially generating the truncated protein p.Val26 fs (Fig. 2A,B). The change c.75C>T generates a new 5′ splice site with a splice score of 0.84 (www.fruitfly.org). The patient had no other changes and the change was absent in Exome Variant Server (www.evs.gs.washinton.edu/EVS/).

image

Figure 2. Functional analysis of the c.75C>T variant change. (A) Comparison of the RT-PCR profile of a healthy control sample (lane 1) and that of patient P8 (lane 2) reveals a smaller band for the latter's lymphoblasts, generated by the activation of a new splice site in exon 1. (B) Genomic DNA sequence (upper panel) and cDNA sequence (lower panel) showing the deletion of 35 bp (r.73_108 del35). (C) Functional analysis using ex vivo splicing assay with minigenes. The constructs harbor the wild-type or mutant exon 1 fused to a fragment of exon 2 of the sepiapterin reductase (SPR) gene. RT-PCR transcriptional profile of cells transfected with each minigene harboring the normal (c.75C) and a mutant sequence (c.75T); lanes 1 and 2 show the activation of the splice site to correlate with the results for the patient's blood and lymphoblast samples.

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To evaluate the effect of this change, a hybrid minigene containing ALDH7A1 exon 1 fused to exon 2 of the SPR gene was constructed (Arrabal et al., 2011). The result of transcriptional profile analysis was similar to that observed previously in the patient's blood sample (Fig. 2C).

Attempts were made to rescue the splicing process using an antisense oligonucleotide targeting the newly activated splice site. This was transfected into patients' lymphoblasts. The RT-PCR pattern and the results of antiquitin Western blot protein analysis revealed the complete rescue of the protein, highlighting that this sequence is recognized during the splicing process, probably via its binding to U1 small nucleolar ribonucleoprotein (U1snRNP) (Fig. 3). The rescued protein was detected in the cytosol and in isolated mitochondria, as suggested by other authors (Wong et al., 2010).

image

Figure 3. Antisense therapy assay designed to block the recognition of the new 5′ splice site in lymphoblasts from patient P8. (A) Diagram of the exonic splice site created in exon 1 and the transcript obtained with and without antisense morpholino oligonucleotide (AMO) transfection. The sequence of the AMO is shown in the figure. (B) Transcriptional profile for healthy control (lane 1) and patient lymphoblasts untreated (lane 2) and treated with 30 μm (lane 3) and 50 μm (lane 4) of AMO, and when treated with a scrambled morpholino (lane 5). (C) Western blotting analysis of antiquitin (ATQ) in control lymphoblasts (C) and patient lymphoblasts untreated (−P8AMO) and treated (+P8AMO) with the morpholino antisense oligonucleotide. Lanes 1, 4, and 7 correspond to cellular total extracts; lanes 2, 5, and 8 correspond to cytosol extracts and lanes 3, 6, and 9 to mitochondrial extracts. Mitochondrial hsp70 (Hsp70mit) and α-Tubulin (α-Tub) was used as loading controls.

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Discussion

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

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.

Acknowledgments

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

This work was funded by grants from the Ministerio de Ciencia e Innovación (PI10/00455 to BP and SAF2010-17272 to LRD) and Fundación Ramón Areces. An institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa is also gratefully acknowledged.

Disclosure

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

None of the authors has any conflict of interest to disclose. 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.

References

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  2. Summary
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
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