Machine learning application for patient stratification and phenotype/genotype investigation in a rare disease.

Alkaptonuria (AKU, OMIM: 203500) is an autosomal recessive disorder caused by mutations in the Homogentisate 1,2-dioxygenase (HGD) gene. A lack of standardized data, information and methodologies to assess disease severity and progression represents a common complication in ultra-rare disorders like AKU. This is the reason why we developed a comprehensive tool, called ApreciseKUre, able to collect AKU patients deriving data, to analyse the complex network among genotypic and phenotypic information and to get new insight in such multi-systemic disease. By taking advantage of the dataset, containing the highest number of AKU patient ever considered, it is possible to apply more sophisticated computational methods (such as machine learning) to achieve a first AKU patient stratification based on phenotypic and genotypic data in a typical precision medicine perspective. Thanks to our sufficiently populated and organized dataset, it is possible, for the first time, to extensively explore the phenotype-genotype relationships unknown so far. This proof of principle study for rare diseases confirms the importance of a dedicated database, allowing data management and analysis and can be used to tailor treatments for every patient in a more effective way.

Conditional targeting in mice reveals that hepatic homogentisate 1,2-dioxygenase activity is essential in reducing circulating homogentisic acid and for effective therapy in the genetic disease alkaptonuria.

Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype. We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a -/- mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy. Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA. This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.

Novel mutations in the homogentisate 1,2 dioxygenase gene identified in Jordanian patients with alkaptonuria.

This study was conducted to identify mutations in the homogentisate 1,2 dioxygenase gene (HGD) in alkaptonuria patients among Jordanian population. Blood samples were collected from four alkaptonuria patients, four carriers, and two healthy volunteers. DNA was isolated from peripheral blood. All 14 exons of the HGD gene were amplified using the polymerase chain reaction (PCR) technique. The PCR products were then purified and analyzed by sequencing. Five mutations were identified in our samples. Four of them were novel C1273A, T1046G, 551-552insG, T533G and had not been previously reported, and one mutation T847C has been described before. The types of mutations identified were two missense mutations, one splice site mutation, one frameshift mutation, and one polymorphism. We present the first molecular study of the HGD gene in Jordanian alkaptonuria patients. This study provides valuable information about the molecular basis of alkaptonuria in Jordanian population.

Development of an in vitro model to investigate joint ochronosis in alkaptonuria.

OBJECTIVES: Alkaptonuria (AKU) is a genetic disorder caused by lack of the enzyme responsible for breaking down homogentisic acid (HGA), an intermediate in tyrosine metabolism. HGA is deposited as a polymer, termed ochronotic pigment, in collagenous tissues. Pigmentation is progressive over many years, leading to CTDs including severe arthropathies. To investigate the mechanism of pigmentation and to determine how it leads to arthropathy, we aimed to develop an in vitro model of ochronosis. METHODS: Osteosarcoma cell lines MG63, SaOS-2 and TE85 were cultured in medium containing HGA from 0.1 µM to 1 mM. Cultures were examined by light microscopy and transmission electron microscopy, and Schmorl's stain was used to detect pigment deposits in vitro, following the observation that this stain identifies ochronotic pigment in AKU tissues. The effects of HGA on cell growth and collagen synthesis were also determined. RESULTS: There was a dose-related deposition of pigment in cells and associated matrix from 33 µM to 0.33 mM HGA. Pigmentation in vitro was much more rapid than in vivo, indicating that protective mechanisms exist in tissues in situ. Pigment deposition was dependent on the presence of cells and was observed at HGA concentrations that were not toxic. There was an inhibition of cell growth and a stimulation of type I collagen synthesis up to 0.33 mM HGA, but severe cell toxicity at 1 mM HGA. CONCLUSION: We have developed an in vitro model of ochronosis that should contribute to understanding joint destruction in AKU and to the aetiology of OA.

An update on molecular genetics of Alkaptonuria (AKU).

Alkaptonuria (AKU) is an autosomal recessive disorder caused by a deficiency of homogentisate 1,2 dioxygenase (HGD) and characterized by homogentisic aciduria, ochronosis, and ochronotic arthritis. The defect is caused by mutations in the HGD gene, which maps to the human chromosome 3q21-q23. AKU shows a very low prevalence (1:100,000-250,000) in most ethnic groups, but there are countries such as Slovakia and the Dominican Republic in which the incidence of this disorder rises to as much as 1:19,000. In this work, we summarize the genetic aspects of AKU in general and the distribution of all known disease-causing mutations reported so far. We focus on special features of AKU in Slovakia, which is one of the countries with an increased incidence of this rare metabolic disorder.

A quantitative assessment of alkaptonuria: testing the reliability of two disease severity scoring systems.

Alkaptonuria (AKU) is due to excessive homogentisic acid accumulation in body fluids due to lack of enzyme homogentisate dioxygenase leading in turn to varied clinical manifestations mainly by a process of conversion of HGA to a polymeric melanin-like pigment known as ochronosis. A potential treatment, a drug called nitisinone, to decrease formation of HGA is available. However, successful demonstration of its efficacy in modifying the natural history of AKU requires an effective quantitative assessment tool. We have described two potential tools that could be used to quantitate disease burden in AKU. One tool describes scoring the clinical features that includes clinical assessments, investigations and questionnaires in 15 patients with AKU. The second tool describes a scoring system that only includes items obtained from questionnaires used in 44 people with AKU. Statistical analyses were carried out on the two patient datasets to assess the AKU tools; these included the calculation of Chronbach's alpha, multidimensional scaling and simple linear regression analysis. The conclusion was that there was good evidence that the tools could be adopted as AKU assessment tools, but perhaps with further refinement before being used in the practical setting of a clinical trial.

Natural history of alkaptonuria revisited: analyses based on scoring systems.

Increased circulating homogentisic acid in body fluids occurs in alkaptonuria (AKU) due to lack of enzyme homogentisate dioxygenase leading in turn to conversion of HGA to a pigmented melanin-like polymer, known as ochronosis. The tissue damage in AKU is due to ochronosis. A potential treatment, a drug called nitisinone, to decrease formation of HGA is available. However, deploying nitisinone effectively requires its administration at the most optimal time in the natural history. AKU has a long apparent latent period before overt ochronosis develops. The rate of change of ochronosis and its consequences over time following its recognition has not been fully described in any quantitative manner. Two potential tools are described that were used to quantitate disease burden in AKU. One tool describes scoring the clinical features that includes clinical assessments, investigations and questionnaires in 15 patients with AKU. The second tool describes a scoring system that only includes items obtained from questionnaires in 44 people with AKU. Analysis of the data reveals distinct phases of the disease, a pre-ochronotic phase and an ochronotic phase. The ochronotic phase appears to demonstrate an earlier slower progression followed by a rapidly progressive phase. The rate of change of the disease will have implications for monitoring the course of the disease as well as decide on the most appropriate time that treatment should be started for it to be effective either in prevention or arrest of the disease.

A molecular spectroscopy approach for the investigation of early phase ochronotic pigment development in Alkaptonuria.

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs due to a deficiency in functional levels of the enzyme homogentisate 1,2-dioxygenase (HGD), required for the breakdown of HGA, because of mutations in the HGD gene. Over time, HGA accumulation causes the formation of the ochronotic pigment, a dark deposit that leads to tissue degeneration and organ malfunction. Such behaviour can be observed also in vitro for HGA solutions or HGA-containing biofluids (e.g. urine from AKU patients) upon alkalinisation, although a comparison at the molecular level between the laboratory and the physiological conditions is lacking. Indeed, independently from the conditions, such process is usually explained with the formation of 1,4-benzoquinone acetic acid (BQA) as the product of HGA chemical oxidation, mostly based on structural similarity between HGA and hydroquinone that is known to be oxidized to the corresponding para-benzoquinone. To test such correlation, a comprehensive, comparative investigation on HGA and BQA chemical behaviours was carried out by a combined approach of spectroscopic techniques (UV spectrometry, Nuclear Magnetic Resonance, Electron Paramagnetic Resonance, Dynamic Light Scattering) under acid/base titration both in solution and in biofluids. New insights on the process leading from HGA to ochronotic pigment have been obtained, spotting out the central role of radical species as intermediates not reported so far. Such evidence opens the way for molecular investigation of HGA fate in cells and tissue aiming to find new targets for Alkaptonuria therapy.

Evaluation of antioxidant drugs for the treatment of ochronotic alkaptonuria in an in vitro human cell model.

Alkaptonuria (AKU) is a rare autosomal recessive disease, associated with deficiency of homogentisate 1,2-dioxygenase activity in the liver. This leads to an accumulation of homogentisic acid (HGA) and its oxidized derivatives in polymerized form in connective tissues especially in joints. Currently, AKU lacks an appropriate therapy. Hence, we propose a new treatment for AKU using the antioxidant N-acetylcysteine (NAC) administered in combinations with ascorbic acid (ASC) since it has been proven that NAC counteracts the side-effects of ASC. We established an in vitro cell model using human articular primary chondrocytes challenged with an excess of HGA (0.33 mM). We used this experimental model to undertake pre-clinical testing of potential antioxidative therapies for AKU, evaluating apoptosis, viability, proliferation, and metabolism of chondrocytes exposed to HGA and treated with NAC and ASC administered alone or in combination addition of both. NAC decreased apoptosis induced in chondrocytes by HGA, increased chondrocyte growth reduced by HGA, and partially restored proteoglycan release inhibited by HGA. A significantly improvement in efficacy was found with combined addition of the two antioxidants in comparison with NAC and ASC alone. Our novel in vitro AKU model allowed us to demonstrate the efficacy of the co-administration of NAC and ASC to counteract the negative effects of HGA for the treatment of ochronotic arthropathy.

Transient pockets as mediators of gas molecules routes inside proteins: The case study of dioxygen pathway in homogentisate 1,2-dioxygenase and its implication in Alkaptonuria development.

Alkaptonuria (AKU) is an ultra-rare disease caused by mutations in homogentisate 1,2-dioxygenase (HGD) enzyme, characterized by the loss of enzymatic activity and the accumulation of its substrate, homogentisic acid (HGA) in different tissues, leading to ochronosis and organ degeneration. Although the pathological effects of HGD mutations are largely studied, less is known about the structure of the enzyme, in particular the pathways for dioxygen diffusion to the active site, required for the enzymatic reaction, are still uninvestigated. In the present project, the combination of two in silico techniques, Molecular Dynamics (MD) simulation and Implicit Ligand Sampling (ILS), was used to delineate gas diffusion routes in HGD enzyme. A route from the central opening of the hexameric structure of the enzyme to the back of the active site trough the protein moiety was identified as the path for dioxygen diffusion, also overlapping with a transient pocket, which then assumes an important role in dioxygen diffusion. Along the route the sequence location of the missense variant E401Q, responsible for AKU development, was also found, suggesting such mutation to be conducive of enzymatic activity loss by altering the flow dynamics of dioxygen. Our in silico approach allowed also to delineate the route of HGA substrate to the active site, until now only supposed.

Mutation spectrum of homogentisic acid oxidase (HGD) in alkaptonuria.

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder, characterized by accumulation of homogentisic acid, leading to darkened urine, pigmentation of connective tissue (ochronosis), joint and spine arthritis, and destruction of cardiac valves. AKU is due to mutations in the homogentisate dioxygenase gene (HGD) that converts homogentisic acid to maleylacetoacetic acid in the tyrosine catabolic pathway. Here we report a comprehensive mutation analysis of 93 patients enrolled in our study, as well as an extensive update of all previously published HGD mutations associated with AKU. Within our patient cohort, we identified 52 HGD variants, of which 22 were novel. This yields a total of 91 identified HGD variations associated with AKU to date, including 62 missense, 13 splice site, 10 frameshift, 5 nonsense, and 1 no-stop mutation. Most HGD variants reside in exons 3, 6, 8, and 13. We assessed the potential effect of all missense variations on protein function, using five bioinformatic tools specifically designed for interpretation of missense variants (SIFT, POLYPHEN, PANTHER, PMUT, and SNAP). We also analyzed the potential effect of splice-site variants using two different tools (BDGP and NetGene2). This study provides valuable resources for molecular analysis of alkaptonuria and expands our knowledge of the molecular basis of this disease.

Homogentisate 1,2-dioxygenase (HGD) gene variants, their analysis and genotype-phenotype correlations in the largest cohort of patients with AKU.

Alkaptonuria (AKU) is a rare metabolic disorder caused by a deficient enzyme in the tyrosine degradation pathway, homogentisate 1,2-dioxygenase (HGD). In 172 AKU patients from 39 countries, we identified 28 novel variants of the HGD gene, which include three larger genomic deletions within this gene discovered via self-designed multiplex ligation-dependent probe amplification (MLPA) probes. In addition, using a reporter minigene assay, we provide evidence that three of eight tested variants potentially affecting splicing cause exon skipping or cryptic splice-site activation. Extensive bioinformatics analysis of novel missense variants, and of the entire HGD monomer, confirmed mCSM as an effective computational tool for evaluating possible enzyme inactivation mechanisms. For the first time for AKU, a genotype-phenotype correlation study was performed for the three most frequent HGD variants identified in the Suitability Of Nitisinone in Alkaptonuria 2 (SONIA2) study. We found a small but statistically significant difference in urinary homogentisic acid (HGA) excretion, corrected for dietary protein intake, between variants leading to 1% or >30% residual HGD activity. There was, interestingly, no difference in serum levels or absolute urinary excretion of HGA, or clinical symptoms, indicating that protein intake is more important than differences in HGD variants for the amounts of HGA that accumulate in the body of AKU patients.

Three-generational alkaptonuria in a non-consanguineous family.

OBJECTIVE: Alkaptonuria (AKU) is a rare inborn error of metabolism of aromatic amino acids and considered to be an autosomal recessive trait caused by mutations in the homogentisate 1,2-dioxygenase (HGD) gene. A dominant pattern of inheritance has been reported but was attributed to extended consanguinity in many cases. However, we have observed a non-consanguineous family segregating AKU in a dominant manner over three generations. RESULTS: All affected individuals presented with typical features of AKU including darkening of the urine, ochronosis, arthropathy, and elevated urinary excretion of homogentisic acid. Sequence analysis of the HGD gene from genomic DNA of two affected individuals, uncle and niece, revealed a heterozygous missense mutation (M368V) in the uncle that was not present in his niece. Microsatellite genotyping demonstrated that both were heterozygous at the HGD locus and shared one haplotype. This haplotype did not contain a detectable HGD mutation. The haplotype was also found in a healthy son of the niece, making a dominant HGD mutation unlikely. Moreover, sequencing of cDNA from lymphoblastoid cells of the niece did not reveal an HGD mRNA with a potentially dominant-negative effect. CONCLUSION: Rare causes of the uncommon AKU inheritance in this family have to be considered, ranging from the coincidence of undetectable HGD mutations to a dominant mutation of a second, hitherto unknown AKU gene.

In vitro inhibition of chick embryo lysyl hydroxylase by homogentisic acid. A proposed connective tissue defect in alkaptonuria.

Homogentisic acid inhibits the in vitro activity of chick embryo lysyl hydroxylase, a microsomal enzyme which catalyzes the transformation of certain lysyl residues in collagen to hydroxylysine. Chick embryo lysyl hydroxylase activity was measured as specific tritium release as tritium water from a [4,5-(3)H]lysine-labeled unhydroxylated collagen substrate prepared from chick calvaria. Kinetic studies revealed a linear, noncompetitive type of inhibition with respect to collagen substrate with a Ki of 120-180 muM. The inhibition by homogentisic acid was reversible in that enzyme activity could be restored after dialysis of preincubated mixtures of homogentisic acid with enzyme or substrate. The inhibition by homogentisic acid was competitive with respect to ascorbic acid, and the addition of reducing agents, such as ascorbic acid or 1,4-dithiothreitol, protected lysyl hydroxylase activity from homogentisic acid inhibition.In organ cultures of embryonic chick calvaria, biosynthesis of hydroxylysine-derived intermolecular collagen cross-links was inhibited in a dose-dependent manner by 0.5-5 mM homogentisic acid. Because homogentisic acid inhibits the formation of hydroxylysine in a cell-free assay and in organ cultures, this compound must pass into the cells of calvaria to inhibit intracellular hydroxylysine formation and subsequently to diminish the reducible intermolecular cross-links of the newly synthesized collagen. We propose that the inhibition of lysyl hydroxylase and the resulting hydroxylsine-deficient, structurally modified collagen may be clinically significant in the defective connective tissue found in alkaptonuric patients.

Toward a generalized computational workflow for exploiting transient pockets as new targets for small molecule stabilizers: Application to the homogentisate 1,2-dioxygenase mutants at the base of rare disease Alkaptonuria.

Alkaptonuria (AKU) is an inborn error of metabolism where mutation of homogentisate 1,2-dioxygenase (HGD) gene leads to a deleterious or misfolded product with subsequent loss of enzymatic degradation of homogentisic acid (HGA) whose accumulation in tissues causes ochronosis and degeneration. There is no licensed therapy for AKU. Many missense mutations have been individuated as responsible for quaternary structure disruption of the native hexameric HGD. A new approach to the treatment of AKU is here proposed aiming to totally or partially rescue enzyme activity by targeting of HGD with pharmacological chaperones, i.e. small molecules helping structural stability. Co-factor pockets from oligomeric proteins have already been successfully exploited as targets for such a strategy, but no similar sites are present at HGD surface; hence, transient pockets are here proposed as a target for pharmacological chaperones. Transient pockets are detected along the molecular dynamics trajectory of the protein and filtered down to a set of suitable sites for structural stabilization by mean of biochemical and pharmacological criteria. The result is a computational workflow relevant to other inborn errors of metabolism requiring rescue of oligomeric, misfolded enzymes.

4-Hydroxyphenylpyruvate Dioxygenase and Its Inhibition in Plants and Animals: Small Molecules as Herbicides and Agents for the Treatment of Human Inherited Diseases.

This review mainly focuses on the physiological function of 4-hydroxyphenylpyruvate dioxygenase (HPPD), as well as on the development and application of HPPD inhibitors of several structural classes. Among them, one illustrative example is represented by compounds belonging to the class of triketone compounds. They were discovered by serendipitous observations on weed growth and were developed as bleaching herbicides. Informed reasoning on nitisinone (NTBC, 14), a triketone that failed to reach the final steps of the herbicidal design and development process, allowed it to become a curative agent for type I tyrosinemia (T1T) and to enter clinical trials for alkaptonuria. These results boosted the research of new compounds able to interfere with HPPD activity to be used for the treatment of the tyrosine metabolism-related diseases.

Tyrosinase, could it be a missing link in ochronosis in alkaptonuria?

The hypothesis that is proposed is that tyrosinase, an enzyme widely found within the human body is implicated in the ochronosis that occurs in alkaptonuria; an autosomal recessive condition first used by Archibald Garrod to describe the theory of "Inborn Errors of Metabolism." The disease results from the absence of a single enzyme in the liver that breaks down homogentisic acid; this molecule becomes systemically elevated in sufferers. The condition is characterised by a clinical triad of symptoms; homogentisic aciduria from birth, ochronosis (darkening) of collagenous tissues (from ∼30years of age) and ochronotic osteoarthropathy in weight bearing joints due to long term ochronosis in them (from ∼40years of age). Tyrosinase, a polyphenol oxidase has been shown in many species to contribute to the darkening of tissues in many organisms; including humans in the production of melanin. Tyrosinase under the right conditions shows alterations in its substrate specificity and may contribute to the darkening seen in AKU where it moves away from polymerising tyrosine but also homogentisic acid, the causative molecule in alkaptonuria, that is present in excess.

Twelve novel HGD gene variants identified in 99 alkaptonuria patients: focus on 'black bone disease' in Italy.

Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T>A), Slovakia (c.500C>T) and France (c.440T>C), three in patients from India (c.469+6T>C, c.650-85A>G, c.158G>A), and six in patients from Italy (c.742A>G, c.614G>A, c.1057A>C, c.752G>A, c.119A>C, c.926G>T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy.

Two novel mutations in the homogentisate-1,2-dioxygenase gene identified in Chinese Han Child with Alkaptonuria.

Alkaptonuria (AKU) is an autosomal recessive disorder of tyrosine metabolism, which is caused by a defect in the enzyme homogentisate 1,2-dioxygenase (HGD) with subsequent accumulation of homogentisic acid. Presently, more than 100 HGD mutations have been identified as the cause of the inborn error of metabolism across different populations worldwide. However, the HGD mutation is very rarely reported in Asia, especially China. In this study, we present mutational analyses of HGD gene in one Chinese Han child with AKU, which had been identified by gas chromatography-mass spectrometry detection of organic acids in urine samples. PCR and DNA sequencing of the entire coding region as well as exon-intron boundaries of HGD have been performed. Two novel mutations were identified in the HGD gene in this AKU case, a frameshift mutation of c.115delG in exon 3 and the splicing mutation of IVS5+3 A>C, a donor splice site of the exon 5 and exon-intron junction. The identification of these mutations in this study further expands the spectrum of known HGD gene mutations and contributes to prenatal molecular diagnosis of AKU.

Molecular diagnosis of alkaptonuria mutation by analysis of homogentisate 1,2 dioxygenase mRNA from urine and blood.

Alkaptonuria (AKU) is caused by lack of homogentisate 1, 2 dioxygenase (HGO) activity. From the complete sequence of a human HGO cDNA, primers were designed in order to obtain reverse transcription-polymerase chain reaction products from tissues with ectopic transcription amenable to diagnostic analysis. A search for mutations in HGO cDNA was performed in an AKU family using urine and blood samples. The results show complete cosegregation (Z = 6.32; theta = 0) between a C-->T transition at position 817 of the human HGO cDNA and AKU. This mutation predicts a Pro-->Ser replacement at amino acid 230, and generates an EcoRV site.