Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is currently facing a major multidrug-resistant tuberculosis (MDR-TB) epidemic and
has one of the highest incidences in the world. Aminoglycoside antibiotics are commonly used in
this country as a treatment against MDR-TB. A well known side-effect of aminoglycosides is
permanent hearing loss and this is thought to have a significant genetic component. To date, at least
six mutations in the mitochondrial genome are known to confer susceptibility to aminoglycosideinduced
hearing loss. It is imperative that we investigate the frequency of these mutations in our
populations and determine whether certain sub-groups are at increased risk. The aim of the present
study was therefore to investigate the genetic aetiology of aminoglycoside-induced hearing loss in
the South African population.
A multiplex method using the ABI Prism® SNaPshotTM Multiplex system was optimised to screen
for 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 were
receiving high doses of either streptomycin, kanamycin or capreomycin were recruited for this
study. Furthermore, 439 control samples, comprising of 93 Afrikaner, 104 Caucasian, 112 Black
and 130 Mixed Ancestry individuals were recruited and screened for the presence of the six
mutations. Identification of novel variants in the MT-RNR1 and the entire mitochondrial genome
was performed using High Resolution Melt analysis (HRM) and whole mitochondrial DNA
sequencing, respectively. A total of 97 family members from a South African family known to
harbour the A1555G mutation were recruited and genotyped using SNaPshot analysis. In addition,
mitochondrial functioning in the presence of different streptomycin drug concentrations, in
transformed lymphoblasts of an individual harbouring the A1555G, was assessed by means of the
MTT colorimetric assay. Detection of heteroplasmic mutations was performed using PCRRestriction
Fragment Length Polymorphism (RFLP) analysis and UN-SCAN-IT software.
We successfully developed a robust and cost-effective method that detects the presence of all six
mutations simultaneously. The method worked equally well on both blood (from adults) and buccal
swabs (from children). The C1494T, T1095C and T1291C mutations were not detected in any of
the MDR-TB or control groups. Alarmingly, the A1555G mutation was detected in 0.9% of the
Black control samples and in 1.1% of the Afrikaner controls (in one sample in the heteroplasmic
state 25%). The A827G mutation was present at a frequency of 0.9% in the MDR-TB patients and
in 1.1% of the Afrikaner controls. The 961delT + insC(n) mutation was found in relatively high
frequencies 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 was
III
detected at high frequencies in the Caucasian (2.9%) and Afrikaner (3.2%) controls. Screening for
novel variants in MT-RNR1 in MDR-TB patients experiencing ototoxicity revealed two novel
variants (G719A and T1040C). However, G719A and T1040C are not likely to be pathogenic since
they 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 eight
MDR-TB patients with ototoxicity. Only five of the 50 variants (one in the MT-TH, ND3, COX3
and two in the CYTB gene) were shown to reside at positions that are evolutionarily conserved
across five species from human to frog, and the four variants in the protein coding genes resulted in
missense 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 irreversible
hearing loss. Genes and variants known to act as genetic modifiers: tRNASer(UCN), homozygous
A10S in TRMU and 35delG in GJB2 were not present in this family. For the MTT assay, decreased
mitochondrial functioning of cells harbouring the A1555G mutation in the presence of streptomycin
were (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 of
concern given the high incidence of MDR-TB in this particular ethnic group. However, future
studies with larger numbers of samples are warranted to determine the true frequencies of the
aminoglycoside deafness mutations in the general South African population. Our data suggests that
the 961delT + insC(n) and T961G variants are common non-pathogenic polymorphisms due to the
high frequencies observed in controls (>1%). The identification of the first novel variants within
protein coding genes that could possibly be associated with aminoglycoside-induced hearing loss
holds great possibilities with regards to the identification of a second gene involved in drug induced
hearing loss. Future studies where the possible effect of these variants on the normal functioning of
these genes could be assessed would contribute greatly to this field of research. All 76 A1555Gpositive
members of the family were given genetic reports and counseled about their risk and that of
their children for developing hearing loss due to aminoglycoside use.
The development of a rapid and cost-effective genetic method facilitates the identification of
individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This
is 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 very
limited or no audiological monitoring. Future studies and greater public awareness is therefore
needed to address this serious problem. / AFRIKAANSE OPSOMMING: Suid Afrika beleef tans „n grootskaalse tuberculose epidemie (veral weerstandige vorme van
tuberculose) (MDR-TB), met een van die hoogste voorkomssyfers in die wêreld. Aminoglikosied
antibiotikums word baie algemeen gebruik in Suid Afrika vir die behandeling van MDR-TB. ‟n
Bekende newe effek van die middels is permanente gehoor verlies en dit is van mening dat dit
gekoppel is aan „n genetiese component. Daar is tans ses mutasies in die mitochondriale genoom
wat vatbaarheid tot aminoglikosied-geinduseerde gehoor verlies veroorsaak. Daarom is dit van
uiterse belang dat die frekwensie van die mutasies in ons populasies bepaal word sodat daar
vasgestel kan word watter groepe „n hoë risiko het om gehoor verlies te kan ontwikkel.
Die ABI Prism® SNaPshotTM Multipleks sisteem is gebruik en geoptimiseer om te toets vir die ses
mutasies in die MT-RNR1: C1494T, T1095C, 961delT+C(n), A827G and T1291C. „n Totaal van 115
MDR-TB pasiente van die Brooklyn Chest Hospital in Kaap Stad is gewerf vir die studie. Hierdie
pasiente ontvang daaglikse hoë dosese van een van die volgende aminoglikosiede: streptomycin,
kanamycin of capreomycin. Verder is „n totaal van 439 kontrole DNA monsters gewerf vanuit die
volgende etniese groepe: 93 Afrikaner, 104 Blank, 112 Swart and 130 Kleurling. Hierdie monsters
is ook getoets vir die ses mutatsies. Hoë Resolusie Smelt analise (HRS) is gebruik om nuwe DNS
volgorde veranderinge in die MT-RNR geen te identifiseer. Die hele mitochondriale genoom is
blootgestel aan DNA volgorde bepaling in „n poging om nuwe DNS volgorde verandering in die
genoom te identifiseer wat moontlik betrokke kan wees by aminoglikosied-geinduseerde gehoor
verlies. „n Total van 97 lede van „n Suid Afrikaanse familie waar die A1555G mutasie teenwoordig
is, is deur middle van die SNaPshot metode gegenotipeer. Verder is die normale funcitoneering van
die mitochondrion in getransformeerde witbloed selle, getoets in die teenwoordigheid van
verskillende konsentrasies streptomycin met behulp van die MTT kleurmetrie toets. Deteksie van
heteroplasmiese mutasies is gedoen deur middle van die PCR-RFLP tegniek en alle analises is
gedoen op die UN-SCAN-IT program.
Ons was suksesvol in die ontwikkeling van „n vinnige, koste effektiewe en kragtige tegniek wat al
ses die mutasies in MT-RNR1 in een reaksie kan optel. Hierdie tegniek het goed gewerk met DNA
monsters van bloed en van selle verkry vanuit die wangholte (geneem van kinders jonger as 12 jaar).
Die C1494T, T1095C en T1291C mutasies is glad nie waargeneem in enige van ons MDR-TB
patiente of kontroles nie. Skrikwekkend is die hoë frekwensie (0.9%) waarby die A1555G mutasie
in die Swart kontrole groep waargeneem is. Hierdie mutasie is ook in 1.1% van die Afrikaner
kontrole groep opgemerk in heteroplasmie van 25%. Die A827G mutasie was teenwoordig in 0.9%
en 1.1% van die MDR-TB patiente en Afrikaner kontrole monsters, onerskeidelik. Die 961delT +
insC(n) mutasie is opgemerk in baie hoë frekwensies in beide die MDR-TB (3.5%) en kontrole
groepe (1.1% van die Afrikaner, 1.5% van die Kleurling en 7.1% van die Swart monsters). Die
T961G mutasie is ook in hoë frekwensies in slegs die Blanke (2.9%) en die Afrikaner (3.2%)
kontrole groepe waargeneem. Nuwe DNS volgorde veranderinge in MT-RNR1 is gesoek in „n groep
MDR-TB patiente wat gehoor verlies ondervind. Slegs twee nuwe verandering is ontdek (G719A en
T1040C). Dit is onwaarskynlik dat hierdie veranderinge patogenies is siende dat hulle teen
frekwensies van 20.7% en 1.8% waargeneem is in die Kleurling en Swart kontrole groepe
onderskeidelik. Tydens die soeke na nuwe DNS volgorde veranderinge wat moontlik geassosieer is
met aminoglikosied-geinduseerde gehoor verlies in die mitochondriale genoom is 50 onbekende
veranderinge ontdek (een in die MT-TH, ND3, COX3 en twee in die CYTB gene). Die veranderinge
is verder ondersoek vir evolusionêre konservasie op beide die nukliotied en amino suur vlak van
mens to padda. Dit is bevind dat 76 uit die 97 familie lede positief is vir die A1555G mutasie en het
dus „n hoë risiko om aminoglikosied-geinduseerde gehoor verlies te ontwikkel as hul bloot gestel
word aan hierdie antibiotikums. Verder is gevind dat hierdie familie op die L0d mitochondriale
haplogroep lê. Geen van die sogenaamde genetiese modifiseerde gene of DNS volgorde
veranderinge in hierdie gene (tRNASer(UCN), A10S in TRMU in homosigotiese vorm en die 35delG in
GJB2) is gevind in die familie nie. Die MTT toets het „n afname in die mitochondriale
funksioneering van selle waar die A1555G mutasie teenwoordig was getoon, alhoewel die verskil
tussen selle wat nie die A1555G mutasie het nie, nie statisties betekenisvol was nie (p-waarde:
0.615-0.999).
Die hoë frekwensie van die A1555G mutasie (0.9%) in die Swart populasie van Suid Afrika is
skrikwekkend siende dat die voorkomssyfer van MDR-TB in hierdie groep baie hoog is.
Toekomstige studies met grooter getalle is nodig om die ware frekwensie van die mutasies
geassosieer met aminoglikosied-geinduseerde gehoor verlies in die algemende Suid Afrikaanse
populasie te bepaal. Ons data dui aan dat die 961delT + insC(n) en die T961G mutasies slegs
algemene nie-patogeniese polimorphismis is siende dat dit in sulke hoë frekwensies (>1%) in
kontroles opgemerk is. Die identifiseering van die eerste DNS volgorde veranderinge in proteïen
kodeerende gene wat moontlik geassosieer is met aminoglikosied-geinduseerde gehoor verlies hou
groot en belowende moontlikehede in, interme van die identifiseering van „n tweede geen.
Toekomstige studies waarin die effek van hierdie veranderinge op die normale funktioneering van
hierdie gene ondersoek word sal „n besondere groot bydrae lewer tot hierdie veld van navorsing. Al
76 van die A1555G positiewe familie lede is voorsien van genetiese verslae en het berading ontvang
in verband met hul risiko en die risiko van hul kinders om aminoglikosied-geinduseerde gehoor
verlies te ontwikkel.
Die ontwikkeling van „n kragtige, vinnige en koste-effektiewe genetiese metode vergemaklik die
vinnige identifiseering van hoë risiko individue vir die ontwikkeling van gehoor verlies voordat
hulle met hul aminoglikosiede behandeling begin. Dit is veral noodsaaklik in „n derde wêreld land
soos Suid Afrika waar, ten spyte van hul gevaarlike newe effekte, aminoglikosied antibiotikums
steeds gebruik sal word. Daarom is grooter publieke bewusmaking nodig om hierdie problem te
probeer oplos en te verhoed.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/3351 |
| Date | 12 1900 |
| Creators | Human, Hannique |
| Contributors | Bardien, Soraya, De Jong, Greetje, University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Stellenbosch |
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