Genetic basis of hyperlysinemia

Hyperlysinemia is an autosomal recessive inborn error of metabolism caused by a defect in the major catabolic pathway of the essential amino acid L-lysine. This pathway is primarily active in the liver and leads to the production of acetyl-CoA (Figure 1A). In the first step, lysine and α-ketoglutarate are converted into saccharopine by lysine-ketoglutarate reductase (LKR, EC 1.5.1.8). Saccharopine is then oxidized to α-aminoadipic semialdehyde and glutamate by saccharopine dehydrogenase (SDH, EC 1.5.1.9). In animals, but also other eukaryotes, both enzyme activities are catalyzed by a single mitochondrial bifunctional enzyme named α-aminoadipic semialdehyde synthase [1], which is encoded by the gene AASS. One mutation in AASS has been reported to cause hyperlysinemia (c.1601_1609del; p.C534X [2]).

Hyperlysinemia is characterized by elevated plasma lysine levels that exceed 600 μmol/L and can reach up to 2000 μmol/L (adult reference range 111–248 μmol/L) [6‐9]. Cases in which the plasma lysine levels are elevated, but remain below 600 μmol/L, are often caused by decreased availability of α-ketoglutarate [10], which occurs in urea cycle disorders, pyruvate carboxylase deficiency, methylmalonic aciduria and propionic aciduria. As a consequence of the accumulating lysine, several alternative biochemical reactions take place. Lysine can be used in place of ornithine in the urea cycle resulting in the production of homoarginine [11]. In addition, N-ε-acetyl-L-lysine, N-α-acetyl-L-lysine and pipecolic acid (Figure 1A) accumulate as a result of the use of alternative or detoxifying biochemical pathways [11, 12]. Hyperlysinemic individuals may also have saccharopinuria, when the SDH is deficient in combination with a preserved LKR activity (also known as hyperlysinemia type 2 [13]).

Although hyperlysinemia was initially associated with neurological damage and mental retardation, later reports questioned this relationship. This was mainly based on a review of 10 individuals identified with hyperlysinemia in newborn screening programs (4 cases), family surveys of a previously diagnosed case (4 cases) and during the investigation of a boy and his family for short stature (2 cases). Cases were rejected if the hyperlysinemia was diagnosed during investigation of a patient presenting with a neurological or intellectual impairment. In none of the identified 10 cases adverse effects could be attributed to hyperlysinemia [14]. Furthermore, it is unclear whether dietary restriction of lysine is beneficial [11, 15], and a child with no clinical manifestations of hyperlysinemia has been born to an affected mother [14]. These observations indicate that hyperlysinemia most likely represents a benign metabolic variant and illustrates that the association of nonspecific clinical signs and symptoms with biochemical abnormalities can be coincidental. Indeed it is currently unknown whether lysine or lysine-derived metabolites are toxic. In vitro, high concentrations of lysine (5 mM) induced oxidative damage to proteins and lipids, decreased oxidized glutathione and inhibited cytosolic creatine kinase activity [16, 17]. Intrastriatal injection of lysine in rats did not affect creatine kinase, but inhibited synaptic Na⁺,K⁺-ATPase activity, induced lipid peroxidation and decreased glutathione level [18]. It is currently unclear how these findings can be related to clinical manifestations in patients with hyperlysinemia.

Hyperlysinemia was diagnosed based on elevated plasma lysine levels in 8 patients with distinct neurological features whose material was sent to a laboratory for metabolic screening and confirmatory testing (Table 1). All patients had no detectable LKR and SDH activity in fibroblasts. For molecular genetic analysis, we sequenced all 24 exons including the flanking exon/intron boundaries of AASS by using standard Sanger sequencing techniques. To determine whether the identified mutations affect the AASS protein levels, we performed immunoblot analysis in fibroblast homogenates (Figure 1B).

Cases 1 and 2 are compound heterozygous for a transition c.194G>A in exon 2, and a transversion c.1256T>G in exon 11. Both substitutions change conserved amino acids, arginine at position 65 into glutamine (p.R65Q) and leucine at position 419 into arginine (p.L419R). In case 3 the c.194G>A mutation was homozygous, consistent with the reported consanguinity of the parents. Immunoblot analysis revealed that AASS protein was absent in case 1 and 3, indicating that both mutations primarily affect protein levels (Figure 1B). In case 4 no mutations were found in exons 2 to 24, but we failed to amplify exon 1 indicating the presence of a deletion. Indeed, no AASS cDNA and protein could be detected in this case (Figure 1B&C), indicating that this deletion interferes with expression of AASS. In case 5 we found two mutations, a heterozygous transition c.460G>A in exon 4 which changes a conserved alanine in the LKR domain into threonine (p.A154T), and a duplication of 1 basepair c.2076dup in exon 19, which creates a frame shift at amino acid position 693 resulting in a stop codon after 9 triplets (p.P693SfsX10). The c.460G>A mutation appeared homozygous at the cDNA level, indicating nonsense-mediated decay of the c.2076dup mRNA (Figure 1D). Indeed no immunoreactive protein with the predicted Mw of the truncated protein (78kDa) was observed upon immunoblotting. The detected AASS with a Mw of 102kDa is therefore the p.A154T mutant protein. In case 6, we found a homozygous transition c.2155A>G in exon 19, which changes threonine at position 719 into alanine (p.T719A). This mutation affects a highly conserved amino acid in the SDH domain of AASS. The activity of LKR is most likely at least partially conserved, which is evidenced by the fact that this patient displayed hyperlysinemia and saccharopinuria. Unfortunately, the available lymphoblasts of this patient were not suited for follow up analysis, because AASS activity and protein are undetectable in this cell type.

In cases 7 and 8 no mutations were found in exons 1 to 19, but we failed to amplify exon 20–24 indicating the presence of a large deletion. Despite the fact that the 5^′ part of the AASS cDNA could be detected (Figure 1C), no AASS protein was present in both cases (Figure 1B). In order to determine the size of this deletion, we performed CGH in case 7. This analysis revealed a homozygous deletion of 126kb at 7q31.32 (karyotype arr7q31.32(121590497–121716631)x0) involving two genes, AASS and PTPRZ1. Although not exactly known, the deletion breakpoint for AASS must be located in intron 19 or exon 20. PTPRZ1 or protein tyrosine phosphatase, receptor-type, Z polypeptide 1 is encoded by 30 exons and spans 193 kbp. The breakpoint for PTPRZ1 lies in exon 2 or intron 2, which will prevent the expression of functional PTPRZ1. PTPRZ1 is a protein tyrosine phosphatase, which is mainly expressed in the developing and mature brain (astrocytes, oligodendrocyte precursors, immature and mature oligodendrocytes). Binding with a variety of cell adhesion and matrix molecules affect intracellular phosphatase activity [20, 21]. Although PTPRZ1 knockout mice have no gross anatomical alterations in their nervous system or other organs, they do display disturbed motor coordination and nociception and have decreased recovery from demyelinating lesions [22, 23]. Thus the physiological role of PTPRZ1 may be in oligodendrogenesis, promotion of neurite outgrowth and glial adhesion [21]. Our data indicate that cases 7 and 8 have a novel contiguous gene deletion syndrome that was biochemically diagnosed as hyperlysinemia. We speculate that the severe neurological disease in these cases is caused by a loss of PTPRZ1 function.

We conclude that in our cohort of 8 cases, hyperlysinemia is caused by mutations in the AASS gene. In two patients originating from one family, the hyperlysinemia was caused by a contiguous gene deletion syndrome affecting AASS and PTPRZ1. Our findings illustrate the importance of detailed biochemical and genetic studies in any hyperlysinemia patient.