Mitochondrial tRNAThr G15927A mutation may modulate the phenotypic manifestation of ototoxic 12S rRNA A1555G mutation in four Chinese families

Abstract

Objective: To investigate the role of mitochondrial modifiers in the development of deafness associated with 12S rRNA A1555G mutation.

Methods: Four Chinese families with nonsyndromic and aminoglycoside-induced deafness were studied by clinical and genetic evaluation, molecular and biochemical analyses of mitochondrial DNA (mtDNA).

Results: These families exhibited high penetrance and expressivity of hearing impairment. Penetrances of hearing loss in WZD31, WZD32, WZD33, and WZD34 pedigrees ranged from 50 to 67% and from 39 to 50%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Matrilineal relatives in these families developed hearing loss at the average of 14, 13, 16, and 15 years of age, respectively, when aminoglycoside-induced deafness was excluded. Mutational analysis of entire mtDNA in these families showed the homoplasmic A1555G mutation and distinct sets of variants belonging to haplogroup B5b1. Of these, the tRNA G15927A mutation locates at the fourth base in the anticodon stem (conventional position 42) of tRNA. A guanine (G42) at this position of tRNA is highly conserved from bacteria to human mitochondria. The lower levels and altered electrophoretic mobility of tRNA were observed in cells carrying A1555G and G15927A mutations or only G15927A mutation but not cells carrying only A1555G mutation. The abolished base pairing (28C-42G) of this tRNA by the G15927A mutation caused a failure in tRNA metabolism, worsening the mitochondrial dysfunctions altered by the A1555G mutation.

Conclusion: The G15927A mutation has a potential modifier role in increasing the penetrance and expressivity of the deafness-associated 12S rRNA A1555G mutation in those Chinese pedigrees.

Fig. 1

Four Han Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing impairment. Hearing impaired individuals are indicated by filled symbols. Arrowhead denotes probands. Asterisks denote individuals who had a history of exposure to aminoglycosides.

Fig. 2

Air conduction audiogram of four affected participants with the A1555G mutation. X, left ear; O, right ear.

Fig. 3

Identification and qualification of the G15927A mutation in the mitochondrial tRNA^Thr gene. (a) Partial sequence chromatograms of tRNA^Thr gene from an affected individual (WZD32-III-2) and a married-in-control (WZD32-III-1). An arrow indicates the location of the base changes at position 15927. (b) The location of the G15927A mutation in the mitochondrial tRNA^Thr. Cloverleaf structure of human mitochondrial tRNA^Thr is derived from the study of Florentz et al. [39]. Arrow indicates the position of the G15927A mutation. (c) Quantification of G15927A mutation in the tRNA^Thr gene of mutants and controls derived from the Chinese families. PCR products around the G15927A mutation were digested with HpaII and analyzed by electrophoresis in a 7% polyacrylamide gel stained with ethidium bromide.

Fig. 4

Northern blot analysis of mitochondrial tRNA. (a) Equal amounts (5 μg) of total mitochondrial RNA from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with digoxigenin (DIG)-labeled oligonucleotide probes specific for the tRNA^Thr. The blots were then stripped and rehybridized with DIG-labeled tRNA^Leu(CUN), tRNA^Ser(AGY), tRNA^Lys, and tRNA^Gly, respectively. (b) Quantification of mitochondrial tRNA levels. Average relative tRNA^Thr, content per cell, normalized to the average content per cell of 16S rRNA in cells derived from an affected individual (WZD32-III-7) carrying A1555G and G15927A mutations, a hearing normal individual (A4) carrying only the G15927A mutation, an individual (WZD1-IV-2) carrying only A1555G mutation, a married-in-control (WZD32-III-1) lacking those mitochondrial DNA mutations. The values for the latter are expressed as percentages of the average values for the control cell line (WZD32-III-1). The calculations were based on three independent determinations of each tRNA content in each cell line and three determinations of the content of reference RNA marker in each cell line.

Fig. 5

In-vivo aminoacylation assays for mitochondrial tRNA. Equal amounts (2 μg) of total mitochondrial RNA purified from various cell lines under acid conditions were treated with electrophoresis at 4°C through an acid (pH 5.1) 10% polyacrylamide/7 mol/l urea gel, electroblotted onto a positively charged nylon membrane, and hybridized with digoxigenin (DIG)-labeled oligonucleotide probes specific for mitochondrial tRNA^Thr. Samples were also deacylated (DA) by heating 10 min at 60°C at pH 8.3. The blots were then stripped and rehybridized with DIG-labeled probes for tRNA^Leu(CUN), tRNA^Ser(AGY), tRNA^Lys, and tRNA^Gly, respectively.