Combined alkaptonuria and osteoporosis contributing to chronic back pain

Alkaptonuria is a rare disorder, an autosomal recessive condition with genetic determinism and hereditary transmission, having a prevalence of 1 per 1 million population in USA. The pathogenesis includes the deficiency of the homogentisate 1,2-dioxygenase (HGD) enzyme, an intermediary enzyme in phenylalanine and tyrosine catabolism. Mutations in HGD gene leads to deficient levels of functional HGD and an excess of homogentisic acid (HGA). Although HGA is rapidly excreted by the kidneys, it slowly accumulates in various tissues. Due to HGA oxidase deficiency, HGA turns into melanin-like pigment which determines: alkaptonuria, accumulation in the connective tissues, in the joints, or can make cardiovascular and genitourinary deposits. The chronic accumulation of HGA destroys the affected tissue, leading to the characteristic black-blue color and to clinical symptoms of alkaptonuria. The aim of this paper is to investigate the particularities of rheumatic manifestations in a rare metabolic disease and to support the correct diagnosis. A 58-year-old male patient was admitted to our clinic in 2019 for bilateral knee and left shoulder pain. In 2008 he was diagnosed with polyarticular ochronosis having dorsal and lumbar pain, mixed scapulohumeral pain, lumbar intervertebral disk calcifications; at that time, a diagnosis of ankylosing spondylitis or Forestier disease was excluded. At the current admission, the patient has been thoroughly reassessed to obtain a proper diagnosis and to determine the severity of the disease. The ochronotic axial damage caused important differential diagnosis problems with ankylosing spondylitis. Pigment deposition in the eyes, ears and skin does not cause problems to patients, but cardiovascular and genitourinary deposition leads to important complications. Kinetotherapy and NSAIDs are beneficial for pain symptoms. There is no specific medication for stopping the disease progression. Conclusions. Ochronosis is a rare disease which can cause a lot of problems regarding a proper diagnosis and treatment. When differential diagnosis with AS is difficult, the HLA-B27 genotyping is recommended. Final diagnosis is based on qualitative and quantitative urinary tests. The treatment includes only symptomatic drugs such as NSAIDs and kinetotherapy to improve joint mobility and muscle toning.

Alkaptonuria (AKU), the first defined human genetic disease with a recessive trait, is caused by mutations within the homogentisate 1,2‐dioxygenase (HGD) gene (3q13.33). This prototypic inborn error of metabolism is characterised by typical bluish‐black pigmentation in connective tissue ochronosis and severe form of osteoarthritis caused by the deposition of ochronotic pigment in the joints. AKU belongs to a group of rare diseases (1:250 000–1:1 000 000), however, several ethnities were reported, where an increased incidence of AKU was observed (Slovakia, Dominican Republic, Jordan and India). Mutation analysis was so far performed in approximately 350 out of more than 650 worldwide reported AKU patients. Rather high heterogeneity was observed with 122 AKU‐causing mutations that are listed together with HGD polymorphisms in the global HGD mutation database ( http://hgddatabase.cvtisr.sk/ ). Because HGD enzyme functions as hexamer, dimer of trimers, genotype/phenotype correlations are difficult to perform in this rare disease. Key Concepts: Alkaptonuria (AKU) is a prototypic inborn error in the metabolism of phenylalanine and tyrosine, characterised by the inability to metabolise homogentisic acid (HGA). The raised HGA levels in plasma and extracellular fluid lead to ochronosis, the deposition of polymers of HGA as pigment (ochronotic pigment) in connective tissues including cartilage, heart valves and sclera. Ochronosis leads to painful destruction of large weight‐bearing joints as well as fusion of the vertebrae, scoliosis and tendon and ligament ruptures. AKU is caused by homozygous or compound heterozygous mutations in the homogentisate‐1,2‐dioxygenase gene ( HGD ) mapping to the chromosome 3q13.33. AKU belongs to a group of rare diseases (1:250 000–1:1 000 000), however, several ethnities were reported, where an increased incidence of AKU was observed (Slovakia, Dominican Republic, Jordan and India). In approximately 350 patients reported worldwide so far 122 different HGD mutations have been reported. It was also shown that AKU is caused also by the apparently partial loss‐of‐function mutations, however, the heterozygous carriers of AKU are healthy. HGD haplotype analysis helps to identify the origin of individual AKU‐causing mutations in different countries. The triketone herbicide nitisinone or Orfadin inhibits the 4‐hydroxyphenylpyruvate dioxygenase enzyme, which produces HGA, thus, it can decrease HGA and should therefore potentially be able to modify AKU. It has been shown that AKU is a novel type II AA amyloidosis, which opens new important perspectives for its therapy, since the control of the underlying inflammatory disorder can result in regression of the disease. Research on ochronosis in this monogenic disease can help to elucidate the molecular pathogenesis of the more common varieties of osteoarthritis, particularly the biochemical and structural changes at its initial stages.

Alkaptonuria (AKU) is a rare autosomal recessive disorder caused by mutations within a gene coding for homogentisate 1,2-dioxygenase (HGD). To date, 251 different variants of this gene have been reported. The metabolic disorder in AKU leads to the accumulation of homogentisic acid (HGA), resulting in ochronosis (pigmentation of the connective tissues) and severe ochronotic spondylo-arthropathy, which usually manifests in the mid-thirties. An earlier genotype–phenotype correlation study showed no differences in serum HGA levels, absolute urinary excretion of HGA, or in the clinical symptoms between patients carrying HGD variants leading to 1% or >30% residual HGD activity. Still, as reported previously, the variance of the excretion of the HGA was smaller within affected siblings that share a common genotype. The present study is the first ever to systematically analyze the baseline clinical data of 24 AKU sibling pairs/groups collected in the SONIA 2 (Suitability Of Nitisinone In Alkaptonuria 2) study to evaluate phenotypical differences between patients carrying the same HGD genetic variants. We show that even between siblings there was considerable variability in the disease severity. This indicates that some other yet unidentified genetic, biomechanical, or environmental modifying factors may contribute to accelerated pigmentation and connective tissue damage observed in some patients.

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.

Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defective homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called ochronosis, which results on the long term in an early-onset and severe osteoarthropathy. HGD’s quaternary structure is known to be easily disrupted by missense mutations, which makes them an interesting target for novel treatment strategies that aim to rescue enzyme activity. However, only prediction models are available providing information on a structural basis. Therefore, an E. coli based whole-cell screening was developed to evaluate HGD missense variants in 96-well microtiter plates. The screening principle is based on HGD’s ability to convert the oxidation sensitive HGA into maleylacetoacetate. More precisely, catalytic activity could be deduced from pyomelanin absorbance measurements, derived from the auto-oxidation of remaining HGA. Optimized screening conditions comprised several E. coli expression strains, varied expression temperatures and varied substrate concentrations. In addition, plate uniformity, signal variability and spatial uniformity were investigated and optimized. Finally, eight HGD missense variants were generated via site-directed mutagenesis and evaluated with the developed high-throughput screening (HTS) assay. For the HTS assay, quality parameters passed the minimum acceptance criterion for Z’ values > 0.4 and single window values > 2. We found that activity percentages versus wildtype HGD were 70.37 ± 3.08% (for M368V), 68.78 ± 6.40% (for E42A), 58.15 ± 1.16% (for A122V), 69.07 ± 2.26% (for Y62C), 35.26 ± 1.90% (for G161R), 35.86 ± 1.14% (for P230S), 23.43 ± 4.63% (for G115R) and 19.57 ± 11.00% (for G361R). To conclude, a robust, simple, and cost-effective HTS system was developed to reliably evaluate and distinguish human HGD missense variants by their HGA consumption ability. This HGA quantification assay may lay the foundation for the development of novel treatment strategies for missense variants in AKU.

The contribution of mouse models in the rare disease alkaptonuria

Alkaptonuria (AKU) is a rare inherited disorder of tyrosine metabolism caused by lack of the enzyme homogentisate 1,2-dioxygenase (HGD). The primary biochemical consequence of HGD deficiency is increased circulating concentration of homogentisic acid (HGA); this is the central cause of the devastating multi-systemic damage observed in AKU. One of the most striking pathophysiological features of AKU is accumulation of ochronotic pigment derived from HGA in tissues throughout the body. HGA has an affinity for cartilaginous tissue. Presence of ochronotic pigment in cartilage of load-bearing joints causes severe early-onset osteoarthropathy; the greatest cause of morbidity in AKU. This thesis addresses two major unanswered questions in AKU. First is the wider metabolic consequences both of HGD deficiency and those observed following treatment with the promising HGA-lowering agent nitisinone. A metabolomics approach was employed to address these questions, with the aim of discovering biomarkers with potential clinical value in monitoring disease in AKU and in assessing the wider metabolic consequences of nitisinone treatment. Second, this thesis explores the nature of ochronotic pigment derived from HGA. The chemical structure of ochronotic pigment is not known, nor are the mechanisms by which HGA interacts with and binds into the cartilage extracellular matrix. Various analytical chemistry techniques were employed to study the chemical properties of ochronotic pigment and pigmented cartilage in patients and mice with AKU. The data from chemical analyses presented in this thesis provide new insights into the nature of ochronotic pigment. The chemical structure of ochronotic pigment remains unknown, but the pigment had long been widely cited as a polymer in the literature. First, these data fundamentally challenge the idea that ochronotic pigment is a polymeric species. Through study of ochronotic tissue samples, the data also indicate for the first time a specific alteration to the cartilage matrix that enables binding of HGA-derived species. An LC-QTOF-MS metabolic phenotyping strategy was developed, enabling identification of hundreds of metabolites simultaneously, based on chemical properties of accurate mass and chromatographic retention time. Application of this technique to AKU showed that the biochemical alterations following a) targeted deletion of the HGD gene in mice to cause AKU, and b) nitisinone treatment, reflect a complex, interconnected network of metabolite changes. The data not only stimulate myriad further lines of research into AKU pathophysiology but also reveal previously uncharacterised association between metabolites at a network level. These studies provide a prime example of how rare diseases such as inborn errors of metabolism can offer unique windows into metabolism and human physiology more generally.

Background & objectives Alkaptonuria (AKU) is an autosomal recessive disease wherein biallelic pathogenic variants in the homogentisate 1,2- dioxygenase (HGD) gene encoding the enzyme homogentisate 1,2 dioxygenase cause high levels of homogentisic acid (HGA) to circulate within the body leading to its deposition in connective tissues and excretion in urine. A homozygous splice donor variant (c.87+1G>A) has been identified to be the founder variant causing alkaptonuria among Narikuravars, a group of gypsies settled in Tamil Nadu. Methods Blood and urine samples of 30 homozygous splice site donor variant individuals (2 groups aged 7-20 and 21-83 yr, with 9 and 21 individuals, respectively), carriers and 30 wild-type individuals from the Narikuravars were collected during field visits after obtaining informed consent. Clinical evaluation and genetic counselling were done. The plasma and urine HGA levels were estimated by high-performance liquid chromatography. RNA was extracted from the peripheral blood and reverse transcribed. Sanger sequencing was done to check the consequence of the splice donor variant. Relative quantification of the cDNA in the three groups was done by real-time qPCR (RT-qPCR) studies using reference genes followed by Pearson's correlation analysis. Results In our cohort, among the affected alkaptonuria individuals, the minimum age for eye pigmentation detected was 23 yr. Similarly, the minimum age for back pain and any joint pain was 30 yr and 38 yr, respectively. Sequencing of the cDNA confirmed exon 2 skipping in affected individuals. In comparison to the normal individuals, the affected individuals showed reduced HGD expression. HGD relative expression showed a significant correlation (P<0.05) with mean plasma HGA levels in the younger (≤22 yr) age group but not in the older one. There was also a significant correlation (P<0.05) of reduced HGD expression with back pain in the 21-37 yr age group. Increasing age showed a positive correlation with circulating mean plasma HGA levels and a negative correlation with excreted HGA. Interpretation & conclusions As per the authors' knowledge, this is the first study to confirm the functional effect by RT-PCR of this highly prevalent founder HGD variant causing alkaptonuria in the Narikuravar community. Both plasma and urinary HGA levels correlated well with the gene expression of this variant and could serve as potential markers of AKU severity for those with this variant.

Alkaptonuria (AKU) is an ultra-rare, autosomal recessive disorder of tyrosine catabolism due to mutations within the homogentisate 1,2-dioxygenase (HGD) gene. The resulting enzyme deficiency leads to accumulation of homogentisic acid (HGA) and deposition of melanin-like pigment polymers in the connective tissues of the body in a process called ochronosis. This leads to debilitating early onset osteoarthropathy, renal damage and aortic valve disease. As a multisystem disorder, AKU results in progressive and chronic pain and severe morbidity. Most management approaches for AKU are palliative and rely largely on analgesia and arthroplasty. Several therapeutic approaches have been tested with low degrees of clinical effectiveness. Nitisinone is a promising drug that blocks the enzyme catalysing the formation of HGA and thus lowers its plasma concentration. HGA lowering therapy has been widely used in another rare inborn error of metabolism, Hereditary Tyrosinemia type 1 (HT-1) for over 20 years. Nitisinone is highly efficacious in terms of its metabolic effect as it decreases HGA to very low levels, but there is limited toxicology data available for its use in AKU. There are also concerns relating to the adverse side effects of elevated tyrosine and potential neurotoxicity if treatment was implemented in children. The work presented within this thesis presents novel findings to inform the future licensing process for the use of nitisinone in AKU and investigates the safety of implementing treatment in younger patients. Nitisinone treatment had no detrimental effect on learning, memory or motor function in young AKU or wild type mice. The thesis also includes new data from mouse dosing studies concerning the correlation between plasma HGA and ochronotic pigmentation and reveals that plasma HGA must be lowered to a critical level before pigmentation is beneficially reduced. Finally, this thesis reports on the lability of the arteriovenous metabolome relating to AKU and initiates a discussion relating to the HPPA to HPLA excretory conversion pathway along with important considerations for collection, analysis and comparison of blood samples in future studies.

Physical therapy (PT) and conservative care are recommended first-line treatments for musculoskeletal (MSK) pain. While essential to high-quality care, these solutions often do not provide immediate or sufficient pain relief. Traditional transcutaneous electronic nerve stimulation (TENS) devices are often recommended; however, there is mixed evidence behind their effectiveness. A novel approach called hybrid form impulse therapy (HFIT) incorporates a priming pulse with a traditional TENS pulse width and frequency. This randomized controlled trial (RCT) aimed to compare the effectiveness of HFIT versus traditional TENS versus usual care among members of a digital MSK program. A three-arm RCT comparing HFIT versus TENS versus usual care was conducted. A total of 325 people with chronic back or knee pain who were members of a digital MSK program consisting of PT-guided exercise therapy, education, and coaching were randomized. Outcomes including pain, function, anxiety, and depression were examined at 1, 2, and 4 weeks (primary endpoint). Engagement was measured through exercise therapy (ET) sessions completed. Unadjusted and adjusted logistic generalized estimating equations were conducted. Adjusted per-protocol results at 4 weeks showed significantly lower odds of achieving pain improvement for both TENS (OR: 0.42, 95% CI: [0.19, 0.92]) and usual care (OR: 0.35, 95% CI: [0.17, 0.72]) groups, compared to HFIT group. Both HFIT and usual care users had significantly higher engagement than the TENS users (p=0.026 and p=0.002, respectively). No adverse events were reported throughout the study. More participants of a digital MSK program who were randomized to the HFIT group experienced meaningful pain improvement at 4 weeks than participants who used TENS and usual care. HFIT can be an effective, non-pharmaceutical solution for relief as a complement to first-line treatments for patients with chronic back and knee pain.

Editor—Alkaptonuria (AKU) is a disorder of the catabolism of aromatic amino acids. A defect of homogentisate 1,2 dioxygenase (HGO) leads to an accumulation of homogentisic acid (HGA) and subsequently to...

Alkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack of active enzyme homogentisate 1,2-dioxygenase (HGD). The primary consequence of HGD deficiency is increased circulating homogentisic acid (HGA), the main agent in the pathology of AKU disease. Here we report the first metabolomic analysis of AKU homozygous Hgd knockout (Hgd−/−) mice to model the wider metabolic effects of Hgd deletion and the implication for AKU in humans. Untargeted metabolic profiling was performed on urine from Hgd−/− AKU (n = 15) and Hgd+/− non-AKU control (n = 14) mice by liquid chromatography high-resolution time-of-flight mass spectrometry (Experiment 1). The metabolites showing alteration in Hgd−/− were further investigated in AKU mice (n = 18) and patients from the UK National AKU Centre (n = 25) at baseline and after treatment with the HGA-lowering agent nitisinone (Experiment 2). A metabolic flux experiment was carried out after administration of 13C-labelled HGA to Hgd−/−(n = 4) and Hgd+/−(n = 4) mice (Experiment 3) to confirm direct association with HGA. Hgd−/− mice showed the expected increase in HGA, together with unexpected alterations in tyrosine, purine and TCA-cycle pathways. Metabolites with the greatest abundance increases in Hgd−/− were HGA and previously unreported sulfate and glucuronide HGA conjugates, these were decreased in mice and patients on nitisinone and shown to be products from HGA by the 13C-labelled HGA tracer. Our findings reveal that increased HGA in AKU undergoes further metabolism by mainly phase II biotransformations. The data advance our understanding of overall tyrosine metabolism, demonstrating how specific metabolic conditions can elucidate hitherto undiscovered pathways in biochemistry and metabolism.

IntroductionWhile numerous studies have emphasized the pivotal involvement of the Interleukin 6 (IL-6) pathway in the development of chronic pain, the causal nature of this relationship remains uncertain.MethodsIn this study, we opted to include genetic variants situated within the locus of the IL-6 receptor (IL-6R) that exhibited associations with C-reactive protein (CRP) levels. CRP serves as a downstream effector in the IL-6 pathway. Utilizing these variants as genetic proxies, we aimed to modulate IL-6 signaling. Employing a two-sample Mendelian randomization (MR) approach, we investigated the potential link between the genetic proxy and seven distinct subtypes of chronic pain, categorized based on their corresponding body locations. Moreover, we examined the relationship between chronic pain and an alternative instrument of IL-6 signaling that was weighted based on s-IL-6R levels. Furthermore, we conducted exploratory analyses to estimate the plausible causal association between CRP, gp130, and the subtypes of chronic pain.ResultsOur analysis showed that genetic proxied downregulation of IL-6 signaling, weighted on CRP levels, was linked to a reduced risk of chronic back and knee pain. The sensitivity analyses across various MR methods confirmed the consistency of the findings and showed no evidence of horizontal pleiotropy or heterogeneity. Moreover, the results remained robust with different sets of instrument variables. A genetically increased level of s-IL-6R was also negatively associated with chronic back and knee pain. However, there was no causal relationship between CRP and gp130 with chronic pain.ConclusionBased on our findings, there is evidence to suggest a potential causal relationship between IL-6 signaling and chronic back and knee pain. Consequently, the downregulation of IL-6 signaling holds promise as a potential therapeutic target for addressing chronic back and knee pain.

A novel mutation in the homogentisate 1,2 dioxygenase gene identified in Chinese Hani pediatric patients with 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.