Abstract
Phenylketonuria (PKU) is caused by phenylalanine hydroxylase (PAH) gene variants. Previous research has identified some PAH mutation hotspots in Chinese patients with PKU. In this study, we introduce a novel MassArray panel for screening the 29 common PAH gene mutations in Chinese patients using iPLEX MALDI-TOF MS. 105 Patients with PKU and known PAH gene mutations were genotyped using this MassArray panel. All of the 29 mutations screened were detected, and MassArray panel results were consistent with those obtained by Sanger sequencing. Fifty patients newly diagnosed with PKU were recruited in the double-blind experiment. PAH gene variants were detected in these 50 patients using the MassArray panel, and the results were verified with Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) methods. Our results show that the mutation detection rate using the MassArray panel with 29 mutations is 74% (95% CI, 65–83%), and the clinical genetic diagnosis rate is 54% (95% CI, 40–68%). This panel can be used as a high throughput, low cost, and rapid method for screening and diagnosing PAH gene mutations. The establishment of this approach provides proof-of-concept for future large-scale PAH mutation carrier screening in areas with high rates of PKU.
1. Introduction
Phenylketonuria (PKU) is one of the most common autosomal recessive metabolic diseases. Phenylalanine (Phe) metabolic disorders in the liver can be caused by phenylalanine hydroxylase (PAH) gene variants. The incidence of PKU varies among ethnic and geographic regions, and PKU affects approximately 1 in 15 000 newborns.1 In mainland China, the average incidence of PKU is 1 in 11 6142.2 However, the newborn screening program indicates that the incidence of PKU level in Gansu, in the northwest region of China, is 1 in 3420.3
More than 1000 PAH mutations have been identified and recorded in the locus-specific database, PAHvdb (http://www.biopku.org/pah/). The frequencies and distributions of PAH mutations also differ among different populations.4 Previous research has identified some PAH mutational hotspots in patients with PKU.5 The higher prevalence of PKU in northwest China may result from a higher frequency of PAH gene mutations. Therefore, it is important to characterize PAH gene mutations in PKU to provide a complete molecular diagnosis, which will contribute to improved disease management. This information will also benefit the screening of at-risk populations and prenatal diagnosis.
The detection of PAH mutations mainly depends on Sanger and next-generation sequencing. These approaches are expensive and time-consuming and are not feasible for screening PAH gene mutations in regions with high rates of PKU. This indicates the need for new ways to detect common PAH mutations. Recently, several high-throughput technologies, such as Denaturing high performance liquid chromatography (DHPLC),6 high resolution melting (HRM),7 SNaPshot,8 and amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR)9 analysis, have been developed. One such promising technology called iPLEX MALDI-TOF MS, which is based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and provided by Sequenom Inc. (San Diego, CA), is also known as MassArray technology. iPLEX MALDI-TOF MS provides rapid measurement of DNA products, with modest multiplexing and minimal assay setup costs owing to the use of unmodified oligonucleotide primers.10 We developed a panel for screening the 29 PAH gene mutations common in Chinese patients using iPLEX MALDI-TOF MS. The power of this panel in detecting PAH mutations was demonstrated by applying it to 50 patients with newly diagnosed PKU.
2. Results
2.1. Design and Establishment of the MassArray Assay
The MassArray assay was developed to detect 29 of 35 selected PAH mutations in two reactions, W1 and W2, and all of PCR and SBE Primers for the 29 mutations are shown in Table S1. The remaining six mutations (c.208_210del, c.694C > T, c.739G > C, c.740G > T, c.482T > C, and c.1252A > C) were excluded from the assay because of their low frequencies and interference of the primers used to detect the mutations with other primers in the multiplex reaction. Reaction W1 covered 21 mutations, and reaction W2 covered 13 mutations. Five common mutations were detected in both reactions (c.611A > G, c.1199G > A, c.782G > A, c.764T > C, c.1174T > A) (Table 1). The molecular weights of all designed and synthesized single-base extended primers, as assessed by mass spectrometry, were consistent with their calculated molecular weights. The preliminary genotyping results were compared with those previously obtained by Sanger sequencing (Figure 1 shows the 10 most common mutations).
Table 1. Design of MassArray for 35 Selected PAH Mutationsa.
| no. | systematic name (DNA level) | trivial name (protein effect) | reaction W1 | reaction W2 |
|---|---|---|---|---|
| 1 | c.728G > A | p.R243Q | - | √ |
| 2 | c.611A > G | p.EX6-96A > G | √ | √ |
| 3 | c.1068C > A | p.Y356* | √ | - |
| 4 | c.442-1G > A | IVS4-1G > A | √ | - |
| 5 | c.1197A > T | p.V399V | - | √ |
| 6 | c.1238G > C | p.R413P | √ | - |
| 7 | c.331C > T | p.R111* | - | √ |
| 8 | c.842+2T > A | IVS7 + 2T > A | √ | - |
| 9 | c.194T > C | p.I65T | √ | - |
| 10 | c.764T > C | p.L255S | √ | √ |
| 11 | c.782G > A | p.R261Q | √ | √ |
| 12 | c.721C > T | p.R241C | - | √ |
| 13 | c.1199G > A | p.R400K | √ | √ |
| 14 | c.526C > T | p.R176* | √ | - |
| 15 | c.208_210delTCT | p.S70del | - | - |
| 16 | c.1222C > T | p.R408W | √ | - |
| 17 | c.781C > T | p.R261* | - | √ |
| 18 | c.1301C > A | p.A434D | √ | - |
| 19 | c.694C > T | p.Q232* | - | - |
| 20 | c.498C > G | p.Y166* | √ | - |
| 21 | c.739G > C | p.G247R | - | - |
| 22 | c.838G > A | p.E280K | √ | - |
| 23 | c.1199G > C | p.R400T | √ | √ |
| 24 | c.473G > A | p.R158Q | √ | - |
| 25 | c.740G > T | p.G247V | - | - |
| 26 | c.910C > A | p.Q304K | - | √ |
| 27 | c.1315+6T > A | IVS12+6T > A | √ | - |
| 28 | c.1045T > C | p.S349A | - | √ |
| 29 | c.482T > C | p.F161S | - | - |
| 30 | c.59_60AG > CC | p.Q20P | √ | - |
| 31 | c.1256A > G | p.Q419R | √ | - |
| 32 | c.505C > A | p.R169S | √ | - |
| 33 | c.1252A > C | p.T418P | - | - |
| 34 | c.1174T > A | p.F392I | √ | √ |
| 35 | c.688G > A | p.V230I | - | √ |
√, this mutation was included in the reaction; -, this mutation was not included in the reaction.
Figure 1.
Results of 10 hotspot mutations of PAH gene by mass spectrometry. (A) cluster point diagram showing the distribution of different typing results; (B) mass spectrum peak diagram showing molecular weight and detection peak of different genotypes; and (C) sequencing peak diagram showing the sequencing peak diagram of the heterozygous mutation sample at this site.
2.2. Clinical Study
The MassArray assay successfully screened 105 patients (with known PAH genotypes) for 29 PAH mutations. Each of the 29 mutations was detected in at least one of the 105 patients screened. The assay was unable to detect mutations in five of the patients. Examination of their corresponding Sanger sequencing results revealed that the mutations present in these patients were not included in the 29 mutations of the assay. MassArray assay results for one patient identified that the patient was homozygous for c.728G > A (using Typer software), while Sanger sequencing showed a compound heterozygous genotype c.728G > A and c.739G > C. A manual check of the original mass spectrometry peak revealed a mini peak in the G position of the c.728G > A mutation (Figure 2).
Figure 2.
Mass spectrometry results of compound heterozygous genotype c.728G > A and c.739G > C. (A) cluster point diagram showing distribution of different typing results of c.728G > A. The red points represent blank controls; (B–E) mass spectrum peak diagram, respectively, for wild type, heterozygous genotype, homozygous genotype, and compound heterozygous genotype c.728G > A and c.739G > C; (F) sequencing peak of compound heterozygous genotype of c.728G > A and c.739G > C.
Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) detection methods detected 98 mutant alleles in 50 newly diagnosed patients recruited in this study, a detection rate of 98% (95% CI: 95.26–100%). The MassArray method detected 74 mutant alleles in 50 patients with a detection rate of 74% (95% CI: 65–83%) (Table 2). All 24 alleles that were detected by Sanger sequencing and MLPA but not detected by MassArray assay were not one of the 29 mutations included in the MassArray assay. The coincidence rate of the detection results using the MassArray assay and using Sanger sequencing and MLPA for the 29 assay mutations was 100%. Homozygous mutations were detected in seven patients using the MassArray, complex heterozygous mutations were detected in 20 patients, a single pathogenic mutation was detected in 20 patients, and no mutation was detected in three patients. Using the MassArray assay, the PKU diagnostic rate reached 54% (95% CI: 40–68%).
Table 2. Results of Clinical Studya.
| Sanger sequencing + MLPA |
MassArray |
|||
|---|---|---|---|---|
| no, | mutation 1 | mutation 2 | mutation 1 | mutation 2 |
| 1 | c.1066-11G > A | c.331C > T | c.331C > T | N |
| 2 | c.194T > C | c.724C > T | c.194T > C | N |
| 3 | c.671T > C | c.722delG | N | N |
| 4 | c.694C > T | c.728G > A | c.728G > A | N |
| 5 | c.728G > A | N | c.728G > A | N |
| 6 | c.1197A > T | c.1147C > G | c.1197A > T | N |
| 7 | c.728G > A | c.1197A > T | c.728G > A | c.1197A > T |
| 8 | c.721C > T | c.1238G > C | c.721C > T | c.1238G > C |
| 9 | c.1315+6T > A | c.911A > G | c.1315+6T > A | N |
| 10 | c.194T > C | c.727C > T | c.194T > C | N |
| 11 | c.842+2T > A | c.1147C > G | c.842+2T > A | N |
| 12 | c.728G > A | c.728G > A | c.728G > A | c.728G > A |
| 13 | c.1238G > C | c.782G > A | c.1238G > C | c.782G > A |
| 14 | c.688A > G | c.782G > A | c.688A > G | c.782G > A |
| 15 | c.1256A > G | c.1045T > G | c.1256A > G | c.1045T > G |
| 16 | c.331C > T | c.728G > A | c.331C > T | c.728G > A |
| 17 | c.194T > A | c.168+5G > C | c.194T > A | N |
| 18 | c.694C > T | c.728G > A | c.728G > A | N |
| 19 | c.721C > T | c.1238G > C | c.721C > T | c.1238G > C |
| 20 | c.1238G > C | EX1-Updel | c.1238G > C | N |
| 21 | c.470G > A | c.1068C > A | c.1068C > A | N |
| 22 | c.721C > T | c.475A > T | c.721C > T | N |
| 23 | c.1238G > C | c.1238G > C | c.1238G > C | c.1238G > C |
| 24 | c.721C > T | c.1301C > A | c.721C > T | c.1301C > A |
| 25 | c.442-1G > A | EX1-Updel | c.442-1G > A | N |
| 26 | c.728G > A | c.1252A > G | c.728G > A | N |
| 27 | c.611A > G | c.728G > A | c.611A > G | c.728G > A |
| 28 | c.212G > A | c.1197A > T | c.1197A > T | N |
| 29 | c.1024G > A | c.1114A > T | N | N |
| 30 | c.728G > A | c.728G > A | c.728G > A | c.728G > A |
| 31 | c.782G > A | c.1068C > A | c.782G > A | c.1068C > A |
| 32 | c.473G > A | c.1238G > C | c.473G > A | c.1238G > C |
| 33 | c.728G > A | c.1315+6T > A | c.728G > A | c.1315+6T > A |
| 34 | c.208-210delTCT | c.1197A > T | c.1197A > T | N |
| 35 | c.526C > T | c.611A > G | c.526C > T | c.611A > G |
| 36 | c.1054G > C | EX1-Updel | N | N |
| 37 | c.611A > G | c.1197A > T | c.611A > G | c.1197A > T |
| 38 | c.442-1G > A | c.1222C > T | c.442-1G > A | c.1222C > T |
| 39 | c.842+2T > A | c.1315+6T > A | c.842+2T > A | c.1315+6T>A |
| 40 | c.194T > C | c.764T > C | c.194T > C | c.764T > C |
| 41 | c.498C > G | c.1068C > A | c.498C > G | c.1068C > A |
| 42 | c.331C > T | c.728G > A | c.331C > T | c.728G > A |
| 43 | c.611A > G | c.755G > A | c.611A > G | N |
| 44 | c.728G > A | c.728G > A | c.728G > A | c.728G > A |
| 45 | c.1197A > T | N | c.1197A > T | N |
| 46 | c.728G > A | c.728G > A | c.728G > A | c.728G > A |
| 47 | c.728G > A | c.1197A > T | c.728G > A | c.1197A > T |
| 48 | c.194T > A | c.194T > A | c.194T > A | c.194T > A |
| 49 | c.475A > T | c.1199G > A | c.1199G > A | N |
| 50 | c.728G > A | c.728G > A | c.728G > A | c.728G > A |
N: no mutation detected.
3. Discussion
PAH gene mutations that cause PKU are diverse and differ between ethnic groups and geographical locations. Comparison of PAH mutational data of different ethnic groups has revealed correlations between the specific mutations and the genetic histories of the investigated populations. Marked differences were identified when comparing PAH mutations of Asian and European populations. The most common mutations are p.R408W and IVS12 + 1G > A in Eastern and Northern Europe, respectively.4 However, these mutations are rarely detected in Asian populations. Five mutations, p.R243Q, p.EX6-96A > G, p.R241C, p.R413P, and IVS4-1G > A, are common in East Asian countries such as China, Japan, and Korea, where they account for 53.76, 69.70, and 62.10% of the total mutations, respectively.13 In the present study, 35 mutations that we previously identified, with frequencies greater than 1%, were selected for MassArray assay design. Finally, 29 mutations were included in the assay, accounting for 66.5–72.6% of Chinese patients with PKU as previously reported5,14,15 (Table 3). The detection rate of 74% in our clinical study by the MassArray assay with 29 mutations was similar to previous reports, but was lower than the results obtained using Sanger sequencing combined with MLPA. However, considering the high frequency of these mutations, the MassArray assay could be performed as a first-pass, low cost, and straightforward approach for mutation detection in patients with PKU and for mutation screening in at-risk populations.
Table 3. Relative Frequency of 29 Common PAH Mutations as Previously Reporteda.
| no. | systematic name (DNA level) | trivial name (protein effect) | RF (%)b n = 185 | RF (%)c n = 212 | RF (%)d n = 796 | RF (%)e n = 495 |
|---|---|---|---|---|---|---|
| 1 | c.728G > A | p.R243Q | 22.2 | 25.43 | 17.53 | 14.84 |
| 2 | c.611A > G | EX6-96A > G | 11.1 | 8.89 | 7.66 | 5.92 |
| 3 | c.1068C > A | p.Y356X | 6.5 | 5.19 | 4.46 | 5.25 |
| 4 | c.442-1G > A | IVS4-1G > A | 3 | 6.42 | 3.77 | 4.58 |
| 5 | c.1197A > T | p.V399V | 3 | 3.21 | 5.84 | 4.35 |
| 6 | c.1238G > C | p.R413P | 6.5 | 5.43 | 4.33 | 5.02 |
| 7 | c.331C > T | p.R111X | 8.7 | 3.7 | 4.77 | 3.13 |
| 8 | c.842+2T > A | IVS7+2T > A | 2.3 | 0.99 | 1.51 | 3.13 |
| 9 | c.194T > C | p.I65T | 0.5 | 0.25 | 0.75 | 2.68 |
| 10 | c.764T > C | p.L255S | 0.3 | 0.74 | 0.44 | 1.79 |
| 11 | c.782G > A | p.R261Q | 0.8 | 0.74 | 1.26 | 2.23 |
| 12 | c.721C > T | p.R241C | 1.1 | 3.7 | 5.4 | 4.35 |
| 13 | c.1199G > A | p.R400K | 0.5 | 0.99 | 0.38 | 2.12 |
| 14 | c.526C > T | p.R176X | 0.3 | 0.49 | 0.82 | 1.23 |
| 16 | c.1222C > T | p.R408W | 0.3 | 0 | 0.5 | 1.00 |
| 17 | c.781C > T | p.R261X | 0 | 0.25 | 0.25 | 0.89 |
| 18 | c.1301C > A | p.A434D | 0.8 | 0.99 | 1.82 | 1.56 |
| 20 | c.498C > G | p.Y166X | 1.9 | 1.73 | 1.07 | 1.12 |
| 22 | c.838G > A | p.E280K | 0.3 | 0.49 | 0.5 | 0.67 |
| 23 | c.1199G > C | p.R400T | 1.1 | 0.99 | 0.44 | 0.56 |
| 24 | c.473G > A | p.R158Q | 0.8 | 0.74 | 0.5 | 0.45 |
| 26 | c.910C > A | p.Q304K | 0 | 0 | 0.06 | 0.45 |
| 27 | c.1315+6T > A | IVS12+6T > A | 0.3 | 0.25 | 0.5 | 1.12 |
| 28 | c.1045T > C | p.S349A | 0.3 | 0.49 | 0.75 | 0.45 |
| 30 | c.59_60AG > CC | p.Q20P | 0 | 0 | 0 | 0.33 |
| 31 | c.1256A > G | p.Q419R | 0 | 0 | 0.57 | 1.12 |
| 32 | c.505C > A | p.R169S | 0 | 0 | 0.06 | 0.45 |
| 34 | c.1174T > A | p.F392I | 0 | 0 | 0.38 | 0.56 |
| 35 | c.688G > A | p.V230I | 0 | 0 | 0.19 | 0.33 |
| 72.6 | 72.1 | 66.51 | 71.65 |
The MALDI-TOF mass spectrometry methodology enables rapid and accurate large-scale mutation screening in high throughput and an automated manner.16 MassArray assays use 384 or 96 chips. Each chip can simultaneously detect up to 384 reactions and each reaction can detect more than 20 mutations.17 In addition, the MassArray assay requires only two steps of multiplex PCR amplification and single-base extension (SBE), the operation process is simple, and it can be completed in one day. The MassArray method directly detects the mass of the extended primer, and directly interprets the typing result through the change in mass, without requiring fluorescence labeling. This avoids the fluorescence labeling quality influencing the result. The single-base extension primers do not require expensive fluorescent labeling, and special probes and primers are not prefabricated on the chip. Because the chip for mass spectrometry detection is universal, different detection methods can be directly adjusted by synthesizing different primers and probes, making this a flexible approach and reducing the reagent cost.
Despite these advantages, MassArray assays have some drawbacks. These include the requirement for specific equipment and the cost of the MassArray instrumentation. Moreover, this approach can only be used for known mutations, and the detection sites are relatively limited, so it can only be used as a screening method for high-frequency mutations. MassArray technology is required to obtain the target DNA product through the design of multiple PCR primers and multiple single-base extension primers. In this study, PAH gene mutations were relatively concentrated on several hotspot mutant exons. On exon 7, for example, there are at least 25 known mutations within a 20-bp range including c.721C > T, c.722delG, c.724C > T, c.727C > T, c.728G > A, c.739G > C, and c.740G > T.11 This made the design of single-base extension primers problematic and led to us abandoning further investigation of some low-frequency sites in the region. Moreover, if variants around the detection site affect the combination of single-base extended primer and template DNA, the results will be inaccurate. In this research, a compound heterozygous sample with c.728G > A/c.739G > C mutation was analyzed during verification of the detection system, and the c.739G > C mutation affected the analysis of c.728G > A. Similar situations could lead to false results.
MassArray technology is a method of microsequencing. It can only perform fixed-point detection for known mutations, which are relatively limited. In this study, 50 patients with an initial PKU diagnosis were screened for 29 hotspot PAH mutations. The mutation detection rate reached 74% (95% CI: 65–83%), and 54% (95% CI: 40–68%) of cases could be diagnosed using this approach. Using MassArray technology, the detection and diagnosis rates are higher than those obtained with other known mutation screening technologies.
Sanger sequencing is still the gold standard for identifying gene mutations owing to its high detection rate and accuracy. With the development of next-generation sequencing technology, scholars will apply multiple sequencing platforms to detect PAH mutations and variants of multiple key genes in phenylalanine metabolism, and the detection rate may reach 100%.18−20 Although second-generation sequencing technology has the advantages of high mutation detection rate and high throughput, it requires complex library construction and sequencing processes, the sequencing cycle is long, and data analysis is complex. These disadvantages limit its scope for clinical application.
In this study, a PAH gene hotspot mutation detection MassArray-based method was established. This method could simultaneously genotype 29 PAH gene hotspot variants with a detection rate of 74% (95% CI: 65–83%). MassArray technology is simple to operate, has high accuracy and specificity, and can be used as a high throughput, low cost, and rapid screening and diagnosis method for PAH gene mutations. The establishment of this scheme also provides a technical basis for large-scale PAH hotspot mutation carrier screening in PKU high-incidence areas in the future.
4. Experimental Section
The study was approved by the Research Ethics Committee of Gansu Provincial Maternity and Child-Care Hospital and followed the approved ethical standards of the declaration of Helsinki. Moreover, written informed consent was obtained from all subjects or from their guardians.
4.1. Patient Recruitment and Sample Preparation
We included 155 unrelated patients with PKU in this study. Of these, 105 had been recruited and genotyped in our previous research.11 Fifty patients with PKU diagnosed from 2016 to 2017 but with unknown genotypes were also included. Blood samples were collected for genetic testing. Genomic DNA was extracted from the leukocytes present in 2–3 mL of peripheral blood using Whole Blood Genomic DNA Extraction Kits (Tiangen, Beijing, China). The purity and concentration of the samples were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific) and the samples were stored at −80 °C until required for analysis.
4.2. Design of MassArray Assay
Thirty-five common PAH gene mutations, reported previously by us and other researchers in China,5,11 were selected for the MassArray assay design. Our mutation-selection criteria are based on our previous research data, the minimum frequency of each mutation is greater than 0.33%, each of the mutations is confirmed in three independent individuals, and finally, 35 mutations are selected. MassArray Assay Designer software (Sequenom Inc., San Diego) was used to design PCR and single-base extension (SBE) primers for all 35 selected common mutations. The optimal amplicon size was set to 80–120 bp. A 10-mer tag (5′-ACGTTGGATG-3′) was added to the 5′ end of each PCR primer to avoid confusion in the mass spectrum, and SBE primers were 5′ tailed with nonhomologous sequences of varying lengths to create large enough mass differences between the different SBE products to allow detection by MALDI-TOF mass spectrometry. To avoid interaction among the primers, the software divided the PCR amplification and SBE primers into multiplex reactions.
4.3. Genotyping by MALDI-TOF Mass Spectrometry
The Sequenom MassArray iPLEX platform (Sequenom Inc., San Diego) was used. Silicon chip and DNA amplification products were used for accurate genotyping by mass spectrometry. Genomic DNA was subjected to iPLEX Gold reactions and the products were transferred to chip wells. The genotypes were detected in situ using MALDI-TOF mass spectrometry.
iPLEX Gold technology consists of an initial locus-specific PCR reaction, followed by SBE using mass-modified dideoxy nucleotide terminators and an oligonucleotide primer which anneals immediately upstream of the polymorphic site of interest.12 The products of these reactions are directly applied to a silicon chip. The mass of the extended primer is determined using MALDI-TOF mass spectrometry. The mass of the primer directly correlates with the mutation of interest and the mass of added bases indicates the alleles present at the polymorphic site. Typer software v4.0 (Sequenom Inc., San Diego, CA) was used to process the raw data obtained from the assays.
More than three blank control samples (sterile water) and negative control samples are included in each batch of tests to ensure the controllability of the experimental tests.
4.4. Clinical Study
To validate the clinical performance of the MassArray assay, 105 patients with PKU were first blindly analyzed for common PAH mutations. The genotypes detected by MassArray assay were then compared with those previously determined by Sanger sequencing. To evaluate the clinical application of this genotyping approach, 50 patients with unknown genotypes were subject to MassArray assay and Sanger sequencing in parallel.
4.5. Statistical Analysis
Statistical analysis was performed using statistical package for social science software (SPSS version 22.0). Mutational frequencies and detection rate were calculated by the counting method.
Acknowledgments
We would like to thank Professor Shangzhi Huang (from the Chinese Academy of Medical Sciences & Peking Union Medical College) for his guidance and suggestions on this research. We thank the patients and their families for participating in this study. We also thank Rebecca Porter, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
Supporting Information Available
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b02955.
PCR and SBE primers for MassArray assay (PDF)
This study was supported by the National Key Research and Development Program of China (Grant No.: 2016YFC1000307, and 2018YFC1002201) and the Natural Science Foundation of Gansu Province (Grant No.: 1606RJZA151).
The authors declare no competing financial interest.
Notes
All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Notes
Informed consent was obtained from all individual participants included in this study.
Supplementary Material
References
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