Homogentisic Acid Oxidase Research Articles

HGA Triggers SAA Aggregation and Accelerates Fibril Formation in the C20/A4 Alkaptonuria Cell Model.

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by mutations in the homogentisate 1,2-dioxygenase (HGD) gene, leading to the accumulation of homogentisic acid (HGA), causing severe inflammatory conditions. Recently, the presence of serum amyloid A (SAA) has been reported in AKU tissues, classifying AKU as novel secondary amyloidosis; AA amyloidosis is characterized by the extracellular tissue deposition of fibrils composed of fragments of SAA. AA amyloidosis may complicate several chronic inflammatory conditions, like rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, chronic infections, neoplasms, etc. Treatments of AA amyloidosis relieve inflammatory disorders by reducing SAA concentrations; however, no definitive therapy is currently available. SAA regulation is a crucial step to improve AA secondary amyloidosis treatments. Here, applying a comprehensive in vitro and in silico approach, we provided evidence that HGA is a disruptor modulator of SAA, able to enhance its polymerization, fibril formation, and aggregation upon SAA/SAP colocalization. In silico studies deeply dissected the SAA misfolding molecular pathway and SAA/HGA binding, suggesting novel molecular insights about it. Our results could represent an important starting point for identifying novel therapeutic strategies in AKU and AA secondary amyloidosis-related diseases.

Redefining a new frontier in alkaptonuria therapy with AI-driven drug candidate design via in- silico innovation.

A rare metabolic condition called alkaptonuria (AKU) is caused by a decrease in homogentisate 1,2 dioxygenase (HGO) activity due to a mutation in homogentisate dioxygenase (HGD) gene. Homogentisic acid is a byproduct of the catabolism of tyrosine and phenylalanine that darkens the urine and accumulates in connective tissues which causes an agonizing arthritis. Employing the use of deep learning artificial intelligence (AI) drug design, this study aims to alleviate the current toxicity of the AKU drugs currently inuse, particularly nitisinone, by utilizing the natural flavanolkaempferol molecule as a 4-hydroxyphenylpyruvate dioxygenase inhibitor. Kaempferol was employed to generate three effective de novo drug candidates targeting the enzyme4-hydroxyphenylpyruvate dioxygenase using an AI drug design tool. We present novel AIK formulations in the present study. The AIK's (Artificial Intelligence Kaempferol) examination of drug-likeliness among the three led to its choice as a possible target. The toxicity assessment research ofAIK demonstrates that it is not only safer to use than othertreatments, but also more efficient. The docking of theAIGT with 4-hydroxyphenylpyruvate dioxygenase, whichrevealed a binding affinity of around-9.099 kcal/mol, highlights the AIK's potential as a therapeutic candidate. Aninnovative approach to deal with challenging circumstances is thus presented in this study by new formulations kaempferol that have been meticulously designed by AI. The results of the invitro tests must be confirmed invivo, even though AI-designed AIK is effective and sufficiently safe as computed.

Treatment of Achilles tendon rupture in a patient with alkaptonuric ochronosis

The treatment result of a patient with spontaneous rupture of the Achilles tendon and features of diagnosing alkaptonuric ochronosis, a rare disease, are presented, providing doctors beneficial information for the prevention and/or delay of possible complications. On March 8, 2021, at about 20:50, while at his workplace, getting up from his chair from the table, patient K, 52 years old, felt a sharp pain in the lower third of his right shin and was urgently taken to the hospital. He was examined by a traumatologist and was initially diagnosed with rupture of the right Achilles tendon. His right lower limb was immobilized with a plaster splint. On March 11, 2021, he entered the military traumatology and orthopedics clinic of the Kirov Military Medical Academy for surgical treatment. Upon admission, the patient complained of pain in the right lower leg and impaired support function of the right lower limb. Based on complaints, medical history, laboratory results, and instrumental studies, subcutaneous rupture of the right Achilles tendon was diagnosed. On March 12, 2021, anchor suture of the right Achilles tendon was performed. Anesthesia — combined, access: two separate incisions on the posterior surface of the lower third of the right leg and in the area of the calcaneal tubercle, each 5 cm long, intraoperatively revealed a complete subcutaneous rupture of the right Achilles tendon. Notably, the tendon was black in color when cut. The postoperative period proceeded without complications, and postoperative wounds of linear shape in the right Achilles tendon healed by primary intention. No signs of disturbances were observed in peripheral circulation and innervation of the right lower limb. To confirm the diagnosis, a simple laboratory test was performed: a change in the color of urine during the day, after its additional alkalization. The color change is due to the polymerization of homogentisic acid, which is excreted in large quantities in the urine. To verify the diagnosis, a molecular genetic technique was performed: sequencing of the clinically significant HGD gene and encoding the enzyme homogentisate-1,2-dioxygenase, which are mutations that lead to alkaptonuria. The analysis results confirmed the presence of an amino acid substitution at the pGLy161Arg position, indicating pathogenicity. The use of simple methods of additional laboratory diagnostics and molecular genetic analysis enabled the diagnosis of alkaptonuric ochronosis. Thus, characteristic diagnostic signs were damage to the Achilles tendon in combination with the obtained anamnestic, clinical, and laboratory data of alkaptonuria.

Alkaptonuric Ochronosis: A case-based review.

Alkaptonuria (AKU) is an extremely rare autosomal recessive metabolic disorder caused by deficiency of homogentisic acid oxidase and resulting in accumulation of homogentisic acid in collagenous structures. It is characterized by a triad of homogentisic aciduria, bluish-black discoloration of connective tissues (ochronosis) and arthropathy of large weight bearing joints. We report on a middle-aged female patient with bilateral severe ochronotic arthritis of both hips and shoulder joints requiring total joint replacements as staged procedures which were done without complications offering a complete pain relief and a satisfactory clinical and functional outcome. Ochronosis can cause severe arthropathy of peripheral joints. Multiple joint affection is common. Total joint replacement can yield persistent pain relief with complete functional recovery in patients with severe ochronotic arthropathy.

Tryptophan Promotes the Production of Xanthophyll Compounds in Yellow Abdominal Fat through HAAO.

Abdominal fat, which in the past was often regarded as waste and discarded, has in recent years been used as a fat source to produce meat by-products. Yellow abdominal fat has higher economic value. Therefore, improving the color of abdominal fat plays an important role in improving the appearance of meat products. This study aimed to identify the contributors and the regulatory network involved in the formation of yellow and white color in abdominal fat. We found that four xanthophyll compounds were significantly different in yellow and white abdominal fat chicken, including zeaxanthin, lutein, canthaxanthin, and β-cryptoxanthin. There were 551 different and 8 common metabolites significantly correlated with these 4 xanthophyll compounds. Similarly, a total of 54 common genes were identified in 4 common related pathways (Complement and coagulation cascades, Metabolic pathways, PPAR signaling pathway, Carbon metabolism) of the 8 common metabolites. The high expression of HAAO in the yellow abdominal fat group leads to the degradation of tryptophan and its intermediate 5-hydroxyindole, and subsequently to the formation of the four xanthophyll compounds. This process is also regulated by tyrosine, kynurenine 3-monooxygenase (KMO), homogentisate 1, 2-dioxygenase (HGD), etc. Together, these findings show the effect of tryptophan on abdominal fat color, as well as a negative regulatory effect of HAAO and 5-hydroxyindole on the production of xanthophyll compounds involved in abdominal fat coloration.

Ochronotic arthropathy knee-arthroscopic diagnosis and treatment – A case report

Ochronotic arthropathy is a rare hereditary disorder found in patients with Alkaptonuria due to a deficiency of Homogentisic acid oxidase. It is associated with the deposition of homogentisic acid derivatives in various connective tissues, causing characteristic pigmentation known as ochronosis. Due to its clinical resemblance to that of a degenerative disorder, the diagnosis of ochronotic arthropathy usually occurs intraoperatively. This case report is of a 56-year-old male who presented with complaints of pain and swelling in the right knee. Clinical examination and MRI findings were correlated to reveal a tear of the lateral meniscus. On arthroscopy, the blackish-brown pigmentation of the meniscus and the articular cartilage led to a diagnosis of ochronotic arthropathy. The pathology and biochemistry studies further support this. Arthroscopy plays an important tool in terms of the management of patients with ochronosis. The characteristic findings, in this case, report may help in establishing the diagnosis of ochronotic arthropathy without other systemic presentations. It can help in delaying inevitable progression and allow patients to be pain-free for longer periods. Arthroscopy delays the need for arthroplasty.

Identification and characterization of methoxy- and dimethoxyhydroquinone 1,2-dioxygenase from Phanerochaete chrysosporium.

White-rot fungi, such as Phanerochaete chrysosporium, are the most efficient degraders of lignin, a major component of plant biomass. Enzymes produced by these fungi, such as lignin peroxidases and manganese peroxidases, break down lignin polymers into various aromatic compounds based on guaiacyl, syringyl, and hydroxyphenyl units. These intermediates are further degraded, and the aromatic ring is cleaved by 1,2,4-trihydroxybenzene dioxygenases. This study aimed to characterize homogentisate dioxygenase (HGD)-like proteins from P. chrysosporium that are strongly induced by the G-unit fragment of vanillin. We overexpressed two homologous recombinant HGDs, PcHGD1 and PcHGD2, in Escherichia coli. Both PcHGD1 and PcHGD2 catalyzed the ring cleavage in methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ). The two enzymes had the highest catalytic efficiency (kcat/Km) for MHQ, and therefore, we named PcHGD1 and PcHGD2 as MHQ dioxygenases 1 and 2 (PcMHQD1 and PcMHQD2), respectively, from P. chrysosporium. This is the first study to identify and characterize MHQ and DMHQ dioxygenase activities in members of the HGD superfamily. These findings highlight the unique and broad substrate spectra of PcHGDs, rendering them attractive candidates for biotechnological applications.IMPORTANCEThis study aimed to elucidate the properties of enzymes responsible for degrading lignin, a dominant natural polymer in terrestrial lignocellulosic biomass. We focused on two homogentisate dioxygenase (HGD) homologs from the white-rot fungus, P. chrysosporium, and investigated their roles in the degradation of lignin-derived aromatic compounds. In the P. chrysosporium genome database, PcMHQD1 and PcMHQD2 were annotated as HGDs that could cleave the aromatic rings of methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ) with a preference for MHQ. These findings suggest that MHQD1 and/or MHQD2 play important roles in the degradation of lignin-derived aromatic compounds by P. chrysosporium. The preference of PcMHQDs for MHQ and DMHQ not only highlights their potential for biotechnological applications but also underscores their critical role in understanding lignin degradation by a representative of white-rot fungus, P. chrysosporium.

Identification of CD8+ T-cell exhaustion signatures for prognosis in HBV-related hepatocellular carcinoma patients by integrated analysis of single-cell and bulk RNA-sequencing

BackgroundHBV infection is the leading risk factor for HCC. HBV infection has been confirmed to be associated with the exhaustion status of CD8+ T cells and immunotherapeutic efficacy in HCC. In this study, we aimed to investigate the prognostic value of the CD8+ T-cell exhaustion signature and immunotherapy response in patients with HBV-related HCC.MethodsWe identified different clusters of HBV-related HCC cells by single-cell RNA sequencing (scRNA-seq) and identified CD8+ T-cell exhaustion-related genes (TERGs) by pseudotime analysis. We conducted differential expression analysis and LASSO Cox regression to detect genes and construct a CD8+ T-cell exhaustion index (TEI). We next combined the TEI with other clinicopathological factors to design a prognostic nomogram for HCC patients. We also analysed the difference in the TEI between the non-responder and responder groups during anti-PD-L1 therapy. In addition, we investigated how HBV induces CD8+ T lymphocyte exhaustion through the inhibition of tyrosine metabolism in HCC using gene set enrichment analysis and RT‒qPCR.ResultsA CD8+ T-cell exhaustion index (TEI) was established with 5 TERGs (EEF1E1, GAGE1, CHORDC1, IKBIP and MAGOH). An AFP level > 500 ng, vascular invasion, histologic grade (G3-G4), advanced TNM stage and poor five-year prognosis were related to a higher TEI score, while HBV infection was related to a lower TEI score. Among those receiving anti-PD-L1 therapy, responders had lower TEIs than non-responders did. The TEI also serves as an independent prognostic factor for HCC, and the nomogram incorporating the TEI, TNM stage, and vascular invasion exhibited excellent predictive value for the prognosis in HCC patients. RT‒qPCR revealed that among the tyrosine metabolism-associated genes, TAT (tyrosine aminotransferase) and HGD (homogentisate 1,2 dioxygenase) were expressed at lower levels in HBV-HCC than in non-HBV HCC.ConclusionGenerally, we established a novel TEI model by comprehensively analysing the progression of CD8+ T-cell exhaustion, which shows promise for predicting the clinical prognosis and potential immunotherapeutic efficacy in HBV-related HCC patients.

An Uncommon Clinical Presentation of a Rare Disease-Alkaptonuria: Case Report

Palmoplantar pigmentation is a rare clinical presentation of alkaptonuria, a rare inborn error of phenylalanine and tyrosine metabolism. Alkaptonuria occurs owing to a deficiency of an enzyme homogentisic acid oxidase inherited as an autosomal recessive disorder. We report a case of alkaptonuria, which presented with palmoplantar pigment pigmentary nail changes and the involvement of the spine and joints. He was advised of ascorbic acid along with symptomatic management and detailed counselling.

From low back pain to ochronosis

Background: Alkaptonuria is a rare metabolic autosomal recessive disorder. Its aetiology involves homogentisate 1,2 dioxygenase (HGD) deficiency resulting in homogentisic acid accumulation in the connective tissues (ochronosis). The classic triad of the disease is: i) homogentisic aciduria, ii) bluish-black pigmentation in tissues such as the sclera, cornea, cartilage and skin, and iii) degenerative arthropathy usually in the fourth decade of life. Case: In this case report, we present a 41-year-old man with diffuse musculoskeletal pain and additional clinical features in tissues such as the ear and urinary tract who was diagnosed late (>30 years). He was diagnosed with alkaptonuria based on clinical findings and elevated urinary homogentisic acid levels. Conclusion: This case report underscores the need for early diagnosis of alkaptonuria, which can help in managing symptoms and improving the quality of life of patients. Further research is needed to develop more targeted therapies for alkaptonuria, which can help slow down the progression of joint degeneration and improve overall patient.

Homogentisate 1,2-dioxygenase (HGD) gene variants in young Egyptian patients with alkaptonuria

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by pathogenic variants in the homogentisate 1,2-dioxygenase (HGD) gene. This leads to a deficient HGD enzyme with the consequent accumulation of homogentisic acid (HGA) in different tissues causing complications in various organs, particularly in joints, heart valves and kidneys. The genetic basis of AKU in Egypt is completely unknown. We evaluated the clinical and genetic spectrum of six pediatric and adolescents AKU patients from four unrelated Egyptian families. All probands had a high level of HGA in urine by qualitative GC/MS before genetic confirmation by Sanger sequencing. Recruited AKU patients were four females and two males (median age 13 years). We identified four different pathogenic missense variants within HGD gene. Detected variants included a novel variant c.1079G > T;p.(Gly360Val) and three recurrent variants; c.1078G > C;p.(Gly360Arg), c.808G > A;p.(Gly270Arg) and c.473C > T;p.(Pro158Leu). All identified variants were properly segregating in the four families consistent with autosomal recessive inheritance. In this study, we reported the phenotypic and genotypic spectrum of alkaptonuria for the first time in Egypt. We further enriched the HGD-variant database with another novel pathogenic variant. The recent availability of nitisinone may promote the need for genetic confirmation at younger ages to start therapy earlier and prevent serious complications.

Clinical presentation of 13 children with alkaptonuria.

Until now, only a few studies have focused on the early onset of symptoms of alkaptonuria (AKU) in the pediatric population. This prospective, longitudinal study is a comprehensive approach to the assessment of children with recognized AKU during childhood. The study includes data from 32 visits of 13 patients (five males, eight females; age 4-17 years) with AKU. A clinical evaluation was performed with particular attention to eye, ear, and skin pigmentation, musculoskeletal complaints, magnetic resonance imaging (MRI), and ultrasound (US) imaging abnormalities. The cognitive functioning and adaptive abilities were examined. Molecular genetic analyses were performed. The most common symptoms observed were dark urine (13/13), followed by joint pain (6/13), and dark ear wax (6/13). In 4 of 13 patients the values obtained in the KOOS-child questionnaire were below the reference values. MRI and US did not show degenerative changes in knee cartilages. One child had nephrolithiasis. Almost half of the children with AKU (5/13) presented deficits in cognitive functioning and/or adaptive abilities. The most frequent HGD variants observed in the patients were c.481G>A (p.Gly161Arg) mutation and the c.240A>T (p.His80Gln) polymorphism. The newly described allele of the HGD gene (c.948G>T, p.Val316Phe) which is potentially pathogenic was identified.

Biochemical and molecular confirmation of alkaptonuria in a Sumatran orangutan (Pongo abelii).

A 6-yr-old female orangutan presented with a history of dark urine that turned brown upon standing since birth. Repeated routine urinalysis and urine culture were unremarkable. Urine organic acid analysis showed elevation in homogentisic acid consistent with alkaptonuria. Sequence analysis identified a homozygous missense variant, c.1081G>A (p.Gly361Arg), of the homogentisate 1,2-dioxygenase (HGD) gene. Familial studies, molecular modeling, and comparison to human variant databases support this variant as the underlying cause of alkaptonuria in this orangutan. This is the first report of molecular confirmation of alkaptonuria in a nonhuman primate.

Structure-Function Relationship of Homogentisate 1,2-dioxygenase: Understanding the Genotype-Phenotype correlations in the Rare Genetic Disease Alkaptonuria.

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs, which occurs because the homogentisate 1,2-dioxygenase (HGD) enzyme is not functional due to gene variants. Over time, HGA oxidation and accumulation cause the formation of the ochronotic pigment, a deposit that provokes tissue degeneration and organ malfunction. Here, we report a comprehensive review of the variants so far reported, the structural studies on the molecular consequences of protein stability and interaction, and molecular simulations for pharmacological chaperones as protein rescuers. Moreover, evidence accumulated so far in alkaptonuria research will be re-proposed as the bases for a precision medicine approach in a rare disease.

ALKAPTONURIA DIAGNOSED IN A 72 YEAR OLD FEMALE AFTER TOTAL KNEE REPLACEMENT

Alkaptonuria is a rare autosomal recessive disorder of metabolism caused by deficiency of homogentisic acid oxidase and resulting in accumulation of homogentisic acid in collagenous structures. It is characterized by homogentisic aciduria, bluish-black discoloration of connective tissues (ochronosis) and arthropathy of large joints. Less common manifestations include cardiovascular abnormalities, renal, urethral and prostate calculi. Bone fractures are unusual in ochronosis. In this report, we describe a woman, 72 years of age, with a history of severe arthropathy requiring total joint replacement in both of her hips and left Knee. During the Left Total Knee Replacement, an intra operative observation of a bluish-black discoloration of the knee joint and the surrounding soft tissue raised the diagnosis of Alkaptonuria. We review the etiology, pathogenesis, clinical presentation, diagnosis and treatment of alkaptonuric ochronosis. Early detection is important for prevention and treatment of multiple systems.

Identification of Potential Inhibitors for the Treatment of Alkaptonuria Using an Integrated In Silico Computational Strategy.

Alkaptonuria (AKU) is a rare genetic autosomal recessive disorder characterized by elevated serum levels of homogentisic acid (HGA). In this disease, tyrosine metabolism is interrupted because of the alterations in homogentisate dioxygenase (HGD) gene. The patient suffers from ochronosis, fractures, and tendon ruptures. To date, no medicine has been approved for the treatment of AKU. However, physiotherapy and strong painkillers are administered to help mitigate the condition. Recently, nitisinone, an FDA-approved drug for type 1 tyrosinemia, has been given to AKU patients in some countries and has shown encouraging results in reducing the disease progression. However, this drug is not the targeted treatment for AKU, and causes keratopathy. Therefore, the foremost aim of this study is the identification of potent and druggable inhibitors of AKU with no or minimal side effects by targeting 4-hydroxyphenylpyruvate dioxygenase. To achieve our goal, we have performed computational modelling using BioSolveIT suit. The library of ligands for molecular docking was acquired by fragment replacement of reference molecules by ReCore. Subsequently, the hits were screened on the basis of estimated affinities, and their pharmacokinetic properties were evaluated using SwissADME. Afterward, the interactions between target and ligands were investigated using Discovery Studio. Ultimately, compounds c and f were identified as potent inhibitors of 4-hydroxyphenylpyruvate dioxygenase.

Rare Case of Ochronosis with Fracture of Intracapsular Neck of Femur and Scoliosis: A Case Report

Ochronosis is a rare syndrome caused by accumulation of homogentisic acid in connective tissue due to deficiency of enzyme homogentisic acid oxidase. It characterized by blue-black pigmentation connective tissues such as sclera, cartilage of ear, and synovium of joints and it causes destruction of joints cartilage and early arthritis. Urine becomes dark coloured on prolonged standing. Some patient may develop rare cardiac manifestation due to accumulation of homogentisic acid on valves. A 56-year-old female admitted with neck of femur fracture after history of fall at home. The patient was having chronic back ache and knee pain. Plain radiograph of knee and spine showed severe arthritic changes. Exposure during surgery was difficult due to hard and brittle tendons and capsule of joint. Femur head and acetabulum cartilage appeared dark brown. Dark brown pigmentation of sclera and hands was found on clinical examination postoperatively. Patients with ochronosis usually develop early osteoarthritis and spondylosis which should be differentiated from other causes of early arthritis such as rheumatoid arthritis and seronegative arthritis. It leads to destruction of joint cartilage and weaking subchondral bone which leads to pathological fracture. And due to the stiffness of soft-tissues around joint, surgical exposure can be challenging.

Untargeted NMR Metabolomics Reveals Alternative Biomarkers and Pathways in Alkaptonuria

Alkaptonuria (AKU) is an ultra-rare metabolic disease caused by the accumulation of homogentisic acid (HGA), an intermediate product of phenylalanine and tyrosine degradation. AKU patients carry variants within the gene coding for homogentisate-1,2-dioxygenase (HGD), which are responsible for reducing the enzyme catalytic activity and the consequent accumulation of HGA and formation of a dark pigment called the ochronotic pigment. In individuals with alkaptonuria, ochronotic pigmentation of connective tissues occurs, leading to inflammation, degeneration, and eventually osteoarthritis. The molecular mechanisms underlying the multisystemic development of the disease severity are still not fully understood and are mostly limited to the metabolic pathway segment involving HGA. In this view, untargeted metabolomics of biofluids in metabolic diseases allows the direct investigation of molecular species involved in pathways alterations and their interplay. Here, we present the untargeted metabolomics study of AKU through the nuclear magnetic resonance of urine from a cohort of Italian patients; the study aims to unravel molecular species and mechanisms underlying the AKU metabolic disorder. Dysregulation of metabolic pathways other than the HGD route and new potential biomarkers beyond homogentisate are suggested, contributing to a more comprehensive molecular signature definition for AKU and the development of future adjuvant treatment.

A robust bacterial high-throughput screening system to evaluate single nucleotide polymorphisms of human homogentisate 1,2-dioxygenase in the context of alkaptonuria

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.

Analysis of the Phenotype Differences in Siblings with Alkaptonuria.

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.