In-Silico analysis of missense SNPs in Human HPPD gene associated with Tyrosinemia type III and Hawkinsinuria

Highlights

• HPPD gene codes a dioxygenase enzyme involved in catalysis of different molecules such as tyrosine and phenylalanine by oxidizing them to produce energy.

• To identify the functional missense SNPs of HPPD gene by using multiple computational tools.

• Narrowing down missense SNPs which are still not confirmed experimentally and demands the confirmation by GWAS data.

• Thus, these missense SNPs could directly or indirectly destabilize the amino acid interactions causing functional deviations of protein.

Abstract

HPPD gene codes a dioxygenase enzyme involved in catalysis of different molecules such as tyrosine and phenylalanine by oxidizing them to produce energy. A single change in protein can trigger serious genetic disorders like Tyrosinemia type III and Hawkinsinuria. This study aims to identify the functional missense SNPs of the HPPD gene by using multiple computational tools. All deleterious missense SNPs retrieved from Ensembl and OMIM database were evaluated through six different software. Ultimately, out of 148 missense SNPs, only 27 were confirmed as diseasecausing SNPs by developing a consensus approach. These damaging SNPs were further examined to evaluate their impact on protein stability and energy including their evolutionary conservation. Native and mutated proteins structures were also designed and superimposed by I-TASSER and PyMol respectively. This work results in narrowing down missense SNPs which are still not confirmed experimentally and demands the confirmation by GWAS data. Thus, these missense SNPs could directly or indirectly destabilize the amino acid interactions causing functional deviations of protein.

Introduction

Single Nucleotide Polymorphism (SNP) is a genetic variation in >1% of the population and the most common source of genetic polymorphism in the human genome (Liu, 2007). Previously, their susceptibility role to cause disease such as thalassemia, sickle cell anemia etc. has been reported (Lettre et al., 2008). These point variations can be divided into coding, non-coding, and intergenic SNPs. Further, coding SNPs are categorized into synonymous (nucleotide change does not affect the protein sequence) and non-synonymous, (change of nucleotide results in an amino acid change in the protein sequence) and the later also known as missense SNPs are considered important for disease point of view. These can alter proteins functions by reducing proteins solubility, destabilizing the proteins structures and expression (Barroso et al., 1999; Chasman & Adams, 2001).

4-Hydroxyphenylpyruvate dioxygenase (HPPD), a non-heme oxygenase is a dimer of subunits and a key enzyme in the catabolism of tyrosine and phenylalanine by catalyzing the 4-hydroxyphenylpyruvate to homogentisate (Hausinger, 2004; Moran, 2005). It typically requires alpha-keto acid and molecular oxygen for oxidation, decarboxylation and rearrangement of the molecules (Hausinger, 2004). This conversion is critical to form fumarate and acetoacetate, molecules used to produce energy, at the end of a series of reactions (Lerner, 2009). In eukaryotes, HPPD plays a role in regulating blood tyrosine levels and in the plant, utilize to produce tocopherol and plastoquinone, essential for plant survival (Mercer, 1998). In plants, some of its inhibitors are already reported and commercialized as herbicides playing important role in crop selectivity (Ndikuryayo et al., 2017; Wang et al., 2015; Wang et al., 2016). However, defects in human HPPD gene lead to severe disorders in the body such as Tyrosinemia type III and Hawkinsinuria (Rüetschi et al., 2000). Although both these disorders are rare with prevalence <1/1000000 but lead to intellectual disability, seizure, the high acid level in body and coordination loss (Tomoeda et al., 2000). However, the prevalence might be higher than expected due to the increasing observation of high degrees of tyrosine levels in newborn babies (Najafi et al., 2018).

Tyrosinemia type III, an autosomal recessive disorder, can be caused by inefficiency in the activity of HPPD enzyme as a consequence blood tyrosine level along with its derivatives such as 4-hydroxyphenylpyruvate, 4-hydroxyphenyllactic acid, and 4-hydroxyphenylacetic acid elevate resulting in mental retardation and convulsion (Rüetschi et al., 2000). In addition, these epoxides can react with either glutathione to form Hawkinsin or with water to form hydroxy cyclohexyl acetic acids (Moran, 2005). This hawkinsin are the characteristics of Hawkinsinuria disease (Lehnert et al., 1999; Wilcken et al., 1981). Because of the central role in converting the aforementioned derivatives, it’s necessary to highlight the important SNPs that can alter the HPPD function.

Several polymorphisms have been found in regulatory, intergenic and coding regions of the HPPD gene. Among them, rs4760099 and rs1916333 are noted in the intergenic and 5-prime-UTR region (Raffler et al., 2015). These both are associated to cause urine problems by affecting the activity of HPPD. Transitions such as A-G and G-A at position 875 and 97 in HPPD leads to the development of Tyrosinemia type III and Hawkinsinuria disorders (Rüetschi et al., 2000; Tomoeda et al., 2000). Taking these considerations and the key role played by the HPPD gene, the present study aimed to anticipate the outcomes of the diseaserelated missense SNPs through bioinformatics tools that may cause disease susceptibility.