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Noninvasive and accurate detection of hereditary hearing loss mutations with buccal swab based on droplet digital PCR Fang Wang, Lingxiang Zhu, Baoxia Liu, Xiurui Zhu, Nan Wang, Tao Deng, Dongyang Kang, Junmin Pan, Wenjun Yang, Huafang Gao, and Yong Guo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01096 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018
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Analytical Chemistry
Noninvasive and accurate detection of hereditary hearing loss mutations with buccal swab based on droplet digital PCR Fang Wang,†,‡ Lingxiang Zhu,†,‡ Baoxia Liu,§ Xiurui Zhu,§ Nan Wang,†,‡ Tao Deng,ǁ Dongyang Kang,⊥ Junmin Pan,†, £ Wenjun Yang,∫ Huafang Gao*,†,‡ and Yong Guo*,§ †
Human Genetic Resource Center, National Research Institute for Health and Family Planning, 12 Da Huisi Raod, Beijing 100081, P. R. China ‡ Chinese Academy of Medical Sciences, Graduate School of Peking Union Medical College, 9 Dongdan Three Road, Beijing 100730, P. R. China § Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, 30 Shuangqing Road, Beijing 100084, P. R. China ǁ Beijing CapitalBio Medical Laboratory, 88 Kechuang Six Street, Beijing 101111, P. R. China Department of Otorhinolaryngology Head Neck Surgery, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, P. R. China £ MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, 30 Shuangqing Road, Beijing 100084, P. R. China ∫ TargetingOne Corporation, 268 Chengfu Road, Beijing 100190, P. R. China *Corresponding authors: Yong Guo, Email:
[email protected] Tel: +86-10-6278 3960 Huafang Gao, Email:
[email protected] Tel: +86-10-6215 4091 ⊥
ABSTRACT: Hereditary hearing loss is a common clinical neurosensory disorder in humans and has a high demand for genetic screening. Current screening techniques using peripheral blood or dried blood spots (DBSs) are invasive. Therefore, this study aims to develop a noninvasive and accurate detection method for eight hotspot deafness-associated mutations based on buccal swab and droplet digital PCR (ddPCR). First, this method was evaluated for analytic performance including specificity, detection limit, dynamic range using plasmid DNA. The specificity was 100% and the detection limit was 5 copies. The dynamic range of this ddPCR-based method was from 10 to 105 copies/µl. Next, the method was found to accurately quantify mitochondrial gene heteroplasmy rate as low as 1% for both m.1494C>T and m.1555A>G sites. Then, we demonstrated that buccal swab was a reliable sample. DNA can be extracted and accurately quantified after a buccal swab had been stored for 90 days at either room temperature or −20°C. Finally, clinical samples (23 DBSs and 42 buccal swabs) were tested to further evaluate the accuracy and clinical applicability of this method. All clinical samples were accurately quantified and genotyped. This noninvasive and accurate method is highly promising as a genetic screening method for deafness-associated mutations due to its high sensitivity and accuracy.
H
earing loss is one of the most common neurosensory disorders in humans. It can be caused by both genetic and environmental factors, among which 50−60% is caused by genetic factors1,2. The incidence of congenital hearing loss is approximately 1−3/per 1,0003,4. In China, nearly 30,000 infants are born with congenital sensorineural hearing loss each year5. The high incidence of hearing loss is due to the 4−5% carrier rate of deafness-associated mutations in Chinese populations2,6. To date, more than 100 genes associated with hearing loss have been identified7. Despite the high genetic heterogeneity, a large proportion (70−80%) of deafness cases in China are caused by several hotspot mutations2,8,9 in a small number of genes including GJB2, SLC26A4, and MTRNR110,11. Therefore, it is feasible to carry out screening for deafnessassociated mutations. Such genetic screenings have high diagnostic value because patients carrying mutations for congenital deafness can be identified early and receive
language rehabilitation training at the proper time, which has a profound effect on language development, communication, mental health, and career planning12. In addition, people carrying mitochondrial gene mutations, once identified, can avoid using aminoglycoside drugs13. Currently, various techniques including microarray14,15, mass spectrometry (MS)16,17, and next generation sequencing (NGS)18,19 have been applied to screen deafness-associated mutations in China. The CapitalBio Deafness Gene Mutation Detection Array (CapitalBio Corporation, Beijing, China) is microarray-based and screens 9 hotspot mutations through 2−3 drops of heel blood from newborns. This array has been used in more than 20 provinces and cities in China and nearly 3 million people have been screened20. Among them, nearly 6,000 children and 60,000 family members carrying mitochondrial gene mutations were identified so they can avoid the risk of deafness caused by the misuse of
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aminoglycoside drugs20. Qi et al.16 tested 20 hotspot mutations with 9317 neonates' dried blood spots via matrix-assisted laser desorption-ionization time-of-flight MS. The sensitivity and specificity of the method were both 99.9%. Wei et al.18 identified 5 novel variants from 104 deafness genes and three micro-RNA regions using peripheral blood samples from 23 unrelated Chinese family probands by targeted genomic capture and massively parallel sequencing. The above techniques have played an important role in deafnessassociated mutation screening, but the DNA samples were isolated from the peripheral blood or dried blood spots (DBSs), which were obtained through invasive sampling methods. Noninvasive detection methods for deafnessassociated mutation screening are in urgent need but have rarely been reported. Buccal swab, an ideal substitute for the blood21,22, is characterized by an easy and noninvasive sampling procedure, convenient transportation, and good performance in genetic research. The collection of buccal swab does not require professional personnel and any participants can collect the buccal swabs by themselves through simple training, without the pain of a needle prick21. Apart from being noninvasive, buccal swabs can be stored at room temperature and transported by mail23,24. In addition, buccal swab has been applied to many genetic research fields and showed satisfactory performance25,26. Adriaanse et al.25 demonstrated that buccal swab is suitable for HLA typing at both outpatient clinics and as self-administrated sampling performed at home. However, the DNA extracted from buccal swabs of different participants varied in yield, integrity and quality, which may affect the accuracy and reproducibility. We hypothesized that buccal swab may be a reliable and suitable source of DNA for droplet digital PCR (ddPCR)-based methods. ddPCR is a new technology that enables absolute quantification of nucleic acids with high analytical sensitivity and precision27. ddPCR splits PCR reagents into tens of thousands of nanoliter or picoliter partitions by a microfludic chip, so that each droplet contains 0 or 1 DNA template. After PCR amplification and fluorescence detection, the target nucleic acids are calculated from the number of positive and negative droplets by Poisson statistics28. Compared to qPCR, ddPCR enables the absolute quantification of nucleic acids without the need of a standard curve28. Moreover, each droplet is an independent and closed reaction environment, which can reduce the possibility of inter-droplet contamination and the effect of PCR inhibitors. ddPCR has been widely applied to molecular diagnostic tests, including measurement of copy number variations29, single nucleotide variant analysis30, cancer biomarker discovery31, and infectious pathogenic microorganism identification32. Huang et al.33 tested 131 HCC patients using ddPCR, and the HBV copy numbers were successfully detected in all clinical samples. Here, based on buccal swab and ddPCR, a noninvasive and accurate method for detecting eight deafness-associated hotspot mutations (GJB2: c.35delG, c.176_191del16, c.235delC, c.299_300delAT; MTRNR1: m.1494C>T, m.1555A>G; SLC26A4: c.2168A>G, c.IVS7-2A>G) was developed and systemically investigated. First, we evaluated the specificity, detection limit, and dynamic range using plasmid DNA. Second, we tested this method in quantifing the heteroplasmy rates for m.1494C>T and m.1555A>G sites. Heteroplasmy rate is one of the factors that are associated with
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the complicated clinical phenotypes of mitochondrialassociated disease34. Few reports have addressed the detection of heteroplasmy rate, especially low level heteroplasmy rate, for aminoglycoside-induced hearing loss. Third, we examined the reliability of buccal swabs under different preservation conditions. Finally, 23 DBSs and 42 buccal swabs were tested to validate the accuracy and clinical applicability of this method. EXPERIMENTAL SECTION Clinical samples. Ethics approval was granted by Ethics Committee of the National Research Institute for Health and Family Planning (Beijing, China). Buccal swabs were collected according to the “Protocol of buccal swab collection” (see Supporting Information). Samples of 10 volunteers from our lab and 32 patients carrying deafness-associated mutations (homozygotes, heterozygotes, or compound heterozygotes) from Chinese PLA General Hospital (Beijing, China) were collected. Genomic DNA was extracted from buccal swab using Hi-Swab DNA Kit (TIANGEN, Beijing, China). DBSs (diameter 6 mm) were provided by Beijing CapitalBio Medical Laboratory (Beijing, China) and stored at room temperature. Genomic DNA was extracted from DBSs using TIANamp Blood Spots DNA Kit (TIANGEN). Genomic DNA was quantified using Qubit 3.0 Fluorometer (Thermo Fishier Scientific, MA). The estimated copy number was calculated by the following equation (1), in which 1 ng human genomic DNA is considered to have 300 copies, E is the estimated copy number/µl (in copies/µl), Y is the total yield of DNA (in ng), and V is the total elution volume (in µl). (1) = × 300 Plasmid preparation. To validate the feasibility of this method, plasmids containing wildtype and mutant DNAs of the eight detection sites were constructed (see Supporting Information). The plasmids were constructed with pGM-T vector and linearized by ScaI-HF (New England Biolabs, MA). Primers and probes. To design primers and probes, we selected sequences of 100–130 bp long of the eight detection sites. Primers and probes were designed and evaluated with Oligo 7 and Primer Express 3.0 software (Table S-2). Primers and probes were synthesized and purified by TSINGKE Biological Technology Corporation (Beijing, China) and Invitrogen (Shanghai, China). Probes were fluorescently labeled (wildtype probes: 5'-FAM/3'-MGB; mutant probes: 5'VIC/3'-MGB). Workflow of ddPCR. We carried out ddPCR with the QX200™ Droplet Digital™ PCR system (Bio-Rad, CA) according to the manufacturer's instructions. After ddPCR mixture preparation and thermal cycling, the plate was loaded on a QX200™ Droplet Reader for fluorescence detection. Finally, data were analyzed using Quantalife Software 3.0 (see Supporting Information). Specificity of ddPCR. To validate the specificity of this method, ddPCR amplification experiments using the wildtype group (WT), mutant group (Mut), heterozygous group (Het), and negative control group (NC) were performed for each detection site. The specificity was verified by analyzing the ddPCR results with the expected ones. Approximately 6,000 copies of wild and mutant plasmid DNA were added to WT and Mut, respectively. Het contained 6,000 copies each of
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Analytical Chemistry wild and mutant plasmid DNA, and sterile water was added to NC. The ddPCR experiment was conducted in triplicate. Dynamic range and detection limit. To examine the dynamic range and detection limit of the method, serial dilutions of plasmid DNA were subjected to ddPCR. Plasmid DNA was quantified using Qubit 3.0 and estimated copy number was calculated. Then, we diluted the plasmid DNA in the following concentrations (copies/µl): 105, 104, 103, 102, 50, 10, 5, 1, and NC. The ddPCR experiments for each detection site were carried out in three groups (WT, Mut and Het), and each group was conducted in triplicate. Heteroplasmy rate. To evaluate the quantification accuracy of the method for mitochondrial gene heteroplasmy rate, standard samples of m.1494C>T and m.1555A>G sites with different mutation loads were prepared and quantified by ddPCR. First, the wildtype and mutant plasmid DNA were diluted to the same concentration (10,000 copies/µl). Next, plasmid DNA was mixed at different volume ratios and standard samples were constructed: 0%, 1%, 2%, 5%, 10%, 30%, 50%, 70%, 90%, 95%, 98%, 99% and 100%. The ddPCR experiment was conducted in triplicate. Heteroplasmy rate was calculated by the following equation (2), in which W is the wildtpye copy number, M is the mutant copy number. (2) % = × 100% Mutation detection and quantification using DBSs. To verify the sensitivity and accuracy of the ddPCR-based method, we quantified DNAs isolated from different sized DBSs using Qubit 3.0 to determine the optimal size and then tested 23 previously genotyped DBSs of the optimal size using ddPCR. DBSs including six wildtype, one c.235delC (homozygote), and one c.299_300delAT (homozygote) were cut into 1/2, 1/4, and 1/8 of the original size. The results of ddPCR were compared with the microarray and Sanger sequencing provided by Beijing CapitalBio Medical Laboratory. Validation of the preservation condition of buccal swab. To validate the reliability of buccal swabs, samples stored under different conditions (temperature and time) were quantified using Qubit 3.0 and ddPCR. First, buccal swabs from 10 volunteers in our lab were collected. The samples from 5 of the volunteers were stored at room temperature, and the remaining samples were stored at room temperature for 7 days and then transferred to −20°C. Then, three buccal swabs of each volunteer were quantified after storage of different periods including 0 day (6 hours), 7 days, 30 days, and 90 days. The optimal preservation conditions were determined according to the total yield of DNA which was quantified using Qubit 3.0 and ddPCR. Moreover, to validate whether a long period of storage causes damages to the genomic DNA, DNA integrity and the amount of the targeted genomic DNA of three volunteers' buccal swabs preserved for different time and at different temperatures were tested by Agilent 2100 (Agilent, CA) and ddPCR. Mutation detection and quantification using buccal swabs. To verify the accuracy and clinical applicability, buccal swabs of 32 patients from Chinese PLA General Hospital and the 10 volunteers were collected and stored at room temperature for about 30 days. Genomic DNA was extracted from buccal swabs and quantified using Qubit 3.0 and ddPCR. The ddPCR results of the 10 volunteers and 32 patients were verified by Sanger sequencing.
Statistical analysis. Some statistical terms were analyzed including dynamic range, detection limit, statistical significance and relative uncertainty. The dynamic range was defined as described a range in which the copy number quantified by ddPCR scales linearly with the estimated copy number by Qubit 3.0 (R2 > 0.98) with an acceptable level of precision (cv ≤ 25%)35. The detection limit was defined as described the lowest concentration in a sample that can be reliably detected in all replicates, but not necessarily quantified35. Statistical significance was regarded as significant when p
