ApreciseKUre: an approach of Precision Medicine in a Rare Disease
- Ottavia Spiga†1Email author,
- Vittoria Cicaloni†1, 2,
- Andrea Bernini1,
- Andrea Zatkova3 and
- Annalisa Santucci1
© The Author(s). 2017
Received: 9 September 2016
Accepted: 1 April 2017
Published: 14 April 2017
Alkaptonuria (AKU; OMIM:203500) is a classic Mendelian genetic disorder described by Garrod already in 1902. It causes urine to turn black upon exposure to air and also leads to ochronosis as well as early osteoarthritis.
Main body of the abstract
Our objective is the implementation of a Precision Medicine (PM) approach to AKU. We present here a novel ApreciseKUre database facilitating the collection, integration and analysis of patient data in order to create an AKU-dedicated “PM Ecosystem” in which genetic, biochemical and clinical resources can be shared among registered researchers. In order to exploit the ApreciseKUre database, we developed an analytic method based on Pearson’s correlation coefficient and P value that generates as refreshable correlation matrix. A complete statistical analysis is obtained by associating every pair of parameters to examine the dependence between multiple variables at the same time.
Employing this analytic approach, we showed that some clinically used biomarkers are not suitable as prognostic biomarkers in AKU for a more reliable patients’ clinical monitoring. We believe this database could be a good starting point for the creation of a new clinical management tool in AKU, which will lead to the development of a deeper knowledge network on the disease and will advance its treatment. Moreover, our approach can serve as a personalization model paradigm for other inborn errors of metabolism or rare diseases in general.
KeywordsAlkaptonuria Rare disease Precision Medicine Database Data analysis Biomarkers
Precision medicine (PM) is an emerging approach for disease prevention, diagnosis and treatment that takes into account individual variability in genes, environment, metabolomics, proteomics and lifestyle . The ability to collect, integrate and analyse relevant data streams is the core for developing a “Precision Medicine Ecosystem” in which genetic, biochemical and clinical resources are shared between researchers, clinicians and patients . Computational modelling and database building can be a useful guide to generate an exhaustive and dynamic picture of the individual and to identify potential new biomarkers. Improvements in the knowledge of disease mechanisms and in the biomarkers identification are opening new opportunities to match therapy to patient, and thus leading to a more personalized medicine for maximizing the benefit-to-risk ratio. Ideally, biomarkers identification should be done as early as possible in the workflow of drug discovery in order to stratify dissimilar patient groups . Nowadays PM is used in many areas of health (e.g., cancer), however, this approach was originally developed to manage and analyze data regarding rare diseases.
Alkaptonuria (AKU) (OMIM:203500) is the very first genetic disorder to be described, as reported by Garrod in 1902 . AKU is an autosomal recessive inborn error of metabolism of the amino acids tyrosine due to a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD). This leads to the accumulation of homogentisic acid (HGA, 2,5-dihydroxyphenylacetic acid) which is excreted in the urine, giving it an unusually dark colour. The accumulation of HGA in connective tissues is responsible for discoloration of bone (a process called ‘ochronosis’) inducing an early-onset painful osteoarthritis. The oxidation of HGA produces free radical species that are associated with tissue oxidative damage and are thought to cause degeneration by inciting inflammation . In some patients, aortic valve stenosis was reported, as well as kidney and prostate stones. Additionally, AKU also implied a secondary amyloidosis, one of the most severe complications of several chronic rheumatic disorders due to the deposition of amyloid in the synovial membranes of the joint or of the tendon sheats . Usually, at early-stages of the disease patients are given anti-inflammatories or pain killers, while for end-stages joint and heart valve replacement is often required. Since 2012, the DevelopAKUre project is underway, aimed to test nitisinone as a possible treatment for AKU. It is believed that it can prevent or slow down progression of AKU .
In order to prevent or minimize the effects of ochronotic arthropathy, a PM approach, tailoring pharmacological, surgical and dietary treatment, would be of great value for AKU patients care. The establishment of a database, which would integrate patient-derived information (quality of life), clinician-derived information (test results, genotypes), and mutational analysis (protein molecular modeling) offers an exhaustive visualization of different informational layers to support clinicians and researchers in PM application to AKU. It is an innovative approach that could not only advance the treatment of AKU, but also to serve as a model for other rare diseases.
Construction and content
The interactive database for AKU, ApreciseKUre database (http://www.sbl.unisi.it/aprecisekure), integrates data on mutations, patient quality of life and clinical outcomes. It is proposed as a supporting online tool for clinicians to be used in order to practice “PM” in the context of AKU. Data has been primarily collected from HGD mutation database (http://hgddatabase.cvtisr.sk/) that includes all HGD variants in AKU patients reported so far . Later we have integrated our results from proteomic studies in AKU, information obtained through collaborations with patients’ organizations in Europe (http://www.aimaku.it/, http://www.akussac.sk/, http://www.alcap.fr/, http://www.akusociety.org/, http://www.dsaku.de/), as well as data from SONIA1 clinical study . A coding system is used to anonymize data. The database integrates different types of information regarding genotype, biomarkers, environment, lifestyle, habit, histopathologic, clinical and therapies of patients. In particular, the database schema is divided into 11 tables (see Additional file 1).
Utility and discussion
We invite users of the ApreciseKUre database not to look at the Pearson’s index only as the numeric value, but to focus also on a deeper understanding of possible biological significance of the compaired markers. This approach can lead into discovery of interesting correlations between biomarkers not considered before and to point out eventual AKU critical points. For example, our analysis of the ApreciseKUre database indicates an inverse statistic correlation between cystatin C (CysC) and cathepsin D (CatD) (Pearson’s index −0.32 and p-value 0.0470).
CysC is produced at a constant rate by all nucleated cells, filtered by the renal glomeruli and catabolized in the kidney tubuli. Generally, when kidneys function properly, they stabilize the levels of CysC in the blood; if the blood levels of CysC are too high, it may indicate kidney disease . CysC appears to have the advantages of being independent of gender and muscle mass, thus it is used in assays for the measurements of the glomerular filtration rate excretion (eGFR) [10, 11]. CatD is an aspartic protease synthesized as a single chain pro-enzyme (412 amino acids) in rough endoplasmic reticulum. It has been found that CatD levels are particularly elevated in muscular dystrophy and arthritis . Additionally, CatD is responsible for different functions: conversion of procollagen to collagen, parathyroid hormone degradation, the splitting of some brain proteins and of CysC degradation .
39 AKU patients show CatD values included between 18,76–185,2 ng/mL (measured with ELISA in serum samples) with a mean and standard deviation 62,54 ± 41,45 ng/mL
CysC mg/L (ELISA)
29 M 11 F
Mean ± SD
M:0.70 ± 0.15
F:0.71 ± 0.14
M:0.86 ± 0.15a
F:0.78 ± 0.20a
CatD ng/mL (ELISA)
Mean ± SD
62.54 ± 41.45
46.68 ± 5.88
HGA μM (ELISA)
Mean ± SD
25.89 ± 8.00
This might indicate, that 1) the current version of the ApreciseKUre database concerns patients who do not show kidney dysfunction, although in all of them kidneys have to cope with excretion of high levels of HGA by both glomerular filtration and tubular secretion that leads the renal parenchyma to develop kidney stones (in 2 of them renal stones are already found) , or, 2) the CysC assay is not suitable for the measurements of the glomerular filtration rate excretion (eGFR) in AKU, since increased CatD can actually degrade CysC and interfere with the results.
It has been shown previously that the presence of HGA in the urine and serum interfere with standard urine creatinine enzymatic assays for kidney dysfunction , our present results might indicate that CysC assays is not suitable in case of AKU too, due to the high CatD. Although these results need further verification, they indicate how the proposed ApreciseKUre database can be used as an useful tool in the future for becoming aware of the failure of biomarkers clinically used and for improving the detection of more exploitable prognostic biomarkers.
PM is an innovative approach that integrates research disciplines and clinical practice to build up a knowledge base that can better guide individualized patient care. In order to favor implementation of PM approach for AKU, rare inborn error of metabolism, we created a novel ApreciseKUre-database that represent suitable “PM Ecosystem” in which genetic, biochemical and clinical AKU patient’s data are shared. It is an innovative approach that gives us the potential: to investigate pathogenic mechanisms at molecular level, to define new AKU biomarkers, to undertake preclinical screening of orphan drugs, to define molecular parameters to be used as outcome measures in ongoing clinical trials and to develop a tool that accurately allows for patient stratification. Including updated case-data and samples from clinicians and patients, the researchers benefit from new information sources and also contribute to improve and increase the knowledge of the disease through data analysis. Given the chronic nature of AKU disease, it may be interesting to monitor the clinical conditions of every patient over time. The tool will be able to supply clinicians with the “weight” of each component of the complex disease (inflammation, oxidative stress, etc.), thus supporting clinical choices. Our approach can be exploited as a model of PM for other rare diseases.
Body Mass Index
Enzyme-linked immunosorbent assay
Glomerular filtration rate
Thanks are due to Dott. Alfonso Trezza for his insightful contribution to this work.
Vittoria Cicaloni is supported by a PhD fellwoship from the Tuscany Region Pegaso PhD School in Biochemistry and Molecular Biology.
The authors thank Telethon Italy grant GGP10058, Toscana Life Sciences Orphan_1 project, and aimAKU – Associazione Italiana Malati di Alcaptonuria (ORPHA263402). The authors also thank Toscana Life Sciences Foundation.
Toscana Life Sciences Foundation, Siena, ITALY
Availability of data and materials
The datasets generated during and analysed during the current study are available in the ApreciseKUre database repository, http://www.sbl.unisi.it/aprecisekure/. after suitable login registration.
OS: corresponding author, designing research studies, analyzing data and writing the manuscript. VC: conducting research, acquiring data, analyzing data and writing the manuscript and graphic section. AB: analyzing data, information technology expert. AZ: genetic and molecular AKU expert, HGD mutation database curator, revising manuscript. AS: supervisor of the research and scientific-technical AKU expert. All the authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Procedures were approved by Siena University Hospital and national Ethics (Comitato Etico Policlinico Universitario di Siena, number GGP10058, date 21/07/2010) in accordance with 1975 Helsinki Declaration, revised in 2000 (52nd WMA General Assembly, Edinburgh, Scotland, October 2000). Informed written consent was obtained from the patient.
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- National Institutes of Health. Precision Medicine Initiative Cohort Program. Precision Medicine Initiative Cohort Program–Frequently Asked Questions. 2015. http://www.nih.gov/precision-medicine-initiative-cohort-program. Accessed 6 Jul 2016.
- Aronson SJ, Rehm HL. Building the foundation for genomics in precision medicine. Nature. 2015. doi:10.1038/nature15816.PubMedGoogle Scholar
- European Commission, DG Research.Biomarkers for patient stratification. 2010. ec.europa.eu/research/health/pdf/biomarkers-for-patient-stratification_en.pdf.Accessed 2010.
- Garrod AE. The incidence of Alkaptonuria: a study in chemical individuality. Lancet. 1902;160(4137):1616–20.View ArticleGoogle Scholar
- Hegedus ZL. The probable involvement of soluble and deposited melanins, their intermediates and the reactive oxygen side-products in human diseases and aging. Toxicology. 2000;145(2–3):85–101.View ArticlePubMedGoogle Scholar
- Millucci L, Spreafico A, Tinti L, Braconi D, Ghezzi L, Paccagnini E, Bernardini G, Amato L, Laschi M, Selvi E, Galeazzi M, Mannoni A, Benucci M, Lupetti P, Chellini F, Orlandini M, Santucci A. Alkaptonuria is a novel human secondary amyloidogenic disease. Biochim Biophys Acta. 2012;1822(11):1682–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Ranganath LR, Milan AM, Hughes AT, Dutton JJ, Fitzgerald R, Briggs MC, Bygott H, Psarelli EE, Cox TF, Gallagher JA, Jarvis JC, van Kan C, Hall AK, Laan D, Olsson B, Szamosi J, Rudebeck M, Kullenberg T, Cronlund A, Svensson L, Junestrand C, Ayoob H, Timmis OG, Sireau N, Le Quan Sang KH, Genovese F, Braconi D, Santucci A, Nemethova M, Zatkova A, McCaffrey J, Christensen P, Ross G, Imrich R, Rovensky J. Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment. Ann Rheum Dis. 2016;75(2):362–7.View ArticlePubMedGoogle Scholar
- Zatkova A, Sedlackova T, Radvansky J, Polakova H, Nemethova M, Aquaron R, Dursun I, Usher JL, Kadasi L. Identification of 11 novel homogentisate 1,2 dioxygenase variants in alkaptonuria patients and establishment of a novel LOVD-based HGD mutation database. JIMD Rep. 2012;4:55–65.View ArticlePubMedGoogle Scholar
- Randers E, Kristensen JH, Erlandsen EJ, Danielsen H. Serum cystatin C as a marker of the renal function. Scand J Clin Lab Invest. 1998;25(7):585–92.View ArticleGoogle Scholar
- Ognibene A, Mannucci E, Caldini A, Terreni A, Brogi M, Bardini G, Sposato I, Mosconi V, Salvadori B, Rotella CM, Messeri G. Cystatin C reference values and aging. Clin Biochem. 2006;39(6):658–61.View ArticlePubMedGoogle Scholar
- Croda-Todd MT, Soto-Montano XJ, Hernández-Cancino PA, Juárez-Aguilar E. Adult cystatin C reference intervals determined by nephelometric immunoassay. Clin Biochem. 2007;40(13–14):1084–7.View ArticlePubMedGoogle Scholar
- Khalkhali-Ellis Z, Hendrix Mary JC. Two faces of cathepsin D: physiological guardian angel and pathological demon. Biol Med (Aligarh). 2014;6(2):1206.View ArticleGoogle Scholar
- Lenarčič B, Krašovec M, Ritonja A, Olafsson I, Turk V. Inactivation of human cystatin C and kininogen by human cathepsin D. FEBS Lett. 1991;280(2):211–5.View ArticlePubMedGoogle Scholar
- Faria B, Vidinha J, Pêgo C, Correia H, Sousa T. Clinical report: impact of chronic kidney disease on the natural history of alkaptonuria. Clin Kidney J. 2012;5:352–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Wolff F, Biaou I, Koopmansch C, Vanden Bossche M, Pozdzik A, Roumeguère T, Cotton F. Renal and prostate stones composition in alkaptonuria: a case report. Clin Nephrol. 2015;84(6):339–42.View ArticlePubMedGoogle Scholar
- Curtis SL, Roberts NB, Ranganath LR. Interferences of homogentisic acid (HGA) on routine clinical chemistry assays in serum and urine and the implications for biochemical monitoring of patients with Alkaptonuria. Clin Biochem. 2014;47(7–8):640–7.View ArticlePubMedGoogle Scholar