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.

Thirty-nine novel neurofibromatosis 1 (NF1) gene mutations identified in Slovak patients.

We performed a complex analysis of the neurofibromatosis type 1 (NF1) gene in Slovakia based on direct cDNA sequencing supplemented by multiple ligation dependent probe amplification (MLPA) analysis. All 108 patients had café-au-lait spots, 85% had axilary and/or inguinal freckling, 61% neurofibromas, 36% Lisch nodules of the iris and 31% optic pathway glioma, 5% suffered from typical skeletal disorders, and 51% of patients had family members with NF1. In 78 of the 86 (90.7%) index patients our analysis revealed the presence of NF1 mutations, 68 of which were small changes (87.2%), including 39 (50%) novel. Among the identified mutations the most prevalent were small deletions and insertions causing frameshift (42.3%), followed by nonsense (14.1%), missense (12.8%), and typical splicing (11.5%) mutations. Type 1 NF1 deletions and intragenic deletions/duplication were identified in five cases each (6.4%). Interestingly, in five other cases nontypical splicing variants were found, whose real effect on NF1 transcript would have remained undetected if using a DNA-based method alone, thus underlying the advantage of using the cDNA-based sequencing. We show that Slovak NF1 patients have a similar repertoire of NF1 germline mutations compared to other populations, with some prevalence of small deletions/insertions and a decreased proportion of nonsense mutations.

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.

Identification of 11 Novel Homogentisate 1,2 Dioxygenase Variants in Alkaptonuria Patients and Establishment of a Novel LOVD-Based HGD Mutation Database.

Enzymatic loss in alkaptonuria (AKU), an autosomal recessive disorder, is caused by mutations in the homogentisate 1,2 dioxygenase (HGD) gene, which decrease or completely inactivate the function of the HGD protein to metabolize homogentisic acid (HGA). AKU shows a very low prevalence (1:100,000-250,000) in most ethnic groups, but there are countries with much higher incidence, such as Slovakia and the Dominican Republic. In this work, we report 11 novel HGD mutations identified during analysis of 36 AKU patients and 41 family members from 27 families originating from 9 different countries, mainly from Slovakia and France. In Slovak patients, we identified two additional mutations, thus a total number of HGD mutations identified in this small country is 12. In order to record AKU-causing mutations and variants of the HGD gene, we have created a HGD mutation database that is open for future submissions and is available online ( http://hgddatabase.cvtisr.sk/ ). It is founded on the Leiden Open (source) Variation Database (LOVD) system and includes data from the original AKU database ( http://www.alkaptonuria.cib.csic.es ) and also all so far reported variants and AKU patients. Where available, HGD-haplotypes associated with the mutations are also presented. Currently, this database contains 148 unique variants, of which 115 are reported pathogenic mutations. It provides a valuable tool for information exchange in AKU research and care fields and certainly presents a useful data source for genotype-phenotype correlations and also for future clinical trials.