The goal of our study was to apply DNA capture and MPS to identify inherited mutations involved in hearing loss. We designed oligonucleotides to capture the exons and regulatory regions of 246 genes involved in hearing loss, in human or in mouse. The inclusion of genes thus far known to be involved in deafness in the mouse is based on the observation that many genes for human deafness are responsible for mouse deafness as well [23
]. Among the genes harboring mutations causing deafness only in the mouse, no deleterious mutations were present in these 11 human families. The mouse genes will be sequenced from DNA of many more human families in the future.
Comprehensive targeted enrichment and MPS has been employed previously for non-syndromic hearing loss [25
]. Our approach targeted more genes (246 versus 54), including in particular genes associated with deafness in the mouse. Our goal in including these genes is to speed future discovery of additional human deafness genes that are orthologues of known mouse genes.
To date, routine clinical diagnostic tests for deafness in the Middle East have consisted of restriction enzyme analysis of the two common GJB2
mutations, and on occasion, DNA sequencing of the GJB2
coding region. In some clinics, screening for the relevant mutations in other genes on the basis of ethnic origin, audiological tests, family history, personal history and findings from physical examination may be performed. Comprehensive testing for genes with mutations common in other populations, such as TMC1
] or SLC26A4
], is not available from health services in the Middle East due to the high cost of testing these genes by Sanger sequencing. The large size of these genes has also precluded their analysis in Middle Eastern research laboratories.
A major challenge for mutation discovery is determining which variants are potentially causative and which are likely benign. This is particularly difficult when sequencing populations that are not well represented in dbSNP. A novel variant may represent a previously undiscovered common population-specific polymorphism or a truly private mutation. Sequencing even a small number of samples (say 100) from the same ethnic background serves as a very effective filter. In our study, many variants not in dbSNP were nonetheless common in our populations and could be ruled out as causative mutations (Additional file 3
). As a result, a smaller fraction of the detected variants had to be verified by Sanger sequencing for segregation in the family.
For the Israeli deaf population of Moroccan Jewish ancestry, this study has substantial clinical implications, as the TMC1
gene was found to be very frequently involved in deafness in this population. Recessive mutations in TMC1
were detected in more than a third (38%) of hearing impaired Jews of Moroccan origin. A single DNA sample of a Moroccan Jewish proband, evaluated by this approach, led to the discovery of four mutations, two of them novel, and solved the cause of hearing loss of an additional 20 families. The TMC1
gene is the sixth most common cause of recessive hearing loss worldwide [27
]. The two novel mutations in Moroccan Jewish deaf individuals add to the 30 recessive mutations that have been reported to date in the TMC1
]. In some populations, including Iran [26
] and Turkey [11
], as Israel, TMC1
is one of the genes most frequently involved in deafness. Based on these results, we recommend that all Israeli Jewish probands of Moroccan ancestry be screened for the four TMC1
mutations, as well as for the most common GJB2
mutations, prior to conducting MPS. An immediate result of these findings is that screening for TMC1
mutations will become routine in Israel for all hearing impaired patients of Moroccan Jewish ancestry.
Novel mutations were identified in multiple other genes - CDH23, MYO15A, WFS1, and TECTA - that are known to be responsible for hearing loss but are not routinely evaluated, largely because of their size. Targeted MPS makes it feasible to screen large genes that have heretofore been largely untested. As sequencing chemistry improves, we believe it will be feasible to multiplex 12 samples per lane and still maintain a high coverage (> 200 ×). It will thus become even more straightforward to screen comprehensively for all known hearing loss genes.
Of the six Palestinian families enrolled in this study, a causative mutation was found in only one. This result is probably due to two factors. First, familial hearing loss in the Palestinian population has been very thoroughly investigated for more than a decade, with the discovery of many critical genes and the characterization of the mutational spectra of these genes as they were identified (for example, [5
]). Therefore, the mutations responsible for hearing loss in many Palestinian families were known before this project was undertaken. Second, as the result of historical marriage patterns, inherited hearing loss in the Palestinian population is likely to be more heterogeneous, at the levels of both alleles and loci, than is inherited hearing loss in the Israeli population. A large proportion of Palestinian families are likely to have hearing loss due to as yet unknown genes. Since the molecular basis of deafness in most of our Palestinian probands was unsolved, we predict that many new genes for hearing loss remain to be found. These may be optimally resolved by exome sequencing in combination with homozygosity mapping, as we previously demonstrated [6