Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations

    Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations Yu Ding, Bo-Hou Xia, Qi Liu, Mei-Ya Li, Shui-Xian Huang, GuangChao Zhuo PII: DOI: Reference:

S0378-1119(16)30539-X doi: 10.1016/j.gene.2016.07.013 GENE 41446

To appear in:

Gene

Received date: Revised date: Accepted date:

13 April 2016 22 June 2016 5 July 2016

Please cite this article as: Ding, Yu, Xia, Bo-Hou, Liu, Qi, Li, Mei-Ya, Huang, Shui-Xian, Zhuo, Guang-Chao, Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations, Gene (2016), doi: 10.1016/j.gene.2016.07.013

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ACCEPTED MANUSCRIPT Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations

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Yu Dinga,b,*, Bo-Hou Xiac, Qi Liud, Mei-Ya Lie, Shui-Xian Huangb,f, Guang-Chao Zhuoa,b

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Central Laboratory, Hangzhou First People’s Hospital, Hangzhou 310006, China

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a

Affiliated Hangzhou Hospital, Nanjing Medical University, Hangzhou 310006,

Department of Pharmacy, Hunan University of Traditional Chinese Medicine,

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c

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China

Changsha 410208, China

Department of Laboratory Medicine, Shaoxing People’s Hospital, Shaoxing Hospital

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e

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of Zhejiang University, Shaoxing 312000, China Analytical Testing Center, Zhejiang University of Traditional Chinese Medicine,

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Hangzhou 310053, China

Department of Otolaryngology, Hangzhou First People’s Hospital, Hangzhou

310006, China

The first two authors have contributed equally for this work

*Corresponding author at: Central Laboratory, Hangzhou First People’s Hospital, Nanjing Medical University, Huan Sha Road No. 261, Hangzhou, China Phone/Fax: +86-0571-87065701; E-mail address: [email protected] (Y. Ding).

ACCEPTED MANUSCRIPT ABSTRACT Mutations in mitochondrial 12S rRNA (MT-RNR1) are the important causes of

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sensorineural hearing loss. Of these mutations, the homoplasmic m.1555A>G or m.1494C>T mutation in the highly conserved A-site of MT-RNR1 gene has been

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found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. Since the m.1555A>G and m.1494C>T mutations

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are sensitive to ototoxic drugs, therefore, screening for the presence of these

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mutations is important for early diagnosis and prevention of deafness. For this purpose, we recently developed a novel allele-specific PCR (AS-PCR) which is able

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to simultaneously detect these mutations. To assess its accuracy, in this study, we

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employed this method to screen the frequency of m.1555A>G and m.1494C>T mutations in 200 deafness patients and 120 healthy subjects. Consequently, four

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m.1555A>G and four m.1494C>T mutations were identified; among these, only one patient with the m.1494C>T mutation had an obvious family history of hearing loss. Strikingly, clinical evaluation showed that this family exhibited a high penetrance of hearing loss. In particular, the penetrances of hearing loss were 80% with the aminoglycoside included and 20% when excluded. PCR-Sanger sequencing of the mitochondrial genomes confirmed the presence of the m.1494C>T mutation and identified a set of polymorphisms belonging to mitochondrial haplogroup A. However, the lack of functional variants in mitochondrial and nuclear modified genes (GJB2 and TRMU) in this family indicated that mitochondrial haplogroup and nuclear genes may not play important roles in the phenotypic expression of the m.1494C>T

ACCEPTED MANUSCRIPT mutation. Thus, other modification factors, such as environmental factor, aminoglycosides or epigenetic modification may have contributed to the high

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penetrance of hearing loss in this family. Taken together, our data showed that this assay is an effective approach that could be used for detection the deafness-associated

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MT-RNR1 mutations.

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Abbreviations

AS-PCR: Allele-specific PCR

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PTA: pure tone audiometry

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mtDNA: mitochondrial DNA

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dBHL: decibels hearing level

ABR: auditory brainstem response

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CI: conservation index

SNPs: single nucleotide polymorphisms

Keywords: Mitochondrial; 1494C>T mutation; Allele-specific PCR; Hearing loss

ACCEPTED MANUSCRIPT 1. Introduction Hearing loss is a very common disorder, affecting one in 700-1000 newborns

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(Morton, 1991). Hearing loss can be caused by hereditary and environmental factors such as ototoxic drugs like aminoglycoside antibiotics. Mutations in mitochondrial

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DNA (mtDNA), especially the 12S rRNA (MT-RNR1) gene, have been associated with both aminoglycoside-induced and non-syndromic hearing loss (Fischel-Ghodsian,

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2005; Ding et al., 2013). Of these, the m.1555A>G or m.1494C>T mutation in the

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highly conserved A-site of MT-RNR1 has been found to be associated with maternally inherited hearing impairment in many families worldwide (Fischel-Ghodsian et al.,

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1993; Prezant et al., 1993; Lu et al., 2010a; Zhao et al., 2004; Wang et al., 2006). In

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the wild type version of MT-RNR1, A1555 and C1494 are in apposition to each other but do not form a base pair (Guan, 2011), whereas the m.1555A>G or m.1494C>T

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mutation creates a new G-C or A-U base pair, making the human mitochondrial ribosomal more like that of bacteria and, consequently, altering binding sites for aminoglycosides. Therefore, the m.1555A>G or m.1494C>T mutation facilitates the binding of these drugs and causes the mistranslation or premature termination of protein synthesis (Chamber and Sande, 1996). Screening for the presence of the m.1555A>G or m.1494C>T mutation is important for early diagnosis and prevention of aminoglycoside-induced deafness, especially for healthy individuals who have a family history of exposure to aminoglycosides. For this purpose, we recently established a novel allele-specific PCR (AS-PCR) to detect the occurrence of m.1555A>G or m.1494C>T mutation (Qi et al., 2016). We

ACCEPTED MANUSCRIPT first designed four primers that specifically binding to the human MT-RNR1 region. After PCR amplification and electrophoresis, we found that samples with the

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m.1555A>G mutation resulted in two specific bands: 736-bp and 226-bp, while the samples with the m.1494C>T mutation resulted in two fragments: 736-bp and 488-bp,

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whereas the samples without these primary mutations resulted in only one fragment: the 736-bp. Thus, this method is a simple, fast, cost-effective way of dual-targets

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identification and can be used for screening the deafness-associated MT-RNR1

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mutations.

In this study, to further evaluate the accuracy of this approach, we screened the

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frequency of the MT-RNR1 mutations in 200 unrelated deafness patients and 120

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healthy controls in Hangzhou area from Zhejiang Province. Consequently, four patients with m.1555A>G mutation and four patients with m.1494C>T mutation were

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identified; however, none of these mutations were found in the control group. Among these, only one patient with m.1494C>T mutation has a typical family history of exposure to aminoglycosides. To further assess the contribution of mitochondrial genetic variants/haplogroup to deafness, we performed PCR-Sanger sequencing for the fragments spanning entire mitochondrial genome.

2. Materials and methods 2.1. Study participants and audiological examinations The study was approved by the ethics committee of Hangzhou First People’s Hospital, and informed consent was obtained from all human subjects or from their

ACCEPTED MANUSCRIPT parents or guardians. As a result, a total of 200 deafness patients (100 females and 100 males, with an average age of 45 years) and 120 healthy subjects (55 females and 65

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males, with an average age of 41 years) were recruited from March 2015 to March 2016. Case library was established, including name, gender, nation, age, family

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history, age of onset, audiological examination, and radiographic testing related information. Audiological examination results such as the pure tone audiometry (PTA)

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and auditory brainstem response (ABR), immittance testing were obtained from the

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hearing examination room in Hangzhou First People’s Hospital. The following are the WHO standards for the classification of the degree of hearing loss: normal hearing, 25

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or less dBHL (decibels hearing level); mild hearing loss, 26-40 dBHL; moderate

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loss, >90 dBHL.

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hearing loss, 41-70 dBHL; severe hearing loss, 71-90 dBHL; profound hearing

2.2. Screening for MT-RNR1 mutations using the AS-PCR Genomic DNA was extracted from blood samples collected from the study participants using the PAXgene Blood DNA Isolation Kits (Qiagen, Valencia, California, USA). The primers, reaction system and conditions for AS-PCR were described in our previous investigation (Qi et al., 2016). The presence of fragments of 736-bp and 226-bp indicated the existence of m.1555A>G mutation. While the presence of fragments of 736-bp and 488-bp suggested the existence of m.1494C>T mutation.

ACCEPTED MANUSCRIPT 2.3. Molecular characterization of one Chinese family with the m.1494C>T mutation 2.3.1. Complete mitochondrial genome analysis

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One Han Chinese family, as shown in figure 1, was ascertained in through the Otology Clinic of Hangzhou First People’s Hospital. The entire mitochondrial

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genome of the matrilineal relatives (II-1, II-4, II-6 and III-3) were amplified by PCR in 24 overlapping fragments by use of sets of light-strand and the heavy-strand

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oligonucleotide primers, as described elsewhere (Rieder et al., 1981). Double-

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stranded automatic sequencing was performed using an ABI PRISMTM 3700 sequencing machine (Applied Biosystems Inc, Foster City, CA, USA). The sequence

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was compared with the human mitochondrial reference sequence (GenBank

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Accession No: NC_012920) (Andrews et al., 1999).

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2.3.2. Phylogenetic analysis

Phylogenetic trees were used to analyze the haplogroup, including the mtDB (http://www.mtdb.igp.uu.se/) and the updated East Asian mtDNA phylogeny (Kong et al., 2006). For the evolutionary conservation analysis, a total of 17 vertebrates’ mtDNA sequences were used in the interspecific analysis. These include: Bos Taurus, Cebus albifrons, Gorilla gorilla, Homo sapiens, Hylobates lar, Lemur catta, Macaca mulatta, Macaca sylvanus, Mus musculus, Nycticebus coucang, Pan paniscus, Pan troglodytes, Pongo pygmaeus, Pongo abelii, Papio hamadryas, Tarsius bancanus and Xenopus laevis (Genbank). The conservation index (CI) was then calculated by comparing the human nucleotide variants with other 16 vertebrates (Ruiz-Pesini and

ACCEPTED MANUSCRIPT Wallace, 2006). Notably, the CI ≥ 70% was considered as having the functional

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potential.

2.3.3. Mutational analysis of GJB2 gene

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The DNA fragments spanning the entire coding region of GJB2 gene were amplified by PCR using the following oligodeoxynucleotides: forward: 5’and

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TATGACACTCCCCAGCACAG-3’;

reverse:

5’-

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GGGGCAATGCTTAAACTGGC-3’. PCR amplification and subsequent sequencing analysis were performed as described in other reference (Li et al., 2004). The results

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were compared with the wild type of GJB2 sequence to identify the potential

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mutations (GenBank Accession No. M86849).

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2.3.4. Mutational analysis of TRMU gene Previous studies showed that TRMU c.28G>T mutation in exon 1 may modulate the phenotypic manifestation of deafness-associated MT-RNR1 mutations (Guan et al., 2006). To see whether TRMU played a putative role in the clinical expression of the m.1494C>T mutation, we screened the c.28G>T mutation in exon 1 of TRMU using the following primer: forward: 5’- ACAGCGCAGAAGAAGAGCAGT-3’, reverse: 5’- ACAACGCCACGACGGACG-3’. The PCR product was purified and analyzed by direct sequencing as described above. The result was compared with the wild type TRMU sequence (GenBank Accession No. AF448221).

ACCEPTED MANUSCRIPT 3. Results 3.1. AS-PCR optimization for detection deafness-associated primary mutations

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The AS-PCR was well established according to our previous investigation (Qi et al., 2016). The PCR results were completely concordant with the sequencing data of the

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eight individuals (four m.1555A>G and four m.1494C>T mutations). To further assess its accuracy, we used this approach to screen the m.1555A>G or m.1494C>T

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mutation in 200 unrelated deafness patients and 120 healthy controls. Consequently,

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four m.1555A>G and four m.1494C>T mutations were identified based on the electrophoresis results (data not shown). However, these primary mutations were

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absent in healthy subjects.

3.2. Clinical features of the Chinese family with the m.1494C>T mutation

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The proband (III-3) was a 33-year-old woman who came from the Hangzhou area of Zhejiang Province, China. She began to suffer bilateral hearing loss at the age of 25, and she did not have a history of exposure to aminoglycosides. As illustrated in figure 2 with a slope-shaped pattern, she had moderate hearing loss (60 dB on the left ear and 30 dB on the right ear). As shown in figure 1, the familiar history was consistent with a maternal inheritance. Notably, this family exhibited a high penetrance of hearing loss, in particular, four of five matrilineal relatives exhibited sensorineural hearing loss as the sole clinical symptom (II-1, II-4, II-6, III-3), thus, the penetrance of hearing loss was 80% when the aminoglycoside was included. However, if the aminoglycoside was excluded,

ACCEPTED MANUSCRIPT there is only one patient with hearing loss (III-3), thus, the penetrance of hearing loss is 20%. In addition, as illustrated in table 1 and figure 2, audiometric studies showed

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variable severity of hearing loss, ranging from severe hearing loss (II-1 and II-4) through mild hearing loss (II-6), to normal hearing (III-2). Furthermore, there is a

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wide range in the age at onset of hearing loss in this family, varying from 25 to 55 years, with an average of 43 years. In addition, other members of this family showed

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dysfunction and neurological disorders.

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no other clinical abnormalities, including diabetes, cardiovascular diseases, visual

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3.3. Mutational screening for the mitochondrial genome

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The maternal transmission of hearing loss in this family suggested the involvement of mitochondrial and led us to analysis the mtDNA mutations of matrilineal relatives

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(II-1, II-4, II-6 and III-3). We performed a PCR-amplification of fragments spanning entire mitochondrial genome and sequenced the PCR products. In addition to the identical m.1494C>T mutation, as shown in table 2, the matrilineal relatives in this family exhibited a set of polymorphisms belonging to human mitochondrial haplogroup A (Kong et al., 2006). Of these, there are 4 variants in D-loop, 2 known variants in MT-RNR1 gene, 2 variants in MT-RNR2 gene, while there are 12 variants in the protein coding genes including 5 missense mutations (Brandon et al., 2005), as well as the COII/tRNALys intergenic 9-bp deletion corresponding with mtDNA at positions 8271-8279. These missense mutations are MT-ND1 3497C>T (p.Ala64Val), MT-ATP6 8860A>G (p.Thr112Ala), MT-ND3 10398A>G (p.Thr114Ala), MT-ND5

ACCEPTED MANUSCRIPT 13928G>C (p.Ser54Thr), MT-CYB 15326A>G (p.Thr194Ala). These variants in RNAs and polypeptides were further evaluated by phylogenetic

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analysis and sequences from 16 other vertebrates, including mouse (Bibb et al., 1981), bovine (Gadaleta et al., 1989) and Xenopus laevis (Roe et al., 1985). None of these

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variants, except for the m.1494C>T mutation (figure 3), were highly conserved

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3.4. Mutational analysis of GJB2 gene

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between different species and implicated to have significant functional consequence.

Mutations in GJB2 have been reported to be a major cause of non-syndromic

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hearing loss (Dai et al., 2015; Adhikary et al., 2015). To examine the role of GJB2

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gene in the phenotypic expression of the m.1494C>T mutation, we performed the mutational screening of GJB2 gene in four affected individuals (II-1, II-4, II-6 and III-

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3), none of variants in GJB2 gene was found in this family with hearing loss, suggested that GJB2 did not play an important role in the clinical expression of the m.1494C>T mutation.

3.5. Analysis of the mutations in TRMU gene Previous studies showed that the TRMU, acted as a nuclear modified gene which was responsible for the 2-thiolation modification for mt-tRNAs, modulated the phenotypic manifestation of deafness-associated MT-RNR1 mutations (Sasarman et al., 2011; Guan et al., 2006). To see the contribution of TRMU c.28G>T mutation in deafness expression, we conducted the mutational screening of TRMU gene in the

ACCEPTED MANUSCRIPT matrilineal members in this family (II-1, II-4, II-6 and III-3). However, we failed to

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detect any nucleotide change in TRMU exon 1.

4. Discussion

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In this study, we employed the previously established AS-PCR method using the four primers that specifically binding the human MT-RNR1 gene to screen the

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presence of m.1555A>G and m.1494C>T mutations (Qi et al., 2016). Many methods

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like PCR-RFLP and SNaPshot have been used to genotype the single nucleotide polymorphisms (SNPs) for decades (Prezant et al., 1993; Zhao et al., 2004; Kokotas et

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al., 2011; Bardien et al., 2009). However, these methods were time-consuming,

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relatively complex and complicated so were not ideal in screening a large sample size. On the other hand, the AS-PCR had been widely used in general diagnosis of

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pathogenic mutations in different diseases because of its speed, simplicity and affordability (Kannim et al., 2009). Scrimshaw et al. (1999) generated an AS-PCR assay to detect the m.1555A>G mutation in patients with deafness which was a simple and reliable method. However, their approach could only detect the m.1555A>G mutation, without the m.1494C>T mutation. By contrast, our study developed a duplex AS-PCR assay to detect the occurrence of the m.1555A>G and m.1494C>T mutations in a single tube. To assess its accuracy, we screened these pathogenic mutations in 200 deafness patients and 120 control subjects; as a result, four patients with m.1555A>G mutation and four patients with m.1494C>T mutation were identified. Therefore, this is a simple, fast and cost-effective AS-PCR method,

ACCEPTED MANUSCRIPT which can correctly and specifically discriminate the deafness-associated MT-RNR1 mutations simultaneously.

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Among these cases carrying the MT-RNR1 mutations, only one patient with m.1494C>T mutation manifestated the maternally inherited pattern of hearing

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impairment. As shown in figure 1, hearing loss as a sole clinical phenotype occurred in matrilineal relatives but not in other members in this family. This Chinese family

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exhibited higher penetrance of hearing loss, which were 80% and 20%, respectively,

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when the aminoglycoside was included or excluded.

We detected the C to T transition at position 1494 in the MT-RNR1 gene, which was

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present in all maternal relatives in nearly homoplasmic form (figure 3), but was absent

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in 120 control subjects, suggested that this mutation was the molecular basis for hearing loss. In fact, m.1494C>T mutation was first identified in a large Chinese

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family with aminoglycoside-induced and non-syndromic hearing loss (Zhao et al., 2004), and subsequently reported in 20 other Chinese pedigrees (Wang et al., 2006; Chen et al., 2007; Yuan et al., 2007; Zhu et al., 2009; Wei et al., 2013) and three Spanish families (Rodriguez-Ballesteros et al., 2006). Moreover, we noticed that three Spanish families appeared to show higher penetrance of hearing loss than these Chinese pedigrees. Functional characterization of cell lines with the m.1494C>T mutation led to only mild mitochondrial dysfunction and sensitive to aminoglycosides (Zhao et al., 2005). In addition, four affected matrilineal relatives in this family exhibited the various severity, age at onset and audiometric configuration of hearing impairment, suggesting the m.1494C>T mutation itself was insufficient to produce the

ACCEPTED MANUSCRIPT clinical phenotypes; other modified factors such as the environmental factors, aminoglycosides, mitochondrial haplogroup, epigenetic modification and nuclear

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genes were involved in deafness expression (Bakhchane et al., 2015; Zhang et al., 2015).

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The mitochondrial variants/haplogroups have been shown to modulate the clinical manifestation of deafness-associated primary mutations (Lu et al., 2010b). For

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example, the tRNAAla (MT-TA) 5628T>C variant was thought to have a modifying

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role in the phenotypic manifestation of m.1494C>T mutation in a Chinese family (Han et al., 2007). Sequence analysis for the entire mitochondrial genome identified a

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set of polymorphisms belonged to haplogroup A (Kong et al., 2006). Despite the

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presence of mtDNA variants, none of them were highly evolutionary conserved and implicated to have functional significance in this family. Thus, mtDNA genetic

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background may not have contributed to the phenotypic expression of the m.1494C>T mutation in this family. Moreover, the absent of any functional variants in nuclear genes (GJB2 and TRMU) suggests that nuclear modified genes may not have a potential role in the development of hearing impairment associated with the m.1494C>T mutation. In this study, although we performed the mutational analysis for the mitochondrial genome and two nuclear genes (GJB2 and TRMU), it cannot explain the phenotypic variability of hearing loss in this family, other factors, such as environmental factors, aminoglycosides, some unidentified nuclear genes may have contributed to the expressivity of deafness-associated m.1494C>T mutation in this family.

ACCEPTED MANUSCRIPT In conclusion, we have optimized the simple, fast and convenience AS-PCR method, which can detect the deafness-associated mitochondrial primary mutations. Moreover,

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a systematic and extended mutational screening for the m.1555A>G and m.1494C>T mutation seemed reasonable, especially in those with an aminoglycoside-induced

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hearing impairment. The main limitation of this study is the small number of cases, further studies including a large cohort of patients with hearing impairment should be

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performed.

5. Conclusions

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This study indicated that our previously established AS-PCR is a high sensitive and

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accurate method which could be used for detecting the deafness-associated MT-RNR1 mutations. In addition, clinical and molecular characterization of the Chinese family

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suggests that the aminoglycosides, environmental and epigenetic factors may contribute to the development of deafness in these subjects carrying the m.1494C>T mutation.

Conflict of interest The authors declare no conflict of interest

Acknowledgements We are grateful to Dr. Lijing Bu from University of New Mexico for critical reading of this manuscript. This work was supported by the Ministry of Public Health of

ACCEPTED MANUSCRIPT Zhejiang Province (2013KYA158), Hangzhou Bureau of Science and Technology

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Shaoxing Bureau of Science and Technology (2013B70066).

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(20150633B16), Hangzhou Health and Family Planning Commission (2015A04),

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hearing loss. The affected members were indicated by filled symbols, arrow denoted the proband.

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Figure 2. Air conduction audiogram of several members in this Chinese family. X, left ear; O, right ear.

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Figure 3. Identification of the m.1494C>T mutation in MT-RNR1 gene, partial

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sequence chromatograms of MT-RNR1 gene from the proband (III-3) and the

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control, arrow indicated the location of the base pair change at position 1494.

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Table 1. Summary of clinical and molecular data of several members in this Chinese family Subjects Gender Age Age Audiomatric Use of PTA PTA Level of at at configuration aminoglycosides (dB) (dB) hearing test onset Left Right loss ear ear II-1 Male 66 55 Slope Yes 110 108 Profound II-4 Female 65 50 Slope Yes 114 118 Profound II-6 Female 60 44 Flat Yes 35 40 Mild III-3 Female 33 25 Slope No 60 30 Moderate III-2 Female 34 / Flat No 19 21 Normal

Presence of MT-RNR1 mutation m.1494C>T m.1494C>T m.1494C>T m.1494C>T m.1494C>T

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Table 2. Mitochondrial sequence variants in this Chinese family with hearing loss Gene Position Replacement Conservation (H/B/M/X)* D-loop 263 A to G 310 T to C 16129 G to A 16519 T to C MT-RNR1 1438 A to G A/A/A/G 1494 C to T C/C/C/C MT-RNR2 2706 A to G A/G/A/A 3109 del N MT-ND1 3497 C to T (Ala to Val) 3970 C to T MT-ND2 4769 A to G 4985 G to A MT-CO1 7028 C to T MT-NC7 8281-8289 Del CCCCCTCTA T/S/L/Q MT-ATP6 8860 A to G (Thr to Ala) T/A/A/T MT-ND3 10398 A to G （ Thr to Ala） MT-ND4 11719 G to A MT-ND5 12561 G to A 13928 G to C (Ser to Thr) S/T/S/T MT-CYB 15326 A to G (Thr to Ala) T/M/I/I 15784 T to C *Conservation of amino acid for polypeptides for rRNAs in human (H), bovine and Xenopus laevis (X).

Previously reported Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes (B), mouse (M)

ACCEPTED MANUSCRIPT Highlights 1. We established a novel AS-PCR for detecting the deafness-associated MT-RNR1

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mutations with high sensitivity.

2. Four patients with m.1555A>G mutation and four patients with m.1494C>T

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mutations were detected by AS-PCR, one patient carrying m.1494C>T mutation had an obvious family history.

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3. Molecular characterization of the Chinese family indicated that environmental

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factors and aminoglycosides may play important roles in deafness expression.