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. 2014 Nov 18:9:168.
doi: 10.1186/s13023-014-0168-7.

Targeted next generation sequencing for molecular diagnosis of Usher syndrome

María J Aparisi  1   2 , Elena Aller  3   4 , Carla Fuster-García  5 , Gema García-García  6   7 , Regina Rodrigo  8   9 , Rafael P Vázquez-Manrique  10   11 , Fiona Blanco-Kelly  12   13 , Carmen Ayuso  14   15 , Anne-Françoise Roux  16 , Teresa Jaijo  17   18 , José M Millán  19   20   21
Affiliations

Targeted next generation sequencing for molecular diagnosis of Usher syndrome

María J Aparisi et al. Orphanet J Rare Dis. .

Abstract

Background: Usher syndrome is an autosomal recessive disease that associates sensorineural hearing loss, retinitis pigmentosa and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous. To date, 10 genes have been associated with the disease, making its molecular diagnosis based on Sanger sequencing, expensive and time-consuming. Consequently, the aim of the present study was to develop a molecular diagnostics method for Usher syndrome, based on targeted next generation sequencing.

Methods: A custom HaloPlex panel for Illumina platforms was designed to capture all exons of the 10 known causative Usher syndrome genes (MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31 and CLRN1), the two Usher syndrome-related genes (HARS and PDZD7) and the two candidate genes VEZT and MYO15A. A cohort of 44 patients suffering from Usher syndrome was selected for this study. This cohort was divided into two groups: a test group of 11 patients with known mutations and another group of 33 patients with unknown mutations.

Results: Forty USH patients were successfully sequenced, 8 USH patients from the test group and 32 patients from the group composed of USH patients without genetic diagnosis. We were able to detect biallelic mutations in one USH gene in 22 out of 32 USH patients (68.75%) and to identify 79.7% of the expected mutated alleles. Fifty-three different mutations were detected. These mutations included 21 missense, 8 nonsense, 9 frameshifts, 9 intronic mutations and 6 large rearrangements.

Conclusions: Targeted next generation sequencing allowed us to detect both point mutations and large rearrangements in a single experiment, minimizing the economic cost of the study, increasing the detection ratio of the genetic cause of the disease and improving the genetic diagnosis of Usher syndrome patients.

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Figures

Figure 1
Figure 1
Mean coverage obtained for the different genes. The blue line shows the lower limit of coverage appropriate to perform CNV analyses (250x).
Figure 2
Figure 2
Mean coverage of all targeted regions included in our custom design.
Figure 3
Figure 3
Images obtained in the validation and quantification of the enriched target DNA with the 2100 Bioanalyzer system. A) Typical image obtained in most patients. B) Atypical image obtained in the patient RP-531, in whom CNV analysis could not be performed.
See this image and copyright information in PMC

References

    1. Espinos C, Millan JM, Beneyto M, Najera C. Epidemiology of Usher syndrome in Valencia and Spain. Community Genet. 1998;1:223–228. doi: 10.1159/000016167. - DOI - PubMed
    1. Keats BJ, Corey DP. The Usher syndromes. Am J Med Genet. 1999;89:158–166. doi: 10.1002/(SICI)1096-8628(19990924)89:3<158::AID-AJMG6>3.0.CO;2-#. - DOI - PubMed
    1. Cohen M, Bitner-Glindzicz M, Luxon L. The changing face of Usher syndrome: clinical implications. Int J Audiol. 2007;46:82–93. doi: 10.1080/14992020600975279. - DOI - PubMed
    1. Millán JM, Aller E, Jaijo T, Blanco-Kelly F, Gimenez-Pardo A, Ayuso C. An update on the genetics of Usher syndrome. J Ophthalmol. 2011;2011:417217. doi: 10.1155/2011/417217. - DOI - PMC - PubMed
    1. Chaïb H, Kaplan J, Gerber S, Vincent C, Ayadi H, Slim R, Munnich A, Weissenbach J, Petit C. A newly identified locus for Usher syndrome type I, USH1E, maps to chromosome 21q21. Hum Mol Genet. 1997;6:27–31. doi: 10.1093/hmg/6.1.27. - DOI - PubMed

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