Adenosine kinase deficiency disrupts the methionine cycle and causes hypermethioninemia, encephalopathy, and abnormal liver function

Abstract

Four inborn errors of metabolism (IEMs) are known to cause hypermethioninemia by directly interfering with the methionine cycle. Hypermethioninemia is occasionally discovered incidentally, but it is often disregarded as an unspecific finding, particularly if liver disease is involved. In many individuals the hypermethioninemia resolves without further deterioration, but it can also represent an early sign of a severe, progressive neurodevelopmental disorder. Further investigation of unclear hypermethioninemia is therefore important. We studied two siblings affected by severe developmental delay and liver dysfunction. Biochemical analysis revealed increased plasma levels of methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) but normal or mildly elevated homocysteine (Hcy) levels, indicating a block in the methionine cycle. We excluded S-adenosylhomocysteine hydrolase (SAHH) deficiency, which causes a similar biochemical phenotype, by using genetic and biochemical techniques and hypothesized that there was a functional block in the SAHH enzyme as a result of a recessive mutation in a different gene. Using exome sequencing, we identified a homozygous c.902C>A (p.Ala301Glu) missense mutation in the adenosine kinase gene (ADK), the function of which fits perfectly with this hypothesis. Increased urinary adenosine excretion confirmed ADK deficiency in the siblings. Four additional individuals from two unrelated families with a similar presentation were identified and shown to have a homozygous c.653A>C (p.Asp218Ala) and c.38G>A (p.Gly13Glu) mutation, respectively, in the same gene. All three missense mutations were deleterious, as shown by activity measurements on recombinant enzymes. ADK deficiency is a previously undescribed, severe IEM shedding light on a functional link between the methionine cycle and adenosine metabolism.

Figure 1

The Methionine Cycle and Its Relationship with the Adenosine/AMP Futile Cycle Met is converted through AdoMet and AdoHcy to Hcy, which is subsequently remethylated back to Met. AdoMet functions as a methyl-group donor in a wide range of transmethylation reactions. The thermodynamics of the SAHH reaction favors condensation of adenosine and Hcy to produce AdoHcy; physiologically, AdoHcy is hydrolyzed when adenosine and Hcy are removed, and increased levels of adenosine therefore cause reversal of the reaction. Adenosine is phosphorylated by ADK to AMP, which can be dephosphorylated back to adenosine by 5′ nucleotidase. This so-called futile cycle, which is disrupted in adenosine kinase deficiency, is considered an important regulator of adenosine and adenine nucleotide levels. MAT, GNMT, SAHH, and CBS are deficient in previously known IEMs that directly interfere with the methionine cycle and result in hypermethioninemia. Abbreviations are as follows: ADK, adenosine kinase; AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; AMP, adenine mononucleotide; CBS, cystathionine beta-synthase; GNMT, glycine N-methyl transferase; Hcy, homocysteine; MAT, methionine adenosyltransferase; Met, methionine; and SAHH, S-adenosylhomocysteine hydrolase.

Figure 2

Sanger-Sequencing Traces of the Three ADK Missense Mutations Causing Adenosine Kinase Deficiency The first column shows results verifying the mutation detected by exome sequencing in two affected Swedish siblings: a homozygous C>A transversion at nucleotide 902 resulted in a p.Ala301Glu amino acid substitution in adenosine kinase. The second and third columns show the two homozygous mutations detected in two Malaysian families with ADK deficiency: an A>C transversion at nucleotide 653 caused a p.Asp218Ala amino acid substitution, and a G>A transition at nucleotide 38 led to a p.Gly13Glu amino acid substitution. In all cases, the control, wild-type sequences are shown on top, a heterozygous parent is shown in the middle row, and the homozygous mutations detected in the affected children are displayed in the bottom row.

Figure 3

Locations of the Mutated Residues in Human ADK (A) Overall view of human ADK, showing the relative position of the three mutated residues in the molecule and the catalytically important Asp300. Two adenosine molecules, one occupying the adenosine-binding site and the other the ATP-binding site, are depicted. (B) Close-up image showing the position of Ala301. The mutated amino acid sits between two crucial residues. Asp300 is ADK's active site responsible for the deprotonation the 5′-OH group of adenosine, which in turn attacks the gamma-phosphate of ATP. Phe302 contributes to the binding of ATP. These three residues are located in an alpha-helix between the two binding sites. Replacing Ala301, a residue with a short, neutral side chain, with glutamic acid, containing a longer, charged side chain, is likely to have deleterious consequences for enzyme function. (C) Close-up image showing the location of residue Asp218 in the central beta-sheet domain of the molecule. Asp218 sits at the N terminus of beta-strand 11, which is part of a beta-sheet domain that forms the core of the overall ADK structure. (D) Close-up image showing the proximity of residue Gly13 to the binding site for adenosine. The substitution of glycine, which has only a hydrogen atom for a side chain, for glutamic acid, which has a long, charged side chain, is likely to compromise the binding of adenosine to ADK. The numbering is based on the short isoform of ADK (isoform a; transcript NM_001123.3; protein NP_001114.2), the crystal structure of which has been resolved.

Figure 4

Adenosine Kinase Activity from Recombinant Wild-Type and Mutant Enzymes Recombinant forms of wild-type ADK, as well as ADK containing each of the mutations detected in individuals with ADK deficiency, were expressed in E. coli, purified, and tested for their capacity to phosphorylate adenosine. The graph shows the mean value + 1 standard deviation of three independent measurements carried out with 5 μM adenosine as described in the Subjects and Methods. All mutants displayed significantly impaired activity in comparison to the wild-type enzyme (two-tailed Student's t test). N.D. denotes not detectable; WT denotes wild-type.