Macrocytic anemia is another typical feature of LND and LNV, which is not widely recognized due to insufficient documentation of its occurrence, intensity, or medical importance in the literature (45,46). The prevalence of macrocytic erythrocytes in subjects with LND and LNV with or without anemia is relatively high. Macrocytic erythrocytes have been shown to occur in 81-92% of subjects with LND and LNV. This high prevalence underscores the significance of macrocytic erythrocytes as a common aspect of the clinical phenotype in individuals with LND and its variants (12).

Plasma hypoxanthine levels are a sensitive parameter for hypoxia in fetuses and newborns. Thus, the hypoxanthine level can be used as a potent indicator of hypoxia. Hypoxia increases purine nucleotide breakdown, generating high levels of hypoxanthine as a by-product. In patients with LND, the combination of hypoxia-induced hypoxanthine generation and HGPRT deficiency exacerbates the accumulation of hypoxanthine, contributing biochemical, renal and neurological manifestations (2,47,48). This highlights the vulnerability of patients with LND to any additional stressors, such as hypoxia, that may further overload the purine metabolism pathway. In vivo, hypoxanthine is formed by the dephosphorylation and deamination of ATP and is a hallmark of hypoxia-induced mitochondrial dysfunction. First, oxygen is required for the proper functioning of the electron transport chain and oxidative phosphorylation in the mitochondria, which ultimately produces ATP. Under low oxygen conditions, these processes are disrupted, resulting in decreased ATP production and adenosine monophosphate (AMP) accumulation. To meet these energy requirements, AMP is degraded to compensate for the loss of ATP. The increased production and degradation of AMP leads to hypoxanthine production (Fig. 1). This is the mechanism through which cells adapt to low oxygen concentrations by altering their metabolic pathways. In addition, during hypoxia, xanthine oxidase is inhibited, leading to the accumulation of hypoxanthine (49,50). The exposure of red blood cells (RBCs) to oxidative stress, whether in vivo or ex vivo, enhances purine deamination through AMP deaminase 3. This process leads to the increased accumulation of hypoxanthine, a deaminated purine. This increase in hypoxanthine levels is accompanied by changes in the morphology of RBCs, followed by increased destruction outside the blood vessels via splenic sequestration and erythrophagocytosis (51). Additionally, low oxygen levels trigger erythropoietin (EPO) gene expression, which codes for the glycoprotein hormone erythropoietin. The kidneys are the main organ in an adult organism that produces EPO (46). The glycoprotein hormone EPO increases the ability of the body to carry oxygen by encouraging the development and differentiation of erythroid precursor cells in the bone marrow, leading to an increase in red blood cell mass and macrocytic anemia.

A previous study demonstrated that RBCs from patients with LNS exhibit an accumulation of glycolytic intermediates upstream of pyruvate kinase along with elevated levels of unsaturated fatty acids and long-chain acylcarnitines. Additionally, there is an increase in highly unsaturated phosphatidylcholines in the RBCs of these patients, while free choline levels are decreased. Furthermore, intracellular concentrations of iron, zinc, selenium and potassium are also reduced in the RBCs of patients with LNS (2). Global proteomic analyses have documented alterations in RBC membrane proteins, hemoglobin, redox homeostasis proteins and the enrichment of coagulation proteins. These changes are accompanied by increased protein glutamine deamidation and methylation in both children with LNS and their carrier mothers. Allopurinol treatment partially reverses these phenotypes. However, these changes have been specifically noted in the context of the HGPRT gene mutation c.485 G>A. Ser162Asn (2). These findings suggest that complementary treatments, in addition to current regimens, such as allopurinol, could involve the supplementation of substrates to activate compensatory regulatory pathways (2). Additionally, the recycling of hypoxanthine by the X-linked HGPRT plays a crucial role in maintaining IMP/GMP homeostasis in RBCs. In patients with LNS, genetic mutations in this enzyme result in various clinical symptoms, including macrocytic anemia (12).

HGPRT deficiency also affects behavioral symptoms, which are a consequence of disrupted dopamine pathways in the basal ganglia (52). Previous research was conducted to elucidate the dysregulation in the development of dopamine neurons in MN9D derived HGPRT-ve (HGPRT-deficient) cell lines (52,53). Two mechanisms were proposed based on the results of that study: i) Microarray analysis of these cell lines revealed the diminished expression of tyrosine hydroxylase. This result is in line with previous experiments conducted to demonstrate the dysregulation of biochemical markers related to the dopamine phenotype (52). ii) Another finding was the overexpression of engrailed genes En 1 and En2, which are transcription factors that play vital roles in neural development, that is, the development and survival of dopamine neurons. The results revealed that En2 was expressed in all fibroblasts and in higher amounts in patients with neurobehavioral problems, suggesting an inverse relationship between HGPRT and En. However, the mechanism by which HGPRT levels regulate the expression of En genes is yet to be determined (53).

Similarly, HGPRT deficiency has also been shown to exhibit a deregulatory effect on guanine metabolism, and hence, it affects G protein-coupled receptor (GPCR) expression. For instance, altered and structurally defective expression of P2Y1 (GPCR) is associated with aberrantly phosphorylated ERK (p-ERK) and cAMP response element-binding protein (CREB) signaling (54). Notably, p-ERK can be transported into the nucleus, where it activates various transcription factors, such as CREB. CREB is crucial for the transcription of numerous neuronal genes, and is essential for long-term synaptic plasticity. Therefore, defects in these genes may affect neuronal development. Compared to LNV, patients with LND exhibit greater reductions in fractional anisotropy across the brain and specific disruptions in the corpus callosum, corona radiata, cingulum, internal capsule and superior longitudinal fasciculus. These deficits in white matter organization are associated with more severe dystonia and cognitive impairments, highlighting the need for the further exploration of the role of white matter in the pathogenesis of LND (14). Emerging studies emphasize that HGPRT deficiency affects neurodevelopment and neurocognitive function through metabolic and cellular disruptions. For example, alterations in white matter integrity, reduced neuronal connectivity and neurobehavioral symptoms have been linked to changes in neurexin expression, genes critical for synaptic function that are associated with autism, schizophrenia, and Alzheimer's and Parkinson's disease (55). NAV3, another identified gene, has been implicated in neurodevelopmental disorders and neuromuscular responses, further suggesting a role in LND-associated neuronal morphogenesis (56). Additionally, hypoxia-induced increases in hypoxanthine levels, a characteristic metabolic disruption in LND, underline the pathophysiological burden on both the central nervous system and peripheral processes. This highlights the interplay between metabolic imbalances and neurobehavioral outcomes in HGPRT deficiency. Stratification studies from analogous conditions, such as traumatic brain injury, suggest that white matter disorganization may serve as a prognostic biomarker for neurocognitive trajectories and may provide a framework for understanding HGPRT-associated symptoms (57). These findings collectively underscore the critical need to elucidate the molecular and structural underpinnings of HGPRT-associated neurobehavioral deficits, aiming to identify targeted therapeutic interventions and improve the quality of life of affected individuals. A previous study revealed that several microRNAs (miRNAs/miRs) from the miR-17 family cluster, along with genes encoding guanine nucleotide exchange factors, are dysregulated in HGPRT deficiency (41). Notably, the EPAC displays a reduced expression in HGPRT-deficient human neuron-like cell lines and fibroblast cells from patients with LNS. Similar alterations have been observed in the cortex, striatum and midbrain of HGPRT knockout mice (41). These dysregulations lead to the impaired activation of the small GTPase RAP1, which is critical for cytoskeletal dynamics. Consequently, HGPRT-deficient cells exhibit an altered motility compared to controls. HGPRT deficiency also results in the dysregulation of miR-181a (58). The expression of miR-181a is elevated in HGPRT-deficient human dopaminergic SH-SY5Y neuroblastoma cells, which in turn leads to the aberrant expression of target genes involved in mammalian central nervous system (CNS) development (59). Utilizing miRNA-based target predictions, researchers have identified critical signaling pathways for potential therapeutic targeting in LNS. A hypothetical model further proposes that HGPRT mRNA transcripts function as competitive endogenous RNAs, engaging in complex regulatory crosstalk with key neural transcripts and miRNAs, potentially amplifying the gene's pleiotropic effects on diverse pathways (60). To further investigate these mechanisms, fibroblasts derived from patients with LNS were reprogrammed into induced pluripotent stem cells (iPSCs) using a combined miRNA/mRNA reprogramming approach (61). This innovative methodology facilitated the development of LNS-specific iPSC lines for in-depth mechanistic and therapeutic research, providing a valuable model system for dissecting the molecular basis of this rare metabolic disorder and guiding the identification of novel biological targets.

Another study was conducted to further elucidate the effects of HGPRT on mitochondrial energy metabolism in the brain (24). The researchers used a CRISPR mouse as their model organism, which carried the same Hgprt1^del8Val mutation as found in humans for LN. This mutation causes the homodimerization of HGPRT, thereby reducing its activity. These findings indicate that there is an inhibitory effect of HGPRT deficiency on complex I, that is, NADH:ubiquinone oxidoreductase. As a result, the rate of consumption of NADH is suppressed, which ultimately results in decrease in mitochondrial membrane potential and increased mitochondrial reactive oxygen species (ROS) production. The increase in mitochondrial ROS production may be due to increased levels of xanthine, leading to the production of superoxide anions by oxidase (62,63). This diminished membrane potential leads to decreased ATP production. The low consumption of NADH by complex I results in NADH accumulation. To compensate for lower levels of ATP, cells acquire glycolysis (anaerobic metabolism) to fulfil their energy requirements, particularly when mitochondrial respiration is compromised owing to the inhibition of complex I. The HGPRT deficiency inhibits complex I-dependent mitochondrial respiration, leading to elevated mitochondrial NADH levels, a reduction in mitochondrial membrane potential, and an increased production rate of ROS in both the mitochondria and cytosol. Despite the heightened ROS production, there is no evidence of oxidative stress, as endogenous glutathione levels remain unaffected. This suggests that the disruption of mitochondrial energy metabolism, rather than oxidative stress, may act as a trigger for the brain pathology characteristic of LNS (24).

Further research using HGPRT-deficient rat B103 neuroblastoma models has identified a significant reduction in adenylate cyclase 2 expression, implying a potentially critical role of adenylate cyclase 2 in LNS-associated neurobehavioral abnormalities (64). However, validation in more advanced models, such as in vivo systems, is necessary to better elucidate the association between adenylate cyclase 2 and the pathogenesis of LNS (65). These findings highlight the critical link between HGPRT deficiency, mitochondrial dysfunction, and the downstream neurological impacts of LNS.

One strategy for the prevention of SIB is through tooth removal. However, this method is not ethical and is not permitted for use by the majority of parents. Personalized/customized intraoral devices and lip bumpers/lip guards are alternative options for the removal of teeth and are used to prevent oral and peri-oral trauma following their application (66,67).

Botulinum is a toxic compound produced by Clostridium botulinum and other related species, such as Clostridium butyrricum, Clostridium barati, and Clostridium argentinensis. Botulinum toxin works by blocking the release of the neurotransmitter acetylcholine, which activates muscle cells and helps them to contract. Thus, when acetylcholine is not released, muscles are in a relaxed state. This toxin is potentially toxic to nerve cells and thus causes paralysis; however, it can be used in optimal or controlled amounts (Fig. 2). BTX-A is also used to reduce the need for more invasive interventions, such as tooth extraction in patients with LNS. To date, there are only a few reported cases of patients with LNS treated with BTX-A; however, these cases vary in terms of dose, site and duration of the injection (Table I) (68,69).

Patients with LNS have low levels of dopamine in the basal ganglia due to the reduced activity of tyrosine hydroxylase, which is the rate-limiting enzyme in dopamine synthesis (70,71). Low levels of dopamine are associated with uncontrolled body movements. Of note, an ~50-63% reduction in the binding of the WIN-35,428 ligand to dopamine transporter was shown in the caudate region and a 64-75% reduction in the putamen region in patients with LNS (72). Therefore, to compensate for the loss of dopamine, levodopa (L-DOPA) is administered to treat movement disorders and SIB. Levodopa is a dopamine precursor, which is metabolized to dopamine in the periphery and in the CNS. Aromatic-L-amino acid decarboxylase (AADC) converts levodopa to dopamine. In contrast to dopamine, levodopa can cross the blood-brain barrier. Therefore, levodopa is prescribed over the direct injection of dopamine. However, the bioavailability of levodopa is low in the CNS. To overcome this issue, levodopa is administered in combination with carbidopa. Carbidopa (L-alpha-methyldopa hydrazine inhibits AADC by binding irreversibly to pyridoxal 5'phosphate, thereby blocking the conversion of levodopa to dopamine in the periphery. However, it does not block conversion in the CNS. Therefore, carbidopa increases the bioavailability of dopamine in the CNS. It also reduces the side-effects associated with the use of levodopa, such as nausea, vomiting and diarrhea. However, the results have been inconsistent and not encouraging (Table II) (73).

Due to these unanticipated complications, which worsen the condition of patients, none of the patients have completed the planned titration phase. Levodopa has no effects on the behavioral aspects, but triggers more adverse motor movements (73-75). These results suggest that the use of levodopa and carbidopa is not advantageous for reducing the symptoms of patients with LNS. It is possible that the mechanism in LND is different than from in DOPA-responsive dystonia, and the responses to medications may not be similar. Therefore, it may be interesting to determine the mechanisms underlying the lack of response to these two drugs in patients with LNS. These types of studies require replica HGPRT-deficient models that can help in determining the cause of drug failure. In addition, instead of combinion therapy, monotherapies of levodopa and carbidopa should be attempted in LNS models. It is essential to consider the fact that carbidopa inhibits AADC activity and, therefore, blocks the conversion of levodopa to dopamine.

SAMe is broken down to yield an adenosyl moiety, which is then converted into AMP (76). This AMP can be converted into either ATP or GTP in the brain, thereby replenishing the nucleotide pool in patients with LND that is otherwise depleted, thereby improving their condition. SAMe supplementation may help reduce the extrapyramidal symptoms associated with dopamine hypersensitivity by boosting the synthesis of GTP, which is crucial for dopamine production, and by enhancing the function of catechol-O-methyltransferase, an enzyme that plays a role in the inactivation of dopamine. Moreover, SAMe is readily taken up into the bloodstream and can cross the blood brain barrier, thereby making purine nucleotides available in the brain. Thus, it can also be used to treat other mental health disorders related to nucleotide depletion. The results of the use if SAMe have been encouraging in patients with LNS (Table III) (7,77,78). From these cases, it is evident that SAMe tends to be more effective in younger patients with LND. However, further research in the context of age-related responses to SAMe is required.

Allopurinol is converted to its active metabolite, oxypurinol, which inhibits xanthine oxidase. Xanthine oxidase is the enzyme that converts hypoxanthine to xanthine and further to uric acid. Therefore, it first stops the production of uric acid and then increases the concentration of both hypoxanthine and xanthine in serum and urine (Fig. 3). The renal clearance of hypoxanthine and xanthine is very rapid compared to that of uric acid, and its plasma concentration is only partly elevated (79). The half-life of allopurinol is ~1-3 h and that of oxypurinol is 12-30 h. When Allopurinol is not used, these oxypurines are secreted through the urine in the form of uric acid. However, when allopurinol is administered, the urinary output is composed of hypoxanthine, xanthine and some amounts of uric acid, thereby reducing the risk of crystalluria, a condition characterized by the presence of uric acid crystals in urine, nephrolithiasis, nephrocalcinosis, and sometimes, acute or chronic kidney impairment (80). These oxypurines are easily cleared by kidneys. Advanced stages of gout are characterized by the formation of tophi, which are yellow-colored lesions that form around joints. The tophi are composed of a uric acid (monosodium urate) core and skin that can become stretched and taut, sometimes to the point of ulceration. Allopurinol aids in the dissolution of tophi (81). However, higher concentrations of allopurinol are responsible for the formation of xanthine stones and oxypurinol-7-riboside, which causes derangement in the de novo synthetic pathway of pyrimidine by blocking the enzyme ornithine decarboxylase (82). As a result, orotidine 5' monophosphate begins accumulating and is rapidly degraded to orotidine, which cannot be degraded further and thus starts accumulating in RBCs and urine (Fig. 4) (83). To avoid xanthine stone deposition, it is advisable to have a high fluid intake to maintain a neutral or alkaline pH. Treatment with allopurinol did not correct the movement disorder or SIB. For example, in a study conducted on two male pediatric patients with a novel LND mutation, allopurinol normalized urate levels in RBCs and lowered serum uric acid levels, thereby lowering the risk of kidney stones, but did not help with movement disorder (2). Although allopurinol is very effective in maintaining urate levels, its downstream products, namely 5-OHisourate and allantoate, do not respond well to allopurinol.

DBS is a neurosurgical procedure that involves implanting electrodes in specific brain regions to control activity. The electrical impulses generated by these electrodes are controlled by an implantable pulse generator, which is fitted to the chest below the clavicle (beneath the skin) (Fig. 5) (84). DBS is used to treat neuropsychotic/neurological disorders, where medication has failed to provide any relief. Depending on the case profile of each individual, a physician/clinician determines the strength of the pulse to be delivered, the duration of the pulse, how long it should last, and how many times it has to be repeated for the patient to obtain optimal results (85). Achieving optimal effectiveness requires adjusting stimulation parameters, including voltage, pulse width and frequency (86). The exact mechanisms through which DBS functions are not yet fully understood. It is generally accepted that it functions by stimulating and/or inhibiting neurons that are in close proximity to electrodes. Low-frequency stimulation can excite the nearby cells. However, high-frequency stimulation tends to have an irreversible effect by lowering local activity (87). Four primary hypotheses have been proposed to explain the beneficial effects of DBS in movement disorders: Depolarization blockade, synaptic inhibition, synaptic depression, and modulation of abnormal oscillations in pathological networks (88). The globus pallidus (GP) is a subcortical triangular structure, present below the cerebral cortex, medial to the putamen. The main function of GP is to control voluntary movements and consciousness (89). Therefore, DBS of the GP is used to treat movement disorders (as in Parkinson's disease) and medication-resistant mental health conditions. Examples of such case studies are presented in Table IV (90,91).

This approach focuses on correcting the mutant HGPRT gene in individuals with LNS. To check the functionality and reliability of this procedure, researchers first developed an LNS disease model using human near-haploid cells (HAP1 cells). Human near-haploid (HAP1) cells were created from the chronic myelogenous leukemia cell line, KBM-7, which was obtained from a male patient; hence, it did not have a Y chromosome; as a result, haploid HAP1 cells have one X chromosome (92). The researchers opted to work with this particular cell line due to the following reasons: i) These are of human origin; hence, the results generated would be much more accurate compared to non-human cell lines; ii) these are haploid in nature; iii) the HAP1 cell line is the optimal model for genetic manipulation as these cells have a high transfection efficiency and responsiveness to CRISPR-based editing techniques; and iv) this allows researchers to induce specific mutations and study the underlying pathophysiology in a controlled environment in vitro.

First, c.430C>T and c.508C>T mutations were introduced in HAP1 and 293T/17 cell lines using cytosine base editors (CBE). CBEs have a cytosine deaminase, which is a catalytically modified cas9 enzyme that can carry out the transition of C:G to T:A, whereas adenine base editors (ABEs) induce the transition of A:T to G:C and contain a catalytically modified cas9 adenine deaminase. These were then successfully corrected using ABEs, demonstrating the potential of base editing for gene therapy (93,94). Not only did it correct the mutation, it also rendered the hgprt gene functional. In HAP1 cells, mutations can be fixed using ABEs.

According to this study, ABEmax-SpG was able to repair the c.508C>T mutation by as much as 5.2% without resulting in bystander mutagenesis (20). ABEmax-xCas9(3.7) also repaired the c.430C>T mutation by up to 3%. These findings demonstrate the efficaciousness of ABEs in correcting particular HGPRT1 mutations in HAP1 cells, while in 293T/17 cells the correction efficiencies were observed to be as high as 3% for the c.430C>T mutation and up to 5.2% for the c.508C>T mutation, with only minimal bystander mutagenesis occurring within the active window of base editing. highlighting their potential for therapeutic gene editing applications.

Second, the c.333_334ins(A) mutation was introduced using prime editors (PEs) in HAP1 cell lines using prime editing guide RNA (pegRNA) designed for this specific mutation. The same mutation was also present in patient-derived fibroblasts. Patient-derived fibroblasts were used to interpret and analyze the applicability of this approach in a clinically relevant context. This pegRNA guides the PE to the site of the gene where changes must be made. Whatever correction/change needs to be induced, the sequence for that is present in the pegRNA. This specific mutation was found in a 9-year-old male pediatric Korean patient with LNS, which mimicked this mutation in the HAP1 cell line and 293T/17 cells, and this was successfully corrected using PE, although the efficiency varied (20). Mutations in 293T/17 cells could be corrected only up to an efficiency of 50%, whereas it was much lower in patient-derived fibroblasts (only 14%). Similarly, it was also shown that adenine and cytosine BEs corrected mutations without DNA cleavage, while improved PEs achieved up to 14% correction in fibroblasts with minimal off-target effects, highlighting their potential as therapeutic tools for this rare genetic disorder (20).