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A rapid method for simultaneous screening of multi-gene mutations associated with hearing loss in the Korean population.

Sagong B, Baek JI, Oh SK, Na KJ, Bae JW, Choi SY, Jeong JY, Choi JY, Lee SH, Lee KY, Kim UK - PLoS ONE (2013)

Bottom Line: The GJB2, MT-RNR1, and SLC26A4 genes have been reported as common causative genes of hearing loss in the Korean population and some mutations of these genes are the most common mutations associated with hearing loss.We found that 4.06% of individuals with normal hearing and 4.32% of neonates were heterozygous carriers.Overall, we successfully developed a robust and cost-effective diagnosis method that detects common causative mutations of hearing loss in the Korean population.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, South Korea.

ABSTRACT
Hearing loss (HL) is a congenital disease with a high prevalence, and patients with hearing loss need early diagnosis for treatment and prevention. The GJB2, MT-RNR1, and SLC26A4 genes have been reported as common causative genes of hearing loss in the Korean population and some mutations of these genes are the most common mutations associated with hearing loss. Accordingly, we developed a method for the simultaneous detection of seven mutations (c.235delC of GJB2, c.439A>G, c.919-2A>G, c.1149+3A>G, c.1229C>T, c.2168A>G of SLC26A4, and m.1555A>G of the MT-RNR1 gene) using multiplex SNaPshot minisequencing to enable rapid diagnosis of hereditary hearing loss. This method was confirmed in patients with hearing loss and used for genetic diagnosis of controls with normal hearing and neonates. We found that 4.06% of individuals with normal hearing and 4.32% of neonates were heterozygous carriers. In addition, we detected that an individual is heterozygous for two different mutations of GJB2 and SLC26A4 gene, respectively and one normal hearing showing the heteroplasmy of m.1555A>G. These genotypes corresponded to those determined by direct sequencing. Overall, we successfully developed a robust and cost-effective diagnosis method that detects common causative mutations of hearing loss in the Korean population. This method will be possible to detect up to 40% causative mutations associated with prelingual HL in the Korean population and serve as a useful genetic technique for diagnosis of hearing loss for patients, carriers, neonates, and fetuses.

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Related in: MedlinePlus

The products of multiplex amplification.Agarose gel electrophoresis pattern of digested PCR products stained with ethidium bromide shows the multiplex amplification products of wild type (lane 1), mutant type (lane 2), 100 bp ladder marker (lane M), and corresponding size (bp) for each band of multiplex PCR products (table).
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pone-0057237-g001: The products of multiplex amplification.Agarose gel electrophoresis pattern of digested PCR products stained with ethidium bromide shows the multiplex amplification products of wild type (lane 1), mutant type (lane 2), 100 bp ladder marker (lane M), and corresponding size (bp) for each band of multiplex PCR products (table).

Mentions: The seven genomic segments containing each mutation were amplified in a single multiplex PCR reaction, after which the amplified DNA fragments were separated and visualized on agarose gel (Figure 1). We developed a genetic diagnostic technique consisting of SNaPshot multiplex minisequencing of seven mutations in three genes. Five positive controls for the mutant allele of the seven mutations and one normal control with wild-type alleles were analyzed and correctly genotyped in all reactions (Figure 2). Normal controls were genotyped to wild-type in every mutant region (Figure 2, first panel). Positive controls were genotyped mutant types in each mutant region (Figure 2, second to sixth panels). Relative differences in peak heights between each allele were observed because fluorescence emission can be directly influenced by interaction between other fluorophores. In addition, the results of SNaPshot minisequencing were compared with those of Sanger sequencing of control samples, and these results were matched exactly.


A rapid method for simultaneous screening of multi-gene mutations associated with hearing loss in the Korean population.

Sagong B, Baek JI, Oh SK, Na KJ, Bae JW, Choi SY, Jeong JY, Choi JY, Lee SH, Lee KY, Kim UK - PLoS ONE (2013)

The products of multiplex amplification.Agarose gel electrophoresis pattern of digested PCR products stained with ethidium bromide shows the multiplex amplification products of wild type (lane 1), mutant type (lane 2), 100 bp ladder marker (lane M), and corresponding size (bp) for each band of multiplex PCR products (table).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585873&req=5

pone-0057237-g001: The products of multiplex amplification.Agarose gel electrophoresis pattern of digested PCR products stained with ethidium bromide shows the multiplex amplification products of wild type (lane 1), mutant type (lane 2), 100 bp ladder marker (lane M), and corresponding size (bp) for each band of multiplex PCR products (table).
Mentions: The seven genomic segments containing each mutation were amplified in a single multiplex PCR reaction, after which the amplified DNA fragments were separated and visualized on agarose gel (Figure 1). We developed a genetic diagnostic technique consisting of SNaPshot multiplex minisequencing of seven mutations in three genes. Five positive controls for the mutant allele of the seven mutations and one normal control with wild-type alleles were analyzed and correctly genotyped in all reactions (Figure 2). Normal controls were genotyped to wild-type in every mutant region (Figure 2, first panel). Positive controls were genotyped mutant types in each mutant region (Figure 2, second to sixth panels). Relative differences in peak heights between each allele were observed because fluorescence emission can be directly influenced by interaction between other fluorophores. In addition, the results of SNaPshot minisequencing were compared with those of Sanger sequencing of control samples, and these results were matched exactly.

Bottom Line: The GJB2, MT-RNR1, and SLC26A4 genes have been reported as common causative genes of hearing loss in the Korean population and some mutations of these genes are the most common mutations associated with hearing loss.We found that 4.06% of individuals with normal hearing and 4.32% of neonates were heterozygous carriers.Overall, we successfully developed a robust and cost-effective diagnosis method that detects common causative mutations of hearing loss in the Korean population.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, South Korea.

ABSTRACT
Hearing loss (HL) is a congenital disease with a high prevalence, and patients with hearing loss need early diagnosis for treatment and prevention. The GJB2, MT-RNR1, and SLC26A4 genes have been reported as common causative genes of hearing loss in the Korean population and some mutations of these genes are the most common mutations associated with hearing loss. Accordingly, we developed a method for the simultaneous detection of seven mutations (c.235delC of GJB2, c.439A>G, c.919-2A>G, c.1149+3A>G, c.1229C>T, c.2168A>G of SLC26A4, and m.1555A>G of the MT-RNR1 gene) using multiplex SNaPshot minisequencing to enable rapid diagnosis of hereditary hearing loss. This method was confirmed in patients with hearing loss and used for genetic diagnosis of controls with normal hearing and neonates. We found that 4.06% of individuals with normal hearing and 4.32% of neonates were heterozygous carriers. In addition, we detected that an individual is heterozygous for two different mutations of GJB2 and SLC26A4 gene, respectively and one normal hearing showing the heteroplasmy of m.1555A>G. These genotypes corresponded to those determined by direct sequencing. Overall, we successfully developed a robust and cost-effective diagnosis method that detects common causative mutations of hearing loss in the Korean population. This method will be possible to detect up to 40% causative mutations associated with prelingual HL in the Korean population and serve as a useful genetic technique for diagnosis of hearing loss for patients, carriers, neonates, and fetuses.

Show MeSH
Related in: MedlinePlus