Association of PRPS1 Mutations with Disease Phenotypes

Phosphoribosylpyrophosphate synthetase 1 (PRPS1) codes for PRS-I enzyme that catalyzes the first step of nucleotide synthesis. PRPS1 gene mutations have been implicated in a number of human diseases. Recently, new mutations in PRPS1 have been identified that have been associated with novel phenotypes like diabetes insipidus expanding the spectrum of PRPS1-related diseases. The purpose of this review is to evaluate current literature on PRPS1-related syndromes and summarize potential therapies. The overexpression of PRPS1 results in PRS-I superactivity resulting in purine overproduction. Patients with PRS-I superactivity demonstrate uric acid overproduction, hypotonia, ataxia, neurodevelopment abnormalities, and postlingual hearing impairment. On the other hand, decreased activity leads to X-linked nonsyndromic sensorineural deafness (DFNX-2), Charcot-Marie-Tooth disease-5 (CMTX5), and Arts syndrome depending on the residual activity of PRS-I. Mild PRS-I deficiency (DFNX-2) results in non-syndromic progressive hearing loss whereas moderate PRS-I deficiency (CMTX5) and severe PRS-I deficiency (Arts syndrome) present with peripheral or optic neuropathy, prelingual progressive sensorineural hearing loss, and central nervous system impairment. Currently, purine replacement via S-adenosylmethionine (SAM) supplementation in patients with Arts syndrome appears to improve their condition. This suggests that SAM supplementation can alleviate symptoms of PRPS1 deficient patients and open new avenues of therapeutic intervention.

In the energy intensive synthesis of purines, PRPP is a substrate for PRPP amidotransferase, the first and ratelimiting step of de novo purine synthesis, ultimately producing purine nucleotides guanine triphosphate (GTP) and adenine triphosphate (ATP) [4,6]. PRPP is also used as a substrate for the "salvage" of purine bases, responsible for converting adenine to adenosine monophosphate (AMP) via adenine phosphoribosyl transferase and converting inosine monophosphate (IMP) to guanine monophosphate (GMP) via hypoxanthine guanine phosphoribosyl transferase (Figure 1) [4,6]. In the pyrimidine nucleotide synthesis, PRPP  is an important cofactor for uridine monophosphate synthetase, which assists in converting orotic acid to UMP, the precursor for all pyrimidine nucleotides [6]. Lastly, PRPP plays a key role in pyridine nucleotide synthesis by nicotinate phosphoribosyl transferase (NAPRT) and nicotinamide phosphoribosyl transferase (NAMPT), which form nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), respectively. Thus, mutations in the PRPS1 can have a profound impact on several vital cell processes, such as nucleic acid synthesis, energy metabolism, and cellular signaling. PRPS1 is expressed in several different tissues [9] including prenatal and postnatal inner ear organs: cochlea, vestibular organs, and utricle [5]. The transcript levels of PRPS1 are regulated by mir-376. MicroRNAs (miRNAs) are short ∼22 nucleotide RNA sequences that bind to complementary sequences in the 3 UTR of multiple target mRNAs, thereby inhibiting protein synthesis or causing mRNA degradation [10][11][12]. Our previous study demonstrated that miR-376a-3p, b-3p, c-3p are present in mouse embryonic inner ears and intensive expression of miR-376a-3p/b-3p was detected in the sensory epithelia and ganglia of both auditory and vestibular portions of the inner ear [8]. In adult inner ear, the expression of miR-376a-3p/b-3p is restricted within ganglion neurons of auditory and vestibular systems as well as the cells in the stria vascularis [8]. Earlier studies from our laboratory also showed continuous expression of the PRPS1 gene in cochlear and vestibular hair cells as well as postnatal expression in the spiral ganglia by in situ hybridization (ISH) [5]. At embryonic day 18.5, PRPS1 was found to be expressed in the following tissues: utricle, crista, and cochlear hair cells, Claudius cells, and the greater epithelial ridge but not in cochlear supporting cells [5]. Postnatal PRPS1 expression was observed in the spiral ganglion cells and the hair cells in the organ of corti [5]. Thus, PRPS1 expression plays a role in fetal inner ear development, and mutations to the PRPS1 gene or to miRNA-376 may result in abnormal organ development.

PRPS1 Structure
The enzymatic active unit is a hexamer, consisting of three homodimers arranged in a propeller-like shape [1,2,17,24]. Each of the homodimers has an active site and two regulatory allosteric sites, I and II. The active site allows for ATP and Disease Markers 3 Arts Syndrome c.856C>T p.R196W Loss of Function Al-Maawali et al. [23] R5P to bind at the junction of two domains within one homodimer. Allosteric site I is located at the interface of the three homodimers and allosteric site II is located at the interface of two monomers within one homodimer [1,2,17,24]. Mutations in the PRPS1 can either result in a gain of function with increased expression, PRPS1 superactivity, or result in a loss of function with a decreased expression, DFN-2, CMTX5, and Arts syndrome.

PRS-I Superactivity
PRS-I superactivity is an X-linked disorder classically associated with hyperuricemia and hyperuricosuria resulting in childhood gout [25]. PRS-I superactivity has been demonstrated to exhibit a wide range of phenotypes. Patients with the milder phenotype of PRS-I superactivity, the more common phenotype, present an overproduction of purines without any neuropathy. Uric acid crystalluria or urinary stones are commonly the first clinical finding followed by gout arthritis if the uric acid levels are not controlled.
Patients with the more severe phenotype exhibit similar earlychildhood gout arthritis that is also accompanied with a combination of neurodevelopment abnormalities, hypotonia, ataxia, and sensorineural hearing loss [17,25]. Two different mechanisms have been proposed that can cause PRS-I superactivity: a gain-of-function point mutations in the open reading frame (ORF) of PRPS1 that results in an altered regulatory site, and an increased expression of PRPS1 that has normal kinetic enzyme properties [1,13,15,[25][26][27][28][29][30][31][32][33][34]. There have been more than 30 patients reported to have a severe form of PRPS1 superactivity. Seven missense mutations in the PRPS have been extracted from these patients [1,3,11,12].
Two of the seven missense mutations (c.154G>C (p.D52H) and c.385C>A (p.L129I)) causing PRS-I superactivity have been found to disrupt one of the two allosteric sites, therefore inhibiting feedback inhibition [1,35]. The p.D52H mutation results in the destabilization of the local structure around Asp52 therefore affecting allosteric site I [1]. The p.L129I mutation results in steric hindrance with the protein backbone at Ala131 and Ile134 therefore destabilizing the local structure, disrupting the allosteric site II [1].
The point mutation c.424G>C (p.V142L), a novel PRPS1 mutation resulting in an increased activity of PRS-I, was recently found in a young boy with the classic findings of uric acid overproduction but with no gout arthritis, as is typically noted in patients with PRS-I superactivity [17].

Disease Markers
The patient also experienced developmental delay, hypotonia, and bilateral hearing loss similar to the severe form of PRS-I superactivity [17]. Moreover, the patient complained of recurrent respiratory infections, myopia, glaucoma, and motor neuropathy; these manifestations mostly occur in Arts syndrome (see below) [17]. Molecular modeling of this mutation suggests that the ATP binding site is affected in addition to the allosteric site II [17]. This novel mutation exhibits a phenotype with characteristics of both gain-offunction and loss-of-function mutations. This is the first missense mutation to connect the two extremes of the PRS-I-related disorders spectrum [17].

X-Linked Nonsyndromic Sensorineural
Hearing Deafness (DFNX-2) The mildest form of loss-of-function PRPS1 activity results in DFNX-2, characterized by only having nonsyndromic hearing loss. These mutations can result in a wide spectrum of phenotypes of hearing loss: bilateral, moderate to profound, progressive or nonprogressive, and prelingual or postlingual [36,37]. Some males showed early-onset (7-20 years of age) moderate hearing loss with an upward sloping audio profile, with low to middle frequency hearing impairment, but retained the ability to hear high frequency sounds, whereas other male patients exhibited profound hearing loss and a flat audio profile with a later onset [36,37]. Female carriers can be normal or abnormal, but if they were abnormal, their hearing impairment was less profound than the affected males [16]. Thus far, four missense mutations have been identified in four separate families: c.193G>A (p.D65N), c.259G>A (p.A87T), c.869T>C (p.I290T), and c.916G>A (p.G306R) [1,5]. None of the identified mutations have any major changes on the overall structure of the enzyme, which explains the mild phenotype of only nonsyndromic hearing loss. Using molecular modeling techniques the D65N mutation has been shown to affect the ATP binding site [1,5]. Similar predictions have been made about the remaining three mutations and their effects on the overall structure. Compared to controls, in vitro enzymatic activity assays of affected patients' erythrocytes and cultured fibroblasts have a 44%-45% decrease in PRS-I activity [5].

Novel Mutations Bridging the Spectrum between DFNX-2 and CMTX5
Four new patients with novel mutations in the PRSP1 have been identified that exhibit symptoms of DFNX-2: c.337G>T (p.All3S), c.343A>G (p.M115V), c.925G>T (p.V309F), and c.362C>G (p.A121G) [18,19]. The p.A113S mutation, being the least severe of the four new mutations, only resulted in postlingual nonsyndromic hearing loss but was met with no signs of clinical or subclinical peripheral neuropathy [18]. This mutation is located at the trimer interface and is predicted, via molecular modeling, to destabilize the surrounding environment therefore affecting the ATP binding site [18].
The patients' p.M115V and p.V309F mutations resulted in postlingual nonsyndromic hearing loss with a downward sloping audio profile. In addition, these patients also showed signs of clinical and subclinical peripheral neuropathy. The mild neuropathy is predominately sensory based and is characterized by absence of deep tendon reflexes, parenthesis, and cramps; no muscle weakness of motor deficits was reported [18]. Both of these mutations are also located at the trimer interface and are predicted to destabilize the surrounding environment affecting both the ATP binding site and the allosteric site I. Lastly, the p.A121G mutation resulted in early-onset bilateral profound sensorineural hearing loss and peripheral neuropathy, resembling CMTX5 syndrome, but no optic atrophy was reported [19]. The peripheral neuropathy, compared to p.M115V and p.V309F, was both motor and sensory related; neurological exams revealed bilateral weakness and atrophy of distal muscles, prominent gait disturbances, lack of deep tendon reflexes, and severely impaired pain and temperature senses [19]. No molecular modeling was done on this mutation but based on PRPS1's primary sequence and the quaternary structure, it is expected to affect the catalytic site (ATP binding site and R5P binding site) thus reducing the enzymatic activity [19].

Charcot-Marie-Tooth Disease (CMTX5) or Rosenberg-Chutorian Syndrome
CMTX5 is an extremely rare X-linked genetic disorder with only two known missense mutations; however, due to the rarity of the disease, it may be underdiagnosed and underrecognized by pediatricians. This disorder is characterized by a "triad" of symptoms: peripheral neuropathy, early-onset (prelingual) bilateral profound sensorineural hearing loss, and optic neuropathy [20,38]. Other reported symptoms include hypotonia, gait disturbances, and loss of deep tendon reflexes [1]. Thus far, two missense mutations have been identified: c.129A>C (p.E43D) and c.344T>C (p.M115T) [30]. Both of these mutations are involved in the PRPS1 trimer interface and affect the ATP binding pocket [1,3,20]. Furthermore, p.M115T mutation is also predicted to interact with the allosteric site I [1,20]. Compared to controls, in vitro enzymatic activity assays in affected patients' cultured fibroblasts have a 62% decrease in PRPS1 activity [5].

Novel Mutations Bridging the Spectrum between Arts Syndrome and CMTX5
A new family with a novel PRPS1 missense mutation, c.830A>C (p.Q277P), has been reported with features resembling both CMTX5 and Arts syndrome. Similar to CMTX5, the patients exhibited a "triad": prelingual hearing loss, optic atrophy with early teen onset, and severe sensorineural motor neuropathy. These symptoms overlap with childhood recurrent infections, progressive muscle weakness, and mild to moderate mental and behavioral deficits, usually seen in Arts syndrome. This myriad of symptoms shows that CMTX5 Disease Markers 5 and Arts syndrome can overlap in patients expressing PRS-I hypoactivity, supporting the theme of a continuous spectrum of phenotypes [21]. The overlapping phenotype exhibited by the p.Q277P mutation can also be shown by the affect it has on PRS-I's quaternary structure. Located at the trimer interface, this mutation affects the catalytic site (similar to other Art syndromes causing mutations), mainly R5P binding site, but does not affect the allosteric sites (similar to CMTX5) [21].

Arts Syndrome
The most severe form of X-linked PRS-I hypofunction results in Arts syndrome. This syndrome is characterized by symptoms that all appear before two years of age, including profound bilateral sensorineural hearing loss, hypotonia, delayed motor development, intellectual disability, ataxia, and increase risk of infection (upper respiratory tract). Peripheral neuropathy and optic atrophy also occur in early childhood. Unfortunately, this disease is lethal with 80% of reported patients with Arts syndrome dying before the age of six [3,39].
Two loss-of-function PRPS1 mutations have been associated with Arts syndrome, namely, c.398A>C (p.Q133P) and c.455T>C (p.L152P) [22]. These mutations impair PRS-I activity most severely, thereby resulting in the most severe form of PRS-I related disorders. The p.Q113P change is predicted to destabilize the homodimer at the dimer interface (allosteric site II) and to severely destabilize the ATP binding pocket [22]. The p.L152P substitution causes interatomic interference that destabilizes the structure predominantly affecting the ATP binding site and hence PRS-I activity [22]. In vitro enzymatic activity assays in affected patients' cultured fibroblasts have a 13-fold decrease in PRS-I activity compared to controls [5].

Recent Novel PRPS1 Mutation
Recently, a novel phenotype has been associated with PRPS1 missense mutation, c.586C>T (p.R196W), in two male siblings leading to decreased PRS-I function [23]. The patients showed prenatal high -fetoprotein (MS-AFP), intrauterine growth restriction, dysmorphic facial features, central nervous system abnormalities, white matter changes, severe intellectual disability, and spastic quadraparesis. These patients also developed Leber's congenital amaurosis and short stature. For the first time, PRPS1 deficiency was associated with diabetes insipidus. This unusual phenotype expands the spectrum of PRPS1-related diseases and demonstrates the crucial role of PRPS1 in nervous system development. The p.R196W mutation affects the interaction of PRS-I with pyrophosphate and destabilizes the transition state resulting in decreased enzyme activity [23]. The identification of additional patients having this PRPS1 mutation will lead to the better characterization of phenotype-genotype correlation.

Gain-of-Function Treatment Options
Excess purine production results in hyperuricemia and hyperuricosuria, ultimately turning into uric acid and subsequently gouty arthritis, if left untreated. This is the classic finding for patients with PRS-I superactivity. The main goal of therapy for these patients is to lower the concentration of purine nucleotides and uric acid production and to prevent or even reverse urate crystal deposition. Treatment requires a dietary change and supplemental medication to be effective. Patients diagnosed with PRS-I superactivity should avoid red meat, shellfish, oily fish, and high-fructose corn syrup while increasing their low-fat dairy intake. In addition, prescribed allopurinol will help decrease uric acid production. Allopurinol is a xanthine oxidase inhibitor, stopping the conversion of oxypurine hypoxanthine and xanthine to uric acid; it also decreases de novo synthesis of purine nucleotides [39].

Loss-of-Function Treatment Options
To counter the decreased concentrations of crucial purine nucleotides, as seen with PRS-I loss-of-function disorders, it is necessary to find a method to replenish the nucleotides. Unlike dietary pyrimidines, which cross the intestinal barrier unharmed, dietary purine nucleotides are usually oxidized to uric acid by intestinal enzymes. However, dietary Sadenosylmethionine (SAM) freely crosses both the intestinal and the blood brain barrier. In an alternate pathway, independent of PRPP, SAM can theoretically replenish GTP and ATP (Figure 1). There are two pathways in the human body in which SAM can be converted into adenosine and indirectly to GTP; methyltransferases convert SAM into Sadenosylhomocysteine, which is then converted into adenosine by S-adenosylhomocysteine hydrolase, and secondly SAM is converted directly into adenosine via the polyamine pathway ( Figure 1). Adenosine can then be converted into AMP via adenine phosphoribosyltransferase (APRT). AMP can be converted into IMP via AMP deaminase, which subsequently can be turned into GTP. SAM treatment has been used to treat 2 patients with Arts syndrome (J Christodoulou et al. unpublished data) and patients with hypoxanthine phosphoribosyltransferase (HPRT) deficiency [40]. It has also been shown to elevate previously low purine nucleotide levels, alleviating the clinical symptoms, and is shown to slow the progression of hearing impairment. With early detection and diagnosis, SAM supplementation is proven to be an effective therapy in slowing down the progression and onset of both neurological and audiologic symptoms in patients with Arts syndrome. Studies are warranted for the effectiveness of SAM supplementation with less severe forms of loss-of-function PRS-I mutations.

Conclusion
PRS-I plays a crucial role in human body by producing the necessary purine and pyrimidine nucleotides. Gain-offunction and loss-of-function mutations in PRPS1 have been found and can result in a wide spectrum disorders with overlapping phenotypes depending on the residual capacity 6 Disease Markers of the PRS-I enzyme. These gene mutations manifest as either altering one of the two allosteric regulation sites (gain of function) or destabilizing the active binding site (loss of function) on the PRS-I enzyme. On one end of the spectrum, increase in PRS-I activity results in PRS-I superactivity with its defining characteristic of childhood gout and variable symptoms of sensorineural hearing loss, hypotonia, and ataxia depending on the severity of the mutation. On the other end, loss of function can result in a spectrum of phenotypes from mild to severe: DFN-2, CMTX5, and Arts syndrome. Decreased PRS-I activity is predominantly characterized by sensorineural hearing loss with variable symptoms of optic atrophy, ataxia, peripheral neuropathy, delayed motor development, and intellectual disability. Currently, treatment options for PRS-I related disorders are limited and not curative and only help slow the progression of the disease. For patients with PRS-I gain-of-function mutations resulting in uric acid overproduction, dietary restrictions and allopurinol are the mainstay treatment options. For loss-of-function mutations resulting in sensorineural hearing loss, SAM supplements in patients with Arts syndrome are able to replenish ATP and GTP concentrations to some extent, therefore slowing the progression of sensorineural hearing loss, and alleviate some of the neurological symptoms. Further large scale studies and clinical trials will help in assessing the therapeutic potential of SAM supplementation in alleviating symptoms due to PRPS1 mutations. Identification of novel biomarkers and detection of manifestations at the prenatal stage will allow the initiation of therapeutic intervention early in the disease course with improved outcomes.