Arts Syndrome Is Caused by Loss-of-Function Mutations in PRPS1

Genetic Mapping of Australian Family F

DNA from family F was extracted from peripheral whole blood by the salting-out method as described by Miller et al.¹⁵ Preliminary X-chromosome mapping in family F was performed using the ABI Prism set version 2, panel 28 (X chromosome), obtained from Applied Biosystems. This set comprised primers for 18 fluorescently labeled microsatellite markers dispersed at intervals of ∼10 cM over the entire X chromosome. Markers for the fine mapping of the candidate regions were spaced at 0.4–5 cM. The forward primers for these microsatellites were fluorescently labeled with Fam, Hex, or Ned. PCR conditions were optimized for individual primers and are available on request.¹⁶ PCRs were performed in a 9600 thermocycler (Applied Biosystems). The programs used were 95°C for 12 min for one cycle, followed by 35 cycles of melting at 94°C for 15 s, annealing at the optimal temperature for 15 s, and then extension at 72°C for 30 s. A final extension was performed at 72°C for 4 min. PCR products were run on an ABI 377 sequencer (Applied Biosystems). Exclusion mapping was used to identify candidate regions on the X chromosome under the following assumptions: alleles not shared between the two affected brothers were excluded, alleles shared between the affected brothers and their unaffected maternal uncle were excluded, and alleles inherited from the maternal grandfather were excluded.

Cell Culturing

Skin fibroblast cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM [Gibco]) supplemented with 20% (v/v) fetal calf serum (Sigma), 1% 10-U/μl penicillin with 10-μg/μl streptomycin (Gibco), 1% GlutaMAX (Gibco), and 1 mM sodium pyruvate (Gibco). Cells were harvested at 80%–90% confluence for both RNA isolation and PRPP synthetase activity analysis by washing once with PBS buffer (8 mM Na₂HPO₄, 2 mM KH₂PO₄, 137 mM NaCl, and 2.7 mM KCl [pH 7.2]), followed by treatment with 0.25% trypsin (Sigma) in PBS. Cells were centrifuged at 200 g for 5 min at room temperature, were washed with PBS, and were pelleted by centrifugation at 200 g for 5 min at room temperature. This last step was repeated for PRPP synthetase activity analysis. Pellets for both RNA isolation and enzyme activity analysis were immediately snap frozen in liquid nitrogen.

Human B-lymphocytes were immortalized by transformation with Epstein-Barr virus in accordance with established procedures.¹⁷ EBV-LCLs from patients and controls were grown to a density of 0.7 million cells per ml RPMI 1640 medium (Gibco) containing 10% (v/v) fetal calf serum (Sigma), 1% 10-U/μl penicillin and 10-μg/μl streptomycin (Gibco), and 1% GlutaMAX (Gibco). A total of 25 million cells were harvested by centrifugation at 200 g for 5 min at room temperature, were washed with PBS, and were pelleted by centrifugation at 200 g for 5 min at room temperature. The pellets for enzyme activity analysis were immediately snap frozen in liquid nitrogen.

RNA Isolation

Total RNA was isolated using the RNeasy Mini Kit (Qiagen) in accordance with the manufacturer’s protocol. To remove residual traces of genomic DNA, the RNA was treated with DNase I (Invitrogen) while bound to the RNeasy column. The integrity of the RNA was assessed on an agarose gel, and the concentration and purity were determined by optical densitometry. Poly-A⁺ RNA was isolated using the Oligotex Direct mRNA Kit (Qiagen) in accordance with the manufacturer’s protocol.

Expression Profiling

Total RNA from eight healthy control males and females was divided into two reference pools consisting of four different samples. These served as references to establish differential gene expression in fibroblast cell lines of the two affected members of family N032. GeneChip expression analysis using the GeneChip Human Genome U133 Plus 2.0 Array was performed in accordance with the manufacturer’s protocol (Affymetrix) by use of total RNA and the one-cycle target-labeling assay. Data from both affected family members were averaged and were compared with the combined reference data by use of Genespring (Agilent Technologies). Probe sets that had a signal value <100 were regarded as “not expressed.” In addition, probe sets with probes in intronic sequences that were not supported by human mRNAs from GenBank or by spliced ESTs were not considered. If a probe set was reduced in expression, the probes in that particular set were checked for redundancy and were discarded if more than half the probes in the set could bind to more than one transcript. When multiple probe sets were present for one transcript, at least two had to be reduced in expression.

First-Strand Synthesis for Quantitative PCR (qPCR)

The poly-A⁺ RNA equivalent of 5 μg of total RNA was transcribed into cDNA as follows. Poly-A⁺ RNA (0.2 μg total RNA/μl), 0.05 U/μl pd(N)₆ (Amersham Biosciences), and 1 mM deoxyribonucleotide triphosphate (dNTP) (Invitrogen) were incubated for 5 min at 65°C. The mixture was then cooled on ice for 1 min. First-strand buffer (Invitrogen), 10 mM dithiothreitol, and 0.5 U/μl RNA Guard (Amersham Biosciences) were added. One-tenth of the reaction mixture served as the minus–reverse transcriptase control. M-MLV reverse transcriptase (Invitrogen) was added for a final concentration of 10 U/μl. This solution was, in subsequent steps, incubated at 25°C for 10 min, at 50°C for 50 min, and at 70°C for 15 min. cDNA was purified using QIAquick columns (Qiagen) in accordance with the manufacturer’s protocol.

qPCR

qPCR was performed by SYBR Green–based quantification in accordance with the manufacturer's protocol (BioRad) by use of an iCycler (MyiQ single-color real-time detection System [BioRad]). Primers were developed using the Primer3 program.¹⁸ In the case of GUSB (MIM 253220) and CUL4B (MIM 300304), the primers were designed on two separate exons. GUSB is stably expressed in fibroblasts and thus is a good reference gene to use.¹⁹ Since PRPS1 and PRPS2 are very similar, primers had to be designed that anneal to the 3′ UTR of these genes. Sequences of the qPCR primers are shown in table 1. PCR products were 80–120 bp in length. All primer pairs were validated in triplicate by use of serial cDNA dilutions that result in end concentrations equivalent to 800, 400, 200, 100, and 50 pg/μl of total RNA input into the first-strand synthesis. Primers that were 100%±5% efficient, which implies a doubling of PCR product in each cycle, were used to quantify mRNA levels. qPCR quantifications were performed with the equivalent of 400 pg/μl of total RNA input into the first-strand synthesis from two separate first-strand syntheses each in duplicate, and they included a water or minus–reverse transcriptase control. The quantification was repeated on separately grown cell lines, and the results were averaged. Experimental threshold cycles (Ct) values were within the range of cDNA dilutions used to validate the primers. The melting curves of all PCR products showed a single PCR product. All controls were negative. Differences in the expression of a gene of interest between two samples were calculated by the comparative Ct or 2^ΔΔCt method.^20,21

Mutation Analysis

Primer sequences for amplification of all exons of CUL4B (GenBank accession number NM_003588.3) and PRPS1 (GenBank accession number NM_002764.2) are shown in table 1. Of the last exon of CUL4B and PRPS1, 210 nt and 750 nt of the 3′ UTR were analyzed, respectively. PCR conditions are available on request. PCR products were sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing version 2.0 Ready Reaction Kit and were analyzed using the ABI PRISM 3730 DNA analyzer (Applied Biosystems). In the case of TRPC5 (GenBank accession number NM_012471.2; MIM 300334), the entire coding region of the candidate gene was amplified using the methods of Sossey-Alaoui et al.²² Automated ABI 377 sequencing was performed using both forward and reverse primers from each PCR.

The segregation of the c.455T→C nucleotide change in PRPS1 in family N032 and its presence in 169 healthy males and 70 healthy females were tested by amplification of exon 4 and subsequent digestion by BsmFI in accordance with the manufacturer’s protocols (Invitrogen). The segregation of nucleotide substitution c.398A→C in PRPS1 in family F and its presence in 154 healthy females were tested by amplification of exon 3 and subsequent digestion with ApoI in accordance with the manufacturer’s protocols (Invitrogen).

Molecular Modeling

The effect of the p.Q133P and p.L152P mutations on the structure of PRS-I was examined using the crystal structure of human PRPS1 from Li et al.²³ (RCSB Protein Data Bank entry 2H06). The altered amino acid side chains in the model were positioned using a backbone-dependent rotamer library as implemented in the Yet Another Scientific Artifical Reality Application (YASARA) program. The models were subsequently refined using the Yamber2 force field, which elsewhere was shown to increase model accuracy.²⁴ Coordinate files are available from the authors on request.

PRPP Synthetase Activity Analysis

PRPP synthetase activity in erythrocytes is based on high-performance liquid chromatography (HPLC) measurement of adenosine monophosphate (AMP), which is produced from the enzyme reaction in equimolar amounts with PRPP. The assay contains diadenosine pentaphosphate (A2P5), which inhibits dephosphorylation of ATP and adenosine diphosphate (ADP) by nonspecific phosphatases. In brief, erythrocytes were washed in saline and were stored frozen until required. For the assay, 100 μl of packed erythrocytes were lysed by freeze thawing in 500 μl buffer comprising 10 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, and 1 mM EDTA. Centrifuged lysate was dialyzed to remove erythrocyte nucleotides, by passing through a Sephadex G-25 column equilibrated with the lysis buffer mentioned above containing 0.9% (w/v) NaCl. The 150-μl assay mixture contained 50 mM Tris-HCl, 0.5 mM ATP, 0.25 mM ribose-5-phosphate, 0.25 mM A2P5, 1 mM dithiothreitol, 5 mM MgCl₂, and inorganic phosphate that varied in concentration from 0 to 32 mM. Each assay was commenced by the addition of 50 μl of dialyzed hemolysate and was incubated for 20 min at 37°C. The reaction was terminated by the addition of trichloroacetic acid, and the supernatant was subjected to ion-pair HPLC to separate the AMP.²⁵

Frozen EBV-LCL and fibroblast pellets were suspended in 300 μl 0.9% (w/v) NaCl and were sonicated three times at 4 W for 10 s, with intervals of 30 s under constant cooling in ice water. After centrifugation (11,000 g at 4°C for 20 min), 250 μl of the supernatant was diluted with 250 μl 0.9% (w/v) NaCl and was concentrated on a Microcon YM-10 filter (Millipore) by centrifugation (14,000 g at 4°C for 60 min). The protein fraction was diluted to a final volume of 500 μl, with 0.9% (w/v) NaCl, and was concentrated again by centrifugation. The final concentrated protein fraction was saved, and the protein concentration was determined with a copper-reduction method using bicinchonic acid, essentially as described by Smith et al.²⁶ The activity of PRPP synthetases in EBV-LCLs was determined in a reaction mixture containing an aliquot of cell sample (0.05–0.65 mg), 40 mM sodium phosphate, 1 mM dithiothreitol, 6 mM MgCl₂, 1.54 mM ATP, 100 μM ribose-5-phosphate, 50 μM P¹,P⁵-di(adenosine-5′)pentaphosphate (Ap5A), and 50 mM Tris/MOPS (pH 7.4). The activity of PRPP synthetases in fibroblasts was determined in a reaction mixture containing an aliquot of cell sample (0.02–0.032 mg), 32 mM sodium phosphate, 1 mM dithiothreitol, 5 mM MgCl₂, 0.5 mM ATP, 150 μM ribose-5-phosphate, 250 μM Ap5A, and 50 mM Tris/HCL (pH 7.4).²⁷ Separation of AMP, ADP, and ATP was performed isocratically (30% 0.0075-mM sodium phosphate to 70% 0.75-mM sodium phosphate [pH 4.55]) at a flow rate of 0.8 ml/min by HPLC on an ion-exchange column (Whatman Partisphere SAX 125×4.6 mm; 5 μm particle size [VWR International]) and a guard column (Whatman Partisphere AX 10×2.5 mm; 5 μm particle size [VWR International]) with online UV detection at 254 nm.

Metabolite Analysis in Urine

Urine and plasma purines and pyrimidines were determined using reversed-phase HPLC analysis as described elsewhere,²⁸ with the modification that a 250×3.1 mm (3μ) Synergi RP80A C18 column (Phenomenex) was used. Purine and pyrimidine metabolites were identified by their retention times and spectra (230–330 nm) by photodiode array detection.

Clinical Description

In this article, two families with Arts syndrome were investigated. Family N032 is the original Dutch family described extensively by Arts et al.¹⁴ In short, the syndrome is defined by mental retardation, early-onset hypotonia, ataxia, delayed motor development, hearing impairment, and optic atrophy (table 2). Susceptibility to infections, in particular of the upper respiratory tract, resulted in an early death, before age 5 years, for 9 of the 10 affected males in this family. Only one patient lived beyond age 5 years, reaching age 18 years. The CNS of one patient was examined at autopsy. In the posterior columns of the spinal cord, an almost complete absence of myelin was found. Carrier females from family N032 show isolated and milder manifestations of symptoms of Arts syndrome, such as perceptive hearing impairment, ataxia, hypotonia, and hyperreflexia.

The Australian family (family F) was ascertained independently (for pedigree, see fig. 1A). The two affected boys, III-2 and III-3, have mental retardation, hypotonia, ataxia, profound congenital sensorineural deafness, progressive peripheral neuropathy, and optic atrophy. They also displayed slowly progressive muscle weakness associated with intermittent acute deterioration in muscle strength during intercurrent illnesses. The acute episodes of muscle weakness resulted in respiratory failure, requiring mechanical ventilation for a period of time. Both boys had delayed motor-nerve conduction velocities and an electromyogram suggestive of denervation, which is consistent with the clinical findings that suggested peripheral neuropathy. In addition, the younger boy had a fibrosing pancreatitis, the cause of which was never established. A sural nerve biopsy in the older boy revealed mild paranodal demyelination. Other investigations with normal findings included urine amino and organic acid screens, blood lactate, liver-function tests, karyotype, magnetic resonance imaging of the brain, screening for the c.35delG mutation in GJB2 (MIM 121011), and, in the older boy, muscle and liver respiratory-chain enzyme studies and testing for several of the common mtDNA point mutations. The affected boys are currently aged 11 and 9 years. Their sister, III-1, is currently aged 13 years, is completely asymptomatic, and has normal hearing. Their parents are both healthy, and the mother has normal hearing, whereas the father has mild adult-onset hearing impairment. One of the maternal uncles, II-1, was thought to have Duchenne muscular dystrophy and died at age 2 years. He never learned to speak, and, in retrospect, it is likely that he, too, had Arts syndrome. Another maternal uncle, II-2, currently aged 27 years, had hydrocephalus of uncertain etiology, which required shunting in infancy. The third maternal uncle, II-3, died of sudden infant death syndrome at age 6 mo.

Linkage Analysis

The genetic defect in both families was linked to the X chromosome. In family N032, the 21-Mb linkage interval was previously described by Kremer et al.³⁰ and is flanked by the polymorphic markers DXS1231 and DXS1001, with a maximum LOD score of 6.97 (fig. 1C). The interval contains 227 annotated genes (NCBI Map Viewer build 36.2). Mutations in known X-linked mental retardation genes ACSL4 (MIM 300157), AGTR2 (MIM 300034), PAK3 (MIM 300142), and UBE2A (MIM 312180) were excluded by direct DNA sequencing.²⁹ Copy-number variations >200 kb were excluded as well by use of array comparative genomic hybridization on a full-coverage X-chromosome BAC array.³¹

For family F, an exclusion-mapping approach was taken, because a recessive X-linked defect was suspected and the family was too small to reach a significant LOD score. The uncle with hydrocephalus, II-2, was presumed to have an unrelated disorder (i.e., was considered unaffected for the purposes of this study), and the mother, II-4, and maternal grandmother, I-2, were presumed to be carriers for the disorder. Therefore, alleles not inherited from the maternal grandmother were excluded, as were alleles shared with the unaffected uncle II-2. In this way, three candidate regions were identified in this family, one of ∼13 cM at Xq23 between markers DXS1106 and DXS8064, which overlaps with the linkage interval of family N032 (fig. 1B), one ∼3-cM region at Xq27 between DXS1227 and DXS8043 and one ∼8-cM region at Xq27 between DXS8043 and DXS8091. The transient receptor potential channel 5 gene (TRPC5), which is located within the Xq23 candidate region identified in family F, was chosen as an initial candidate gene. TRPC5 is expressed exclusively in the adult and developing CNS, and it has proposed that it forms receptor-mediated nonselective cation channels in CNS cells.^22,32–34 It is thus a candidate for mental retardation and other developmental disorders, such as Arts syndrome.²² One change, c.919-42A→G, was identified in the affected boys in family F and in their normal father and maternal grandfather. It is therefore likely to be a common polymorphism.

Expression Profiling in Family N032

Fibroblast cell lines from two affected members of family N032, IV-2 and V-5 (for patient designation, see the work of Arts et al.¹⁴), were analyzed by expression profiling with the use of the GeneChip Human Genome U133 Plus 2.0 Array. The combined expression profile of the two affected family members was compared with the combined expression profile of two pools of four separate cell lines from healthy control individuals (see the tab-delimited ASCII file [online only], which can be imported into a spreadsheet). The three genes that were most reduced in expression in the linkage interval were CUL4B, C1GALT1C1 (MIM 300611), and PRPS1 (table 3); they were reduced in expression 1.9-fold, 1.4-fold, and 1.4-fold, respectively. P values corrected for multiple testing by use of the Benjamini-Hochberg method³⁵ were all >.5, which was probably the result of using only four hybridizations. C1GALT1C1 was considered an unlikely candidate gene, because somatic loss-of-function mutations in this gene cause Tn syndrome, a rare autoimmune disease in which subpopulations of blood cells of all lineages carry an incompletely glycosylated membrane glycoprotein.³⁶ The reduction in expression of CUL4B and PRPS1 was validated by qPCR, in which the average expression of the gene of interest in the cell lines from the two affected individuals was compared with the average expression in eight separate cell lines from different control individuals. In family members IV-2 and V-5, PRPS1 was reduced in expression 1.3-fold (P=.68) and 2.4-fold (P=.22), respectively. Although these values are only marginally lower than those of controls, they were reproducible by qPCR, and they do confirm the results of the expression array. The expression of CUL4B in fibroblast cell lines could not be assessed by qPCR, most likely because of its low expression level in these cells.

CUL4B and PRPS1 Mutation Analysis

Recently, Tarpey et al.³⁷ reported that mutations in CUL4B cause an X-linked mental retardation syndrome associated with aggressive outbursts, seizures, relative macrocephaly, central obesity, hypogonadism, pes cavus, and tremor. Although this syndrome appears to be clearly distinct from Arts syndrome and although we could not confirm the reduction in expression by qPCR, we nevertheless analyzed all exons, intron-exon boundaries, and branch sites of the protein-coding sequence of CUL4B in the two affected members of family N032, IV-2 and V-5. No changes were identified (results not shown).

PRPS1 encodes PRS-I, an enzyme that catalyzes the first step in the purine metabolic pathway converting ribose-5-phosphate to PRPP. Direct DNA sequencing of the seven exons, intron-exon boundaries, and branch sites in the two affected family members IV-2 and V-5 revealed one change, c.455T→C (fig. 2C), that results in the substitution of a proline for a leucine at position 152 (p.L152P). This change introduces a BsmFI restriction site. Restriction analysis by BsmFI digestion showed that this change segregated with the disease in the family (fig. 2D) and was absent in 301 X chromosomes from healthy Dutch individuals (data not shown). Subsequent mutation analysis of PRPS1 in the two affected boys from family F revealed a second mutation, c.398A→C (fig. 2A), which results in the substitution of proline for a glutamine at position 133 (p.Q133P). This change removes an ApoI restriction site. ApoI restriction analysis revealed that this change segregated with the disease in the family (fig. 2B) and was absent in 308 X chromosomes from healthy Australian individuals (data not shown).

Molecular Modeling

PRS-I amino acid residues Q133 and L152 are completely conserved among species, from human to zebrafish (UCSC Genome Browser).³⁸ According to the Sorting Intolerant From Tolerant (SIFT) program,³⁹ which can be used to predict whether amino acid residues at a specific position in a protein can be altered, neither mutation would be tolerated at their positions. Models of PRS-I with p.L152P or p.Q133P were built on the recent crystal structure of human PRS-I²³ (fig. 3). PRS-I is believed to be physiologically functional as a hexamer, which consists of three homodimers arranged in a propeller-like shape (fig. 3A and 3B). Q133 is positioned at the interface of the two monomers of a homodimer and forms a hydrogen bond to D98 from the other unit in the homodimer (fig. 3A). Thus, the p.Q133P mutation disrupts two of the intermolecular hydrogen bonds between the two monomers (fig. 3C and 3E). As a consequence, both the homodimer and the allosteric site would be destabilized. In addition, Q133 and especially its hydrogen-bonding partner D98 are in close proximity to the ATP-binding pocket. The neighboring amino acid residues R96, Q97, K99, and D101 all interact with the incoming ATP molecule; therefore, the loss of the hydrogen-bonding partner of D98 could also destabilize the ATP-binding pocket and thus impair PRS-I activity. L152 is positioned in the middle of one of the α-helixes (fig. 3D). Replacement of this leucine with a proline will break this α-helix configuration (fig. 3F) and, consequently, will destabilize the protein structure.

PRPP Synthetase Activity in Erythrocytes, Fibroblasts, and EBV-LCLs

PRPP synthetase activity was measured in erythrocytes from the two patients, III-2 and III-3; the mother, II-4; the sister who is not a carrier, III-1; and the father, II-5, from family F (table 4). There was no activity in erythrocytes from the affected boys. The activity in their mother, 18 nmol/mg/h, was outside the normal range (24–48 nmol/mg/h). In both family members without the mutation, the PRPP synthetase activity was normal. A repeat analysis of the test gave comparable results.

PRPP synthetase activity was measured in cultured skin fibroblasts from three affected members of family N032, IV-2, V-5, and V-27, and from one affected member of family F, III-2. The PRPP synthetase activity in the patient fibroblasts was compared with that in fibroblasts from eight unrelated healthy control individuals. The activity in the three patients from family N032 was, on average, 13-fold reduced (P<.05, calculated by Student’s t test) compared with the average PRPP synthetase activity in fibroblasts from controls (table 5). The activity in fibroblasts from the patient in family F was equally reduced.

PRPP synthetase activity was also determined in EBV-LCLs from two carrier females, IV-28 and IV-36, and an unaffected male, V-10, from family N032. The enzyme activities in EBV-LCLs from two unrelated healthy control individuals were analyzed as additional controls. In carrier females, the PRPP synthetase activity was 486 and 392 nmol/mg/h, which was lower than the activity in the unaffected male family member, 664 nmol/mg/h, and that in the two controls, 519 and 636 nmol/mg/h. This reduction in PRPP synthetase activity was not statistically significant.

Metabolite Analysis

Serum uric acid levels lower than average were found in patients and carrier females in family F, with values at the lower limit of the reference range for age and sex (table 4). Purine urinary profiles of the Australian boys were unusual, in that hypoxanthine was below the level of detection, whereas xanthine was relatively normal. Urine uric acid excretion was within the normal range for age. It should be noted that excreted uric acid, unlike hypoxanthine and xanthine, arises from dietary sources as well as from endogenous metabolism. All other purine and pyrimidine metabolites appeared to be within the normal ranges. An unidentified metabolite with a spectral maximum at ∼265 nm was noticed in urine from the affected boys, but its origin was undetermined.