Investigation of mitochondrial sequence variants associated with aminoglycoside-induced ototoxicity in South African TB patients on aminoglycosides

Abstract

A known side effect of aminoglycoside antibiotics is the development of permanent hearing loss. As South Africa is currently facing a tuberculosis (TB) epidemic, with an increasing number of multi-drug resistant tuberculosis (MDR-TB) infections, the use of aminoglycosides is on the increase. It is therefore important to determine whether the mitochondrial mutations associated with aminoglycoside-induced hearing loss occur at high frequencies in particular ethnic groups in our population. A total of 115 mainly MDR-TB patients all on aminoglycosides and 439 controls representative of the main ethnic groups in South Africa were screened for six mutations using the SNaPshot technique. Furthermore, the mitochondrial genomes of eight patients with ototoxicity were sequenced. Homoplasmic mutations were found in controls (A1555G in 0.9% of Black controls and A827G in 1.1% of Afrikaner controls) which reveal that a significant proportion of the South African population is genetically predisposed to developing aminoglycoside-induced hearing loss. The 961delT+insC_(n) and T961G variants were found at frequencies of >1% indicating that both are probably non-pathogenic polymorphisms. Sequencing of the entire mitochondrial genome in eight patients did not reveal any mutations in the MT-RNR1 gene. However, two potentially pathogenic variants, T10114C (I19T in MT-ND3) and T15312C (I189T in MT-CYB) were found that may impact on the oxidative phosphorylation capacity and warrant further investigation for their possible role in this disorder. It is imperative that the genetic basis of this potentially preventable condition be investigated, particularly in countries where aminoglycosides are still commonly used, in order to identify individuals and/or ethnic groups who are at risk for this type of hearing loss.

Introduction

South Africa is currently facing a major tuberculosis (TB) and multi-drug resistant tuberculosis (MDR-TB) epidemic, and has been ranked 4th among the 22 high-burden TB countries worldwide [1]. MDR-TB, defined as the resistance of Mycobacterium tuberculosis (Mtb) bacillus to at least Isoniazid and Rifampacin, which are the two most powerful first-line drugs, is thought to be a ‘man made’ problem [2]. In the majority of cases, MDR-TB emerges when either a TB patient receives inappropriate treatment which results in the naturally occurring resistant TB bacteria to survive and multiply or because of poor compliance of the TB patient to their treatment. It is estimated that approximately 2.6% of all TB cases are diagnosed as MDR-TB and there are more than 10,000 new MDR-TB cases in South Africa per year [1].

In South Africa, according to the World Health Organization’s (WHO) guidelines, MDR-TB is treated with a number of second-line drugs including the quinolones (Ciprofloxacin, Ofloxacin and Sparfloxacin) and aminoglycosides (Streptomycin, Kanamycin and Amikacin). The second-line drugs are much more costly and more toxic than the first-line drugs [3]. Regardless of the effectiveness of aminoglycosides against bacterial infections, serious toxicity is a major limitation in the usefulness of these drugs [4]. The same spectrum of toxicity is shared by all members of the aminoglycoside family of which the most notable is ototoxicity and nephrotoxicity [5]. Ototoxicity is caused as a result of damage to the outer hair cells in the cochlea and vestibular labyrinth leading to permanent hearing loss in patients hypersensitive to these drugs. Aminoglycoside ototoxicity is recognized by a distinctive pattern of hearing loss starting in the high frequency range (4000–8000 Hz) with lower frequencies only affected later.

Mutations in mitochondrial genes have been associated with increased susceptibility to hearing loss in individuals who receive aminoglycoside therapy. Hearing loss due to aminoglycoside exposure has been identified in a number of families from all over the world [6], [7], [8], [9]. Numerous mutations (including A1555G, C1494T, T1095C, 961delT+insC_(n), T961G, A827G and T1291C) have been identified in the mitochondrial MT-RNR1 gene that encodes the human 12S rRNA ribosomal subunit. It has been shown that the A1555G mutation causes increased binding of aminoglycosides to the 12S rRNA ribosomal subunit [10] which results in the disruption of mitochondrial protein synthesis in the cell. All proteins synthesized in the mitochondria are involved in oxidative phosphorylation and the disruption thereof will ultimately lead to death of the cell.

As the incidence of MDR-TB increases in South Africa, the use of aminoglycosides will also increase. Therefore, there is a need for a rapid genetic screening method that is easy, cost effective and high throughput, so that it may be used in a diagnostic setting. This method can potentially be incorporated into the global TB strategy in order to detect these mutations in MDR-TB patients prior to them starting their aminoglycoside therapy. Recently, we have developed such a method based on the SNaPshot multiplex system that can detect six of these mutations in a single reaction [11], [12]. The application of this rapid screening test in clinical practice would aid in identifying susceptible individuals prior to the start of their aminoglycoside treatment therapy thereby potentially lowering the incidence of aminoglycoside-induced hearing loss in South Africa. There are certain precautionary measures that can be applied to minimize the progression of cochlear and vestibular damage in mutation-positive individuals which include avoidance of drugs with synergistic ototoxic effects, reducing therapy time and regular audiological monitoring throughout treatment.

Currently, there is no known data on the incidence of aminoglycoside-induced hearing loss in South Africa. As aminoglycosides are relatively commonly used here, the population of this country is a unique resource to study both the short and long term effects of these drugs on the auditory system and the genetic etiology of aminoglycoside-induced hearing loss. In the present study we investigated the frequencies of six known MT-RNR1 mutations in a group of MDR-TB patients on aminoglycosides as well as controls, and screened for novel pathogenic mutations in the mitochondrial genome.