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Elevated serum dihydroorotate in miller syndrome: Biochemical, diagnostic and clinical implications, and treatment with uridine

John A. Duley, Michael G. Henman, Kevin H. Carpenter, Michael J. Bamshad, George A. Marshall, Chee Y. Ooi, Bridget Wilcken, Jason R. Pinner

PII: S1096-7192(16)30116-0 DOI: doi: 10.1016/j.ymgme.2016.06.008 Reference: YMGME 6069

To appear in: Molecular Genetics and Metabolism

Received date: 13 May 2016 Revised date: 13 June 2016 Accepted date: 13 June 2016

Please cite this article as: Duley, J.A., Henman, M.G., Carpenter, K.H., Bamshad, M.J., Marshall, G.A., Ooi, C.Y., Wilcken, B. & Pinner, J.R., Elevated serum dihydroorotate in miller syndrome: Biochemical, diagnostic and clinical implications, and treatment with uridine, Molecular Genetics and Metabolism (2016), doi: 10.1016/j.ymgme.2016.06.008

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Elevated serum dihydroorotate in Miller syndrome: biochemical, diagnostic and clinical implications, and treatment with uridine.

John A. Duleya, Michael G. Henmanb, Kevin H. Carpenterc, Michael J. Bamshadd, George A. Marshalle, Chee Y. Ooif, Bridget Wilckeng, Jason R. Pinnerh

aSchool of Pharmacy and Mater Research Institute, The University of Queensland, Brisbane, QLD 4102, Australia. [email protected] bDepartment of Pathology, Mater Health Services, Brisbane, QLD 4101, Australia. [email protected] cNSW Biochemical Genetics Service, The Children's Hospital at Westmead, Disciplines of Genetic Medicine & Child and Adolesecent Health, The University of Sydney, NSW 2145, Australia. [email protected] dDepartments of Pediatrics and Genome Sciences, University of Washington, and the Division of Genetic Medicine at Seattle Children’s Hospital, Seattle, WA 98195, USA. [email protected] eDepartment of Pathology, Mater Health Services, Brisbane, QLD 4101, Australia. [email protected] fSchool of Women's and Children's Health, School of Medicine, UNSW, and Sydney Children's Hospital, Sydney NSW 2031, Australia. [email protected] g Department ofACCEPTED Medical Genetics, Sydney MANUSCRIPT Children's Hospital, University of Sydney, NSW 2031; Australia. [email protected] hDepartment of Medical Genomics, Royal Prince Alfred Hospital, The University of Sydney, NSW 2050. [email protected]

Correspondence to: Dr John Duley, School of Pharmacy, PACE Building, Cornwall Street, Woolloongabba, Brisbane, 4102 QLD, Australia. [email protected] tel: +61 7 3346 1900, fax: +61 7 3346 199

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Abstract

Background: Miller syndrome (post-axial acrofacial dystosis) arises from gene mutations for the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). Nonetheless, despite demonstrated loss of enzyme activity dihydroorotate (DHO) has not been shown to accumulate, but paradoxically urine orotate has been reported to be raised, confusing the metabolic diagnosis. Methods: We analysed plasma and urine from a 4-year-old male Miller syndrome patient. DHODH mutations were determined by PCR and Sanger sequencing. Analysis of DHO and orotic acid (OA) in urine, plasma and blood-spot cards was performed using liquid chromatography-tandem mass spectrometry. In vitro stability of DHO in distilled water and control urine was assessed for up to 60 hours. The patient received a 3-month trial of oral uridine for behavioural problems. Results: The patient had early liver complications that are atypical of Miller syndrome. DHODH genotyping demonstrated compound-heterozygosity for frameshift and missense mutations. DHO was grossly raised in urine and plasma, and was detectable in dried spots of blood and plasma. OA was raised in urine but undetectable in plasma. DHO did not spontaneously degrade to OA. Uridine therapy did not appear to resolve behavioural problems during treatment, but it lowered plasma DHO. Conclusion: This case with grossly raised plasma DHO represents the first biochemical confirmation of functional DHODH deficiency. DHO was also easily detectable in dried plasma and blood spots. We concluded that DHO oxidation to OA must occur enzymaticallyACCEPTED during renal secretion.MANUSCRIPT This case resolved the biochemical conundrum in previous reports of Miller syndrome patients, and opened the possibility of rapid biochemical screening.

Keywords: dihydroorotate-dehydrogenase; Miller syndrome; POADS; mitochondria; screening

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1. Introduction

Dihydroorotate dehydrogenase (DHODH1, OMIM #126064) oxidises dihydroorotate (DHO) to orotic acid (OA) during pyrimidine de novo synthesis. The enzyme is located on the inner mitochondrial membrane, with flavin mononucleotide as the electron acceptor. Thereafter, electrons are passed to ubiquinone (coenzyme Q10) then cytochrome C and oxygen as final electron acceptors (Fig. 1). DHODH directly links carbamoyl-phosphate metabolism to pyrimidine nucleotide synthesis and to the production of energy via mitochondrial oxidative phosphorylation (1). Miller syndrome, also known as Genee-Wiedemann or Wildervanck-Smith syndromes (exhibiting post-axial acrofacial dystosis, POADS) was the first Mendelian disorder whose genetic basis was identified using whole exome sequencing, when it was shown to be caused by mutations in the DHODH gene (2). However, despite the description of a number of DHODH mutations that have been demonstrated to cause loss of enzyme activity in vitro or in silico (3), DHO has not been shown to accumulate, at least not in urine: in the only two cases where metabolic analyses of Miller syndrome patients have been described, urinary OA was raised but DHO absent. This is the reverse of the predicted metabolite pattern, and has presented a conundrum for metabolic diagnosis. We report here the presence of grossly raised concentrations of DHO in the plasma and urine of a patient with Miller syndrome. DHO was also easily detectable in dried blood and dried plasma on filter paper. OA was elevated in urine but it was undetectable ACCEPTEDin plasma. We examined theMANUSCRIPT distribution of DHO between plasma and blood cells. We propose that loss of DHODH activity leads to the systemic accumulation of DHO, which is converted to OA during renal excretion, by either residual DHODH activity or an alternative oxidative enzyme mechanism.

1 Abbreviations: AST: aspartate transaminase; ALT: alanine transaminase; CYP: cytochrome- P450; Cyt C: Cytochrome C; DHO: dihydroorotic acid; DHODH: DHO dehydrogenase; EDTA: ethylenediamine-tetraacetic acid; FMN: Flavin mononucleotide; GGT: gamma- glutamyl transferase; LC-MS/MS: high performance liquid chromatography-tandem mass spectrometry; OA: orotic acid; POADS: post-axial acrofacial dystosis; UMP: uridine monophosphate; UQH2: reduced ubiquinone.

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2. Methods

2.1. Patient Our 4-year-old patient was born prematurely at 27/40 weeks, in Sydney, with bilateral absence of the 5th rays of his hands, cleft palate and a peri-membranous ventricular septal defect. He had prominent eyes, a short nose with upturned nares, a low nasal bridge, high columella, a deeply grooved philtrum, prominent infraorbital creases, single palmar creases, broad thumbs and short, broad halluces as well as under-riding 4th toes (hypoplastic 4th rays on X-ray) (Fig. 2). There was no contributing family history and his parents were non-consanguineous. His neonatal course was complicated by respiratory distress syndrome, heart failure and cholestasis. The ventricular septal defect closed spontaneously. He had uncomplicated repairs to a bilateral inguinal hernia and cleft palate at 13 months.

He developed bilateral radioulnar synostosis and has myopic astigmatism corrected with glasses. His height and weight continue around the third percentiles. He has had mild speech delay and some behavioural problems. His facial features are similar to other reported cases, including sparse eyebrows, almond shaped eyes with up-slanting palpebral fissures, malar hypoplasia, long philtrum, small mouth, and low- set, normally-shaped ears. He has unusual cruciform palmar creases. Our case thus has the characteristic features of Miller syndrome, including cleft palate, congenital heart disease, oligodactyly and consistent dysmorphic features. His neonatalACCEPTED cholestasis was initially MANUSCRIPT thought to be due to total parenteral nutrition. Due to persistence of cholestasis despite cessation of parenteral nutrition and normalisation of oral feeds, he was extensively investigated but no aetiology was identified. An intraoperative cholangiogram and liver histopathology excluded biliary atresia. Liver biopsy revealed unusual generalised feathery degeneration of hepatocytes, mild fibrosis and cholestasis. Immunohistochemical staining for familial cholestatic diseases was negative, and typing of his alpha-1-antitrypsin, PiMM, was normal. The conjugated hyperbilirubinaemia resolved at 6 months of age while receiving ursodeoxycholic acid, but the patient’s liver parameters remained abnormal, with fluctuating elevated transaminases (AST 87-228 U/L, reference range 10-50;

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ALT 132-433 U/L, ref. 0-45; GGT 78-263 U/L, ref. 0-45) and progressive hepatomegaly (9.5 cm). Albumin levels have been normal. He continued treatment with ursodeoxycholic acid 250mg bd. Following approval from the local Human Research Ethical Committee, he was provided with a 3-month trial of oral uridine, 100 mg/kg/day, as a dietary supplement.

2.2. Patient Samples Blood and plasma (EDTA anticoagulant) and urine samples were initially collected in Sydney during October 2013, for biochemical diagnosis of the patient. The uridine trial began with baseline samples (t = 0 on Fig.4) being taken in mid- April 2015, approximately 80 weeks after diagnosis and 2 weeks before commencement of uridine. Samples were then collected routinely until the end of July 2015. Blood samples were cooled, and plasma and urine were frozen then immediately transported to the Pathology Department of Mater Hospital, Brisbane, for metabolite analysis. Prior to analysis, patient plasma was deproteinised by centrifuging through a 50 kilodalton Amicon ultrafilter (Merck Pty. Ltd., Kilsyth, VIC, Australia), while urine was diluted to yield a creatinine concentration of 0.5 mmol/L. These were then mixed with equal volumes of isotope-labelled internal standard solution as previously described (4).

2.3. Extraction of dried blood and plasma spots Whole EDTA blood and plasma from the patient and a normal subject were spotted and dried on filter paper ('Guthrie') cards, to test recovery of DHO and practicability ACCEPTEDof transporting dried samples MANUSCRIPT for diagnosis via mail. Blood and plasma from the normal subject were spiked with 10-40 µmol/L DHO followed by spotting and drying onto the filter paper cards. All dried blood and plasma spots were cut out using the 1 cm Guthrie card guidelines, chopped into small pieces in a 1.5 mL capped Epppendorf tube, and mixed with 500 µL of 50:50 v/v acetonitrile:water, then 25 µL isotope-labelled internal standard solution was added. Methanol:water (10:80 v/v) was also tested for metabolite extraction from the Guthrie cards. Each tube with filter paper card and solvent was rotated for 30 min, then the extract was centrifuged briefly to remove fibres, dried under nitrogen at 50oC, redissolved in 50 µL water, and analysed by mass spectrometry.

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2.4. Analysis of metabolites by mass spectrometry Separation and identification of OA, DHO and other purine and pyrimidine nucleosides and bases as well as creatinine in plasma, urine, filter paper extracts and red cell lysates was achieved using high-performance liquid chromatography with an electrospray interface linked to an ABI-4000 QTrap mass spectrometer (LC-MS/MS) (AB SCIEX, Mulgrave, VIC, Australia) in multiple reaction mode, as reported previously (4), with the addition of an ion transition for DHO (156.9/112.8). Isotopic 15 [ N2]-OA was used as the internal standard for DHO and OA; injection volume was 5 µL in all cases. All data were quantified using AB SCIEX Multiquant v.2.1 software. Ultraviolet detection is unsuitable for DHO because its maximum wavelength is below 230 nm, which is non-specific for metabolite screening.

2.5. Distribution of DHO between red cells and plasma Fresh EDTA blood from a normal subject was spiked with small volumes of 1 mmol/L DHO to yield 10 and 20 µmol/L final concentrations. The blood was incubated at room temperature with slow rotation and sampled at 1 h and 2.5 h. Each incubated sample was then centrifuged for 2 min at 10,000 rpm in a microfuge and the plasma and packed red cell fractions separated. The packed red cell fraction was mixed with an equal volume of distilled water then freeze/thawed twice to produce a red cell lysate. Both plasma samples and red cell lysates were microfuged in 0.5 mL Amicon ultrafilters to remove proteins, producing clear non-coloured ultrafiltrates that were then mixed with isotope-labelled internal standard solution, for injection into the LC-MS/MS. ACCEPTED MANUSCRIPT 2.6. Stability of dihydroorotic acid DHO stability was tested by adding (1 mmol/L final concentration) to distilled water and to control urine. These were maintained at room temperature and 5 oC, with sampling at 24 h and 60 h. Both aqueous and urine samples were then analysed for DHO and OA by LC-MS/MS as above, to assess non-enzymatic or bacterial (in urine) oxidation of DHO. DHO (25 umol/L final concentration in phosphate-buffered saline) was also incubated for 24 h at room temperature with purified bovine milk xanthine oxidase (Sigma-Aldrich, Castle Hill, NSW, Australia). A positive control substrate for xanthine oxidase incubations was the purine base xanthine. These incubates were then

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deproteinised by ultrafiltration and isotope labelled before injection into the LC- MS/MS.

2.7. DHODH gene sequencing The 5'-untranslated region and each coding exon of the DHODH gene were amplified by polymerase chain reaction from 20 ng of gDNA and the products were enzymatically purified using Exonuclease I (New England Biolabs, Ipswich, MA, USA) and Shrimp Alkaline Phosphatase (Affymetrix, Santa Clara, CA, USA), then bidirectionally sequenced with the Big Dye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA), according to manufacturer instructions. Electropherograms were produced on an ABI 3130xl Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) and assembled using Codon Code Aligner Software (CodonCode Corporation, Centerville, MA, USA). Sequences were then compared against reference sequence NM_001361.4 to identify variants.

3. Results

3.1. Urine, plasma and dried spot analyses Fig. 3 shows the LC-MS/MS chromatograms of the patient’s plasma (left) and urine (right). The ion transition for DHO (156.9/112.8) was the same as that for the 15 internal standard, [ N2]-OA, but the peaks were sufficiently resolved (DHO at 3.1 min, OA at 3.8 min) to accurately measure these metabolites. ACCEPTED MANUSCRIPT Table 1 shows the initial pyrimidine profile of the patient in plasma and urine samples (Week 0, Fig. 4). DHO was raised in plasma (3.5 µmol/L, reference < 0.5 µmol/L) and grossly elevated in urine. OA was undetectable in plasma, but was significantly raised in urine. Other pyrimidines – uracil, thymine, uridine, pseudouridine, deoxyuridine and thymidine – were normal in urine and plasma. Plasma amino acids were normal. Fig. 4 shows monitoring of the patient for almost 2 years, including the 3- month uridine therapy. Initially his plasma DHO was in excess of 3 µmol/L, but following commencement of uridine therapy his plasma DHO exhibited a sustained decrease until it was almost undetectable (0.6 µmol/L). Urine DHO fluctuated and

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remained grossly raised, between 33-110 µmol/mmol creatinine (reference range < 1). Despite uridine supplementation, both plasma and urinary uridine remained normal (plasma < 10 µmol/L, urine 0.9-2.4 µmol/mmol creatinine; not shown). On the other hand, urinary uracil, an excretion product of uridine, increased gradually during therapy to a mean of 40 µmol/mmol (reference < 25). The plasma OA values prior to and during the uridine trial were at or below our 0.1 µmol/L limit of detection (i.e. 0.1, 0.15, <0.1, 0.1, <0.1, 0.1 µmol/L). These were well within the normal range (< 1 µmol/L). In contrast, urine OA, presumably derived from DHO, was raised and fluctuated around a mean of 27 µmol/mmol (reference < 5). Plasma and urine uric acid remained within the normal range for age throughout uridine therapy, as did other purines and pyrimidines.

Table 1 Pyrimidines in samples of patient plasma and urine, and dried spots.

Urine Plasma Dried plasma Dried blood Metabolite (µmol/mmol (µmol/L) spot (µmol/L) spot (µmol/L) creatinine)

DHO 3.5 [<0.5 a] 39 [<0.5 a] 2.3b 0.43 b

OA <1 [<1 a] 33 [<5] <1 <1 Uracil <1 [<1] 42 [<25] - - Uridine 5 [<10] 2.4 [<5] 4.4 12.9 c Creatinine 30 [15-50] 0.67 (mmol/L) - -

Plasma/urine andACCEPTED dried plasma/blood spots wereMANUSCRIPT sampled at week 0 (see Fig. 3). DHO: Dihydroorotate; OA: Orotic acid; [ ] Reference ranges. a Normally undetectable. b Corrected for creatinine = 30, recovery ~65% from plasma spots , ~25% from blood spots. c Uridine in dried blood was raised due to breakdown of red cell uridine-diphophate-glucose.

DHO was easily detectable in dried plasma spots of the patient, and to a lesser extent in dried blood spots. Using our LC-MS/MS system the lower limit of quantification for DHO in extracts of dried EDTA plasma and blood spots using spiked control samples was approximately 0.5 µmol/L for both, although sensitivity will vary between mass detectors. However, recovery of DHO from dried spots of control plasma and blood spiked with DHO showed large differences: recovery from

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dried plasma was 41-68% (mean 58%), but it was 23-26% (mean 25%) from dried blood. Recovery using methanol:water extraction was similar to acetonitrile:water.

An extract of the patient's plasma dried onto filter paper yielded DHO concentration of 9.0 µmol/L, corrected to 2.2 µmol/L based on the patient’s plasma creatinine of 30 µmol/L, a recovery of 63% from the original plasma value of 3.5 µmol/L (see Table 1). A blood spot extract from the same patient sample yielded a mean DHO concentration of 1.41 µmol/L uncorrected, or 0.43 µmol/L corrected for creatinine (Table 1). Assuming a DHO recovery of about 25% (calculated from the dried control blood studies) the patient's original whole blood DHO concentration would have been about 1.7 µmol/L, i.e. approximately half of the plasma concentration. This raised the question of whether DHO was excluded from red cells. When DHO was added to fresh whole blood and incubated for up to 2.5 h, the DHO was recovered entirely from the plasma fraction (results not shown), i.e. it was not taken up by red cells. Therefore the concentration of DHO in whole blood can be predicted to be approximately half of the plasma concentration, corresponding with our observation of DHO in the patient's blood spots. OA did not appear during the incubation, so the absence of DHO from red cells could not be explained by its metabolism. DHO did not spontaneously convert to OA in either distilled water or urine, when incubated for 60 hours at both room temperature and refrigerated (5oC), even in high micromolar concentrations. Purified xanthine oxidase did not convert DHO to OA but readily oxidised the purine substrate xanthine to uric acid. ACCEPTED MANUSCRIPT 3.2. Genotyping Molecular confirmation of the patient's clinical diagnosis was provided by sequencing of the DHODH gene, which was shown to be compound heterozygous for the frameshift and missense mutations: c.165-166delCA {mat}, p.His55Glufs*21 and c.925C>T {pat}, p.Arg309Trp (Fig. 5). A microarray was normal.

3.3. Clinical The patient's liver enzymes remain modestly elevated. His liver ultrasound has remained unchanged, and no further biopsies have been undertaken. While on the 3- month trial of uridine, no clear alterations in the patient’s behaviour were noted by his

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mother or commented on by his school teachers, the latter being unaware of any trial taking place. However, at an outpatient visit six months after the end of the trial his behaviour showed a very marked improvement (co-operative, interactive, and not hyperactive) which, his mother spontaneously commented, had been present for some time.

4. Discussion

4.1. A metabolic conundrum solved Pyrimidine excretion has previously been detailed for two out of five other Miller patients (1): both had elevated urinary OA, while DHO was undetectable. Plasma was not analysed. Expression studies of DHODH mutations, confirmed to underlie Miller syndrome in HeLa cells and E. coli, demonstrated DHODH enzyme activity to be partially but not fully inactivated (1, 3). Rainger and coworkers (1) suggested that complete deficiency of DHODH is probably not compatible with life and that a narrow range of residual activity, or "disease permissive window" from about 24-36% of normal activity, results in Miller syndrome. This presented an unusual problem for the diagnosis of a metabolic disorder, as it appeared that urinary DHO could not confidently be used as a metabolic marker for low DHODH activity. Furthermore, the presence of elevated urinary OA in two out of five patients tested appeared "paradoxical", as the DHODH reaction is assumed to be greatly reduced (1). We have demonstrated here for the first time that functional mutations in the DHODH gene lead to plasma accumulation of DHO, not OA, in a Miller syndrome patient. This ACCEPTEDplaces the focus firmly back MANUSCRIPT onto DHODH as causative of the clinical phenotype.

4.2. Renal excretion of orotate Both DHO and OA were grossly elevated in the patient’s urine. However, we found that DHO in urine (and water) is stable, suggesting that post-renal degradation (in the urinary tract or bladder) does not explain the paradoxically raised urine OA. We conclude that the appearance of OA in the urine of Miller syndrome patients must occur via enzymatic oxidation during renal secretion. We excluded xanthine oxidase from this mechanism. Residual renal DHODH activity could not be excluded,

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although we regard it unlikely that the gene mutations would knock out DHODH in liver and other tissues but not kidney. However, there are other examples of renal metabolism induced by metabolic disorders: particularly relevant to DHODH deficiency is dihydropyrimidine dehydrogenase deficiency. In this disorder uracil and thymine (5-methyluracil) accumulate in both plasma and urine, however thymine becomes an alternative substrate for an unidentified renal enzyme that oxidises thymine to 5-hydroxy- methyluracil (5), which then appears in urine but not in plasma. We thus hypothesise that in Miller syndrome DHO accumulates in plasma where it becomes an alternative substrate for an enzyme which oxidises DHO to OA: we suggest a renal isoform of cytochrome P450 (CYP). We specifically propose a role for CYP because this class of enzymes has diverse genetic variance in humans. Thus we predict that Miller patients who have high renal CYP activity would oxidise DHO completely during renal secretion, as seen in previously reported patients, while others such as our patient who have low renal CYP would only partially convert DHO to OA. Further research is required to test this hypothesis. An unusual property that we discovered of DHO is its distribution in blood: we found it is excluded from red cells and recovered only in plasma. This was surprising because its metabolite OA is rapidly taken up by red cells, then converted via UMP synthase to pyrimidine nucleotides (6).

4.3. Miller syndrome phenotype The molecular mechanism to explain how a reduction in DHODH activity results in the ACCEPTEDcharacteristic dysmorphic phenotypeMANUSCRIPT of Miller syndrome is not currently understood. However, treatment of pregnant mice with the selective DHODH inhibitor leflunomide has been demonstrated to cause limb and craniofacial malformations consistent with Miller syndrome in humans (7). This provided elegant biochemical evidence for the pathogenesis of the characteristic Miller syndrome structural anomalies, arising from low or absent DHODH. Leflunomide is also associated with inhibition of the liver nuclear factor NF-κB (2), demonstrating a molecular link between DHODH deficiency and essential cell-signalling mechanisms. The clinical phenotype of Miller syndrome significantly overlaps with several facial dysostosis syndromes including Treacher Collins, Guion-Almeida (mandibulo facial dysostosis with microcephaly) and Nager syndromes. A suggestion of a

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common molecular pathogenesis involving RNA was supported by the discovery that genes causing these conditions are involved in RNA transcription or splicing: Treacher Collins syndrome arises from mutations in TCOF1, POLR1D, and POLR1C, which are all involved in pre-rRNA transcription as components of the spliceosome complex (8-11). How the molecular defect in Miller syndrome produces an overlapping phenotype with these conditions remains to be determined. The pyrimidine synthetic pathway is highly active during embryogenesis, so reduction of OA synthesis in Miller patients can be predicted to affect key biosynthetic mechanisms for which pyrimidines are essential: lipoproteins and glycoproteins hence membrane assembly (via pyrimidine nucleotide lipid/sugar esters), glycogen production (utilising uridine diphosphate-glucose), and of course DNA and RNA synthesis. On the other hand, OA is converted into pyrimidine nucleotides via the bi-functional enzyme UMP synthase: inherited deficiency of UMP synthase is not associated with severe developmental defects, suggesting that blockage of pyrimidine nucleotide synthesis is insufficient, per se, to explain the anomalies in developmental morphology observed in Miller syndrome. We found that for our patient all other pyrimidine metabolites downstream from OA were essentially normal, in particular uridine and uracil. Thus, although the pyrimidine nucleotide synthesis pathway is partially blocked at DHODH, residual pyrimidine synthesis combined with absorption of dietary pyrimidines may suffice to maintain essential cellular pyrimidine nucleotides. It has also been suggested that intracellular DHO or an alternative metabolite may adverselyACCEPTED affect the mitogenic response MANUSCRIPT to fibroblast growth factor signalling in proliferating zones, resulting in malformations (1, 12). A more direct toxic effect of DHO cannot be excluded. Finally, the energy generation by DHODH feeding electrons into the oxidative phosphorylation chain is not trivial during rapid cell division, as demonstrated by Fang and coworkers (3), so low DHODH may lead to highly localised energy depletion in tissues during early foetal development. The hepatic features of our patient may have their origin in either defective pyrimidine metabolism or in mitochondrial dysfunction, but for the present we assume this to be a rare feature of Miller syndrome, and we continue to monitor the patient's progress.

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4.4. Screening and early diagnosis Confirmation of the presence of DHO in the plasma of a Miller syndrome patient is important because it provides a simple means of screening for the disorder, particularly with blood or plasma spots that can be mailed to tertiary referral centers providing metabolite screening. The incidence of Miller syndrome is unknown but it is considered to be rare, however the majority of children born with a POADS phenotype are not screened for DHODH deficiency, so under-diagnosis cannot be excluded. Early diagnosis of Miller syndrome may provide the possibilities, on one hand, of early dietary supplementation with uridine for the child to assist development, and on the other hand families may be able to avoid having further affected children by providing the mother with a uridine-supplemented diet.

4.5. Uridine therapy We undertook a 3-month uridine trial for our patient to assess if it could correct behavioural changes that had arisen, however, no obvious behavioural improvements were noted during the trial. There were no untoward effects of the uridine, nor any changes in his routine biochemistry. Exogenous uridine treatment reduced plasma DHO to near-normal, perhaps by a feedback mechanism, although urinary DHO excretion did not decrease: further study is required. Early screening may be important for reasons extending beyond simply providing a patient diagnosis. The teratogenicity of leflunomide producing a POADS- type phenotype in mice was found to be reversible by a high-uridine diet for the females during pregnancy (7). DietaryACCEPTED uridine, from RNA and uridineMANUSCRIPT nucleotides, is readily taken up by the gut and salvaged (i.e. metabolically recycled). Uridine supplementation has long been established as safe as long-term therapy of UMP synthase deficiency (13), but more recently it has been trialled in the treatment of adolescents with bipolar disorders (14). Maternal uridine treatment to prevent morphological anomalies of Miller syndrome presents an exciting possibility. As treatment would have to commence in the first trimester, the risk of uridine exposure to an unaffected foetus would be 75%. Treatment could then be rationalised in the second trimester using prenatal diagnosis for previously identified DHODH mutations. However, consideration of teratogenicity versus safety of uridine is important. Evidence for the safety of dietary uridine supplementation has been provided by the cases of two women (with disorders

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unrelated to Miller syndrome), who between them had six successful pregnancies for which five of the offspring were normal; one child had multiple anomalies due to a maternally-derived chromosomal anomaly for which the mother was a balanced carrier (13, 15).

5. Conclusions

We conclude that the DHODH deficiency that produces Miller syndrome is indeed functional, resulting in accumulation of DHO in plasma. At high intracellular concentrations DHO may then act as an alternative substrate for another oxidative enzyme during renal secretion, perhaps a cytochrome P450 isoform, producing urinary OA. The lowered functionality of the pyrimidine de novo synthetic pathway during foetal development is not a complete explanation for the clinical defects observed for Miller syndrome: other possible mechanisms include compromised cell signalling, perturbation of pyrimidine nucleotides synthesising RNA, or mitochondrial dysfunction leading to localised tissue energy deficits during embryological development. Early screening and biochemical diagnosis of DHODH deficiency should be feasible and reliable using dried spots of blood or plasma, but not urine. Potential uridine treatment in early pregnancy to prevent morphological anomalies presents an exciting but untested possibility.

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Acknowledgments The authors wish to acknowledge Professor Deon Venter, Director of Pathology at Mater Health Services, for facilitating the metabolite analyses for this work.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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References

1. Rainger J, Bengani H, Campbell L, Anderson E, Sokhi K, Lam W, et al. Miller (Genee-Wiedemann) syndrome represents a clinically and biochemically distinct subgroup of postaxial acrofacial dysostosis associated with partial deficiency of DHODH. Hum Mol Genet. 2012;21(18):3969-83. 2. Ng SB, Buckingham KJ, Lee C, Bigham AW, Tabor HK, Dent KM, et al. Exome sequencing identifies the cause of a mendelian disorder. Nat Genet. 2010;42(1):30-5. 3. Fang J, Uchiumi T, Yagi M, Matsumoto S, Amamoto R, Saito T, et al. Protein instability and functional defects caused by mutations of dihydro-orotate dehydrogenase in Miller syndrome patients. Biosci Rep. 2012;32(6):631-9. 4. Al-Shehri S, Henman M, Charles BG, Cowley D, Shaw PN, Liley H, et al. Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2013;931:140-7. 5. Brockstedt M, Jakobs C, Smit LM, van Gennip AH, Berger R. A new case of dihydropyrimidine dehydrogenase deficiency. J Inherit Metab Dis. 1990;13(1):121-4. 6. Berman P, Harley E. Orotate uptake and metabolism by human erythrocytes. Adv Exp Med Biol. 1984;165 Pt A:367-71. 7. Fukushima R, Kanamori S, Hirashiba M, Hishikawa A, Muranaka R, Kaneto M, et al. Inhibiting the teratogenicity of the immunosuppressant leflunomide in mice by supplementationACCEPTED of exogenous uridine. MANUSCRIPT Toxicol Sci. 2009;108(2):419-26. 8. Group. TTCSC. Positional cloning of a gene involved in the pathogenesis of Treacher Collins syndrome. The Treacher Collins Syndrome Collaborative Group. Nat Genet. 1996;12(2):130-6. 9. Dauwerse JG, Dixon J, Seland S, Ruivenkamp CA, van Haeringen A, Hoefsloot LH, et al. Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome. Nat Genet. 2011;43(1):20-2. 10. Lines MA, Huang L, Schwartzentruber J, Douglas SL, Lynch DC, Beaulieu C, et al. Haploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly. Am J Hum Genet. 2012;90(2):369-77.

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11. Bernier FP, Caluseriu O, Ng S, Schwartzentruber J, Buckingham KJ, Innes AM, et al. Haploinsufficiency of SF3B4, a component of the pre-mRNA spliceosomal complex, causes Nager syndrome. Am J Hum Genet. 2012;90(5):925-33. 12. Corson LB, Yamanaka Y, Lai KM, Rossant J. Spatial and temporal patterns of ERK signaling during mouse embryogenesis. Development. 2003;130(19):4527-37. 13. Webster DR, Becroft DM, van Gennip AH, van Kuilenburg ABP. Hereditary orotic aciduria and other disorders of pyrimidine metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. II: Medical Publishing Division, McGraw-Hill, New York, USA; 2001. p. 2663-702. 14. Kondo D, Sung Y, Hellem T, Delmastro K, Jeong E, Kim N, et al. Open-label uridine for treatment of depressed adolescents with bipolar disorder. J Child and Adolescent Psychopharmacology. 2011;21(2):5. 15. Bensen JT, Nelson LH, Pettenati MJ, Block SM, Brusilow SW, Livingstone LR, et al. First report of management and outcome of pregnancies associated with hereditary orotic aciduria. Am J Med Genet. 1991;41(4):426-31.

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Legends for Figures

Fig. 1. Diagrammatic representation of cell cytosol and mitochondrial membrane showing dihydroorotate dehydrogenase (DHODH) located on the inner mitochondrial membrane, passing electrons (e-) from flavin mononucleotide (FMN) coenzyme to reduce ubiquinone

(UQH2), ultimately reducing cytochrome C (Cyt C) and thus feeding into the oxidative phosphorylation complex which produces energy. UMP: uridine monophosphate.

Fig. 2. Physical features of patient (with parental permission). a preterm neonate. b six months of age. c two years. d Oligodactyly of hands with cruciform palmar creases and 4th ray hypoplasia of feet. e X-rays showing oligodactyly of hands and 4th ray hypoplasia of feet.

Fig. 3. LC-MS/MS of urine and plasma from a Miller syndrome patient. The DHO peak is at 3.1 min, OA is at 3.8 min. The large peak at 3.8 min in (a) and (b) is [15N2]-OA internal standard as its ion transition overlapped with that of DHO. a DHO in urine. b DHO in plasma. c OA in urine. d OA was absent in plasma (peak in background noise).

Fig. 4. Plasma and urine metabolites before and during uridine therapy. X-axis (weeks) shows consecutive plasma/urine samples, from ~18 months (-80 weeks) prior to the uridine trial, commencing week 2, until week 15, week 0 samples were for baseline. Y-axis shows concentration units: plasma - µmol/L, urine - µmol/mmol creatinine; Dashed lines indicate the upper limits of normal ranges. a plasma DHO.ACCEPTED b urine OA (derived from DHOMANUSCRIPT metabolism). c urine DHO. d urine uracil (from uridine catabolism).

Fig. 5. Mutations in the DHODH gene of the patient. a) gene structure location of mutations in exons 2 and 7; b) proband allele, exon 2. c) paternal allele, exon 2. d) proband allele, exon 7. e) maternal allele, exon 7.

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Figure 3

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Figure 4

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Figure 5

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Highlights – Miller syndrome, Duley et al

 First report of raised dihydroorotate in plasma and urine for Miller syndrome  Biochemical confirmation of functional deficiency of the enzyme  Diagnosis is simplified using plasma or dried blood/plasma spots  Urine orotate proposed to arise from alternative metabolism of dihydroorotate  Oral uridine did not resolve behavioural problems but lowered plasma dihydroorotate

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