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Screening of genetic alterations related to non-syndromic hearing loss using MassARRAY iPLEX® technology.

Svidnicki MC, Silva-Costa SM, Ramos PZ, dos Santos NZ, Martins FT, Castilho AM, Sartorato EL - BMC Med. Genet. (2015)

Bottom Line: The generated results were validated using conventional techniques, as direct sequencing, multiplex PCR and RFLP-PCR.To optimize these results, the failed tests were re-designed and new primers were synthesized.However, we demonstrated that optimization is required to increase the genotyping success and accuracy.

View Article: PubMed Central - PubMed

Affiliation: Human Molecular Genetics Laboratory, Molecular Biology and Genetic Engineering Center (CBMEG), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. mcarolinasvid@gmail.com.

ABSTRACT

Background: Recent advances in molecular genetics have enabled to determine the genetic causes of non-syndromic hearing loss, and more than 100 genes have been related to the phenotype. Due to this extraordinary genetic heterogeneity, a large percentage of patients remain without any molecular diagnosis. This condition imply the need for new methodological strategies in order to detect a greater number of mutations in multiple genes. In this work, we optimized and tested a panel of 86 mutations in 17 different genes screened using a high-throughput genotyping technology to determine the molecular etiology of hearing loss.

Methods: The technology used in this work was the MassARRAY iPLEX® platform. This technology uses silicon chips and DNA amplification products for accurate genotyping by mass spectrometry of previous reported mutations. The generated results were validated using conventional techniques, as direct sequencing, multiplex PCR and RFLP-PCR.

Results: An initial genotyping of control subjects, showed failures in 20 % of the selected alterations. To optimize these results, the failed tests were re-designed and new primers were synthesized. Then, the specificity and sensitivity of the panel demonstrated values above 97 %. Additionally, a group of 180 individuals with NSHL without a molecular diagnosis was screened to test the diagnostic value of our panel, and mutations were identified in 30 % of the cases. In 20 % of the individuals, it was possible to explain the etiology of the HL. Mutations in GJB2 gene were the most prevalent, followed by other mutations in in SLC26A4, CDH23, MT-RNR1, MYO15A, and OTOF genes.

Conclusions: The MassARRAY technology has the potential for high-throughput identification of genetic variations. However, we demonstrated that optimization is required to increase the genotyping success and accuracy. The developed panel proved to be efficient and cost-effective, being suitable for applications involving the molecular diagnosis of hearing loss.

No MeSH data available.


Related in: MedlinePlus

Steps in genotyping using MassARRAY® iPLEX System. A schematic of the genotype reaction of an A-to-G SNP. a. Locus-specific amplification reaction b. Treatment with SAP enzyme to neutralize unincorporated dNTPs. c. Locus-specific primer extension reaction (iPLEX assay). In this reaction, an oligonucleotide primer anneals immediately upstream of the polymorphic site being genotyped, the primer and amplified target DNA are incubated with mass-modified dideoxynucleotide terminators. The primer extension is made according to the sequence of the variant site. d. The products of the reactions are spotted on a spectroCHIP. The CHIP is placed into the mass spectrometer and each spot is then shot with a laser under vacuum by the matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) method. A laser beam serves as desorption and ionization source in MALDI mass spectrometry. Once the sample molecules are vaporized and ionized, they are transferred electrostatically into a time-of-flight mass spectrometer (TOF-MS), where they individually detected. The mass of the extended primer is determined. e. Sequenom supplies software that automatically translates the mass of the observed primers into a genotype for each reaction
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Fig1: Steps in genotyping using MassARRAY® iPLEX System. A schematic of the genotype reaction of an A-to-G SNP. a. Locus-specific amplification reaction b. Treatment with SAP enzyme to neutralize unincorporated dNTPs. c. Locus-specific primer extension reaction (iPLEX assay). In this reaction, an oligonucleotide primer anneals immediately upstream of the polymorphic site being genotyped, the primer and amplified target DNA are incubated with mass-modified dideoxynucleotide terminators. The primer extension is made according to the sequence of the variant site. d. The products of the reactions are spotted on a spectroCHIP. The CHIP is placed into the mass spectrometer and each spot is then shot with a laser under vacuum by the matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) method. A laser beam serves as desorption and ionization source in MALDI mass spectrometry. Once the sample molecules are vaporized and ionized, they are transferred electrostatically into a time-of-flight mass spectrometer (TOF-MS), where they individually detected. The mass of the extended primer is determined. e. Sequenom supplies software that automatically translates the mass of the observed primers into a genotype for each reaction

Mentions: The iPLEX Gold technology consists of an initial locus-specific PCR reaction, followed by single base extension (SBE) using mass-modified dideoxynucleotide terminators of an oligonucleotide primer, which anneals immediately upstream of the polymorphic site of interest (Fig. 1) [15]. The product of these reactions are directly applied in a silicon chip. The mass of the extended primer is determined by means of MALDI-TOF mass spectrometry. The mass of the primer indicates the mutation of interest and the mass of added bases indicate the alleles present at the polymorphic site. Sequenom supplies a software (SpectroTYPER) that automatically translates the mass of the observed primers into a genotype for each reaction [16].Fig. 1


Screening of genetic alterations related to non-syndromic hearing loss using MassARRAY iPLEX® technology.

Svidnicki MC, Silva-Costa SM, Ramos PZ, dos Santos NZ, Martins FT, Castilho AM, Sartorato EL - BMC Med. Genet. (2015)

Steps in genotyping using MassARRAY® iPLEX System. A schematic of the genotype reaction of an A-to-G SNP. a. Locus-specific amplification reaction b. Treatment with SAP enzyme to neutralize unincorporated dNTPs. c. Locus-specific primer extension reaction (iPLEX assay). In this reaction, an oligonucleotide primer anneals immediately upstream of the polymorphic site being genotyped, the primer and amplified target DNA are incubated with mass-modified dideoxynucleotide terminators. The primer extension is made according to the sequence of the variant site. d. The products of the reactions are spotted on a spectroCHIP. The CHIP is placed into the mass spectrometer and each spot is then shot with a laser under vacuum by the matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) method. A laser beam serves as desorption and ionization source in MALDI mass spectrometry. Once the sample molecules are vaporized and ionized, they are transferred electrostatically into a time-of-flight mass spectrometer (TOF-MS), where they individually detected. The mass of the extended primer is determined. e. Sequenom supplies software that automatically translates the mass of the observed primers into a genotype for each reaction
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4581412&req=5

Fig1: Steps in genotyping using MassARRAY® iPLEX System. A schematic of the genotype reaction of an A-to-G SNP. a. Locus-specific amplification reaction b. Treatment with SAP enzyme to neutralize unincorporated dNTPs. c. Locus-specific primer extension reaction (iPLEX assay). In this reaction, an oligonucleotide primer anneals immediately upstream of the polymorphic site being genotyped, the primer and amplified target DNA are incubated with mass-modified dideoxynucleotide terminators. The primer extension is made according to the sequence of the variant site. d. The products of the reactions are spotted on a spectroCHIP. The CHIP is placed into the mass spectrometer and each spot is then shot with a laser under vacuum by the matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) method. A laser beam serves as desorption and ionization source in MALDI mass spectrometry. Once the sample molecules are vaporized and ionized, they are transferred electrostatically into a time-of-flight mass spectrometer (TOF-MS), where they individually detected. The mass of the extended primer is determined. e. Sequenom supplies software that automatically translates the mass of the observed primers into a genotype for each reaction
Mentions: The iPLEX Gold technology consists of an initial locus-specific PCR reaction, followed by single base extension (SBE) using mass-modified dideoxynucleotide terminators of an oligonucleotide primer, which anneals immediately upstream of the polymorphic site of interest (Fig. 1) [15]. The product of these reactions are directly applied in a silicon chip. The mass of the extended primer is determined by means of MALDI-TOF mass spectrometry. The mass of the primer indicates the mutation of interest and the mass of added bases indicate the alleles present at the polymorphic site. Sequenom supplies a software (SpectroTYPER) that automatically translates the mass of the observed primers into a genotype for each reaction [16].Fig. 1

Bottom Line: The generated results were validated using conventional techniques, as direct sequencing, multiplex PCR and RFLP-PCR.To optimize these results, the failed tests were re-designed and new primers were synthesized.However, we demonstrated that optimization is required to increase the genotyping success and accuracy.

View Article: PubMed Central - PubMed

Affiliation: Human Molecular Genetics Laboratory, Molecular Biology and Genetic Engineering Center (CBMEG), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. mcarolinasvid@gmail.com.

ABSTRACT

Background: Recent advances in molecular genetics have enabled to determine the genetic causes of non-syndromic hearing loss, and more than 100 genes have been related to the phenotype. Due to this extraordinary genetic heterogeneity, a large percentage of patients remain without any molecular diagnosis. This condition imply the need for new methodological strategies in order to detect a greater number of mutations in multiple genes. In this work, we optimized and tested a panel of 86 mutations in 17 different genes screened using a high-throughput genotyping technology to determine the molecular etiology of hearing loss.

Methods: The technology used in this work was the MassARRAY iPLEX® platform. This technology uses silicon chips and DNA amplification products for accurate genotyping by mass spectrometry of previous reported mutations. The generated results were validated using conventional techniques, as direct sequencing, multiplex PCR and RFLP-PCR.

Results: An initial genotyping of control subjects, showed failures in 20 % of the selected alterations. To optimize these results, the failed tests were re-designed and new primers were synthesized. Then, the specificity and sensitivity of the panel demonstrated values above 97 %. Additionally, a group of 180 individuals with NSHL without a molecular diagnosis was screened to test the diagnostic value of our panel, and mutations were identified in 30 % of the cases. In 20 % of the individuals, it was possible to explain the etiology of the HL. Mutations in GJB2 gene were the most prevalent, followed by other mutations in in SLC26A4, CDH23, MT-RNR1, MYO15A, and OTOF genes.

Conclusions: The MassARRAY technology has the potential for high-throughput identification of genetic variations. However, we demonstrated that optimization is required to increase the genotyping success and accuracy. The developed panel proved to be efficient and cost-effective, being suitable for applications involving the molecular diagnosis of hearing loss.

No MeSH data available.


Related in: MedlinePlus