Gene:

MT-RNR1

mitochondrially encoded 12S RNA

Introduction

Mitochondria are the powerhouses of eukaryotic cells, generating energy in the form of adenosine triphosphate (ATP) through a process known as oxidative phosphorylation. Mitochondria are unique from other organelles within the cell in that they contain their own genetic system, with mitochondrial DNA (mtDNA) that undergoes replication, transcription and translation. Proteins created from mtDNA remain in the mitochondria, where they take part in a range of essential tasks. Mitochondria also contain proteins coded for by nuclear DNA that are imported into the organelle [Cooper (2000)]. The generation of ATP by mitochondria is critical for survival, and genetic mutations that affect mitochondrial function can lead to a variety of serious and incurable diseases. Genetic variations within one particular mitochondrial gene, MT-RNR1, have been strongly linked with the development of hearing loss following administration of aminoglycoside antibiotics.

The mitochondrial genetic system

Mitochondria are ellipsoid organelles, with both an outer and inner membrane. The outer membrane is a simple phospholipid bilayer enclosing the entire mitochondria, while the inner membrane consists of a series of deep folds known as cristae. The proteins mediating oxidative phosphorylation are bound to the inner membrane, with the folds allowing for a larger surface area on which oxidative phosphorylation can take place. The inner membrane encloses the matrix, where the mtDNA and other genetic machinery are located [Alberts (2002)]. The number of mitochondria in a cell varies depending on the cell’s energy requirements, with some cells containing hundreds or even thousands of the organelles [Alberts (2002)].

mtDNA is circular, double-stranded, and exists in multiple copies within the mitochondrion. The number of mtDNA can vary between mitochondria. Since the number of mitochondria varies between cells, copies of mtDNA within a cell can number in the thousands [Article:15861210]. mtDNA consists of only 37 genes, with 13 coding for proteins involved in cellular respiration and 22 coding for transfer RNAs (tRNA), proteins essential for translation. The remaining two genes, MT-RNR1 and MT-RNR2, code for ribosomal RNAs (rRNA) [Cooper (2000)]. rRNA is the main constituent of ribosomes, the organelles in which translation takes place; mitochondria have their own ribosomes as part of their genetic system. Ribosomes and the rRNA within them are designated in svedbergs (S), a measure of the rate of sedimentation during centrifugation that reflects a molecule’s size and shape. Ribosomes are composed of two subunits, referred to as the large (39S) and the small (28S) subunit [Article:25837512][Lodish et al. (2000)]. These two subunits fit together and are involved in protein translation, moving along the mRNA and catalyzing the assembly of amino acids into protein chains [Lodish et al. (2000)]. The MT-RNR1 gene codes for the rRNA present in the small ribosomal subunit, known as 12S rRNA; the MT-RNR2 gene codes for the rRNA present in the large ribosomal subunit, known as 16S rRNA [Article:25837512].

Mitochondrial DNA is maternally inherited: males can inherit a mitochondrial condition, but cannot pass it on to following generations, while females with a mitochondrial condition can pass it on to their children [Article:16815381]. In most individuals, all copies of their mtDNA will be identical, a state known as homoplasmy. However, some individuals may have a mixture of alleles at one or more positions within the mtDNA, a state known as heteroplasmy [Article:15861210] ¹. Heteroplasmy can occur due to a person inheriting heteroplasmic mtDNA from their mother, or due to the accumulation of somatic mutations over time - the process of oxidative phosphorylation produces free radicals that can damage mtDNA, and the mitochondrial DNA repair system is much less effective than its nuclear counterpart [Articles:16815381, 16050976][Genetics Home Reference]. The concept of heteroplasmy is important to consider in the context of mitochondrial disease: individuals with a mitochondrial condition are typically heteroplasmic for damaging mutations within the mtDNA, though since so many copies of mtDNA exist within the body, a certain proportion with a pathogenic genetic variation must exist before the disease develops [Article:15861210]. Though homoplasmy for a highly damaging genetic mutation is possible, such embryos are unlikely to survive past the early development stages, due to the significant impact on energy production [New South Wales Government Health - Centre for Genetics Education].

Mitochondrial disease

Since mitochondria play such a critical role in cellular energy production, any form of genetic variation that affects the normal operation of mitochondria can result in a significant, negative impact on health. Mitochondrial diseases often present with varied clinical phenotypes, though organs and tissues that require large amounts of energy, such as the brain, heart and other muscles, are often affected [Genetics Home Reference]. Myoclonic epilepsy with ragged-red fibers (MERRF) and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), two mitochondrial conditions, both present with a variety of muscular and neurological abnormalities, such as myopathy, cerebellar ataxia, seizures and dementia [Chinnery (2014)]. An additional feature common to both diseases is bilateral deafness [Chinnery (2014)]. Sensory cells in the inner ear known as hair cells are critical for the transduction of sound waves into an electrical signal that can be sent to the brain for interpretation. Since hair cells have a high demand for energy and are rich in mitochondria, hearing loss is a common symptom across many mitochondrial conditions [Article:25792261][Chinnery (2014)]. Indeed, the same variants in the MT-RNR1 gene that are linked with aminoglycoside antibiotic-induced hearing loss are also associated with hearing loss unrelated to antibiotic use.

Anatomy of the ear and hearing loss

The process of hearing involves a complex series of steps in which sound waves are transduced into electrical signals, which are then transmitted via the auditory nerve to the brain for processing. Briefly, the human ear is composed of an outer, middle and inner section. Sound waves enter through the outer ear and travel down the ear canal, striking the eardrum and causing it to vibrate. These vibrations are transmitted to small bones called ossicles in the middle ear. From the ossicles, the vibrations are further transmitted to the inner ear, to a fluid-filled, snail-shaped organ known as the cochlea. Inside the cochlea are sensory cells known as hair cells, named for the rows of cilia that project from their surface. Once the sound waves reach the cochlea, the hair cells transduce the sound waves into the electrical signal sent to the brain [National Institute on Deafness and Other Communication Disorders (NIDCD)].

All the structures and processes that compose the human ear are integral for its proper function. Damage to any of these structures can result in hearing loss, either temporary or permanent. Hearing loss can be defined based on several categories, one of which is the section of the ear that is affected. Conductive hearing loss occurs when sounds cannot reach the inner ear, such as when the ear canal or eardrum is damaged. Sensorineural hearing loss is due to injury to the hair cells within the cochlea, the auditory nerve, or the brain itself. Mixed hearing loss is a combination of the two. Conductive hearing loss can often be treated, either with medication or surgery, but sensorineural hearing loss can rarely be corrected, and is often permanent [Centers for Disease Control and Prevention (CDC)][St. Jude Children's Research Hospital]. Hair cells do not regenerate, and can be permanently damaged by loud noises, conditions such as high blood pressure and diabetes, or certain drugs, including aminoglycoside antibiotics and some chemotherapeutic agents [NIDCD].

In addition to sensorineural, conductive or mixed hearing loss, hearing loss can also be defined as unilateral or bilateral, depending on whether it occurs in one or both ears, and pre- or post-lingual, depending on when the hearing loss occurs with regard to language acquisition. It can also be nonsyndromic if it occurs in the absence of any other symptoms, or syndromic, when it is accompanied by other clinical findings. The severity of hearing loss is measured in decibels (dB HL), and can range from mild to profound, as shown in Table 1. Severity levels and dB HL ranges may vary among authorities [Smith et al. (2014)]. Individuals who are “deaf” mostly have profound hearing loss [World Health Organization (WHO)].

Table 1. Levels of hearing loss. The thresholds in decibels indicate the softest sound that an individual with that level of hearing loss can hear. For a typical person with normal hearing, the softest sound that can be hear is 0 decibels. The softest sound that can be perceived by someone with severe hearing loss may be above 71 decibels [Smith et al. (2014)].

MT-RNR1 and nonsyndromic hearing loss

Nonsyndromic hearing loss can be caused by a variety of factors, both environmental and genetic [Genetics Home Reference]. A small percentage of cases of nonsyndromic hearing loss are due to mutations within mtDNA, specifically within the MT-RNR1 and MT-TS1 genes (MT-TS1 codes for a tRNA molecule) [Pandya (2014)]. Perhaps the most common cause of mitochondrial hearing loss is the 1555A>G variant within the MT-RNR1 gene (rs267606617). The 1555G allele is strongly associated with the development of aminoglycoside antibiotic-induced hearing loss. However, it is also associated with nonsyndromic hearing loss independent of aminoglycoside use, albeit at a lower penetrance. It has been found in individuals of numerous different ethnicities worldwide. Many studies looking at the link between 1555A>G and nonsyndromic hearing loss have been done by analyzing the matrilineal line within a pedigree: the matrilineal relatives of a proband with the 1555G allele will likely all also carry the 1555G allele. Penetrance of the mutation has varied widely between different cohorts – one family in South Africa had eight individuals homoplasmic for the 1555A>G variation across three generations, none of whom had any form of hearing loss [Article:9391883]. However, it is important to note that the median age of onset of hearing loss for an individual with the 1555G variant that has not received aminoglycoside antibiotics is estimated to be around 20 years old [Pandya (2014)]. Though the ages of family members were not given in this study, it is possible that there were individuals in the most recent generation who were still children, and who may have gone on to develop hearing loss later in life. In contrast to the South African cohort, penetrance in a large Arab-Israeli family and in 19 Spanish pedigrees, all without any history of aminoglycoside use, was ~65% and ~54%, respectively [Articles:9490575, 9632174]. In one of the largest studies, encompassing 43 Chinese pedigrees, excluding those with aminoglycoside-induced hearing loss, 111 individuals out of 715 exhibited hearing impairment (15.5%). Within each family, the penetrance ranged from 0 to 47.8%, with the age of onset of hearing loss ranging from 5 to 30 years old [Article:19818876]. It is thought that some sort of modifying factor, such as variation within nuclear-encoded genes or mitochondrial haplotypes, may be responsible for the variability in penetrance [Article:24339937].

In most studies on hearing loss and the 1555G variation, individuals are homoplasmic for the G allele. However, several studies have found that the G allele present was in heteroplasmy in their subjects. Studies by del Castillo et al. and Ballana et al. found a correlation between the level of heteroplasmy and the presence and severity of hearing loss [Articles:12920080, 17999439]. In both studies, those with percentage of copies of the G allele of below ~20-25% were asymptomatic. The level at which individuals began showing hearing loss varied between studies: del Castillo et al. found that those with a mutation load above ~50% had hearing loss, while Ballana et al. found this level to be 80%. Both studies included less than 20 individuals, with del Castillo et al. analyzing 19 subjects across six Spanish families and Ballana et al. analyzing 13 subjects of one Spanish family [Articles:12920080, 17999439]. Due to the size of the studies, any conclusions about correlation should be drawn lightly. Some exceptions existed in these studies, such as the two individuals within the del Castillo et al. study who were homoplasmic for the 1555G allele but asymptomatic, a finding that supports the idea of a modifying factor affecting the development of hearing loss [Article:12920080].

Other MT-RNR1 variants associated with nonsyndromic hearing loss (both with and without aminoglycoside use) include 1494C>T (rs267606619) [Article:17698030] and 1095T>C (rs267606618) [Article:15555598], though these are less common and have not been studied as extensively as the 1555A>G variant. Within Chinese populations, excluding individuals who received aminoglycosides, the 1494T variant appears to have an average penetrance of around 18%, with the severity of hearing loss ranging anywhere from mild to profound. However, the penetrance within individual pedigrees can vary greatly, with some families showing 0% penetrance of the T allele [Article:19682603] and others showing close to 40% [Article:14681830]. Within one study in Spanish family that included 13 individuals with the 1494T allele who did not take aminoglycosides, the penetrance reached 77% [Article:17085680]. Currently, no studies have included individuals that are heteroplasmic for the 1494T allele. Only a handful of individuals with the 1095C allele have been found to develop nonsyndromic hearing loss [Articles:11079536, 11313749, 15555598]. Heteroplasmy has been seen in these individuals [Articles:11313749, 11079536], though it is not clear if there is any correlation between level of heteroplasmy and severity or age-of-onset of nonsyndromic hearing loss.

Pharmacogenetics

Multiple variations within the MT-RNR1 gene have been associated with the development of hearing loss in patients who receive aminoglycoside antibiotics. Aminoglycosides are a class of antibiotics that includes drugs such as streptomycin, kanamycin, gentamycin and tobramycin, among others. They consist of several, usually three, carbon rings, onto which amino groups are attached [Article:10686428]. Aminoglycosides are highly effective against gram-negative bacteria, such as Escherichia coli, Klebsiella, and Salmonella; they are also used to treat tuberculosis [Article:10686428]. Aminoglycosides work by binding to16S rRNA within the small ribosomal subunit of bacteria, specifically the mRNA decoding site (A site) - this causes miscoding or premature termination of protein synthesis [Articles:8910275, 17489842, 22121370]. While aminoglycosides are the most commonly used antibiotic worldwide, they can cause toxic side effects, particularly ototoxicity and nephrotoxicity [Article:10686428]. Individuals who carry MT-RNR1 variations such as 1555A>G (rs267606617) and 1494C>T (rs267606619) are highly susceptible to hearing loss after receiving the drug, regardless of dose or length of treatment. The prevalence of the deafness-associated MT-RNR1 variants is unclear and varies by population, but may be about 1-2% [Articles:19371214, 24100002].

^{1 There is a form of heteroplasmy known as microheteroplasmy, or the presence of hundreds of independent mutations with each mutation found in approximately 1-2% of mitochondrial genomes. This type of low-level heteroplasmy is thought to be relatively common, but can only be detected through lengthy methods. Microheteroplasmy has been implicated in the development of aging and age-related disease.}