Alkaptonuria Research Articles

Idiopathic Intervertebral Disc Calcification in a Child: An Atypical Case Involving Many Segments.

A seven-year-old girl presented to the emergency department with severe neck pain and restricted movement of sudden onset. A basic neurological examination supplemented by radiography showed calcification within the intervertebral disc at the C6-C7 level. Computed tomography and magnetic resonance imaging of the spine confirmed the diagnosis and revealed additional calcification within the spine at the C3-C4, C7-T1, T1-T2, T4-T5, and T5-T6 levels. Idiopathic intervertebral disc calcification is a rare condition in children, and its etiology remains unclear. In this report, we present a rare case of idiopathic intervertebral disc calcification at numerous levels in a seven-year-old girl. The clinical and radiographic findings, laboratory examination, and follow-up are discussed.

Acute fatal metabolic complications in alkaptonuria.

Alkaptonuria (AKU) is a rare inherited metabolic disorder of tyrosine metabolism that results from a defect in an enzyme called homogentisate 1,2-dioxygenase. The result of this is that homogentisic acid (HGA) accumulates in the body. HGA is central to the pathophysiology of this disease and the consequences observed; these include spondyloarthropathy, rupture of ligaments/muscle/tendons, valvular heart disease including aortic stenosis and renal stones. While AKU is considered to be a chronic progressive disorder, it is clear from published case reports that fatal acute metabolic complications can also occur. These include oxidative haemolysis and methaemoglobinaemia. The exact mechanisms underlying the latter are not clear, but it is proposed that disordered metabolism within the red blood cell is responsible for favouring a pro-oxidant environment that leads to the life threatening complications observed. Herein the role of red blood cell in maintaining the redox state of the body is reviewed in the context of AKU. In addition previously reported therapeutic strategies are discussed, specifically with respect to why reported treatments had little therapeutic effect. The potential use of nitisinone for the management of patients suffering from the acute metabolic decompensation in AKU is proposed as an alternative strategy.

Estimate of heritability and genetic trend of intervertebral disc calcification in Dachshunds in Finland.

BackgroundIntervertebral disc disease (IDD) is a hereditary condition particularly common in Dachshunds. The breed is predisposed to early intervertebral disc degeneration and intervertebral disc calcification (IDC). When calcified, these severely degenerated discs are visible in spinal radiographs. Since the number of calcified discs (NCD) is associated with IDD, spinal radiography can be utilized in screening programmes in attempts to diminish the incidence of IDD in Dachshunds. Our aims were to estimate the heritability and genetic trend of NCD in Dachshunds in Finland and to explore the effect of age at the time of radiographic screening. Since the NCD has a highly skewed distribution, a log-transformed NCD (lnNCD) was also used as an analysed trait. The variance components for both traits were estimated, using the restricted maximum likelihood method. The fixed effects of breed variant, sex, as well as year of screening and the random effects of litter and animal were included in the model. The genetic trends in the NCD and lnNCD were assessed from the estimated breeding values (EBVs) of individual dogs by comparing the mean EBV of dogs born in different years. The breeding values were estimated, using the best linear unbiased prediction animal model. The pedigree in the genetic analyses included a total of 9027 dogs, of which 1567 showed results for NCDs.ResultsThe heritability estimates of the NCD and lnNCD in Dachshunds were high (0.53 and 0.45, respectively). Small genetic improvements were seen as the mean EBVs increased from 100 to 104 and 105 over a 15-year period. The gain in the entire Dachshund population in Finland may differ from that observed, since less than 10 % of the Dachshunds registered have a screening result for NCD. Age at the time of the screening did not significantly affect the NCD or lnNCD.ConclusionsWe recommend systematic radiographic screening for IDC in Dachshunds and adopting EBVs as a tool for selecting breeding dogs. Age at the time of the radiographic screening may not be as important as previously suggested.

Acute spinal cord injury associated with multilevel pediatric idiopathic intervertebral disc calcification: case report.

Pediatric idiopathic intervertebral disc calcification (PIIVDC) is a rare condition; most cases are reported to be selflimited with conservative management. In this study, we describe a case of PIIVDC presenting with acute incomplete spinal cord injury with Brown-Séquard-plus syndrome that was treated with surgery and demonstrate the subsequent rehabilitation time course.

Acute Spinal Cord Injury Associated with Multi-level Pediatric Idiopathic or Inflammatory Intervertebral Disc Calcification (PIIVDC)

Acute Spinal Cord Injury Associated with Multi-level Pediatric Idiopathic or Inflammatory Intervertebral Disc Calcification (PIIVDC)

Precision of spinal radiographs as a screening test for intervertebral disc calcification in Dachshunds

Among dog breeds, the Dachshund has the highest lifetime incidence of intervertebral disc disease (IVDD). Intervertebral disc (IVD) calcification is an indicator of severe degeneration that predisposes to disc herniation. IVDD is heritable in Dachshunds, and in some countries, breeding candidates are screened to reduce IVDD occurrence by selecting dogs according to their score of radiographically detectable intervertebral disc calcification (RDIDC) and excluding dogs with ≥5 RDIDCs from breeding. This study evaluated the precision of scoring spinal radiographs for IVD calcification and subsequent classification of Dachshund dogs for breeding based on their RDIDC score. Digital radiographs of the spine were obtained in 19 clinically healthy, young adult Dachshunds, and scored for RDIDC independently by five scorers with varying levels of experience, three times each. Within scorer (repeatability) and between scorer (reproducibility) variability was estimated both at the individual IVD level and at the whole dog level for breeding classification purposes.At the IVD level, some degree of scorer effect was supported by the pairwise repeatability (92.3%; 95% CI: 88.8–94.7%) being marginally higher than the reproducibility (89.2%; 95% CI: 85.7–91.8%). Scorer-specific patterns confirmed the presence of scorer subjectivity. Repeatability significantly increased with scorer experience but the reproducibility did not. RDIDC scoring repeatability and reproducibility substantially decreased at the cervicothoracic spine region, likely due to anatomical superimpositions. At the dog level, a breeding classification could be repeated by the same scorer for 83.6% (95% CI: 73.8–90.2%) of the dogs, and was reproduced between two scorers for 80.2% (95% CI: 66.6–89.1%) of the dogs. The repeatability of breeding classification also seemed to increase with scorer experience but not the reproducibility. Overall, RDIDC scoring revealed some degree of inconsistency explained by scorer subjectivity and inexperience, and anatomical superimpositions. Scorer training and experience is strongly recommended to improve test precision and ensure appropriate classification of Dachshunds for breeding.

Thoracic intervertebral disc calcification and herniation in adults: a report of two cases

Acute paraplegia due to thoracic intervertebral disc protrusion and calcification is rare. The purpose of this study was to report two cases with acute paraplegia due to a calcified thoracic disc prolapse, and discuss its clinical diagnosis and surgical treatment with literature reviews. These two cases were verified by patient history, physical examination, laboratory examination, CT and MRI studies, and pathological findings. CT scan revealed disc calcification and protrusion at the T11-12 level in case 1 and at the T10-11 level in case 2, respectively. MRI images revealed severe spinal cord compression with a hyperintense central core and surrounding hypointense area in two cases, which were directly connected to the calcified intervertebral nucleus pulposus. Pathological examination revealed calcium deposition. Patients underwent discectomy followed by interbody fusion, and satisfactory therapeutic outcomes were obtained. We suggest that decompression surgery should be carried out as early as possible for patients with early spinal myelopathy or paraplegia caused by a calcified protruded disc.

Osteoarticular cells tolerate short-term exposure to nitisinone-implications in alkaptonuria.

Alkaptonuria (AKU) is a rare genetic disease resulting in severe, rapidly progressing, early onset multi-joint osteoarthropathy. A potential therapy, nitisinone, is being trialled that reduces the causative agent; homogentisic acid (HGA) and in a murine model has shown to prevent ochronosis. Little is currently known about the effect nitisinone has on osteoarticular cells; these cells suffer most from the presence of HGA and its polymeric derivatives. This led us to investigate nitisinone's effect on chondrocytes and osteoblast-like cells in an in vitro model. Human C20/A4 immortalized chondrocytes, and osteosarcoma cells MG63 cultured in DMEM, as previously described. Confluent cells were then plated into 24-well plates at 4 × 10(4) cells per well in varying concentrations of nitisinone. Cells were cultured for 7 days with medium changes every third day. Trypan blue assay was used to determine viability and the effect of nitisinone concentration on cells. Statistical analysis was performed using analysis of variance, and differences between groups were determined by Newman-Keuls post-test. Analysis of C20/A4 chondrocyte and MG63 osteoblast-like cell viability when cultured in different concentrations of nitisinone demonstrates that there is no statistically significant difference in cell viability compared to control cultures. There is currently no literature surrounding the use of nitisinone in human in vitro models, or its effect on chondrocytes or osteoblast like cells. Our results show that nitisinone does not appear detrimental to cell viability of chondrocytes or osteoblast-like cells, which adds to the evidence that this therapy could be useful in treating AKU.

Ca2+ Regulates the Expression of Type II Collagen and Aggrecan in Intervertebral Disc Cells by Activating the Extracellular Calcium-Sensing Receptor

Introduction Intervertebral disc (IVD) calcification frequently occurs with age as demonstrated by radiographic analysis of elderly spines and is enhanced in patients with scoliosis. In one study by Chanchairujira K. et al (Spine 2004;230:499–503), the degree of disc degeneration as measured by disc space height was significantly correlated with IVD calcification. Several systemic disorders such as hyperparathyroidism and chondrocalcinosis, which are related to calcium handling, arthritis, trauma, vertebral fusion, and infection, have been associated with IVD calcification. Our preliminary data suggest that ionic calcium content and expression of the extracellular calcium-sensing receptor (CaSR) are increased in NP and AF cells of human Thompson graded discs, however, a role in disc degeneration remains unknown. Material and Methods Cells: Human NP and AF cells were incubated in culture media containing various concentrations of calcium (1.0, 1.5, 2.5, and 5.0 mM) a CaSR agonist (5 µM), or IL-1β (10 ng/mL) for 7 days. Lysates were extracted and the expression of aggrecan and type II collagen (Col II) were measured by Western blotting. IVDs: Caudal IVDs from the tails of 20- to 24-month-old steers were isolated and the vertebral bone was removed. IVDs were cultured for 4 weeks in culture medium containing calcium (1.0, 2.5, or 5.0 mM), or a CaSR agonist (5 µM). NP and AF tissues were subjected to guanidium extraction and Western blotting was performed to determine the expression of aggrecan and Col II. Histological sections were prepared to determine degree of mineralization by von Kossa staining and expression of alkaline phosphatase. Results The expression of aggrecan and Col II decreased dose-dependently in both NP and AF cells following incubation with calcium or the CaSR agonist. A similar phenomenon was observed in the expression of aggrecan and Col II in IVDs following calcium incubation. In addition to decreases in Col II and aggrecan, increase in mineralization and expression of alkaline phosphatase was observed in calcium-incubated IVDs. Conclusion Our results suggest that changes in the local concentrations of calcium are not benign, and that activation of CaSR may be a contributing factor in IVD degeneration.

Free calcium induces degenerative changes in the intervertebral disc through the calcium sensing receptor

Introduction: Degenerative disc disease (DDD) is a common cause of lower back pain. Calcification of the intervertebral disc (IVD) has been correlated with DDD. The role of IVD calcification in the development DDD is unknown. We noticed an increase in ionic calcium content and expression of the extracellular calcium-sensing receptor (CaSR) in the degenerate discs, however, its role in DDD remains unclear. Material and Methods: IVD Cells: Bovine and human NP and AF cells were incubated in culture media supplemented with various concentrations of calcium, and a CaSR agonist IVD Cultures: IVDs from bovine tails were isolated and the vertebral bone was removed. IVDs were cultured for 6 weeks in culture medium supplemented with calcium (1.0, 2.5, or 5.0 mM), or a CaSR agonist. Western blotting was performed on extracts to assess for aggrecan and Col II. Results: The expression of aggrecan and Col II decreased dose-dependently in both NP and AF cells, as well as in the organ culture model following supplementation with calcium or the CaSR agonist. Conclusion: Our results suggest that changes in the local concentrations of calcium are not benign, and that activation of the CaSR may be a contributing factor in IVD degeneration.

Gene diagnosis and analysis of alkaptonuria in a Chinese pedigree

Objective To study the clinical feature and genetic mutations of alkaptonuria(AKU). Methods One patient was diagnosed with AKU disease via gas chromatography mass spectrometry(GC/MS) in the investigated family.All exon of homogentisate 1, 2 dioxygenase(HGD) gene were amplified in the family by means of polymerase chain reaction(PCR) and followed by using direct DNA sequencing and Polyphen software was used to predict protein function. Results The infant only had red-brown urine, with no skin, joints, or viscera lesion, and GC/MS suggested AKU.Two heterozygous mutations of AKU were identified c. 34A>C(p.N12H) in exon 2 c. 240A>T(p.Q80H)in exon 4 and c. 910A>G(p.K304E)in exon 12 from the proband.The heterozygous change c. 34A>C(p.N12H) in exon 2 was found in the proband's mother and sister with normal phenotype.The proband's father had the heterozygous change c. 910A>G(p.K304E) in exon 12. Conclusions The proband is hetero-zygous compound AKU disease patient carrying on one allele with the c. 34A>C(p.N12H) mutation inherited from his mother and the other allele with the c. 910A>G(p.K304E) from his father.The parents and his sister are heterozygous carrier with normal phenotype.The p. N12H and p. K304E mutations are novel mutations not reported around world yet, which has the earliest onset age of AKU with the only symptom of dark urine. Key words: Alkaptonuria; Homogentisate 1, 2 dioxygenase; Gene mutation; Gene diagnosis

Amyloidosis in alkaptonuria.

Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces an HGA-melanin ochronotic pigment, of hitherto unknown composition. Besides the accumulation of HGA, the potential role and presence of unidentified proteins has been hypothesized as additional causal factors involved in ochronotic pigment deposition. Evidence has been provided on the presence of serum amyloid A (SAA) in several AKU tissues, which allowed classifying AKU as a novel secondary amyloidosis. In this paper, we will briefly review all direct and indirect lines of evidence related to the presence of amyloidosis in AKU. We also report the first data on abnormal SAA serum levels in a cohort of AKU patients.

Old treatments for new insights and strategies: proposed management in adults and children with alkaptonuria.

Alkaptonuria (AKU) is caused by deficiency of the enzyme homogentisate 1,2 dioxygenase. It results in an accumulation of homogentisate which oxidizes spontaneously to benzoquinone acetate, a highly oxidant compound, which polymerises to a melanin-like structure, in a process called ochronosis. Asymptomatic during childhood, this accumulation will lead from the second decade of life to a progressive and severe spondylo-arthopathy, associated with multisystem involvement: osteoporosis/fractures, stones (renal, prostatic, gall bladder, salivary glands), ruptures of tendons/muscle/ligaments, renal failure and aortic valve disease. The pathophysiological mechanisms of AKU remain poorly understood, but recent advances lead us to reconsider the treatment strategy in AKU patients. Besides the supporting therapies (pain killers, anti-inflammatory drugs, physiotherapy, joints replacements and others), specific therapies have been considered (anti-oxidant, low protein diet, nitisinone), but clinical studies have failed to prove efficiency on the rheumatological lesions of the disease. Here we propose a treatment strategy for children and adults with AKU, based on a review of the latest findings on AKU and lessons from other aminoacipathies, especially tyrosinemias.

Adipocytic Templating of Bone Matrix Deposition in Osteoarthropathies

Alkaptonuria (AKU), the first human disorder recognised to conform to Mendelian inheritance, is accompanied by early onset, rapidly progressing osteoarthropathy. Novel anatomical and microanatomical structures found in AKU in joint tissues have subsequently been seen in other human arthropathy samples. One type of trabecular excrescence shows a close relationship with adipocytes, incongruent with typical views of osteoblastic bone formation.Joint samples, obtained with ethical approval were fixed and prepared routinely for histological, immunohistochemical and ultrastructural examination.Histology and ultrastructural analysis of such excrescences show their scalloped surfaces to be continuous with marrow adipocytes fitting the contours of the bone surface. Immunohistochemistry shows excrescences and their marrow surfaces to contain collagen VI, an ECM protein produced by adipocytes but not seen in mature bone matrix.These results demonstrate a variety of bone matrix deposition templated and modulated by adipocytes. The data provide strong support for the view that adipocytes rather than inactive osteoblasts are de facto lining cells on some bone surfaces

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.

Five Cases of Alkaptonuria Among Two Generations of Single Family in Dharwad, Karnataka (India)

Alkaptonuria (AKU) is a disorder of tyrosine metabolism. A rare disorder due to accumulation of homogentisic acid leading to progressive deposition of grey to bluish black pigment causing degenerative changes in cartilage. Medical interest of AKU is due to medical triad arthropathy, Ochronosis and Homogentisic aciduria. A 38 ½ years old male presented with complain of back pain, which persisted since last 8 months. Hyper-pigmentation of skin of chick, discolouration on ear cartilage and pigmentation of the conjunctiva on both eyes were observed. X-ray findings were normal except inter-vertebral disc calcification. Based on the results obtained from Clinical examination, X-ray findings and Biochemical investigations, diagnosis was confirmed as AKU. On screening family members additional four persons have been detected positive but, without any clinical symptoms. AKU, though rare it may occur in cluster among the family members.

Chondroptosis in alkaptonuric cartilage.

Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of treatment is palliative and little is known about AKU physiopathology. Chondroptosis, a peculiar type of cell death in cartilage, has been so far reported to occur in osteoarthritis, a rheumatic disease that shares some features with AKU. In the present work, we wanted to assess if chondroptosis might also occur in AKU. Electron microscopy was used to detect the morphological changes of chondrocytes in damaged cartilage distinguishing apoptosis from its variant termed chondroptosis. We adopted histological observation together with Scanning Electron Microscopy and Transmission Electron Microscopy to evaluate morphological cell changes in AKU chondrocytes. Lipid peroxidation in AKU cartilage was detected by fluorescence microscopy. Using the above‐mentioned techniques, we performed a morphological analysis and assessed that AKU chondrocytes undergo phenotypic changes and lipid oxidation, resulting in a progressive loss of articular cartilage structure and function, showing typical features of chondroptosis. To the best of our knowledge, AKU is the second chronic pathology, following osteoarthritis, where chondroptosis has been documented. Our results indicate that Golgi complex plays an important role in the apoptotic process of AKU chondrocytes and suggest a contribution of chondroptosis in AKU pathogenesis. These findings also confirm a similarity between osteoarthritis and AKU. J. Cell. Physiol. 230: 1148–1157, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Investigating the Robustness and Diagnostic Potential of Extracellular Matrix Remodelling Biomarkers in Alkaptonuria.

Alkaptonuria (AKU) clinical manifestations resemble severe arthritis. The Suitability of Nitisinone in Alkaptonuria 1 (SONIA 1) study is a dose-finding trial for nitisinone treatment of AKU patients. We tested a panel of serum and urinary biomarkers reflecting extracellular matrix remodelling (ECMR) of cartilage, bone and connective tissue in SONIA 1 patients to identify non-invasive and diagnostic biomarkers of tissue turnover in AKU. Fasted serum and urine were retrieved from 40 SONIA 1 patients and 44 healthy controls. Established biomarkers of bone remodelling (CTX-I, P1NP, OC), cartilage remodelling (CTX-II, C2M, AGNx1) and inflammation (CRPM) as well as exploratory biomarkers of ECMR (C6M, VCANM, MIM, TIM) were measured at baseline in serum and urine by means of enzyme-linked immunosorbent assays (ELISAs) or automated systems (Elecsys 2010). The levels of bone resorption (CTX-I) and cartilage degradation (C2M) were elevated in AKU patients as compared to controls (p > 0.0001 and p = 0.03, respectively). Also tissue inflammation (CRPM) was elevated in AKU patients (p = 0.01). In addition all four exploratory biomarkers of ECMR (C6M, VCANM, MIM, TIM) were elevated in AKU patients compared to healthy controls. CTX-II was the only biomarker to be reduced in AKU patients. TIM was the only marker that showed a higher concentration than the normal assay range in AKU patients. We have identified new potential biomarkers for assessment of cartilage, bone and cardiovascular remodelling in AKU and demonstrated the robustness of the assays used to measure the biomarker concentration in biological fluids.

Relationship Between Serum Concentrations of Nitisinone and Its Effect on Homogentisic Acid and Tyrosine in Patients with Alkaptonuria.

Alkaptonuria (AKU) is a serious genetic disease due to a defect in tyrosine metabolism, leading to increased serum levels of homogentisic acid (HGA). Nitisinone decreases HGA in AKU, but the concentration-response relationship has not been previously reported. To determine the relationship between serum concentrations of nitisinone and the effect on both HGA and tyrosine; secondly to determine steady-state pharmacokinetics of nitisinone in AKU patients. Thirty-two patients with AKU received either 1, 2, 4, or 8mg nitisinone daily. Urine and serum HGA and serum tyrosine and nitisinone were measured during 24h at baseline (before first dose) and after 4 weeks of treatment. Nitisinone pharmacokinetics (area under the curve [AUC] and maximum concentrations [C max]) were dose proportional. The median oral clearance determined in all patients, irrespective of dose, was 3.18mL/h·kg (range 1.6-6.7).Nitisinone decreased urinary excretion of HGA in a concentration-dependent manner, with a maximum effect seen at average nitisinone concentrations of 3μmol/L. The association between nitisinone and tyrosine concentrations was less pronounced. Serum levels of HGA at Week 4 were below the limit of quantitation in 65% of samples, which prevented determination of the relationship with nitisinone concentrations. Nitisinone exhibits dose-proportional pharmacokinetics in the studied dosage interval. Urinary excretion of HGA decreases in a concentration-dependent manner, while the increase in tyrosine is less clearly related to nitisinone concentrations.

The Pigment in Alkaptonuria Relationship to Melanin and Other Coloured Substances: A Review of Metabolism, Composition and Chemical Analysis.

The pigments found in plants, animals and humic substances are well described and classified. In humans considerable progress has been made with the main pigment melanin in defining its biochemistry, the different types and function. However, analytical techniques to show these differences in vivo are still not readily available. NMR and IR spectroscopy are relatively insensitive and reveal only major structural differences. Techniques utilising MS are useful in determining elemental content but require further studies to optimise conditions for accurate mass analysis. How the components may be structurally organised seems to be the most problematic with scanning TEM and the improved FTIR of use in this respect. As regards understanding the nature of the pigment related to HGA seen in patients with Alkaptonuria (AKU), it is still thought of as a melaninlike pigment simply because of its colour and likewise thought to be a polymer of undetermined size. It is important that detailed analysis be carried out to define more accurately this pigment. However, observations suggest it to be the same as the HGA-derived pigment, pyomelanin, produced by bacteria and containing both quinone and phenolic groups. The interesting developments in alkaptonuria will be to understand how such a polymer can cause such profound collagen and connective tissue damage and how best to reverse this process.