A rapid method for detection of five known mutations associated with aminoglycoside-induced deafness

Abstract

Background: South Africa has one of the highest incidences of multidrug-resistant tuberculosis (MDR-TB) in the world. Concomitantly, aminoglycosides are commonly used in this country as a treatment against MDR-TB. To date, at least five mutations are known to confer susceptibility to aminoglycoside-induced hearing loss. The aim of the present study was to develop a rapid screening method to determine whether these mutations are present in the South African population.

Methods: A multiplex method using the SNaPshot technique was used to screen for five mutations in the MT-RNR1 gene: A1555G, C1494T, T1095C, 961delT+C(n) and A827G. A total of 204 South African control samples, comprising 98 Mixed ancestry and 106 Black individuals were screened for the presence of the five mutations.

Results: A robust, cost-effective method was developed that detected the presence of all five sequence variants simultaneously. In this pilot study, the A1555G mutation was identified at a frequency of 0.9% in the Black control samples. The 961delT+C(n) variant was present in 6.6% of the Black controls and 2% of the Mixed ancestry controls. The T1095C, C1494T and A827G variants were not identified in any of the study participants.

Conclusion: The frequency of 0.9% for the A1555G mutation in the Black population in South Africa is of concern given the high incidence of MDR-TB in this particular ethnic group. Future larger studies are warranted to determine the true frequencies of the aminoglycoside deafness mutations in the general South African population. The high frequencies of the 961delT+C(n) variant observed in the controls suggest that this change is a common non-pathogenic polymorphism. This genetic method facilitates the identification of individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This is important in a low-resource country like South Africa where, despite their adverse side-effects, aminoglycosides will continue to be used routinely and are accompanied with very limited or no audiological monitoring.

Figure 1

SNaPshot analysis of the five mutations [C1494T, T1095C, A1555G, 961delT+C(n) and A827G]. Peaks are shown that represent wild-type alleles, for one individual, at all five loci. The smaller non-specific peaks in the figure are all either below a certain threshold peak height value or do not fall into specific bins and are therefore not genotyped by the GeneMapper software. * The SNaPshot extension primers for 961 delT+C(n) and A827G are in the reverse orientation. In the SNaPshot figure, the genotypes are represented as follows: For C1494T, either a black peak (representing the C allele) or a red peak (T allele) is present, in their respective bin positions. For T1095C, either a red peak (T allele) or a black peak (C allele) is present, in their respective bin positions. For A1555G, either a green peak (A allele) or a blue peak (G allele) is present, in their respective bin positions. For 961delT+C(n) (as the primer anneals to the reverse strand), either a green peak (A allele; T on forward strand) or a blue peak (G allele; C on forward strand) is present, in their respective bin positions. For A827G (as the primer anneals to the reverse strand), either a red peak (T allele; A on forward strand) or a black peak (C allele; G on forward strand) is present, in their respective bin positions.

Figure 2

SNaPshot analysis of an individual who harbours wild-type alleles at four of the loci and the mutant allele (delT) for 961delT+C(n). The five peaks each represent an allele as described in detail in the legend to Figure 1. * The SNaPshot extension primers for 961 delT+C(n) and A827G are in the reverse orientation.

Figure 3

Sequence alignments of the MT-RNR1 gene across diverse species indicating that the A1555, A827, T1095 and C1494 alleles are all evolutionarily conserved but that the T allele at 961 is not.

Figure 4

Representative sequencing results confirming the presence of the 961delT+C(n) variant as detected by SNaPshot analysis.