Alkaptonuria.

Alkaptonuria is a rare inborn error of metabolism caused by the deficiency of homogentisate 1,2-dioxygenase activity. The consequent homogentisic acid (HGA) accumulation in body fluids and tissues leads to a multisystemic and highly debilitating disease whose main features are dark urine, ochronosis (HGA-derived pigment in collagen-rich connective tissues), and a painful and severe form of osteoarthropathy. Other clinical manifestations are extremely variable and include kidney and prostate stones, aortic stenosis, bone fractures, and tendon, ligament and/or muscle ruptures. As an autosomal recessive disorder, alkaptonuria affects men and women equally. Debilitating symptoms appear around the third decade of life, but a proper and timely diagnosis is often delayed due to their non-specific nature and a lack of knowledge among physicians. In later stages, patients' quality of life might be seriously compromised and further complicated by comorbidities. Thus, appropriate management of alkaptonuria requires a multidisciplinary approach, and periodic clinical evaluation is advised to monitor disease progression, complications and/or comorbidities, and to enable prompt intervention. Treatment options are patient-tailored and include a combination of medications, physical therapy and surgery. Current basic and clinical research focuses on improving patient management and developing innovative therapies and implementing precision medicine strategies.

Combination of expert guidelines-based and machine learning-based approaches leads to superior accuracy of automated prediction of clinical effect of copy number variations.

Clinical interpretation of copy number variants (CNVs) is a complex process that requires skilled clinical professionals. General recommendations have been recently released to guide the CNV interpretation based on predefined criteria to uniform the decision process. Several semiautomatic computational methods have been proposed to recommend appropriate choices, relieving clinicians of tedious searching in vast genomic databases. We have developed and evaluated such a tool called MarCNV and tested it on CNV records collected from the ClinVar database. Alternatively, the emerging machine learning-based tools, such as the recently published ISV (Interpretation of Structural Variants), showed promising ways of even fully automated predictions using broader characterization of affected genomic elements. Such tools utilize features additional to ACMG criteria, thus providing supporting evidence and the potential to improve CNV classification. Since both approaches contribute to evaluation of CNVs clinical impact, we propose a combined solution in the form of a decision support tool based on automated ACMG guidelines (MarCNV) supplemented by a machine learning-based pathogenicity prediction (ISV) for the classification of CNVs. We provide evidence that such a combined approach is able to reduce the number of uncertain classifications and reveal potentially incorrect classifications using automated guidelines. CNV interpretation using MarCNV, ISV, and combined approach is available for non-commercial use at https://predict.genovisio.com/ .

APOC3 and ABCA1 variants in unusual combined hypolipidaemia showing premature peripheral vascular disease.

BACKGROUND: Familial combined hypolipidaemia is a condition characterised by very low concentrations of circulating very-low-density lipoprotein (VLDL), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL). It is thought that low LDL/combined hypolipidaemia can protect from cardiovascular disease (CVD), but this is not what we found in a case we present. OBJECTIVE: We report on a 57-years-old male patient with combined hypolipidaemia who presented with premature peripheral vascular disease. We investigated also his two sons, 32- and 27-years-old, who manifested a tendency to low lipid levels. METHODS AND RESULTS: We used Illumina exome analysis in all three individuals and in all of them we could exclude the major effect of the variants within the genes most frequently mutated in hypolipidaemia, including recently reported LIPC gene variant. Instead, in all three individuals we identified a novel ABCA1 variant, possibly responsible for the decreased HDL levels. The proband and one of his sons also share the splicing APOC3 variant rs138326449, known to be associated with decreased TG levels. CONCLUSION: The heterogeneous nature and the risk of atherosclerosis in combined hypolipidaemia seems to be variable, based on an interplay between low HDL and LDL levels, and it depends on the combination of variants that cause it (Tab. 2, Ref. 38).

Nutritional interventions for patients with alkaptonuria: A minireview.

Alkaptonuria (AKU, OMIM, No. 203500) is a rare, slow-progressing, irreversible, multisystemic disease resulting from a deficiency of the homogentisate 1,2-dioxygenase enzyme, which leads to the accumulation of homogentisic acid (HGA) and subsequent deposition as pigment in connective tissues called ochronosis. As a result, severe arthropathy of large joints and spondyloarthropathy with frequent fractures, ligament ruptures, and osteoporosis develops in AKU patients. Since 2020, the first-time treatment with nitisinone has become available in the European Union. Nitisinone significantly reduces HGA production and arrests ochronosis in AKU patients. However, blocking of the tyrosine metabolic pathway by the drug leads to tyrosine plasma and tissue concentrations increase. The nitisinone-induced hypertyrosinemia can lead to the development of corneal keratopathy, and once it develops, the treatment needs to be interrupted. A decrease in overall protein intake reduces the risk of the keratopathy during nitisinone-induced hypertyrosinemia in AKU patients. The low-protein diet is not only poorly tolerated by patients, but over longer periods, leads to a severe muscle loss and weight gain due to increased energy intake from carbohydrates and fats. Therefore, the development of novel nutritional approaches is required to prevent the adverse events due to nitisinone-induced hypertyrosinemia and the negative impact on skeletal muscle metabolism in AKU patients.

Breakpoints characterisation of the genomic deletions identified by MLPA in alkaptonuria patients.

Until recently, mainly DNA sequencing has been used to identify variants within the gene coding for homogentisate dioxygenase (HGD, 3q13.33) that cause alkaptonuria (AKU), an autosomal recessive inborn error of metabolism of tyrosine. In order to identify possible larger genomic deletions we have developed a novel Multiplex Ligation-dependent Probe Amplification (MLPA) assay specific for this gene (HGD-MLPA) and tested it successfully in healthy controls and in patients carrying two known previously identified HGD deletions. Subsequently, we analysed 22 AKU patients in whom only one or none classical HGD variant was found by sequencing. Using HGD-MLPA and sequencing, we identified four larger deletions encompassing from 1 to 4 exons of this gene and we defined their exact breakpoints: deletion of exons 1-4 (c.1-8460_282 + 6727del), deletion of exons 5 and 6 (c.283-9199_434 + 1688del), deletion of exon 11 (c.775-1915_879 + 1293del), and deletion of exon 13 (c.1007-1709_1188 + 1121del). We suggest including MLPA in the DNA diagnostic protocols for AKU in cases where DNA sequencing does not lead to identification of both HGD variants.

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.

Radiological evolution of spinal disease in alkaptonuria and the effect of nitisinone.

OBJECTIVES: Ochronotic spondyloarthropathy represents one of the main clinical manifestations of alkaptonuria (AKU); however, prospective data and description of the effect of nitisinone treatment are lacking. METHODS: Patients with AKU aged 25 years or older were randomly assigned to receive either oral nitisinone 10 mg/day (N=69) or no treatment (N=69). Spine radiographs were recorded yearly at baseline, 12, 24, 36 and 48 months, and the images were scored for the presence of intervertebral space narrowing, soft tissue calcifications, vacuum phenomena, osteophytes/hyperostosis and spinal fusion in the cervical, thoracic and lumbosacral segment at each of the time points. RESULTS: At baseline, narrowing of the intervertebral spaces, the presence of osteophytes/hyperostosis and calcifications were the three most frequent radiographic features in AKU. The rate of progression of the five main features during the 4 years, ranked from the highest to lowest was as follows: intervertebral spaces narrowing, calcifications, vacuum phenomena, osteophytes/hyperostosis and fusions. The rate of progression did not differ between the treated and untreated groups in any of the five radiographic parameters except for a slower rate of progression (sum of all five features) in the treatment group compared with the control group (0.45 (1.11) nitisinone vs 0.74 (1.11) controls, p=0.049) in the thoracic segment. CONCLUSION: The present study shows a relatively slow but significant worsening of radiographic features in patients with AKU over 4 years. Our results demonstrate a modest beneficial effect of 10 mg/day of nitisinone on the slowly progressing spondylosis in AKU during the relatively limited follow-up time. TRIAL REGISTRATION NUMBER: NCT01916382.

Molecularly confirmed pontocerebellar hypoplasia in a large family from Slovakia with four severely affected children.

BACKGROUND: Pontocerebellar hypoplasia type 1 (PCH1) is characterized by a central and peripheral motor dysfunction associated with anterior horn cell degeneration, similar to spinal muscular atrophy (SMA). OBJECTIVES: We analysed three probands (later discovered to be siblings) suspected to have severe SMA, however, not confirmed by genetic test. METHODS: Clinical-exome analysis (Illumina) was performed to identify causative variants, followed by Sanger sequencing confirmation in probands and other 10 family members. RESULTS: Homozygous pathogenic variant c.92G>C (p.(Gly31Ala)) in the Exosome Component 3 (EXOSC3) gene was found in all 3 probands, thus confirming the diagnosis of a severe form of PCH1B. The parents and six siblings were carriers, while one sibling was homozygous for a reference allele. This variant is frequent in the Czech Roma population, where it is considered a founder mutation. Haplotype analysis in this largest reported PCH1B family showed that our patients inherited from their father (of Roma origin) a haplotype identical to that found in the Czech Roma population, thus indicating these alleles have a common origin. CONCLUSION: This EXOSC3 variant is rare among the general population but most likely frequent also among Roma people in Slovakia. PCH1B should be considered for a differential diagnosis in infants manifesting SMA-like phenotype, especially if of Roma origin (Tab. 1, Fig. 1, Ref. 22). Text in PDF www.elis.sk Keywords: pontocerebellar hypoplasia, PCH1B, EXOSC3, SMA plus syndromes, pathogenic sequence variant.

Alkaptonuria in Russia.

Alkaptonuria is characterized by the accumulation of homogentisic acid (HGA), part of which is excreted in the urine but the excess HGA forms a dark brown ochronotic pigment that deposits in the connective tissue (ochronosis), eventually leading to early-onset severe arthropathy. We analyzed a cohort of 48 Russian AKU families by sequencing all 14 exons (including flanking intronic sequences) of the homogentisate 1,2-dioxygenase gene (HGD) and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. Nine novel likely pathogenic HGD variants were identified, which have not been reported previously in any other country. Recently, Bychkov et al. [1] reported on the variant spectrum in another cohort of 49 Russian AKU patients. Here we summarize complete data from both cohorts that include 82 Russian AKU families. Taken together, 31 different HGD variants were found in these patients, of which 14 are novel and found only in Russia. The most common variant was c.481G>A (p.(Gly161Arg)), present in almost 54% of all AKU alleles.

Efficacy and safety of once-daily nitisinone for patients with alkaptonuria (SONIA 2): an international, multicentre, open-label, randomised controlled trial.

BACKGROUND: Alkaptonuria is a rare, genetic, multisystem disease characterised by the accumulation of homogentisic acid (HGA). No HGA-lowering therapy has been approved to date. The aim of SONIA 2 was to investigate the efficacy and safety of once-daily nitisinone for reducing HGA excretion in patients with alkaptonuria and to evaluate whether nitisinone has a clinical benefit. METHODS: SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study done at three sites in the UK, France, and Slovakia. Patients aged 25 years or older with confirmed alkaptonuria and any clinical disease manifestations were randomly assigned (1:1) to receive either oral nitisinone 10 mg daily or no treatment. Patients could not be masked to treatment due to colour changes in the urine, but the study was evaluator-blinded as far as possible. The primary endpoint was daily urinary HGA excretion (u-HGA24) after 12 months. Clinical evaluation Alkaptonuria Severity Score Index (cAKUSSI) score was assessed at 12, 24, 36, and 48 months. Efficacy variables were analysed in all randomly assigned patients with a valid u-HGA24 measurement at baseline. Safety variables were analysed in all randomly assigned patients. The study was registered at ClinicalTrials.gov (NCT01916382). FINDINGS: Between May 7, 2014, and Feb 16, 2015, 139 patients were screened, of whom 138 were included in the study, with 69 patients randomly assigned to each group. 55 patients in the nitisinone group and 53 in the control group completed the study. u-HGA24 at 12 months was significantly decreased by 99·7% in the nitisinone group compared with the control group (adjusted geometric mean ratio of nitisinone/control 0·003 [95% CI 0·003 to 0·004], p<0·0001). At 48 months, the increase in cAKUSSI score from baseline was significantly lower in the nitisinone group compared with the control group (adjusted mean difference -8·6 points [-16·0 to -1·2], p=0·023). 400 adverse events occurred in 59 (86%) patients in the nitisinone group and 284 events occurred in 57 (83%) patients in the control group. No treatment-related deaths occurred. INTERPRETATION: Nitisinone 10 mg daily was well tolerated and effective in reducing urinary excretion of HGA. Nitisinone decreased ochronosis and improved clinical signs, indicating a slower disease progression. FUNDING: European Commission Seventh Framework Programme.

Alkaptonuria: Current Perspectives.

The last 15 years have been the most fruitful in the history of research on the metabolic disorder alkaptonuria (AKU). AKU is caused by a deficiency of homogentisate dioxygenase (HGD), the enzyme involved in metabolism of tyrosine, and is characterized by the presence of dark ochronotic pigment in the connective tissue that is formed, due to high levels of circulating homogentisic acid. Almost 120 years ago, Sir Archibald Garrod used AKU to illustrate the concept of Mendelian inheritance in man. In January 2019, the phase III clinical study SONIA 2 was completed, which tested the effectiveness and safety of nitisinone in the treatment of AKU. Results were positive, and they will serve as the basis for the application for registration of nitisinone for treatment of AKU at the European Medicines Agency. Therefore, AKU might become a rare disease for which a cure will be found by 2020. We understand the natural history of the disease and the process of ochronosis much more, but at the same time there are still unanswered questions. One of them is the issue of the factors influencing the varying severity of the disease, since our recent genotype-phenotype study did not show that differences in residual homogentisic acid activity caused by the different mutations was responsible. Although nitisinone has proved to arrest the process of ochronosis, it has some unwanted effects and does not cure the disease completely. As such, enzyme replacement or gene therapy might become a new focus of AKU research, for which a novel suitable mouse model of AKU is available already. We believe that the story of AKU is also a story of effective collaboration between scientists and patients that might serve as an example for other rare diseases.

Homogentisic acid is not only eliminated by glomerular filtration and tubular secretion but also produced in the kidney in alkaptonuria.

The clinical effects of alkaptonuria (AKU) are delayed and ageing influences disease progression. Morbidity of AKU is secondary to high circulating homogentisic acid (HGA) and ochronosis. It is not known whether HGA is produced by or processed in the kidney in AKU. Data from AKU patients from four studies were merged to form a single AKU group. A control group of non-AKU subjects was generated by merging data from two non-AKU studies. Data were used to derive renal clearance and fractional excretion (FE) ratios for creatinine, HGA, phenylalanine (PHE) and tyrosine (TYR) using standard calculations, for comparison between the AKU and the control groups. There were 225 AKU patients in the AKU group and 52 in the non-AKU control group. Circulating HGA increased with age (P < 0.001), and was significantly associated with decreased HGA clearance (CLHGA ) (P < 0.001) and FEHGA (P < 0.001). CLHGA and FEHGA were increased beyond the theoretical maximum renal plasma flow, confirming renal production and emphasising the greater contribution of net tubular secretion than glomerular filtration to renal elimination of HGA. The kidneys are crucial to elimination of HGA. Elimination of HGA is impaired with age resulting in worsening disease over time. The kidney is an important site for production of HGA. Tubular secretion of HGA contributes more to elimination of HGA in AKU than glomerular filtration.

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.

A new integrated and interactive tool applicable to inborn errors of metabolism: Application to alkaptonuria.

This paper describes our experience with the development and implementation of a database for the rare disease Alkaptonuria (AKU, OMIM: 203500). AKU is an autosomal recessive disorder caused by a gene mutation leading to the accumulation of homogentisic acid (HGA). Analogously to other rare conditions, currently there are no approved biomarkers to monitor AKU progression or severity. Although some biomarkers are under evaluation, an extensive biomarker analysis has not been undertaken in AKU yet. In order to fill this gap, we gained access to AKU-related data that we carefully processed, documented and stored in a database, which we named ApreciseKUre. We undertook a suitable statistical analysis by associating every couple of potential biomarkers to highlight significant correlations. Our database is continuously updated allowing us to find novel unpredicted correlations between AKU biomarkers and to confirm system reliability. ApreciseKUre includes data on potential biomarkers, patients' quality of life and clinical outcomes facilitating their integration and possibly allowing a Precision Medicine approach in AKU. This framework may represent an online tool that can be turned into a best practice model for other rare diseases.

ApreciseKUre: an approach of Precision Medicine in a Rare Disease.

BACKGROUND: Alkaptonuria (AKU; OMIM:203500) is a classic Mendelian genetic disorder described by Garrod already in 1902. It causes urine to turn black upon exposure to air and also leads to ochronosis as well as early osteoarthritis. Our objective is the implementation of a Precision Medicine (PM) approach to AKU. We present here a novel ApreciseKUre database facilitating the collection, integration and analysis of patient data in order to create an AKU-dedicated "PM Ecosystem" in which genetic, biochemical and clinical resources can be shared among registered researchers. In order to exploit the ApreciseKUre database, we developed an analytic method based on Pearson's correlation coefficient and P value that generates as refreshable correlation matrix. A complete statistical analysis is obtained by associating every pair of parameters to examine the dependence between multiple variables at the same time. SHORT CONCLUSIONS: Employing this analytic approach, we showed that some clinically used biomarkers are not suitable as prognostic biomarkers in AKU for a more reliable patients' clinical monitoring. We believe this database could be a good starting point for the creation of a new clinical management tool in AKU, which will lead to the development of a deeper knowledge network on the disease and will advance its treatment. Moreover, our approach can serve as a personalization model paradigm for other inborn errors of metabolism or rare diseases in general.

The first Slovak Legius syndrome patient carrying the SPRED1 gene mutation.

Autosomal dominant disorder Legius syndrome (NF1- like syndrome) shows phenotype features that overlap with neurofibromatosis type 1 (NF1), such as CALMs, freckling, macrocephaly and learning disability. Mutation analysis provides an important tool in order to distinguish two entities that have different clinical implications. We analyzed SPRED1 gene by cDNA and/or gDNA sequencing in a cohort of 46 Slovak patients in whom previously NF1 mutation was excluded. In one case we identified a nonsense mutation c.46C>T (p.Arg16*) in exon 2 of SPRED1 gene, confirming diagnosis of Legius syndrome. This mutation was reported previously.

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.

Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.

BACKGROUND: Alkaptonuria (AKU) is a serious genetic disease characterised by premature spondyloarthropathy. Homogentisate-lowering therapy is being investigated for AKU. Nitisinone decreases homogentisic acid (HGA) in AKU but the dose-response relationship has not been previously studied. METHODS: Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1) was an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study. The primary objective was to investigate the effect of different doses of nitisinone once daily on 24-h urinary HGA excretion (u-HGA24) in patients with AKU after 4 weeks of treatment. Forty patients were randomised into five groups of eight patients each, with groups receiving no treatment or 1 mg, 2 mg, 4 mg and 8 mg of nitisinone. FINDINGS: A clear dose-response relationship was observed between nitisinone and the urinary excretion of HGA. At 4 weeks, the adjusted geometric mean u-HGA24 was 31.53 mmol, 3.26 mmol, 1.44 mmol, 0.57 mmol and 0.15 mmol for the no treatment or 1 mg, 2 mg, 4 mg and 8 mg doses, respectively. For the most efficacious dose, 8 mg daily, this corresponds to a mean reduction of u-HGA24 of 98.8% compared with baseline. An increase in tyrosine levels was seen at all doses but the dose-response relationship was less clear than the effect on HGA. Despite tyrosinaemia, there were no safety concerns and no serious adverse events were reported over the 4 weeks of nitisinone therapy. CONCLUSIONS: In this study in patients with AKU, nitisinone therapy decreased urinary HGA excretion to low levels in a dose-dependent manner and was well tolerated within the studied dose range. TRIAL REGISTRATION NUMBER: EudraCT number: 2012-005340-24. Registered at ClinicalTrials.gov: NCTO1828463.

Mutation screening of the HGD gene identifies a novel alkaptonuria mutation with significant founder effect and high prevalence.

Alkaptonuria (AKU) is an autosomal recessive disorder; caused by the mutations in the homogentisate 1, 2-dioxygenase (HGD) gene located on Chromosome 3q13.33. AKU is a rare disorder with an incidence of 1: 250,000 to 1: 1,000,000, but Slovakia and the Dominican Republic have a relatively higher incidence of 1: 19,000. Our study focused on studying the frequency of AKU and identification of HGD gene mutations in nomads. HGD gene sequencing was used to identify the mutations in alkaptonurics. For the past four years, from subjects suspected to be clinically affected, we found 16 positive cases among a randomly selected cohort of 41 Indian nomads (Narikuravar) settled in the specific area of Tamil Nadu, India. HGD gene mutation analysis showed that 11 of these patients carry the same homozygous splicing mutation c.87 + 1G > A; in five cases, this mutation was found to be heterozygous, while the second AKU-causing mutation was not identified in these patients. This result indicates that the founder effect and high degree of consanguineous marriages have contributed to AKU among nomads. Eleven positive samples were homozygous for a novel mutation c.87 + 1G > A, that abolishes an intron 2 donor splice site and most likely causes skipping of exon 2. The prevalence of AKU observed earlier seems to be highly increased in people of nomadic origin.