Investigation of the genetic aetiology of aminoglycoside-induced hearing loss in South African populations

[摘要] ENGLISH ABSTRACT: South Africa is currently facing a major multidrug-resistant tuberculosis (MDR-TB) epidemic andhas one of the highest incidences in the world. Aminoglycoside antibiotics are commonly used inthis country as a treatment against MDR-TB. A well known side-effect of aminoglycosides ispermanent hearing loss and this is thought to have a significant genetic component. To date, at leastsix mutations in the mitochondrial genome are known to confer susceptibility to aminoglycosideinducedhearing loss. It is imperative that we investigate the frequency of these mutations in ourpopulations and determine whether certain sub-groups are at increased risk. The aim of the presentstudy was therefore to investigate the genetic aetiology of aminoglycoside-induced hearing loss inthe South African population.A multiplex method using the ABI Prism® SNaPshotTM Multiplex system was optimised to screenfor six mutations in the MT-RNR1: A1555G, C1494T, T1095C, 961delT+C(n), A827G and T1291C.A total of 115 MDR-TB patients from the Brooklyn Chest Hospital in Cape Town who werereceiving high doses of either streptomycin, kanamycin or capreomycin were recruited for thisstudy. Furthermore, 439 control samples, comprising of 93 Afrikaner, 104 Caucasian, 112 Blackand 130 Mixed Ancestry individuals were recruited and screened for the presence of the sixmutations. Identification of novel variants in the MT-RNR1 and the entire mitochondrial genomewas performed using High Resolution Melt analysis (HRM) and whole mitochondrial DNAsequencing, respectively. A total of 97 family members from a South African family known toharbour the A1555G mutation were recruited and genotyped using SNaPshot analysis. In addition,mitochondrial functioning in the presence of different streptomycin drug concentrations, intransformed lymphoblasts of an individual harbouring the A1555G, was assessed by means of theMTT colorimetric assay. Detection of heteroplasmic mutations was performed using PCRRestrictionFragment Length Polymorphism (RFLP) analysis and UN-SCAN-IT software.We successfully developed a robust and cost-effective method that detects the presence of all sixmutations simultaneously. The method worked equally well on both blood (from adults) and buccalswabs (from children). The C1494T, T1095C and T1291C mutations were not detected in any ofthe MDR-TB or control groups. Alarmingly, the A1555G mutation was detected in 0.9% of theBlack control samples and in 1.1% of the Afrikaner controls (in one sample in the heteroplasmicstate 25%). The A827G mutation was present at a frequency of 0.9% in the MDR-TB patients andin 1.1% of the Afrikaner controls. The 961delT + insC(n) mutation was found in relatively highfrequencies in both the MDR-TB patients (3.5%) and control groups (1.1% of the Afrikaner, 1.5%of the Mixed Ancestry and 7.1% of the Black samples). Similarly, the T961G mutation wasIIIdetected at high frequencies in the Caucasian (2.9%) and Afrikaner (3.2%) controls. Screening fornovel variants in MT-RNR1 in MDR-TB patients experiencing ototoxicity revealed two novelvariants (G719A and T1040C). However, G719A and T1040C are not likely to be pathogenic sincethey were detected in ethnic-matched controls: Mixed Ancestry (20.7%) and Black (1.8%) controls.Furthermore, a total of 50 novel variants were identified within the mitochondrial genome of eightMDR-TB patients with ototoxicity. Only five of the 50 variants (one in the MT-TH, ND3, COX3and two in the CYTB gene) were shown to reside at positions that are evolutionarily conservedacross five species from human to frog, and the four variants in the protein coding genes resulted inmissense changes. A total of 76 of the 97 family members recruited were found to be A1555Gpositive(on mitochondrial haplogroup L0d) and are therefore at risk of developing irreversiblehearing loss. Genes and variants known to act as genetic modifiers: tRNASer(UCN), homozygousA10S in TRMU and 35delG in GJB2 were not present in this family. For the MTT assay, decreasedmitochondrial functioning of cells harbouring the A1555G mutation in the presence of streptomycinwere (compared to wild type) observed but this was not statistically significant (p-value: 0.615-0.999).The high frequency of the A1555G mutation (0.9%) in the Black population in South Africa is ofconcern given the high incidence of MDR-TB in this particular ethnic group. However, futurestudies with larger numbers of samples are warranted to determine the true frequencies of theaminoglycoside deafness mutations in the general South African population. Our data suggests thatthe 961delT + insC(n) and T961G variants are common non-pathogenic polymorphisms due to thehigh frequencies observed in controls (>1%). The identification of the first novel variants withinprotein coding genes that could possibly be associated with aminoglycoside-induced hearing lossholds great possibilities with regards to the identification of a second gene involved in drug inducedhearing loss. Future studies where the possible effect of these variants on the normal functioning ofthese genes could be assessed would contribute greatly to this field of research. All 76 A1555Gpositivemembers of the family were given genetic reports and counseled about their risk and that oftheir children for developing hearing loss due to aminoglycoside use.The development of a rapid and cost-effective genetic method facilitates the identification ofindividuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. Thisis of critical important in a low-resource country like South Africa where, despite their adverse sideeffects,aminoglycosides will be continue to be used routinely and are accompanied with verylimited or no audiological monitoring. Future studies and greater public awareness is thereforeneeded to address this serious problem.

[发布机构] Stellenbosch University