Purine Functions

Purines are key players in multiple fundamental cellular processes that are ubiquitously present in cells of the human body. For example, the adenine-based nucleotide ATP is the principal cellular carrier of free energy and is essential for muscle contraction and other cellular movements, active transport of molecules, and biosynthesis of (macro-) molecules from simple precursors [1]. It should be noted, however, that in addition to being synthesized and salvaged through the metabolic pathways described above, ATP is primarily synthesized during cellular respiration. Second, the purinergic ribonucleotides (i.e. ATP and GTP) and deoxyribonucleotides (i.e. dATP and dGTP) are two of the four substrates for RNA and DNA synthesis, respectively. Third, GTP and GDP are essential in signal transduction, particularly pathways involving G proteins coupled with a cell-membrane-bound receptor [2]. In this mechanism, binding of a ligand to the G-protein-coupled receptor complex triggers an allosteric change in the G protein, causing a bound GDP to be replaced by GTP. Subsequently, the GTP activates the alpha subunit of the G protein, causing it to dissociate from the G protein and act as a downstream effector. A final generic function of purines has been proposed for the purine breakdown product, uric acid, which may act as an endogenous antioxidant.

Obviously, these universal purine functions are important for the nervous system as well. For example, the brain’s energy demand is disproportionally high, and therefore ATP consumption is large [3, 4]. In addition, many different G-protein-coupled receptors regulate synaptic communication, and the antioxidant uric acid may be relevant in brain damage. But in addition to these more common functions, purines play also very specific roles in the nervous system, as adenine nucleotides and nucleosides mediate intercellular signaling [5]. As such, ATP has been identified as a co-neurotransmitter in both sympathetic and parasympathetic nerves. Moreover, multiple purinergic receptor subtypes are widely distributed throughout the central nervous system, both in neurons and glia. Four G-protein-coupled receptors for adenosine have been recognized, as well as multiple ATP receptors that include both classic cationic ligand-operated channels for Na⁺, K⁺, and Ca²⁺ and G-protein-coupled receptors. Over the last decades, it has become clear that these purinergic signaling pathways are involved in many functional aspects of the nervous system, including the interaction between neuronal and glial networks, neural development and plasticity, regeneration, as well as specialized sensory mechanisms in the retina, inner ear, olfactory and taste system, and in pain transmission [5]. Consequently, purinergic pathways have been associated with multiple aspects of behavior, including feeding, locomotion, sleep, mood, and motivation.

Phosphoribosyl Pyrophosphate Synthase 1 Deficiency and Superactivity

PRS-1 (EC 2.7.6.1), one of three PRPP synthetases in humans, is encoded by the PRPS1 gene. PRS-1 catalyzes the first step of nucleotide synthesis, i.e. the formation of PRPP from ATP and ribose-5-phosphate.

Deficiency and superactivity of PRS-1 may result in different clinical syndromes that appear to depend on residual enzyme activity, with an X-linked recessive inheritance [9]. Overall, the mutations resulting in the severest symptoms are predicted to have the most impact on the protein structure. Mutations in the PRPS1 gene that lead to a complete absence of PRS-1 activity have been linked with Arts syndrome, characterized by developmental delay, intellectual disability, early-onset hypotonia, ataxia, hearing impairment, and optic atrophy. In addition, PRPS1 mutations that cause a partial reduction in PRS-1 activity have been linked with X-linked sensorineural deafness (DFNX1) and X-linked recessive Charcot–Marie–Tooth disease-5 (CMTX5). DFNX1 patients have mild to moderate hearing impairment, or prelingual-onset hearing loss. CMTX5 patients have peripheral sensorimotor neuropathy, sensorineural hearing loss and optic neuropathy. Finally, mutations in the PRPS1 gene that increase the activity of PRS-1, due to the lack of regulatory control, lead to hyperuricemia and sensorineural hearing loss, with or without intellectual disability to varying degrees.

It has been hypothesized that the clinical features due to PRPS1 mutations primarily result from reduced levels of GTP and possibly other purine nucleotides including ATP. However, the neurological manifestations linked with PRS may result from, perhaps additional, alterations in non-purine pathways, as PRPP is an important cofactor in pyrimidine and pyridine nucleotide metabolism as well.

Myoadenylate Deaminase Deficiency

In humans, three genes have been identified that encode AMP deaminase (AMPD, EC 3.5.4.6) that catalyzes the irreversible deamination of AMP to IMP, freeing ammonia (NH₃). Deficiency of myoadenylate deaminase (mAMPD) is an inherited autosomal-recessive disorder caused by mutations in the AMPD1 gene, the isoform predominantly involved in skeletal muscle purine metabolism [11].

The vast majority of individuals with mAMPD deficiency are asymptomatic, but non-progressive exercise intolerance accompanied by myalgia and cramping that begins in late childhood or adulthood may occur [7]. Serum creatine kinase can be increased, sometimes only after exercise. Electromyography may be normal. Mutations in the AMPD1 gene may coincide with other genetic metabolic deficiencies, having a synergistic effect on myopathic symptoms.

The myopathic symptoms are thought to arise from interruption of the purine nucleotide cycle between AMP and IMP. During exercise, the flux through this metabolic cycle is increased in skeletal muscle. mAMPD deficiency is thought to disturb one or more of the proposed functions of this cycle, including maintaining the myocytic adenylate energy charge, enhancing the glycolysis rate, providing citric acid cycle intermediates, and preserving the IMP pool to replenish ATP during recovery [12].

Deficiency of mAMPD is often diagnosed with a forearm lactate-ammonia blood test, in which skeletal muscle does not accumulate NH₃ as occurs in normal subjects. Additionally, histochemical staining of biopsied skeletal muscle, genotyping, and enzyme assay can also be used for diagnosis or confirmation.

There is currently no reliable effective treatment for symptomatic mAMPD deficiency. Avoidance of triggers such as excessive exercise is generally recommended.

Other Purine Enzyme Deficiencies with Neurological Sequelae

Several other, often very rare, purine enzyme deficiencies have been reported, for which the neurological manifestations are less well characterized (Table 27.1).

First, purine nucleoside phosphorylase (PNP, EC 2.4.2.1) deficiency is a rare autosomal-recessive immunodeficiency disorder, with decreased T-cell function. Approximately half of the patients exhibit neurological features with a broad range of severity, ranging from severe psychomotor retardation to mild motor or cognitive abnormalities.

Second, adenosine deaminase (ADA, EC 3.5.4.4) deficiency most often presents with severe combined immunodeficiency, with lethal infections in infancy or early childhood. Children who survive may develop neurobehavioral difficulties such as attention deficits, hyperactivity, aggression and social problems, as well as bilateral sensorineural hearing loss.

Third, deficiency of the mitochondrial enzyme deoxyguanosine kinase (DGK, EC 2.7.1.113) causes a severe form of mitochondrial DNA depletion. Patients typically present with liver failure, and have severe psychomotor retardation with nystagmus, cerebral atrophy, microcephaly, and hypotonia. Fatal multi-organ system failure is common in the first year of life.

Fourth, AICAR transformylase (EC 2.1.2.3) and IMP cyclohydrolase (EC 3.5.4.10) enzymes are present in one protein (ATIC). ATIC deficiency is associated with intellectual deficiency, epilepsy, dysmorphic features, and congenital blindness. The enzyme’s substrate AICAR and its dephosphorylated form, AICA-riboside, are increased in patients’ cells and urine, respectively.

Finally, mutations in the gene encoding IMP dehydrogenase (IDH, EC 1.1.1.205) have been linked with retinitis pigmentosa type 10 and some cases of Leber congenital amaurosis.

Movement Disorder in HGPRT Deficiency

The neurobehavioral phenotype of classic LND includes a characteristic motor syndrome with only minor phenotypic variability. The most prominent feature of the motor syndrome, universally present in all patients, is a generalized action dystonia (Figure 27.2) [15, 16]. Dystonia of the upper limbs usually prevents their functional use for activities of daily living, and patients use wheelchairs because lower limb dysfunction prohibits walking or standing unassisted. Some patients have severe opisthotonus or truncal arching. Although dystonia is universal and always the most severe extrapyramidal disorder in LND, many patients also have other extrapyramidal movement disorders, including choreoathetosis or ballismus (Figure 27.2). In virtually all patients, these motor features are superimposed on hypotonia when fully relaxed [15]. Corticospinal signs may sometimes occur, such as pathological reflexes or spasticity, but they are typically mild and often limited to the legs.

Figure 27.2 The movement disorder associated with HGPRT deficiency. The prevalence of both extrapyramidal and pyramidal signs in patients with classic LND and those with partial enzyme defects (LNV) are expressed as a percentage of the patient population studied. Data from Jinnah HA, Ceballos-Picot I, Torres RJ, Visser JE, Schretlen DJ, Verdu A, et al. Attenuated variants of Lesch-Nyhan disease. Brain. 2010;133(Pt 3):671–89; and from Jinnah HA, Visser JE, Harris JC, Verdu A, Larovere L, Ceballos-Picot I, et al. Delineation of the motor disorder of Lesch–Nyhan disease. Brain. 2006;129(Pt 5):1201–17.

If hyperuricemia exists along with some degree of neurological dysfunction and/or cognitive deficits, hence without self-injury, patients are described as having HGPRT-related neurological disease (HND, Table 27.2) [14]. The motor features of HND can vary among these partial phenotypes, from mild clumsiness to severe handicap similar to classic LND.

Non-Motor Features in HGPRT Deficiency

Most LND patients also have a mild or moderate intellectual disability. Reliable cognitive testing is challenging in this population, due to the motor handicap, behavioral disorder with variable cooperation, and usually limited education. With these challenges in mind, IQ scores have been estimated to fall in the 60–80 range. The most affected domains are attention and mental flexibility [17, 18]. Significant generalized cognitive impairment is not an invariable characteristic of LND, and severe intellectual deficiency is uncommon.

In HND patients, some cognitive impairment may be present that is usually less severe than in classic LND patients. The majority of HND patients have some type of learning impairment, and difficulties with attention – similar to LND – is usually the most prominent problem.

The neurobehavioral phenotype of LND includes recurrent severe self-injurious behavior, such as self-biting, self-hitting, eye poking, and others [19, 20]. This behavior is universal in LND, and considered a hallmark of the disease (Figure 27.3). Self-injurious behavior usually emerges before 4 years of age, but may not be present before the second decade of life. In addition to self-injury, patients may exhibit other difficult behaviors, such as spitting at or hitting others, use of foul language, or oppositional defiant behavior. The nature of these disinhibited behaviors is enigmatic, but appears different from purposeful aggression, as patients often apologize afterwards.

Figure 27.3 Self-injury in LND. Characteristic self-inflicted tissue damage to the lip (a) and the fingers (b) in LND patients. The challenging management of self-injury in LND requires covering any hard surfaces or sharp parts of the wheelchair with soft padding, in combination with protective restraints to prevent hitting or biting. (c) Reproduced with permission from Visser JE, Bar PR, Jinnah HA. Lesch–Nyhan disease and the basal ganglia. Brain Res Brain Res Rev. 2000;32(2–3):449–75.

By definition, HND patients do not exhibit self-injurious behavior. However, they may have impulsivity and other undesirable behaviors.

Genetic Background

LND was the first neurogenetic disorder for which the responsible gene, HPRT1, located in the q26-27 region on the X chromosome, was identified [21]. Since then, many different HPRT1 mutations causing disease have been reported, and these may appear throughout the gene. The mutations are heterogeneous, including missense, nonsense and splicing mutations, deletions, insertions, partial duplications, non-coding regulatory mutations, and more complex changes [22]. The list of known mutations in the HPRT1 gene is maintained at www.lesch-nyhan.org. It is generally believed that the majority of mutations occur de novo, since classic LND males do not reproduce.

Several comprehensive studies of genotype–phenotype correlations suggest that the most relevant factor for disease severity is the effect of the HPRT1 mutation on the residual HGPRT enzyme activity, rather than specific mutation types or sites [22, 23]. As such, less severe clinical manifestations result from mutations predicted to allow some degree of residual enzyme function. These less severely affected patients with HGPRT deficiency typically have the LNV (i.e. HRH or HND) phenotype, rather than the more severe classic LND phenotype.

Biochemical Background

HGPRT drives the recycling of hypoxanthine and guanine into their respective nucleotide pools (Figure 27.1), using the high-energy phosphate bonds of PRPP to catalyze the reaction. HGPRT pyrophosphorylates hypoxanthine into IMP, the central precursor for both adenine and guanine nucleotides, and also guanine directly into GMP. In the absence of HGPRT, hypoxanthine and guanine are degraded to uric acid. The reduced purine salvage, together with an accompanying activation of de novo purine synthesis, causes the uric acid overproduction. The subsequent hyperuricemia, if untreated, is the cause of the nephrolithiasis, gouty arthritis and tophi that is seen in HGPRT deficiency, as explained above.

Neuropathological Findings

Overall, the most frequent abnormality among imaging and autopsy studies that has been reported is a relatively small reduction in brain volume, that may escape notice in routine studies. Quantitative MRI studies showed a 17% reduction of total cerebral volume and 12–34% reduction in volumes of the basal ganglia [24]. In another cross-sectional voxel-based morphometric MRI study, LND patients showed a 17% volume reduction in gray matter compared to controls, particularly in the basal ganglia, frontotemporal, and limbic regions [25]. Moreover, the white matter volume was reduced in LND by 26%, particularly frontal white matter adjoining limbic and temporal regions and the motor cortex [26].

Autopsy studies have not revealed any signs suggestive of a degenerative process or other consistent abnormalities in any brain region. However, neurons of the substantia nigra from the LND cases showed reduced melanization and reduced immunoreactivity for tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis [27]. These findings suggest an important relationship between purine recycling pathways and the neurochemical integrity of the dopamine neurons.