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Diagnostic application of targeted resequencing for familial nonsyndromic hearing loss.

Choi BY, Park G, Gim J, Kim AR, Kim BJ, Kim HS, Park JH, Park T, Oh SH, Han KH, Park WY - PLoS ONE (2013)

Bottom Line: Each proband carried 4.8 variants that were not synonymous and had the occurring frequency of less than three among the 20 probands.These variants were then filtered out with the inheritance pattern of the family, allele frequency in normal hearing 80 control subjects, clinical features.Finally NSHL-causing candidate mutations were identified in 13(65%) of the 20 probands of multiplex families, bringing the total solve rate (or detection rate) in our familial cases to be 78.1% (25/32) Damaging mutations discovered by the targeted resequencing were distributed in nine genes such as WFS1, COCH, EYA4, MYO6, GJB3, COL11A2, OTOF, STRC and MYO3A, most of which were private.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Seoul National University Bundang Hospital, Seongnam, Korea.

ABSTRACT
Identification of causative genes for hereditary nonsyndromic hearing loss (NSHL) is important to decide treatment modalities and to counsel the patients. Due to the genetic heterogeneity in sensorineural genetic disorders, the high-throughput method can be adapted for the efficient diagnosis. To this end, we designed a new diagnostic pipeline to screen all the reported candidate genes for NSHL. For validation of the diagnostic pipeline, we focused upon familial NSHL cases that are most likely to be genetic, rather than to be infectious or environmental. Among the 32 familial NSHL cases, we were able to make a molecular genetic diagnosis from 12 probands (37.5%) in the first stage by their clinical features, characteristic inheritance pattern and further candidate gene sequencing of GJB2, SLC26A4, POU3F4 or mitochondrial DNA. Next we applied targeted resequencing on 80 NSHL genes in the remaining 20 probands. Each proband carried 4.8 variants that were not synonymous and had the occurring frequency of less than three among the 20 probands. These variants were then filtered out with the inheritance pattern of the family, allele frequency in normal hearing 80 control subjects, clinical features. Finally NSHL-causing candidate mutations were identified in 13(65%) of the 20 probands of multiplex families, bringing the total solve rate (or detection rate) in our familial cases to be 78.1% (25/32) Damaging mutations discovered by the targeted resequencing were distributed in nine genes such as WFS1, COCH, EYA4, MYO6, GJB3, COL11A2, OTOF, STRC and MYO3A, most of which were private. Despite the advent of whole genome and whole exome sequencing, we propose targeted resequencing and filtering strategy as a screening and diagnostic tool at least for familial NSHL to find mutations based upon its efficacy and cost-effectiveness.

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Related in: MedlinePlus

Validation of candidate mutations by PCR-Sanger sequencing.Candidate mutations in 9 autosomal dominant and 4 autosomal recessive NSHL families were shown in chromatogram of Sanger sequencing.
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pone-0068692-g002: Validation of candidate mutations by PCR-Sanger sequencing.Candidate mutations in 9 autosomal dominant and 4 autosomal recessive NSHL families were shown in chromatogram of Sanger sequencing.

Mentions: We selected rare single nucleotide variations (SNV) or indels following five steps of filtering to find candidate mutations related to hearing loss in each patient (Table 2). In a basic filtering step, variations with a quality score of less than 20 were discarded, and for heterozygous alleles, only the alleles with a ratio (coverage of variant over the total coverage) of 20% or more were included. The average number of variants was 4.8±0.42 per patient after basic filtering. As a second step, we checked inheritance pattern of multiplex family of each proband (Fig. 1), and excluded the variants which were not matched with the patient's inheritance pattern. According to the information on the inheritance, we could significantly reduce the average numbers of candidate mutations to 1.95±0.29 per patient (t-test p = 2.8×10−6). In three families, all the mutations were not matched with the inheritance pattern. In the third step, we validated 39 variants from the 18 probands by Sanger sequencing and confirmed 36 variants (92.3%) (Fig. 2). When we checked 80 normal hearing control subjects for the variants by Sanger sequencing, seven variants were also found in Korean population. As a final step for the filtering, we investigated segregation and/or phenotype matching to confirm the causality of the variant for deafness. Nineteen variants were examined in nine families by Sanger sequencing in all the family members to exclude 8 variants. We also examined patient's audiogram to match the candidate genes with the patient's phenotype and ruled out eleven variants, too. Especially, in cases where the segregation study could not be performed, we relied upon the audiogram configuration. Molecular genetic diagnosis was made in four subjects (SB61-109, SB55-102, SB50-94 and SB47-91) despite the lack of segregation study results, since their audiograms were well matched the previously reported characteristic audiogram configuration. Finally, we were able to find a most likely causative mutation in 13 out of 20 multiplex hearing loss families (Table 3).


Diagnostic application of targeted resequencing for familial nonsyndromic hearing loss.

Choi BY, Park G, Gim J, Kim AR, Kim BJ, Kim HS, Park JH, Park T, Oh SH, Han KH, Park WY - PLoS ONE (2013)

Validation of candidate mutations by PCR-Sanger sequencing.Candidate mutations in 9 autosomal dominant and 4 autosomal recessive NSHL families were shown in chromatogram of Sanger sequencing.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750053&req=5

pone-0068692-g002: Validation of candidate mutations by PCR-Sanger sequencing.Candidate mutations in 9 autosomal dominant and 4 autosomal recessive NSHL families were shown in chromatogram of Sanger sequencing.
Mentions: We selected rare single nucleotide variations (SNV) or indels following five steps of filtering to find candidate mutations related to hearing loss in each patient (Table 2). In a basic filtering step, variations with a quality score of less than 20 were discarded, and for heterozygous alleles, only the alleles with a ratio (coverage of variant over the total coverage) of 20% or more were included. The average number of variants was 4.8±0.42 per patient after basic filtering. As a second step, we checked inheritance pattern of multiplex family of each proband (Fig. 1), and excluded the variants which were not matched with the patient's inheritance pattern. According to the information on the inheritance, we could significantly reduce the average numbers of candidate mutations to 1.95±0.29 per patient (t-test p = 2.8×10−6). In three families, all the mutations were not matched with the inheritance pattern. In the third step, we validated 39 variants from the 18 probands by Sanger sequencing and confirmed 36 variants (92.3%) (Fig. 2). When we checked 80 normal hearing control subjects for the variants by Sanger sequencing, seven variants were also found in Korean population. As a final step for the filtering, we investigated segregation and/or phenotype matching to confirm the causality of the variant for deafness. Nineteen variants were examined in nine families by Sanger sequencing in all the family members to exclude 8 variants. We also examined patient's audiogram to match the candidate genes with the patient's phenotype and ruled out eleven variants, too. Especially, in cases where the segregation study could not be performed, we relied upon the audiogram configuration. Molecular genetic diagnosis was made in four subjects (SB61-109, SB55-102, SB50-94 and SB47-91) despite the lack of segregation study results, since their audiograms were well matched the previously reported characteristic audiogram configuration. Finally, we were able to find a most likely causative mutation in 13 out of 20 multiplex hearing loss families (Table 3).

Bottom Line: Each proband carried 4.8 variants that were not synonymous and had the occurring frequency of less than three among the 20 probands.These variants were then filtered out with the inheritance pattern of the family, allele frequency in normal hearing 80 control subjects, clinical features.Finally NSHL-causing candidate mutations were identified in 13(65%) of the 20 probands of multiplex families, bringing the total solve rate (or detection rate) in our familial cases to be 78.1% (25/32) Damaging mutations discovered by the targeted resequencing were distributed in nine genes such as WFS1, COCH, EYA4, MYO6, GJB3, COL11A2, OTOF, STRC and MYO3A, most of which were private.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Seoul National University Bundang Hospital, Seongnam, Korea.

ABSTRACT
Identification of causative genes for hereditary nonsyndromic hearing loss (NSHL) is important to decide treatment modalities and to counsel the patients. Due to the genetic heterogeneity in sensorineural genetic disorders, the high-throughput method can be adapted for the efficient diagnosis. To this end, we designed a new diagnostic pipeline to screen all the reported candidate genes for NSHL. For validation of the diagnostic pipeline, we focused upon familial NSHL cases that are most likely to be genetic, rather than to be infectious or environmental. Among the 32 familial NSHL cases, we were able to make a molecular genetic diagnosis from 12 probands (37.5%) in the first stage by their clinical features, characteristic inheritance pattern and further candidate gene sequencing of GJB2, SLC26A4, POU3F4 or mitochondrial DNA. Next we applied targeted resequencing on 80 NSHL genes in the remaining 20 probands. Each proband carried 4.8 variants that were not synonymous and had the occurring frequency of less than three among the 20 probands. These variants were then filtered out with the inheritance pattern of the family, allele frequency in normal hearing 80 control subjects, clinical features. Finally NSHL-causing candidate mutations were identified in 13(65%) of the 20 probands of multiplex families, bringing the total solve rate (or detection rate) in our familial cases to be 78.1% (25/32) Damaging mutations discovered by the targeted resequencing were distributed in nine genes such as WFS1, COCH, EYA4, MYO6, GJB3, COL11A2, OTOF, STRC and MYO3A, most of which were private. Despite the advent of whole genome and whole exome sequencing, we propose targeted resequencing and filtering strategy as a screening and diagnostic tool at least for familial NSHL to find mutations based upon its efficacy and cost-effectiveness.

Show MeSH
Related in: MedlinePlus