High-throughput detection of mutations responsible for childhood hearing loss using resequencing microarrays.
BACKGROUND
Despite current knowledge of mutations in 45 genes that can cause nonsyndromic sensorineural hearing loss (SNHL), no unified clinical test has been developed that can comprehensively detect mutations in multiple genes. We therefore designed Affymetrix resequencing microarrays capable of resequencing 13 genes mutated in SNHL (GJB2, GJB6, CDH23, KCNE1, KCNQ1, MYO7A, OTOF, PDS, MYO6, SLC26A5, TMIE, TMPRSS3, USH1C). We present results from hearing loss arrays developed in two different research facilities and highlight some of the approaches we adopted to enhance the applicability of resequencing arrays in a clinical setting.


RESULTS
We leveraged sequence and intensity pattern features responsible for diminished coverage and accuracy and developed a novel algorithm, sPROFILER, which resolved >80% of no-calls from GSEQ and allowed 99.6% (range: 99.2-99.8%) of sequence to be called, while maintaining overall accuracy at >99.8% based upon dideoxy sequencing comparison.


CONCLUSIONS
Together, these findings provide insight into critical issues for disease-centered resequencing protocols suitable for clinical application and support the use of array-based resequencing technology as a valuable molecular diagnostic tool for pediatric SNHL and other genetic diseases with substantial genetic heterogeneity.
Kothiyal
Prachi
P
1Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.
Cox
Stephanie
S
Ebert
Jonathan
J
Husami
Ammar
A
Kenna
Margaret A
MA
Greinwald
John H
JH
Aronow
Bruce J
BJ
Rehm
Heidi L
HL
D000465:Algorithms; D001482:Base Composition; D000072259:Connexin 26; D017630:Connexins; D004252:DNA Mutational Analysis / Q000379:methods*; D006319:Hearing Loss, Sensorineural / Q000235:genetics*; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012680:Sensitivity and Specificity
D017630:Connexins; C000607042:GJB2 protein, human; D000072259:Connexin 26