Twelve novel HGD gene variants identified in 99 alkaptonuria patients: focus on ‘black bone disease’ in Italy

Abstract

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.557T4A), Slovakia (c.500C4T) and France (c.440T4C), three in patients from India (c.469+6T4C, c.650-85A4G, c.158G4A), and six in patients from Italy (c.742A4G, c.614G4A, c.1057A4C, c.752G4A, c.119A4C, c.926G4T). 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 pathogenic missense mutations using protein stability predictors

“…Although computational stability predictors have not been specifically designed to identify pathogenic mutations, they are very commonly used when assessing candidate disease mutations. For example, publications reporting novel variants will often include the output of stability predictors as evidence in support of pathogenicity [22][23][24][25] . This relies essentially upon the assumption that the molecular mechanism underlying many or most pathogenic mutations is directly related to the structural destabilisation of protein folding or interactions [26][27][28][29] .…”

Demographic, Phenotypic and Genotypic Features of Alkaptonuria Patients: A Single Centre Experience

“…Recently, a high number of AKU cases were also found in Jordan (23) and India (18). Nemethova et al (24) reported 99 AKU patients with 12 novel mutations from Italy. In our study, 8 different AKU mutations with two new mutations were identified.…”

Combating mutations in genetic disease and drug resistance: understanding molecular mechanisms to guide drug design

“…Two comprehensive analysis on AKU causing mutations were carried out in an attempt to characterize the potential molecular mechanisms on which mutations could disruption enzyme activity 17,18 .…”