Clinical, biochemical, and molecular studies in pyridoxine-dependent epilepsy. Antisense therapy as possible new therapeutic option.
OBJECTIVE
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.


RESULTS
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.


CONCLUSIONS
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.
Center of Diagnosis of Molecular Diseases, Center of Molecular Biology UAM-CSIC, Center for Biomedical Network Research on Rare Diseases, Institute for Health Research, IDIPAZ, Autonomous University of Madrid, Madrid, Spain. bperez@cbm.uam.es
Belén
B
Pérez
Luis González
LG
Gutiérrez-Solana
Alfonso
A
Verdú
Begoña
B
Merinero
Patricia
P
Yuste-Checa
Pedro
P
Ruiz-Sala
Rocio
R
Calvo
Anil
A
Jalan
Laura López
LL
Marín
Oscar
O
Campos
Maria Ángeles
MÁ
Ruiz
Marta
M
San Miguel
Maria
M
Vázquez
Margarita
M
Castro
Isaac
I
Ferrer
Rosa
R
Navarrete
Lourdes Ruiz
LR
Desviat
Pablo
P
Lapunzina
Magdalena
M
Ugarte
Celia
C
Pérez-Cerdá
D000444:Aldehyde Dehydrogenase / Q000235:genetics; D002460:Cell Line; D004252:DNA Mutational Analysis; D004827:Epilepsy / Q000188:drug therapy* / Q000209:etiology / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D020167:Hyperlysinemias / Q000652:urine; D007223:Infant; D007231:Infant, Newborn; D008214:Lymphocytes / Q000187:drug effects; D008297:Male; D009154:Mutation / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D020641:Polymorphism, Single Nucleotide; D012326:RNA Splicing; D012443:Saccharopine Dehydrogenases / Q000172:deficiency / Q000652:urine; D053719:Tandem Mass Spectrometry; D026681:Vitamin B 6 Deficiency / Q000150:complications*
D016376:Oligonucleotides, Antisense; C089507:ALDH7A1 protein, human; D000444:Aldehyde Dehydrogenase; D012443:Saccharopine Dehydrogenases