Massively Parallel DNA Sequencing Facilitates Diagnosis of Patients with Usher Syndrome Type 1

Abstract

Usher syndrome is an autosomal recessive disorder manifesting hearing loss, retinitis pigmentosa and vestibular dysfunction, and having three clinical subtypes. Usher syndrome type 1 is the most severe subtype due to its profound hearing loss, lack of vestibular responses, and retinitis pigmentosa that appears in prepuberty. Six of the corresponding genes have been identified, making early diagnosis through DNA testing possible, with many immediate and several long-term advantages for patients and their families. However, the conventional genetic techniques, such as direct sequence analysis, are both time-consuming and expensive. Targeted exon sequencing of selected genes using the massively parallel DNA sequencing technology will potentially enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis.Using this technique combined with direct sequence analysis, we screened 17 unrelated Usher syndrome type 1 patients and detected probable pathogenic variants in the 16 of them (94.1%) who carried at least one mutation. Seven patients had the MYO7A mutation (41.2%), which is the most common type in Japanese. Most of the mutations were detected by only the massively parallel DNA sequencing. We report here four patients, who had probable pathogenic mutations in two different Usher syndrome type 1 genes, and one case of MYO7A/PCDH15 digenic inheritance.This is the first report of Usher syndrome mutation analysis using massively parallel DNA sequencing and the frequency of Usher syndrome type 1 genes in Japanese. Mutation screening using this technique has the power to quickly identify mutations of many causative genes while maintaining cost-benefit performance. In addition, the simultaneous mutation analysis of large numbers of genes is useful for detecting mutations in different genes that are possibly disease modifiers or of digenic inheritance.

Novel and Recurrent MYO7A Mutations in Usher Syndrome Type 1 and Type 2

“…Similarly, multiple splice site mutations of the MYO7A gene have been reported previously [36], [56], [57], and two of those mutations were proved to alter the natural splicing through hybrid minigene assays [58]. In our case, in an attempt to understand the possible protein change resulted from c.1343+1G>A, we applied four online prediction software to evaluate the effect of c.1343+1G>A on splicing.…”

Partial USH2A deletions contribute to Usher syndrome in Denmark

“…The remaining 35 patients had no function-affecting sequence variants identified before this investigation. The eight patients with two sequence variants in USH2A were included, as patients with three different function-affecting sequence variants located in two different genes 19,23 as well as patients with three different sequence variants located in the same gene, including USH2A, have been published previously. 15,24 The project was approved by the local ethics committee (H-3-2011-070) and carried out in accordance with the Declaration of Helsinki.…”

Deafness Gene Variations in a 1120 Nonsyndromic Hearing Loss Cohort

“…[3][4][5][6][7][8][9][10][11][12][13][14][15][16] We applied MPS technology to (1) discover causative mutations in relatively rare causative genes 12,13 and (2) clarify the molecular epidemiology. [3][4][5][6][7][8][9][10][11][12][13][14][15][16] We applied MPS technology to (1) discover causative mutations in relatively rare causative genes 12,13 and (2) clarify the molecular epidemiology.…”

“…11,13,16,17 In the current study, on the basis of our PCR-based technologies in combination with MPS, 13,17 we increased the number of patients (1120 cases of nonsyndromic hearing loss) to establish a database for clinical molecular diagnosis and to confirm the molecular epidemiology of deafness. Hybridization-based capture is commonly used for genomic target enrichment, but for clinical application, polymerase chain reaction (PCR)-based technologies in combination with MPS have also been proposed.…”