GENETIC TESTING AND MOLECULAR BIOMARKERS
Volume 14, Number 5, 2010
ª Mary Ann Liebert, Inc.
Pp. 1–6
DOI: 10.1089/gtmb.2010.0011
ORIGINAL ARTICLE
Role of the Mitochondrial Mutations, m.827A>G
and the Novel m.7462C>T, in the Origin of Hearing Loss
Daniela Tiaki Uehara,1 Daniel Rincon,1 Ronaldo Serafim Abreu-Silva,1
Maria Teresa Balester de Mello Auricchio,1 Alfredo Tabith Junior,2
Fernando Kok,1,3 and Regina Célia Mingroni-Netto1
Samples from 30 deaf probands exhibiting features suggestive of syndromic mitochondrial deafness or from
families with maternal transmission of deafness were selected for investigation of mutations in the mitochondrial
genes MT-RNR1 and MT-TS1. Patients with mutation m.1555A>G had been previously excluded from this
sample. In the MT-RNR1 gene, five probands presented the m.827A>G sequence variant, of uncertain pathogenicity. This change was also detected in 66 subjects of an unaffected control sample of 306 Brazilian individuals from various ethnic backgrounds. Given its high frequency, we consider it unlikely to have a pathogenic
role on hereditary deafness. As to the MT-TS1 gene, one proband presented the previously known pathogenic
m.7472insC mutation and three probands presented a novel variant, m.7462C>T, which was absent from the
same control sample of 306 individuals. Because of its absence in control samples and association with family
history of hearing impairment, we suggest it might be a novel pathogenic mutation.
There are many reports on novel mutations in mitochondrial DNA that could be responsible for hearing loss. In some
cases, lack of population information leads to controversy on
the pathogenic significance of these mutations (Yao et al.,
2006). One of the most controversial questions regards the
MT-RNR1 m.827A>G mutation (Li et al., 2005; Xing et al.,
2006a, 2006b; Chaig et al., 2008).
To investigate the contribution of MT-RNR1 and MT-TS1
mutations to hearing loss in a Brazilian sample, we searched
for mutations in 30 unrelated probands. Our work contributes
to the discussion on the pathogenic effect of mitochondrial
mutations on hereditary deafness.
Introduction
H
earing loss, of environmental and/or genetic etiology, is the most common sensorial disorder. Mitochondrial mutations are estimated to be responsible for at
least 1% of all hereditary hearing loss cases. The two most
important mitochondrial genes associated with nonsyndromic hearing loss are the 12S rRNA gene (MT-RNR1)
and the tRNASer(UCN) gene (MT-TS1). The m.1555A>G nucleotide change in the MT-RNR1 gene has been reported as
the most frequent mitochondrial mutation causative of
hearing loss (Prezant et al., 1993; Matthijs et al., 1996; Pandya
et al., 1997; Abe et al., 1998; Estivill et al., 1998). In a Brazilian
unselected sample of 200 individuals with hearing impairment, it was present in 2% of probands (Abreu-Silva et al.,
2006). Other MT-RNR1 mutations related to nonsyndromic
hearing loss are m.827A>G, m.961delTinsC(n), m.1095T>C,
and m.1494C>T (Bacino et al., 1995; Thyagarajan et al., 2000;
Zhao et al., 2004). The MT-TS1 mutations m.7445A>G,
m.7472insC, m.7510T>C, and m.7511T>C have been detected in association with nonsyndromic hearing loss (Reid
et al., 1994; Tiranti et al., 1995; Hutchin et al., 2000; Chapiro
et al., 2002). Occasionally, additional MT-RNR1 gene and
MT-TS1 gene mutations have been found in patients with
hearing loss and syndromic features.
Patients and Methods
Subjects
We selected 30 affected individuals for molecular investigation of mitochondrial genes, from genealogies with familial
hearing loss, compatible with maternal inheritance (22 cases),
or presenting symptoms frequent in syndromic forms of mitochondrial deafness, such as neurological disorders and diabetes (6 cases) or fulfilling both criteria (2 cases). These
individuals were ascertained in our genetic counseling unit,
Centro de Estudos do Genoma Humano, Universidade de São
1
Departamento de Genética e Biologia Evolutiva, Centro de Estudos do Genoma Humano, Instituto de Biociências, Universidade de São
Paulo, São Paulo, Brazil.
2
Divisão de Educação e Reabilitação de Distúrbios da Comunicação, Pontifı́cia Universidade Católica, São Paulo, Brazil.
3
Departamento de Neurologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.
1
2
UEHARA ET AL.
Table 1. Frequency of Detected Variants
in MT-RNR1 and MT-TS1 Genes
Gene
MT-RNR1
MT-TS1
Sequence variant
Patients
Control sample
m.827A>G
m.7472insC
m.7462C>T
5/30
1/30
3/30
66/306
—
0/306
Paulo, Brazil. In all probands, the most frequent mutations
associated with hearing loss, m.1555A>G in the mitochondrial MT-RNR1 gene, c.35delG and c.167delT in the nuclear
GJB2 gene, and del(GJB6-D13S1830) and del(GJB6-D13S1854)
in the GJB6 gene, had been previously excluded as being the
cause of the hearing impairment.
A control sample was also screened for specific variants. It
consisted of 306 Brazilian unaffected individuals of various
ethnic backgrounds: 133 subjects were predominantly of European ancestry, 165 were of predominantly African ancestry,
and 8 were of Asian ancestry. Part of the control sample is
described in Abreu-Silva et al. (2010). The Brazilian population is highly admixed and subjects, with either European or
African ancestry, may present high frequencies of Native
American lineages of mitochondrial DNA (Bortolini et al.,
1997; Alves-Silva et al., 2000; Marrero et al., 2005).
Written informed consent was obtained from all normal
controls, patients, and their relatives participating in the study.
Methods
Total DNA was extracted from peripheral blood using
standard procedures. The two mitochondrial genes, MTRNR1 and MT-TS1, were sequenced in all 30 probands. The
MT-RNR1 gene was amplified by polymerase chain reaction
(PCR) in two fragments using the following primers: 50 ATG
TAG CTT ACC TCC TCA AAG C 30 and 50 TTA AGC TGT
GGC TCG TAG TG 30 , and 50 TGC TTA GCC CTA AAC CTC
AA 30 and 50 GGT TTA GCT CAG AGC GGT CA 30 . For the
MT-TS1 gene, the primers used were 50 GGA TGC CCC CCA
CCC TAC C 30 and 50 CCT ACT TGC GCT GCA TGT GCC 30 .
Each fragment was purified and submitted to sequence
analysis in the MegaBACE 1000 DNA Analysis System
(Amersham Biosciences).
To determine whether the m.7462C>T and m.7472insC
mutations in the MT-TS1 gene are in a homoplasmic or het-
eroplasmic state, PCR–restriction fragment length polymorphism (RFLP) analysis was performed with enzymes TaqI for
m.7462C>T and XcmI for m.7472insC. For the latter mutation,
the detection of the C insertion was obtained using a modified
forward primer (50 GAA GGA ATC GAA CCA CCC 30 ), in
which the wild-type C was replaced by an A at position 7469
(underlined). This change creates an artificial XcmI restriction
site [CCA(N)9TGG] at position 7472, when the mutated sequence is present. The products of PCR-RFLP were submitted
to electrophoresis in 6% acrylamide gels and silver staining.
The probands whose sequence analysis showed m.827A>G
and m.7462C>T mutations had their mitochondrial haplogroup determined by sequencing of the hypervariable region
I with primers and protocols previously described by AlvesSilva et al. (2000). Over 95% of the mitochondrial DNA lineages
can be assigned by this method (Marrero et al., 2007).
The control sample was screened by PCR-RFLP for the
m.827A>G (MseI), m.7462C>T (TaqI), and m.13263A>G
(HincII) substitutions. The latter substitution allows the
identification of individuals with haplogroup C. Individuals
from the control sample with m.827A>G also had their hypervariable region I sequenced for haplogroup identification.
Results and Discussion
The results are summarized in Table 1.
Mutational analysis of the MT-RNR1 gene
The known m.961delTinsC(n), m.1095T>C, and
m.1494C>T mutations in the MT-RNR1 gene were not detected in our probands. However, five probands presented
the m.827A>G mutation. Aminoglycoside exposure was not
reported in these cases. This mutation was first reported in
a sporadic case of deafness, in 2004 (Li et al., 2004). In 2005,
Li et al. analyzed 128 Chinese pediatric patients with aminoglycoside-induced and nonsyndromic hearing loss; they
found the m.827A>G mutation in five patients, and considered it as a putative pathogenic mutation. Xing et al. (2006a,
2006b) studied two nonrelated Chinese families with hearing
loss in which the same mutation was present in all maternal
relatives, but not all the carriers of the mutation were affected.
This supposed incomplete penetrance led the authors to
conclude that m.827A>G mutation alone is not sufficient to
produce the clinical phenotype, which would require the involvement of modifying factors. In 2008, Chaig et al. detected
FIG. 1. Pedigree of a family with m.7472insC mutation in the MT-TS1 gene. Hearing-impaired individuals are indicated by
full symbols, arrow denotes the proband, and asterisks denote individuals with mitochondrial DNA studied.
ROLE OF M.827A>G AND M.7462C>T IN HEARING LOSS
FIG. 2. Partial sequence analysis of MT-TS1 gene in the
7462 region. (a) The proband with the variant. (b) Control
individual. Arrows indicate the position of the nucleotide
change.
the same mutation in an Argentinean family with hearing loss
after aminoglycoside treatment. The contribution of the variant m.827A>G to hearing loss is, however, controversial:
indeed, together with m.15535C>T, this variant defines the
subhaplogroup B4bd of the haplogroup B mitochondrial lineage (Bandelt et al., 2003; Kong et al., 2003; Tanaka et al., 2004;
3
Yao et al., 2006), and as noted by Bandelt et al. (2005), a variant
that defines a whole major or minor subhaplogroup is very
unlikely to be a primary disease mutation. In fact, these
studies that associated m.827A>G mutation to hearing loss
were performed in Chinese and Argentinean families, and the
haplogroup B was found in both East Asia and South America
(Torroni et al., 1993; Kong et al., 2003; Mishmar et al., 2003).
Moreover, in the study of Li et al. (2005), this variant was also
found in 2 of 144 controls, but this issue was not subjected to
further discussion.
The five probands carrying the m.827A>G mutation detected in our study had their mitochondrial haplogroup determined as haplogroup B, which is in agreement with
previous haplogroup studies (Bandelt et al., 2003; Kong et al.,
2003; Tanaka et al., 2004). The Argentinean family studied by
Chaig et al. (2008) also had haplogroup B. Further, in our
control sample composed of 306 individuals with normal
hearing, we detected 66 (21%) individuals carrying this sequence variant. Interestingly, other haplogroups, in addition
to haplogroup B, such as the Native American A and C and
African haplogroups L1 and L3, were found to be associated
with m.827A>G mutation among these 66 individuals, indicating it is not exclusive to subhaplogroup B4bd. Given its
high frequency in the hearing population, it seems unlikely
that m.827A>G is the cause of hereditary hearing loss, even
with a low penetrance.
Mutational analysis of the MT-TS1 gene
The previously reported MT-TS1 mutations, m.7445A>G,
m.7510T>C, and m.7511T>C, were not detected in our sample. One of the probands carrying an m.827A>G mutation in
FIG. 3. Pedigrees a, b and c of the families with m.7462C>T mutation in the MT-TS1 gene. Hearing-impaired individuals
are indicated by filled symbols, arrows denote the probands, asterisks denote individuals with mitochondrial DNA studied,
and interrogation marks denote individuals whose hearing status is unknown.
4
UEHARA ET AL.
the MT-RNR1 gene also presented the known pathogenic
m.7472insC mutation in the MT-TS1 gene, apparently in
homoplasmy after PCR-RFLP and sequencing. The pedigree
shows a pattern of transmission of hearing loss compatible
with mitochondrial inheritance (Fig. 1). At clinical examination,
a proband (IV-10) presented ataxia and sensorineural hearing
loss, noticed at the age of 6 years. Her maternal aunt (III-4)
presents deafness but no ataxia, and heteroplasmy was not
detected after PCR-RFLP and sequencing. Other affected
family members had their hearing loss detected approximately
at the age of 25–30 years, and ataxia was not found either. This
mutation was first described in 1995 (Tiranti et al., 1995) in six
patients whose deafness was accompanied by ataxia, dysarthria, and focal myoclonus. Subsequently, it was identified in
two subjects presenting ataxia, myoclonic epilepsy, and mental
retardation ( Jaksch et al., 1998). Therefore, though the proband
also has the m.827A>G variant, her hearing impairment and
ataxia are most probably due solely to m.7472insC mutation.
We also detected a novel variant in the MT-TS1 gene,
m.7462C>T (Fig. 2), in three unrelated probands; we did not
detect heteroplasmy in either case after PCR-RFLP and sequencing. All probands had nonsyndromic hearing loss and
belong to genealogies in which maternal inheritance of deafness was present (Fig. 3). Figure 4 shows tonal audiograms of
FIG. 4.
all individuals with m.7462C>T who had their mitochondrial
DNA examined. In one case (Fig. 3a), the proband (II-7) was
examined at the age of 30 years, presenting bilateral, mild, and
sensorineural hearing loss. Hearing loss was noticed at the
age of 26 years. Her older daughter (III-4) presented bilateral,
moderate-to-severe, and sensorineural hearing loss, whereas
her younger daughter (III-5) presented bilateral, moderate,
and sensorineural hearing loss (Fig. 4). In the second case (Fig.
3b), the proband (IV-3) was examined at the age of 14 years
and also presented bilateral, moderate, and sensorineural
hearing loss, whereas his mother (III-5) has mild-to-moderate
hearing loss. Finally, the third proband carrying the
m.7462C>T mutation (Fig. 3c; V-3) was examined at the age of
6 years and presented bilateral moderate hearing loss. Onset
of deafness was postlingual in all cases.
The m.7462C>T mutation was not previously reported in
databases such as Mitomap (2009), mtDB (2009), and GenBank (2009). We determined the mitochondrial haplogroups
and found that all three probands had mitochondrial DNA of
haplogroup C of Native American origin. Therefore, the
m.7462C>T variant could be a polymorphism of haplogroup
C. However, this variant has never been reported as a polymorphism in the few studies of the coding region of mitochondrial genes in haplogroup C (Tamm et al., 2007; Achilli
Tonal audiograms of the individuals with m.7462C>T mutation in the MT-TS1 gene, from pedigrees a–c in Figure 3.
ROLE OF M.827A>G AND M.7462C>T IN HEARING LOSS
5
et al., 2008). In addition, our control sample of 306 subjects (58
of which were classified in haplogroup C) was tested for
m.7462C>T with restriction enzyme TaqI and none presented
this mutation. These data strongly suggest that the
m.7462C>T variant is rare and might be either a very rare
polymorphism of haplogroup C or a novel pathogenic mutation leading to hearing impairment.
It is a hard task to speculate on mechanisms that lead to
phenotype when the product is a transfer RNA that does not
allow a precise prediction of the effect of a substitution. According to McFarland et al. (2004), two criteria have to be
considered when assessing the pathological significance of a
novel mitochondrial tRNA variant: the location of the mutation
in the secondary structure of the tRNA molecule and the disruption it causes to Watson–Crick base pairing. By comparing
the characteristics of neutral variants with those of pathogenic
mutations, they concluded that the hotspots for pathogenic
mutations occur in stems, especially in the acceptor and anticodon stems. The 7462 position in the mitochondrial tRNASer(UCN) molecule is located at the TCC loop; however, it is also
the case of the pathogenic mutations m.3291T>C in the
tRNALeu(UUR) gene (MT-TL1) and m.8344A>G in the tRNALys
gene (MT-TK). So, its location does not rule out the possibility
of being pathogenic.
In summary, our data suggest that the m.827A>G variant
in the MT-RNR1 gene is unlikely to be pathogenic. The
m.7462C>T variant in the MT-TS1 gene might appear as a
mutation leading to hearing impairment. Nevertheless, the
definitive classification of m.7462C>T as a pathogenic variant
requires further investigation, namely the finding of this
variant in mitochondrial genomes belonging to haplogroups
other than haplogroup C, as well as functional studies to assess its effects on the tRNASer(UCN) molecule. Our results also
reflect the importance of using a suitable population approach
in clinical studies, to avoid premature claims as to the pathogenic role of mutations. Such studies are particularly appropriate in an admixed population such as the Brazilian.
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Acknowledgments
The cooperation of all the hearing-impaired patients, their
relatives, and all the control individuals is acknowledged. The
authors thank the professionals from DERDIC for helping in
the clinical evaluation of individuals. The authors also thank
Dr. Paulo A. Otto, Dr. Angela Vianna-Morgante, and Lilian
Dluhosch for critical reading of the manuscript. This research
was supported by CEPID-FAPESP and CNPq.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Regina Célia Mingroni-Netto, Ph.D.
Departamento de Genética e Biologia Evolutiva
Centro de Estudos do Genoma Humano
Instituto de Biociências
Universidade de São Paulo
Rua do Matão, 277–Cidade Universitária
São Paulo 55-11
E-mail: [email protected]