Clinical Chemistry 65:10 Molecular Diagnostics and Genetics 1295–1306 (2019)

Cytidine Diphosphate-Ribitol Analysis for Diagnostics and Treatment Monitoring of Cytidine Diphosphate-L- Ribitol Pyrophosphorylase A Muscular Dystrophy Walinka van Tol,1,2 Monique van Scherpenzeel,2 Mohammad Alsady,1 Moniek Riemersma,1 Esther Hermans,1 Else Kragt,2 Giorgio Tasca,3 Erik-Jan Kamsteeg,4 Maartje Pennings,4 Ellen van Beusekom,4 Jeroen R. Vermeulen,5† Hans van Bokhoven,4 Nicol C. Voermans,1 Miche` l A. Willemsen,6 Angel Ashikov,1

and Dirk J. Lefeber1,2* Downloaded from https://academic.oup.com/clinchem/article/65/10/1295/5608232 by guest on 28 September 2021

BACKGROUND: Many muscular dystrophies currently CONCLUSIONS: Sensitive detection of CDP-ribitol with remain untreatable. Recently, dietary ribitol has been LC-MS allows fast diagnosis of patients with severe suggested as a treatment for cytidine diphosphate and mild CRPPA myopathy. Ribose offers a readily (CDP)-L-ribitol pyrophosphorylase A (CRPPA, ISPD), testable dietary therapy for CRPPA myopathy, with fukutin (FKTN), and fukutin-related protein (FKRP) possible applicability for patients with FKRP and myopathy, by raising CDP-ribitol concentrations. Thus, FKTN myopathy. Evaluation of CDP-ribitol in blood to facilitate fast diagnosis, treatment development, and is a promising tool for the evaluation and monitoring treatment monitoring, sensitive detection of CDP-ribitol of dietary therapies for CRPPA myopathy in a patient- is required. specific manner. © 2019 American Association for Clinical Chemistry METHODS: An LC-MS method was optimized for CDP- ribitol in human and mice cells and tissues. Muscular dystrophies (MDs)7 are a heterogeneous group RESULTS: CDP-ribitol, the product of CRPPA, was de- of genetic disorders characterized by progressive muscle tected in all major human and mouse tissues. Moreover, weakness and atrophy, potentially accompanied with CDP-ribitol concentrations were reduced in fibroblasts functional or structural brain involvement such as mental and skeletal muscle biopsies from patients with CRPPA retardation or cobblestone lissencephaly (1). The high myopathy, showing that CDP-ribitol could serve as a genetic, clinical, and biochemical heterogeneity compli- diagnostic marker to identify patients with CRPPA with cates identification of treatment strategies. A subgroup severe Walker–Warburg syndrome and mild limb-girdle involves the muscular dystrophy-dystroglycanopathy muscular dystrophy (LGMD) phenotypes. A screen for syndromes (MDDG). Patients with MDDG have limb- potentially therapeutic monosaccharides revealed that ri- girdle muscular dystrophy (LGMD) with or without bose, in addition to ribitol, restored CDP-ribitol concen- mental retardation, muscle–eye–brain disease (MEB, trations and the associated O-glycosylation defect of OMIM ID:253280), or Walker–Warburg syndrome (WWS, ␣-dystroglycan. As the effect occurred in a mutation- OMIM ID:236670). MEB and WWS are characterized by dependent manner, we established a CDP-ribitol blood severe congenital muscular dystrophy with brain and eye test to facilitate diagnosis and predict individualized abnormalities. The pathophysiological hallmark is hypo- treatment response. Ex vivo incubation of blood cells glycosylation of O-mannosyl glycans on ␣-dystroglycan with ribose or ribitol restored CDP-ribitol concentra- (␣DG), resulting in aberrant binding to extracellular ma- tions in a patient with CRPPA LGMD. trix components (2). Mutations in cytidine diphosphate

1 Department of Neurology, Donders Institute for Brain, Cognition and Behavior, Radboud * Address correspondence to this author at: Geert Grooteplein Zuid 10, 6525 GA Nijme- University Medical Center, Nijmegen, the Netherlands; 2 Translational Metabolic gen, the Netherlands. Fax +31-(0)24 3668754; e-mail [email protected]. Laboratory, Department of Laboratory Medicine, Radboud Institute for Molecular Life †Current affiliation: Department of Neurology, Maastricht University Medical Center, Sciences, Radboud University Medical Center, Nijmegen, the Netherlands; 3 Unita` Op- Maastricht, the Netherlands. erativa Complessa di Neurologia, Dipartimento di Scienze dell’Invecchiamento, Neuro- Received April 9, 2019; accepted June 27, 2019. logiche, Ortopediche e della Testa-Collo, Fondazione Policlinico A. Gemelli IRCCS, Roma, Previously published online at DOI: 10.1373/clinchem.2019.305391 Italy; 4 Department of Human Genetics, Donders Institute for Brain, Cognition and Be- © 2019 American Association for Clinical Chemistry havior, Radboud University Medical Center, Nijmegen, the Netherlands; 5 Department 7 Nonstandard abbreviations: MD, muscular dystrophy; MDDG, muscular dystrophy- of Child Neurology, VUmc, Amsterdam, the Netherlands; 6 Department of Pediatric Neu- dystroglycanopathy;LGMD,limb-girdlemusculardystrophy;MEB,muscle-eye-braindis- rology, Amalia Children’s Hospital, Donders Institute for Brain, Cognition and Behavior, ease; WWS, Walker–Warburg syndrome; ␣DG, alpha-dystroglycan; CDP, cytidine Radboud University Medical Center, Nijmegen, the Netherlands. diphosphate; Rbo5P, ribitol-5-phosphate; P, patient.

1295 8 (CDP)-L-ribitol pyrophosphorylase A (CRPPA, ISPD) MAPPING CRPPA MUTATIONS ON THE 3D MODEL OF CRPPA have been suggested as the second most common cause of Mutations were mapped with structure 4CVH from the MDDG (3, 4). CRPPA synthesizes CDP-ribitol, which RCSB Protein Data Bank (5) in YASARA software (10), is used by the glycosyltransferases fukutin-related protein and the effect of missense mutations was predicted with (FKRP) and fukutin (FKTN) to add ribitol 5-phosphate HOPE (11). (Rbo5P) moieties on ␣DG (5–8). Recent studies have shown that ribitol can restore deficient ␣DG IMMUNOHISTOCHEMISTRY, SDS-PAGE, AND WESTERN O-mannosylation in CRPPA fibroblasts (6). In addition, BLOTTING ␣ Immunohistochemistry, SDS-PAGE, western blotting,

dietary ribitol restores the O-mannosylation of DG in Downloaded from https://academic.oup.com/clinchem/article/65/10/1295/5608232 by guest on 28 September 2021 P448L FKRP-mutant mice (9). Whether ribitol is effec- and laminin overlay of muscle biopsies and HAP1 or fibroblast glycoproteins enriched with wheat germ agglu- tive for all mutations and can be safely administered to tinin agarose were performed as described (12, 13). Pri- patients remains to be investigated. These advances illus- mary antibodies were desmin (1:100, ab8470, Abcam) trate the need for the analytically sensitive detection of and ␤-dystroglycan (1:250, NCL-b-DG, Novacastra). CDP-ribitol in patient specimens to facilitate diagnosis Secondary antibodies were horse radish peroxidase- and treatment monitoring, paving the way for future conjugated polyclonal goat antirabbit or goat antimouse clinical trials. Here, we applied LC-MS for the detection (1:5000, Pierce). of CDP-ribitol in patient blood cells, fibroblasts, and muscle tissue. POLAR METABOLITE EXTRACTIONS A detailed description of metabolite extractions is pro- Materials and Methods vided in the Materials file in the Data Supplement that accompanies the online version of this article at http:// D-ribose, D-ribulose, and D-ribitol were purchased from www.clinchem.org/content/vol65/issue10). In short, Carbosynth. D-galactose was from Merck Millipore. Cy- samples were incubated for 5 min with ice-cold (Ϫ20 °C) tidine triphosphate, D-xylulose, D-xylose, L-arabinose, 2:2:1 (v:v:v) methanol:acetonitrile:water and centrifuged doxycycline, and 1 mol/L triethylammonium acetate at 16000g for 3 min at 4 °C. Supernatants were dried were from Sigma. CMP-N-acetylneuraminic acid, UDP- with a centrifugal vacuum concentrator (SpeedVac, galactose, UDP-glucose, UDP-N-acetylgalactosamine, Thermo Fisher Scientific) and stored at Ϫ80 °C before UDP-N-acetylglucosamine, GDP-mannose, GDP-fucose, LC-MS. ADP-ribose, UDP-xylose, and UDP-glucuronic acid were from Sigma or Carbosynth. LC-MS OF NUCLEOTIDE SUGARS Rbo5P was produced as previously described (5). Nucleotide sugars were analyzed by reversed-phase ion- CDP-ribitol was synthesized in vitro from Rbo5P and pairing chromatography (Agilent Technologies 1290 In- ϫ cytidine triphosphate with recombinantly produced hu- finity LC) with a HSS T3 column (Waters, 2.1 150 ␮ man CRPPA enzyme from Escherichia coli (kind gift of mm i.d., 1.8 m particle size) coupled to a triple quad- Dr. W. Yue, Oxford) (5). The internal standard, a 13C- rupole mass spectrometer operating in negative ion mode 13 (Agilent Technologies 6490 Triple Quad MS) (see Ma- yeast extract containing [U- C]-UDP-glucose, was a terials file in the online Data Supplement). kind gift of Dr. G. Hermann, Vienna.

PATIENT SPECIMENS GENETIC ANALYSIS OF PATIENTS WITH CRPPA Informed consent was obtained from patients or their Whole-exome sequencing, Sanger sequencing, and copy- legal representatives for use of diagnostic samples. Spec- number variant analysis of CRPPA (NM_001101426.3) imens were obtained and used in accordance with the were performed as described (4). Reverse transcription PCR Declaration of Helsinki. was performed with fibroblast cDNA by use of standard procedures. Results

ANALYSIS OF CDP-RIBITOL For high-throughput analysis of CDP-ribitol, we applied a targeted mass spectrometry method based on ion-pair

8 Human Genes: CRPPA (ISPD), CDP-L-ribitol pyrophosphorylase A; FKTN, fukutin; FKRP, reversed-phase separation of polar metabolites, coupled fukutin related protein; POMT1, protein O-mannosyltransferase 1; LARGE1, LARGE with a triple quadrupole mass spectroscopy system. A xylosyl- and glucuronyltransferase 1; PGM1, phosphoglucomutase 1; MPI, mannose CDP-ribitol standard was synthesized from Rbo5P with phosphate isomerase; SLC35C1, solute carrier family 35 member C1; TMEM165, trans- membrane protein 165; SLC35A2, solute carrier family 35 member A2; SLC39A8, solute recombinant CRPPA enzyme (5) and was used to gener- carrier family 39 member A8. ate standard curves for quantification of CDP-ribitol in

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Fig. 1. CDP-ribitol detection and quantification. (A), Laminin overlay (LO) of ␣DG from HAP1 wildtype (WT) and CRPPA knockout (KO) cells with ␤DG as loading control. (B), Relative peak area of CDP-ribitol in HEK293 with doxycycline (dox)-induced CRPPA overexpression and HAP1 CRPPA KOs. (C), CDP-ribitol quantification in polar metabolite extracts of C57 mouse and human tissues per mg wet weight. Bars represent mean (SD) ofn=3.ND,notdetectable. (D), Relative peak area of nucleotide sugars in tissues. GlcA, glucuronic acid; HexNAc, N-acetylhexosamine. Bars represent mean (SD) ofn=3.(E), CDP-ribitol concentrations in human tissues left at 4 °C for different time-points. patient materials (see Materials file and Fig. 1 in the HAP1 CRPPA knockout clones (Fig. 1B). This result online Data Supplement). For validation, we evaluated demonstrated that CDP-ribitol concentrations were de- relative CDP-ribitol concentrations in 2 cellular models: pendent on CRPPA and that CRPPA is a nonredundant (a) HEK293 cells with doxycycline-induced overexpres- cytidyltransferase for CDP-ribitol biosynthesis in HAP1 sion of CRPPA and (b) 2 HAP1 CRPPA knockout clones cells. with defective O-mannosylation of ␣DG, as demon- strated by defective laminin-binding of ␣DG (Fig. 1A) CDP-RIBITOL IS FOUND PREDOMINANTLY IN BRAIN, SKELETAL (5). Relative CDP-ribitol concentrations were increased MUSCLE, AND LIVER 9-fold in HEK293 cells after 48 h of doxycycline induc- CDP-ribitol has been identified in mouse skeletal muscle tion, whereas CDP-ribitol was undetectable in both and brain (6, 9). However, it remains unknown if CDP-

Clinical Chemistry 65:10 (2019) 1297 ribitol occurs ubiquitously in human tissues. Nucleotide 4 of his children were healthy. A muscle biopsy showed a sugar LC-MS analysis of different mouse or human tis- myopathy with myofibrillar changes including increased sues revealed that CDP-ribitol was present ubiquitously fiber-size diameter and number of internal nuclei. Immu- in most of the tissues assessed (Fig. 1C). In line with nohistochemistry with IIH6 demonstrated a signal re- dystroglycanopathy phenotypes, we found relatively high duction compared to laminin (Fig. 2F). Laminin overlay (Ͼ1 pmol/mg) concentrations of CDP-ribitol in mouse analysis showed reduced laminin-binding of muscle brain and skeletal muscle. CDP-ribitol concentrations of ␣DG (Fig. 2E). Mutations in P1–P3 were all predicted to 2.36 Ϯ 0.17 pmol/mg were found in mouse liver, al- be disease-causing by Mutation Taster (16) and Poly- though the role of CDP-ribitol in this tissue is currently phen (17) and to alter cytidyltransferase activity or pro- unknown. We did not detect CDP-ribitol in mouse and tein structure by HOPE (11) (Fig. 3, A and B). Downloaded from https://academic.oup.com/clinchem/article/65/10/1295/5608232 by guest on 28 September 2021 human kidneys, although hypoglycosylated ␣DG in kid- ney of chimeric FKTN (a Rbo5P transferase) mice has REDUCED CDP-RIBITOL CONCENTRATIONS IN PATIENTS WITH been observed (14). Fig. 1D shows the relative abun- CRPPA dance of the nucleotide sugars in the investigated tissues. To study if the analysis of CDP-ribitol potentially facil- Postmortem time or time before freezing might affect itates functional confirmation of CRPPA mutations, we nucleotide sugar concentrations. Further analyses re- analyzed the polar metabolites from fibroblasts and mus- vealed that CDP-ribitol concentrations were stable in cle biopsies from 9 patients with CRPPA, including P1, skeletal muscle but decreased in heart and liver over time P2, and P3, and 6 previously described patients with (Fig. 1E). Hence, CDP-ribitol concentrations in tissues CRPPA (P4, siblings P5.1 and P5.2, siblings P6.1 and that are not immediately frozen after biopsy might be P6.2, P7) (4, 18) (Table 1). Patients carried mutations in underestimated. Taken together, CDP-ribitol was pres- different domains of the CRPPA enzyme (Fig. 3, A and ent in most tissues but was found predominantly in the B), and covered the full range of phenotypes from severe tissues primarily affected in dystroglycanopathy patients WWS and MEB to adult-onset LGMD phenotypes. and in liver. Skeletal muscle samples were immediately frozen after biopsy, and 3–10 mg was used for metabolite extractions. THREE NEW PATIENTS WITH CRPPA CDP-ribitol was absent in biopsies from P4 (WWS), Patients 1 (P1) (homozygous, p.Arg205His) and 2 (P2) (ho- siblings P6.1 and P6.2 (LGMD), and P7 (LGMD) (Fig. mozygous, p.His312_Ser373del/Thr344_Ser373del) (Fig. 3C). Relative CDP-ribitol concentrations in P3 were 2, A and B) were nonconsanguineous and presented with slightly reduced, in line with the mild LGMD pheno- abnormalities compatible with WWS. MRI revealed hydro- type observed in this patient. CDP-ribitol was clearly cephalus, cobblestone lissencephaly and abnormal anatomy detected in 6 different control fibroblast lines (Fig. of the brainstem and cerebellum (Fig. 2C). Serum creatine 3D) but was reduced in fibroblasts from P3 and was kinase activities were highly increased at 10.754 and 86.390 undetectable in fibroblasts of patients with CRPPA U/L (normal Ͻ175 U/L) for P1 and P2, respectively. Mus- with a WWS or MEB phenotype (Fig. 3E). Further- cle biopsy investigations of P1 revealed muscular dystrophy more, we evaluated if reduced relative CDP-ribitol and defective O-mannosylation of ␣DG, as shown by re- concentrations were specific for CRPPA deficiency duced IIH6 labeling and laminin-binding on western blot and assessed these concentrations in fibroblasts from (Fig. 2, D and E). P2 carries a splice-site mutation resulting dystroglycanopathy patients with mutations in POMT1 in transcripts lacking exon 7 or exon 7 and 8 (Fig. 2B) that (homozygous, p.Asn144Lys), POMGNT1 (homozy- are part of the CRPPA C-terminal domain suggested to be gous, p.Arg488*), and LARGE1 (compound heterozy- essential for proper cytidyltransferase activity (4).At1 gous: p.Val378_Asn429del, p.Leu405Pro) (see Table 1 in month of age, both patients received a ventriculoperitoneal the online Data Supplement). Relative CDP-ribitol concen- shunt. They developed epileptic seizures and hardly showed trations in fibroblasts from these patients were normal. any psychomotor development. P1 died at 4 years of age of Thus, a decrease in CDP-ribitol concentrations could be respiratory insufficiency; P2 died at 1 year of pneumonia. used for diagnostics of CRPPA deficiency. Patient P3 (homozygous, p.Arg184Gly, Fig. 2A) is a 64-year-old patient, the oldest patient with mildest RIBOSE AS A POTENTIAL DIETARY THERAPY FOR CRPPA CRPPA myopathy reported thus far, who developed MYOPATHY proximal weakness in his lower limbs in his late 50s and The biosynthesis route of Rbo5P, the substrate of showed normal creatine kinase activity of 437 U/L (nor- CRPPA, remains unknown so far. Ribitol was previously mal for adult Asian men, 47–641 U/L) (15). Motor shown to increase CDP-ribitol concentrations in mouse function in his arms was normal, and he had never expe- myoblasts, CRPPA overexpressing HEK293, and in rienced myoglobinuria. Furthermore, he had a large mus- mouse brain, heart, and muscle (6, 9). In gram-positive cle mass despite the absence of training. He had 7 sib- bacteria, CDP-ribitol is formed by TarJ after reduction of lings, 3 of whom had died from an unknown reason and ribulose 5-phosphate to Rbo5P (19). We therefore

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Fig. 2. Clinical and biochemical analysis of patients with CRPPA myopathy. (A), Nucleotide and amino acid conservation in CRPPA exon 3. Mutations of P1 (p.Arg205His) and 3 (p.Arg184Gly) are indicated with red and green boxes, respectively. (B), cDNA sequencing analysis of patient P2 reveals that c.1027–3T>G leads to skipping of exon 8 (left panel) and of exon 7 and 8 (right panel). C, control. (C), MRI of patient P1 at 3 weeks. Left: Sagittal T2W image showing dilated lateral ventricles, minimal parenchyma of the brain, hypoplastic cerebellum, and kinked brainstem. Right: Axial FLAIR image showing severe hydrocephalus and lissencephaly. (D), IIH6 immunohistochemistry of patient P1 skeletal muscle. Spectrin was used as control staining to show integrity of the sarcolemma. (E), Laminin overlay (LO) assays using patient (P) and control (C) skeletal muscle homogenates. Desmin staining was used as loading control. (F), Immunohistochemistry of skeletal muscle biopsy of patient P3 with IIH6, spectrin, and laminin.

Clinical Chemistry 65:10 (2019) 1299 Downloaded from https://academic.oup.com/clinchem/article/65/10/1295/5608232 by guest on 28 September 2021

Fig. 3. The effect of CRPPA mutations on protein function and relative CDP-ribitol concentrations. (A), Schematic visualization of CRPPA with locations of the patient mutations. P5.1 and P5.2 are siblings, and P6.1 and P6.2 are siblings. Red boxes, WWS/MEB patients; blue boxes, LGMD patients. (B), Mutations (red) of patients P1 to P7 were mapped on human CRPPA monomer (RCSB Protein Data Bank 4CVH) (5) in YASARA structure software (11). The effect of missense mutations on protein function was predicted with HOPE (12). Left: Mutation p.Arg205His potentially affects dimer formation. Mutations p.Gly54Ala, p.Arg184Gly, and p.Ala122Pro are located in the cytidyltransferase domain, differ in charge and hydrophobicity, and potentially affect cytidyltransferase activity. The p.Ala216Asp mutation is located in the core of the cytidyltransferase domain. The mutant residue is more hydrophobic, probably altering CRPPA structure andstability.(C),RelativeCDP-ribitolconcentrationsinskeletalmuscleand(D)incontrolfibroblasts.Foldchangeswerecalculatedtothemean of controls (skeletal muscle,n=5;fibroblasts,n=6,dashed lines). SD of this value is depicted with gray shading. Bars represent mean (SD) ofn=3.(E), CDP-ribitol concentrations in fibroblasts from patients with CRPPA relative to C1. Bars represent mean (SD) ofn=3.ND,not detectable.

1300 Clinical Chemistry 65:10 (2019) oioigCPRbtlCnetain nCPAMyopathy CRPPA in Concentrations CDP-Ribitol Monitoring

Table 1. Clinical data summary for CRPPA patients.

Case P1 P2 P3 P4 P5.1 P5.2 P6.1 P6.2 P7

Sex Female Male Male Male Female Female Male Female Female Zygosity Homozygous Homozygous Homozygous Compound Homozygous Homozygous Homozygous Homozygous Homozygous heterozygous Nucleotide change Chr7[GRCh38] g.16376162C>T g.16258485A>G g.16376226G>C g.16406231C>G g.16376129G>T g.16376129G>T g.16421162C>G g.16421162C>G g.16258393_ 16258395del NM_001101426.3 c.614G>A c.1027–3T>C c.550C>G c.364G>C c.647C>A c.647C>A c.161G>C c.161G>C c.1114_1116del g.16301454G>A c.802C>T Protein change p.Arg205His p.His312_Ser373del/ p.Arg184Gly p.Ala122Pro p.Ala216Asp p.Ala216Asp p.Gly54Ala p.Gly54Ala p.Val372del Thr344_ Ser373del p.Arg268X Affected exon(s) 3 7 and 8 3 2 and 5 3 3 1 1 8 Diagnostic category WWS WWS LGMD MEB WWS/MEB WWS/MEB LGMD no MR LGMD no MR LGMD no MR Brain MRI Lissencephaly Lissencephaly No brain MR Pachyria and Cobblestone Cobblestone Gliotic Gliotic Normal Hydrocephalus Hydrocephalus polymicrogyria Lissencephaly Lissencephaly Pontocerebellar Pontocerebellar Hydrocephalus Hydrocephalus atrophy Kinking of atrophy Kinking of the brainstem the brainstem Kinking of the ischemic ischemic brainstem lesions lesions lnclChemistry Clinical Ophthalmologic Yes Not available Not available Yes, retinal Yes, bilateral Yes, bilateral None None None abnormalities detachment, congenital congenital optic atrophy, cataract, cataract, glaucoma buphthalmos, shallow Peter anomaly anterior chamber Muscle biopsy Dystrophic, reduced Not available Dystrophic, mildly Not available Not available Dystrophic, Dystrophic, Dystrophic, Dystrophic, laminin and IIH6 reduced IIH6 reduced IIH6 reduced IIH6 reduced IIH6 reduced staining staining staining staining staining IIH6 staining

51 (2019) 65:10 Creatine kinase (U/L) 10 754 86 390 437 9366 6543 104 769 960–1445 1004 975–1504 Epileptic seizures Yes Yes No Unknown Unknown Unknown None None None Survival No, age of death: 4 No, age of death: 1 Yes, >63 years Yes, at least 5.5 No, age of death: 13 No, age of death Yes, >56 years Yes, >54 years Yes, >40 years year years months neonatal years period Reference This article This article This article WWS-163 (4) WWS-25.22 (4) WWS-25.19 (4) A-V:5 (19) A-V:6 (19) B-II:2 (18)

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Fig. 4. Ribitol and ribose increase CDP-ribitol concentrations and restore O-mannosylation of ␣DG. Relative CDP-ribitol concentrations of (A) fibroblasts cultured in the presence of 10 mmol/L of ribitol or different monosaccharides for 24 h, (B–C) fibroblasts cultured in the presence of different ribitol or ribose concentrations for 24 h, (D) fibroblasts after 24, 48, or 72 h incubation in medium supplemented with ribose or ribitol, and (E) HAP1 and (F) fibroblasts from patients with CRPPA cultured for 24 h in the presence of ribose or ribitol. WT, wild type; KO, knockout; ND, not detectable. Fold changes were calculated to the mean of controls (dashed line). Gray shading depicts SD of this value. Bars represent mean (SD) ofn=3.(G), Laminin overlay (LO) assay and ␤DG immunolabeling of wheat germ agglutinin-enriched lysates of fibroblasts cultured in medium supplemented with 10 mmol/L ribose for 72 h. (H), CDP-ribitol quantification in healthy donor blood and P3 blood cells from patients with CRPPA. Values were normalized with [U-13C]-UDP-glucose as internal standard, and absolute concentrations were calculated with the standard curve of CDP-ribitol (see Fig. 1 in the online Data Supplement). The dashed line and gray shading present the mean and SD of controls (n = 3), respectively. Bars represent mean (SD) of n = 2 for C1 and P3 and mean (SD) of n = 3 for C2, C3, and C4. (I), P3 and healthy donor blood were incubated with ribose, ribitol, or 0.9% NaCl (negative control) for 24 h. Fold change wascalculatedrelativelytothenegativecontrolinhealthydonorbloodforeachtimepoint.Barsrepresentmean(SD)ofn=3.(J),Visualization of how CDP-ribitol LC-MS can potentially be used for diagnostics and personalized ribose or ribitol treatment for CRPPA patients. WES, whole-exome sequencing. Continued on page 1303 hypothesized that pentoses could also restore CDP- ribitol, ribulose, arabinose, xylulose, xylose, or galactose ribitol concentrations. We performed a monosaccharide for 24 h. Xylulose Ͼ1 mmol/L was toxic for fibroblasts screen of potentially therapeutic monosaccharides and (data not shown), and this compound was excluded incubated fibroblasts of LGMD patient P3 and WWS for further analysis. Relative CDP-ribitol concentra- patient P4 in medium containing 10 mmol/L of ribose, tions increased on ribitol treatment (Fig. 4A). Ribu-

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Fig. 4. Continued.

lose supplementation increased CDP-ribitol concen- knockouts supplemented with 10 mmol/L ribitol or ri- trations slightly in control fibroblasts but not in cells bose (Fig. 4E). Therefore, we developed an assay to ad- from patients. Interestingly, ribose increased relative dress the potential response of individual patients with CDP-ribitol concentrations in control fibroblasts CRPPA to dietary therapy. Furthermore, we questioned and in fibroblasts from the LGMD and the WWS whether the recovery of CDP-ribitol concentrations in patient. patient fibroblasts after ribose treatment was sufficient to To investigate the effect of ribitol and ribose on rescue the laminin-binding capacity of ␣DG. Therefore, CDP-ribitol concentrations in more detail, we supple- we incubated all available CRPPA patient fibroblast lines mented fibroblasts from controls and CRPPA P1 and with 10 mmol/L ribose or ribitol, evaluated relative P4 with different concentrations of ribitol or ribose for CDP-ribitol concentrations, and assessed the laminin- 24 h. Nucleotide sugar analysis confirmed that relative binding capacity of ␣DG of ribose-treated cells. Consis- CDP-ribitol concentrations increased in a dosage- tent with our earlier findings, relative concentrations of dependent manner (Fig. 4, B and C). Incubation with CDP-ribitol increased in ribose-treated fibroblasts of P1, 200 ␮mol/L ribitol doubled the amount of CDP- P3, and P4 (Fig. 4F). In line with this recovery, laminin- ribitol produced, whereas 1 mmol/L of ribose accom- binding in P1 and P4 was restored and laminin-binding plished the same effect. Furthermore, we found a in P3 fibroblasts was slightly increased after ribose treat- marked increase of CDP-ribitol after incubation with ment (Fig. 4G). CDP-ribitol concentrations and ␣DG 10 mmol/L and 50 mmol/L of ribose. Ribitol or ribose laminin binding were not restored in P2 fibroblasts, treatment induced the formation of CDP-ribitol in which carry a mutation resulting in transcripts lacking cells from CRPPA P4 and P1, suggesting that there exon 7 or exon 7 and 8. Similarly, relative CDP-ribitol was residual cytidyltransferase activity in these cells. concentrations in siblings P5.1 and P5.2 were not re- CDP-ribitol content was maximized after 24 h (ribose, stored. They carried a missense mutation in exon 3 fold change ϭ 0.77; ribitol, fold change ϭ 0.76) and (p.Ala216Asp), probably affecting stability and folding of longer incubation times did not increase relative con- the protein (Fig. 3B) (5). Ribose or ribitol treatment did centrations further (Fig. 4D). not restore deficient O-mannosylation in LARGE- We hypothesized that residual CRPPA activity is deficient fibroblasts, because they lack the xyloglucuro- required for efficient ribitol and ribose treatment. In- nan polysaccharide responsible for laminin binding (20). deed, CDP-ribitol was not synthesized in HAP1 CRPPA Supplementation with ribose or ribitol did not affect the

Clinical Chemistry 65:10 (2019) 1303 concentrations of other analyzed nucleotide sugars, al- is not gene specific, and a muscle biopsy is an invasive pro- though ADP-ribose was increased in some (see Fig. 2 in cedure. Using our LC-MS technology, we found that CDP- the online Data Supplement). ribitol, the enzymatic product of CRPPA, was reduced in extracts from CRPPA fibroblasts, blood cells, and skeletal PERSONALIZED PREDICTION OF DIETARY TREATMENT muscle. CDP-ribitol was undetectable in patients with RESPONSE OF PATIENTS WITH CRPPA MYOPATHY WWS/MEB and was less severely affected in samples from For future clinical trials, there is a need for the analyti- patients with LGMD, demonstrating a potential relation cally sensitive detection of CDP-ribitol in patient speci- between clinical severity and biochemical status. Taken to- mens to monitor treatment at the biochemical concen- gether, the assessment of CDP-ribitol concentrations in tration. In addition, such a method would allow blood or fibroblasts facilitates functional confirmation when Downloaded from https://academic.oup.com/clinchem/article/65/10/1295/5608232 by guest on 28 September 2021 assessment of therapeutic potential in a patient-specific CRPPA myopathy is suspected. manner. Thus, we developed a polar metabolite extrac- In dystroglycanopathy, skeletal muscle and brain are tion procedure from whole blood that could be used for predominantly affected. Interestingly, we found rela- diagnostics, for prediction of treatment response, and for tively high concentrations of CDP-ribitol in these tissues, treatment monitoring. We confirmed that CDP-ribitol confirming an important role for this compound in these was detectable in whole blood samples, red blood cells, tissues. In liver, we found a relatively high amount of and peripheral mononuclear blood cells of 2 healthy do- CDP-ribitol, but liver ␣DG is produced at low concen- nors (data not shown). Metabolic assays require a rapid trations and is unable to bind laminin (21, 22). Thus, the sample preparation, and we therefore proceeded using role of CDP-ribitol in this tissue remains unclear, but it whole-blood samples. CDP-ribitol concentrations were might be involved in the glycosylation of other proteins. analyzed from whole blood of P3 and 4 healthy donors. Surprisingly, we did not detect CDP-ribitol in human or CDP-ribitol was reduced in the blood of P3 (0.15 pmol/ mouse kidney. However, Kojima et al. (14) demon- ␮L) compared to healthy donors (0.24–0.31 pmol/␮L) strated hypoglycosylated ␣DG in kidney of chimeric (Fig. 4H). Therefore, CDP-ribitol concentrations in FKTN (Rbo5P transferase) mice, implying that CDP- blood could potentially serve as diagnostic marker, but ribitol and thus Rbo5P moieties on ␣DG are present in further confirmation with more blood samples from pa- kidney. Our samples consisted of a pool of kidney cells, tients with CRPPA is required. not only podocytes, potentially explaining why CDP- Subsequently, we developed an assay to mimic the ribitol was not detected in our extracts. Further studies in effect of dietary ribose or ribitol ex vivo. Whole-blood different kidney cells are required to understand the role aliquots (100 ␮L) were incubated for 24 h with either 1 of dystroglycan and FKTN in kidney function and de- mmol/L or 10 mmol/L of ribose or ribitol. Incubations velopment. In all other tissues, cell lines and blood sam- with 10 mmol/L ribose or 1 mmol/L or 10 mmol/L of ples of healthy donors that we have analyzed, CDP- ribitol increased relative CDP-ribitol concentrations ribitol was present, in concordance with the finding that considerably in both healthy donor and patient blood CRPPA is expressed in most tissues (3). samples (Fig. 4I). Collectively, our findings support the The biosynthesis pathway of the CRPPA substrate, hypothesis that besides ribitol, dietary supplementation Rbo5P, remains to be elucidated. In a study of pentose– of ribose is a potential therapy for patients with CRPPA. phosphate pathway defects, fibroblasts excreted ribitol Furthermore, we showed that blood polar metabolite after ribose supplementation (23), suggesting the pres- analysis is a promising tool for diagnostics, personalized ence of a ribose reductase. Recently, it was shown that a assessment of potential treatment response, and treat- sorbinil-sensitive reductase affects CDP-ribitol synthesis, ment monitoring (Fig. 4J). but the responsible enzyme has not been identified (6). Possibly, Rbo5P is formed by a reduction of ribulose Discussion 5-phosphate, as observed in gram-positive bacteria that have phosphodiester-linked polyol repeats in their cell walls (19). Using an analytically sensitive LC-MS method, we dem- Furthermore, the transporter responsible for CDP-ribitol onstrated that CDP-ribitol, a nucleotide sugar, is a bio- transfer into the Golgi lumen remains to be identified. marker for CRPPA myopathy, the second most common Novel technologies like LC-MS can be used to unravel the form of dystroglycanopathy (4). Furthermore, we inves- mammalian biosynthesis pathway of Rbo5P and CDP- tigated treatment options for patients with CRPPA my- ribitol, potentially revealing new targets for therapy. opathy and found that, besides ribitol, ribose restored the Besides exploring new diagnostic tools, we investi- relative concentrations of CDP-ribitol and the glycosyl- gated potential therapies for CRPPA myopathy. In other ation defect of ␣DG. MDs, a range of therapeutic approaches have been ex- Traditionally, functional confirmation of dystrogly- plored, including gene therapy, cell therapy, and many canopathy gene mutations is performed with IIH6 label- different therapeutic compounds. In Duchenne MD, an- ing on skeletal muscle. Unfortunately, this methodology tisense oligonucleotides inducing exon skipping and res-

1304 Clinical Chemistry 65:10 (2019) Monitoring CDP-Ribitol Concentrations in CRPPA Myopathy

toration of the open reading frame of dystropin have was predicted to alter the structure and stability of received the most attention. This therapy partially res- CRPPA. All other missense mutations were predicted to cues the dystropin production (24, 25), and a recent affect cytidyltransferase activity, but the presence of re- clinical trial in patients with Duchenne MD demon- sidual activity is plausible. P2 carries an intronic muta- strated that eteplirsen, which induces skipping of exon tion that induces exon skipping in the C-terminus, which 51, caused a less severe disease progression (26).Inthe is essential for CRPPA activity (4). Indeed, ribose treat- dystroglycanopathies, recent studies focused on adeno- ment was ineffective in P2 fibroblasts. Subsequently, we associated virus-mediated gene therapy for Fukuyama developed an assay to predict the individual response to congenital muscular dystrophy (27, 28). The beneficial dietary treatment in whole blood samples. Thus, LC-MS effect of the therapy in mice was shown to be dependent of polar metabolites in blood cells is a promising tech- Downloaded from https://academic.oup.com/clinchem/article/65/10/1295/5608232 by guest on 28 September 2021 on the dosage and the disease progression, and further nique to monitor patients during clinical trials and future studies are required in this field. Dietary sugar therapies treatment. are available for other congenital disorders of glycosyla- In conclusion, we showed that besides ribitol, ribose tion (CDG) such as PGM1-CDG, MPI-CDG, and therapy is a potential treatment for CRPPA myopathy. SLC35C1-CDG (29–31). Dietary interventions are Interestingly, dietary galactose therapy, which is effective cheaper and readily available for patients, and ribitol sup- for PGM1-CDG, is also applicable in other galactosyla- plementation has recently gained much attention (6, 9). tion disorders like TMEM165-CDG, SLC35A2-CDG, In agreement with the findings of Gerin et al. (6) and and SLC39A8-CDG (39). Although our study focused Cataldi et al., (9) our monosaccharide screen demon- on treatment opportunities for CRPPA deficiency, pa- strated that ribitol supplementation restored CDP- tients with residual activity of FKTN or FKRP, the 2 ribitol concentrations in fibroblasts. However, clinical Rbo5P Golgi glycosyltransferases, could also benefit trials are required to assess the long-term safety of ribitol from ribose treatment. This hypothesis is supported by administration in humans. Accumulation of polyols, the positive effect of ribitol in FKRP-deficient mice (9). such as sorbitol and galactitol, is associated with diabetic Dietary sugar therapies are cheaper, safer, and more cataract (32, 33). We found that ribulose was unable to quickly available for patients, facilitating a faster route enter cells efficiently, consistent with the findings of toward clinical trials than other therapies. Furthermore, Huck et al. (23). However, ribose treatment successfully we expect that modern mass spectroscopy technologies in increased CDP-ribitol concentrations and restored the combination with a growing interest in personalized ␣ functional O-mannosylation of DG. Ribose has been therapies will lead to the discovery of treatment opportu- administered safely to humans, including patients with nities for other neuromuscular disorders. Duchenne MD and Type V glycogen storage disease, with the hypothesis of increasing ATP pools (34, 35). We postulate that the proposed effect of ribose is due to the increase in CDP-ribitol. High concentrations of ri- Author Contributions: All authors confirmed they have contributed to bose administration have been reported to lead to hypo- the intellectual content of this paper and have met the following 4 require- glycemia, whereas the formation of advanced glycation ments: (a) significant contributions to the conception and design, acquisi- end products has been reported in mice but not in a study tion of data, or analysis and interpretation of data; (b) drafting or revising with ribose-treated horses (36–38). In addition, we the article for intellectual content; (c) final approval of the published article; found that ribose or ribitol treatment resulted in in- and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the creased concentrations of ADP-ribose in some fibroblast article are appropriately investigated and resolved. lines, although the biological relevance remains elusive. Taken together, future studies are warranted to assess the E.-J. Kamsteeg, provision of study material or patients; J.R. Vermeulen, provision of study material or patients; H. van Bokhoven, financial safety, dosage, and efficacy of ribitol and ribose supple- support, administrative support, provision of study material or patients; mentation in patients with CRPPA. W. van Tol, Experimental design, method development, data acquisi- The rapid development of metabolomics technolo- tion, data interpretation and writing of the manuscript, description of gies allows the development of personalized treatment. clinical data CRPPA patients; M. van Scherpenzeel, method develop- Ribitol or ribose did not increase relative CDP-ribitol ment, data interpretation; M. Alsady, data interpretation, writing man- uscript; E. Hermans, method development, data acquisition; E. Kragt, concentrations in all patient fibroblasts, indicating that method development, data acquisition; M. Riemersma, description of dietary treatment only aids patients with residual CRPPA clinical data CRPPA patients; N.C. Voermans, description of clinical activity. For example, ribose treatment increased CDP- data CRPPA patients; E.-J. Kamsteeg, G. Tasca, M.A. Willemsen, E. ribitol concentrations in fibroblasts with missense muta- van Beusekom, H. van Bokhoven, J.R. Vermeulen, and M. Pennings, tions in the cytidyltransferase domain from P1, P3, and provided CRPPA patient samples, and clinical and genetic details of CRPPA patients; A. Ashikov, experimental design, data interpretation; P4, but not in P5.1 and P5.2. The siblings P5.1 and P5.2 D.J. Lefeber, experimental design, data interpretation, writing of the are homozygous for the p.Ala216Asp mutation in the manuscript, supervision of the study. All co-authors have critically eval- core of the cytidyltransferase domain, and the mutation uated the manuscript.

Clinical Chemistry 65:10 (2019) 1305 Authors’ Disclosures or Potential Conflicts of Interest: Upon man- fonds (Grant W.OR17-15), ERA-NET Cofund action N° 643578 uscript submission, all authors completed the author disclosure form. Dis- (EUROCDG-2). closures and/or potential conflicts of interest: Expert Testimony: None declared. Patents: None declared. Employment or Leadership: M. van Scherpenzeel, GlycoMScan B.V. Consultant or Advisory Role: None declared. Role of Sponsor: The funding organizations played no role in the Stock Ownership: None declared. design of study, choice of enrolled patients, review and interpretation of Honoraria: None declared. data, preparation of manuscript, or final approval of manuscript. Research Funding: The European Union’s Horizon 2020 research and innovation program under the ERA-NET Cofund action N° 643578 Acknowledgments: The authors thank Dr. Gerrit Hermann from ISO- (EUROCDG-2). H. van Bokhoven, the Prinses Beatrix Spierfonds topic solutions in Vienna for providing them with the 13C-yeast metabolite 13 (Grant W.OR17-15); D.J. Lefeber, The Netherlands Organization for standard containing [U- C]-UDP-glucose. The authors thank Dr. Chris- Downloaded from https://academic.oup.com/clinchem/article/65/10/1295/5608232 by guest on 28 September 2021 Scientific Research (VIDI Grant 91713359), the Prinses Beatrix Spier- tian Bu¨ll for kindly providing them with snap-frozen mouse tissues. References

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