BMJ Innov: first published as 10.1136/bmjinnov-2018-000324 on 28 September 2019. Downloaded from Diagnostics

Original research Subclinical ochronosis features in : a cross-sectional study

Trevor Cox,1 Eftychia Eirini Psarelli,2 Sophie Taylor,3 Hannah Rose Shepherd ‍ ,4 Mark Robinson,5 Gabor Barton ‍ ,5 Alpesh Mistry,6 Federica Genovese ‍ ,7 Daniela Braconi ‍ ,8 Daniela Giustarini,9 Ranieri Rossi,9 Annalisa Santucci ‍ ,8 Milad Khedr ‍ ,10 Andrew Hughes,11 Anna Milan,11 Leah Frances Taylor ‍ ,12 Elizabeth West,13 Nicolas Sireau,14 Jane Patricia Dillon ‍ ,15 Nicholas Rhodes ‍ ,16 James Anthony Gallagher,15 Lakshminarayan Ranganath17

►► Additional material is Abstract characterised by high circulating homo- published online only. To view Background Alkaptonuria (AKU) is present gentisic acid (HGA).1 The frequency please visit the journal online (http://dx.​ ​doi.org/​ ​10.1136/​ ​ from birth, yet clinical effects are considered to of AKU is around 1 in 250 000 to 1 in bmjinnov-2018-​ ​000324). appear later in life. Morbidity of AKU, considered 1 000 000 in most populations worldwide. irreversible, is secondary to ochronosis. Age of Deposition of HGA in connective tissue For numbered affiliations see 2 end of article. ochronosis onset is not clearly known. Nitisinone as pigment in AKU is termed ochronosis. profoundly lowers (HGA), Debilitating manifestations of AKU are Correspondence to the metabolic defect in AKU. Nitisinone also due to ochronosis including premature Dr Lakshminarayan Ranganath, arrests ochronosis and slows progression of AKU. arthritis, cardiac valve disease, fractures, Clinical Biochemistry and 3 Metabolic Medicine, Royal However, tyrosinaemia post-nitisinone has been and muscle and tendon ruptures. Despite Liverpool University Hospital, associated with corneal keratopathy, rash and the presence of the defect from birth, Liverpool L7 8XP, UK; ​lrang@​ cognitive impairment in HT 1. The optimal time apart from dark urine, and sometimes liv.ac.​ ​uk to start nitisinone in AKU is unknown. renal stones, there are few osteoarticular

4 http://innovations.bmj.com/ Received 10 October 2018 Methods In an open, cross-sectional, single- symptoms until around age 25 years. Revised 4 April 2019 site study, 32 patients with AKU were to be A recent study has shown that nitisinone, Accepted 11 August 2019 recruited. The primary outcome was presence of an inhibitor of p-hydroxyphenylpyruvate ochronosis in an ear biopsy. Secondary outcomes dioxygenase (EC:1.13.11.27), is effective included analysis of photographs of eyes/ears, in AKU.5–8 Nitisinone decreases circu- serum/urine HGA, markers of tissue damage/ lating HGA, and inhibits ochronosis in inflammation/oxidation, MRI imaging, gait, AKU mice and humans, slowing progres- quality of life and Alkaptonuria Severity Score sion of human AKU, but is expensive.9 10 Index (qAKUSSI). It is an imperfect treatment producing Results Thirty patients, with mean age (SD) a different metabolic block, character- on September 29, 2019 by guest. Protected copyright. 38 (14) years, were recruited. Percentage ised by tyrosinaemia; toxic consequences pigmentation within ear biopsies increased such as corneal keratopathy, eczema-like with age. Ear pigmentation was detected in a skin rash and cognitive impairment can 20-year-old woman implying ochronosis can ensue.11 start in patients before the age of 20. Gait and AKU is not fully reversible and would qAKUSSI were outside the normal range in all benefit from early treatment. Mouse © Author(s) (or their the patients with AKU. studies show that nitisinone can prevent employer(s)) 2019. No Conclusions Ochronosis can be present before onset of ochronosis when started soon commercial re-use. See rights age 20 years. after birth and arrest ochronosis when and permissions. Published by 9 10 BMJ. started later. Mouse studies revealed ochronotic pigment laid down in knee To cite: Cox T, Psarelli EE, 9 Taylor S, et al. BMJ Innov joints as early as 15 weeks. These find- Epub ahead of print: Background ings highlight the concern that subclinical [please include Day Month Alkaptonuria (AKU) (OMIM #203500) ochronosis could be damaging connec- Year]. doi:10.1136/ is a rare genetic deficiency of homo- tive tissues early in life. Starting nitis- bmjinnov-2018-000324 gentisate dioxygenase (EC:1.13.11.5), inone later in AKU is expected to result

Cox T, et al. BMJ Innov 2019;0:1–10. doi:10.1136/bmjinnov-2018-000324 1 BMJ Innov: first published as 10.1136/bmjinnov-2018-000324 on 28 September 2019. Downloaded from DIAGNOSTICS in residual disease as AKU is irreversible.4 Identifying Disease questionnaires were used to calculate when ochronosis takes hold in AKU is important, as the modified questionnaire-based qAKUSSI (online this information would allow optimal use of nitisinone supplementary table S4).19 in terms of when to begin treatment. Fasting blood samples and 24-hour urine samples The SOFIA (Subclinical Ochronosis Features In were collected to measure metabolites and biomarkers Alkaptonuria) study was designed to identify the of inflammation, cartilage, bone and tissue. Serum and earliest age when ochronosis, microscopic and macro- urine HGA were quantitated by liquid chromatography– scopic, can be detected in patients and at what age it tandem mass spectrometry methods.20 21 Serum amyloid might be appropriate to begin nitisinone treatment. A (SAA) was analysed using a commercial assay. Quan- titative determination of serum protein thiols and S-thi- 22 Methods olated proteins was carried out. Serum protein thiols Patients were measured by colorimetric reaction with 5,5′-dith- Patients with AKU, verified by elevated HGA levels, iobis-(2-nitrobenzoic acid).23 S-Thiolated proteins and at least 16 years old were eligible for inclusion. A were measured.24 Protein Thiolation Index (PTI) was non-AKU control group was also selected. None of the calculated as the molar ratio between total S-thiolated patients in this study received nitisinone at the time of proteins (RSSP, where RS is cysteine (CySSP), cysteinyl- participation. (CyGlySSP), homocysteine (HcySSP), γ-glutam- ylcysteine (γGluCySSP) and glutathione (GSSP)) and the Study design concentration of protein thiols.25 SOFIA was an open, cross-sectional, single-site (Royal The rate of connective tissue remodelling was Liverpool University Hospital) study; 32 patients with analysed by the measurement in serum and urine of AKU were to be recruited (two males, two females biomarkers of collagen and other extracellular matrix in each age interval: 16–20, 21–25, 26–30, 31–35, protein formation and degradation using competitive 36–40, 41–45, 46–50, over 50) covering the age and sandwich ELISAs developed at Nordic Bioscience spread of non-ochronotic and ochronotic groups. (online supplementary table S5). The assays were run The primary outcome was the amount of ochronosis according to the standard procedure detailed in the measured in ear biopsies. The secondary outcomes references in online supplementary table S5. were visible ochronosis quantification in eyes and QoL was assessed using HAQ, SF-36 and Knee ears, MRI examination, deviation in gait, modified injury and Osteoarthritis Outcome Score (KOOS) qAKUSSI (questionnaire Alkaptonuria Severity Score questionnaires.26–28 Index), circulating and urine HGA, inflammation, cartilage damage, bone and other tissue biomarkers, Statistical analysis

and quality of life (QoL). Multiple linear and LOESS regression were the main http://innovations.bmj.com/ analyses used. Outcome variables were plotted against Assessments age and regression analyses carried out. Comparisons Ear cartilage: a 4 mm diameter, 1–2 mm thick biopsy was with controls were made as appropriate. All analyses taken from the conchal bowl of the ear and stored using were conducted using SAS V.9.3 and R V.3.3.2. a standardised protocol. Ochronosis was measured by percentage of light absorbance in microscope photo- graphs (non-ochronotic tissue=0, completely ochro- Results notic tissue=100) (see online supplementary material). Thirty patients with AKU (15 male, 15 female) were recruited. Mean age was 39 years for males and 37

Standardised photographs of eyes and ears were on September 29, 2019 by guest. Protected copyright. years for females; mean body mass index (BMI) was scored for ochronosis: for eyes using qualitative and 2 2 quantitative systems; for ears, a qualitative system 25 kg/m for males and 23 kg/m for females (online (online supplementary figure S1, tables S1, S2). supplementary tables S6–S8). Modified Pfirrmann Grading System and Spondy- loarthritis Research Consortium of Canada (SPARCC) Ear cartilage biopsy, eye and ear photographs scores of the spine and whole-organ MRI scores Ear biopsies from 28 samples were processed. Figure 1 (WORMS) of the knee were calculated (online supple- shows ear and eye ochronosis measured by biopsy and mentary figures S2–S5, table S3).12–14 Institutional photographs plotted against age, together with regres- MRI protocols were followed. sion lines. Figure 1A shows percentage of pigmenta- 3D gait analysis was performed for 36 patients with tion of whole-ear biopsy. Fitted regression lines were: AKU (29 from SOFIA, 7 from the UK National Alkap- % ear pigmentation=−23.5+1.43×age for males tonuria Centre) and for a control group of 10 volun- (age: p=0.001, gender: p=0.742, R2=0.39). teers free from gait problems.15 16 Mean deviation of % ear pigmentation=−26.9+1.43×age for females. AKU gait from normality (MDP ) was calculated Age was statistically significant (p=0.001), but not mean using the MDP program (online supplementary mate- gender. The fit of this model, and some of the following rial, online supplementary figure S6).17 18 models, was not particularly good, but it does give a

2 Cox T, et al. BMJ Innov 2019;0:1–10. doi:10.1136/bmjinnov-2018-000324 BMJ Innov: first published as 10.1136/bmjinnov-2018-000324 on 28 September 2019. Downloaded from DIAGNOSTICS

Figure 1 (A) Ochronosis score from ear biopsy plotted against age with regression lines, (B) ear ochronosis measured from photographs plotted against age with LOESS regression line, (C) eye ochronosis measured from photographs plotted against age with regression lines, (D) total quantitative eye pigmentation expressed as pixel value score plotted against total qualitative eye pigmentation score. http://innovations.bmj.com/ strong indication of the increase in pigmentation with in figure 2B. Knee scores are shown in figure 2C. age. Increases of the MRI scores with age are clearly not Assessment of pigment intensity by measuring light linear; LOESS regressions are shown for the two spine absorption of whole cartilage biopsy revealed pigmen- scores. Both show significant increases starting in the tation in a patient aged 20. third decade of life. The WORMS generally stay low Figure 1B shows ear ochronosis measured from over the age range with a few exceptions. photographs. Ochronosis was not detected until the Figure 2D shows the mean movement deviation on September 29, 2019 by guest. Protected copyright. middle of the third decade; a LOESS (Local Regres- profile (MDP ) plotted against age for patients with mean sion) reflects this. Figure 1C shows qualitative eye AKU and controls together with a linear regression line ochronosis scores. Regression lines were: for controls and a LOESS regression for the patients Eye pigmentation=−7.3+0.36×age for males (age: with AKU. Deviation from normal gait was found even p<0.001, gender: p=0.448, R2=0.50). in the younger patients, and a steep incline after 50 Eye pigmentation=−8.7+0.36×age for females. years. Age was statistically significant, but gender was not. MDP =2.44–0.01×age for controls. mean Figure 1D shows the correlation of the qualitative and Online supplementary figures S7 and S8 show a quantitative eye ochronosis scores (R2=0.83). linear clustering and a 2D visualisation of the patients with AKU and controls. MRI scans and gait analyses MRI spine and left knee imaging were performed on Modified qAKUSSI 27 and 26 patients with AKU, respectively. Disc degen- Figure 3A shows total modified qAKUSSI plotted eration, measured using the modified Pfirrmann score, against age; figure 3B–D shows its component parts is shown in figure 2A and marrow oedema results, of clinical features, spine rheumatology and non-spine indicated by the modified SPARCC score, are shown rheumatology. The corresponding regressions are:

Cox T, et al. BMJ Innov 2019;0:1–10. doi:10.1136/bmjinnov-2018-000324 3 BMJ Innov: first published as 10.1136/bmjinnov-2018-000324 on 28 September 2019. Downloaded from DIAGNOSTICS

Figure 2 (A) MRI Pfirrmann scores plotted against age, (B) Spondyloarthritis Research Consortium of Canada (SPARCC) scores plotted against age, (C) whole-organ MRI score (WORMS) plotted against age, (D) mean movement deviation profile (MDP ) mean plotted against age. AKU, alkaptonuria. http://innovations.bmj.com/

qAKUSSI=−8.53+1.15×age for males HGA_clearance=1322–11.8×age for males (age: (age: p<0.001, gender: p=0.780, R2=0.47); p=0.007, gender: p=0.934, R2=0.26). qAKUSSI=−10.41+1.15×age for females. HGA_clearance=1313–11.8×age for females. qAKUSSI_clin=−7.31+0.67×age for males (age: Gender was not significant, but serum HGA signifi- 2 p=0.001, gender: p=0.316, R =0.36); qAKUSSI_ cantly increased and HGA clearance significantly clin=−12.35+0.67×age for females. decreased with age. Online supplementary figure S9 qAKUSSI_spine=−1.32+0.22×age for males (age: shows 24-hour urine HGA plotted against age. 2 p=0.002, gender: p=0.076, R =0.35); qAKUSSI_ Figure 4C–E shows SAA, CyGlySSP and PTI plotted on September 29, 2019 by guest. Protected copyright. spine=1.91+0.22×age for females. against age and fitted regression lines. SAA and PTI qAKUSSI_nonspine=0.05+0.26×age for males 2 significantly increased with age, but with no signifi- (age: p=0.017, gender: p=0.995, R =0.20); cant difference between groups. The regression lines qAKUSSI_nonspine=0.03+0.26×age for females. for CyGlySSP showed significantly different slopes There were no significant differences for gender; all and intercepts for groups.19–22 regressions showed a significant increase with age. SAA=8.5+0.43×age for AKU (age: p=0.047, 2 HGA, biomarkers and metabolite analyses group: p=0.667, R =0.07)* Figure 4 shows some results obtained for HGA SAA=10.8+0.43×age for controls measurements, the inflammation biomarker, SAA and CyGlySSP=22.1–0.20×age for AKU (age: p<0.001, connective tissue damage markers. Figure 4A,B shows group: p=0.005). serum HGA and HGA clearance against age. The CyGlySSP=25.4+0.04×age for controls 2 regression equations are: (age×group interaction: p=0.002, R =0.25). Serum_HGA=17.4+0.28×age for males (age: PTI=0.31+0.002×age for AKU (age: p=0.015, p=0.006, gender: p=0.465, R2=0.26). group: p=0.090, R2=0.14). Serum_HGA=19.2+0.28×age for females. PTI=0.28+0.002×age for controls.

4 Cox T, et al. BMJ Innov 2019;0:1–10. doi:10.1136/bmjinnov-2018-000324 BMJ Innov: first published as 10.1136/bmjinnov-2018-000324 on 28 September 2019. Downloaded from DIAGNOSTICS

Figure 3 Modified qualitative Alkaptonuria Severity Score Index (qAKUSSI): (A) Total modified qAKUSSI; (B) qAKUSSI, clinical features; (C) aAKUSSI, spine rheumatology; (D) qAKUSSI, non-spine rhematology.

*Two outliers removed. Quality of life http://innovations.bmj.com/ Online supplementary figure S10 shows extra Figure 5 shows 6 of the 33 QoL measurements: Health inflammatory and oxidative markers. assessment questionnaire (HAQ) Disability Index, Figure 4F–H shows uC1M_cr, TIM and P1NP HAQ Pain score, Short-Form Health Survey (SF)-36 plotted against age together with regression lines. Physical Functioning score, SF-36 General Health The regression lines for the two groups for uC1M_ score, KOOS Sport and Recreation score and KOOS cr have marginally significantly different slopes and Quality of Life score. The regression equations were: significantly different intercepts. TIM has significantly HAQ_DI=−0.73+0.03×age for males (age: different slopes and intercepts for the two groups. p<0.001, gender: p=0.676, R2=0.61). on September 29, 2019 by guest. Protected copyright. P1NP significantly decreases with age for patients with HAQ_DI=−0.68+0.03×age for females AKU and controls at the same rate, but with mean HAQ_Pain=−0.81+0.05×age for males (age: P1NP higher for patients with AKU. uC1M_cr=3.59– p<0.001, gender: p=0.533, R2=0.71) 0.05×age for AKU (age: p=0.004, group: p=0.004, HAQ_Pain=−0.65+0.05×age for females. uC1M_cr=1.65–0.03×age for controls (age×group SF36_PF=109.8–1.17×age for males (age: interaction: p=0.056, R2=0.30). p<0.001, gender: p=0.390, R2=0.36) TIM=157+4.09×age for AKU (age: p=0.047, SF36_PF=117.3–1.17×age for females. group: p=326). SF36_GH=83.3–1.03×age for males (age: TIM=223+0.52×age for controls (age×group p=0.001, gender: p=0.775, R2=0.35) interaction: p=0.039, R2=0.30). SF36_GH=85.5–1.03×age for females. P1NP=172–1.43×age for AKU (age: p=0.022, KOOS_Sport_Rec=144.6–2.18×age for males (age: group: p=0.002, R2=0.22). p<0.001, gender: p=0.938, R2=0.60). P1NP=121–1.43×age for controls. KOOS_Sport_Rec=145.3–2.18×age for females. Online supplementary figure S11 shows the KOOS_QoL=115.1–1.37×age for males (age: biomarkers not covered in the main text, plotted p<0.001, gender: p=0.815, R2=0.45) against age for patients with AKU and controls. KOOS_QoL=117.0–1.37×age for females.

Cox T, et al. BMJ Innov 2019;0:1–10. doi:10.1136/bmjinnov-2018-000324 5 BMJ Innov: first published as 10.1136/bmjinnov-2018-000324 on 28 September 2019. Downloaded from DIAGNOSTICS http://innovations.bmj.com/

Figure 4 (A) Serum homogentisic acid (HGA) (μmol/L), (B) HGA clearance (mL/min), (C) serum amyloid A (SAA) (ng/mL), (D) CyGlySSP (μM), (E) Protein Thiolation Index (PTI), (F) urine C1M_cr (ng/μmol), (G) serum TIM (ng/mL), (H) serum P1NP (ng/mL). AKU, alkaptonuria.

All these measures show significant worsening of regions appeared at 8–10 years. Dark brown to black on September 29, 2019 by guest. Protected copyright. health with age but no difference between genders. staining of ear cerumen was present even in childhood. The other QoL measures are reported in online Ear cartilage pigmentation in a 12-year-old patient, supplementary figures S12–S14. and scleral pigmentation in a 13-year-old patient, was reported, although no photographs were taken. The Discussion reported youngest age of was 24 years. The SOFIA study objective was to identify the earliest One 6-month-old patient presented with a kidney age when ochronosis, microscopic and macroscopic, 31 can be detected in patients with AKU. First, we review stone. the published information in childhood AKU. It is We are the first to report on direct tissue studies important to clarify that none of the patients in this in AKU, reasoning that it might be possible to iden- study received nitisinone at the time of participation. tify pigmentation in tissue that could not be identi- A natural history study in 2002 reported on 64 fied through overlying skin. Percentage pigmentation patients (ages 4 to 80 years), but described no clin- within ear biopsies increases with age, females lagging ical features in childhood.29 A Slovak study described behind males. Difference between genders was not childhood AKU 35 years ago in 39 patients.30 Dark statistically different, probably due to the small sample urine was present in all. Pigmentary changes in axillary size. Pigmentation was detected in a 20-year-old

6 Cox T, et al. BMJ Innov 2019;0:1–10. doi:10.1136/bmjinnov-2018-000324 BMJ Innov: first published as 10.1136/bmjinnov-2018-000324 on 28 September 2019. Downloaded from DIAGNOSTICS http://innovations.bmj.com/

Figure 5 Quality of life (QoL) scores: (A) HAQ Disability Index, (B) HAQ Pain score, (C) SF-36 Physical Functioning score, (D) SF-36 General Health score, (E) Knee injury and Osteoarthritis Outcome Score (KOOS) Sport and Recreation score, (F) KOOS Quality of Life score. on September 29, 2019 by guest. Protected copyright. female, so ochronosis can start in patients with AKU and PTI (oxidative stress marker) increased with age before the age of 20. Eye ochronosis increased with with no significant difference between patients with age and was first detected at age 22 years. Externally, AKU and controls, confirming previous findings in visible ear ochronosis was only detected after age 34 AKU.32 However, the highest SAA concentrations years. were found in younger subjects (CTR 29 years, SAA Patients with AKU had 24-hour urine HGA and 132 mg/L; AKU 20 years, SAA 121 mg/L). Addition- serum HGA values much higher than those of ally, in both groups, SAA was above the reference controls, showing no correlation with age or gender. threshold of 10 mg/L in 21/30 subjects.33 Since no Serum HGA increased with age, but with no differ- additional data apart from gender and age were made ence between genders, coexisting with worsening available on control subjects, we cannot rule out the AKU. Conversely, HGA clearance decreased with age. hypothesis of underlying inflammatory conditions, The inflammation biomarkers, SAA and serum protein however unlikely, raising SAA levels also in controls, thiols, increased with age with no significant difference for example, BMI32; this, however, again excludes a between patients with AKU and controls. SAA by itself major role for SAA in AKU. was not a discriminative marker between AKU and uC1M, a marker of collagen type I degradation controls. Both circulating SAA (inflammation marker) measured in urine, decreased with age in patients with

Cox T, et al. BMJ Innov 2019;0:1–10. doi:10.1136/bmjinnov-2018-000324 7 BMJ Innov: first published as 10.1136/bmjinnov-2018-000324 on 28 September 2019. Downloaded from DIAGNOSTICS AKU, and P1NP, a marker of collagen type I forma- matched to a normal cohort for age and gender. Plans tion measured in serum, decreased with age in both for this further study are advanced and likely to begin AKU and control groups. Bones are the main source of shortly in the SOFIA-Paediatric study. collagen type I in the human body; therefore, the results The modified Pfirrmann spine scores on MRI analysis suggest the rate of bone remodelling decreases with show patients with AKU are unlikely to demonstrate age. C1M measured in urine could reflect the remod- degenerative lesions before age 30 years; however, elling of the renal tissue, suggesting the degradation of above 35 years, they frequently show degenerative disc collagen type I in the kidney is decreased compatibly changes and overall worse Pfirrmann scores.12 Marrow with an increase in fibrogenesis in the kidneys.34 This oedema lesion results (modified SPARCC scores) also hypothesis needs further investigation. TIM, a marker appear to significantly rise from age of 30 years.13 of MMP-mediated titin degradation describing cardiac Degeneration of the knee (WORMS) did not appear to remodelling, increased with age in patients with AKU be associated with age up to the middle of the fourth but not in controls, indicating increased remodelling decade.14 Despite the lack of change in spine and of heart muscle with disease progression. At present, joints at ages younger than 30 years, the gait analysis it is not possible to establish a precise age at which was abnormal early on. The mean deviation profiles the biomarkers change dramatically. Further studies (MDPs) for gait was significantly higher for patients are needed with more patients at younger age with with AKU than for controls.17 18 The younger patients matched controls. with AKU all had high MDP values indicating that gait Identifying ochronosis externally by examining the is affected at this early age. qAKUSSI scores increase eyes and ears does not tell us about what is happening with age, the mean increasing at a rate of 1.14 units inside the body. It is possible that highly stressed tissues per year. All qAKUSSI components (clinical, spine and of the musculoskeletal system could be affected by non-spine) increase with age. Even at the ages between ochronosis earlier but not easily accessible to assess- 16 and 20, scores well above zero have been observed. ment. Conventional imaging is not sensitive enough Clinically, this means that as patients with AKU get to identify internal ochronosis early as the changes older, they score higher on qAKUSSI regardless of are late; newer approaches such as Raman spectros- their gender, reflecting a worsening disease burden. copy which are non-invasive may provide solutions. Three QOL questionnaires showed pain increases We were hoping to find biomarkers that could inform and general QoL decreases with age for patients with on potential changes in connective tissue in this study. AKU. For the HAQ questionnaire, deterioration starts The usefulness of such markers is limited in advanced around age 30 years, but this can be at the start of the AKU where there are other revealing features. In second decade for some domains for some patients. early AKU, that is, in young patients, biomarkers are For the SF-36 questionnaire, physical functions steadily

potentially more likely to prove valuable to monitor decrease with age. For the KOOS questionnaire, the http://innovations.bmj.com/ the disease, as well as inform when the disease has functions decrease with age. Overall, the QoL appears taken hold. Unfortunately, in the young, the muscu- to seriously deteriorate from the third decade. loskeletal system is changing enormously due to The overall conclusion is that this study has shown physiological growth, as well as the gender differ- that ochronosis starts at an early age, before adult- ences in growth, maturation and development. In hood, but was unable to assess the earliest age that it the present study, the low numbers of young patients may start. Further data are needed from a paediatric with AKU did not allow a clear distinction between study if this start point is to be established. AKU and non-AKU controls, even though there was The difficulties of carrying out a human natural

an apparent difference. The formation and deposi- history study of AKU can be overcome to some extent on September 29, 2019 by guest. Protected copyright. tion of the ochronotic pigment in the cartilaginous by studying mouse AKU.9 10 With biochemistry similar tissue renders the cartilage stiffer and would cause an to humans, mice with AKU also develop ochronosis, increased remodelling of proteins from the connective results suggesting that ochronosis begins a short time tissue of cartilage, bone and possibly other soft tissues. after weaning and progresses linearly over time. The The examined markers of connective tissue remodel- use of nitisinone from early on in the life of an AKU ling, however, fail to show a direct correlation with mouse completely prevented the appearance of ochro- initiation of ochronosis. TIM, a marker of titin degra- nosis. When nitisinone was administered after ochro- dation reflecting a remodelling of the cardiac sarco- nosis was established, it arrested further progression. mere, could be an indirect marker of ochronosis of The reason for carrying out this AKU study is the cardiac valve, causing impairment in the cardiac because of the availability of a HGA-lowering and a function. AKU is an ultrarare disease with a frequency disease-modifying therapy in the form of an enzyme of 1 in 250 000. This is the first study to examine a inhibitor nitisinone. To consider using nitisinone early wide variety of AKU features in a cohort of sufficient in life requires justification in the form of early ochro- size to provide reliable data. Bone, cartilage and tissue nosis or early morbidity. The present study suggests markers are likely to be beneficial if they can be vali- the presence of apparently irreversible ochronosis and dated in a larger young cohort of patients with AKU morbidity early in life.

8 Cox T, et al. BMJ Innov 2019;0:1–10. doi:10.1136/bmjinnov-2018-000324 BMJ Innov: first published as 10.1136/bmjinnov-2018-000324 on 28 September 2019. Downloaded from DIAGNOSTICS

Author affiliations Nicholas Rhodes http://​orcid.​org/​0000-​0002-​5173-​7032 1Molecular and Clinical Cancer Medicine, Liverpool University, Liverpool, UK 2LCTU, University of Liverpool, School of Life Sciences, Liverpool, UK 3Physiotherapy, Royal Liverpool University Hospital, Liverpool, Merseyside, UK References 4Research Institute for Sport and Exercise Sciences, Liverpool John Moores 1 LA DU BN, Zannoni VG, Laster L, et al. The nature of the University, Liverpool, UK defect in metabolism in alcaptonuria. J Biol Chem 5School of Sport and Exercise Sciences, Liverpool John Moores University, 1958;230:251–60. Liverpool, UK 6Musculoskeletal Radiology, Royal Liverpool University Hospital, Liverpool, UK 2 Zannoni VG, Lomtevas N, Goldfinger S. Oxidation of 7Nordic Bioscience A/S, Herlev, Denmark homogentisic acid to ochronotic pigment in connective 8 Biotecnologie Chimica e Farmacia, Universita degli Studi di Siena Dipartimento tissue. Biochimica et Biophysica Acta (BBA) - General Subjects di Biotecnologie Chimica e Farmacia, Siena, Italy 1969;177:94–105. 9Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy 10 3 O’Brien WM, La Du BN BJJ. Biochemical, pathologic and Department of Clinical Chemistry and Metabolic Medicine, Royal Liverpool University Hospital, Liverpool, UK clinical aspects of allcaptonuria, ochronosis and ochronotic 11Clinical Biochemistry, Royal Liverpool and Broadgreen University Hospitals arthropathy: review of world literature (1584–1962). Am J NHS Trust, Liverpool, UK Med 1963;34:813–38. 12 College of Clinical and Biomedical Science, University of Central Lancashire, 4 Ranganath LR, Cox TF. Natural history of alkaptonuria Preston, UK 13Dermatology, Royal Liverpool University Hospital, Liverpool, UK revisited: analyses based on scoring systems. J Inherit Metab 14AKU Society, Cambridge, UK Dis 2011;34:1141–51. 15Musculoskeletal Biology, University of Liverpool, Institute of Ageing and 5 Ranganath LR, Khedr M, Milan AM, et al. Nitisinone arrests Chronic Disease, Liverpool, UK ochronosis and decreases rate of progression of alkaptonuria: 16Musculoskeletal Biology, University of Liverpool, Liverpool, UK evaluation of the effect of nitisinone in the United 17Clinical Biochemistry and Metabolic Medicine, Royal Liverpool University Hospital, Liverpool, UK Kingdom National Alkaptonuria Centre. Mol Genet Metab 2018;125:127–34. Acknowledgements The authors would like to thank Emily 6 Ranganath LR, Jarvis JC, Gallagher JA. Recent advances in Luangrath and Helen Bygott for their contribution towards ethics management of alkaptonuria (invited review; best practice submissions and logistics of carrying out the study on site. article). J Clin Pathol 2013;66:367–73. Contributors TC, JAG and LR: design of the study and manuscript 7 Introne WJ, Perry MB, Troendle J, et al. A 3-year randomized writing. NS and NR: grant writing and conduct of the study. JAG therapeutic trial of nitisinone in alkaptonuria. Mol Genet and JPD: analysis of ear biopsy and qualitative scoring of eye Metab 2011;103:307–14. ochronosis. JAG and LFT: quantitative analysis of eye ochronosis. EW: ear biopsies. AnM and AH: assay of HGA. AlM: scoring of 8 Ranganath LR, Milan AM, Hughes AT, et al. Suitability of MRI. GB, HRS and MR: gait analysis. FG: bone and cartilage nitisinone in alkaptonuria 1 (SONIA 1): an international, biomarkers. DB, DG, RR, AS: inflammatory markers. MK: quality multicentre, randomised, open-label, no-treatment controlled, of life assessments. ST: analysis of questionnaires. AH: HGA assays. parallel-group, dose-response study to investigate the effect TC and EEP: statistical analysis. TC: first drafting and revision of of once daily nitisinone on 24-h urinary homogentisic acid the manuscript. NR was involved in obtaining grant funding and writing of the manuscript. All authors contributed to analysis of the excretion in patients with alkaptonuria after 4 weeks of data, edited the manuscript and approved the final version. treatment. Ann Rheum Dis 2016;75:362–7.

Funding This work was supported by European Commission 9 Preston AJ, Keenan CM, Sutherland H, et al. Ochronotic http://innovations.bmj.com/ Seventh Framework Programme funding granted in 2012 osteoarthropathy in a mouse model of alkaptonuria, and its (DevelopAKUre, project no. 304985). Additional support was inhibition by nitisinone. Ann Rheum Dis 2014;73:284–9. received from the UK National AKU Centre, funded by NHS 10 Keenan CM, Preston AJ, Sutherland H, et al. Nitisinone arrests England Highly Specialized Services. but does not reverse ochronosis in alkaptonuric mice. JIMD Disclaimer The authors confirm independence from the sponsors; Rep 2015;24:45–50. the content of the article has not been influenced by the sponsors. The funding source was not involved in the study design, 11 McKiernan PJ. Nitisinone for the treatment of hereditary collection, analysis and interpretation of data, the writing of type I. Expert Opin Orphan Drugs 2013;1:491–7. the manuscript or in the decision to submit the manuscript for 12 Griffith JF, Wang Y-XJ, Antonio GE, et al. Modified Pfirrmann publication. grading system for lumbar intervertebral disc degeneration. Competing interests None declared. Spine 2007;32:E708–E712. on September 29, 2019 by guest. Protected copyright. Patient consent for publication Obtained. 13 Maksymowych WP, Dhillon SS, Park R, et al. Validation of the spondyloarthritis research consortium of Canada magnetic Ethics approval UK Research Ethics Committee (REC) no. 15/ NW/0749. Integrated Research Application System (IRAS) Project resonance imaging spinal inflammation index: is it necessary to ID: 180968. score the entire spine? Arthritis Rheum 2007;57:501–7. Provenance and peer review Not commissioned; externally peer 14 Peterfy CG, Guermazi A, Zaim S, et al. Whole-Organ magnetic reviewed. resonance imaging score () of the knee in osteoarthritis. Data availability statement All data relevant to the study are Osteoarthritis Cartilage 2004;12:177–90. included in the article or uploaded as online supplementary 15 Davis RB, Õunpuu S, Tyburski D, et al. A gait analysis information. data collection and reduction technique. Hum Mov Sci 1991;10:575–87. ORCID iDs Hannah Rose Shepherd http://​orcid.​org/​0000-​0002-​8596-​3601 16 Vesanto J, Himberg J, Alhoniemi E, et al. Som toolbox for Gabor Barton http://​orcid.​org/​0000-​0002-​7214-​1967 Matlab 5. Espoo: Helsinki University of Technology, 2000. Federica Genovese http://​orcid.​org/​0000-​0003-​1984-​1881 17 Barton GJ, Hawken MB, Scott MA, et al. Movement deviation Daniela Braconi http://​orcid.​org/​0000-​0002-​9657-​4169 profile: a measure of distance from normality using a self- Annalisa Santucci http://​orcid.​org/​0000-​0001-​6976-​9086 Milad Khedr http://​orcid.​org/​0000-​0002-​4998-​2397 organizing neural network. Hum Mov Sci 2012;31:284–94. Leah Frances Taylor http://​orcid.​org/​0000-​0002-​4595-​4890 18 Barton GJ, Hawken MB, Holmes G, et al. A gait index may Jane Patricia Dillon http://​orcid.​org/​0000-​0002-​7055-​5664 underestimate changes of gait: a comparison of the movement

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