Transaldolase haploinsufficiency in subjects with acetaminophen-induced liver failure

Abstract

Transaldolase (TAL) is an enzyme in the pentose phosphate pathway (PPP) that generates NADPH for protection against oxidative stress. While deficiency of other PPP enzymes, such as transketolase (TKT), are incompatible with mammalian cell survival, mice lacking TAL are viable and develop progressive liver disease attributed to oxidative stress. Mice with homozygous or heterozygous TAL deficiency are predisposed to cirrhosis, hepatocellular carcinoma (HCC) and acetaminophen (APAP)-induced liver failure. Both mice and humans with complete TAL deficiency accumulate sedoheptulose 7-phosphate (S7P). Previous human studies relied on screening patients with S7P accumulation, thus excluding potentially pathogenic haploinsufficiency. Of note, mice with TAL haploinsufficiency are also predisposed to HCC and APAP-induced liver failure which are preventable with oral N-acetylcysteine (NAC) administration. Based on TALDO1 DNA sequencing, we detected functional TAL deficiency due to novel, heterozygous variations in two of 94 healthy adults and four of 27 subjects with APAP-induced liver failure (P = .022). The functional consequences of these variations were individually validated by site-directed mutagenesis of normal cDNA and loss of activity by recombinant enzyme. All four patients with TAL haplo-insufficiency with APAP-induced liver failure were successfully treated with NAC. We also document two novel variations in two of 15 children with previously unexplained liver cirrhosis. Examination of the National Center for Biotechnology Information databases revealed 274 coding region variations have been documented in 1125 TALDO1 sequences relative to 25 variations in 2870 TKT sequences (P < .0001). These findings suggest an unexpected prevalence and variety of genetic changes in human TALDO1 with relevance for liver injury that may be preventable by treatment with NAC.

Keywords: acetaminophen; liver disease; prevalence; transaldolase deficiency; variations.

Figure 1

Variations in the TAL coding sequence and resultant amino acid changes that affect enzymatic activity of the translated protein. The nucleotide sequence is based on the TAL cDNA with a GenBank accession number L19437.2,1, 10 which is encoded by the TALDO1 locus in region 15.5 of chromosome 11 with GenBank accession number AF058913.11 The wild‐type cDNA is comprised of 1319 nucleotides that encode 337 AMino acids, with the first methionine (M) being at base position 51. In the two healthy control subjects (C1 and C2) with reduced TAL activities, C → T transition at nucleotide position 225 and 59‐base deletion between positions 272‐330 (deleted nucleotides are underlined; potential alternative splice donor GT and splice acceptor AG sites are bolded) resulted in early termination codon at amino acid positions 39 and 94, respectively. In two subjects with congenital liver fibrosis from Minnesota (CLF‐MN) and Turkey (CLF‐TU), an insertion of nucleotide G after base position 102 resulted in a frame shift at amino acid position 18 (L18R) and a termination codon at position 81 and a C610T point variation causes serine to phenylalanine substitution at amino acid residue 187 (S187F), respectively. In four subjects with APAP‐induced liver failure, single nucleotide polymorphisms caused amino acid substitutions D202N, A246T, Y221C, and A248P

Figure 2

Reduced enzymatic activity of TAL in patients with APAP‐induced liver failure. A, Western blot detection of recombinant TAL with amino acid substitutions D202N, A246T, Y221C, and A248P. The functional consequences of these genetic variations were individually regenerated by site‐directed mutagenesis of recombinant human TAL cDNA cloned into the pGEX‐2 T expression vector. The presence of intended mutations and the absence of unintended mutations were confirmed by sequencing of both strands of the expression vectors. Recombinant proteins were expressed in Escherichia coli, affinity purified by binding to glutathione‐coated agarose beads, and cleaved by thrombin from GST in parallel with wild‐type TAL. B, Enzymatic activities of wild‐type and mutated recombinant TAL proteins were tested in parallel in three independent experiments. *P < .05 when comparing individual mutant proteins to wild‐type TAL; horizontal lines reflect differences in enzymatic activities between mutant proteins at P < .05

Figure 3

Frequency of variations in nucleotide sequences of TALDO1 and TKT submitted to NCBI. A, 274 variations were reported within the open reading frame (ORF) of TALDO1 in 1125 human TALDO1 sequences deposited in NCBI (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=6888; Table S1). In comparison, 25 variations were reported within the ORF of TKT in 2870 sequences deposited in NCBI (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=7086; Table S2). B, Bar chart representation of 274 coding sequence variations in the 1011‐nucleotide‐long ORF of TALDO1 in comparison to 25 variations in the 1893‐nucleotide long ORF of TKT. C, Bar chart representation of 140 AMino acid changes documented within the 337 residue‐long peptide of TAL in comparison to detection of 12 AMino acid changes in the 631‐residue TKT protein. P values indicate differences using two‐tailed chi‐square analyses