Pyridoxine responsiveness in novel mutations of the PNPO gene

Abstract

Objective: To determine whether patients with pyridoxine-responsive seizures but normal biomarkers for antiquitin deficiency and normal sequencing of the ALDH7A1 gene may have PNPO mutations.

Methods: We sequenced the PNPO gene in 31 patients who fulfilled the above-mentioned criteria.

Results: We were able to identify 11 patients carrying 3 novel mutations of the PNPO gene. In 6 families, a homozygous missense mutation p.Arg225His in exon 7 was identified, while 1 family was compound heterozygous for a novel missense mutation p.Arg141Cys in exon 5 and a deletion c.279_290del in exon 3. Pathogenicity of the respective mutations was proven by absence in 100 control alleles and expression studies in CHO-K1 cell lines. The response to pyridoxine was prompt in 4, delayed in 2, on EEG only in 2, and initially absent in another 2 patients. Two unrelated patients homozygous for the p.Arg225His mutation experienced status epilepticus when switched to pyridoxal 5'-phosphate (PLP).

Conclusions: This study challenges the paradigm of exclusive PLP responsiveness in patients with pyridoxal 5'-phosphate oxidase deficiency and underlines the importance of consecutive testing of pyridoxine and PLP in neonates with antiepileptic drug-resistant seizures. Patients with pyridoxine response but normal biomarkers for antiquitin deficiency should undergo PNPO mutation analysis.

Figure. Mutations found in the human PNPO gene

(A) Left column: deletion c.279_290del (p.Ser93Ser, Ala94_Leu97del) in exon 3. Right column: restriction digest test: PCR with loss of restriction site (restriction site alteration—Cac8l). Patient 3 and his father are heterozygous. (B) Left column: missense mutation c.421C>T (p.Arg141Cys) in exon 5. Right column: restriction digest test: PCR with loss of restriction site upon introduction of a mismatch primer (restriction site alteration—MvnI*). Patient 3 and his mother are heterozygous. (C) Left column: missense mutation c.674G>A (p.Arg225His) in exon 7. Right column: restriction digest test: PCR with introduction of a restriction site (restriction site alteration + BspMI). Patients 1b, 2b, and 2c (4a, 4b, 5, 6, and 7 not shown) were homozygous. Heterozygote (Hz) state as identified in parents of families 1, 2, 4, 5, 6, and 7. F = father; M = mother; MW = molecular weight; P = patient; un = undigested PCR; wt = wild-type.