Targeted next generation sequencing for molecular diagnosis of Usher syndrome

Abstract Background: The USH2A gene encodes usherin, a basement membrane protein that is involved in the development and homeostasis of the inner ear and retina. Mutations in USH2A are linked to Usher syndrome type II (USH II) and non-syndromic retinitis pigmentosa (RP). Molecular diagnosis can provide insight into the pathogenesis of these diseases, facilitate clinical diagnosis, and identify individuals who can most benefit from gene or cell replacement therapy. Here, we report 21 pathogenic mutations in the USH2A gene identified in 11 Chinese families by using the targeted next-generation sequencing (NGS) technology. Methods: In all, 11 unrelated Chinese families were enrolled, and NGS was performed to identify mutations in the USH2A gene. Variant analysis, Sanger validation, and segregation tests were utilized to validate the disease-causing mutations in these families. Results: We identified 21 pathogenic mutations, of which 13, including 5 associated with non-syndromic RP and 8 with USH II, have not been previously reported. The novel variants segregated with disease phenotype in the affected families and were absent from the control subjects. In general, visual impairment and retinopathy were consistent between the USH II and non-syndromic RP patients with USH2A mutations. Conclusions: These findings provide a basis for investigating genotype–phenotype relationships in Chinese USH II and RP patients and for clarifying the pathophysiology and molecular mechanisms of the diseases associated with USH2A mutations.

Download Full-text

Targeted next-generation sequencing for molecular diagnosis of endometriosis-associated ovarian cancer

Journal of Molecular Medicine ◽

10.1007/s00109-016-1395-2 ◽

2016 ◽

Vol 94 (7) ◽

pp. 835-847 ◽

Cited By ~ 33

Author(s):

Tze-Kiong Er ◽

Yu-Fa Su ◽

Chun-Chieh Wu ◽

Chih-Chieh Chen ◽

Jing Wang ◽

...

Keyword(s):

Ovarian Cancer ◽

Next Generation Sequencing ◽

Molecular Diagnosis ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Generation Sequencing

Download Full-text

Molecular diagnosis of inherited peripheral neuropathies by targeted next-generation sequencing: molecular spectrum delineation

BMJ Open ◽

10.1136/bmjopen-2018-021632 ◽

2018 ◽

Vol 8 (10) ◽

pp. e021632 ◽

Cited By ~ 13

Author(s):

Juliette Bacquet ◽

Tanya Stojkovic ◽

Amandine Boyer ◽

Nathalie Martini ◽

Frédérique Audic ◽

...

Keyword(s):

Next Generation Sequencing ◽

Molecular Diagnosis ◽

Sanger Sequencing ◽

Diagnostic Yield ◽

Motor Neuropathy ◽

Peripheral Neuropathies ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Targeted Ngs ◽

Generation Sequencing

PurposeInherited peripheral neuropathies (IPN) represent a large heterogenous group of hereditary diseases with more than 100 causative genes reported to date. In this context, targeted next-generation sequencing (NGS) offers the opportunity to screen all these genes with high efficiency in order to unravel the genetic basis of the disease. Here, we compare the diagnostic yield of targeted NGS with our previous gene by gene Sanger sequencing strategy. We also describe several novel likely pathogenic variants.Design and participantsWe have completed the targeted NGS of 81 IPN genes in a cohort of 123 unrelated patients affected with diverse forms of IPNs, mostly Charcot-Marie-Tooth disease (CMT): 23% CMT1, 52% CMT2, 9% distal hereditary motor neuropathy, 7% hereditary sensory and autonomic neuropathy and 6.5% intermediate CMT.ResultsWe have solved the molecular diagnosis in 49 of 123 patients (~40%). Among the identified variants, 26 variants were already reported in the literature. In our cohort, the most frequently mutated genes are respectively:MFN2,SH3TC2,GDAP1,NEFL,GAN,KIF5AandAARS. Panel-based NGS was more efficient in familial cases than in sporadic cases (diagnostic yield 49%vs19%, respectively). NGS-based search for copy number variations, allowed the identification of three duplications in three patients and raised the diagnostic yield to 41%. This yield is two times higher than the one obtained previously by gene Sanger sequencing screening. The impact of panel-based NGS screening is particularly important for demyelinating CMT (CMT1) subtypes, for which the success rate reached 87% (36% only for axonal CMT2).ConclusionNGS allowed to identify causal mutations in a shorter and cost-effective time. Actually, targeted NGS is a well-suited strategy for efficient molecular diagnosis of IPNs. However, NGS leads to the identification of numerous variants of unknown significance, which interpretation requires interdisciplinary collaborations between molecular geneticists, clinicians and (neuro)pathologists.

Download Full-text