HGDiscovery: an online tool providing functional and phenotypic information on novel variants of homogentisate 1,2- dioxigenase

Abstract

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in the body. Affected individuals lack enough functional levels of an enzyme required to breakdown HGA. Mutations in the HGD gene cause AKU and they are responsible for deficient levels of functional homogentisate 1,2-dioxygenase (HGD), which, in turn, leads to excess levels of HGA. Although HGA is rapidly cleared from the body by the kidneys, in the long term it starts accumulating in various tissues, especially cartilage. Over time (rarely before adulthood), it eventually changes the color of affected tissue to slate blue or black. Here we report a comprehensive mutation analysis of 111 pathogenic and 190 non-pathogenic HGD missense mutations using protein structural information. Using our comprehensive suite of graph-based signature methods, mCSM complemented with sequence-based tools, we studied the functional and molecular consequences of each mutation on protein stability, interaction and evolutionary conservation. The scores generated from the structure and sequence-based tools were used to train a supervised machine learning algorithm with 84% accuracy. The empirical classifier was used to generate the variant phenotype for novel HGD missense mutations. All this information is deployed as a user friendly freely available web server called HGDiscovery (http://biosig.unimelb.edu.au/hgdiscovery/).

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

“…AKU is an example of a rare monogenic autosomal-recessive disorder caused by a mutation in the HGD gene coding for the HGD hexameric protein, an enzyme involved in the tyrosine degradation pathway 7 , 34 , 47 . Several mutations within the HGD gene are known to be at the basis of AKU disease onset 14 , 37 , 40 . At present, 251 unique AKU-causing variants have been reported and summarized in the HGD mutation database 36 from which 65% are missense mutations 11 , 17 .…”

“…At present, 251 unique AKU-causing variants have been reported and summarized in the HGD mutation database 36 from which 65% are missense mutations 11 , 17 . So far, the impact of these missense variants on residual HGD enzyme activities has not been quantified yet 37 . With the aim to bridge this knowledge gap, we present here an in vitro HTS system to rank AKU-causing missense variants based upon their ability to consume the oxidation-sensitive substrate HGA and the obtained results indicate that substitutions to positively charged Arg-residues have a stronger HGD activity reducing effect than substitutions to Ala or Val.…”

“…So far, the impact of these missense variants on protein stability, folding and conformational flexibility can be analyzed by biophysical tools ( i.e. mCSM-Stability, DUET and DynaMut) 37 . However, no methods exist to estimate the possible residual activities of HGD missense variants 14 .…”